Regulation of Coronary Blood Flow PDF

M. Inoue· M. Hori· S. Imai· R.M.
Berne
(Eds.)
Regulation of
Coronary Blood Flow
With 149 Illustrations
Springer Japan KK
Michitoshi Inoue, M.D., Ph.D.
Professor, Department of Medical Information Sciences, Osaka University Hospital,
Osaka, 553 Japan
Masatsugu Hori, M.D., Ph.D.
Assistant Professor, The First Department of Medicine, Osaka University School of
Medicine, Osaka, 553 Japan
Shoichi /mai, M.D., Ph.D.
Professor, Department of Pharmacology, Niigata University School of Medicine, Niigata,
951 Japan
Robert M. Berne, M.D.
Alum ni Professor, Department of Physiology, University of Virginia School of Medici ne,
Charlottesville, VA 22908, US
On the frontcover: Scanning electron micrographs of polycarbonate fii ters through which 1 : 5
diluted whole blood has been passed for 30s after the addition of 2 nM PAF. See p. 174.
The publication was supported in part by the grant-in-aid for scientific research of the Japanese
Ministry of Education, Science and Culture.
ISBN 978-4-431-68369-8 ISBN 978-4-431-68367-4 (eBook)

DOI 10.1007/978-4-431-68367-4
Library of Congress Cataloging-in-Publication Data
Regulation of coronary blood ftow 1 M. Inoue ... [et al.] (eds.). p. cm. Based on the satellite
symposium of the 4th International Symposium on Adenosine and Adenine Nucleotides, in Kobe
in 1990. Includes bibliographical references and index. ISBN 978-4-431-68369-8
l. Coronary circulation-Regulation-Congresses. 2. Coronary circulation-Measurement-
Congresses. 3. Adenosine-Physiological effect-Congresses. 4. Adeno·sine-Receptors-Con-
gresses. 5. Coronary heart disease-Pathophysiology-Congresses.
1. Inoue, Michitoshi, 1937- . Il. International Symposium on Adenosine and Adenine Nucleo-
tides (4th: 1990:Yamanaka Lake, Japan) [DNLM: l. Adenosine-physiology-congresses.
2. Coronary Circulation-physiology-congresses. 3. Endothelium-physiology-congresses.
WG 300 R344 1990], QPIOS.R45 1991, 612.1'7-dc20, DNLM/DLC, for Library of Congress.
91-5083
©Springer Japan 1991

Originally published by Springer-Verlag Tokyo in 1991
Softcover reprint of the hardcover 1st edition 1991
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Preface
The importance of the physiology and pathophysiology of coronary circulation

has been increasing over the last eighty years since Dr. James Herrick first
described in 1912 the fact that acute myocardial infarction is attributed to the
obstruction of the coronary arteries. Although extensive research has been
done to prevent coronary arterial disease, the death rate due to heart disease
is still high: 35% of total deaths in the United States, and 20% in Japan. In
recent decades, cardiovascular researchers have mainly focused their efforts on
the physiology of coronary circulation, and extensive and elegant physiological
studies have been accomplished in animals. Recent progress in biochemistry is
opening up new fields, with research focusing on the novel substances involved
in the regulation of coronary circulation, the roles of endothelial cells and
interactions between the circulating blood cells. In the last decade, cardio-
vascular researchers seem to have moved from the mechanical to humoral
regulation of coronary blood flow. Another big impact is technical progress in
clinical intervention for reperfusion and angioplasty in ischemic heart disease,
including PTCR and PTCA.
We have had the timely opportunity to hold the Satellite Symposium of the
4th International Symposium on Adenosine and Adenine Nucleotides in Kobe
in 1990, focusing on "Regulation of Coronary Blood Flow." R.M. Berne of
Virginia University summarized the role of adenosine in regulation of coronary
blood flow, and E.O. Feigl of Washington University overviewed neural
control, in their special lectures. This book is based upon the fruitful outcome
of this meeting, as the proceedings of the symposium. The volumes are divided
into six parts. Part 1 focuses on methods of measuring coronary blood flow.
Part 2 deals with basic neural control mechanisms of coronary blood flow.
Recently, the contribution of endothelial cells in coronary flow regulation
is found to be important. Part 3 emphasizes the roles of endothelial cellular
functions. Part 4 centers on metabolic controls of coronary blood flow. Parts 5
and 6 consider the consequences due to abnormalities in coronary circulation.
Although they do not cover the whole spectra of the physiological and patho-
physiological states, I believe this volume provides readers with a unique
aspect of the regulatory mechanisms of coronary blood flow.
v
VI Preface
I am most grateful to the contributors for their efforts in providing the

manuscripts in a timely fashion. Finally my acknowledgments are cordially
given to the publisher Springer-Verlag, Tokyo for their great help with this
publication and also to the Ichiro Kanehara Foundation for their financial
support.
Michitoshi Inoue
for Editors
Contents
Preface......................................................... V
List of Contributors .............................................. XI
A New Approaches for Coronary Flow Measurement

1. A Doppler Catheter Technique Using Fast Fourier Spectrum
Analysis for the Assessment of Coronary Flow Dynamics
Akira Kitabatake, fun Tanouchi, Masaaki Uematsu, Yasuji Doi,
and Masatsugu Hori .......................................... 3
2. A Study of Coronary Circulation by Laser Doppler Velocimetry
Fumihiko Kajiya, Osamu Hiramatsu, Yasuo Ogasawara,
Keiichiro Mito, and Katsuhiko Tsujioka . . . . . . . . . . . . . . . . . . . . . . . . . . 11
3. Direct Observation of the Coronary Microvasculature in a Beating
Heart by the Floating Objective System
Hiroshi Kanatsuka, Kouichi Ashikawa, Nobuyo Sekiguchi,
Tatsuya Komaru, Toshimi Suzuki, and Tamotsu Takishima . . . . . . . . . 24
4. PET Measurement of Myocardial Blood Flow
Nagara Tamaki, Yoshiharu Yonekura, and funji Konishi. . . . . . . . . . . 34
B Neural Control of Coronary Blood Flow
1. Autonomic Control of Coronary Blood Flow

Eric O. Feigl. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
2. Role of Alpha-Adrenoceptor Activity in Regulation of Coronary
Blood Flow During Myocardial Ischemia
Masatsugu Hori, Masafumi Kitakaze, Takenobu Kamada,
Akira Kitabatake, and Michitoshi Inoue. . . . . . . . . . . . . . . . . . . . . . . . . . 61
VII
VIII Contents
3. Vasoacative Monoamines in the Regulation of Arterial Tone

Mitsuhiro Yokoyama and Hozuka Akita. . . . . . . . . . . . . . . . . . . . . . . . . 78
4. Coronary Vasomotion During Exercise Influence of the Geometry
of Stenosis
K. Eid, O.M. Hess, Th. Suter, A. Bortone, 1. Grimm,
and H. P. Krayenbuehl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
C Role of Adenosine in Regulation of Coronary Blood Flow
1. The Role of Adenosine in the Metabolic Regulation of Coronary

Blood Flow
Robert M. Berne ............................................. 109
2. Adenosine Receptors in the Heart
Masayuki Ueeda and Ray A. Olsson. . . . . . . . . . . . . . . . . . . . . . . . . . . .. 123
3. Role of Ecto-5' -Nucleotidase on Hypoxia-Induced Adenosine
Formation in the Perfused Guinea Pig Heart
Mikio Nakazawa, Hiromasa lin, Hiroto Matsuda, and Shoichi Imai .. 133
4. Energy Charge as a Cytosolic Signal for Adenosine Release
Mark W. Gorman, Miao-Xiang He, and Harvey V. Sparks ......... 147
5. The Role of Adenosine on Myocardial Reactive Hyperemia
Daiji Saito, Tsutomu Mima, Kazuyoshi Hina, Shinji Uchida,
Naotsugu Ohbayashi, Morio Marutani, and Shoichi Haraoka 160
6. Inhibition of PMN and Platelets in the Coronary System by
Endothelium-Derived Adenosine, PGE 1 and PGE 2
Stephan Nees and Andreas Dendorfer. . . . . . . . . . . . . . . . . . . . . . . . . . .. 169
7. Effects of Exogenous Adenosine on Human Coronary Circulation
Mario Marzilli, Gerald Klassen, Paolo Marraccini,
Maria Giovanna Trivella, Paolo Camici, and Antonio L'Abbate . . . .. 179
D Role of Endothelial Cells in Coronary Circulation
1. Endothelial Cell P 2 Purinoceptors

Jeremy D. Pearson and Thomas D. Carter 195
2. The Metabolic Barrier of the Coronary Endothelium as a
Determinant of Flow Responses
Bernhard F. Becker, Birgit Leipert, Lisa Schwartz,
and Eckehart Gerlach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 206
3. Regulation of Vascular Tone by Endothelium-Derived Contracting
Factor (EDCF)
Takayuki Ito, Toshio Kato, Yoshio Iwama, Masahito Muramatsu,
Kiyokazu Shimizu, Hiroshi Asano, Kenji Okumura,
Hidekazu Hashimoto, and Tatsuo Satake . . . . . . . . . . . . . . . . . . . . . . . .. 217
Contents IX
4. Flow-Induced Calcium Response in Cultured Vascular Endothelial

Cells
Joji Ando, Shigenobu Araya, Youichi Katayama, Akira Ohtsuka,
and Akira Kamiya . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 230
5. Endothelin and Vasoconstriction
Hisashi Kai, Mayuko Kodama, Hiromichi Yamamoto,
and Hideo Kanaide ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 242
6. Cascade of Pathophysiological Events Leading to Spasm of
Coronary Arteries
Hitonobu Tomoike, Kensuke Egashira, Yusuke Yamamoto,
Hiroaki Shimokawa, Yasuo Hayashi, Akira Yamada,
Kazushige Nagasawa, Wataru Mitsuoka, Shogo Egashira,
Takeshi Kuga, Hirofumi Tagawa, and Motoomi Nakamura. . . . . . . .. 254
E Ischemia and Reperfusion Injury in the Experimental and

Clinical Studies
1. Coronary Blood Flow in Reperfused Myocardium

Thomas Aversano ............................................ 261
2. Continuity of Myocardial Stunning-Latent Myocardial Damage
After Coronary Occlusion
Mamoru Miura, Takashi Saito, and Tomohiro Kanazawa .......... 271
3. Complement-Induced Myocardial Ischemia: Neutrophil and
Vascular Mechanisms
Robert L. Engler, Ughetta del Balzo, and Bruce R. Ito. . . . . . . . . . . .. 280
4. Reoxygenation-Induced Heart Microvasculature Endothelial Cell
Injury and Neutrophil Hyperreaction: Role of Arachidonate
Lipoxygenase Metabolism
Tsunehiko Kuzuya, Youngjoon Kim, Shiro Hoshida, Masashi Nishida,
Hisakazu Fuji, Masatsugu Hori, Akira Kitabatake, and
Michihiko Tada ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 291
5. Possible Mechanisms of the Beneficial Effects of Nitroglycerin in
Patients with Effort Angina: Potential Roles of Collateral
Circulation
Kazuhisa Kodama, Yasushi Okazaki, Shinsuke Nanto,
Masayoshi Mishima, Atsushi Hirayama, Hiroshi Sato,
Masafumi Kitakaze, Masatsugu Hori, and Michitoshi Inoue. . . . . . . .. 299
6. Importance of Collateral Circulation in Acute Myocardial
Infarction
Shigetake Sasayama, Masatoshi Fujita, and Tadakazu Hirai. . . . . . . .. 315
Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 325
List of Contributors
Akita, H. 78 Hirayama, A. 299 Leipert, B. 206

Ando, J. 230 Hori, M. 3, 61, 291, Marraccini, P. 179
Araya, S. 230 299 Marutani, M. 160
Asano, H. 217 Hoshida, S. 291 Marzilli, M. 179
Ashikawa, K. 24 Imai, S. 133 Matsuda, H. 133
Aversano, T. 261 Inoue, M. 61,299 Mirna, T. 160
Becker, B.F. 206 Ito, B.R. 280 Mishima, M. 299
Berne, R.M. 109 Ito, T. 217 Mito, K. 11
Bortone, A. 91 Iwama, Y. 217 Mitsuoka, W. 254
Camici, P. 179 Jin, H. 133 Miura, M. 271
Carter, T.D. 195 Kai, H. 242 Muramatsu, M. 217
del Balzo, U. 280 Kajiya, F. 11 Nagasawa, K. 254
Dendorfer, A. 169 Kamada, T. 61 Nakamura, M. 254
Doi, Y. 3 Kamiya, A. 230 Nakazawa, M. 133
Egashira, K. 254 Kanaide, H. 242 Nanto, S. 299
Egashira, S. 254 Kanatsuka, H. 24 Nees, S. 169
Eid, K. 91 Kanazawa, T. 271 Nishida, M. 291
Engler, R.L. 280 Katayama, Y. 230 Ogasawara, Y. 11
Feigl, E.O. 47 Kato, T. 217 Ohbayashi, N. 160
Fuji, H. 291 Kim, Y. 291 Ohtsuka, A. 230
Fujita, M. 315 Kitabatake, A. 3, 61, Okazaki, Y. 299
Gerlach, E. 206 291 Okumura, K. 217
Gorman, M.W. 147 Kitakaze, M. 61, 299 Olsson, R.A. 123
Grimm, J. 91 Klassen, G. 179 Pearson, J.D. 195
Haraoka, S. 160 Kodama, K. 299 Saito, D. 160
Hashimoto, H. 217 Kodama, M. 242 Saito, T. 271
Hayashi, Y. 254 Komaru, T. 24 Sasayama, S. 315
He, M.-X. 147 Konishi, J. 34 Satake, T. 217
Hess, O.M. 91 Krayenbuehl, H.P. 91 Sato, H. 299
Hina, K. 160 Kuga, T. 254 Schwartz, L. 206
Hirai, T. 315 Kuzuya, T. 291 Sekiguchi, N. 24
Hiramatsu, O. 11 L' Abbate, A. 179 Shimizu, K. 217
XI
XII List of Contributors
Shimokawa, H. 254 Tamaki, N. 34 Uematsu, M. 3

Sparks, H.V. 147 Tanouchi, J. 3 Yamada, A. 254
Suter, Th. 91 Tomoike, H. 254 Yamamoto, H. 242
Suzuki, T. 24 Trivella, M.G. 179 Yamamoto, Y. 254
Tada, M. 291 Tsujioka, K. 11 Yokoyama, M. 78
Tagawa, H. 254 Uchida, S. 160 Yonekura, Y. 34
Takishima, T. 24 Ueeda, M. 123
A
New Approaches for Coronary
Flow Measurement
1
A Doppler Catheter Technique Using
Fast Fourier Spectrum Analysis for the
Assessment of Coronary Flow
Dynamics
Akira Kitabatake, fun Tanouchi, Masaaki Uematsu, Yasuji Doi,
and Masatsugu Hori 1
Summary. The recent development of a catheter-tipped Doppler probe has

enabled the measurements of coronary flow velocity in humans. However, the
conventional Doppler catheter system with zero-cross signal processing (ZC)
contains some limitations in accuracy and reproducibility. Thus, the coronary
Doppler catheter was used together with a fast Fourier transform signal
processor (FFf), and the resultant system was validated in animal experiments.
Further more, this Doppler system was applied to the beat-to-beat assessment
of coronary flow dynamics in various clinical settings.
The FFf Doppler catheter system was validated by monitoring the coronary
flow in the canine coronary artery with an electromagnetic flowmeter (EMF).
The phasic flow velocity obtained with the FFf system remained constant
regardless of the sample volume position (range, 2-lOmm from the probe).
The flow velocities obtained by the FFT system agreed well with those
estimated by EMF, while the velocities obtained by ZC significantly
underestimated those by EMF even at the optimal sample volume position.
Phasic coronary flow velocity patterns changed in various clinical settings. For
example, in patients with aortic regurgitation, the systolic peak often exceeded
the diastolic peak flow, differing from the normal pattern of predominance in
diastole over systole. In patients with dilated cardiomyopathy, the time from
the initiation of the diastolic flow to the diastolic peak flow was longer than
that in normal subjects, suggesting an attenuation in early diastolic flow increase.
Hence, the catheter-tipped Doppler probe together with FFf has potential in
evaluating various coronary flow dynamics in catheterization laboratories.
1 The First Department of Medicine, Osaka University School of Medicine, Osaka, 553
Japan
3
4 A. Kitabatake et al.
FIG. 1. A commercially available coronary Doppler catheter (DC-WI , Millar , size 3F)
and a guiding catheter (SF)
Introduction
Coronary flow dynamics is a subject of great interest not only for physiologists
but also for clinicians, since myocardial ischemia often plays a pivotal role in
many cardiovascular diseases . Real-time measurement of phasic coronary
artery flow in humans, however, had been limited to measurement during
cardiac surgery [1]. The recent advent of the 3F Doppler catheter probe (Fig.
1) has enabled us to measure coronary artery flow selectively in cardiac
catheterization laboratories [2-4] .
Doppler signals derived from a coronary catheter probe have been processed
with a zero-cross frequency detector. However, this detector has been known
to have some limitations in accuracy, especially when the signal-to-noise ratio
of the originated signals is low. Therefore , we used a specially designed on-line
fast Fourier transform (FFT) processor to analyze Doppler signals in our
coronary Doppler velocimetric system, and validated the Doppler catheter
technique with FFT signal processing for coronary flow velocity measurements
in animal experiments [5] . In addition, we applied this technique to coronary
flow velocity measurements in a catheterization laboratory [6-8].
Effects of the Range Gate on

Coronary Flow Velocity Patterns
Coronary flow velocity patterns obtained with the FFT system were compared
with those obtained with the conventional zero-cross system, with the range
gates being between 2 mm and 10 mm from the catheter tip in the canine left
anterior descending coronary artery (Fig. 2). The velocity patterns obtained
with the FFT system remained unchanged regardless of the changes in the
range gate; the velocity patterns obtained with the conventional zero-cross
method changed drastically: the peak velocity apparently decreased as the
sample position became more distant from the catheter tip. The velocity
1. A Doppler Catheter Technique Using Fast Fourier Spectrum Analysis 5
Sample posit ion from the catheter tip

2 3 4 6 8 10 mm
Zero- :---1- 1--.--1- f----1_ -'---1-

cross , .' -20 em Is
"
".:
,
I
I I j f
" ,
' 'I 1" , 111 ;'
I L ;" '~ , .j,-"
I
0
( I Ii " j
-, (AWAY I
FFT
-25 em / s
o
- I
FIG. 2. Effects of range gates on the coronary flow velocity patterns obtained with the
FFT technique and those with the zero-cross technique. From top to bottom, the range
gates, i.e., sample position from the catheter tip in mm , electrocardiogram, phasic
coronary flow velocity patterns obtained with the zero-cross system, and those with the
FFT system
patterns obtained with the FFT system and those with the zero-cross were
almost identical only in the range of 2-3 mm from the catheter tip. It is thus
necessary to set the sample position, i.e., range gate, within 2-3 mm to obtain
an accurate value using the conventional coronary Doppler velocimeter with
the zero-cross processor. However, if the sample position is too close to the
catheter tip, the complicated flow exerted by the catheter may affect the flow
velocity pattern.
Influence of Catheter Insertion Direction on Coronary Flow

Therefore, we examined the influence of coronary catheter insertion on the
flow in the canine coronary artery. In this experiment, a Doppler catheter
probe was inserted in two different ways. One way was to insert the catheter
from the carotid artery to the left anterior descending coronary artery, i.e., in
an antegrade direction; the other was insertion from the diagonal branch to the
left anterior descending artery in a retrograde direction. The coronary flow-
velocity profile was obtained using a cuff-type multigate pulsed Doppler
velocimeter (carrier frequency: 20 MHz, pulse repetition frequency 50 KHz)
[9] . This multigate velocimeter enabled us to measure 80 point-flow velocities
Distance from 2mm 4mm

catheter- tip 2mm
,
Retrograde Antegrade
J
B !~0c.~ <; B l~k'"
I ' -'-'"
I•• ~ • I :, 8", .' ••'.:: 5.Bm
FIG . 3. Effects of catheter insertion on coronary flow velocity profiles in the canine
coronary artery. Upper panels, schematic illustrations of the catheter insertion
directions and lower panels, flow velocity profiles obtained by the cuff-type 20 MHz
multigate Doppler velocimeter
along the ultrasound beam simultaneously within the depth of 15 mm , thereby

providing instantaneous flow velocity profiles across the vessel in real time .
The upper panels in Fig. 3 describe the catheter insertion directions and the
lower panels show the flow velocity profiles obtained by the cuff-type multigate
(em/sec)
100
FFT
y =o. 88x +9. 7
r =0.93, p<O.OOl
\..
<II
8:o 50
o
ZERO - CROSS
y =O. 23x +l. 6
r =0.82, p<O.OOl
o 100 (em / see)
Electromagnetic flowmeter
FIG. 4. Comparison of the coronary flow volumes obtained with the two Doppler signal
processing techniques, FFf and zero-crossing, using an electromagnetic flowmeter as a
reference in a canine experiment. The coronary flow volume obtained by the
electromagnetic flowmeter and that by the FFf Doppler catheter seemed to be
identical. However, the conventional zero-cross Doppler system apparently under-
estimated the flow volume even at the optimal sample position
Doppler velocimeter. The velocity profile was parabolic in the retrograde

insertion, demonstrating the characteristics of coronary artery flow. Thus, the
retrograde catheter insertion exerted no influence on the flow upstream to the
catheter. In comparison, in the ante grade insertion, the profile was M-shaped
at 2 mm distal to the catheter tip. At 4 mm distal to the catheter tip, the profile
became parabolic again even in the antegrade insertion . As in the clinical
setting, a coronary catheter is always inserted in the antegrade direction and
the sample position should be set at least 4 mm apart from the catheter tip.
However, the conventional zero-cross method provides accurate velocity
patterns only in the range gates less than 3 mm, as discussed previously . Thus,
the FFf system has the advantage of obtaining a reliable output over a wider
range of the sample position than the zero-cross system.
Comparison of the Volumetric Data Obtained by the

FFf Method and Those by the Zero-cross Method
Coronary flow volumes obtained with the two Doppler signal processing
techniques, FFf and zero-crossing, were compared in a canine experiment
using an electromagnetic flowmeter as a reference. Doppler-derived coronary
Normal control em l s
Severe AR em I s
FIG. 5. Left anterior descending coronary artery flow velocity pattern in a normal
subject (upper panel) compared with that in a patient with severe aortic regurgitation
(lower panel)
flow minute volume was calculated as the cross-sectional area multiplied by the
time velocity integral and the heart rate. In this canine experiment, the cross-
sectional area was echocardiographically measured with a 7.5 MHz linear
scanner. The time velocity integral was measured with the Doppler catheter
velocimeter, both with the zero-cross and the FFT methods . As a result, the
coronary flow volume obtained by the electromagnetic flowmeter and that by
the FFT Doppler catheter seemed to be identical. However, the conventional
zero-cross Doppler system apparently underestimated the flow volume even at
the optimal sample position (Fig. 4) .
Thus, the conventional zero-cross detector system has limitations in assessing
coronary flow dynamics; in contrast, the Doppler catheter velocimeter
equipped with the FFT signal processor is a useful tool for the accurate
measurement of coronary flow [5] .
Clinical Applications
Phasic coronary flow velocity patterns in various clinical settings were
evaluated with the FFT Doppler technique. Figure 5 demonstrates an
abnormal left anterior descending coronary artery flow velocity waveform in a
patient with severe aortic regurgitation compared with that in a normal subject.
The abnormal velocity waveform in patients with aortic regurgitation was
characterized by higher peak systolic velocity, more rapid diastolic decelera-
tion, and a smaller diastolic fraction than that in normal subjects, indicating
that the diastolic coronary flow is disturbed in patients with aortic regurgitation
[6].
- 25
Doppler - Oem/s
ECG
DCM T.T. GOM
FIG .6. Left anterior descending coronary artery flow velocity pattern in a patient with
dilated cardiomyopathy
In patients with dilated cardiomyopathy, coronary flow acceleration in early

diastole was slow, resulting in a prolonged time from the onset to the peak flow
velocity in diastole. In addition, the diastolic flow velocity deceleration was
rapid in patients with dilated cardiomyopathy, especially in the cases of
elevated left ventricular pressure [7] (Fig. 6). Furthermore, we demonstrated a
positive correlation between a reference of left ventricular relaxation tau and
the time from the onset to the peak on the diastolic coronary flow velocity
waveform [8]. These findings suggest that impaired left ventricular function
may deleteriously affect diastolic coronary flow dynamics.
The Doppler catheter can also be used to evaluate large epicardial coronary
arterial lesions. The severity of this disease has been usually diagnosed
morphologically with coronary arteriography. With the advent of the coronary
Doppler catheter, functional assessment of the severity of coronary stenosis is
now possible by (1) assessing pharmacological coronary flow reserve, for
example, by administrating intracoronary papaverine hydrochloride [2,3], and
(2) assessing severity of the stenosis by applying the equation of continuity [4].
Conclusions
The catheter-tipped Doppler probe together with the on-line FFT signal
processor has a great advantage over the conventional zero-cross detector
system. It enables us to analyze phasic coronary flow velocity waveforms
accurately. Furthermore, it provides us with a functional assessment of the
severity of epicardial coronary stenoses.
References
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Brody M (1981) Measurements of coronary velocity and reactive hyperemia in the
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Armstrong ML, Marcus ML, White CW (1985) Transluminal, subselective
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2
A Study of Coronary Circulation by
Laser Doppler Velocimetry
Fumihiko Kajiya, Osamu Hiramatsu, Yasuo Ogasawara, Keiichiro Mito,
and Katsuhiko Tsujioka 1
Summary. Our laser Doppler velocimeter (LDV) with an optical fiber is a

powerful tool for the measurement of both coronary artery and vein flow
velocities because of its excellent accessibility to coronary vessels of the beating
heart. We designed four different accesses of a fiber probe to measure blood
velocities, depending upon the objectives of the measurements, i.e., epicardial
large coronary vessels, the epicardial small artery and vein, the intramyocardial
artery and vein, and a laser catheter. The blood flow velocities showed a
diastolic-predominant pattern in the coronary artery and a systolic-
predominant pattern in the coronary vein. The phase opposition between
arterial and venous flows was more remarkable in intramyocardial vessels, i.e.,
the systolic reverse flow in the artery showed a reciprocal relation to the
systolic forward flow in the vein. In veins, suction of blood from superficial
veins to a deeper portion may occur during diastole. The laser Doppler
catheter was found to be useful for monitoring coronary vein flows in the
coronary sinus and the great cardiac vein. We found that the four different
routes of access of optical fiber probe are useful for the evaluation of coronary
flows.
Key words: Blood-flow velocity-Small epicardial coronary artery and vein-
Intramyocardial coronary artery and vein-Laser Doppler velocimeter-
Optical fiber
Introduction
The phasic flow in the left coronary artery is diastolic-predominant. Scaramucci
(1689, cited by Porter [1]) hypothesized that the deeper coronary vessels are
squeezed by the contraction of the muscle fiber around them, which displaces
I Department of Medical Engineering and Systems Cardiology, Kawasaki Medical

School, Kurashiki, Okayama, 701-01 Japan
11
12 F. Kajiya et al.
the intramyocardial blood into coronary veins, and that the vessels are refilled
from the aorta during diastole. To prove this hypothesis, it is necessary to
investigate coronary arterial inflow and venous outflow of the myocardium.
Since Anrep et al. [2] investigated the circulation in the coronary artery and
vein more than sixty years ago by making blood flow measurements using a hot
wire method, there have been few reports [3-5] describing arterial inflow and
venous outflow simultaneously. This is partly because the measurement of
coronary venous flow using conventional methods, including the electro-
magnetic flowmeter, has until recently been difficult, and also because the vein
was regarded as the only conduit of coronary venous outflow. Investigation of
another matter of great interest, the direct measurement of intramyocardial
blood velocity, has been hampered by the difficulty to find an appropriate
route to access a transducer into myocardial vessels of the beating heart.
The laser Doppler velocimeter (LDY) with an optical fiber is a powerful new
tool for the measurement of both coronary arterial and venous flows [6-8].
The most important advantage of the LDY method over conventional
velocimeters is its excellent accessibility to the vessel, even when a vessel is
easily collapsible, as is a vein, or when the vessel is located in a deeper portion
of myocardium. In this paper, we describe the optical arrangement of our LDY
with an optical fiber and the routes of access of the fiber probe to various
coronary vessels. We also clarify characteristics of the phasic blood flow
pattern in large epicardial coronary arteries and veins, in small epicardial
coronary arteries and veins of ventricles, and in intramyocardial arteries and
veins.
Principle of Fiber Optic Laser Doppler Velocimetry
Figure 1 shows the newly revised system of our LDY. The He-Ne laser beam
(632.8nm, 5mW) is divided by a beam splitter (BS). A greater part of the
initial light passed by the BS is focused onto the entrance of a graded-index
multimode fiber (crad diameter: 125 or 62.5 11m, core diameter: 50 11m) and
transmitted through the fiber into a blood stream. A part of the light back-
scattered by flowing erythrocytes is collected by the same fiber and is
transmitted back to its entrance. The other part of the initial light divided by
the BS is used as a reference beam. Two frequency shifters (82 and 78 MHz)
are interposed on the path of the incidence and reference beams to
differentiate the forward from the reverse flow. Thus, the difference of the
shifter frequencies (82-78 = 4 MHz) indicates zero flow velocity. When a
Doppler-shift frequency is greater than 4 MHz, the direction of blood flow is
toward the fiber tip, and when it is less than 4MHz, the direction is away from
the fiber tip. The optical heterodyning is accomplished by mixing the back-
scattered light from the moving erythrocytes with the reference beam. The
photocurrent from the photodetector (APD) is fed into a spectrum analyzer to
detect the Doppler-shift frequency.
2. Laser Doppler Velocimetry 13
He-Ne laser P.B.S. Shifter P.B.S.
-
o
"t:I
o
III
=:
C"
APD ...
CD
or
PMT
Polarizer
Blood
H
Flo~
FiG. 1. Schematic diagram of the laser Doppler velocimeter with optical tiber (revised
type). P.B.S., Polarization beam splitter; M, M2, and M3, mirrors; APD, avalanche
photodiode; PMT, photomultiplier
The back-scattered light signal has a Doppler-shift frequency Ai from 4 MHz

given by
Ai = 2n V cose/,,-
where V is the blood flow velocity; n is the refractive index of blood,
approximately 1.33; e is the angle between the fiber axis and the axis of the
blood stream, and A is the He-Ne laser wavelength of 632.8 nm in a free space.
When e is 60°, the Doppler-shift frequency of 1 MHz corresponds
approximately to blood velocity of 48 cm/s. The sample volume of our system is
approximately 1t x 0.05 2 x 0.1 mm3 and the temporal resolution is 8ms [9-12].
Four Different Routes of Access of the Fiber Probe to

Coronary Vessels
Our laser Doppler velocimeter provides excellent accessibility to coronary
vessels of the beating heart. We used four different routes of access for the
fiber probe depending upon the measuring objectives (Fig. 2). First (access
route 1), we placed a cuff around the vessel and inserted the fiber into the
vessel through a small hole in the vessel wall for the blood flow velocity
measurements in large and middle-sized epicardial coronary arteries and veins
[13]. The fiber tip was moved stepwise across the vessel to obtain the velocity
14 F. Kajiya et al.
(1) epicardial large (2) epicardial small

vessels vessels
Opt ical fibe r
~
b l ood
b l ood
-~
(3) intra myocardial (4) clinical application
ll~Art,~
{
vessels
Optical fiber catheter
'~~ii!k':::' I
~~
~: Vein
FIG. 2. Four routes of access of the fiber probe to blood vessels. Route 1 for the blood
velocity measurements in large- and middle-sized epicardial coronary arteries and veins.
Route 2 for the blood velocity measurements in small epicardial coronary arteries and
veins . Route 3 for the blood velocity measurements in intramyocardial arteries and
veins. Route 4 for the blood flow monitoring for clinical use
profile across the vessel. Second (access route 2), we placed the fiber tip on the
outer surface of the vessel and fixed it by a drop of cyanoacrylate for the blood
flow velocity measurements in small epicardial arteries and veins whose walls
were thin enough to be transparent for laser light [14]. Third (access route 3),
we inserted the fiber into the vascular lumen from a position just penetrating
into the myocardium and introduced the fiber into a deeper portion for the
blood flow velocity measurements in intramyocardial arteries and veins [15].
Fourth (access route 4), we fabricated a catheter-type laser Doppler
velocimeter for clinical monitoring of blood flow in the coronary vein . The
Doppler catheter was inserted into the coronary sinus or the great cardiac vein
through the right atrium.
C'%.c
50
....>-
u
0 .r....
.
~
(' ,
w j •
>
",
0
j.
()..... I
~'V~
~ «'......
~~
30
....>-
u
o
~
w
>
FIG. 3. a Three-dimensional display of the blood flow velocity in proximal and b distal
portions of the left circumflex coronary artery of a mongrel dog. This is reconstructed
from the velocity waveforms at more than 20 sampling points across the vessel by keying
on the R wave in ECG. (From [12] with permission of the American Society of
Mechanical Engineers)
Blood Flow Velocity Waveforms and Profiles in Proximal

and Distal Left Coronary Arteries: Access Route 1
[7,9-11,13]
The coronary blood flow velocity measurements were performed by access
route 1 (Fig, 2). The left circumflex coronary artery (LCX) was isolated at its
proximal and distal portions. The fiber tip was inserted into the vascular lumen
at an angle of 60° with the aid of a small plastic cuff selected from several types
of different diameters (0.8-3.6mm) to fit the vessel snugly. The fiber tip was
traversed stepwise from the near to the far wall in order to measure the local
blood velocity at each sampling point. The position of the vessel wall was
determined as the place where the Doppler signals disappeared. Coronary
16 F. Kajiya et al.
blood flow velocity was recorded on a tape recorder (TEAC R-21O). By keying
on the R wave in the ECG, the velocity profiles in the proximal and distal
portions of the left circumflex coronary artery were reconstructed during one
cardiac cycle.
Representative velocity profiles in the proximal and distal portion of the left
circumflex coronary artery (LCX) are shown in Fig. 3. The characteristics of
coronary arterial velocity profiles are readily comprehensible using the three-
dimensional display. The velocity waveform showed a diastolic-predominant
pattern which is a characteristic of the coronary arterial flow. The velocity
profiles across the vascular lumen were flat near the axial region and declined
abruptly at the vicinity of the vessel wall. Compared with the velocity profile in
the proximal portion, the magnitude of blood-flow velocity was smaller in the
distal portion throughout the cardiac cycle, especially during systole. Reverse
flow was frequently observed during early systole. The velocity profiles across
the vascular lumen were more parabolic in the distal portion.
Blood Velocity Waveforms for the Stenotic Coronary

Artery: Access Route 1
We measured blood flow velocity in the stenotic coronary artery by using a
double core fiber probe [16]. The double core fiber probe was inserted into the
LAD at an angle of 60° with the aid of a small cuff. The position of the fiber
insertion was 2-3 cm distal to the occluder and the fiber tip was placed near the
central axial region in the vessel. Figure 4 shows a typical example of the blood
flow velocity waveform near the central region under control conditions and
during a transient coronary artery stenosis. The velocity waveforms were
obtained by keeping the cursor level at half of the peak power of the Doppler
spectrum. The spectra of post-stenotic blood velocities were much wider than
those of non-stenotic velocities, indicating the presence of disturbed flow in the
post-stenotic region.
Blood Velocity Waveforms in the Epicardial Small Artery

Under Control Conditions and During Proximal Artery
Stenosis: Access Route 2
For the investigation of phasic myocardial perfusion, it is necessary to evaluate
the phasic blood-flow velocities in the small epicardial coronary artery at a
position just before their penetration into the myocardium, since the proximal
coronary arterial flow pattern is influenced by the capacitance effect of large
epicardial arteries. Thus, we measured the phasic flow velocity in the small
epicardial arteries at a position just before their penetration into the myo-
cardium by access route 2 (Fig. 2). Figure 5 shows an example of the velocity
waveforms in the small epicardial artery of the left ventricle under control
conditions. The velocity waveform in the peripheral epicardial artery was
CONTROL STENOSIS
I .. ." .. ;.' ~
_~£ ~.n ....... ~., ~ .
- ~ -' - .
. . r~ ·
1
frequency
velocity
100] \,
I: ~ i.....
L ~··1f\~~!~~1
(cm / sec) ~~ .; ~l ;d~F
',1 nr r '''ll
o ~:1.i~. •• J.:..
i!~ ,
I
.!o.
I
systole diastoleI
FIG . 4. The blood flow velocity waveform and Doppler spectra under control condition
and following transient coronary stenosis. Arrows indicate the timing when the Doppler
spectra were obtained
almost exclusively diastolic and the reverse flow was frequently observed in the
early half of systole . These observations are consistent with those by Chilian
and Marcus [17].
We also measured the blood flow velocity in the small epicardial coronary
artery during proximal coronary stenosis or complete occlusion by access route
2. Figure 6 shows a representative tracing of the blood velocity waveforms for
different degrees of stenoses and for complete occlusion in a small epicardial
coronary artery. The magnitude of diastolic blood velocity decreased with the
severity of stenosis, whereas the systolic reverse flow increased. Thus, the net
inflow into the myocardium greatly decreased with stenosis. After complete
occlusion, the diastolic velocity area became almost equal to the systolic
velocity area, indicating a "to-and-fro" velocity between epicardial arteries and
intramyocardial vessels.
Blood Velocity Waveforms in Intramyocardial Arteries and

Veins: Access Route 3
Because of a possible difference of hemodynamics in epicardium and
intramyocardium, the flow in epicardial coronary vessels may not provide
direct information of intramyocardial flow dynamics, even when the velocity
ECG
20
LV small artery
velocity
(cm/sec)
o
FIG. 5. An example of velocity waveforms in a small epicardial artery of the left

ventricle just before its penetration into the myocardium. Arrows indicate the reverse
flows
Control
Blood flow
Velocity
15J
(cm/sec) 0
15
Blood flow ]
Velocity
(cm/sec) 0
Blood flow
Velocity
15 J Severe Stenosis
(cm/sec) 0
Blood flow15 ]
Velocity
(cm/sec) 0
Diastole
FIG. 6. A representative tracing of blood flow velocity waveforms under control
conditions, and for two different degrees of stenoses and complete occlusion in a small
coronary artery just before its penetration into the myocardium
18
2. Laser Doppler Ve10cimetry 19
ECG
20
Septal artery
velocity
(cm/sec)
20
Intramyocardial
small vein
velocity
(cm/sec)
o
1 sec
FIG. 7. An example of the velocity patterns in the septal artery (18 mm depth from its
orifice) and in a small intramyocardial vein during isoproterenol administration. Three
different arrows ( i ' n, !) indicate the early systolic and mid-systolic reverse flows in
septal artery, and diastolic reverse flow in the intramyocardial small vein, respectively
waveforms are measured in small arteries and veins. Therefore, measurements

of intramyocardial blood flow are needed for better understanding of coronary
circulatory physiology. However, measurements of phasic blood velocities in
the deeper myocardial vessels have been hampered due to methodological
limitation, although the trans-illumination method is a powerful tool for
evaluating superficial intramyocardial hemodynamics. Recently, we measured
the blood velocity in the intramyocardial small arteries and veins and in the
deep sites of the septal artery by access route 3 [18-20] (Fig. 2).
Figure 7 shows an example of the velocity patterns in the septal artery and in
an intramyocardial small vein of the dog. Measurements in these examples were
obtained during isoproterenol administration in order to enhance the effect of
myocardial compressive force on the velocity waveforms. The depth of the
measured position of the septal artery in this case was 18 mm from the orifice
of the septal artery, and that of the intramyocardial vein was about 2 mm
beneath the cardiac surface. The most prominent characteristic of the septal
arterial velocity waveform was the systolic reverse flow, i.e., the reverse flow
was divided into two components, isovolumic and mid-and/or late-systolic
reverse flows. As for the venous flow in contrast to the arterial flow, the
forward flow was exclusively systole and began at the onset of the left
ventricular contraction. Furthermore, the systolic forward flow wave was
20 F. Kajiya et al.
ECG
GCV
Velocity
(cm/sec)
AoP 150 ]
(mmHg) 0
100 ]
CBF(LAO)
(mllmin)
o
0.5 sec
FIG. 8. Phasic blood velocity waveform in the central axial portion of the great cardiac
vein monitored by a laser Doppler catheter. The velocity waveform was characterized
by a prominent systolic flow wave
divided into two components. Thus, it was concluded that the systolic forward
flow shows a reciprocal relation with the reverse flow in the intramyocardial
artery. During diastole, there may be suction of the blood in superficial veins
into the deeper portions, since diastolic reverse flow was frequently observed in
the intramyocardial coronary veins. This reverse flow was also observed in the
epicardial small veins just after its appearance in the myocardium.
Blood-Flow Velocity Waveforms in the Coronary Vein:

Access Route 4
A catheter-type LOY has been developed to monitor coronary vein flow [21].
An optical fiber is set inside a SF catheter and eight elastic silicon rubber spikes
are arranged radially toward the vessel wall to fix the catheter tip near the
central region. Figure 8 shows a typical trace of the blood flow velocity in the
great cardiac vein. The velocity waveform showed a systolic-predominant
pattern as that in the intramyocardial vein. The blood velocity incr~ased
around the onset of left ventricular ejection and decreased gradually after the
peak formation at the end of ejection. It should be noted that there was a
phase delay from the intramyocardial venous flow to the great cardiac venous
flow, i.e., in the intramyocardial vein, the onset of the intramyocardial venous
flow was earlier, the flow acceleration was higher, and the diastolic reverse flow
was observed in most cases. Nevertheless, the measurements of coronary
IVEIN OUTFLOW I
Extravascular myocardial
itance vessels
FIG. 9. A schematic drawing of possible blood flow directions in intramyocardial

arteries and veins during one cardiac cycle postulated by our observations and earlier
reports
venous flow by access route 4 may be effective for clinical monitoring of

coronary flows.
Conclusions
Figure 9 illustrates possible blood flow directions in one cardiac cycle based
upon our own observations [19,20] and earlier reports [17,22]. In early diastole
(left), the blood flow direction from both superficial and deeper portions is
reversal in intramyocardial arteries, whereas the direction is forward in both
portions in intramyocardial veins . However, in the arteries during mid-systole,
the blood may be translocated from a deeper to a superficial layer, in which the
extravascular compressive force may be much smaller. The vein flow direction
may remain unchanged during systole. During diastole , the direction of arterial
flow is forward in both superficial and deeper portions. However, in the
intramyocardial veins , there may be translocation of blood from superficial to
deeper vessels by the suction effect due to myocardial relaxation.
References
1. Porter WT (1898) The influence of the heart-beat on the flow of blood through the
walls of the heart. Am J Physiol 1:145-163
2. Anrep GV, Cruickshank EWH, Downing AC, Sabba RA (1927) The coronary
circulation in relation to the cardiac cycle. Heart 14:111-133
22 F. Kajiya et al.
3. Chilian WM, Marcus ML (1984) Coronary venous outflow persists after cessation of
coronary arterial inflow. Am J Physiol 247:H984-H990
4. Spaan JAE (1982) Intramyocardial compliance studies by venous outflow at arterial
occlusion (abstract). Circulation 66: II -42
5. Kajiya F, Hiramatsu 0, Mito K, Tadaoka S, Ogasawara Y, Tsujioka K (1990)
Evaluation of coronary blood flow by fiber-optic laser Doppler velocimeter. In:
Kajiya F, Klassen GA, Spaan JAE, Hoffman HE (eds) Coronary circulation.
Springer-Verlag, Tokyo, pp 43-53
6. Tanaka T, Benedek GB (1975) Measurement of the velocity of blood flow (in vivo)
using a fiber optic catheter and optical mixing spectroscopy. Appl Optics 14:189-
196
7. Kajiya F, Hoki N, Tomonaga G, Nishihara H (1981) A laser-Doppler-velocimeter
using an optical fiber and its application to local velocity measurement in the
coronary artery. Experientia 37:1171-1173
8. Kilpatric D, Linderer T, Sievers RE, Tyberg JV (1982) Measurement of coronary
sinus blood flow by fiber-optic laser Doppler anemometry. Am J PhsioI242:H1111-
HI114
9. Kajiya F, Mito K, Ogasawara Y, Tsujioka K, Tomonaga G (1984) Laser Doppler
blood flow velocimeter with an optical fiber and its applications to detailed
measurements of the coronary blood flow velocities. Proc SPIE 494:25-31
10. Kajiya F, Hiramatsu 0, Mito K, Ogasawara Y, Tsujioka K (1987) An optical-fiber
laser Doppler velocimeter and its application to measurements of coronary blood
flow velocities. Med Prog Technol 12:77-85
11. Kilpatrick D, Kajiya F, Ogasawara Y (1988) Fibre optic laser Doppler
measurement of intravascular velocity. Australas Phys Eng Sci Med 11:5-14
12. Kjiya F (1991) Characteristics and possible origins of blood velocity waveforms of
the epicardial and intramyocardial coronary circulation in the ventricles and the
atria. In: Nakamura M, Vanhoutte PM (eds) Coronary circulation in physiological
and pathophysiological states. Springer-Verlag, Tokyo, pp 1-19
13. Kajiya F, Tomonaga G, Tsujioka K, Ogasawara Y, Nishihara H (1985) Evaluation
of local blood flow velocity in proximal and distal coronary arteries by laser
Doppler method. Trans ASME J Biomech Eng 107:10-15
14. Kajiya F, Tsujioka K, Ogasawara Y, Hiramatsu 0, Wada Y, Goto M,Yanaka M
(1989) Analysis of the characteristics of the flow velocity waveforms in left atrial
small arteries and veins in the dog. Circ Res 65: 1172-1181
15. Kajiya F, Tsujioka K, Ogasawara Y, Mito K, Hiramatsu 0, Goto M, Wada Y,
Matsuoka S (1989) Mechanical control of coronary artery inflow and vein outflow.
Jpn Circ J 53:431-439
16. Kajiya F, Hiramatsu 0, Ogasawara Y, Mito K, Tsujioka K (1988) Dual-fiber laser
Doppler velocimeter and its application to the measurements of coronary blood
velocity. Biorheology 25:227-235
17. Chili an WM, Marcus ML (1984) Effects of coronary and extravascular pressure on
intramyocardial and epicardial blood velocity. Am J Physiol 248:H170-H178
18. Mito K, Ogasawara Y, Hiramatsu 0, Wada Y, Goto M, Tadaoka S, Tsujioka K,
Kajiya F (1987) Evaluation of velocity waveform in an intramyocardial small artery
and vein by laser Doppler method (abstracts). Circulation 76:IV-386
19. Mito K, Ogasawara Y, Hiramatsu 0, Wada Y, Tsujioka K, Kajiya F (1988)
Evaluation of blood flow velocity waveforms in intramyocardial artery and vein by
laser Doppler velocimeter with an optical fiber. In: Manabe H, Zweifach BW,
Messmer K (eds) Microcirculation in circulatory disorders. Springer-Verlag, Tokyo,
pp 525-528
20. Hiramatsu 0, Mito K, Kajiya, F (1990) Evaluation of the velocity waveform in

intramyocardial small vessels. In: Kajiya F, Klassen GA, Spaan JAE, Hoffman HE
(eds) Coronary circulation. Springer-Verlag, Tokyo, pp 179-172
21. Mito K, Ogasawara Y, Hiramatsu 0, Tsujioka K, Kajiya F (1990) A laser Doppler
catheter for monitoring both phasic and mean coronary vein flow. Heart Vessels
6:1-8
22. Ashikawa K, Kanatsuka H, Suzuki T, Takishima T (1986) Phasic blood flow
velocity pattern in epimyocardial microvessels in the beating canine left ventricle.
Circ Res 59:704- 711
3
Direct Observation of the Coronary
Microvasculature in a Beating Heart by
the Floating Objective System
Hiroshi Kanatsuka l , Kouichi Ashikawa2 , Nobuyo Sekiguchil,
Tatsuya Komaru l, Toshimi SuzukP, and Tamotsu Takishima l
Summary. Direct and continuous visualization of the coronary microcircula-

tion has been almost impossible in the beating mammalian heart. To achieve
this purpose, we have developed a new microscopic system which we termed
the "floating objective system", and which consists of a pair of convex lenses
vertically aligned on the same optical axis. The real image on the front focus of
a lens facing the heart (floating lens) is transmitted to the back focus of another
convex lens fixed to the stage of a standard microscope. When the floating lens
moves in unison with cardiac motion, the transmitted real image is not affected
by the change in the distance between these two convex lenses. Therefore, in a
beating heart, it is possible to observe coronary microvasculature with a
standard microscope by observing the transmitted real image. The advantage
of this system is that, in a beating heart, continuous observation of coronary
microcirculation is possible throughout a cardiac cycle and resolution is suf-
ficient to measure red cell velocity. The red cell velocity in epimyocardial
microvessels reached the peak at midsystole in small arterioles and capillaries,
and at late systole in small venules. In all vessels, red cell velocity gradually
declined during diastole. Momentary cessation or reverse flow was observed
during the pre-ejection period in all microvascular channels in the epimyo-
cardium. Cyclic variation of diameter was observed in small venules but not in
small arterioles.
Key words: Red cell velocity-Intravital microscope-High-speed motion
picture camera-SIT camera-High-speed video camera
1 The First Department of Internal Medicine, Tohoku University School of Medicine,

Sendai, 980 Japan
2 Department of Cardiology, National Sendai Hospital, Sendai, 983 Japan
3 Internal Medicine, Tohoku Rosai Hospital, Sendai, 981 Japan
24
3. Direct Observation of Coronary Microvasculature 25
Introduction
Direct assessment of the microcirculation has facilitated the understanding of
the physiology and pathophysiology of many organs. However, this approach
has been rarely employed in the coronary microcirculation because of
methodological difficulties; in particular, continuous observation of coronary
microcirculation has been almost impossible in a beating mammalian heart.
Martini and Honig reported the first direct visualization method in 1969 [1].
They took pictures of coronary microcirculation with a high-speed motion
picture camera in a freely beating heat, then selected several well-focused
frames from among several hundred frames. Bing et al. developed the first
continuous visualization method available for the turtle heart, which has a very
slow heart rate (20-30 bpm) [2]. Tillmanns, who initially worked with Bing,
started continuous observation using a standard microscope with his regional
immobilization method [3]. He inserted 5-7 tiny needles into the mid-
myocardium of a rat heart and completely stopped the regional cardiac motion.
After these trials, Nellis et al. developed an ingenious stroboscopic illumina-
tion method that allowed intermittent observation of the coronary micro-
circulation in a beating rabbit heart [4]. This technique has been improved by
Chilian et al. [5]. These techniques kindled substantial interest in the
physiology and pathophysiology of coronary microcirculation, but continuous
observation in a beating mammalian heart was still impossible.
The main purpose of this report is to describe a new technique for
continuous observation of coronary microcirculation in a beating mammalian
heart [6,7].
General Preparation
Small mongrel dogs of both sexes weighing 3-9 kg were anesthetized with
a-chloralose (a-chloralose 60 mg/kg + sodium borate 50 mg/kg, iv.). Additional
doses were given as needed throughout the experiment. A left thoracotomy
was performed in the fifth intercostal space under mechanical ventilation at an
end-expiratory pressure of 3-5 cm H 2 0. The pericardium was opened, and the
heart was suspended in a pericardial cradle. The exposed cardiac surface was
kept moist by a continuous drip of warm physiological solution ([mM] NaCl
118.2, KCI 4.7, CaCl2 2.5, MgS04 1.2, KH 2 P0 4 1.2, NaHC0 3 25, calcium
disodium EDTA 0.026, and glucose 5.5, maintained at 37°C and pH 7.40).
Heart rate was kept constant (120-140 bpm) with left atrial pacing after sinus
node suppression by local injection of formaldehyde (0.3-0.5 ml). Catheters
were introduced into the aortic arch through the left carotid artery, and into
the left ventricle through the apex for measurements of aortic and left
ventricular pressures, respectively. A lead II ECG was monitored.
Microscopic System
The major difficulty in the continuous observation of coronary microcirculation
is to maintain fine focus of the microscopic image in the beating heart. For this
26 H. Kanatsuka et al.
High Speed Camera & Monitor TV FiG. 1. A schematic illustration of the floating
objective and the method of trans-illumination
of the epimyocardium. (From [7] with per-
Standard Microscope mission of the American Heart Association,
Inc.)
EPimYQCar"~-----------------~~~~-;~-~-~;d~r-- tight
<art Stabilizing Needles
purpose, we have developed a new microscope system, the "floating objective

system" [6,7]. The floating objective consists of a pair of convex lenses aligned
on the same optical axis, and transmits a real image of the epimyocardium of
the beating heart to a fixed position without any change in magnification (Fig.
1). The real image of the object at the front focus of a convex lens facing the
heart is transmitted to the back focus of another convex lens . .The transmitted
real image is not affected by the change in distance between those two convex
lenses. This transmitted real image is then observed with a standard
microscope. A convex lens facing the heart is mounted in a thin alumium tube
and supported by six low-resistance ball bearings and by a weight-adjusting coil
spring. The total weight of the lens and the aluminum tube is 16 g and its static
balance was kept at less than 4 g by lifting it with the weight-adjusting coil
spring. The combination of using this minimal weight, the reduction of the
inertial force in vertical movement, and the use of low-resistance ball bearings
and a weight-adjusting coil spring permits the floating objective to move easily
in unison with the cardiac motion. The distance between the lens and cardiac
surface was adjusted to the focal distance of this lens. Resolution of the
floating objective system is approximately 211m.
Partial Restraint of Card;iac Motion

The floating objective follows vertical motion of a heart, but it is necessary to
restrict excessive horizontal movement in order to keep the desired area in the
microscopic field of view. For the restriction of horizontal cardiac motion, two
24-gauge steel needles were inserted horizontally (5-7 mm apart) through the
midmyocardium of the left ventricel. Both ends of each needle were fixed to a
TV motion
analyzer
highly sensitive
TV camera
(SIT) D
o 0
VTR
video
manlpUator
o
mercury~ standard microscope
[J
videographk: printer
floating objective
FIG. 2. A method for epiilumination of epimyocardium and the image acquisition

system with the SIT TV camera. (From [8] with permission of the American Heart
Association, Inc.)
needle holder held with coil springs. This apparatus allowed the heart to move
perpendicularly but limited excessive horizontal movement, thereby keeping
the desired area in the microscopic field of view.
Illumination Technique
The epimyocardium of the left ventricle was transilluminated with a xenon arc
lamp. A light-conducting glass fiber (0.6 mm in diameter), which was intro-
duced through the lumen of a 20-gauge stainless needle, was inserted into the
subepimyocardial muscle layer (Fig. 1). The needle was mounted in a needle
holder that is designed to move its tip up and down in unison with the cardiac
motion. The needle holder has an arm (objective lifter) that lifts the floating
objective. The height of the objective lifter was controlled with a micro-
manipulator to keep the floating objective just above the cardiac surface (Fig.
1). This procedure prevented compression of tissue with the floating objective.
In our floating objective system, an epiillumination technique is also available
by using a dichroic mirror [8] (Fig. 2). This technique has been employed
mostly in fluorescence coronary microangiography.
Image Acquisition
The images obtained with this microscope system were recorded using three
different systems: a high-speed motion picture camera, a SIT TV camera, and
a high-speed video camera. In several cases, an image-intensifier was combined
to increase the sensitivity of the system. A high-speed motion picture camera
(model DBM-5D, Milliken, Arcadia, Calif.) can take consecutive pictures at
500 frames/which allows us to measure red cell velocity in the coronary
microcirculation [7]. A SIT TV camera (type C1000-12, Hamamatsu-Photo-
nics, Hamamatsu, Japan) has very high sensitivity that allows us to take images
of fluorescence coronary micro angiography at 30-60 frames/s by injecting
fluorescein isothiocyanate-dextran (MW: 154, 200, 20 mg/ml in 0.2 ml of saline)
into the left atrium via the catheter. It is also possible to assess the leakage of
high molecular weight substances in the coronary microcirculation using
fluorescein isothionate-dextran of different molecular weights. A high-speed
video camera (model MHS-200, Nac, Tokyo), in combination with an image-
intensifier (model C3100, Hamamatsu-Photonics), can be used to take
consecutive video frames at 200 frames/s with sufficient resolution and sen-
sitivity to obtain fluorescent images of the coronary microvasculature. In this
system, f1uorescently labeled latex microspheres (Fluoresbrite carboxilate
microsphere: 2.13!lm in diameter, Polyscience) were also used to measure
microvascular flow velocity rather than red cell velocity. The microspheres (5
X 108 ) were suspended in 0.5 ml saline with Tween 80, shaken for 5 min and
injected into the catheter placed in the right atrium. By using these procedures,
aggregated microspheres were reduced and trapped in the lung. A stroboscopic
epiillumination was employed to avoid any blur caused by the rapid movement
of the microspheres. In fluorescence coronary micro angiography , the maximal
wave length of the illuminating light was 495 nm, obtained by using a B2
excitation filter. The emitted light was then passed through a 510 nm filter.
Image Analysis and Velocity Measurement

High-speed motion picture images were analyzed on a projection screen.
Images stored on the video tapes were analyzed on the screen of a high
resolution TV monitor with a video manipulator (model C2117, Hamamatsu-
TABLE 1. Phasic change of internal diameter in small arterioles and venules

Time from the onset of QRS (ms)
n 25 75 125 175 225 275 325 385
Arterioles 17 Mean 24.9 25.5 25.5 25.6 25.4 25.3 24.9 25.0
SD 11.0 11.7 11.4 11.6 11.0 11.1 11.6 11.0
Venules 10 Mean 27.7 30.4 30.7 32.2' 31.1 29.3 28.2 27.3
SD 11.6 13.2 13.6 14.1 14.0 12.7 11.7 11.3
, P < 0.05 vs 25 and 385 ms; n, Number of measurements; SD, standard deviation.
(From [7) with permission of the American Heart Association, Inc.)
Photonics) or on prints produced by a video graphic printer (model UP-811 ,

Sony, Tokyo). Red cell or microsphere velocity was measured by frame-to-
frame analysis from the distance of red cell or microsphere progression on a
screen and the number of frames required. The motion of reference points,
such as a branch point of vessels, caused by cardiac movement was subtracted
from that of the red cell or microsphere to get the true movement of red cell or
microsphere.
Change of Microvascular Diameter During the Cardiac Cycle

The internal diameters of small arterioles (mean diameter = 24.9 ± 11.0/lm, n
= 17) and venules (mean diameter = 27.7 ± 11.6/lm, n = 10) were measured
in the epimyocardium of beating left ventricles [7] (Table 1). In small
arterioles, no significant change in diameter was observed during a cardiac
cycle under normal conditions. On the other hand, diameters of small venules
significantly changed during a cardiac cycle, with the maximal diameter being
observed in late systole. In contrast with these observations, diameters of
arterioles gradually decreased during a single long diastole when aortic
pressure was allowed to decline [9] (Fig. 3). The reduction of diameter was
greatest at aortic pressures less than 35 mmHg. Diameters of venules did not
change significantly during a long diastole.
Phasic Change of Blood Flow in Coronary Microcirculation

Red cell velocities were measured in terminal arterioles (mean diameters =
12.8 ± 4.1 /lm, n = 6), collecting venules (mean diameter = 16.8 ± 6.5 /lm, n
= 13), and in capillaries (n = 11) every 20-40ms [7] (Fig. 4). In the left
ventricular epimyocardium, the peak red cell velocity of arterioles and
capillaries occurred in midsystole, followed by a decrease during diastole.
Abrupt cessation of red cell movement or momentary reverse flow was
observed in these vessels during the pre-ejection period. In small venules, peak
velocity was observed in late systole followed by a gradual decline during
diastole. Momentary cessation or reverse flow was also observed in small
venules.
Comparison of Microsphere Velocity and Red Cell Velocity

In order to examine whether or not microsphere velocity reflects capillary flow
velocity, microsphere velocity was compared with red cell velocity during
diastole. Although absolute microsphere velocity differed from red cell velocity
in the resting condition (1,111 ± 33/lm/s vs 2,412 ± 311 /lm/s, P < 0.05), the
percent of changes in these velocities produced by an adenosine potentiator
(dilazep, 50/lg/kg iv. or dipyridamole 400/lg/kg iv.) were equivalent (23.0 ±
7.2 vs 25.0 ± 3.0%, NS).
30 H. Kanatsuka et aJ.
250
00
•
200
J ....
..... . •••
o
r 000
° a°
oOm
CIl:tlJ
•
•• •
-
E
.:; 150
o
o &CO
oo 0Cl .tI. -
o 0
.--
......
...
-
.. ..
.. ..
... .... .. ..
Q)
Q)
E "... 1'" 0«>0 0000
00
ctI o <X>. 000<0
o
-
,...0 0
ctI ./l<v 0
-
c
100
Q;
c .
50
o~--~----~--~----~--~--~
o 20 40 60 80 100 120
Aortic Pressure (mmHg)
FIG. 3. The relation between aortic pressure and internal diameters of arterial
microvessels (n = 12) during long diastoles. Each symbol represents 1 of 12 micro-
vessels. Internal diameters of all arterial microvessels decreased as aortic pressure fell.
(From [9] with permission)
Discussion
There are two important advantages of the floating objective system: (1)
continuous observation of the coronary microcirculation is possible throughout
the cardiac cycle in a beating mammalian left ventricle, and (2) resolution is
a
b
~ 7
E 6
"~
52 5 ~EN
.;. R=r·····l
~ 4 t
..
0;
> 3
0;
u
..
2 u 2
~
II: 1 ..
~
II: 1
o 100 200 300 400 ( mS<!'C ) (ms~c)

-I
-I
-2
FIG. 4a,b. Phasic red cell velocity in epimyocardial microvessels. ART, arterioles
(n = 6); CAP, capillaries (n = 11); VEN, venules (n = 13); AP, aortic pressure; LVP,
left ventricular pressure. (From [7] with permission of the American Heart Association,
Inc.)
sufficient for the analysis of red cell or fluorescent microsphere velocity. It is

also possible, with a SIT TV camera or an image intensifier, to examine the
leakage of the molecular weight substances by fluorescence labeling of those
substances.
In our experimental preparation, mechanical factors which might affect
coronary microcirculation were minimized as much as possible. In particular,
the floating objective did not contact the cardiac surface, and restraint of the
cardiac muscle was minimized by a needle holder that allowed vertical motion
but restrained horizontal motion. However, it is possible that the inserted
illumination fiber and two heart stabilizing needles might have had some
influence on the microcirculation by producing a small trauma or by changing
intramyocardial pressure . Previous reports from our laboratory indicated that
arterial microvessels respond well to a low dosage of an adenosine potentiator
(dilazep, 50llg iv.) [7] and to a-blockers (phentolamine : 2mg/kg iv. or
lOOllg/kg into LAD [10]. Also, arterial microvessels clearly showed reactive
hyperemia after 20s of complete occlusion of the coronary artery [11]. These
results indicated well-preserved viability of the vessels and related receptors
that were located on the illumination fiber. Myocardial blood flow in the area,
partially restrained with the four stabilizing needles, was examined using the
radioactive microsphere technique [12]. In the resting condition, the flow in
epi-, mid- and endomyocardium were not affected by the insertion of the four
stabilizing needles, and the maximal blood flow in those layers (flow reserve:
adenosine l.Omg/kg iv.) was comparable with the flow in non-restrained areas.
Although our technique is quite useful in understanding the physiology and
pathophysiology of coronary microcirculation, the implication of the results
obtained with this technique should be carefully assessed. In this technique, the
coronary microcirculation was observed in the superficial layer of the
epimyocardium. The responses to the several different stimuli may differ in the
different myocardial layers, as observed in myogenic responses [13]. The direct
observation of endomyocardial microcirculation in the beating heart is still an
issue for the future.
References
1. Martini J, Honig CR (1969) Direct measurement of intercapillary distance in
beating rat heart in situ under various conditions O 2 supply. Microvasc Res 1:244-
256
2. Tillmanns H, Ikeda S, Hansen H, Sarma JSM, Faubel J, Bing RJ (1974)
Microcirculation in the ventricle of the dog and turtle. Circ Res 34:561-569
3. Tillmanns H, Steinhausen M, Leinberger H, Thedern H, Kuber W (1981) Pressure
measurements in the terminal vascular bed of the epimyocardium of rats and cats.
Circ Res 49: 1202-1211
4. Nellis SH, Liedtke AJ, Whitesell L (1981) Small coronary vessel pressure and
diameter in an intact beating rabbit heart: Fixed position and free-motion
techniques. Circ Res 49:342-353
5. Chilian WM, Eastham CL, Marcus ML (1986) Microvascular distribution of
coronary vascular resistance in beating left ventricle. Am J Physiol 251 :H779-
H788
6. Ashikawa K, Kanatsuka H, Suzuki T, Takishima T (1984) A new microscopic
system for the continuous observation of the coronary microcirculation in the
beating canine left ventricle. Microvasc Res 28:387-394
7. Ashikawa K, Kanatsuka H, Suzuki T, Takishima T (1986) Phasic blood flow
velocity pattern in epimyocardial microvessels in the beating canine left ventricle.
Circ Res 59:704-711
8. Komaru T, Ashikawa K, Kanatsuka H, Sekiguchi N, Suzuki T, Takishima T (1990)
Neuropeptide Y modulates vasoconstriction in coronary microvessels in the beating
canine heart. Circ Res 83:774-777
9. Kanatsuka H, Ashikawa K, Komaru T, Suzuki T, Takishima T (1990) Diameter
change and pressure-red blood cell velocity relations in coronary microvessels
during long diastole in the canine left ventricle. Circ Res 66:503-510
10. Sekiguchi N, Ashikawa K, Komaru T, Suzuki T (1989) Effect of a-adrenergic
blockade on coronary microvessels in the anesthetized dogs (abstract). Circulation
80 (Suppl 11):11-310
11. Kanatsuka H, Sekiguchi N, Akai K, Sato K, Wang Y, Ashikawa K, Takishima T

(1990) The site in coronary microcirculation responsible for reactive hyperemia
(abstract). Circulation 82 (Suppl III):III705
12. Kanatsuka H, Lamping KG, Eastham CL, Dellsperger KC, Marcus ML (1989)
Comparison of the effects of increased myocardial oxygen consumption and
adenosine on the coronary microvascular resistance. Circ Res 65:1296-1305
13. Kuo L, Davis MJ, Chili an WM (1988) Myogenic activity in isolated subepicardial
and subendocardial coronary arterioles. Am J Physiol 255:HI558-HI572
4
PET Measurement of Myocardial
Blood Flow
Nagara Tamaki, Yoshiharu Yonekura, and Junji Konishi l
Abstract. Positron emission tomography (PET) has potentials for quantifica-

tion of regional myocardial blood flow and metabolism in vivo. PET perfusion
images can be obtained using N-13 ammonia, a generator-produced rubidium-
82, and 0-15 water. PET provides higher quality perfusion images than the
widely used thallium-201 perfusion images. Therefore, the qualitative PET
perfusion study can yield higher sensitivity and specificity for diagnosing
coronary artery disease than the thallium-201 imaging. In addition, quantitative
analysis of flow reserve by PET may further enhance the identification of a
mild coronary perfusion abnormality. The tracers and techniques of PET
perfusion imaging as well as the clinical value of PET perfusion study are
discussed.
Key words: Positron emission tomography-Coronary artery disease-N-13
ammonia-Rubidium-82-0-15 Water
Introduction
Positron emission tomography (PET) has the unique capability of quantitating
various physiological and biochemical processes in vivo. In the field of
cardiology, PET study has been applied to measure myocardial blood flow and
to assess energy metabolism [1-3]. Myocardial blood flow can be measured by
PET by using suitable radiotracers.
The tracers of blood flow are classified as extractable particles and diffusible
tracers. The diffusible tracers consist of extractable diffusible tracers, such as
N-13 ammonia and rubidium-82 and possibly diffusible tracers, classified as
0-15 water.
I Department of Nuclear Medicine, Kyoto University Faculty of Medicine, Kyoto, 606

Japan
34
4. Myocardial Flow by PET 35
We will describe several characteristics of these tracers for measuring

myocardial blood flow.
Assessment of Myocardial Blood Flow
1. N-13 Ammonia
This radioisotope is produced by the cyclotron and has a physical half-life of
10 min. Its first pass extraction fraction by the myocardium is approximately
80%. It is trapped in the myocardium by glutamine synthetase reaction. Since
this retention fraction decreases with higher flow, an underestimation of flow
by this tracer occurs, resulting in a nonlinear relationship between flow and
N-13 ammonia uptake [4]. However, quantification of regional myocardial
blood flow has been attempted by serial dynamic PET imaging using N-13
ammonia and the arterial input function [5]. On the other hand, the perfusion
images seem to be the best among all of the PET perfusion tracers. We have
been using N-13 ammonia as a perfusion agent for assessing coronary artery
disease in conjunction with F-18 fluorodeoxyglucose (FOG) as a tracer for
exgenous glucose utilization [6-10] .
PRE-f'TCA
REST STRESS
(S.K.>
POST-PTCA
FIG . 1. Rest (left) and stress (right) perfusion images before (top) and after (bottom)
PTCA using N-13 ammonia and positron emission tomography in a patient with anterior
wall myocardial infarction. Hypoperfusion in the anterior wall with stress-induced
ischemia in large anterior areas is well demonstrated before PTCA , where perfusion is
strikingly improved without stress-induced ischemia after PTCA
36 N. Tamaki et al.
STRESS
N-13 I=H'OIIA
F"-18 F (;
FIG. 2. Rest (top left) and stress (top right) perfusion images using N-13 ammonia and
glucose metabolic image using F-18 deoxyglucose (FDG) in a patient with anterior wall
myocardial infarction . Mild hypoperfusion with stress-induced ischemia in anterior wall
is well illustrated in these perfusion images. Enhanced glucose utilization is observed in
the same area, suggestive of ischemia
The diagnostic accuracy for identifying coronary artery disease is very high
[6,7]. In addition, resting and stress perfusion studies seem to be useful for
differentiating ischemic from infarcted myocardium [8]. Figure 1 illustrates
resting and stress perfusion images before and after percutaneous transluminal
coronary angioplasty (PTCA) in a patient with anterior wall myocardial
infarction. These images show resting hypoperfusion with stress-induced
hypoperfusion in the anterior region. The post-PTCA images clearly show
improvement in perfusion in the same area without stress-induced ischemia. In
such areas of stress-induced ischemia, the perfusion is most likely to improve
after intervention.
We performed rest-stress N-13 ammonia perfusion imaging on 30 patients
receiving coronary bypass grafting [9]. Those showing stress-induced ischemia
were most likely to improve in regional perfusion and wall motion after
surgery, whereas those without stress-induced ischemia were least expected to
improve in regional function. The predictive values for improvement in
regional function were very high. In particular, the negative predictive value
FiG. 3. The fraction of the segments

showing positive FDG uptake in
relation to presence of stress-induced
ischemia on N-13 ammonia study
p(0.05
55'
50
39'
O~---L----~~--~
stress-induced stress-Induced
ischemia (+1 ishcemia (-I
(110) (80)
10 %
p<0.005
p<O.OOl
65'
FIG. 4. The fraction of the segments

showing positive FDG uptake in rela- mild moderate severe
tion to severity of hypoperfusion on hypoperfusion hypoperfusion hypoperfusion
N-13 ammonia study (lOS) (62) (37)
was significalty higher than that obtained by stress-delayed thallium-201

imaging [8,9].
Figure 2 (top) shows resting and stress N-13 ammonia perfusion images and
FDG exogenous glucose utilization image (bottom) of a patient with anterior
wall myocardial infarction. N-13 ammonia images indicate resting hypo-
perfusion with stress-induced ischemia in the anterior and septal regions. The
FDG image shows enhanced FDG uptake, indicating increased utilization of
glucose. These areas may suggest the presence of ischemic myocardium
[10,11].
We focused on this FDG uptake in the areas indicated by ECG of infarcted
myocardium in relation to perfusion and regional wall motion abnormality. In
38 N. Tamaki et al.
peO.OOl FIG. 5. The fraction of the segments

p<O.OOl showing positive FDG uptake in relation
79% to severity of wall motion abnormality on
contrast ventriculography
36%
normal akinesis dyskinesis

hypokinesis
(58) (44) (16)
the study of 37 patients with myocardial infarction, an increase in FDG uptake

was observed more frequently in the areas exhibiting stress-induced ischemia
than those without stress-induced ischemia (Fig. 3). When FDG uptake was
compared to the severity of hypoperfusion at rest, FDG uptake was observed
more often in the areas showing mild hypoperfusion than in those exhibiting
moderate to severe hypoperfusion (Fig. 4), indicating that preserved perfusion
may be required to maintain energy metabolism in the infarcted myocardium.
However, Fig. 4 also indicates occasional FDG uptake in the areas with severe
hypoperfusion. Thus, there often exists a discrepancy between perfusion
abnormality and metabolic derangement.
FDG uptake was also compared with the regional wall motion abnormality
in the infarcted myocardium. The regional wall motion abnormality seems to
be less severe in the areas showing increased FDG uptake than in those
without FDG uptake (Fig. 5), indicating the FDG-positive areas to be of less
compromised ischemic myocardium. However, approximately 30% of those
patients showing akinesis or even dyskinesis had persistent FDG uptake. These
data may indicate that regional wall motion analysis may be important for
assessing tissue viability, it should be noted that the segments showing severe
wall motion abnormality may often have ischemic myocardium.
2. Rubidium-82
Rubidium-82 is an alternative to N-13 ammonia as a PET perfusion tracer.
Since this is a generator-produced tracer, PET perfusion study can be
performed without an on site cyclotron [12]. Thus, PET perfusion imaging
using rubidium-82 has potentials for wide application in evaluating coronary
artery disease in clinical settings [12]. In addition, the short physical half-life
(75 s) of rubidium-82 permits repeated serial measurements, such as various
interventions or drugs in the same patient. Figure 6 shows serial PET perfusion
images of a canine model at control, during coronary occlusion, and after
FIG. 6. The canine myocardial perfusion images at control (left), during LAD occlusion
(middle), and after reperfusion (right) using rubidium-82 and positron emission
tomography
reperfusion. The perfusion defect during LAD occlusion with partial improve-
ment after reperfusion is clearly demonstrated. Such repeated measurements of
myocardial perfusion are particularly useful for detecting "silent ischemia"
[13], or serial perfusion study during mental stress.
The tracer kinetic models are presently being tested for application in clinical
studies to quantitate regional coronary blood flow and flow reserve [14,15].
The impairment of flow reserve seems to be the most sensitive functional value
for use in the detection of coronary artery disease [16] .
3. 0-15 Water
Myocardial uptake of N-13 ammonia and rubidium-82 is theoretically
influenced by factors other than blood flow. In contrast, nearly 100% of 0-15
water is extracted by the myocardium in wide range of myocardial blood flow,
and the extraction is not affected by metabolism [17]. On the other hand, the
high concentration of 0-15 water in the blood and lung requires subtraction of
blood pool activity from the original water image for accurate measurement of
tissue tracer concentration [18,19] .
Figure 7 illustrates 0-15 water, 0-15 carbon monoxide, and regional myo-
cardial blood flow images after subtraction of these two images in a patient
with anterior wall myocardial infarction. Since the 0-15 water image contains
both myocardial blood flow and blood pool activity, the latter activity should
be subtracted using the blood pool image after inhalation of 0-15 carbon
monoxide. Although the functional image of regional myocardial blood flow
can be derived from this technique, the image quality is not as good as that of
N-13 ammonia or rubidium-82 because of the subtraction procedure.
40 N. Tamaki et al.
100
H2O IMF6E CO IMFGE
ANT MI
SUBTRACTION
RMBF IMAGE
FIG. 7. The 0-15 water image (top left) and blood pool image (top right) using 0-15
carbon monoxide in a patient with anterior wall myocardial infarction. Regional
myocardial blood flow (RMBF) functional image (bottom) is created after subtraction of
these two images. (Unit : ml/min /IOO g)
Clinical Applications
Recent clinical studies employing exercise or dipyridamole PET perfusion
imaging using either N-13 ammonia or rubidium-82 demonstrated high
sensitivity and specificity in detecting coronary artery disease (Table 1). The
diagnostic accuracy for detecting individual stenosis seems to be higher than
the routinely performed stress thallium-201 imaging [20,21] . These data have
been derived from qualitative perfusion analysis in selected patient populations.
A larger prospective clinical study using quantitative analysis may be warranted
for further definition of the diagnostic accuracy of PET perfusion imaging.
PET is suitable for quantification of the tracer concentration. Thus, the
appropriate tracer kinetic models permit quantification of regional myocardial
blood flow. The impairment of coronary .flow reserve may prove to be the most
sensitive functional value for detection of coronary artery disease [12].
Identification of viable but compromized ischemic myocardium has been of
great importance in the study of coronary artery disease. The patients who may
benefit from revascularization need to be identified. The presence or absence
TABLE l. Diagnostic accuracy of positron emission tomography for detection of coronary

artery disease
Authors No. of patients Tracer Stress Sensitivity Specificity
Schelbert 45 NH3 Dipyridamole 97% 100%
et al. [16]
Tamaki 25 NH3 Exercise 95% 100%
et al. [6]
Yonekura 60 NH3 Exercise 96% 100%
et al. [7]
Gould 50 RB Dipyridamole 95% 100%
et al. [12]
Stewart 81 RB Dipyridamole 84% 88%
et al. [20]
Go 202 RB Dipyridamole 93% 78%
et al. [21]
of redistribution on thallium-201 imaging has played a major role for this

purpose [22]. However, the redistribution analysis may often fail to detect
severely ischemic but viable myocardium [23]. In this respect, metabolic
imaging, such as FDG-PET shows promise for assessing tissue viability
[10',11,23]. In the PET perfusion study, stress-induced ischemia has been more
often detected by stress-rest N-13 ammonia PET study than the stress-delayed
thallium-201 imaging [8]. This technique predicts reversible ischemic myo-
cardium which improves regional function after revascularization more
accurately than the thallium-201 imaging [9]. This may be due to two separate
injections at rest and during stress and analysis on high-quality images.
It should be noted that PET is an expensive tool which has been installed in
only a limited number of institutes. To make this elegant technique a clinically
available tool, an easy and efficient application of PET is needed. Isotope
production will be simplified by minicyclotrons with automated isotope
production or generator-produced tracers, such as rubidium-82 or copper-62.
Future Roles of Positron Tomography

PET provides two major functions: a challenging tool for cardiovascular
research and a clinical tool for wide and useful applications. The high
sensitivity of labeling compounds in tracing physiological processes with
quantitative measurements offers unique research opportunities. It will playa
major role for providing insights into pathophysiology in cardiovascular
diseases.
In the clinical setting, PET competes with less expensive noninvasive
techniques, such as thallium-201 imaging and echocardiography. The success of
PET will greatly depend upon its ability to quantitate regional myocardial
blood flow and to define metabolic and other biochemical processes in the
myocardium. The combined study of flow and glucose metabolism identifies
42 N. Tamaki et al.
viable tissue more specifically than any other technique. However, future
modification of the PET study using generator-produced or minicyclotron-
produced tracers will further enhance the value of PET as a clinical tool for
evaluating cardiovascular diseases.
References
1. Phelps ME, Hoffman EJ, Coleman RE, (1976) Tomographic images of blood pool
and perfusion in brain and heart. J Nucl Med 17:603-612
2. Torizuka K, Yonekura Y, Tamaki N, (1985) Noninvasive evaluation of regional
myocardial perfusion with positron emission computed tomography. Jpn Circ J
39:719-726
3. Schelbert HR, Schwaiger M (1986) PET studies of tjhe heart In: Phelps ME,
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myocardial blood flow. Circulation 63:1259-1272
5. Krivokapich J, Smith GT, Huang SC, (1989) 13N-ammonia myocardial imaging at
rest and with exercise in normal volunteers: Quantification of absolute myocardial
perfusion wth dynamic positron emission tomography. Circulation 80: 1328-1337
6. Tamaki N, Yonekura Y, Senda M, (1985) Myocardial positron computed
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246-251
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13N-ammonia and high-resolution positron emission computed tomography. Am
Heart J 113:645-654
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9. Tamaki N, Yonekura Y, Yamashita K, (1989) Value of rest-stress myocardial
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766-778
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12. Gould KL, Goldstein RA, Mullani NA, (1986) Noninvasive assessment of coronary
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dilation: VIII. Clinical feasibility of positron cardiac imaging without a cyclotron
using generator-produced rubidium-82. J Am Coli Cardiol 7:775-789
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relation to symptom and heart rate changes Lancet 1983 (13) 1986
14. Mullani NA, Godstein RA, Gould KL, (1983) Perfusion imaging with rubidium-82:
I. Measurement of extraction and flow with external detectors. J Nucl Med 24:
898-906
15. Goldstein RA, Mullani NA, Marani SK, (1983) Perfusion imaging with rubidium-
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24:907-915
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coronary stenosis by myocardial imaging during pharmacologic coronary vaso-
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ammonia and positron computed tomography. Am J CardioI49:1197-1207
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blood flow in vivo with H2 1S 0. Circulation 70:724-733
18. Huang SC, Schwaiger M, Carson RE, (1985) Quantitative measurement of
myocardial blood flow with oxygen-15 water and positron computed tomography:
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64:860-865
B
Neural Control of Coronary Blood
Flow
1
Autonomic Control of Coronary Blood
Flow
Eric O. Feig[l
Summary. Activation of parasympathetic fibers to the heart results in

bradycardia, a fall in blood pressure with a concomitant decrease in myocardial
oxygen consumption, that results in a decrease in coronary blood flow by a
local metabolic mechanism. If the heart is electrically paced at a constant rate,
then a clear increase in coronary blood flow is observed during parasym-
pathetic activation. Baroreflex parasympathetic cholinergic coronary
vasodilation has been demonstrated in response to carotid sinus hypertension,
and carotid body chemoreceptor activation by hypoxia results in transient
parasympathetic coronary vasodilation. Coronary vasodilation secondary to the
intracoronary infusion of acetylcholine depends upon functioning endothelial
cells to produce endothelium-derived relaxing factor. Normal human epicardial
coronary arteries dilate with acetylcholine infusions, but vessels in patients with
atherosclerosis constrict, suggesting that the loss of endothelial function is an
early marker for atherosclerosis.
Activation of sympathetic fibers to. the heart results in tachycardia and
augmented contractility that produce an increase in myocardial oxygen
consumption, which results in an increase in coronary blood flow by a local
metabolic mechanism. Paradoxically, there is a concomitant alpha-receptor-
mediated coronary vasoconstrictor effect that competes with the metabolic
vasodilation and limits the increase in coronary flow. Adrenergic coronary
vasoconstriction has been demonstrated during carotid sinus baroreflexes,
emotion, and exercise. Adrenergic coronary vasoconstriction helps maintain
a uniform transmural blood flow in the left ventricular wall during the
tachycardia of exercise. This beneficial effect may explain why paradoxical
adrenergic coronary vasoconstriction is activated during cardiovascular
stress.
1 Department of Physiology and Biophysics, University of Washington, School of

Medicine, Seattle, WA 98195, USA
47
48 E.O. Feigl
Key words: Sympathetic-Parasympathetic-Reflex-Alpha receptors-Beta

receptors
Introduction
Study of the autonomic control of the coronary circulation is complicated by
the interaction with aortic pressure and myocardial metabolism, which also
determine coronary blood flow. Arterial pressure is the result of cardiac output
and the peripheral resistance, but arterial pressure is also the afterload for the
left ventricle and thus an important variable in determining myocardial
metabolism. Since parasympathetic and sympathetic activation have chrono-
tropic and inotropic cardiac effects that alter cardiac output and myocardial
metabolism, it has often been difficult to separate the direct action of auto-
nomic activation on the coronary vessels from the indirect effects acting via
changes in coronary (aortic) perfusion pressure and local metabolic control of
coronary vessels. For this reason, experiments are often done with controlled
coronary pressure and heart rate.
The literature prior to 1981 has been extensively reviewed, and many of the
pioneer papers will not be cited here [1]. This review will emphasize research
on the control of coronary blood flow, since the work on isolated vessels is
covered elsewhere.
Parasympathetic
Cholinergic Effects
Activation of parasympathetic fibers to the heart results in bradycardia, a fall
in blood pressure with a concomitant decrease in myocardial metabolism. The
diminished myocardial oxygen consumption results in a decrease in coronary
blood flow by a local metabolic mechanism. When the heart was electrically
paced at a constant rate in dogs, vagal stimulation results in an increase in
coronary flow independent of the negative chronotropic and inotropic effects of
parasympathetic activation [2]. Intracoronary infusion of acetylcholine pro-
duces coronary vasodilation in dogs when the heart was paced at a constant
rate [3]. Acetylcholine also dilates large canine epicardial coronary arteries [4].
The effects of vagal stimulation and acetylcholine are blocked by atropine,
indicating that muscarinic receptors are involved.
In species other than dogs, the coronary effects of acetylcholine are
complicated [5]. In baboons, low doses of acetylcholine result in an increase in
coronary blood flow and coronary venous oxygen tension without a change in
myocardial oxygen consumption, indicating a clear vasodilation [3]. At high
doses, the negative inotropic effect of acetylcholine is dominant and a net
decrease in coronary blood flow is observed [3,6,7]' although coronary venous
oxygen tension still increased [3]. At very high doses of acetylcholine, a
1. Autonomic Coronary Control 49
decrease in coronary blood flow and myocardial oxygen consumption was

observed without a change in coronary venous oxygen tension. This result is
ambiguous and may indicate a direct vasoconstriction combined with a negative
inotropic effect.
In goats, a weak coronary vasodilation was observed at low doses of
acetylcholine, and a marked decrease in coronary flow and myocardial oxygen
consumption was found with high doses [8]. In calves, intracoronary bolus
doses of nicotine or acetylcholine resulted in prompt transient decreases in
coronary flow; however, myocardial oxygen consumption was not determined,
so it is difficult to interpret the relative importance of coronary vascular and
myocardial effects [9]. In pigs, intracoronary bolus doses of acetylcholine also
produce transient decreases in coronary flow, but myocardial oxygen
consumption was not measured [10].
The vasodilator action of acetylcholine in the coronary circulation depends
on endothelium-derived relaxing factor (EDRF) [11,12]. The action of
acetylcholine in the human coronary circulation appears to be exquisitely
sensitive to the functional state of the coronary endothelium. In normal
subjects, intracoronary infusion of acetylcholine produced epicardial coronary
artery dilation [13-15], but this is reversed to a paradoxical vasoconstriction in
atherosclerotic arteries, even when the atherosclerotic involvement is slight
[13,15,16].
The epicardial coronary arteries of transplanted human hearts undergo an
accelerated diffuse atherosclerosis and also exhibit paradoxical constriction
with acetylcholine [17,18]. Thus, the conversion from cholinergic dilation to
paradoxical constriction of epicardial coronary arteries is an early marker of
human atherosclerosis. Whether the loss of endothelial function as indicated by
paradoxical constriction is part of the causal chain in atherosclerosis, or only
associated with it, is unknown at this time. The possible role of acetylcholine in
the syndrome of coronary spasm is discussed by Tomoike in Chapter D-6.
The response of human coronary resistance vessels to acetylcholine is
vasodilation with an increase in blood flow, whether the epicardial vessels
dilate or constrict [19,20].
In summary, acetylcholine in moderate doses causes vasodilation and
increases coronary blood flow in dogs, baboons, and humans. Normal
epicardial coronary arteries dilate in response to acetylcholine, but a
paradoxical constriction is observed in atherosclerotic vessels.
Parasympathetic Reflex Control of Coronary Flow

Carotid sinus hypertension produces a reflex inhibition of sympathetic dis-
charge to the heart and systemic resistance vessels and an activation of para-
sympathetic fibers to the heart, resulting in bradycardia. In addition to vagal
bradycardia, there is a reflex parasympathetic coronary vasodilation [21].
Reflex parasympathetic coronary vasodilation has also been demonstrated by
afferent stimulation of the carotid sinus nerve [22-24].
50 E.O. Feigl
Excitation of carotid body chemoreceptors produces a reflex hyperpnea and

parasympathetic bradycardia. A transient reflex vagal coronary vasodilation is
part of the carotid body chemoreceptor reflex during hypoxia [25] or to the
intracarotid injection of nicotine or serotonin [26-28]' There is also a coronary
vasodilation secondary to intracarotid nicotine injection by an unexplained
mechanism not involving parasympathetic or sympathetic nerves [29].
The Bezold-larisch reflex response of vagal bradycardia and inhibition of
adrenergic peripheral vascular tone is produced by the intracoronary injection
of veratrum alkaloids. Interestingly, the Bezold-larisch reflex activates para-
sympathetic coronary vasodilation [30,31], even when the coronary circulation
is underperfused [32]. The coronary vascular response also includes inhibition
of adrenergic coronary vasoconstrictor tone [32]. A similar parasympathetic
reflex coronary vasodilation can be elicited by intracoronary injection of
ouabain [33].
Activation of pulmonary C-fibers with injections of capsaicin results in reflex
bradycardia and systemic hypotension. This pulmonary reflex also elicits
parasympathetic coronary vasodilation [34,35].
In summary, reflex parasympathetic coronary vasodilation has been
demonstrated in dogs in baroreceptor and chemoreceptor reflexes where vagal
bradycardia is elicited. A resting parasympathetic vasodilator tone has not thus
far been observed.
Cholinergic Control of Transmural Flow

The inner layer of the left ventricle normally receives 10% - 20% more blood
flow than the outer layer in unstressed hearts [1]. However, the inner layer of
the left ventricle is much more susceptible to underperfusion than the outer
layer, and a fall in the inner/outer flow ratio to values below approximately 0.9
usually indicates subendocardial ischemia. Therefore, agents which selectively
dilate the coronary circulation in the inner layer of the left ventricle might have
therapeutic potential. The intracoronary infusion of acetylcholine increases the
inner/outer flow ratio [36-38]. However, the vasodilation produced by vagal
activation increases coronary blood flow uniformly across the left ventricular
wall [32,38].
Sympathetic
Adrenergic Effects
Activation of sympathetic fibers to the heart results in tachycardia and
increased contractility mediated by beta-adrenergic receptors. This increase in
cardiac activity results in an augmented myocardial oxygen consumption that
produces coronary vasodilation by a local metabolic mechanism. However,
sympathetic activation also results in a relative coronary vasoconstriction
mediated by alpha-adrenergic receptors. Usually the net effect of sympathetic
activation to the heart is an increase in coronary blood flow, indicating that the
metabolic vasodilation is the dominant mechanism; however, alpha vaso-
constriction competes with the metabolic vasodilation by as much as 20%-
30%, thus increasing oxygen extraction across the coronary circulation [39].
The cardiac effects of sympathetic activation can be blunted by beta receptor
blockade that unmasks alpha receptor-mediated coronary vasoconstriction, and
a net decrease in coronary blood flow is observed [40-42]. Alpha receptor-
mediated vasoconstriction occurs primarily in large arterioles with internal
diameters greater than 100 11m, in contrast to metabolic vasodilation that occurs
generally throughout the microvasculature, including arterioles smaller than
100l1m [43]. The interesting interaction between adenosine and alpha
adrenergic coronary mechanisms is covered by Hori in Chapter B-2.
In the presence of beta receptor blockade, canine epicardial coronary
arteries constrict in response to sympathetic activation [44-47] or selective
alpha-1 agonists [45,48]. In beta-receptor blocked calves, epicardial coronary
arteries vasoconstrict in response to selective alpha-2 agonists [6].
Alpha Receptor Subtype

Post junctional alpha receptors in coronary resistance vessels are a mixture of
alpha-1 and alpha-2 types. Activation of sympathetic fibers to the heart in
previously beta-receptor blocked canine preparations results in coronary
vasoconstriction that can be blocked by selective alpha-1 or alpha-2 antagonists
in various proportions [42,45,47,49-51]. The vasoconstriction due to the
infusion of norepinephrine in beta-receptor blocked preparations is also
variously prevented by selective alpha-1 and alpha-2 antagonists [51-54].
Coronary vasoconstriction can also be elicited with selective alpha-1 and alpha-
2 agonists [45,52].
Beta Receptor Subtype

In vitro studies with isolated rings or strips of large epicardial coronary arteries
have usually been interpreted to indicate that beta-1 receptors mediate
relaxation without significant beta-2 effects. In contrast, in vivo experiments
have demonstrated beta-2 receptor dilation in coronary resistance vessels in
response to isoproterenol after cardiac beta-1 receptor blockade [55-58].
However, prior beta-1 receptor blockade to prevent cardiac effects rules out
the possibility of finding coronary vascular beta-1 effects (an extensive review
appears in [1]).
The difficulty in determining coronary beta-1 effects in vivo has been to
avoid the metabolic vasodilation that occurs secondary to activating myocardial
beta-1 receptors that produces tachycardia and increased cardiac con-
tractility. Therefore, a nonbeating cardiac preparation without chronotropic or
inotropic effects is required. Recently, the long asystoles that follow the
cessation of pacing in hearts with atrioventricular block were used to study beta
receptor vasodilation. Intracoronary injections of isoproterenol during asystole
52 E.O. FeigJ
when there were no inotropic or chronotropic effects resulted in coronary

vasodilation. The isoproterenol vasodilation was blocked by either beta-lor
beta-2 selective antagonists, thereby indicating that coronary resistance vessels
have both beta-l and beta-2 receptors [59].
In summary, the coronary circulation has both alpha vasoconstrictor and
beta vasodilator adrenergic receptors. Vasoconstriction is mediated by both
alpha-l and alpha-2 subtype receptors. Interestingly, there are both beta-l and
beta-2 receptors on coronary vascular smooth muscle, in contrast to peripheral
vessels, where only beta-2 receptors are found.
Sympathetic Reflex Control of Coronary Flow

Carotid sinus hypotension results in reflex sympathetic activation to the heart
to produce tachycardia and an increase in contractility. There is also reflex
activation .of sympathetic fibers to the peripheral vessels that results in an
increase in peripheral resistance. These adrenergic actions result in an
augmented myocardial oxygen metabolism that is accompanied by a local
metabolic coronary vasodilation. However, a concomitant reflex activation of
sympathetic fibers to the coronary circulation results in a relative vasoconstric-
tion mediated by alpha receptors. There is a net increase in coronary blood
flow, but the alpha-mediated constrictor influence restricts the metabolic
vasodilation by about 30% [39]. The alpha-receptor-mediated baroreceptor
reflex coronary vasoconstriction may be "unmasked" by prior beta-receptor
blockade that blunts the cardiac effects [60-63].
Adrenergic coronary vasoconstriction has been demonstrated in dogs during
a conditioned emotional response to a sound that precedes an unavoidable
noxious stimulus [64]. A marked decrease in coronary flow in an artificially
stenosed coronary artery has been observed during the post-anger state in
dogs [65].
Placing a human subject's hand in cold water results in a generalized
sympathetic activation with an increase in coronary blood flow secondary to the
augmented myocardial metabolism [66]. Interestingly, a subset of patients with
angina pectoris demonstrated a decrease in coronary sinus blood flow during a
cold pressor reflex [67-70].
In summary, reflex sympathetic coronary vasoconstriction that competes
with the concomitant metabolic vasodilation and reduces the net increase in
coronary flow has been observed in the carotid sinus baroreceptor and cold
pressor reflexes and during emotional states.
Sympathetic Control of Coronary Blood Flow

During Exercise
Generalized sympathetic activation to the heart and peripheral circulation is
part of the exercise response, with large increases in cardiac and skeletal
muscle oxygen consumption [71]. An alpha-receptor-mediated coronary vaso-
constriction that limits metabolic vasodilation has been demonstrated in many
canine exercise studies [72-86]. There is also evidence of adrenergic coronary

constriction in humans during exercise, although the measurements are often
indirect [87-89].
Sympathetic coronary vasoconstriction seems paradoxical during exercise,
when coronary blood flow must increase to meet the large increase in
myocardial oxygen consumption. The problem appears particularly acute for
the inner layer of the left ventricle, which is only perfused during diastole
because of the large compressive forces acting on subendocardial vessels during
systole [90]. Flow to the inner layer of the left ventricle begins in diastole, is
stopped by compressive forces during systole, and is then restarted with the
next diastole. The tachycardia of exercise exacerbates the problem, because
the diastolic periods become very brief with increasing heart rate.
Adrenergic Control of Transmural Flow

The infusion of norepinephrine results in a uniform vasoconstriction across the
wall of the left ventricle, indicating that there is not a greater density of
post junctional alpha receptors in the vessels of the outer layer than those of the
inner layer [75,91-93]. In contrast, electrical activation of sympathetic fibers to
the heart results in a somewhat greater vasoconstriction in the outer layer than
in the inner layer of the left ventricle, suggesting that innervation may be
denser in the outer layer than in the inner layer [91,94]'
Huang and Feigl [81] observed that adrenergic alpha-receptor blockade in
the circumflex region of the left ventricle produced the expected increase in
coronary blood flow during exercise, but the transmural distribution of flow
worsened with a decrease in the inner/outer flow ratio. In the absence of
alpha-receptor blockade, the inner/outer flow ratio is normally greater in the
circumflex region than in the left anterior descending region. However, with
alpha blockade in the circumflex region, during high levels of exercise, the
inner/outer flow ratio is worse (lower) than in the comparison anterior
descending region of the same heart. Thus, alpha-receptor vasoconstrictor
activation during exercise helps maintain a more uniform transmural distribu-
tion of blood flow in the wall of the left ventricle. A greater alpha-receptor-
mediated vasoconstriction in the outer layer than in the inner layer of the left
ventricle is probably not a complete explanation of the beneficial effect of
adrenergic vasoconstriction during exercise observed by Huang and Feigl, since
an impedance matching between coronary vascular admittance and heart rate
may be involved.
The postulated mechanism is that alpha vasoconstriction stiffens intra-
myocardial vessels so there is less wasted back and forth "sloshing" of coronary
flow during systole and diastole. Spaan has provided evidence that there is a
concealed backflow in coronary arteries at the beginning of each systole [95].
In the systolic-diastolic interaction hypothesis of Hoffman and Spaan, the
systolic force in the wall of the left ventricle acts as an "intramyocardial
pump" not only to impede flow by narrowing intramyocardial vessels, but also
by causing retrograde flow that is diverted to vessels in the outer layers in the
54 E.O. Feigl
left ventricle, as well as expanding the epicardial coronary arteries [96]. Thus, a
portion of arterial inflow fills coronary vessels in the inner layers of the left
ventricle at the beginning of diastole before nutritive flow through the
capillaries can be established during the remainder of diastole. At the onset of
the next systole, the intramyocardial arteries are squeezed and there is a
retrograde flow from the inner layer to the outer layer and epicardial arteries.
This oscillatory flow is wasteful, since the intramyocardial vessels must be filled
before capillary flow can begin. When diastole becomes very brief during
tachycardia, there may not be adequate time for capillary flow if the oscillatory
flow is large. Adequate perfusion of the inner layer of the left ventricle is a
special problem during exercise, when heart rate and myocardial oxygen
consumption are both maximal.
The "anti-slosh" hypothesis of Huang and Feigl is that the wasted flow that
occurs between systole and diastole is lessened during exercise by adrenergic
coronary vasoconstriction of vessels larger than approximately 100 ~m in
diameter. The effect is to stiffen the intramyocardial vessels so that there is less
to-and-fro sloshing of blood during diastole and systole. The hypothesis is that
alpha adrenergic vasoconstriction "tunes" the coronary input impedance to the
heart rate during exercise, thereby preserving flow to the vulnerable inner layer
of the left ventricle. The input impedance of an electrical transmission line is
dependent upon resistance, capacitance, and inductance, and must be carefully
adjusted to the frequency of the current to be carried if large losses are to be
avoided. Similarly, the hypothesis is that sympathetic activation of coronary
vascular smooth muscle adjusts the coronary input impedance to match the
heart rate during exercise.
Whether adrenergic coronary vasoconstriction is beneficial under all cir-
cumstances is controversial. Heusch found that, in the presence of a flow-
limiting stenosis, alpha-2-mediated vasoconstriction is potentiated with adverse
effects [97]. It is possible that the sympathetic coronary vasoconstriction that
evolved to maintain flow to the inner layer of the left ventricle during exercise
may be detrimental in the presence of a stenosis [98].
In summary, the paradox of alpha coronary vasoconstriction that is observed
whenever there is generalized sympathetic activation to the cardiovascular
system may be resolved by the postulated "anti-slosh" hypothesis. During
tachycardia and augmented myocardial oxygen consumption that result from
sympathetic activation, there may be a concomitant stiffening of coronary
vessels that tunes the coronary input impedance to the heart rate so that
oscillatory systolic-diastolic flow is lessened and flow to the vulnerable
sub endocardium is maintained.
Acknowledgment. This work was supported by NIH grant HL 16910.

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60 E.O. Feigl
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76:737-745
2
Role of Alpha-adrenoceptor Activity in
Regulation of Coronary Blood Flow
During Myocardial Ischemia
Masatsugu Hori, Masafumi Kitakaze, Akira Kitabatake,
Takenobu Kamada I , and Michitoshi Inoue 2
Summary. We discuss the beneficial roles of alpha-adreno-ceptor actIvIty

on the pathogenesis of ischemic heart disease. The major effect of alpha-
adrenoceptor stimulation in the coronary arteries was thought to be vasocon-
striction, which may restrict myocardial oxygen supply and worsen myocardial
ischemia. However, this is not necessarily true, because (1) aiphal-adrenoceptor
stimulation favorably maintains endocardial coronary flow at the expense of
epicardial flow reduction, (2) alphal-adrenoceptor stimulation enhances the
release of adenosine, and alphaz-adrenoceptor stimulation increases the release
of EDRF and histamine, and (3) alphaz-adrenoceptor stimulation increases
the sensitivity of the effects of adenosine. These beneficial effects of alpha-
adrenoceptor stimulation may conversely attenuate the severity of myocardial
ischemia and reperfusion injuries. Further clinical studies are necessary for
better understanding of the roles of alpha-adrenoceptor activity in coronary
flow regulation and myocardial function in human ischemic hearts.
Key words: Alpha-adrenoceptor-Ischemia-Adenosine-Coronary blood flow

-Reperfusion injury.
Introduction
During myocardial ischemia, norepinephrine is released from the sympathetic
nerve terminals and acts on both alpha- and beta-adrenergic receptors in
ischemic myocardium and coronary arteries [1-3]. When myocardial ischemia
is prolonged, norepinephrine spills into the circulating blood to affect the
systemic vascular tone [4]. Accordingly, the release of norepinephrine during
I The First Department of Medicine, Osaka University School of Medicine, and the
2Department of Medical Information Sciences, Osaka University Hospital, Osaka, 553
Japan
61
62 M. Hori et al.
myocardial ischemia may modulate the severity of ischemia; beta-adrenergic

stimulation may enhance myocardial oxygen demand, and alpha-adrenergic
stimulation may cause vasoconstriction. Alpha-adrenoceptor stimulation is
particularly worthy of study, given the multiplicity of its vascular effects. It is
well known that alpha-adrenoceptor stimulation increases the tone of coronary
arteries [5-7]. In addition to this direct effect, alpha-adrenoceptor stimulation
interacts with other vasoactive substances that may regulate coronary blood
flow; alpha-adrenoceptor stimulation is reported to enhance releases of
endothelium-dependent relaxant factors (EDRF) [8], histamine [9] and
adenosine [10], and inhibit releases of norepinephrine. Alpha-adrenoceptor
stimulation also increases the sensitivity of adenosine receptors of coronary
arteries [11-13]. However, there is no clear consensus whether these coronary
effects caused by alpha-adrenoceptor stimulation are beneficial or deleterious
to ischemic injury. We shall discuss the individual and overall effects of alpha-
adrenoceptor activity on myocardial ischemia.
Norepinephrine Release During Myocardial Ischemia
Myocardial ischemia is known to be a potent trigger of norepinephrine release

from the sympathetic nerve terminals [3,4,14]. Deleterious sequelae, such as
the occurrence of fetal arrhythmias [15,16] and the acceleration of myocardial
cell damage [17,18]' have been attributed to the excessive stimulation of both
alpha- and beta-adrenergic receptors of myocardium and coronary arteries.
Accumulation and release of norepinephrine are predominantly induced at the
local nerve endings in the ischemic myocardium. The activation of the nerve
ending during ischemia is reported not to be influenced by the central
sympathetic nerve activity [3]. Sch6mig et al. [3] demonstrated that a massive
amount of norepinephrine is released during ischemia in the Langendorff
preparation of rat hearts, and showed that the amount of the released
norepinephrine is increased as the ischemic period is prolonged. They also
reported that the release of catecholamine occurs within 10 min after the onset
of ischemia. Wollenberger and Shaab [1] also observed that 3-4 min of
ischemia causes the overflow of a large amount of norepinephrine in isolated
rabbit hearts. Similarly, in in vivo dog hearts, a coronary arterial occlusion of
2.5 min also results in a net overflow of norepinephrine [19]. The consistency of
the timing of norepinephrine release in the isolated and in vivo hearts may
support the idea that the ischemia-induced norepinephrine release is regulated
with local factors rather than with central nervous system regulations.
Some hormones and neurotransmitters are released in the exocytotic process
which depends upon Ca2 + concentrations in the local environment. However,
release of norepinephrine from the sympathetic nerve terminals is considered
to be independent of extracellular calcium and has been characterized as a
nonexocytotic release in the ischemic myocardium [3]. Rather, accumulations
of sodium within the sympathetic neurons seem to trigger the release of
2. Alpha-adrenoceptor activity and CBF 63
norepinephrine [20]. Schomig et al. [20] reported that Na +- H+ exchange is

identified as the predominant pathway of sodium entry into the sympathetic
nerve ending during ischemia, because treatments with amiloride and
ethylisopropylamiloride, inhibitors of Na+ -H+ exchange, markedly suppress
the ischemia-induced norepinephrine release. The extrusion of protons which
may be accumulated during ischemia is coupled with sodium entry, leading to
intracellular sodium accumulation. During ischemia, decreases in intracellular
adenosine 5 ' -triphosphate (ATP) inhibits Na+ -K+-ATPase activity, which
may also contribute to sodium accumulation.
The Responses of Coronary Arteries to

Alpha-adrenoceptor Stimulation
Alpha-adrenoceptor stimulation contracts the isolated coronary arteries [5-7].
Zuberbuhler and Bohr [5] showed that exposure to norepinephrine under
beta-adrenoceptor blockade exerts the potent contraction of the isolated dog
coronary artery, concluding that alpha-adrenoceptor stimulation mediates
coronary vasoconstriction. The vasoconstrictive action caused by alpha-
adrenoceptor stimulation was more potent in the large coronary arteries than
in the smaller coronary arteries [5]. Vatner and Murray [6,7] confirmed by the
direct measure of coronary arterial diameters using an ultrasonic dimension
gauge that alpha-adrenoceptor stimulation caused by exogenous and endo-
genous norepinephrine causes potent vasoconstriction of large epicardial
coronary arteries in conscious dogs. On the other hand, Kelly and Feigl [21]
reported that increases in coronary vascular resistance during the exposure of
norepinephrine under the treatments with propranolol are due to the
vasoconstriction both in large and small coronary arteries. Indeed, Chilli an et
al. [22] directly observed that 100-300 11m of small coronary arteries responded
well to alpha-adrenoceptor stimulations. The contribution of small coronary
arteries to the increase in coronary vascular resistance during alpha-adreno-
ceptor stimulation is reported to be 40% [21]. These findings show that small as
well as large coronary arteries participate in the regulation of coronary blood
flow when sympathetic nerves are stimulated. Heusch et al. [23] clarified the
contributions of alphar and alpha2-adrenoceptor activities to vasoconstriction
using an alphaJ-adrenoceptor agonist and antagonist, i.e., methoxamine and
prazosin, and an alphaz-adrenoceptor agonist and antagonist, i.e., BHT 920
and rauwolscine in open-chest dogs. An intracoronary administration of
methoxamine increased the large coronary arterial resistance by 19% and
end-diastolic coronary resistance by 10%, whereas BHT 920 did not affect the
large vessel resistance but increased the end-diastolic coronary vascular
resistance by 38%. End-diastolic coronary vascular resistance is known to be
mainly determined by the tones of small coronary arteries. Left cardiac
sympathetic nerve stimulation increased the large vessel resistance by 11 % and
end-diastolic coronary resistance by 32%. This increase in the large vessel
64 M. Hori et al.
resistance was not prevented by rauwolscine but was almost inhibited by

prazosin. The increase in end-diastolic resistance was hardly prevented by
prazosin but was completely inhibited by rauwolscine. These observations
indicate that alpha-adrenoceptor-mediated vasoconstriction of large coronary
arteries is mediated exclusively by alphal-adrenoceptor activity, whereas alpha-
adrenergic vasoconstriction of small coronary arteries is mediated partially by
alphal-, but predominantly by alpharadrenoceptor activities. Another aspect
of alpha-adrenoceptor-mediated vascular effects is the redistribution of
intramyocardial flow, since it is reported that alpha-adrenoceptor stimulation
favors an increase in endocardial flow at the expense of epicardial flow
reduction during coronary hypoperfusion [24,25]. On the other hand, alphal-
adrenoceptors are located mainly at the outer part of the coronary arteries,
whereas alpharadrenoceptors are predominantly located at the inner part of
the coronary arteries. Since the neurons are only penetrated into the outer
one-third of medial layers of the coronary arteries and arterioles, alphal-
adrenoceptor may be activated by norepinephrine secreted from sympathetic
nerve endings, and alpharadrenoceptors may be mainly activated by
circulating catecholamines.
The second messengers of alphal-adrenoceptors in cells are recognized as
being 1,2-diacyl glycerol (DG) and inositol phosphates (IP) [26]. DG activates
protein kinase C, which phosphorylates intracellular and membrane-bound
enzymes. Inositol-l,4,5-trisphosphate (IP3 ), a member of the IP family, is
reported to release Caz+ from the sarcoplasmic reticulum. Intracellular Caz+ is
capable of activating the myosin light chain kinase in the myofilament in
smooth muscle cells, which triggers phosphorylation of the myosin light chain
and thereby causes vascular contraction [27,28]. Although activation of protein
kinase C also phosphorylates myosin light chain kinase, it relaxes the coronary
smooth muscles contracted by the myosin light chain kinase. This difference is
thought to be attributed to the differences of the sites in which myosin light
chain kinase and protein kinase C phosphorylate: myosin light chain kinase
phosphorylates the serine residue of the myosin light chain and protein kinase
C phosphorylates the threonine residue [27-29]. On the contrary, adenosine
3'5' -cyclicphosphate cyclic (cAMP), which is increased by beta-adrenoceptor
stimulation, causes vasorelaxation because cAMP may decrease Caz+
concentrations through enhancement of Caz+ uptake into the sarcoplasmic
reticulum. Alpharadrenoceptor stimulation is reported to decrease the
intracellular cAMP content which is responsible for vasoconstriction [26].
Furthermore, alphaz-adrenoceptor stimulation is reported to activate Na +- H+
exchange [30] which, in turn, stimulates the reverse Na+ -Ca2+ exchange and
thereby accumulates the intracellular Caz+. These increases in Caz+ may also
contribute to the vasoconstriction which is present during alpharadrenoceptor
stimulations.
Since these recent advances have clearly revealed the roles of alphal- and
alphaz-adrenoceptors upon coronary smooth muscles, we should investigate the
physiological roles of these differences of location and receptor subtypes and
their contribution to coronary flow regulation during ischemia.
Interactions Between Alpha-adrenoceptor Activities

and Vasoactive Substances
Alpha-adrenoceptor activity is reported to enhance releases of various
vasoactive substances in the heart which may affect the severity of myocardial
ischemia. Alpharadrenoceptor stimulation enhances the release of EDRF
from the endothelial cells [8]. Two experiments provide evidence for this. First,
(mmHg) a
8:() ~::E
50
o
1::[
(ml/mln)
~ _/ ~----
() o[
L propranolol (0.3 mg/kg,l.c.)
I I
adenosine (2,ug/kg/min,i.c.)
30s
(mmHg) b
8:() ~::E
50
o
(ml/min)
LL lOOt
CD 50
()
o Lpropranoiol (0.3 mg/kg,l.c.)
yohimbine (9,ug/kg/mln,l.c)
I
adenosine (2,ug/kg/mln,l.c.)
30s
FIG. 1. Representative records of coronary perfusion pressure (CPP) and coronary

blood flow (CBF) before and during infusion of adenosine with or without moderate
alpharadrenergic attenuation. a hyperemic response of CBF during infusion of
adenosine (2Ilg/kg/per min, i.c.). b This hyperemic response was markedly attenuated
when treated with yohimbine (9Ilg/kg/min, i.c.). In each experiment, propranolol
(0.3 mg/kg) was pretreated. Note that baseline CBF was not altered by moderate
alpharadrenergic attenuation. Propranolol per se did not alter either baseline CBF or
hyperemic flow. (From [11] with permission)
66 M. Hori et al.
when resting tone is low, norepinephrine concentration-vascular contraction

curves in the absence of endothelium are shifted to the left and have an
increased maximal response compared to the endothelium-intact arteries.
Second, an exposure to norepinephrine relaxes the precontracted arteries
under beta-adrenoceptor blockade. This response is blocked by alphaz-
adrenoceptor antagonists and mimicked by c10nidine but not by methoxamine.
The release of EDRF by alphaz-adrenoceptor stimulation is also observed in
the small coronary arteries and increases the coronary blood flow [8], although
physiological significance during exercise and ischemia are yet to be elucidated.
Recent reports suggest that nitric oxide (NO) is an essential of EDRF which
increases cyclic GMP and relaxes coronary smooth muscles [31].
Alphaz-adrenoceptor stimulation is known to inhibit norepinephrine release
from the sympathetic nerve endings [26,32], resulting in a decrease in the
coronary arterial tone. It is also known that alpha2-adrenoceptor stimulation
increases release of histamine in the skeletal muscle [9]. If histamine is released
due to alpha2-adrenoceptor stimulation in the heart, this substance may
increase coronary blood flow.
Recently our laboratory reported that alpha2-adrenoceptor stimulation
increases coronary vasodilatory effects of adenosine [11-13]. Adenosine is
known to be an endogenous substance which is released during exercise and
ischemia and which increases coronary blood flow [33]. Figure 1 shows that
yohimbine, an alphaz-adrenoceptor antagonist, attenuated adenosine-induced
coronary vasodilation in open-chest dogs. Clonidine, an alphaz-adrenoceptor
agonist, conversely affected adenosine-induced coronary vasodilation. These
observations, as suggested in Fig. 2, are consonant with the results of Nayler et
al. [34]. On the other hand, alphal-adrenoceptor stimulation augmented the
release of adenosine in the ischemic myocardium. Figure 3 depicts the effects
of alpha- and beta-adrenoceptor activities on adenosine release from the
ischemic myocardium in open-chest dogs. Administration of a low dose of
prazosin, which does not affect basal coronary blood flow, reduced adenosine
release [10]. The results obtained in the ischemic myocardium [10,35]
contrasted well to those in the non-ischemic myocardium [36]. In the non-
ischemic hearts, propranolol inhibited release of adenosine, while prazosin did
not inhibit it during an intracoronary infusion of norepinephrine. In the rat
isolated cardiomyocytes, intracellular adenosine production during hypoxia was
increased by alpha\-adrenoceptor stimulation. The enhanced production of
adenosine was mimicked by phorbol 12-myristate 13-acetate (PMA) , a
stimulator of protein kinase C, and was inhibited by H-7, an inhibitor of
protein kinase C, indicating that activity of protein kinase C plays a crucial role
in adenosine production in the ischemic heart [37]. These results suggest that
protein kinase C may affect the enzymes responsible for adenosine production
and degradation, or production of AMP. It should be noted, however, that
enhanced release of adenosine by alpha I-adrenergic stimulation was only
observed during ischemia. Thus, the underlying mechanism for this
phenomenon may be different from that of adenosine release when beta-
adrenoceptors are stimulated.
moderate alpha, ·attenuatlOn patent alpha, -attenuatIOn

a .D. orooranolOl 0 J mll/klll C (mill_111m."
b
.c. lII'_enoIol Olmg/klllC
2~0 .m. oroo<anolOl 0 J mg/kgl C 750 . . lII'_enoIDI 0 1mg/kll IC

yohImbIne g.g/kg/mon I t yohorlOne ''-lI/kll/m.. I C
~
200 n-8 £ n"
"8
.Q
.c
...>-
CII
C
0
~
u
InfUSion rate of adenosine InfusIOn rate of adenosine
moderate alpha, -stimulatIOn patent alpha,-stmulatlOn

C .i5.. propranolOl 0 1mg/kgl C d a. o<_enoIDI 0 1mg/kgl C
..... propranolOl 0 1mg/kgl C

1101'--....
__ pr_anoIoI Dlmg/klllC
Iml/l.g/m."
noreoonepl1rone 0 Ol.g/kg/m,n I C Dl.g/kll/m..IC
OralOSln 6... g/kg/m.n I C prlZOSon 1,"/kl/mon I C
200
1~0
..
100 ..
~o
o 0.5
,nfuSlOn rate of adenosine InfusIOn rate of adenoSine
FIG. 2. Hyperemic coronary blood flow during intracoronary infusion of 3 doses of

adenosine in a moderate or b potent alphaz-adrenoceptor attenuation, and c moderate
or d potent alphaz-adrenoceptor stimulation. Moderate and potent alphaz-adrenoceptor
attenuation were produced by intracoronary infusion of 9 and 20I1g/kg/min i.c, yohim-
bine, respectively, and moderate and potent alphaz-adrenoceptor stimulation were
produced by norepinephrine (0.03 and 0.3I1g/kg/min i.c., respectively) with prazosin
(6I1g/kg/min i.e.). *P < 0.05; ** P < 0.001 vs propranolol treated condition (open
column). Values are means ± SE. (From [11] with permission)
Effects of Alpha-adrenoceptor Activity on

_Myocardial Ischemia
Considering the effects of alpha-adrenoceptor activity on myocardial and
coronary functions, it is unclear whether alpha-adrenoceptor stimulation is
beneficial or deleterious to myocardial ischemia. Since alpha!" and alphaz-
68 M. Hori et al.
(n mol/IDOl/min)
20 t means±SE
• untreated (n=9)
o alpha, - attenuation (n=10)
c nonselective alpha-attenuation (n=8)
• alpha,-attenuation (n=8)
15 • beta-attenuation (n=8)
* p<0.05. ** p<O.OI vs. the untreated
\I) I---·t*......
en
:'"-····r·····t··
r"'--'" '-
'/,-r. ······rnuu......-- -- _____ u...~
aJ 10
Q)
Q)
a:
"
Q)
c:
'w0
c:
Q)
'0
5
,, ,,/'1
,' ,,
<2: :: i
r --
** **
-1' . -'. L --_I. -. ----1 .. --. -.---___ **
~.-.f·---·±-'-'- t----. r------ -t-------------t~ ~ .. ---.-----~-.:J*
If." .. ' 1*
I
o I!· ** ** ** ** **
i i i i ,
con trOt 3 5 7 10 15 20
(min)
onset Time after Onset of Hypoperfusion
of
hypoperfusion
FIG. 3. Adenosine release during coronary hypoperfusion under alpha]- (prazosin),

alphar (yohimbine + propranolol), non-selective alpha- (phentolamine + propranolol),
and beta-adrenoceptor (propranolol) attenuations. Note that both prazosin and
phentolamine markedly reduced the release of adenosine. n, Number of measurements.
(From [10] with permission)
adrenoceptor actIVIties exert different cardiovascular effects, dose-related

actions may modify the overall effects.
When circulating norepinephrine is not high, alphaz-adrenoceptor stimula-
tion provides a beneficial effect for ischemic injury [13]. During coronary
hypoperfusion, moderate alpharadrenoceptor stimulation, which does not
cause direct coronary vasoconstriction, increased coronary blood flow of the
ischemic area and restored the myocardial contractile dysfunction (Fig. 4). An
increase in coronary blood flow may be attributed to the release of histamine
or EDRF, enhancement of sensitivity of adenosine receptors, and attenuation
a (m
J60
J40
I~
b (rnl/lCDclmin)
40
l
1:1)
}20
10
(~) 1t it • m•• ns±SE

C
n-7
."<0.05
10 +"<0.005
·rj
t ++"<0.001
.s. the vllue .t IOmin
15
~
clonidine (0.24PI/kl/min. ic)
00 10 20 30
(min)
Time after the Onset of Ischemia
FIG. 4. a Changes in coronary perfusion pressure, b coronary blood flow, and c

fractional shortening during intracoronary infusion and withdrawal of clonidine infusion
in coronary hypoperfusion. Three to five min after clonidine infusion, coronary blood
flow was gradually increased (b) and fractional shortening recovered (c), indicating that
intracoronary infusion of clonidine improves mechanical myocardial function during
ischemia. During this protocol, reduced coronary perfusion pressure was kept constant
(a). (From [131 with permission)
of release of norepinephrine from the sympathetic nerve. We also observed the

beneficial effects of clonidine even in denervated hearts, showing that the
attenuation of release of norepinephrine by alpharadrenoceptor stimulation is
not involved. It is of interest that the beneficial effects of alpharadrenoceptor
on myocardial ischemia were abolished under treatment with 8-phenyl-
70 M. Hori et al.
a
. (mmHg) d (ml/l00g/min)
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~
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0/1 100 6
ll.
c
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ll. 2 :E
...
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III
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0
0
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0 0
b (ml/l00g/min) e (%)
.2 -20
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)(
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4 £ means±SE
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n=5
0 0
ischemia ischemia ischemia ischemia
with clonidine with clonidine
FIG. 5. a Changes in coronary perfusion pressure, b coronary blood flow, c fractional

shortening, d myocardial oxygen consumption, e lactate extraction ratio, and f
adenosine release by intracoronary infusion of cionidine during coronary hypoperfusion.
Adenosine receptors were antagonized by intracoronary infusion of 8-phenyl-
theophylline. (From [13J with permission)
theophylline, an antagonist of adenosine receptors. Figure 5 shows that the

beneficial effects of clonidine on both myocardial and coronary functions were
completely prevented by the treatment with 8-phenyltheophylline. Therefore,
we can hypothesize that adenosine-induced coronary vasodilation is primarily
involved in the beneficial effects of alpharadrenoceptor stimulation. However,
if the dose of clonidine is high, ischemia may be worsened, because high doses
of clonidine conversely blunt the adenosine-induced coronary vasodilation

through its direct vasoconstriction.
Indeed, Heusch and Deussen [38] demonstrated that postsynaptic alphaz-
adrenoceptor stimulation mediated coronary vasoconstriction produced by
sympathetic nerve stimulation during critical reduction of coronary perfusion
pressure, and concluded that alphaz-adrenoceptor stimulation is deleterious for
myocardial ischemia. Seitelberger et al. [39] also demonstrated that alpha-
adrenoceptor blockade attenuated the severity of the exercise-induced myo-
cardial ischemia. Both exercise and sympathetic nerve stimulation elevated the
systemic norepinephrine level more than 1,000 pg/ml, which is a two- to three-
fold higher concentration than that during regional ischemic condition.
Norepinephrine concentration in the systemic blood is 200-300pg/ml even
when the LAD coronary flow was reduced to one-third of the control, and
fractional shortening was reduced to 5% from 25% in the control condition.
These observations indicate that if alphaz-adrenoceptor stimulation is potent,
enhancement of adenosine-induced vasodilation by alphaz-adrenoceptor
stimulation is masked by direct vasoconstriction. In potent alphaz-adrenoceptor
stimulation, platelet aggregation may also occur and contribute to the pre-
cipitation of myocardial ischemia since alphaz-adrenoceptors of the platelets
mediate their activation and aggregation, although adenosine released from the
ischemic myocardium is known to potently inhibit platelet aggregation [40,41].
In contrast, under low norepinephrine levels in the systemic arterial blood,
alphaz-adrenoceptor stimulation may enhance the adenosine-induced coronary
vasodilation which exerts the beneficial effect.
Alphal-adrenoceptor stimulation is also reported to be beneficial for
myocardial ischemia [10,35]. During coronary hypoperfusion (coronary per-
fusion pressure before and during ischemia: 105 vs 38 mmHg), administration
of a low dose of prazosin, which does not cause direct vasodilation but inhibits
the release of adenosine, further decreased coronary blood flow and worsened
the severity of ischemia (Table 1). When a larger dose of prazosin was
employed, direct coronary vasodilation was exerted, and thereby attenuated
the extent of ischemia [42]. Thus, it is most likely that mild to moderate alphar
adrenoceptor stimulation exerts beneficial effects on the ischemic myocardium
through the augmentation of adenosine release, whereas potent alpharadreno-
ceptor stimulation causes the deleterious effects via direct coronary vaso-
constriction.
Effects of Alpha-adrenoceptor Activity on
Reperfusion Injury
Contractile dysfunction after a brief period of ischemia is defined as myocardial
stunning [43,44]. Although the pathophysiology of myocardial stunning has
been extensively discussed, the role of alpha-adrenoceptor activity in this
abnormality is hardly known. Our recent observation revealed that administra-
tion of methoxamine attenuated myocardial stunning associated with enhanced
release of adenosine, and that treatment with 8-phenyethophylline completely
abolished this beneficial effect of methoxamine [45]. These results suggest that
-.)
N
TABLE 1. Changes in coronary hemodynamics, myocardial metabolism, and adenosine release after onset and withdrawal of alpha,-adrenoceptor
attenuation during hypoperfusion
Coronary
hemodynamics Myocardial metabolism Adenosine
CBF MV0 2 AdR
CPP (mliJOO AV02 D (mllJOO FS LER AVAdD (nmol/JOO
(mmHg) glmin) (mlldl) glmin) (%) (%) (pmollml) glmin) ~
Control 106 ± 4 102 ± I 7.81 ± 0.39 7.1 ±0.4 22.7 ± 1.5 19.8 ± 1.8 21.3 ± 5.9 I.X ± 0.6
::r:
0
:J.
Hypoperfusion ~
Untreated 37 ± I 27 ± 2 9.48±0.19 2.6 ± 0.5 2.7 ± 1.2 54.7±6.1 296.7 ± 40.1 X.O ± 1.2 e:..
ul-Attenuation 37 ± I 21 ± 2** 9.62 ± 0.36 2.1 ± 0.3** -1.6 ± 1.0** -50.6 ± 5.9 65.2 ± 15.9** 1.5 ± 0.4**
Withdrawal of
ul-attenuation 36 ± I 27 ±2 9.64 ± 0.56 2.6 ± 0.3 2.0 ± 1.2 -56.9 ± X.4 2X3.2 ± 49.7 7.4 ± 1.5
** P < 0.01 (n = 8) compared with the untreated condition

Values are means ± standard error of the means (SEM). CPP, coronary perfusion pressure; CBF, coronary blood flow; AV02 D, coronary arteriovenous oxygen
difference; MV0 2 , myocardial oxygen consumption; FS, fractional shortening; LER, lactate extraction ratio; AVAdD, coronary arteriovenous adenosine
concentration difference; AdR, adenosine release into coronary vein. (From [10] with permission)
alphal-adrenoceptor stimulation can attenuate the severity of myocardial

stunning through the enhanced release of adenosine. This is compatible with
the other studies supporting the protective effects of adenosine on myocardial
stunning [46,47]. However, it should be noted that the high dose of methoxa-
mine blunted the beneficial effects of methoxamine. Preservation of A TP may
not provide a mechanism for the beneficial effects of adenosine since the
replenishment of ATP does not necessarily restore contractile function [48,49]'
although this hypothesis can not be completely excluded [50,51].
Instead, calcium overload may be a primary mechanism of myocardial
stunning [52-56]. If this is the case, the alpha I-mediated release of adenosine
may attenuate myocardial stunning by inhibiting calcium influx through
stimulation of adenosine AI-receptors [57,58]. The reason that the higher doses
of methoxamine blunt the beneficial effects of alpha-adrenoceptor stimulation
may be attributed to the fact that the higher dose of methoxamine increases
intracellular Ca2 + during ischemia and reperfusion [59]. A report that prazosin
attenuates Ca2 + increases during reperfusion may support this hypothesis [60].
Alphal-adrenoceptor-mediated release of adenosine also attenuates the
activation of neutrophils [61,62]: Free radical generation is inhibited through
adenosine Az-receptor stimulation, and adherence to endothelial cells is
attenuated through adenosine AI-receptor stimulation [63]. Platelet aggrega-
tion may cause reperfusion injury since aggregated platelets impede the micro-
circulation of the coronary vessels. It is known that adenosine also inhibits
platelet aggregation through Az-receptor stimulation [40,41], and that endo-
genous adenosine released from the ischemic myocardium inhibits platelet
aggregation in the blood-perfused canine heart [64]. When platelet aggregation
occurs during ischemia and reperfusion periods, adenosine 5' -diphosphate
(ADP), serotonin, and several prostaglandins are released to provoke vaso-
constriction which may further damage the myocardium [40]. Thus, inhibition
of platelet aggregation by endogenous adenosine released from ischemic myo-
cardium may attenuate reperfusion injury. Since 'adenosine is a potent vaso-
dilator, massive release of adenosine during ischemia and reperfusion may
attenuate microcirculatory disturbances [65,66]. Thus, an improvement of the
coronary microcirculation by adenosine [67,68] enhanced by alpha I-adreno-
ceptor stimulation may largely contribute to the prevention of reperfusion
injury.
In contrast, there is no clear consensus whether alphaz-adrenoceptor stimula-
tion is beneficial in attenuating myocardial stunning. Although alphaz-adreno-
ceptor stimulation may enhance adenosine-induced coronary vasodilation,
there is no close relation between the severity of myocardial stunning and the
extent of coronary vasodilation. Alpharadrenoceptor stimulation may cause
platelet aggregation which may worsen myocardial stunning. This question
remains to be elucidated by further investigation.
Acknowledgment. This work is supported by a Grant-in-Aid for Scientific

Research (No. 0367044a) and on Priority Areas (No. 62624005) from the
Ministry of Education, Science, and Culture, Japan.
74 M. Hori et al.
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3
Vasoactive Monoamines in the
Regulation of Arterial Tone
Mitsuhiro Yokoyama 1 and Hozuka Akita 1
Summary. Autacoids such as serotonin and histamine are potent vasoactive

substances. In vivo and in vitro studies indicate that these monoamines are
potent vasoactive substances, and that the endothelium modulates vasomotor
responses to these amines.
We examined the effect of serotonin on coronary vasculature and cardiac
performance in anesthetized open chest dogs. Without coronary stenosis,
intracoronary infusion of serotonin caused concentration-dependent increases
in coronary blood flow. During fixed coronary stenosis, serotonin, except the
highest concentration, failed to alter coronary blood flow. During dynamic
cornary stenosis, intracoronary serotonin decreased coronary blood flow
accompanied by the deterioration of left ventricular function. Thus, serotonin
produced opposing effects on coronary vasculature, that is, small coronary
artery dilation and large coronary artery constriction.
The mechanisms of the vascular action of serotonin and histamine
in the regulation of arterial tone were examined in isolated rabbit aortic
preparations. These substances elicited vascular contractions due to direct
stimulation of serotonergic and histaminergic receptors of vascular smooth
muscle cells. The data using the 32p_ or 3H-inositol labeling methods indicate
that islet-activating protein (lAP), sensitive guanine nucleotide regulatory
protein, and phosphoinositide turnover are involved in the transduction of a
signal between the amine receptors and intracellular Ca2+ mobilization in
vascular smooth muscle cells. In arterial preparations, removal of the endo-
thelium and addition of methylene blue, a guanylate cyclase inhibitor, poten-
tiated vascular contractions and augmented phosphoinositide turnover. These
results suggest the self-suppression of phosphoinositide turnover and contrac-
tion of vascular smooth muscle cells by stimulating the release of endothelium-
derived relaxing factor in response to these amines.
1 The First Department of Medicine, Kobe University School of Medicine, Kobe, 650
Japan
78
3. Vasoactive Monoamines in the Regulation of Arterial Tone 79
Atherosclerotic arteries exhibited hyperreactivity to the constrictor effects of

serotonin and histamine partly due to endothelial dysfunction. In addition,
serotonin exhibited significant phosphoinositide turnover in diseased arteries
by subthreshold concentrations of this amine, indicating supersensitivity of
these arteries to this substance.
The above observations imply that serotonin and histamine may play an
important role in the pathophysiology of atherosclerotic artery disease.
Key words: Serotonin-Histamine-Vascular contraction-Endothelium-
derived relaxing factor (EDRF)-Atherosclerotic vessels-Coronary stenosis
Introduction
Monoamines, such as norepinephrine, serotonin and histamine, act as
neurotransmitters and autacoids. There is some evidence that monoamines
modulate vascular tone in animals and humans. In vivo and in vitro studies
indicate that these monoamines are the constrictors and/or dilators of vessels,
and that the endothelium modulates vasomoter responses to these amines.
The objective of this study was to examine the effects of various mono-
amines on vasculature and to clarify the mechanisms mediating the vascular
actions of monoamines to interpret their pathophysiologic role.
Effects of Monoamines on Coronary Vasculature and

Cardiac Performance in Dogs with Fixed and
Dynamic Coronary Stenosis
Our understanding of the pathogenesis of angina pectoris is based on the
current concept of fixed and dynamic coronary stenosis. Recent observations
indicate that epicardial coronary vasomotion contributes to the clinical mani-
festations of myocardial ischemia in many patients with angina pectoris. We
have developed in vivo canine models of two different types of coronary
stenosis that seem to be relevant to the clinical features of angina pectoris:
dynamic coronary stenosis, which preserves stenosis vasomobility, and fixed
coronary stenosis, which precludes it. We evaluated the effects of vasoactive
agents, including serotonin, on coronary vasculature and cardiac performance
in these experimental models [1-4].
Methods
We used open-chest dogs anesthetized with alpha-chloralose (100mg/kg, iv.)
and ventilated by a mechanical respirator. The left circumflex artery was
autoperfused from the left common carotid artery through Silastic tubing.
Dynamic coronary stenosis was created by inflating a specially-made micro-
balloon occluder that had been inserted through the side arm of the perfusion
80 M. Yokoyama and H . Akita
tubing and advanced into the proximal circumflex coronary artery. Fixed
coronary stenosis was produced with an externally applied screw-type metal
constrictor device around the circumflex coronary artery . The degree of
coronary stenosis was adjusted to obtain a pressure gradient across the stenosis
of approximately 30 mmHg. Stenosis resistance was calculated by dividing
mean pressure gradient across the stenosis by mean coronary blood flow.
Measurements of heart rate , aortic pressure , distal coronary pressure , left
ventricular pressure and its dP /dt, and circumflex coronary blood flow were
continuously recorded. The intracoronary infusion of serotonin (0.001, 0.01,
Serotonin (I ·Oug I ~g l m i n) I
Nitrogl yc.er in (10 , ug l m,n)
- - I
i-rr+-";
I
Coronary Blood
Flow
I
! ,
(ml lmin) 3~[ V
- J~ -
If- . - +H-h
Di stal Coronary ,
-
± H--
,
+
Prl'ssurl'
(mmHg)
r
[
>-
..L. - ., i H~
-1 I I
I
I" !
- t- .1. ~ .I. ~
Aort i c I. , !
10~[.~_m~~~if~;:E~~$~*i~~~~$S,~-=- =-t:.~i~;.
~
Prl'ssure
(m mHg)
t i _r_ I; i :
+
LV Pr essure
(mmHg)
LV dPl dt
(mmHgls)
LV EDP
(mmHg)
FIG. !' Effects of intracoronary serotonin (1.0 Ilg/kg per min) in dynamic coronary
stenosis . Serotonin greatly decreased coronary blood flow and distal coronary pressure
in association with an elevation of left ventricular (LV) end-diastolic pressure (EDP)
and reduction in left ventricular dP/dt. Additive intracoronary infusion of nitroglycerin
(10 Ilg/min) reversed the detrimental effects of serotonin
0.1, 1.0 ~g/kg per min) for 40 s was performed to avoid the influence of
systemic hemodynamic alterations.
Results and Discussion

In the absence of coronary stenosis, intracoronary infusion of serotonin
resulted in an increase in coronary blood flow without any changes in aortic
pressure, distal coronary pressure, left ventricular pressure and its dP/dt. In
the presence of dynamic coronary stenosis, intracoronary serotonin produced
decreases in coronary blood flow, distal coronary pressure and subsequently
LV dP/dt and an elevation of left ventricular end-diastolic pressure (Fig. 1). In
the presence of fixed coronary stenosis, serotonin at any concentration except
the highest one failed to alter coronary and systemic hemodynamic variables.
Figure 2 summarizes the effects of intracoronary serotonin on coronary blood
flow. Without coronary stenosis, serotonin increased coronary blood flow in a
concentration-dependent manner due to dilation of small coronary arteries.
With dynamic coronary stenosis, serotonin decreased coronary blood flow
due to intensification of stenosis severity as a result of constriction of large
coronary arteries.
Mechanisms Responsible for Monoamine-Induced

Vasomotion
To clarifty the mechanisms mediating the vascular action of monoamines, we
measured isometric tension and phosphoinositide turnover of isolated rabbit
aortic strips with and without endothelium. In particular, the role of
endothelium-derived relaxing factor (EDRF) on its action was examined in this
study.
Methods
The conventional method to measure isometric tension of rabbit thoracic aorta
in an organ bath was used in this study. Phosphoinositide turnover was studied
using [32 p] Pi and myo-[2-3H] inositol labelling methods in rabbit thoracic aorta
e
[5,6]. 2 p] Pi incorporation into phosphatidylinositol was measured in aortic
rings. The small aortic rings (2 mm wide, wet weight 25 mg) were incubated for
e
60 min in a plastic tube with the oxygenated buffer. After incubating for 30 min
in the same buffer containing 2 p] Pi (30 ~Ci ml-l), each tissue was stimulated
with histamine or vehicle (buffer) for 30 min. The reaction was then stopped by
adding 3 ml chloroform/methanol/concentrated HCl (30: 60: 0.6, by volume).
Tissues were homogenised and 2 ml chloroform 2 mol 1-1 KCl (1: 1, by volume)
was added. After centrifuging at 1700g for 5 min, the upper phase was re-
moved and the lower phase was dried with N z and was redissolved in 50 ~l
chloroform/methanol (2: 1, by volume). Phospholipids and standards were then
chromatographed on thin layer chromatography plates impregnated with
82 M. Yokoyama and H. Akita
Owithout coronary st.nosis

g wi th fill.d coronary stMosis
~with dynamic coronary st.nosis **
**
-•
, ,!
-
<I
u..
m
u
L---I L---I
0·01 0·1
S.rotanin (JIg/kg/min)
FIG. 2. Percent changes in coronary blood flow (CBF) in response to serotonin without
coronary stenosis and with dynamic and fixed coronary stenosis. Vertical bars indicate
SEM; * and **, compared with serotonin at the dose of O.OOlllg/kg per min, P < 0.05
and P < 0.01, respectively
potassium oxalate (1%) in a solvent system [chloroform/acetate/methanol!

acetic acid/H20 (10: 4 : 2 : 2 : 1, by volume)]. Finally the spots were identified by
autoradiography and counted.
Primary cultures of vascular smooth muscle cells (VSMC) were obtained
from male Japanese white rabbit thoracic aortae by the explant method.
Intimal-medial tissues were placed in a 75-cm2 flask and grown in Eagle's
minimal essential medium (EMEM) containing 10% fetal calf serum (FCS).
After 2 weeks of incubation at 37°C in a humidified atmosphere of 5% CO2
and 95% air, cultured cells that had migrated from the explanted tissue were
harvested in 0.1% trypsin solution containing 0.05% ethylenediaminetetra-
acetic acid (EDTA) and grown in the same medium for 3 days. The cells in the
secondary cultures were trypsinized, seeded into 35-mm dishes at a density of 2
x lOs cells/dish and grown in 2.5 ml of the same medium containing 2.5 !lCi/ml
of myo-[2- 3H] inositol for 72 h.
The prepared cells were washed twice with serum-free EMEM and incubated
in 1 ml of the same medium containing 2.5 !lCi/ml of myo-[2_3H] inositol for
48 h at 37°C. After incubation, they were incubated with balanced salt solution
(BSS) containing 10 mM glucose, 1 mg/ml bovine serum albumin (BSA) and
10 mM LiCI for 15 min. In some experiments, the cells were pretreated with
pertussis toxin for varied periods before stimulation of the cells with serotonin.
The cells were then stimulated by serotonin at 37°C. The reaction was
terminated by rapid aspiration of assay buffer and addition of 1 ml of ice-cold
15% trichloroacetic acid (TCA). The phosphorylated inositols were separated
by passing each sample through a column containing 1 ml of Dowex AG 1 x 8
(formate form). [3H]-inositol and [3H]-glycerophosphoinositol were eluted with
8 ml of water and with 16 ml of 5 mM sodium tetraborate/60 mM sodium
formate, respectively. [3H]-IP I was eluted with lOml of 0.1 M formic acid/
0.2 M ammonium formate. The column was washed with 6 ml of the same
buffer and eH]-IP2 was eluted with 10 ml of 0.1 M formic acid/O.4 M
ammonium formate. The column was washed with 6 ml of the same buffer and
[3H]-IP3 was eluted with 10 ml of 0.1 M formic acid/l.O M ammonium formate.
Fractions of 2 ml each were collected. The radioactivity of each fraction
containing IPJ, IP2 and IP3 was determined.

Histamine caused concentration-dependent contraction of intact rabbit aortic
strips (Fig. 3). The concentration-response curve was shifted competitively to
the right by pretreatment with diphenhydramine, an HI antagonist. Cimeti-
dine, an H2 antagonist, significantly potentiated the maximal contraction by
18% without any change in threshold concentration. Histamine produced an
increased incorporation of e 2p] Pi into phosphatidylinositol. The concentra-
tion-response relation for phosphatidylinositol labelling was similar to the
contraction curve. Diphenhydramine significantly suppressed phosphati-
dylinositollabelling (Fig. 4). However, cimetidine had no effect on histamine-
induced phosphatidylinositol labelling. In aortic preparations, which were
pretreated with diphenhydramine and then pre contracted with phenylephrine,
histamine produced concentration-dependent relaxation in strips both with and
without endothelium.
Removal of endothelium potentiated the histamine-induced maximal
contraction by 15%, but had no effect on its threshold concentration (Fig. 5).
In endothelium intact preparations which were pretreated with phentolamine
(1 !lM) and methylene blue (10 !lM), an inhibitor of EDRF, the maximal
contractions induced by histamine were potentiated by 14% as compared with
the control (Fig. 6). However, in endothelium-denuded preparations, methy-
lene blue had no significant effects on the histamine-induced contractions. In
the phosphatidylinositol labelling study, both endothelium-denuded
a o Control
• Diphenhydramine 0·1 pM
t:, Diphenhydramine 1 pM __0 .
/ /o/?
100
....c:
j!'
80
Q)
u
....
R
;/
Q)
c.
60
c:
.Q
....u
....c:....'" 40
o
u
1/
20
o r - - -••-~ .--"r-----..,
5 4 3
Histamine -Log (M)
b
120 o Control *
• Cimetidine 10pM
100
....c:
~
.... 80
Q)
c.
c:
.Q
....u 60
....'"....c:
8 40
20
O~-.--~--r-----.-----~-----
7 6 5 4
Histamine -Log (M)
FIG. 3a,b. Effects of diphenhydramine (a) and cimetidine (b) on histamine-induced

contractions in endothelium-intact aortic preparations. The concentration response
curve for histamine was shifted to the right by 30 min pretreatment with
diphenhydramine (O.l~mol·I-I, n = 6; l~mol'l-l, n = 5). Cimetidine (lO~mol'l-l,
n = 7) significantly potentiated histamine-induced maximal contractions by 18% of
control values. Data are means; bars, SEM; *P < 0.01 v control
250
0>
C
.D Antagonist (-)
Qj I ~ Diphenhydramine 10tlM
.D
~ 200 _ Cimetidine 10tlM
0..
e.....c:
-
o
u 150
o
.....
c:
OJ
*
~
OJ
0.. 100
(-) 7 6 5 4
Histamine -Log (M)
FIG. 4. Effects of diphenhydramine and cimetidine on histamine induced e 2p] Pi

incorporation into phosphatidylinositol (PI) in endothelium-intact aortic rings. Rabbit
thoracic aortic rings were preincubated with diphenhydramine (lO/lmo!· 1-1) or
cimetidine (lO/lmol· 1-1) for 30min before the addition of each concentration of
histamine. Diphenhydramine suppressed PI labelling induced by histamine 10/lmol and
0.1 mmol . 1-1. Cimetidine showed no significant effects on histamine induced PI
e
labelling with 2 p] Pi' Each column is the mean of five to nine experiments; bars, SEM;
*P < 0.01 v antagonist( -) group
preparations and pretreatment of intact aortic strips with methylene blue

showed significant augmentation of histamine-induced phosphatidylinositol
labelling (Table 1). In endothelium-denuded aortic rings, however, methylene
blue had no influence on the amine-induced phosphatidylinositollabelling. The
calcium ionophore A23187 (0.01-0.1 /lM), which is known to release EDRF
from intact endothelium, produced concentration-dependent relaxations in
intact aortic strips precontracted by histamine or phenylephrine. A23187
(0.01/lM) depressed histamine-induced 2 p] e
Pi incorporation into
phosphatidylinositol significantly in intact aortic rings, but not in endothelium-
denuded ones.
Thus, histamine· contracted rabbit thoracic aorta through HI receptors, and
dilated through H2 receptors activation of vascular smooth muscle cells. The
former response was associated with the turnover of phosphoinositides. EDRF
released by histamine showed negative feedback depression on phosphoino-
sitide hydrolysis as well as vascular contractions.
Using the [3H]-inositol labelling method, we demonstrated that stimulation
of cultured rabbit aortic vascular smooth muscle cells with serotonin induced a
rapid generation of inositol phosphates [IP I, IP 2 , IP3] from receptor-mediated
hydrolysis of inositol phospholipids [6]. Pretreatment of these cells with
a
o Endothelium (-)
'·0
• Endothelium (+)
0·8
<C
~ 0·6
01
0-4
0·2
i i
7 5 4
Histamine -Log (M)
*
b
300
o Endothelium (+)
~ Endothelium (-)
01
C
a; 250
.0
'"
a...
ec
+-' 200
o
u
.....
o
+-'
~
u
150
.....
Q.l
a...
100
(._.) 7 4
Histamine -Log (M)
FiG. 5a,b. Modification of histamine-induced contraction by removal of the

endothelium (a) and [32 p] Pi incorporation into phosphatidylinositol (PI) (b). Removal
of the endothelium potentiated the maximal contraction by 15% over control. PI
labelling was also significantly potentiated in denuded rings at histamine concentrations
of 10 Ilmol . 1-1 and 0.1 mmol ,1- 1. Columns are means, bars, SEM of contraction
experiments (control, n = 6; denudation, n = 9) and 6-9 PI experiments; * P < 0.05 v
endothelium( +) group
* **•__**i
120
o Control
• MB lO/lM i--
100 :/ 0-0
_/2
/ /Q--
...c 80
f2
CI.l
u
n;
..s-
c
0
.;; 60
u
...c
I)j
~
0
u 40
20
0 ~
J2 i i i i
7 6 5 4 3
Histamine -Log (M)
FIG. 6. Effects of methylene blue (MB) on histamine induced contractions. Methylene

blue (10 ~mol . I-I) was added 5 min before the contraction study in phenylephrine
(1 ~mol .1- 1) pretreated aortic preparations with intact endothelium. MB potentiated
histamine induced contraction by 14% at its maximum concentration. Values are
means; bars, SEM (n = 5); *P < 0.05; **P < 0.01 v control
TABLE 1. Effects of methylene blue (MB) on histamine (HA)-induced phosphati-

dylinositol (PI) labelling in aortic rings with or without endothelium
Percent change from control
Methylene blue Histamine HA +MB
Control (lOf-lmol· litre-I) (50f-lmol'litre-l) (50 + 10 f-lmol . litre-I)
+E 100 113.8 (10.7) 213.3 (16.3) 272.0 (12.1)*
-E 100 101.7 (3.7) 277.7 (35.2) 274.7 (34.6)
Aortic rings with (+ E) or without (- E) endothelium, which were preincubated with phentolamine
111mol . 1-1 for 30 min to exclude the effect of noradrenaline released from intramural nerve
endings, were exposed to methylene blue 10 I1mol· 1-1 for 10 min. Thereafter, vessels were
incubated with histamine 10 I1mol·I-1 for 30 min. Control, PI labelling in phentolamine treated
aortic rings; Values are means (SEM), n = 6-9; 'P < 0.01 v HA group
pertussis toxin (500 ng/ml, 24 h) prior to addition of serotonin, reduced sero-

tonin-induced formation of inositol phosphates. This suggests that a guanine
nucleotide regulatory protein may couple serotonergic receptors to
phospholipase C in vascular smooth muscle cells.
a b
~ o Control (n = 8)
__ I--!...*l
.v
...J
-,/
~
U
-l...-!l
...J " MB 10J-'M (n = 8)
U :0::
:0:: o Endothelium(.) (n=8)
~
~
0
E 100
• Endotheiium(-)(n=8) /
-1 (/
2 -2
0
N
E
100 -- P < 0.01
_ P<0.05
({,/~-2
!
1/
N
'0
'0 - P<0.05
-!
/
~ !.,
c
- -
c: 0
0
J;
50 v 50
v
'II
'II
.... ....
c0
I
c:
0
u u
~ ?,,2
8 6 5 8 6 5
St'rotoni n -logM Serotonin -log 1101
FIG. 7a,b. Effect of removal of the endothelium (a) and methylene blue (MB) (b) on
serotonin-induced contractions. Values are means; bars, SEM
o Control (n =10)
150 • WHHL (n = 8)
150
...J
o Control = 12)
~
...J (n U • P<O.OS
U :0::
:0:: • WHHL (n = 12)
~
~
E E
o
2 100 N
100
c: c:
o o
v 50 u 50
...
'II
...
'II
c: c:
o o
u u
10 6 5 7 5 4
Serotonin -log 1101 Histamine -log 1101
FiG. 8. Concentration-response relations FIG.9. Concentration-response relations

for serotonin in control and WHHL rabbit for histamine in WHHL rabbits·. Values
aortas. Values are means; bars, SEM are means; bars, SEM
Effects of Monoamines on Normal and Atherosclerotic

Vessels
Serotonin, which is released when platelets aggregate at atherosclerotic lesions
where endothelial injury and plaque rupture or ulceration occur, may produce
marked localized vasoconstriction [7]. It is reported that atherosclerotic
arteries are particularly supersensitive to vasoconstrictor stimuli of serotonin
and histamine [8]. Thus, this study was designed to clarify the underlying
mechanisms of altered contractile properties of atherosclerotic vessels in
response to serotonin and histamine.
Methods
We used the methods described in the last section. Control and atherosclerotic
aortae were obtained from age-matched, normal Japanese white rabbits and
Watanabe hereditary hyperlipidemic rabbits (WHHL).

The measurement of isometric tension of endothelium-intact aortic strips from
normal rabbits revealed that endothelial removal enhanced the maximal
vasoconstrictor response to serotonin by 31 % (Fig. 7.a,b). The pretreatment of
aortic strips with methylene blue (10 11M) potentiated serotonin-induced
contraction by 31 %. Concentration-response relations for serotonin in normal
and atherosclerotic aortae revealed that the curve of atherosclerotic aorta was
shifted to the left and upper direction with significantly lower values of
threshold concentrations and EDso (half maximum effective dose) and
augmented maximal contraction by 38% (Fig. 8). The concentration-response
curves for histamine in normal and atherosclerotic aortae showed that the
maximal contraction of atherosclerotic aorta was augmented without any
changes in threshold concentratoin (Fig. 9). We also demonstrated that the
phosphatidylinositol labelling curve for serotonin was shifted to the left.
Thus, the augmented maximal contraction of atherosclerotic arteries was due
to endothelial dysfunction which failed to produce endothelium-dependent
relaxation. The shift of threshold concentration to the left reflects the
supersensitivity of atherosclerotic vascular smooth muscle cells to serotonin.
References
1. Sakamoto S, Yokoyama M, Fukuzaki H (1986) Regulation of coronary blood flow by
counteraction of coronary vascular u- and ~-adrenergic activation during experi-
mental pliable coronary stenosis. Jpn Circ J 50:416-425
2. Sakamoto S, Yokoyama M, Kashiki M, Fukuzaki H (1987) Comparative effects of
intracoronary vasodilators on restoring coronary perfusion during flow-reducing
coronary stenosis in the dog. J Am Coll Cardiol 9:119-126
3. Ichikawa Y, Yokoyama M, Akita H, Fukuzaki H (1989) Constriction of a large

coronary artery contributes to serotonin-induced myocardial ischemia in the dog with
pliable coronary stenosis. J Am Coll Cardiol 14:449-459
4. Yatani A, Yokoyama M, Akita H, Fukuzaki H (1990) Endothelium-dependent
vasodilating effect of substance P during flow-reducing coronary stenosis in the dog.
J Am Coll CardioI15:1374-1384
5. Nishimoto T, Yokoyama M, Fukuzaki H (1990) Self-suppression of phosphoin os it ide
turnover and contraction by stimulating release of endogenous endothelium-derived
relaxing factor in vascular action of histamine. Cardiovasc Res 24:364-372
6. Go M, Yokoyama M, Akita H, Fukuzaki H (1988) Phorbol ester modulates
serotonin-stimulated phosphoinositide breakdown in cultured vascular smooth
muscle cells. Biochem Biophy Res Commun 153:51-58
7. Awano K, Yokoyama M, Fukuzaki H (1989) Role of serotonin, histamine, and
thromboxane A2 in platelet-induced contractions of coronary arteries and aortae
from rabbit. J Cardiovasc Pharmacol 13:781-792
8. Henry P, Yokoyama M (1980) Supersensitivity of atherosclerotic aorta to
ergonovine: mediation by a serotonergic mechanism. J Clin Invest 66:306-313
4
Coronary Vasomotion During
Exercise: Influence
of the Geometry of Stenosis
K. Eid, O.M. Hess, Th. Suter, A. Bartone, J. Grimm,
and H.P. Krayenbuehl
Summary. Coronary vasomotion of normal and stenotic coronary arteries was

evaluated in 17 patients with coronary artery disease and stable exercise-
induced angina pectoris. Luminal areas of 16 normal and 21 stenotic vessel
segments were determined by biplane quantitative coronary arteriography at
rest, at submaximal exercise, and 5 min after 1.6 mg sublingual nitroglycerin
administered at the end of the exercise test. Patients were subdivided (a) into 2
groups according to stenosis severity of mild (~50% area reduction) and
moderate to severe (>50% area reduction) lesions, (b) into 2 groups according
to vessel size of small (~1.37mm2) and large stenosis areas (>1.37mm 2), and
(c) into 2 groups according to length with short (~4.20mm) and long stenotic
segments (>4.20mm).
Normal vessels showed coronary vasodilation with all % increase (P < 0.05
vs rest) in the luminal area during submaximal exercise and a 27% increase (P
< 0.001 vs rest) after administration of sublingual nitroglycerin. In contrast,
stenotic vessel segments showed coronary vasoconstriction during dynamic
exercise with a 26% decrease (P < 0.01 vs rest) of the minimal luminal area
during exercise but a 9% increase (NS vs rest) after administration of
sublingual nitroglycerin. In patients with mild coronary stenoses, the minimal
luminal area decreased slightly but not significantly (- 7%, NS vs rest) during
exercise, whereas it decreased significantly (-33%, P < 0.001 vs rest) in
patients with moderate to severe stenoses. Small stenotic arteries showed
significantly (P < 0.05) more exercise-induced vasoconstriction (-34%) than
large stenotic arteries (- 17% ). Short and long stenoses showed virtually no
difference in the behavior of coronary vasomotion during exercise. There was a
significant inverse correlation between percent change in the luminal area
during exercise and percent area stenosis (r = -0.85, P < 0.001, n = 37).
1 Medical Policlinic, Cardiology, University Hospital, 8091 Zurich, Switzerland
91
92 K. Eid et al.
It is concluded that exercise-induced percent stenosis narrowing is dependent

upon the severity and the size of the stenotic lesion, i.e., the more severe and
the smaller the stenotic lesion is, the more severe is the exercise-induced
stenosis narrowing. The exact mechanism is not clear, but it might be related
to a passive collapse of the disease-free vessel wall at the site of the stenosis
(Venturi mechanism), endothelial dysfunction, increased sensitivity to
catecholamines, or turbulent blood flow with platelet aggregation and release
of vasoconstrictive substances.
Key Words: Quantitative coronary arteriography-Coronary artery disease-
Coronary vasomotion-Supine bicycle exercise-Stenosis vasoconstriction-
Geometry of stenosis
Introduction
Coronary vasomotion plays an important role in the regulation of coronary
perfusion at rest and during exercise [1-3]. Both normal and stenotic coronary
arteries show coronary vasomotion at the site of coronary stenosis, since
approximately 70% of all coronary stenoses have a normal musculo-elastic wall
segment within the stenosis [4-5]. Previous studies have reported that during
isometric exercise, both normal and stenotic coronary arteries show coronary
vasoconstriction [6], probably due to enhanced sympathetic stimulation.
Recently, it was shown that dynamic exercise is associated with coronary
vasodilation of the normal and coronary vasoconstriction of the stenotic
coronary arteries. The exact mechanism of exercise-induced stenosis narrow-
ing is not clear but probably involves several mechanisms [1].
The purpose of the present study was to evaluate the influence of the
severity of stenosis in terms of percent area of stenosis, stenotic vessel size, and
length of stenosis on coronary vasomotion in patients with coronary artery
disease and stable exercise-induced angina pectoris.
Patients and Methods

Seventeen patients with a mean age of 53 years (range: 37-67) were included
in the present study. Patients were selected on a consecutive basis when there
was (1) a history of stable, exercise-induced angina pectoris with no signs of
coronary vasospasm or angina at rest and (2) a clearly visible coronary stenosis
for quantitative evaluation. Left ventricular biplane ejection fraction was 60%.
Prior to cardiac catheterization, an upright exercise test was carried out
while the patient was on his regular medication. Of the 17 patients, 14 were
on a regimen with beta-blocking agents, 9 on calcium antagonists, and 10 on
nitrates. All drugs were stopped at least 12-24h prior to cardiac
catheterization.
4. Coronary Vasomotion During Exercise 93
FIG . la-c. Coronary angiograms of a 44- a

year-old male with a severe stenosis (arrow)
of the left circumflex coronary artery (patient
No. 17). Quantitative evaluation was carried
out at a rest (minimal luminal area 1.0mm 2 ),
b during submaximal exercise with 125 watts
(minimal luminal area 0.7 mm 2), and c after
sublingual administration of 1.6 mg nitro-
glycerin (minimal luminal area 1.0 mm 2 ).
Exercise was terminated because of angina
pectoris
b
Quantitative Coronary Arteriography (Fig. 1)

Biplane coronary arteriography was performed after an interval of at least
10 min after the last diagnostic coronary angiogram. Baseline coronary arterio-
graphy was carried out after the patient's feet had been attached to the bicycle
ergometer [3,7]. Repeated biplane coronary arteriography with concurrent
aortic and pulmonary artery pressure recordings was carried out at the end of
each exercise level. Exercise was begun at 50-75 Wand increased every 2 min
in increments of 25-50 W, and was terminated because of anginal pain,
fatigue, or ST-segment depression of more than 0.2mV. Immediately after the
exercise test, 1.6 mg sublingual nitroglycerin was administered and, 5 min later,
biplane coronary arteriography was repeated.
Quantitative evaluation of biplane coronary arteriography was carried out in
a blinded fashion. Tracings were made manually from both projections during
diastasis or end-diastole. Each vessel segment was traced and analysed 4-6
94 K. Eid et al.
times and the results were averaged to reduce the sampling error [3,7]. A
section of the catheter of known dimensions was traced as a scaling factor. The
tracings of the coronary vessel segments were analyzed on a PDP 11/34
computer [3]. Interobserver variability was 9.3% of mean vessel area for
monoplane and 7.9% for biplane measurements [3,7]. Monoplane angiographic
assessment was used in 65% of the stenotic and in 25% of the normal vessel
segments because of overlying vessels or contrast reflux into the aorta. Similar
data for the stenotic vessel segments have been reported by others, namely,
61 % [3], 64% [8] and 76% [9], respectively. The standard error of estimate
between two observers was slightly larger for monoplane than for biplane
evaluation because of the eccentric location of most coronary arterial stenoses
[4]. However, the correlation between monoplane and biplane data was
exellent (r = 0.979) [3,7]. Therefore, the observed relative changes during
exercise and after nitroglycerin can be considered to be representative even
with monoplane assessment.
The luminal area of a normal and a stenotic vessel segment was calculated in
each patient and expressed in absolute values and in percent of the resting
value (see Tables 4, 5). The length of the stenosis was calculated from the
digitized angiograms, assuming that the stenosis begins when the area of the
normal prestenotic segment falls below 90% and ends when the vessel area
increases above 90% of the normal poststenotic segment [10].
PATIENT GROUPS. The patients were analyzed separately for the influence on
coronary vasomotion of stenosis severity expressed in percent area reduction,
stenotic vessel size, and length of stenosis.
Severity of Stenosis Three groups of coronary vessels were evaluated:
normal vessels (n = 15), stenotic vessels (n = 5) with mild stenosis (:::::::50% area
reduction), and stenotic vessels (n = 13) with moderate to severe stenosis
(>50% area reduction).
Vessel Size Four groups of coronary vessels were studied: normal vessels
larger than 4.22mm2 (n = 8), normal arteries smaller or equal to 4.22mm2 (n
= 7), stenotic vessels larger than 1.37 mm2 (n = 9) and stenotic vessels smaller
or equal to 1.37 mm 2 (n = 9). The cutoff point for normal and stenotic vessels
was chosen arbitrarily in order to obtain two groups with an equal or a similar
number of vessels.
Length of Stenosis Stenotic vessel segments were divided into 2 groups:
vessels with short stenosis length :::::::4.20mm (n = 9) and vessels with long
stenosis length :::::::4.20mm (n = 9). Again, the cutoff point was chosen
arbitrarily to obtain 2 groups with the same number of vessels.
Statistics
Statistical comparisons of angiographic data in response to a first and a second
exercise level and to sublingual nitroglycerin were carried out by two-way
analysis of variance for repeated measurements. Comparisons between 2
groups or subgroups were performed by Student's t-test. In Figs. 2, 3, and 4
mean values of ± 1 standard error are reported.
TABLE 1. Patient characteristics

No. Age (years) CAD NYHA AP MI
1. 37 3 II +
2. 67 3 II + + (I)
3. 57 3 II +
4. 57 3 II +
5. 52 2 (RCA, LAD) III + + (I)
6. 52 3 II +
7. 54 1 (LCX)
8. 54 3
9. 50 3 II + + (A-L)
10. 54 2 (RCA. LCX) 1 + (A-S)
11. 55 3 III + +
12. 39 1 (LCX) II + + {A-L)
13. 46 1 (LAD) II +
14. 61 1 (LAD) II +
15. 56 1 (LCX) II + (I-P)
16. 45 1 (LAD) II + + (A-S)
17. 44 1 (LCX) III +
Mean 50 2.0 12/17 9117
SD 7.7 0.6
CAD, Coronary artery disease; NYHA. New York Heart Association classification; AP, angina
pectoris; MI, myocardial infarction (I. inferior; A-L, antero-lateral; A-S, antero-septal; I-P, infero-
posterior); RCA, right coronary artery; LCX, left circumflex artery; LAD, left anterior descending
coronary artery
TABLE 2. Exercise data

Upright bicycle exercise Supine bicycle exercise
WL % HR BP WL % HR BP
Mean 136 87 124 163176 98 63 106 155/82
SD 34 21 18 27112 34 20 15 24112
WL, Workload at maximal exercise in watts; %, workload as a percent of predicted working

capacity; HR, heart rate (bpm); BP, blood pressure (mmHg)
Results
Clinical and Exercise Data (Tables 1, 2)

One-vessel disease was present in seven patients, two-vessel disease in two,
and three-vessel disease in eight. The functional classification according to the
New York Heart Association was 2.0 (median). A history of angina pectoris
was present in 12 patients. Previous myocardial infarction was reported in nine
patients (two inferior, two antero-Iateral, two antero-septal, one inferio-
posterior, and two small non-localized infarctions).
Angina pectoris occurred in 7 out of 17 patients during upright exercise and
in 10 out of it during the supine exercise test. The total group of 17 patients
achieved a mean work load of 136 W during the upright exercise (87% of the
96 K. Eid et al.
predicted age-, sex-, and height-corrected physical working capacity) and of 98

W (63% of predicted physical working capaCity, P < 0.01 vs the upright
exercise test) during the supine bicycle exercise test. The maximal achieved
mean heart rate was 124 and 106 beats/min, respectively. The rate-pressure
product amounted to 202mmHg X 102 /min during the upright and to
164 mmHg x 102 /min during the supine bicycle exercise test (NS vs the upright
exercise test).
Hemodynamic Data (Table 3)

Mean pulmonary arterial pressure and mean aortic pressure increased
significantly (P < 0.001) during supine bicycle exercise. After sublingual
administration of nitroglycerin, both pulmonary and aortic pressure decreased
significantly when compared to that during exercise, but remained unchanged
compared to the data obtained at rest.
Quantitative Coronary Angiography (Table 4, 5)

Severity of Stenosis (Fig. 2) Normal coronary arteries showed coronary
vasodilation during exercise (+ 11 %, P < 0.01 vs rest) and vasodilation was
maximal after sublingual administration of nitroglycerin (+27%, P < 0.001 vs
rest). However, stenotic vessels showed exercise-induced vasoconstriction,
which was more pronounced in severely stenotic than in mildly stenotic
coronary arteries. At the second exercise level, mild coronary stenoses showed
TABLE 3. Hemodynamic data

HR Mean AoP Mean PAP
R EX NTG R EX NTG R EX NTG
Mean 62 106 73 95 113 92 25 46 23
SD 9 15 10 17 17 19 6 6 7
P (vs R) '" " '*' NS *" NS
, = P < 0.05; '* = P < 0.01; **' = P < 0.001

HR, Heart rate; AoP, aortic pressure (mm Hg); PAP, pulmonary artery pressure (mm Hg); R,
rest; Ex, exercise; NTG, sublingual administration of nitroglycerin; NS, not significantly different
TABLE 4. Luminal area of normal coronary arteries

R EX] EX2 NTG
Mean 4.6 (100) 5.0 (105) 5.1 (111) 5.8 (129)
SD 2.4 (-) 2.4 (8) 2.6 (14) 2.9 (18)
P (vs R) (NS) (*) ('*')
• = P < 0.05; •• = P < 0.01; . n = P < 0.001
R, Rest; Ex 1, first exercise level; Ex 2, second (= submaximal) exercise level; NTG, sublingual
administration of nitroglycerin; NS, not significantly different; data in brackets represent percent
change
TABLE 5. Luminal area of stenotic coronary arteries

%St R EX 1 EX2 NTG
Mean 61 1.7 (100) 1.3 (78) 1.2 (74) 1.9 (109)
SD 18 1.0 ( -) 0.7 (17) 0.7 (17) 1.1 (23)
P (vs R) (**) (**) (NS)
%Sl, Percent area stenosis; data in brackets represent percent change
FIG. 2. Influence of severity of stenosis on coronary vasomotion in patients with

coronary artery disease. Patients were divided into 2 groups with mild (:::;50%) and
severe (>50%) percent area stenosis. Severely stenotic lesions showed significantly
more exercise-induced stenosis narrowing than mildly stenotic lesions or normal
vessels. Ex I, Ex 2, First and second level of exercise; NTG s.l., 5min after sublingual
administration of 1.6 mg nitroglycerin
only minor coronary vasoconstriction compared with the resting state (- 7%,
NS vs rest), but severe coronary stenoses revealed significant coronary
vasoconstriction during dynamic exercise (-33%, P < 0.001 vs rest). After
sublingual administration of nitroglycerin, both mildly and severely stenotic
98 K. Eid et al.
% of control area
150
normal: 0--0 small (s 4.22 mm 2)
---
0--0 large (> 4.22 mm 2)
140
130
stenosis:
....... small (s 1.37 mm 2)
large (> 1.37 mm 2)
120
110
100
90
80
!
70
60 mean ± 1 SEM * p < 0.05

** P < 0.01
T *** p<O.OOl
O~~----~----~--~
Rest Ex 1 Ex2 NTG sol.
FiG. 3. Influence of vessel size on coronary vasomotion in patients with coronary

arterial disease. Patients were divided into 2 groups with small and large coronary
luminal areas of the normal and stenotic vessel segments. Normal vessels showed no
difference between small (=:::;4.22mm2) and large (>4.22mm 2) arteries during exercise.
In contrast, small stenotic (=:::;1.37 mm 2) vessel segments elicited significantly more
exercise-induced stenosis narrowing than large (> 1.37 mm 2) stenotic vessels. Abbrevia-
tions are as in Fig. 2
vessel segments showed coronary vasodilation (+2% and + 12% respectively,

both NS vs rest).
Vessel Size (Fig. 3) The luminal area of normal coronary arteries had no
influence on coronary vasomotion of the normal vessel segment. In contrast,
the small stenotic arteries showed significantly more exercise-induced stenosis
vasoconstriction (-34%, P < 0.001 vs rest) than that of large stenotic arteries
(-17%, NS vs rest). After sublingual administration of nitroglycerin, coronary
vasodilation of small and large stenotic arteries was similar (+ 11 % NS vs rest
and +7% NS vs rest, respectively) but significantly less than normal coronary
arteries.
Length of Stenosis (Fig. 4) Long and short stenotic vessel segments showed
similar changes in the area of stenosis during exercise and after sublingual
administration of nitroglycerin.
% of control area
150
0-0 normal vessel
140 e---e short stenosis (s 4.20 mm)
~ long stenosis (> 4.20 mm)
130
120
l
110
100
90 *** ***
80
n=8
J
*
!
70 p < 0.05
** P < 0.01
60 mean ± 1 SEM *** P < 0.001
Rest Ex 1 Ex 2 NTG s.1.
FIG. 4. Influence of length of stenosis on coronary vasomotion in patients with coronary

artery disease. Patients were divided into 2 groups with short (~4.20 mm) and long
(>4.20mm) stenotic vessel segments. Length of stenosis and percent change in minimal
luminal area was calculated at rest, during a first (Ex 1) and second (Ex 2) level of
exercise as well as 5 min after sublingual administration of l.6 mg nitroglycerin (NTG
s.l.). Both groups with short and long stenotic segments showed exercise-induced
stenosis vasoconstriction to a similar extent but significantly more than normal coronary
arteries. Abbreviations are as in Fig. 2
Correlations (Figs. 5, 6) There was a good correlation (r = -0.85, P <

0.001) between the severity of stenosis (percent area stenosis) and percent
change in minimal luminal area during exercise (Fig. 5). The absolute values of
minimal luminal area and the change in minimal luminal area during exercise
(Fig. 6) also showed a significant although weak correlation (r = 0.56, P <
0.001). The smaller the minimal luminal area was, the more exercise-induced
vasoconstriction of the stenotic vessel segments was observed and vice versa.
However, 3 of the 16 normal vessel segments also showed mild exercise-
induced vasoconstriction.
100 K. Eid et al.
140
IV
...
Q)
IV
y = 111.77 - 0.609 x r = 0.847
-
120
...0 • o normal
I: • stenosis
-
0
u 100
0
Q) 80
C)
I:
IV
.c:
u
:;,!!
60
• ••
•
0
40
0 20 40 60 80 100
% area stenosis
FIG. 5. Relationship between severity of stenosis (percent area stenosis) and percent
change in minimal luminal area during exercise. Twenty-one stenotic (dots) and 16
normal vessel segments (circles) are plotted. There is a good correlation between these
2 parameters with a correlation coefficient of -0.85 (P < 0.001). The zero line is
crossed at an area stenosis of 20%, which indicates that exercise-induced stenosis
narrowing is mainly observed in vessels with moderate to severe coronary lesions
-...
E 2,---------------------------------~
E o
IV Y= - 0.442 + 0.159 x r = 0.556 o normal
Q)
o • stenosis
IV o
o
o
.0 0
________ Il ____________________________ _
I: o
E o
o
Q)
•
C)
I:
.c:
u
IV
o 2 4 6 8 10
minimal luminal area
FIG. 6. Relationship between minimal luminal area and the absolute change in luminal
area during exercise in the 17 patients with coronary artery disease. The correlation
coefficent was 0.56 (P < 0.001). The zero line is crossed at a minimal luminal area of
2.8 mm 2 , which corresponds to a vessel diameter of approximately 1.7 mm
Discussion
The geometry of coronary artery stenoses influences hemodynamic severity,
coronary vasomotion, and, ultimately, coronary blood flow of the stenotic
vessel segment [11]. Irregular and complex coronary arterial stenoses have
been reported with increasing frequency in patients with unstable angina
pectoris [12-14]. Plaque rupture and coronary thrombotic lesions are thought
to be responsible for the irregular appearence of the lesion. The effect of
morphology on coronary vasomotion has also been studied in patients with
exercise-induced angina pectoris [15]. It was found that stenotic arteries elicit
coronary vasoconstriction during exercise, whereas irregularly shaped, non-
stenotic arteries showed no or minimal vasomotion during exercise. Apparently,
the morphology of a coronary artery stenosis is an important determinant of
coronary vasomotion and might influence not only myocardial perfusion but
also clinical symptomatology in patients with both unstable and stable angina
pectoris.
The purpose of the present study was, therefore, to evaluate the geometry of
stenosis and its influence on coronary vasomotion in patients with stable,
exertional angina pectoris.
Geometry of Stenosis and Coronary Vasomotion

Quantitative coronary arteriography was used to assess the geometry of
stenosis in patients with coronary artery disease. The minimal luminal area,
percent area stenosis, vessel size, and length of stenosis were used to define the
geometry of coronary stenosis in the present analysis. Each stenotic lesion was
traced manually 4-6 times to minimize observer-related variability.
The severity of stenosis influences coronary vasomotion significantly (Fig. 2)
because severe stenotic lesions (percent area of stenosis >50%) are
accompanied by exercise-induced stenosis narrowing, which is significantly
more pronounced than in mildly stenotic vessels (~50%). This effect could be
explained by an increase in coronary blood flow velocity during exercise with
an increase in pressure gradient and, thus, a drop in coronary distending
pressure within the stenotic segment (Venturi mechanism), resulting in a
passive collapse of the disease-free portion of the vessel wall [1,3]. This is
supported by the fact that a significant correlation exists between the severity
of stenosis and percent change in minimal luminal area (Fig. 5) and between
minimal luminal area and the absolute change in luminal area during exercise
(Fig. 6). Since metabolic factors, such as circulating catecholamines,
endothelium-derived relaxing factor (EDRF), serotonin, endothelin,
prostaglandins, adenosine, etc. [16-20] influence coronary vasomotion of the
stenotic vessel segment to a great extent, it appears rather unlikely that
exercise-induced stenotic narrowing is a purely passive phenomenon.
Vessel size of the stenotic segment influences coronary vasomotion signi-
ficantly (Fig. 3) because small vessel segments reveal more exercise-induced
stenosis narrowing than do large segments. An exaggerated response of small,
102 K. Eid et al.
non-stenotic epicardial vessels to various drugs [21-22] or exercise [23] has

been also reported by others. The mechanism of the differing vasodilatory
responses of small and large stenotic arteries to exercise is not clear. Decreased
extravascular compression, increased vessel capacitance, and different
responses of the smooth vasculature to vasodilatory stimuli have been dis-
cussed [21]. In the present study, small stenotic coronary arteries showed more
exercise-induced stenosis narrowing than that of large stenotic arteries. This
observation could be explained by a higher blood flow velocity during exercise
in the small coronary arteries compared to the large ones, and a higher
transstenotic pressure gradient in the arteries with a small absolute lumen
cross-sectional area compared to the stenotic arteries with a larger cross-
sectional area.
The effect of the length of stenosis on coronary vasomotion was evaluated in
the present analysis (Fig. 4). No direct influence of the length of stenosis on
coronary vasomotion was observed because vasoconstriction was similar during
exercise in both short and long stenotic lesions. According to the equation of
Bernoulli, the critical dimensions are the diameter of the stenosis which is
raised to the fourth power and length of the stenosis which is raised to the first
power. Thus, a small decrease in the diameter affects flow by a fourth power
term, whereas length has a proportionately much lesser effect [24].
Pathophysiologic Mechanisms
A different response of normal and stenotic coronary arteries to exercise has
been reported by several authors [3,7,8,15,25]. The possibility of passive
collapse at the site of stenosis has been raised [1,3] but Gordon and coworkers
[15] have demonstrated in patients without significant stenoses but with
irregularly shaped vessel segments that coronary constriction can occur as well.
In this subgroup of patients, it was postulated that passive collapse due to
diminished distending pressure (Venturi mechanism) is not the predominating
mechanism for constriction of the stenotic vessel segments during exercise.
Considerable experimental evidence from animal and human studies indicates
that atherosclerosis is associated with enhanced vasoconstriction in response to
acetylcholine, catecholamines, serotonin, histamine, and ergonovine [16-20].
It is possible that coronary constriction of atherosclerotic arteries during
exercise occurs because of increased sensitivity of the smooth vasculature to
catecholamines. Recent angiographic studies have demonstrated vasodilation
of normal but vasoconstriction of minimally diseased or stenotic vessels after
intracoronary administration of acetylcholine [15,20]. These studies have
postulated that endothelium-dependent vasodilation exists in normal vessels via
the release of endothelium-derived relaxing factor (EDRF), but is replaced by
paradoxical vasoconstriction in both early and advanced atherosclerosis. In this
respect, 3 of the 16 normal vessel segments also showed exercise-induced
vasoconstriction (Fig. 5) probably due to the fact that these vessel-segments
were not truly normal although coronary arteripgraphy revealed no luminal
irregularities or stenotic lesions. Apparently, coronary atherosclerosis causes
disturbances in endothelium-dependent vasomotor function. Thus, the normal

dilator effect of the healthy endothelium is lost in the diseased state and
constrictor influences may predominate during exercise. Another possibility
includes enhanced platelet aggregation due to turbulent blood flow during
exercise with release of thromoboxane A2 and serotonin, both of which are
potent vasoconstrictors [3]. Which of these mechanisms-passive collapse,
increased sensitivity to catecholamines, insufficient production of EDRF, Or
enhanced platelet aggregation-is responsible for the exercise-induced vaso-
constriction of the stenotic vessel segment cannot be determined from the
present study.
Limitations of the Study

Accuracy of quantitative coronary arteriography has been well established in
our laboratory [3,7] and in reported validation studies [26,27]. Brown et al.
[26] have found that the accuracy of quantitative coronary arteriography is
within 0.08mm for measurements of known dimensions and O.lOmm for
minimal diameter estimates. The changes observed in our study are small but
clearly larger than the reported angiographic resolution.
In the present study, ionic contrast material (amidotrizoate: Urographin
76%) was used in 11 of the 17 patients and non-ionic contrast material
(iopamidol 755 mg/ml, trometamol 1 mg/ml: Iopamiro 370) in 6 patients. The
effect of ionic and non-ionic contrast material on coronary vasomotion has
been studied previously [28]. There were no significant differences in coronary
vasomotion of the normal and stenotic vessel segments with ionic and non-ionic
contrast material, although exercise-induced vasodilation of the normal
segments was mOre pronounced after injection of the ionic contrast material.
This tendency was not observed for stenotic vessel segments [28]. Thus, the
effect of ionic and non-ionic contrast material on coronary vasomotion seems
to be small.
Clinical Implications
Although a variety of factors may determine vasomotion of epicardial coronary
arteries during exercise, vasoconstriction and vasodilation seem to be primarily
related to the presence Or absence of atherosclerotic changes of the vessel wall.
The loss of normal vasodilation and the appearence of coronary vasoconstric-
tion during exercise is dependent upon the geometry of the stenotic vessel
segment and might have important implications for the understanding of the
pathophysiology of exercise-induced myocardial ischemia.
References
1. Brown BG, Bolson EL, Dodge HT (1984) Dynamic mechanisms in human coronary
stenosis. Circulation 70:917-922
104 K. Eid et al.
2. Mates RE, Gupta RL, Bell AC, Klocke FJ (1978) Fluid dynamics of coronary
artery stenosis. Cirs Res 42:152-162
Vasoconstriction of stenotic coronary arteries during dynamic exercise in patients
with classic angina pectoris: Reversibility by nitroglycerin. Circulation 73:865-876
4. Freudenberg H, Lichtlen PR (1981) The normal wall segment in coronary
stenosis-a postmortal study. Z Kardiol 70:863-869
5. Saner HE, Grobel FL, Salmononwitz E, Erlien DA, Edwards JE (1985) The
disease-free wall in coronary atherosclerosis: Its relation to degree of obstruction. J
Am ColI Cardiol 6:1096-1099
6. Brown BG, Lee AB, Bolson EL, Dodge HT (1984) Reflex constriction of signi-
ficant coronary stenosis as a mechanism contributing to ischemic left ventricular
dysfunction during dynamic exercise. Circulation 70:18-24
7. Gaglione A, Hess OM, Corin WJ, Ritter M, Grimm J, Krayenbuehl HP (1987) Is
there coronary vasoconstriction after intracoronary beta-adreneric blockade in
patients with coronary artery disease? J Am ColI Cardiol 10:299-310
8. Nonogi H, Hess OM, Ritter M, Bortone AS, Corin WJ, Grimm J, Krayenbuehl HP
(1988) Prevention of coronary vasoconstriction by diltiazem during dynamic
exercise in patients with coronary artery disease. J Am ColI Cardiol 12:892-899
9. Brown BG, Josephson MA, Petersen RB (1981) Intravenous dipyrimdamole
combined with isometric handgrip for near maximal acute increase in coronary flow
in patients with coronary artery disease. Am J CardioI48:1077-1085
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Hosp Pract [Off] 19:160-175
11. Fedele FA, Sharaf B, Most AS, Gewirtz H (1989) Details of coronary stenosis
morphology influence its hemodynamic severity and distal flow reserve. Circulation
80:632-642
12. Ambrose JA, Winters SL, Stein A, Eng C, Teichholz LE, Gorlin R, Fuster V
(1985) Angiographic morphology and the pathogenesis of unstable angina pectoris.
J Am ColI Cardiol 5:609-616
13. Levin DC, Fallon JT (1982) Significance of the angiographic morphology of
localized coronary stenoses: Histopathologic correlations. Circulation 66:316-320
14. Vetrovec GW, Leinbach RC, Gold HK, Cowley MJ (1982) Intracoronary throm-
bolysis in syndromes of unstable ischemia: Angiographic and clinical results. Am
Heart J 104:946-952
15. Gordon JB, Ganz P, Nabel EG, Fish RD, Zebede J, Mudge GH, Alexander RW,
Selwyn AP (1989) Atherosclerosis influences the vasomotor response of epicardial
coronary arteries to exercise. J Clin Invest 83: 1946-1952
16. Freiman PC, Mitchell GG, Heistad DD, Armstrong ML, Harrison DD (1986)
Atherosclerosis impairs endothelium-dependent vascular relaxation to acetylcholine
and thrombin in primates. Circ Res 58:783-789
17. Heistad DD, Armstrong ML, Marcus ML, Piegors DJ, Mark AL (1984) Aug-
mented responses to vasoconstrictor stimuli in hypercholesteremic and athero-
sclerotic monkeys. Circ Res 54:711-718
18. Shimokawa H, Tomoike H, Nabeyama S, Tamamoto H, Araki H, Nakamura M
(1983) Coronary artery spasm induced in atherosclerotic miniature swine. Science
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19. Schroeder JS, Bolen JL, Quint RA, Clark DA, Hayden WG, Higgins CB, Wexler
L (1977) Provocation of coronary spasm with ergonovine maleate. Am J Cardiol
40:487-491
20. Ludmer PL, Selwyn AP, Shook TL, Wayne RR, Mudge GH, Alexander RW, Ganz
P (1986) Paradoxical vasoconstriction induced by acetylcholine in atherosclerotic
coronary arteries. N Engl J Med 315:1046-1051
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small coronary arteries by nitroglycerin. Circulation 64:324-333
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c
Role of Adenosine in Regulation of
Coronary Blood Flow
1
The Role of Adenosine in the
Metabolic Regulation of Coronary
Blood Flow
Robert M. Berne l
Summary. Coronary blood flow is closely related to the metabolic activity of

the heart. When the oxygen supply is insufficient for the oxygen needs of the
myocardium, adenosine formation is increased and its release into the
interstitial fluid dilates the coronary resistance vessels. A key to understanding
the role of adenosine as mediator of coronary dilation is an accurate estimate
of the interstitial fluid adenosine concentration. Since direct measurement is
still technically impossible, indirect methods have been used. These include
pericardial infusates and small fluid-filled chambers on the surface of the dog
heart, collection of cardiac transudates from rat and guinea pig hearts, and the
application of small porous disks to the epicardial and endocardial surfaces of
the left ventricle of the isolated perfused guinea pig heart. Interventions that
increase the oxygen requirements or decrease the oxygen supply augment the
levels of adenosine in the interstitial fluid, as reflected by these indirect
methods. The porous disk technique reveals that the ventricular endocardial
adenosine concentrations are about 12-fold greater than the epicardial
concentrations. The high endocardial adenosine levels may be responsible for
the lower intravascular resistance of the endocardial vessels relative to the
epicardial arterial vessels in the face of a greater extravascular resistance in the
endocardium.
Key words: Coronary resistance-Oxygen supply/demand-Transmural
adenosine gradient-Interstitial fluid
Historical Background
Over the years many substances have been proposed as mediators of the
adjustments of coronary blood flow to the metabolic needs of the myocardium.
In the classic studies of Hilton and Eichholtz in 1925 [1], the authors attributed
1 Department of Physiology, University of Virginia School of Medicine, Charlottesville,

VA 22908, USA
109
110 R.M. Berne
the coronary vasodilation associated with severe hypoxia to a direct effect of a

reduced blood oxygen tension on the vasculature. These conclusions were
based upon the observations in the dog heart-lung preparation that deoxy-
genated arterial blood or the administration of cyanide elicited maximal
coronary dilation. These studies [1] and those of Eckenhoff et al. [2], which
showed a striking parallelism between myocardial oxygen consumption and
coronary blood flow, prompted us to further investigate the role of oxygen in
the regulation of coronary blood flow.
In the open-chest dog we studied the effect of hypoxemia on coronary
vascular resistance. In these experiments, blood was partly deoxygenated by
passing it through the lungs of a donor dog during ventilation of those lungs
with 95% N, 5% CO2 [3]. This partially deoxygenated blood was then pumped
into the left coronary artery at different perfusion pressures. Perfusion of the
left coronary artery with hypoxic blood at control perfusion pressure elicited
vasodilation. However, if the oxygen needs of the myocardium were met by a
greater perfusion (high perfusion pressure), hypoxemia did not cause coronary
vasodilation, and coronary sinus blood oxygen content stayed above 5.5 mIldl.
These studies [3] indicated that arterial oxygen tension was not a critical factor
in changes in coronary resistance, but that oxygen supply to the myocardium is
of paramount importance in the regulation of coronary blood flow. Coronary
vasodilation associated with a diminished oxygen supply, but not related to
arterial blood oxygen tension, suggested the release of a vasodilator substance
when oxygen supply was inadequate for myocardial needs. However, arterial
infusion of coronary venous blood collected during hypoxic perfusion and then
reoxygenated failed to produce a decrease in resistance in the vascular bed of a
test coronary artery [4]. This observation indicates that either no vasodilator
substance is released into the venous effluent of the hypoxic heart or, that if
released, it is very labile and disappears during the reoxygenation of the blood.
In light of these findings, it seems unlikely that stable substances, such as
potassium or osmotically active products of metabolism, playa significant role
in mediating metabolically associated vasodilation. With respect to carbon
dioxide and hydrogen ions, the picture is cloudy. A local increase in CO2 and a
decrease in pH can elicit vasodilation [5] but a cause and effect relationship
between PC02 and pH and coronary vascular resistance has not been
established. In fact, levels of CO2 and H+ that exceed those found with
hypoxia and enhanced metabolic activity fail to elicit vasodilation of the
magnitude seen under physiological conditions [1,5].
Since our studies with reoxygenated venous blood were negative, we looked
for a labile substance in venous effluent of hypoxic hearts. Drury and Szent-
Gyorgyi [6] had shown that adenosine and AMP were potent coronary
vasodilators, an observation subsequently confirmed by other investigators
[7-9]. Therefore, it seemed logical to determine whether either or both of
these substances were released from hypoxic hearts. In our first series of
experiments, we found only inosine and hypoxanthine in effluents from anoxic
isolated perfused cat hearts [10,11] and in the coronary sinus blood of open-
chest dogs during asphyxia [11]. Subsequently, we were able to detect
1. The Role of Adenosine in the Metabolic Regulation 111
adenosine in the venous effluent of the isolated perfused guinea pig heart in the
presence of an inhibitor of adenosine deaminase [12] and in the coronary sinus
blood of the open-chest dog during reactive hypermia [13].
Although adenosine release could be readily demonstrated with a reduced
oxygen supply, as in ischemia or hypoxia, in order to be of physiological
significance its release should also occur with an increase in cardiac work. An
adenosine response to increased cardiac work was observed in the open-chest
rat in which aortic constriction produced an increase in myocardial adenosine
levels [14]. Constriction of the ascending aorta resulted in a twofold increase in
pressure proximal to the constriction and hence a higher coronary perfusion
pressure. Despite the greater myocardial perfusion, the increased work load
elicited greater adenosine production. Even within a single cardiac cycle the
adenosine levels of the myocardium fluctuated; tissue concentrations of
adenosine were highest during systole and lowest during diastole [15].
With respect to basal coronary blood flow and autoregulation of coronary
blood flow, the bulk of evidence is against a role for adenosine as mediator of a
decrease in coronary resistance with a reduction in perfusion pressure. In some
studies, infusion of adenosine deaminase [16,17] or adenosine antagonists [18]
failed to affect autoregulation of blood flow, whereas in other studies the
effects of transient ischemia [19] or hypoxia [20] were attenuated by these
agents. In no instance was autoregulation abolished by adenosine deaminase or
adenosine antagonists, which indicates that other factors operate in
autoregulation and that adenosine may play only a minor role or no role in this
phenomenon. In view of the clear demonstration of a myogenic response in
isolated coronary arterioles [21], it seems probable that the mechanisms for
autoregulation in the coronary circulation are myogenic, and that the coronary
blood flow changes that occur during disparities between oxygen supply and
oxygen demand are mediated by adenosine. Also, the facts that blood pressure
is fairly constant in the normal state and that adjustments in coronary blood
flow are linked to the changes in metabolic activity of the heart, provide
support for the concept that release of an endogenous vasodilator, such as
adenosine, is regulated by the oxygen supply-to-demand ratio and serves as the
messenger from the parenchymal tissue to the vascular smooth muscle of the
resistance vessels [11 ,22,23].
Interstitial Fluid Adenosine

Many of the early cardiac adenosine experiments involved measurements of
myocardial tissue levels of the nucleoside. Since a major fraction of intra-
cellular adenosine is tightly bound to S-adenosylhomocysteine hydrolase [24]
and is, therefore, not available for metabolic vasodilation, total tissue
adenosine values may mask the free adenosine concentrations. Venous effluent
adenosine concentrations may also be misleading because the endothelial cells
avidly take up adenosine [25] and the interstitial fluid concentration of
adenosine (adenosine in contact with the vascular smooth muscle) may be
112 R.M. Berne
TABLE 1. Effects of dipyridamole on cardiac adenosine release and coronary blood flow
Control Dipyridamole
CBF (ml/min/loo g) 39.7 ± 1.6 50.9 ± 1.7**
MVOz (mllmin/loo g) 6.6 ± 0.4 6.5 ± 0.4
PCI ADO (pmoles/ml) 45.4 ± 5.2 80.6 ± 7.0**
CVR (pru/100 g) 2.68 ± 0.14 1.94 ± 0.13**
O 2 EXT (mlldl) 16.8 ± 1.0 12.9 ± 0.8*
CS Oz (mlldl) 3.8 ± 0.1 8.0 ± 0.7**
Difference between control and dipyridamole treatment: *P < 0.5, ** P < 0.01
Values are mean ± standard error.
CBF, Coronary blood flow; MV0 2 , myocardial oxygen consumption; PCI ADO, pericardial
infusate adenosine concentration; CVR, coronary resistance; O2 EXT, oxygen extraction by the
heart; CS Oz, coronary sinus blood oxygen levels
much higher than that of the venous effluent. Therefore, we experimented with
methods designed to obtain an index of the interstitial fluid concentration of
adenosine under control and experimental conditions.
The first attempt in this direction was to infuse 25-40 ml of Krebs-Henseleit
solution into the intact pericardial sac of the dog and then remove it after
4 min for quantification of adenosine and its degradative products. These
studies were carried out in the anesthetized open-chest dog and in the trained
unanesthetized dog. In the open-chest dog, interventions, such as cardiac
sympathetic nerve stimulation, atrial pacing, administration of norepinephrine
or calcium, and aortic constriction, significantly increased the adenosine
concentration of the fluid placed in the pericardial sac [26,27]' Under basal
conditions (Table 1) and during interventions that enhanced myocardial
metabolic rate, dipyridamole produced increases in coronary blood flow and
pericardial infusate adenosine concentrations without affecting myocardial
oxygen consumption. In the unanesthetized dog, mild to moderate exercise,
feeding, and excitement all produced a large increment in the pericardial
infusate adenosine concentration (Fig. 1). The concentrations of adenosine
reached with these various interventions in all likelihood reflect directional
changes in the cardiac interstital fluid adenosine levels, but underestimate the
true extracellular space adenosine concentration because of the large fluid
volume relative to the surface area of the epicardium.
In order to obtain a better estimate of the epicardial interstitial fluid
adenosine concentration, a small chamber (1 sq. cm) was placed on the surface
of the left ventricle of open-chest dogs and made leakproof with a thin layer of
vaseline [28]. One hundred III of Krebs-Henseleit solution were added to the
chamber and left in contact with the epicardium for 4 min, which was longer
than the time necessary for equilibrium to be reached. This technique was
similar to the well technique used by Hanley et al. [29], but avoided the use of
cyanoacrylic cement on the adjacent epicardium and the scraping of the
360 ,
*
320 t--
280 - r+-
E
"- *
In
Q) 240 - *
0 r-r--
E rr--
a. 200 - *
w r-r--
z 160 ~
(f)
0
z 120 '-
r+
w
0
« 80 r + +
40 ~
CON 2MPH 4 MPH CON EXC CON FEED
FIG. 1. Pericardial infusate adenosine concentrations in the conscious dog. Values are
means ± SE. CON, Control; 2 MPH and 4 MPH, treadmill exercise at 2 and 4 mph at a
10% grade; EXC, excitement produced by loud noises; FEED, sight, smell and con-
sumption of food. *Denotes significant differences compared to control. (Reproduced
by permission of the American Physiological Society from [27])
o CONTROL
... DOBUTAMINE
225
o RECOVERY
200
175
1 l
L Y 174 il-e- O 845t)
c 150
125
78 il-e -1 :58t)
0 Y
0 100
«
u 75
w
50 y 57 il-e- 1 293t)
25
0
0 2 3 4 5 6 7 8
TIME (mInutes)
FIG. 2. Epicardial chamber (EC) adenosine (ADO) influx before, during and after
dobutamine (5-15 ).!g/kg per min) infusion. Monoexponential curves (equations in-
dicated) were generated by nonlinear least-squares regression analysis. (Reproduced by
permission of the American Physiological Society from [28])
114 R.M. Berne
epicardium to remove the mesothelial layer. The levels of adenosine in the

cardiac chamber increased significantly during dobutamine administration and
returned to control levels when the infusion was stopped (Fig. 2). The increase
in chamber adenosine concentration produced by catecholamines was
associated with proportionate increases in coronary blood flow and dP/dt
(Fig. 3).
The next step in pursuit of a more accurate index of interstitial adenosine
was the use of nylon disks [30]. Porous nylon hydrophilic disks 6mm in
diameter were saturated with Krebs-Henseleit solution and applied to the
epicardial surface of the left ventricle of the isolated perfused guinea pig heart.
After 2 min, the disks were removed and stored at -80°C until their contents
were analyzed for adenosine by reversed phase gradient HPLC. A time study
indicated that a plateau concentration of adenosine was reached in 30 sand
remained constant thereafter. During control periods, the epicardial disk
adenosine concentration was 0.28 ± O.03I1M and increased to 1.19 ±
0.0911M in the presence of dipyridamole and erythro-4-(2-hydroxy-3-nonyl)
adenine hydrochloride (EHNA), whereas the venous effluent adenosine
concentration rose from 0.004 ± 0.001 to 0.027 ± 0.004 with dipyridamole and
EHNA administration (Fig. 4). With infusion of 6 and 12 11M adenosine, very
little of the adenosine appeared in either the epicardial disks or venous,effluents
(Fig. 5). However, during adenosine infusion in the presence of dipyridamole
and EHNA, the concentrations of the nucleoside in the disks and venous
effluent equaled that in the perfusion fluid (Fig. 5). When a non-metabolizable
adenosine analogue 9-~-D-arabinofuranosyl hypoxanthine (Ara-H) was added
to the perfusion liquid, the concentration in the disks and venous effluent
equaled that in the perfusion fluid with and without dipyridamole (Fig. 6).
These results indicate that the epicardial interstitial fluid adenosine concentra-
tion, as represented by the concentration of the nucleoside in the epicardial
disks, is considerably greater than that of the venous effluent in the absence of
an adenosine transport blocker and adenosine deaminase inhibitor, and that
infused adenosine is taken up in the heart, presumably by the endothelial cells
[25]. When cellular uptake of adenosine is blocked, the nucleoside is evenly
distributed throughout the extracellular fluid of the heart and the epicardial
porous disks provide a reliable index of the epicardial interstitial fluid adenosine
concentration. The rapid attainment of a steady-state adenosine concentration
in the disks reflects the large surface area to volume ratio of the disks com-
pared to the epicardial chamber which required and eightfold longer contact
time to reach a steady-state adenosine concentration.
The porous disk technique is similar in concept to the collection of droplets
of transudate from the epicardial surface of the isolated perfused rat and
guinea pig heart [31-35]. However, the disk technique permits measurement
of adenosine concentrations in epicardial fluid from specific areas of the heart
surface and does not necessitate the use of high perfusion pressures or suction
to accelerate transudate formation. The technique also can be used in the
blood perfused heart in an open-chest preparation and also enables the
determination of the endocardial interstitial fluid adenosine concentration.
a
D 225 *
~ 200
175
o
o
« 150
u 125
w
W 100
I-
« 75
I-
(Jl
50
>-
o
« 25
w
I-
(Jl
CONTROL OOBUTAMINE RECOVERY
b 01
g
D 225 100
200
* *
o
o
«
u
w ~
o
W -.J
I- LL
« o
I-
(Jl o
o
>- -.J
o OJ
« >-
w
I- IT
(Jl «
CONTROL NOREPINEPHRINE RECOVERY z
o
IT
o
U
FIG. 3. a Steady-state epicardial chamber (EC) adenosine (ADO) concentrations

collected during different steady-state levels of cardiac work (dP/dt) induced by
intravenous dobutamine. *Significant increase (P < 0.05) compared with control and
recovery. b Steady-state EC Ado concentrations collected during different steady-state
levels of coronary blood flow induced by intravenous norepinephrine (0.OS-0.12 mg/kg
per min). *Significant increase (P < 0.05) compared with control and recovery. (Re-
produced by permission of the American Physiological Society from [2S])
In a recent series of experiments, single epicardial disks were applied to the

endocardial surface of the free wall of the left ventricle in the isolated guinea
pig heart perfused at constant flow [36]. After myocardial perfusion was started
by the Langendorff technique, the left atrium was opened and the exposed
leaflets of the mitral valve were carefully excised. The right ventricle was
cannulated via the inferior vena cava for collection of the coronary effluent,
116 R.M. Berne
t::;,,:, il VENOUS
_DISCS
1.5
1.25
~
::I
1.00
lJJ
Z
U) 075
0
Z
lJJ 050
0
<t
025
000
CONTROL DIP + EHNA
FIG. 4. Effect of dipyridamole (DIP) and EHNA on endogenous adenosine con-
centrations in venous effluent and nylon disks applied to the epicardial surface of the
left ventricle of the guinea pig heart (n = 7). Note: low venous effluent adenosine
concentration under control conditions (0.004IlM) is too small to appear on this
ordinate scale. (Reproduced by permission of the American Physiological Society
from [30])
o ARTERIAL
14 ~ VENOUS
_DISCS
12
~I
W
~
(/)
~ G
w
~ 4
~_ _ _12_u_M-'1 I GuM 12uM I

CONTROL DIP + EHNA
FIG. 5. Effect of dipyridamole (DIP) and adenosine concentrations in epicardial disks

and venous effluent of the guinea pig heart during infusion of 6 and 12 11M adenosine in
absence and presence of 0.51lM DIP and 51lM EHNA (n = 6). (Reproduced by
permission of the American Physiological Society from [30])
and the pulmonary artery was tied off. These procedures were followed by a
45-min equilibration period. Porous 6 mm disks were then placed on the
epicardial and endocardial surfaces of the left ventricle and removed after
2 min of contact. Oxygen tension of arterial and venous perfusion fluid and
perfusion pressure were continuously monitored. Oxygen consumption, coro-
c:J ARTERIAL
~ VENOUS
6 _DISCS
:I:
I
«
«a:: 2
CONTROL DIPYRIDAMOLE
FIG. 6. Concentrations of Ara-H in epicardial disks and venous effluent of the guinea
pig heart during arterial infusion of Ara-H in the absence and presence of O.SI!M
dipyridamole (n = 6). (Reproduced by permission of the American Physiological
Society from [30])
nary resistance, and the oxygen supply/demand ratio were unchanged during
the 45-min experimental periods. Analysis of the adenosine content of the
endocardial and epicardial disks at 0, 15,30, and 45 min revealed a several-fold
greater concentration of adenosine, inosine, and hypoxanthine in the
endocardium. All values remained essentially unchanged for the duration of
the experiment (Fig. 7). With reduction in flow, the epicardial and endocardial
levels of these purine compounds increased. The ratio of endocardial to
epicardial concentrations of these purine compounds is shown in Fig. 8. This
large gradient for these substances across the left ventricular wall indicates that
measurements of epicardial adenosine and related compounds is not repre-
sentative of their global interstitial fluid concentration. The higher oxygen
consumption of the endocardium and the greater extravascular compression of
the endocardial vessels in the face of an equal microvascular distribution and a
slightly greater endocardial than epicardial blood flow are consonant with the
high endocardial concentration of adenosine. The lower intravascular resis-
tance of the endocardial vessels could be attributed to this high level of
adenosine.
Based upon results with infused adenosine, maximal coronary dilation occurs
with about 111M adenosine [37]. Extrapolating these observations to the
concentrations we find in the endocardium (3.6IlM) would indicate that an
adenosine concentration greatly in excess of that required to produce maximal
dilation of the coronary resistance vessels is present in the endocardium.
However, a value of 0.3 11M for the epicardium and 3.6 11M for the endocard-
ium is not inconsistent with the average transmural value of 111M found in the
blood perfused dog heart by the micro dialysis technique [38]. Furthermore, the
fact that the endocardial/epicardial ratios of the various purine compounds
118 R.M. Berne
500
Epicardial
i 400
.s
= 300
0
:I
....f
= 200
~
~
8 100
0
0 15 30 45
5000
Endocardial
i
.s 4000
.......= 3000
0
....f
= 2000
~
~
0 1000
C)
0
0 15 30 45
200
Venous
j 160
=
0
:I
120
! 80
= ~
C)
~ 40
0
0 15 30 45
Time (min)
FIG. 7. Epicardial, endocardial, and venous effluent purines during 45 min of constant
flow perfusion. All purine concentrations were significantly higher in endocardial
samples compared to epicardial samples at all times (P < 0.05). All values shown are
means ± SEM (n = 9-11). D Adenosine; • inosine; ~ hypoxanthine. (Reproduced with
revision by permission of the Proceedings of the National Academy of Sciences from
[36])
FIG. 8. Endocardial/epicardial 20,---------------------------,

ratios for adenosine, inosine, and
hypoxanthine during 45 min of 16
constant flow perfusion. All
values shown are means ± stan-
12
dard error of the mean (SEM)
(n = 9-11). 0 Adenosine;
• inosine; ~ hypoxanthine . 8
(Reproduced with revision by
permission of the Proceedings 4
of the National Academy of
Sciences from [36]) o
o 15 30 45
Time (min)
75
•
0
VSM RespOnse
Endothelial Response
60
-
C
0 45
m
><
m
Q)
a: 30
0~
15
o +-~~L-~-r~--~~_.--~~
-7 -6 - 5 - 4 - 3 - 2
Log Molar Concentration
FIG . 9. Endothelial-dependent and -independent responses to adenosine in guinea pig

aorta. Response of rubbed rings is assumed to represent direct vascular smooth muscle
relaxation (VSM response). Endothelial-dependent relaxation was calculated as the
difference between responses in rubbed and unrubbed rings . Values are means ± SO; n
= 18 animals. (Reproduced by permission of the American Physiological Society
from [39])
differ and that the disks did not produce tissue damage, as evaluated by lactate
dehydrogenase levels, militate against artifactual values. Infusion of 1211M
adenosine only increased the epicardial concentration to O.4I1M [30], and
diffusion distances to the vascular smooth muscle are different with exogenous
and endogenous adenosine. It is also possible that sensitivity to adenosine is
120 R.M. Berne
different for the endocardial and epicardial resistance vessels. Finally, results
with infused adenosine may not be applicable to those with endogenous
adenosine because there is an endothelial component to arterial dilation with
adenosine [39] (Fig. 9).
Since direct measurement of the interstitial fluid in the different layers of the
heart is not yet possible, indirect methods, such as the nylon porous disk
technique, are the best ones available. Their use in examining the transmural
purine gradient under different physiological conditions will, hopefully, shed
more light on this interesting observation.
References
1. Hilton R, Eichholtz F (1925) The influence of chemical factors on the coronary
circulation. J Physiol 59:413-425
2. Eckenhoff JE, Hafkenschiel JH, Landmesser CM, Harmel M (1947) Cardiac
oxygen metabolism and control of the coronary circulation. Am J PhysiolI49:634-
649
3. Berne RM, Blackmon JR, Gardner TH (1957) Hypoxemia and coronary blood
flow. J Clin Invest 36:1101-1106
4. Jelliffe RW, Wolf CR, Berne RM, Eckstein RW (1957) Absence of vasoactive and
cardiotropic substances in coronary sinus blood of dogs. Circ Res 5:382-387
5. Berne RM, Rubio R (1979) Coronary circulation. In: Handbook of physiology.
Section 2: The cardiovascular system-the heart, vol 1. American Physiological
Society, Bethesda, Md., pp 873-952
6. Drury AN, Szent-Gyorgyi A (1929) The physiological activity of adenine
compounds with especial reference to their action upon the mammalian heart. J
Physiol 68:213-237
7. Wedd AM, Drury AN (1934) The action of certain nucleic acid derivatives on the
coronary flow in the dog. J Pharmacol Exp Ther 50: 157 -164
8. Winbury MM, Papierski DH, Hemmer ML, Hambourger WE (1953) Coronary
dilator action of the adenine-ATP series. J Pharmacol Exp Ther 109:255-260
9. Wolf MM, Berne RM (1956) Coronary vasodilator properties of purine and
pyrimidine derivatives. Circ Res 4:343-348
10. Jacob MI, Berne RM (1960) Metabolism of purine derivatives by the isolated cat
heart. Am J Physiol 198:322-326
11. Berne RM (1963) Cardiac nucleotides in hypoxia: Possible role in regulation of
coronary blood flow. Am J Physiol 204:317-322
12. Katori M, Berne RM (1966) Release of adenosine from anoxic hearts. Relationship
to coronary flow. Circ Res 19:420-425
13. Rubio R, Berne RM, Katori M (1969) Release of adenosine in reactive hyperemia
of the dog heart. Am J Physiol 216:56-62
14. Foley DH, Herlihy JT, Thompson CI, Rubio R, Berne RM (1978) Increased
adenosine formation by rat myocardium with acute aortic constriction. J Mol Cell
CardioI1O:293-300
15. Thompson CI, Rubio R, Berne RM (1980) Changes in adenosine and glycogen
phosphorylase activity during the cardiac cycle. Am J Physiol 238:H389- H398
16. Gewirtz H, Olsson RA, Most AS (1984) Role of adenosine in mediating coronary
autoregulation under basal conditions (abstract). Circulation 70 (Suppl 11):14
17. Kroll K, Feigl EO (1985) Adenosine is unimportant in controlling coronary blood

flow in unstressed dog hearts. Am J Physiol 249:H1176-H1187
18. Bittar N, Pauly TJ (1971) Myocardial reactive hyperemia responses in the dog after
aminophylline and lidoflazine. Am J Physiol 220:812-815
19. Curnish RR, Berne RM, Rubio R (1972) Effect of aminophylline on myocardial
reactive hyperemia. Proc Soc Exp Bioi Med 141:593-598
20. Merrill GF, Downey HF, Yonekura S, Watanabe N, Jones CE (1988) Adenosine
deaminase attenuates regional myocardial hypoxia in the dog. Cardiovasc Res
22:345-350
21. Kuo L, Davis MJ, Chilian WM (1988) Myogenic activity in isolated subepicardial
and subendocrdial coronary arterioles. Am J Physiol 244:H1558-H1562
Circ Res 47:807-813
23. Bardenheuer H, Schrader J (1986) Supply-to-demand ratio for oxygen determines
formation of adenosine by the heart. Am J PhysioI250:H173-H180
24. Hershfield MS, Kredich NM (1978) S-adenosylhomocysteine-hydrolase is an
adenosine-binding protein: A target for adenosine toxicity. Science 202:757-
760
25. Nees S, Herzog V, Becker BF, Bock M, DesRosiers Ch, Gerlach E (1985) The
coronary endothelium: A highly active metabolic barrier for adenosine. Basic Res
Cardiol 80:515-529
26. Watkinson WP, Foley DH, Rubio R, Berne, RM (1979) Myocardial adenosine
formation with increased cardiac performance in the dog. Am J Physiol 236:H13-
H21
27. Bacchus AN, Ely SW, Knabb RM, Rubio R, Berne RM (1982) Adenosine and
coronary blood flow in conscious dogs during normal physiological stimuli. Am J
Physiol 243:H628- H633
28. Gidday JM, Hill HE, Rubio R, Berne RM (1988) Estimates of left ventricular
interstitial fluid adenosine during catecholamine stimulation. Am J Physiol
254:H207-H216
29. Hanley FL, Messina LM, Baer RW, Uhlig, RN, Hoffman JIE (1983) Direct
measurement of interstitial adenosine. Am J Physiol 245:H327-H335
30. Tietjan CS, Tribble CG, Gidday JM, Phillips CL, Belardinelli L, Rubio R, Berne
RM (1990) Interstitial adenosine in guinea pig hearts: An index obtained by
epicardial disks. Am J PhysioI259:H1471-H1476
31. DeDeckere EAM, Ten Hoor P (1977) A modified Langendorff technique for
metabolic investigations. Pfluegers Arch 370: 103-105
32. Fenton RA, Dobson JG (1987) Measurement by fluorescence of interstitial
adenosine levels in normoxic, hypoxic, and ischemic perfused rat hearts. Circ Res
60:177-184
33. Decking UKM, Juengling E, Kammermeir H (1988) Interstitial transudate
concentration of adenosine and inosine in rat and guinea pig hearts. Am J Physiol
254:H1125-H1132
34. Heller LJ, Mohrman DE (1988) Estimates of interstitial adenosine of isolated rat
hearts from surface exudates. J Mol Cell Cardiol 20:509-523
35. Imai S, Chin WP, Jin H, Nakazawa M (1989) Production of AMP and adenosine in
the interstitial fluid compartment of the isolated perfused normoxic guinea pig
heart. Pfluegers Arch 414:443-449
36. Zhu Q, Headrick JP, Berne RM (1991) Transmural distribution of extracellular
purines in isolated guinea pig heart. Proc Natl Acad Sci USA 88:657-660
122 R.M. Berne
37. Schrader J, Haddy FJ, Gerlach E (1977) Release of adenosine inosine and
hypoxanthine from the isolated guinea pig heart during hypoxia, flow-auto-
regulation and reactive hyperemia. Pflugers Arch 369:1-6
38. Van Wylen DGL, Willis J, Sodhi J, Weiss RJ, Lasley RD, Mentzer RM (1990)
Cardiac microdialysis to estimate interstitial adenosine and coronary blood flow.
Am J PhysioI258:H1642-H1649
39. Headrick JP, Berne RM (1990) Endothelium-dependent and independent relaxa-
tions to adenosine in guinea pig aorta. Am J Physiol 259:H62-H67
2
Adenosine Receptors in the Heart
Masayuki Ueeda 1 and Ray A. Olsson 1 ,2
Summary. Al and A2 adenosine receptors (AlAR, A2AR) mediate the

biological actions of this nucleoside. The ligand-binding peptide of the AlAR
has an Mr of 35-38 kd and that of the A2AR an Mr of 45 kd. Both are
glycoproteins. Alkylxanthines, such as theophylline, competitively inhibit
ligand binding at both receptors. GTP-binding transduction proteins couple the
receptors to their effectors, G j (and Go?) for the AJAR and G s for the A2AR.
Effectors which are known to be coupled to cardiac AJARs include adenyl ate
cyclase (inhibition), muscarinic K+ channels (activation), and ATP-sensitive
K+ channels (activation). AJARs coupled to phospholipase C apparently do
not occur in the heart. Stimulation of adenylate cyclase is the only known
effect of A2AR activation. Adenosine, formed in the heart and acting through
AIARs coupled to K+ channels, slows the rate of SA node firing, retards
conduction through the A V node, and reduces the force of atrial but not
ventricular contraction. Acting through AJARs coupled to adenylate cyclase,
adenosine antagonizes the cardiostimulatory actions of catecholamines and
other agonists that stimulate cyclic AMP production. A2ARs in the coronary
arteries mediate vasodilation. One source of adenosine is cytosolic AMP,
linked through myokinase to the cytosolic ATP phosphorylation potential, an
index of cellular energy state. Adenosine from this source is thought to couple
coronary blood flow rate to the cellular energy state, thus constituting the
metabolic component of coronary flow control. The actions of adenosine at
cardiac muscle AIARs tend to reduce energy consumption, whereas the action
at coronary A 2ARs increases energy supply. In concert, these actions are
cardioprotective.
Key words: Coronary flow-Negative chronotropic-Negative dromotropic-
Negative inotropic-Adenyl ate cyclase-K+ Channels
1Departments of Internal Medicine and

2Biochemistry and Molecular Biology, University of South Florida, Tampa, FL 33612,
USA
123
124 M. Ueeda and R.A. Olsson
Introduction
Vertebrate hearts contain adenosine receptors (ARs) that mediate the negative
chronotropic, dromotropic, and inotropic as well as the coronary vasodilatory
actions of this autacoid. This review summarizes what is known about the
sources of the cardiac adenosine pool, the properties of the two major kinds of
ARs, and the roles of these receptors in cardiac and coronary physiology.
Adenosine Metabolism
The size of the cardiac adenosine pool is 1-2 nmole/g wet weight in the guinea
pig and dog [1,2] and 4-6 nmole/g wet weight in the rat [3]. The adenosine
pool is highly compartmentalized, the bulk residing intracellularly as a complex
with S-adenosylhomocysteine hydrolase [4,5]. The cytosolic free adenosine
concentration is on the order of 80 nM [6]. Because the size of the interstitial
adenosine compartment cannot be measured directly, its size is uncertain.
Neither the destruction of interstitial adenosine by means of adenosine
deaminase [7-11] nor the blockade of ARs with theophylline [7] affects basal
coronary flow, evidence that the interstitial concentration of adenosine is below
the threshold of vasoactivity, which is 50-100 nM [12-14]. The high affinity
and capacity of the nucleoside transporter that facilitates the diffusion of
adenosine into and out of cells [15] raises the possibility that the concentration
of adenosine in the interstitium is not greatly different from that in the cytosol.
Three enzymatic pathways generate the cardiac adenosine pool. The
hydrolysis of adenosine-monophosphate (AMP) by a cytosolic 5 ' -nucleotidase
[16,17] generates adenosine at a rate inversely proportional to the cytosolic
adenosine triphosphate (ATP) phosphorylation potential [18,19]. The hydro-
lysis of S-adenosylhomocysteine, a by-product of biological methylactions, is a
second source of adenosine [5] and the hydrolysis of extra-cellular AMP by an
ecto-5' -nucleotidase is a third [20-22]. ATP released along with autonomic
neurotransmitters appears to be the source of the AMP that fuels the ecto-5'-
nucleotidase pathway. The cytosolic 5'-nucleotidase pathway is quantitatively
the most important of the three and, perhaps in combination with K+ and
CO2, might account for the "metabolic" component of coronary tone [23].
Adenosine Receptors
The coupling of adenosine receptors to adenylate cyclase served as the basis of
the original classification of adenosine receptors as Al (or R i ) and A2 (Ra)
[24,25]. Table 1 summarizes the properties of the two types of receptors.
Secondary characteristics, such as the potency rank order of adenosine
analogues and the stereo-selective recognition of certain N6-substituted
adenosines by the AlAR [26], also aided classification. The AI/A2 terminology
has survived the discovery of receptors coupled to effectors other than
2. Adenosine Receptors in the Heart 125
TABLE l. Characteristics of Al and A2 adenosine receptors
Action of adenylate cyclase Inhibit Stimulate

Location in cell Surface Surface
Mr Of ligand-binding pctpide, kd 35-38 45
Alkylxanthine inhibition Yes Yes
GTP Dependence Yes Yes
Transduction protein G i , Go? G,
Toxin for NAD+ riboxylation of the G
protein PTX CTX
Ligand affinity -log KD 8-10 5-6
Selective agonists S-ENBA, CPA CGS 21680, DPMA
Selective antagonist CPDPX,XAC PD 115,199, CGS
15,943
R-PIA/S-PIA Potency ratio 50-100 <10
PTX, Toxin of Bordetella pertussis; CTX, toxin of Vibrio cholerae. Structures of agonists and
antagonists are shown in Figs. I and 2
adenylate cyclase, and now, thanks to newer, highly selective adenosine

analogues, the activity profile to these agonists is the primary criterion for
receptor classification. Antagonists are not as selective as agonists and are,
therefore, less useful in classification. Figures 1 and 2 show, respectively, the
structures of several agonists and antagonists commonly used in research.
Figure 3 summarizes bioassay data from our laboratory On the selectivity of
several of these agonists for the A I- and A2AR. Similarity of pharmacological
profiles is, of course, nO assurance that two receptors are identical. Ultimately,
the amino acid sequence of the ligand-binding peptide will replace the semi-
empirical approach of receptor classification that is currently in use. The
purification of the AlAR to homogeneity [27] and the substantial enrichment
of the A2AR [28] are encouraging steps toward elucidating the amino acid
sequences of these peptides and establishing a receptor classification based
upon the molecular structure of the receptor.
There are now several examples of AlAR coupled to effectors other than
adenylate cyclase (Table 2). At the present time, it appears that the only
AIARs in cardiocytes are those coupled to adenyl ate cyclase, to the muscarinic
K+ channel, and to the ATP-sensitive K+ channel. Adenylate cyclase is the
only known effector for the A2AR.
Adenosine Receptors in Heart Muscle
Adenosine slows the heart rate, retards conduction through the A V node and,
in atrial but not ventricular muscle, reduces the force of contractility [29].
These are referred to as the direct actions of adenosine [30], and AIARs
coupled to muscarinic K + channels mediate all three direct effects [31- 34].
Adenosine slows sinoatrial (SA) node firing rate by a combination of hyper-
R1NH
;O:~)
R'0
1-0 OH
(Rl =H, R2=H, R3=CH 2OH Adenos i ne)
1) N6 Substituted Agonists
~
S-ENBA
q
(R2=H) (R2=C I) ~
CPA CCPA
R1 = OCH3
03:
R-PIA
3 ~s
R-PBA
2) C2 Substituted Agonists
o~/ ~ Y)
~~/
H3C
H (R3=-CQ\IHC2HS) H
CV1808 CGS 21,680 PMPEA
3) 5' Substituted Agonists

-CQ\IH-<]
NCPCA
FIG. 1. Structures of commonly used adenosine receptor agonists. S-ENBA, CPA
CCPA, CHA, R-PIA, and R-PBA are selective for the AlAR. DPMA, CV-1808, CGS
21,680 and PMPEA are selective for the A2AR. NECA and NCPCA are, like 2-
choroadenosine (not shown), unselective agonists
polarization and a decrease in the slope of phase 4 of the action potential [35].
In the atrioventricular (AV) node, adenosine acts on the cells of the N zone,
delaying the onset and reducing the amplitude of the action potential [34]. The
negative atrial inotropic effect of adenosine is due to a shortening of the action
potential duration [31]. Although ventricular muscle contains AIARs coupled
to ATP-sensitive K+ channels [36], their physiological significance is unknown.
yH3
~~~S03H
H3C"'N~~
o
8-(p-Sulfophenyl)
Theophy I line
~ ~
~NyN>-O
~~I
~ CPDPX
) 0
PACPX
NH2
FIG. 2. Structures of commonly used adenosine receptor antagonists. Theophylline,

caffeine, and 8-(p-sulfophenyl) theophythine are unselective agonists. CPDPS, PACPX,
and XAC are more potent at some AIARs and PD 115,199 and CGS 15,943 are more
potent at some A 2 ARs
In some but not all species, adenosine antagonizes the positive inotropic
action of agonists that stimulate adenylate cyclase, the indirect action [30].
AIARs coupled to adenylate cyclase mediate this effect, which is evident only
when the cyclase is stimulated. The antiadrenergic action of adenosine is
prominent in rodent hearts [37,38] but appears to be absent in the dog [39],
despite evidence that dog cardiocytes contain AIARs [40].
Two recent reports [41,42] describe evidence that cardiocytes contain A2ARs
in addition to A\ARs. The functional significance of these receptors remains to
be established.
A1
9 .......... i ......................... i ......................... ;......................... ;...
S-~NBA: ! '
~ ..: .
..:
8 ··········:·······················:·······i=f·PBA····:·........................ ~.. .
.--.. 'CPA ' -, ,
a: I R-~I~ I :
a.. :
0 7
C/)
a
r;C:~NECArT
It)
0
6 ......... '1'··· ................... ··l············· ........... ,j......................... ~.. .
---
LU
0>
0
• : DPMA
I ADO ~ • •
5 ··········i···········~~~~~·~························r·~······PMPE1··
: : C~S21680:
•.•.•..••• j .••••••.•••••••••••••.••. j ........ , ................ '0 ........................ ~ .. .
4
7 8 9
-log(ECSOCF)
FiG. 3. Bioassay in guinea pig Langendorff preparation of several analogues listed in
Fig. 1. Abscissa EC so of coronary vasodilation, mediated by A2ARs; ordinate EC so of
prolongation of A V node conduction time (SQPR), mediated by AlARs
TABLE 2. Effectors coupled to the AlAR

Effector Reference
Adenylate cylcase [24]
[25]
Guanylate cyclase J Bioi Chem 262:6296, 1987
Muscarinic K + channel [3 i]
[32]
ATP-sensitive K+ channel Nature (London) 305:147, 1983
Ca 2 + Channel J Physiol 373:47, 1986
Phosphiolipase A2 Biochem Biophys Res Commun 152:886, 1988
Phospholipase C potentiator Biochem Biophys Acta 847:207, 1986
Phospholipase C antagonizer Eur J Pharmacol 137:269, 1987
Glucose transporter FEBS Lett 201:246, 1986
Coronary Artery Adenosine Receptors

The agonist potency profile of receptor-selective adenosine analogues suggests
that an AzAR mediates the coronary vasoactivity of adenosine [43,44]. Recent
work calls for a re-examination of the conventional view that vasodilation
represents a direct action of adenosine on coronary smooth muscle and that

cyclic AMP mediates relaxation. At least in the case of exogenous adenosine,
there is evidence that adenosine receptors in the coronary endothelium rather
than smooth muscle might initiate relaxation. Autoradiographic studies show
that the administration of labeled adenosine in concentrations in the vasoactive
range «111M) results in selective uptake by the endothelium, suggesting that
the nucleoside never reaches adenosine receptors in the underlying smooth
muscle [45]. The relaxation of isolated coronary arteries in vitro is at least
partially endothelium-dependent [46,47]. Although coronary endothelial cells
contain an AzAR [48] that presumably mediates vasodilation, the nature of
events subsequent to activation of this receptor is uncertain. "P site" inhibition
of adenylate cyclase does not affect the coronary vasoactivity of adenosine [49],
an observation difficult to reconcile with the large body of evidence that
indicates that adenyl ate cyclase is the only effector system coupled to the
AzAR. We find that NG-nitro-L-arginine, an inhibitor of the biosynthesis of
nitric oxide which is an endothelium-derived relaxing factor [50-52], inhibits
the coronary vasoactivity of adenosine (M. Ueeda et a1., unpublished work).
Such a result suggests that activation of an endothelial cell AzAR might
stimulate nitric oxide production. Whether the coronary vasoactivity of
endogenous adenosine is also endothelium-dependent is not clear.
Acknowledgements. Recent reports describe the nucleotide sequences of two

AIARs (Nucleic Acids Res 18:1915 1990; Mol Pharmacol 40:1 1991) and of
one AzAR (Nucleic Acid Res 18:1914 1990; Biochem Biophys Res Commun
173: 1169 1990). These discoveries provide the amino acid sequences of two
receptors, the molecular basis for receptor classification. The authors wish to
thank Mrs. Germaine Jones for the preparation of this manuscript. This work
was supported by the Ed C. Wright Chair in Cardiovascular Research, Univer-
sity of South Florida, and NIH HL-30391.
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3
Role of Ecto-5' -Nucleotidase on
Hypoxia-Induced Adenosine
Formation in the Perfused Guinea
Pig Heart
Mikio Nakazawa, Hiromasa lin, Hiroto Matsuda, and Shoichi Imai 1
Summary. The role of adenosine in the transmyocardial effluent, which was

obtained by the method of De Deckere and Ten Hoor on the regulation of
coronary flow, was examined in the perfused guinea pig heart. EHNA, an
adenosine deaminase inhibitor, was used for changing adenosine concentration
in the fluid. It was found that there was a good correlation between adenosine
concentration in the transmyocardial effluent and the coronary flow. Using the
same method, the role of ecto-5' -nucleotidase on the hypoxia (60% O 2 )-
induced adenosine formation was reinvestigated. In hypoxia, the adenosine
concentration in the transmyocardial effluent was increased together with the
coronary flow, and the regression line representing the relation between the
adenosine concentration and the coronary flow coincided with the same correla-
tion obtained after EHNA treatment under normoxia, indicating that adeno--
sine in the transmyocardial effluent plays an important role in the regulation of
the coronary flow. Since the hypoxia-induced increase in the adenosine forma-
tion was inhibited by AOPCP, a specific inhibitor of the ecto-5'-nucleotidase, it
was suggested that the majority of the hypoxia-induced adenosine formation in
the guinea pig heart was via the pathway of ecto-5' -nucleotidase. Furthermore,
it was found that AOPCP caused an increase in AMP concentration in the
transmyocardial effluent which was un associated with an increase in coronary
flow. Thus, it seems very likely that endogenous AMP has only a very weak or
no vasodilatatory activity and its apparent vasoactivity was due to its con-
version to adenosine.
Key words: Ecto-5'-nucleotidase-Adenosine-Coronary flow-AMP
I Department of Pharmacology, Niigata University School of Medicine, Niigata, 951

Japan
133
134 M. Nakazawa et al.
Introduction
The vasodilator effect of adenosine has been known for many years and the
hypothesis proposed by Berne [1] that the substance plays an important role in
the regulation of coronary flow has been studied extensively. Although suppor-
tive evidence has been accumulated [2-4], the enzyme(s) responsible for its
formation has not yet been clarified.
There are two enzymes which may be responsible for adenosine formation.
One is ecto-5'-nucleotidase [5-8] and the other is cytosolic-5'-nucleotidase
[9-13]. Schutz et al. [14] and Frick and Lowenstein [6] reported that a, ~
methylene adenosine 5'-diphosphate (AOPCP), a potent and specific inhibitor
of ecto-5' -nucleotidase [11,15], did not produce any inhibition of adenosine
release into the pulmonary effluent during hypoxia in the perfused hearts.
Meghji et al. [16] stated that the antibody to ecto-5' -nucleotidase had no
effects on adenosine release during the adenosine 5' -triphosphate (ATP)
catabolic state induced by metabolic inhibitors in neonatal rat cardiomyocytes.
On the contrary, Bukoski and Sparks [17] reported an effective inhibition by
AOPCP of adenosine release from the adult rat cardiomyocytes induced by
metabolic inhibition. Headrick and Willis [18] suggested an important role of
ecto-5' -nucleotidase in the formation of extracellular adenosine during iso-
prenaline infusion, graded hypoxia, and graded underperfusion. Dendorfer et
al. [19] suggested that the guinea pig cardiomyocyte itself has no ecto-5'-
nucleotidase. However, Imai et al. [20] reported attenuation by AOPCP of
adenosine formation during a transient ischemia in the isolated perfused heart
preparations of the guinea pig.
In view of this discrepancy, we reinvestigated the effect of AOPCP on
hypoxia-induced adenosine formation in the perfused guinea pig heart using a
special perfusion method described by De Deckere and Ten Hoor [21], which
enabled us to collect not only the coronary venous effluent but also the
interstitial fluid (transmyocardial effluent) separately; Schutz et al. [14] and
Frick and Lowenstein [6] analyzed hypoxia-induced adenosine formation only
in the coronary effluent.
A significant inhibition by AOPCP of hypoxia-induced adenosine formation
in the transmyocardial effluent was found, suggesting an important role being
played by ecto-5' -nucleotidase in hypoxia-induced adenosine formation in the
perfused guinea pig heart. It was also found that adenosine 5' -monophosphate
(AMP) itself did not produce any vasodilatation either in the perfused guinea
pig heart or in the isolated porcine coronary artery.
Materials and Methods

Preparation and Perfusion of Isolated Guinea Pig Hearts
Experiments were performed in the isolated perfused heart preparation of
male guinea pigs weighing 300-350 g.. The method employed in the present
3. Role of Ecto-5'-Nucleotidase on Hypoxia-Induced Adenosine Formation 135
experiment was similar to the one previously described [22]. In brief, under
ether anesthesia, the heart was rapidly excised and the ascending aorta was
cannulated. Retrograde perfusion with a modified Krebs-Ringer bicarbonate
solution from a reservoir 75 cm above the heart was begun immediately. The
adherent mediastinal tissues were cleaned off, and a cannula was introduced
into the pulmonary artery to draw out the coronary venous effluent. After
carefully tying off the caval and pulmonary veins to avoid leakage, the small
amount of fluid dripping from the apex (transmyocardial effluent) was collected
as described by De Deckere and Ten Hoor [21].
The composition of the modified Krebs-Ringer bicarbonate solution used
was (in mM): NaCI 125.2, KCI 4.7, CaCl2 2.5, KH2P04 1.2, NaHC0 3 24.9,
sodium pyruvate 2.0, and glucose 5.5. The solution was oxygenated with 95%
O 2 + 5% CO 2 to ensure P02 values higher than 600 mmHg, and kept at a
temperature of 38°C. Experiments were performed after a stabilization period
of 50-60 min. Drugs were infused directly into the perfusate inflow line near
the aortic cannula by means of an infusion pump (Harvard Apparatus infusion/
withdrawal pump 940, South Natick). The volume of infusion was limited to
less than 0.1 mllmin.
An adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine,
was used to assess the relationship between adenosine concentration in the
transmyocardial effluent and the coronary flow.
In order to study the effects of hypoxia on the coronary flow and adenosine
concentration in the transmyocardial effluent, the perfusate was changed from
the one equilibrated with 95% O 2 + 5% CO2 to the one equilibrated with 60%
O 2 + 5% CO 2 + 35% N2 . AOPCP, a potent and specific inhibitor of ecto-5'-
nucleotidase, was used for examining the contribution of ecto-5' -nucleotidase
to the production of adenosine during hypoxia.
Analyses
Samples of the transmyocardial and coronary effluents were heated in the
water bath at lOO°C for 3 min immediately after collection. Analysis of
adenosine and related compounds was performed with high performance liquid
chromatography. A Radial-Pak I!Bondapak CIS column (Waters, Milford)
was used as the stationary phase and a mixture of 92.5% ammonium phosphate
buffer (0.01 M) and 7.5% methanol, pH 4.0 was used as the mobile phase for
isocratic analysis of adenosine and inosine. For analysis of AMP, adenosine
5'-diphosphate (ADP) , and ATP, 0.025% trihydroxyfuran, 0.05M KH 2P04 ,
0.001 M tetrabutylammonium hydrogen sulfate, and 4.5% acetonitrile (pH
6.25) was used as the mobile phase. The absorbance was measured at 254 nm.
From ·100 to 200 I!l of the sample was injected directly onto the column.
Identification of the compounds was carried out on the basis of retention times
and enzymatic transformation, and the concentrations were quantitated from
peak height measurements and calculated from standard runs.
136 M. Nakazawa et at.
The Continuous Measurement of the External Diameter of

the Porcine Coronary Artery
The method employed in this experiment was the one already reported by
Busse et al. [23]. In brief, a branch of the left circumflex coronary artery
isolated from the porcine heart obtained from a local slaughter house and
dissected free of loose connective tissue from the external surface was
cannulated and perfused. With the aid of a special apparatus, it was possible to
regulate intra- and extraluminal perfusion separately. The hydrostatic
perfusion pressure in the segments was adjusted to 50 mmHg. The rate of flow
of the perfusate at the luminal side was 0.75 mllmin, while the rate of flow at
the extraluminal bath side was 14 mllmin. The external diameter of the
segment was measured at its midpoint using a photoelectric device. The
Tyrode's solution which was used had the following composition in mM: NaCi
132.1, KCI3.64, KH 2 P0 4 0.36, MgCIz 1.0, CaCI 2 1.6, NaHC0 3 11.9, ethylene-
diaminetetraacetic acid (EDTA) 0.025, and glucose 5.6 and was equilibrated
with 95% O 2 + 5% CO 2 (37°C).
After the vessel segment was allowed to equilibrate for 60 min, it was con-
tracted with 50 mM KCI to study the relaxation caused by purine compounds.
Chemicals and Drugs

The chemicals and drugs used were: ethanol (for HPLC grade), tetra-
hydroxyfuran (Wako Pure Chemical Industries, Japan), adenosine, AOPCP
(Sigma Chemicals, St. Louis), tetrabutylammonium hydroxy sulphate (Aldrich
Chemicals, Milwaukee), and erythro-9-(2-hydorxy-3-nonyl)adenine (EHNA)
(Burroughs Wellcome, Research Triangle Park). All other chemicals were
obtained form Wako Pure Chemical Industries.
Statistics
Data were expressed as a mean ± standard error of the mean (SEM). The
paired and unpaired Student's {-test were used for analyses of statistical
differences and P values of less than 0.05 were considered significant.
Results
Does the Transmyocardial Effluent Adenosine

Concentration Correlate with the Coronary Flow?
In order to assess whether there is a correlation between the adenosine
concentration in the transmyocardial effluent and the coronary flow, adenosine
concentration in the transmyocardial effluent was increased with EHNA (a
potent inhibitor of adenosine deaminase). EHNA was infused into the
perfusion line at doses of 56.5, 142, 285, 565, and 1420nmollmin which
1500 ,-------,-------.---~ 15 ,-----,------,------,-------,
o
c o
~
~ 1000
c
CIl
u
c TME
o
u
Y=2.3IogX + 5.3
CIl
c ~ r=0.94
'00 500 o
o c
c o
L-
CIl
-0
o
« U
5'--____..L..-_ _ _ _..L..-_ _ _ _..L..-_ _- - - J

o 50 100 150 30 100 300 1000 3000
EHNA concentration (J.I. M) Adenosine in TME (nM)
FiG. 1. Effects of EHNA on adenosine FIG. 2. The relationship between the

concentration in transmyocardial (TME, adenosine concentration in transmyo-
n = 4) and pulmonary effluent (Pul- cardial effluent (TME) and the coronary
monary, n = 4) flow under EHNA infusion
corresponded to around 6,15,30,60, and 150 11M, respectively. EHNA caused

a concentration-dependent increase in adenosine concentration in the
transmyocardial effluent (Fig. 1). No such concentration-dependent increase
was observed in the pulmonary effluent (Fig. 1). EHNA infusion produced a
concentration-dependent increase in the coronary flow and a significant linear
correlation (P < 0.001) was observed between the adenosine concentration in
the trans myocardial effluent and the coronary flow (Fig. 2).
Effects of Hypoxia on the Formation of Adenosine and

the Coronary Flow
In Table 1 are listed the hypoxia (60% 02)-induced changes in adenosine,
inosine, and AMP concentrations in the transmyocardial and pulmonary
effluents. Changes in the coronary flow are also listed in Table 1. During
hypoxia, adenosine concentrations in the transmyocardial and pulmonary
effluents increased by 590% and 380%, respectively. Inosine concentrations
also increased, by 283% in the transmyocardial effluent and by 182% in the
pulmonary effluent. AMP concentration increased by 153% in the trans-
myocardial effluent and by 22% in the pulmonary effluent. It was clear that the
hypoxic changes in the transmyocardial effluent were greater than those in the
pulmonary effluent. Additionally, hypoxia increased the coronary flow rate by
20% (Table 1).
The rate of release of adenosine, inosine, and AMP is listed in Table 2. In
both fluid compartments, hypoxia induced an increase in rate of release of
adenosine, inosine, and AMP.
......
\.N
00
TABLE1. Hypoxia (60% 02)-induced changes in adenosine (Ads), inosine (Ins) and AMP concentration in the transmyocardial effluent
(TME) and in the pulmonary effluent (PE) (n = 6)
Normoxia 1 Normoxia 2 Hypoxia Hypoxic changes I
Control 47.0 ± 6.6 48.4 ± 7.4 334.5 ± 50.0b 286.0 ± 44.8 I

Ads
(nM)
AOPCP 49.8 ± 17.0 37.8 ± 5.8 68.5 ± 17.0** 31.1 ± 12.9**
Control 0.36 ± 0.04 0.36 ± 0.04 1.38 ± O.13 b 1.01 ± 0.13

Ins
TME
(JlM) AOPCP 0.36 ± 0.06 0.72 ± 0.04*** 0.78 ± 0.02** 0.05 ± 0.06**
Control 26.6 ± 4.3 27.4 ± 6.7 69.4 ± 13.8a 42.0 ± 16.5

AMP ~
(nM) 485.0 ± 70.4***
AOPCP 58.5 ± 12.1 560.3 ± 131.1* 75.3 ± 82.4 Z
~
:>;"
Control 29.6 ± 2.7 32.4 ± 3.5 156.0 ± 18.0b 123.7 ± 14.9 ~

~
Ads ~
~
(nM) 42.0 ± 13.1 51.7 ± 17.9** 9.7 ± 7.3***
AOPCP 23.2 ± 9.2 ~
:::..
Control 0.34 ± 0.02 0.34 ± 0.02 0.96 ± O.ose 0.61 ± 0.07
Ins
PE
(JlM) AOPCP 0.28 ± 0.04 0.67 ± 0.18 0.43 ± 0.06*** -0.23 ± 0.13***
Control 24.8 ± 5.2 22.4 ± 4.6 37.1 ± 4.5 a 14.7 ± 4.6

AMP
(nM) 66.4 ± 11.4* 81.2 ± 18.2 14.8 ± 13.4
AOPCP 37.2 ± 0.51
Control 8.9 ± 0.4 8.9 ± 0.3 10.7 ± 0.2a 1.8 ± 0.2

Coronary flow
(mllmin)
AOPCP 9.3 ± 0.6 9.0 ± 0.7 12.1 ± 0.6 3.1 ± 0.4
- - - - '---------- ------
*P < 0.05; **P < lUll; ***P < 0.001 vs control hearts (non-paired I-test), "P < 0.05, hp < O.OI,"P < 0.001 vs Normoxia 2 (paired I-test), Sampling
details are descrihed in the legend of Fig. 3
Effects of AOPCP on the Hypoxic Changes in Adenosine,

Inosine, AMP, and Flows
In order to examine the contribution of ecto-5' -nucleotidase to hypoxia-
induced adenosine formation, its inhibitor, AOPCP, was used. In the normoxic
state, AOPCP decreased the concentration of adenosine and coronary inflow,
and increased inosine and AMP concentration (Table 1). AOPCP significantly
reduced the rate of release of adenosine and inosine into the transmyocardial
effluent to 13% and 30% of the normoxic value, respectively (Table 2).
Release of adenosine into the pulmonary effluent was also reduced by AOPCP
to 54% of the normoxic value, although the decrease was not statistically
significant. These results indicate that ecto-5'-nucleotidase contributed to the
hypoxia-induced adenosine formation.
The Relationship Between Hypoxia-Induced Increase in

Adenosine Concentration in the Transmyocardial Effluent
and Coronary Flow Rate
The relationship between the hypoxia-induced increase in adenosine con-
centration in the transmyocardial effluent and the changes in coronary flow is
depicted in Fig. 3. In the hearts not treated with AOPCP, a good correlation
was observed between hypoxia-induced changes in the adenosine concentration
and those in the coronary flow. The regression line corresponded to the line
obtained with EHNA infusion. However, the data obtained after treatment
with AOPCP deviated from the regression line obtained under the normoxic
condition using EHNA infusion (Fig. 3). The increase in coronary flow was
much greater than was expected from the increase in adenosine. These results
indicate that after AOPCP, some other substance(s) came into play and
contributed to the hypoxia-induced increase in the coronary flow.
As mentioned above, under the normoxic condition, AOPCP caused a
marked increase in AMP concentration in the transmyocardial effluent with a
small decrease in adenosine concentration and a slight decrease in coronary
flow. The changes in adenosine concentration in the transmyocardial effluent
and those in the coronary flow under this condition could be correlated by a
regression line obtained with EHNA (Fig. 3). Due to the increase in AMP, the
sum of adenosine and AMP after AOPCP was around 10 times higher than
that of the non-treated group (Table 1). Nevertheless, there a decrease in
coronary flow was definitely present. Thus, it must be concluded that AMP
itself has no vasodilatory effect, although Biinger et al. [27] reported that AMP
was a vasodilator in the perfused guinea pig heart.
The Effects of Extra- and Intraluminal Applications of AMP

and Adenosine on the Porcine Coronary Artery
In order to examine the effects of extra-( adventitial side) and intraluminal
(luminal side) applications of adenine nucleotides on the tone of the coronary
TABLE 2. Hypoxia (60% Oz)-induced changes in adenosine (Ads), inosine (Ins) and AMP release in the transmyocardial effluent (TME) ......
and in the pulmonary effluent (PE) o+>-
Control (n = 6) AOPCP (n = 6)
Normoxia 1 268.0 ± 46.8 243.0 ± 96.9
Ads Normoxia 2
(pmol/min per gram dry wt) 300.0 ± 52.3 149.5 ± 36.9*
Hypoxia 2688 ± 410 b 323.3 ± 102.1***
Normoxia 1 2.07 ± 0.32 1.66 ± 0.45

Ins Normoxia 2
TME (nmol/min per gram dry wt) 2.30 ± 0.36 2.71 ± 0.41
Hypoxia 11.23 ± 1.39' 3.34 ± 0.51 **
Normoxia 1 141.2 ± 13.5 201.4 ± 30.5 ~

AMP Normoxia 2 391**
(pmol/min per gram dry wt) 164.1 ± 35.3 1854 ± Z
I'l
:>;"
Hypoxia 552.9 ± 115.6" 2670 ± 918 ~
I'l
Normoxia 1 1535 203 1057 285
:;:
± ± I'l
Ads Normoxia 2 ~
(pmol/min per gram dry wt) 1662 ± 234 1702 ± 159
e:..
Hypoxia 9448 ± 1235" 5138 ± 1843
Normoxia 1 17.75 ± 1.96 17.21 ± 2.67
PE Ins Normoxia 2 17.68 1.67
(nmol/min per gram dry wt) ± 30.18 ± 7.29
Hypoxia 57.76 ± 3.84c 37.62 ± 6.79*
Normoxia 1 1226 ± 207 1953 ± 337
AMP Normoxia 2 397**
1120 ± 183 2893 ±
(pmol/min per gram dry wt)
Hypoxia 2231 ± 267" 5002 ± 1209
*P < 0.05; **P < 0.01; ***P < 0.001 vs control hearts
aP < 0.05; b P < 0.01; c P < 0.001 vs Normoxia 2 (paired t-test)
Sampling details are described in the legend of Fig. 3
3. Role of Ecto-S'-Nucleotidase on Hypoxia-Induced Adenosine Formation 141
15
r--.
C
E
"'-
E
'-"
Hypoxia + AOPCP /
~
/
10
--
0 /
~
0
c
0
I...
0
U
5
10 100 1000
Ads in TME (nM)
FIG. 3. The relationship between the adenosine concentration in the trans myocardial
effluent (Ads in TME) and the coronary flow before and after hypoxia (60% 02)' The
transmyocardial effluent (TME) was collected at three sequential periods. After
stabilization of the preparation at around SOmin, the first samples were obtained as the
controls (Normoxia 1, closed and open circles). Then, the infusion of AOPCP (O.Sllmoll
min) or vehicle (distilled water 0.OS7mllmin) was begun and continued during the
experiment. Four minutes later, second samples (Normoxia 2, closed upper triangle for
non-treated and open upper triangle for AOPCP-treated) were obtained to assess the
effects of AOPCP under the normoxic state. After the sampling, the perfusate buffer
was switched to the hypoxic one (equilibrated with 60% O 2 + 3S% N2 + S% CO 2),
and, at the same time, third samples were obtained during 3 min of hypoxia (Hypoxia,
closed downward triangle for non-treated heart; AOPCP-treated heart, open downward
triangle). The linear line drawn was the regression line of Fig. 2
artery, a special photoelectric device developed by Busse et al. [23] was used,
and the changes in the external diameter of the coronary artery were
continuously recorded.
The effects of AMP and adenosine are depicted in Fig. 4. Adenosine and
AMP induced a dose-related relaxation irrespective of whether they were
administered at the extraluminal or intraluminal side. The relaxations produced
by intraluminal application of both compounds were smaller than those by
intraluminal application. These results suggest that the endothelium may play
an important role in the relaxant effects of adenosine and AMP.
AMP Adenosine
ore~"'::--"*""=~iIr---,
(%)
c
0
:.;:i
0 25
x
0 \
-
v
L- \
0
\
.......
c
v 50
\~
u
L-
v
a..
75
7 6 5 4 3 7 6 5 4 3
Concentration (-log[MJ)
FIG.4. Effects of AMP and adenosine on the precontracted porcine coronary artery
(n = 6-9). Circles, Intraluminal applications: triangles extra luminal applications; open
symbols, preparations with endothelium; closed symbols, preparations without
endothelium
Therefore, we examined the effects of the removal of endothelium on the

relaxant effects of these substances. After the removal of endothelium, the
dose-response curves of intra- and extraluminal applications of adenosine and
AMP were shifted to the right (Fig. 4). Thus, it was concluded that the
relaxations induced by adenosine and AMP were partly dependent upon the
presence of endothelium.
A dose of 50 11M of AOPCP caused a rightward shift of the dose-response
curve to the intraluminal application of AMP (Fig. 5). Because of the competi-
tive nature of inhibition, the maximal response by AMP was not changed. This
result suggests that AMP itself is neither a vasodilator nor merely a very weak
vasodilator, and that the observed relaxation was due to its conversion to
adenosine. The ED50 values of relaxation were 6.9 x 1O- 6 M and 4.5 x 10- 5 M
(control and AOPCP-treated, respectively). If we assume that the relaxant
effects to AMP is entirely due to its conversion to adenosine, the calculated
rate of inhibition of the ecto-5'-nucleotidase by AOPCP amounts to 85%.
FIG. 5. Effect of AOPCP o

(50 ~M) on the dilatation of the
porcine coronary artery pro- (%)
duced by intraluminal application
of AMP (n = 7). Open circles,
c
Without AOPCP; closed circles, o
with AOPCP :;:;
o 20
X
o
-
Ql
L..
o
~
C
Ql 40
()
L..
Ql
a...
60
7 6 5 4 3
AMP concentration -log(M)
Discussion
In the present experiments, which were designed to clarify the contribution of
ecto-5'-nucleotidase to the hypoxia-induced formation of adenosine in the
perfused guinea pig heart, we found that AOPCP, a potent and specific
inhibitor of ecto-5'-nucleotidase [11,15], caused an 87% reduction of hypoxia-
induced adenosine release into the transmyocardial effluent (P < 0.001) and a
46% reduction of the release into the pulmonary effluent (not significant). This
means that the major part of hypoxia-induced adenosine formation is through
ecto-5' -nucleotidase.
Schutz et al. [14] reported that AOPCP caused no reduction of adenosine
formation in the coronary effluent during hypoxia. The differences between the
present study and the study by Schutz et al. were the preparation used and the
Mg2+ composition in the perfusion buffer. They used the buffer with 1.1 mM of
Mg2+ , while we used the one without Mg2+. It is well known that the Mg2+ ion
is an activator of ecto-5'-nucleotidase [24]. However, at the same time, it is
known that this substance can reverse the inhibition of the enzyme [24]. Schutz
et al. showed an inhibition of breakdown of exogenous AMP of about 85%
with AOPCP (50 ~M). Nevertheless, we have to consider that the compartment
in which ecto-5'-nucleotidase resides in the heart is not one but at least three.
There are intra-vascular (endothelium), extra-vascular (smooth muscle), and
interstitial (myocytes) compartments. Exogenous AMP, i.e., AMP administered

from outside to the heart, distributes mainly in the intra-vascular compart-
ment and faces mainly to endothelial ecto-5' -nucleotidase. On the other
hand, endogeneous AMP faces to extra-vascular and/or interstitial ecto-5'-
nucleotidase. Therefore, it does not seem likely that the true rate of inhibition
of degradation of endogenous AMP by AOPCP can be measured by using
exogenous AMP. It is possible that SchUtz et al. [14] over-estimated the
inhibition by ecto-enzyme and that the actual inhibition of the degradation of
the endogenous AMP was much less than they assumed. In order to get a more
complete inhibition of degradation of endogenous AMP, a higher concentra-
tion of AOPCP must have been necessary under their conditions (with
1.1mMMg2 +). Moreover, the difference of the site of sampling used may be
the cause of divergent results. Even under the present experiment, the
inhibition by AOPCP was less in the pulmonary effluent than in the trans-
myocardial effluent.
In the experiments in which EHNA, a potent inhibitor of adenosine
deaminase [25] was used, it was found that the coronary flow was a function of
adenosine concentration in the transmyocardial effluent, i.e., in the interstitial
space compartment. We did not use the injection of exogenous adenosine to
assess the relationship between adenosine concentration and coronary flow
because, as previously noted, the effects of exogenous adenosine might differ
from that of endogenous adenosine. Because of the high activity of adenosine
deaminase, endogenous adenosine is rapidly degraded to inosine, which has no
vasodilatatory activity. Thus, inhibition of adenosine deaminase causes an
increase in endogenous adenosine concentration and in the coronary flow. The
compartment in which these changes in adenosine occurred should be the same
in which the concentration of adenosine changes during the pathophysiologic
state. Indeed, the adenosine concentration in the transmyocardial effluent in
hypoxia was increased together with the coronary flow, and the line depicting
the relation between adenosine and coronary flow coincided with the line
obtained after EHNA infusion under normoxia (Fig. 3). This indicates that
adenosine in the transmyocardial effluent plays an important role in the
regulation of the coronary flow not only under normoxia but also under
hypoxia.
After AOPCP treatment under normoxia, the line depicting the relation
between the adenosine concentration and the coronary flow almost coincided
with the one obtained during hypoxia without AOPCP. However, in the
AOPCP-treated heart, the increase of coronary flow during hypoxia was much
greater than was expected from adenosine formation. We have no explanation
for this discrepancy at present. Presumably, an additional regulator sub-
stance(s) comes into play under this condition [26]. The problem merits further
study.
Under the normoxic condition, AMP concentration in the transmyocardial
effluent increased approximately tenfold after AOPCP. However, no increase
in coronary flow was observed (Table 1, Fig. 3). This was very strange, because
it had been reported that exogenous administration of AMP caused a definite
vasodilation in the perfused guinea pig heart [27]. If AMP has a vasodilatatory
activity, the coronary flow should increase after AOPCP treatment. As a
matter of fact, the coronary flow after AOPCP treatment did not increase, but
was just a function of the adenosine concentration in the transmyocardial
effluent (Fig. 3). From these results, we concluded that the endogenous AMP
had only very weak or no vasolilatatory activity. Its apparent vasoactivity was
due to its conversion to adenosine, a potent vasodilator, by ecto-5'-nucleo-
tidase. Indeed, we found that the vasodilation caused by exogenously
administered AMP in the perfused guinea pig heart to be inhibited by AOPCP
(data not shown).
In order to clarify this point further, we studied the relaxant effects of AMP
and adenosine on the coronary arterial smooth muscle, using a special
apparatus which enabled us to monitor the external diameter of the porcine
coronary artery and to separately administer drugs either from the intraluminal
or extraluminal side [23]. With an intact endothelium, the artery which had
been preconstricted with 50mM KCl was dilated with AMP and adenosine.
Without endothelium, these dilatations were diminished. Thus, dilatations by
adenosine and AMP were partially dependent upon the presence of
endothelium.
In this preparation, AOPCP caused an inhibition of the dilatatory effects of
AMP. The ED50 values of dilatation of AMP were 6.9 x 1O-6 M and 4.5 x
10- 5 M without and with 50 11M AOPCP, respectively. If AMP itself has no
vasdilatatory activity, the inhibition of ecto-5'-nucleotidase should be 85%.
This value was very similar to the results of Schutz et al. [14] who used the
same concentration of AOPCP.
References
1. Berne RM (1963) Cardiac nucleotides in hypoxia: Possible role in regulation of
coronary blood flow. Am J Physiol 204:317-322
Circ Res 47:807-813
3. Imai S, Nakazawa M, Imai H, Jin H (1987) 5/ -nucleotidase inhibitors and the
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(eds) Topics and perspectives in adenosine research. Springer-Verlag, Berlin
Heidelberg, pp 416-424
blood flow in microembolization. Am J PhysioI250:H509-H518
5. Rubio R, Berne RM, Dobson JG Jr (1973) Sites of adenosine production in cardiac
and skeletal muscles. Am J Physiol 225:938-953
6. Frick GP, Lowenstein JM (1976) Studies of 5'-nucleotidase in perfused rat heart. J
Bioi Chern 251:6372-6378
7. Frick GP, Lowenstein JM (1978) Vectorial production of adenosine by 5'-
nucleotidase in the perfused rat heart. J Bioi Chern 253: 1240-1244
8. Newby AC, Luzio JP, Hales N (1975) The properties and extracellular location of
5'-nucleotidase of the rat fat-cell plasma membrane. Biochem J 146:625-633
9. Hoh R, Usami C, Nishino T, Tsushima K (1978) Kinetic properties of cytosol

5'-nucleotidase from chicken liver. Biochim Biophys Acta 526:154-162
10. Van Den Berghe G, Van Pottelsberghe C, Hers H-G (1977) A kinetic study of the
soluble 5'-nucleotidase of rat liver. Biochem J 162:611-616
11. Worku Y, Newby AC (1983) The mechanism of adenosine production in rat
polymorphonuclear leucocytes. Biochem J 214:325-330
12. Newby AC (1888) The pigeon heart 5'-nucleotidase responsible for ischaemia-
induced adenosine formation. Biochem J 253:123-130
13. Troung VL, Collinson AR, Lowenstein JM (1988) 5'-nucleotidase in rat heart.
Evidence for the occurrence of two soluble enzymes with different substrate
specificities. Biochem J 253: 117 -121
14. Shiitz W, Schrader J, Gerlach E (1981) Different sites of adenosine formation in the
heart. Am J Physiol 240:H963-H970
15. Naito Y, Lowenstein JM (1985) 5'-nucleotidase from rat heart membranes.
Inhibition by adenine nucleotides and related compound. Biochem J 226:645-651
16. Meghji P, Holmquist CA, Newby AC (1985) Adenosine formation and release from
neonatal-rat heart cells in culture. Biochem J 229:799-805
17. Bukoski RD, Sparks HV Jr (1986) Adenosine production and release by adult rat
cardiocytes. J Mol Cell Cardiol 18:596-605
18. Headrick JP, Willis RJ (1989) 5'-nucleotidase activity and adenosine formation in
stimulated, hypoxic and underperfused rat heart. Biochem J 261:541-550
19. Dendorfer A, Lank S, Schaff A, Nees S (1987) New insights into the mechanism of
myocardial adenosine formation In: Gerlach E, Becker BF (eds) Topics and
perspectives in adenosine research. Springer-Verlag, Berlin Heidelberg, pp 170-
185
20. Imai S, Nakazawa M, Imai H, Jin H (1987) 5'-nucleotidase inhibitors and the
myocardial reactive hyperemia and adenosine content In: Gerlach E, Becker BF
(eds) Topics and perspectives in adenosine research, Springer-Verlag, Berlin
Heidelberg, pp 416-424
21. De Deckere EAM, Ten Hoor P (1977) A modified Langendorff technique for
metabolic investigation. Pflugers Arch 370: 103-105
22. Imai S, Chin W-P, Jin H, Nakazawa M (1989) Production of AMP and adenosine in
heart. Pflugers Arch 414:443-449
23. Busse R, F6rstermann U, Matsuda H, Po hI U (1984) The role of prostaglandins in
the endothelium-mediated vasodilatory response to hypoxia. Pflugers Arch 401:
77-83
24. Sullivan JM, Alpers JB (1971) In vitro regulation of rat heart 5'-nucleotidase by
adenine nucleotides and magnesium. J BioI Chern 246:3057-3063
25. Acterberg PW, Harmsen E, De Jong JW (1985) Adenosine deaminase inhibition
and myocardial purine release during normoxia and ischaemia. Cardiovasc Res
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26. Edlund A, Fredholm BB, Patrignani P, Patrono C, Wennmalm A, Wennalm M
(1983) Release of two vasodilators, adenosine and prostacyclin, from isolated rabbit
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Pfliigers Arch 358:213-224
4
Energy Charge as a Cytosolic Signal for
Adenosine Release
Mark W. Gorman, Miao-Xiang He, and Harvey V. Sparks
Summary. Studies in isolated cells and with purified cytosolic 5 ' -nucleo-
tidase have suggested that adenosine formation may be regulated by the
cytosolic energy charge. We have used isolated guinea pig and rat hearts to test
this hypothesis measuring in vivo high energy phosphate concentrations with
31p_NMR. Several different interventions were used to lower energy charge
(and phosphorylation potential), including norepinephrine (NE) infusion,
hypoperfusion, hypoxia, and 2-deoxyglucose (2DG) infusion. Caffeine was also
used in order to elevate adenosine release during NE infusion. We tested for a
biphasic relationship between adenosine release and energy charge as had been
found in in vitro studies. We found such a relationship during hypoperfusion
and 2DG treatment, but not during hypoxia. During hypoperfusion and 2DG
treatment, adenosine release began to decline at energy charges much higher
than those observed in vitro. In addition, caffeine elevated adenosine release
more than predicted by the change in phosphorylation potential. We concluded
that regulation of cytosolic 5 ' -nucleotidase by energy charge or phosphoryla-
tion potential cannot explain adenosine formation in all circumstances. The
availability of free cytosolic AMP is also not a sufficient explanation. These
results further suggested that (1) different cytosolic 5' -nucleotidases account
for the differences between in vitro and in vivo adenosine formation during
decreases in energy charge, and (2) cardiac 5 ' -nucleotidase is inhibited during
severe hypoperfusion but not during severe hypoxia.
Key words: Adenosine-Energy charge-Phosphorylation potential-5' -

nucleotidase
Department of Physiology, Giltner Hall, Michigan State University, East Lansing, MI

48824, USA
147
148 M.W. Gorman et al.
Introduction
Adenosine is an important extracellular messenger in the heart. It causes
vasodilation, inhibits norepinephrine release from sympathetic nerves, and has
negative chronotropic and dromotropic effects as well as anti-~-adrenergic
effects [1-4]. ·The regulation of cardiac adenosine formation remains in-
completely understood. Recent evidence suggests that basal adenosine release
rates can be accounted for by the activity of S-adenosyl homocysteine hydro-
lase, but that elevated adenosine formation during periods of oxygen supply/
demand imbalance results from the action of cytosolic 5' -nucleotidase on
adenosine 5'-monophosphate (AMP) [5-7]. Intracellularly formed adenosine is
then transported into the extracellular fluid by a membrane nucleoside carrier.
What is the biochemical mechanism linking the oxygen supply/demand ratio
to adenosine formation? One candidate is the free intracellular AMP con-
centration. Free AMP concentration varies inversely with adenosine 5'-
triphosphate (ATP) concentration, so adenosine formation rate could be
regulated by substrate availability [8]. An additional possibility is that adeno-
sine formation is regulated by changes in 5' -nucleotidase activity. Several
different cytosolic 5' -nucleotidases have been isolated, as reviewed by
Skladanowski and Newby [9]. These can be classified into an inosine mono-
phosphate (IMP)-preferring form with a high Km for AMP, and an AMP-
preferring form with a low Km for AMP. Because the activity of both forms is
sensitive to adenine nucleotide concentrations, several studies have examined
adenosine formation as a function of the energy charge, ([ATP] + 1f2[ADP])/
([ATP] + [ADP] + [AMP]). Adenosine formation by polymorphonuclear
leukocytes showed a biphasic dependence on energy charge [10]. At high
energy charges, the formation of adenosine increased as the energy charge fell.
As the energy charge was further decreased, adenosine formation reached a
peak and then fell. A purified high Km 5' -nucleotidase from chicken and rat
hearts also displayed biphasic activity vs energy charge [11]. Rubio et al.
demonstrated that adenosine release from isolated guinea pig hearts correlated
inversely with energy charge (calculated from tissue concentrations) during
hypoxia [12]. Energy charge (or a related variable) might, therefore, regulate
adenosine formation in vivo.
Calculation of energy charge in the heart involves several assumptions. The
free intracellular concentration of A TP is roughly the same as the tissue
concentration in clamp-frozen hearts [13], but the free adenosine diphosphate
(ADP) and AMP concentrations are now known to be far lower than what is
calculated from their tissue contents, due to protein binding and subcellular
compartmentation [8,14]. Free concentrations of ADP and AMP must, there-
fore, be calculated from the creatine kinase and myokinase equilibrium con-
stants, respectively. These calculations also require knowledge of creatine
phosphate (PCr) concentration as well as the intracellular free [Mg+2] and pH.
The resulting ADP and AMP concentrations are so much lower than the ATP
concentration that, in cardiac muscle, energy charge deviates very little from
unity even during severe ischemia or hypoxia. A more useful index of cardiac
4. Energy Charge as a Cytosolic Signal for Adenosine Release 149
energy status in vivo is the adenine nucleotide phosphorylation potential,

[ATP]/[ADPHPd. This quantity changes much more than the energy charge
and requires fewer assumptions for its calculation. It also has an obvious
biochemical meaning because the logarithm of the phosphorylation potential
determines the free energy derived from the hydrolysis of A TP.
Several studies support an inverse correlation between the phosphorylation
potential and cardiac adenosine formation in vivo. Nuutinen et al. found such a
correlation between adenosine release in isolated rat hearts exposed to hypoxia,
amytal, and dinitrophenol [15,16). Kiviluoma et al. measured tissue adenosine
content and high energy phosphates in isolated rat hearts paced at different
rates [17]. They found that elevated heart rates increased MV0 2 , decreased
phosphorylation potential, and increased tissue adenosine content. An inverse
linear correlation between phosphorylation potential and adenosine release or
adenosine plus inosine release has been reported in rat hearts during hypoxia,
hypoperfusion, or isoproterenol infusion [18,19].
The purpose of the studies reported here was to more severely test the
hypothesis that phosphorylation potential or energy charge regulates adenosine
formation in vivo. If this hypothesis is true, then the time course of adenosine
formation should match the time course of changes in phosphorylation
potential. Furthermore, the studies on the high Km cytosolic 5' -nucleotidase
suggest that there should be a biphasic relationship between energy charge and
adenosine formation if this enzyme is responsible for adenosine formation in
vivo. As energy charge is progressively lowered, adenosine formation should
first increase and then decrease at a very low energy charge. In order to lower
the energy charge and phosphorylation potential, we used several interventions,
including norepinephrine infusion, hypoperfusion, and hypoxia. Cardiac
adenosine release was also modulated by caffeine treatment in order to deter-
mine whether changes in adenosine release would be accompanied by the
expected changes in phosphorylation potential.
We used an isolated guinea pig heart preparation modified slightly for use in a
31p_NMR magnet [14). The perfusate was a low phosphate Krebs-Henseleit
solution containing (mM) NaCl 127, KCI 5.8, KH2 P04 0.1, MgS04 1.1,
NaHC03 25, CaCh 2.5, glucose 5.5, and Na pyruvate 2.0. The perfusate was
equilibrated with 95% O 2 /5% CO 2 under all conditions except hypoxia, where
O 2 was replaced with N2 . A latex balloon was placed in the left ventricle for
measurement of left ventricular pressure (LVP) and (dP/dt). The pulmonary
artery was cannulated for collection of coronary venous effluent. Effluent
adenosine concentration was measured by (HPLC). Adenosine release was
calculated as the product of venous adenosine concentration and coronary
flow. Coronary flow was adjusted to yield a basal perfusion pressure of
46 mmHg (60 cm H2 0). Resting flows averaged 4-5 mllmin per g wet weight.
Phosphorous compound concentrations (ATP, PCr, Pi) were measured via

31p_NMR using a Bruker AM400 spectrometer (9.4 Tesla) and a 20mm broad-
band probe. Spectra were collected every minute using a 1 s interval between
pulses. The resulting free induction decays were multiplied by an exponential
corresponding to a 15 Hz line broadening before Fourier transformation. The
resulting peaks were integrated and multiplied by empirically determined
saturation factors in order to determine relative concentrations. In order to
transform these relative concentrations into absolute concentrations, the
average ~-ATP peak area under resting conditions in each heart was assumed
to represent the concentration determined chemically from clamp-frozen hearts
under identical conditions (7.6 mM for 1 kg guinea pigs, 10.6 mM for 500 g
guinea pigs, and 10.5 mM for 300 g rats). Intracellular [Mg+2] was calculated
from the chemical shift of the ~-ATP peak relative to the PCr peak [20], and
intracellular pH was calculated from the chemical shift of the Pi peak relative
to PCr [21]. Cytosolic free ADP concentration was calculated from the creatine
kinase reaction assuming equilibrium conditions, and cytosolic AMP con-
centration was similarly calculated from the myokinase reaction equilibrium:
[ADP] = ([ATP][Cr]Keq)/([PCr][H+])
Log ([H+]/Keq) = -0.87 pHi + 8.31
[AMP] = Kmyk[ADPJZ/[ATP]
where Cr is creatine and Keq and Kmyk are the equilibrium constants of
creatine kinase and myokinase, respectively. Kmyk was assumed to be 1.12 [22].
[Cr] was measured chemically in hearts clamp-frozen under control conditions
and other experimental conditions, and the resulting sum of [PCr] + [Cr]
(37 mM in both rats and guinea pigs) was found to be constant under all
conditions except severe hypoxia. [Cr] was measured chemically in frozen
hearts at the conclusion of the severe hypoxia protocol, and we assumed a
linear decline in total creatine content during the period of severe hypoxia.
Norepinephrine Series (n = 5)
In this series we examined the time course of adenosine release and phos-
phorylation potential during norepinephrine (NE) infusion (6 X 10- 8 M per-
fusate concentration). After control spectra were collected, NE was infused for
20 min while flow was maintained at the resting level.
Hypoperfusion Series (n = 8)
In this series we sought to lower phosphorylation potential and energy charge
by a combination of NE infusion and graded flow reductions. Following the
control period, NE infusion was begun and maintained for the duration of the
experiment. The flow was first held constant at the resting level, increased to
regain the control perfusion pressure, and then lowered in 3 steps correspond-
ing to mild, moderate, and severe hypoperfusion. During the severe hypo-
perfusion period the flow was 0.2 ± 0.03 mt/min per g. A reperfusion period at
the original control flow level followed. Each experimental period lasted 20
min, and data from the final 5 min of each period were used, representing
steady-state hemodynamics and adenosine releases.
Hypoxia Series (n = 6)
This series employed mild (30% O 2 ) and severe (0% O 2 ) hypoxia in combina-
tion with 6 X 10- 8 M NE infusion in order to lower phosphorylation potential
and increase adenosine release. Flow was held constant at the resting level
throughout the procedure. A control period was followed by an NE infusion
that continued during the rest of the experiment. Following 20 min of NE
infusion, mild and then severe hypoxia periods followed, each lasting 30min.
Steady-state data were taken during the final 5 min of the NE infusion and
control periods, and during the final 15 min of the hypoxic periods. In a
separate series of hearts, HCl infusion (9 mM) was superimposed during severe
hypoxia using the same protocol in order to create intracellular acidosis.
2-Deoxyglucose Series (n = 5)
High energy phosphates were depleted in this series by infusion of 2-deoxy-
glucose (2DG). Because guinea pig hearts were relatively insensitive to this
treatment, we used rat hearts in this series. NE was not infused. Following a
control period, the perfusate was switched to one containing 5 mM 2DG.
Glucose was removed from the perfusate but pyruvate remained. Four VII
insulin were present in both the control and 2DG perfusates. Perfusion with
2DG continued for 60min while 31p_NMR spectra and venous adenosine
samples were collected.
Caffeine Series (n = 5)
Caffeine greatly increases cardiac adenosine release during NE infusion [23].
This series examined the changes in phosphorylation potential that accompany
caffeine treatment. NE was first infused under control conditions for 10 min.
31p_NMR spectra and venous adenosine samples were collected every minute.
Following a 10 min recovery period, the perfusate was switched to one contain-
ing 0.2mM caffeine. Following 10 min of exposure to caffeine, the NE infusion
was repeated. Flow was varied in order to maintain a constant perfusion
pressure in this series, and no left ventricular balloon was present.
Results
Norepinephrine Series
Figure 1 shows the time course of adenosine release and phosphorylation
potential during NE infusion. A fall in phosphorylation potential preceeded the
3.5
....
Q.
.~ 2.5
300
Gl .....
eI) CI
III .... 200
! .~
a: ::::
g~ 100
-c:.e
o
r-"..
0 20
II---r
40 70
0• 2• 4• •
6• 8• 10 12
• 14 16 18 20
I I I I
~.I-
Control
NE Perfusion
.I-r'
Recovery
Time (min)
FiG. 1. The effect of norepinephrine (NE) infusion on phosphorylation potential (top)

and on adenosine release (bottom) in isolated guinea pig hearts perfused at constant
flow. Units of phosphorylation potential are mM- 1 • *P < 0.01 vs control. Time points
between points with asterisks are also P < 0.01 vs control. (From [14] with permission)
increase in adenosine release. As reported previously, adenosine release is

phasic despite constant NE concentration and steady-state flow and oxygen
consumption [24]. The decline in adenosine release after 7 min was accom-
panied by a slight return of phosphorylation potential towards control. When
adenosine release was plotted vs phosphorylation potential (Fig. 2) the rela-
tionship was hyperbolic.
The results of several other series of experiments have been combined in
Fig. 2. All interventions reduced phosphorylation potential and increased
adenosine release, as expected. During hypoperfusion and 2DG infusion the
2000
,--..". hypoxia
OJ '0 >-A NE+caffeine
j
"--... 1 T +HCI
c
1
E
"--...
0 1000
E
0... !Q
-......./
\J
0
0
0::::
hYPOP~.~~
'. NE b,.~ ,
0 -->-
3 4 5 6
LOG [ATP]/[ADP][P j ]
FIG. 2. Adenosine release as a function of phosphorylation potential during NE infusion
alone (ll), graded hypoperfusion + NE (.), graded hypoxia + NE (0), caffeine + NE
(.&), and HCI infusion during severe hypoxia + NE (D). All measurements were made
during steady-state hemodynamics and adenosine release 10- 20 min following each
intervention. Units of phosphorylation potential are M- 1 . As phosphorylation potential
declines adenosine release increases hyperbolically, except during hypoperfusion where
adenosine release declines at lower phosphorylation potentials. Caffeine in combination
with NE produces the highest adenosine releases for a given phosphorylation potential.
Reduction of intracellular pH by addition of HCl during severe hypoxia does not change
the relationship between adenosine release (Rado) and phosphorylation potential.
relationship between adenosine release and energy charge was biphasic (Fig. 3)
as predicted by studies of cytosolic 5' -nucleotidase in vitro. In these cases,
adenosine release declined from peak levels at very low phosphorylation
potentials. During graded hypoxia, however, adenosine release continued to
increase as phosphorylation potential fell. This occurred even though the
phosphorylation potentials reached during hypoxia were as low as those
achieved during severe hypoperfusion. During severe hypoxia, the intracellular
pH declined from a control level of 7.16 ± 0.01 to 6.99 ± 0.01, while during
severe hypoperfusion the corresponding decline was from 7.17 ± 0.01 to 6.63
± 0.05. HCl infusion during severe hypoxia reduced intracellular pH to
6.75 ± 0.02, but had no apparent effect on the relationship between adenosine
release and phosphorylation potential (Fig. 2).
In the 2DG series, rat hearts were used instead of guinea pig hearts. Because
adenosine release was much higher from the former, the 2DG series data were
plotted against a different scale in Fig. 3. Although it is difficult to compare
500
____ HVPOPERf'USION
400
0 300
~
<
~
200
100
0
0.98 0.99 1.00
ENERGY CHARGE
4000
-0- 2·00
3000
0
Q
<I( 2000
~
1000
O+-------~--------_r--------~------_,
0.ge 0.91 1.00
ENERGY CHARGE
FiG. 3. Adenosine release as a function of energy charge during graded hypoperfusion
in guinea pig hearts (top) and during 2-deoxyglucose infusion in rat hearts (bottom).
The units of adenosine release (Rado) are pmollmin per g wet weight. Adenosine
release was much higher in rat hearts and was plotted against a different scale. Both
species display a biphasic relationship between adenosine release and energy charge
under these conditions
absolute rates of adenosine release in this case, the biphasic nature of adenosine
release vs phosphorylation potential was similar to that in guinea pig hearts
during hypoperfusion.
Caffeine produced much higher adenosine releases during NE infusion, and
this was accompanied by a significant reduction in phosphorylation potential
compared to the control NE infusion (P < 0.05). When adenosine release was
plotted vs phosphorylation potential, however, caffeine appeared to alter the
relationship between adenosine formation and phosphorylation potential (Fig.
2).
Discussion
The objective of these studies was to test the hypothesis that cardiac adenosine
formation can be explained in terms of changes in energy charge or phos-
phorylation potential. Furthermore, we were interested in obtaining information
to aid in choosing between the two well-described cytosolic 5' -nucleotidases,
which will be referred to as the high and low Km forms of the enzyme. To
achieve these objectives, we (1) compared the time course of changes in
phosphorylation potential and adenosine release, (2) tested whether adenosine
release shows a biphasic pattern vs phosphorylation potential as indicated by
studies of purified 5' -nucleotidase, and (3) modulated adenosine release via
caffeine treatment and measured the associated changes in phosphorylation
potential.
The time course of adenosine release during catecholamine infusion is
known to be phasic in this preparation [24]. The changes in phosphorylation
potential observed during NE infusion (Fig. 1) are consistent with the hypo-
thesis that phosphorylation potential regulates adenosine formation. A fall in
phosphorylation potential precedes the increase in adenosine release, and the
decline in adenosine release after 7 min is accompanied by a slight rise in
phosphorylation potential. Although this rise in phosphorylation potential is
small, small changes in phosphorylation potential in this region are associated
with large changes in adenosine release (Fig. 2).
In order to determine whether there is a biphasic relationship between
adenosine release and phosphorylation potential, it was necessary to deplete
ATP stores. A biphasic pattern of adenosine release was observed during
hypoperfusion and 2DG treatment, but not during hypoxia (Figs. 2, 3). The
hypoxia results indicate that regulation of cytosolic 5'-nucleotidase activity by
phosphorylation potential or energy charge is not the sole controller of adeno-
sine formation in vivo. The caffeine experiments provide an additional argu-
ment against an exclusive role for energy charge or phosphorylation potential.
Although caffeine changed the phosphorylation potential in the expected
direction, the reduction in phosphorylation potential does not seem sufficient
to account for all the increase in adenosine release in this case. If it were
sufficient, then we would expect the caffeine point in Fig. 2 to fall on the same
line as the hypoxia and/or hypoperfusion data. Since it is significantly above
these lines, we conclude that caffeine elevates cardiac adenosine release by a
mechanism in addition to reduced phosphorylation potential.
Our data also allow us to test the hypothesis that adenosine formation is
regulated by the availability of free intracellular AMP. As indicated in Fig. 4,
NE alone, hypoxia, and hypoperfusion all increase free [AMP] as phosphoryl a-
'i!
·:1
1
,,--_::::::+-
/-"'-
....=
.......
E
Ii:
;:::;
0
Ii: +-----
,,
0 o.s ,,,
Q
,,
~
~----~----~----~~"~'I----~--~
o 2 4 6 10 20
AMP(UM)
FIG. 4. Adenosine release (Rado) as a function of free intracellular AMP concentration

during NE infusion alone (i',.), graded hypoperfusion + NE (e), and graded hypoxia +
NE (0) in isolated guinea pig hearts. Increasing [AMP] stimulates adenosine release
monotonically during hypoxia, but during severe hypoperfusion Rado falls despite
increasing [AMP]
tion potential decreases, yet adenosine release declines at high [AMP] during
hypoperfusion but not during hypoxia. We conclude from this that AMP
availability is not a sufficient explanation by itself for the rate of adenosine
formation. An examination of Fig. 4 suggests that the Km for AMP is in the
micro molar range. This observation argues against the high Km form of the
enzyme being responsible for adenosine formation.
Although phosphorylation potential was reduced considerably by the inter-
ventions in this study, changes in energy charge were much smaller. Typical
resting energy charges were 0.998, and the lowest energy charge achieved
(during severe hypoxia) was 0.973. Lower energy charges have been reported
in previous in vivo studies, but those calculations were based upon tissue
nucleotide contents rather than on the free intracellular concentrations. Peak
adenosine formation rates were achieved at energy charges of approximately
0.6 in the purified 5'-nucleotidase study [11] and at 0.8 in isolated polymor-
phonuclear leukocytes [10]. When we observed biphasic adenosine release
during hypoperfusion or 2DG infusion, peak adenosine release occurred at an
energy charge of about 0.996. This discrepancy between energy charges in vivo
and in vitro suggests that different forms of 5'-nucleotidase are responsible.
For example, it could be that the low Km 5'-nucleotidase dominates in vivo and
accounts for our results, and the biphasic relationship of adenosine release and
energy charge is correlated to the high Km enzyme [9,11].
Even though we could not lower energy charge to the levels achieved in the
in vitro studies, we nevertheless observed a biphasic pattern of adenosine
release under some conditions (hypoperfusion and 2DG). If the high Km
enzyme is not responsible for the biphasic relationship, what accounts for this
pattern? Figure 4 suggests that the active 5' -nucleotidase is inhibited during
severe hypoperfusion but not during severe hypoxia. The difference is not due
to differences in intracellular pH because HCI infusion during severe hypoxia
reduced pH without much effect on adenosine release. Elevated [Pd can be
eliminated because biphasic adenosine release was seen during 2DG treatment
despite low [PJ Furthermore, HCI infusion during severe hypoxia increased
[Pd without lowering adenosine release. These observations are consistent with
the weak effect of Pi on the low Km 5' -nucleotidase isolated from pigeon heart
[25]. Free intracellular [Mg+2], which stimulates 5'-nucleotidases, was approxi-
mately 1 mM and did not change enough to be a factor in any of these studies.
Inhibition of adenosine release at low phosphorylation potential is also not an
artifact of low flow in the hypoperfusion series, because inhibition occurred in
the 2DG series at constant flow. In summary, the mechanism of this apparent
inhibition remains unknown.
Throughout this discussion we have been using adenosine release as an index
of adenosine formation rate. The release rate is dependent upon the formation
rate and the activity of several degradative and uptake pathways. The failure of
hypoxia-induced adenosine release to decline at low phosphorylation potential
might, therefore, be due to inhibition of adenosine degradation or inhibition of
uptake and rephosphorylation. If adenosine degradation is inhibited by
hypoxia, we would expect to see a decrease in inosine release during hypoxia.
Instead, inosine release increased in parallel with adenosine (data not shown).
Reuptake and rephosphorylation of adenosine might become saturated during
hypoxia and thus lead to a dissociation between adenosine formation and
adenosine release. Although adenosine release is higher during hypoxia, the
venous adenosine concentration is actually higher during severe hypoperfusion
than during hypoxia. Since saturation of reuptake/rephosphorylation should be
a function of concentration, we conclude that this mechanism cannot explain
the different patterns of adenosine release during hypoperfusion and hypoxia.
Lloyd and Schrader [6] found an increase in adenosine release when adenosine
kinase was inhibited during hypoxia, which also suggests that adenosine kinase
is not saturated under these conditions.
A speculative hypothesis which could explain many of our results is that
adenosine inhibits its Own formation via a receptor-mediated mechanism.
According to this hypothesis, caffeine increases adenosine release by blocking
adenosine receptors, and severe hypoperfusion (but not hypoxia) produces
adenosine concentrations high enough to stimulate these receptors. If
adenosine 3' ,5' -cyclicphosphate (cAMP) is involved in stimulating adenosine
formation, these effects might be mediated by Al receptors. This hypothesis
remains to be tested.
References
1. Drury AN, Szent-Gyorgyi A (1929) The physiological activity of adenine
compounds. J Physiol (Lond) 68:213-237
2. Verhaeghe RH, Vanhoutte PM, Shepherd JT (1977) Inhibition of sympathetic
neurotransmission in canine blood vessels by adenosine and adenine nucleotides.
Circ Res 40:208-215
3. Belardinelli L, West A, Crampton R, Berne RM (1983) Chronotropic and
dromotropic effects of adenosine. In: Berne RM, Rail TW, Rubio R (eds)
Regulatory function of adenosine. Nijhoff. The Hague, pp 377-396
4. Dobson JG, Ordway RW, Fenton RA (1986) Endogenous adenosine inhibits
catecholamine contractile responses in normoxic hearts. Am J Physiol 251:H455-
H462
5. Schutz W, Schrader J, Gerlach E (1981) Different sites of adenosine formation in
the heart. Am J Physiol 240:H963- H970
6. Lloyd HGE, SChrader J (1987) The importance of the transmethylation pathway
for adenosine metabolism in the heart. In: Gerlach E, Becker BF (eds) Topics and
perspectives in adenosine research. Springer-Verlag, Berlin, pp 199-207
7. Newby AC, Worku Y, Meghji P (1987) Critical evaluation of the role of ecto- and
cytosolic 5' -nucleotidase in adenosine formation. In: Gerlach E, Becker BF (eds)
Topics and perspectives in adenosine research. Springer-Verlag, Berlin, pp 155-169
8. Bunger R, Soboll S (1986) Cytosolic adenylates and adenosine release in perfused
working heart. Eur J Biochem 159:203-213
9. Skladanowski AC, Newby AC (to be published) 5'-Nucleotidases involved in
adenosine formation. In: Proceedings of the 4th International Symposium on
Adenosine and Adenine Neucleotides. May, 1990, Japan
10. Worku Y, Newby AC (1983) The mechanism of adenosine production in rat
11. Itoh R, Ozasa H (1986) Regulation of rat heart cytosol 5 ' -nucleotidase by adenylate
energy charge. Biochem J 235:847-851
12. Rubio R, Berne RM, Dobson JG (1973) Sites of adenosine production in cardiac
and skeletal muscle. Am J Physiol 225:938-953
13. Dawson MJ, Gadian DG, Wilkie DR (1977) Contraction and recovery of living
muscles studied by 31P nuclear magnetic resonance. J Physiol (Lond) 267:703-735
14. He MX, Wangler RD, Dillon PF, Romig GD, Sparks HV (1987) Phosphorylation
potential and adenosine release during norepinephrine infusion in guinea pig heart.
Am J Physiol 253:H1184- H1191
15. Nuutinen EM, Nishiki K, Erecinski M, Wilson DF (1982) Role of mitochondrial
oxidative phosphorylation in regulation of coronary blood flow. Am J Physiol
243:HI59- H169
16. Nuutinen EM, Nelson D, Wilson DF, Erecinska M (1983) Regulation of coronary
blood flow: Effects of 2,4-dinitrophenol and theophylline. Am J Physiol 244:H396-
H405
17. Kiviluoma KR, Peuhkurinen KJ, Hassinen IE (1986) Role of cellular energy state
and adenosine in the regulation of coronary flow during variation in contraction
frequency in an isolated perfused heart. J Mol Cell CardioI18:1133-1142
18. Headrick JP, Willis RJ (1990) Adenosine formation and energy metabolism: A
31P-NMR study in isolated rat heart. Am J Physiol 258:H617-H624
19. Headrick J, Clarke K, Willis RJ (1989) Adenosine production and energy
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21:1089-1100
20. Kushmerick MJ, Dillon PF, Meyer RA, Brown TR, Krisanda JM, Sweeney HL
(1986) 31P-NMR spectroscopy, chemical analysis, and free Mg+2 of rabbit bladder
and uterine smooth muscle. J BioI Chern 261:14420-14429
21. Petroff OAC, Prichard JW, Behar KL, Alger JR, den Hollander JA, Shulman RG
(1985) Cerebral intracellular pH by 31P nuclear magnetic resonance spectroscopy.
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22. Veech RL, Lawson JWR, Cornell NW, Krebs HA (1979) Cytosolic
phosphorylation potential. J BioI Chern 254:6538-6547
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without a change in the 02 supply/demand ratio (abstract). FASEB J 2:Al714
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adenosine during steady state metabolic stimulation in the isolated guinea pig heart.
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J PhysioI260:H917-H926
5
The Role of Adenosine on Myocardial
Reactive Hyperemia
Daiji Saito!, Tsutomu Mima 2, Kazuyoshi Hina 2, Shinji Uchida 2,
Naotsugu OhbayashP, Morio MarutanP, and Shoichi Haraoka 1
Summary. The present study was conducted to test the hypothesis that
activation of adenylate cyclase participates in myocardial reactive hyperemia by
release of adenosine during brief coronary occlusions using forskolin and
8-phenyltheophylline (8-PT). We also examined the possible contribution of
membrane-bound 5'-nucleotidase to the synthetizing of adenosine. Intra-
coronary infusion of a, p-methylene adenosine 5-diphosphate (AOPCP) was
used for this purpose. Forskolin increased flow debt repayments by about 25%
following 15-, 20- and 30-s coronary occlusions, but with 8-PT, forskolin-induced
increments in the flow debt repayments diminished significantly (P < 0.05).
Further, AOPCP infusion attenuates flow debt repayment by about 30%
following coronary occlusions of 15 s or longer (P < 0.05). Neither forskolin,
8-PT, nor AOPCP affected blood pressure, heart rate, or myocardial oxygen
consumption. These results suggest that adenosine is involved in myocardial
reactive hyperemia through an activation of adenyl ate cyclase, and that ecto-5'-
nucleotidase participates in producing adenosine of the interstitial myocardial
space.
Key words: Adenosine-Ecto-5' -nucleotidase-Reactive hyperemia-Forskolin
Introduction
Many lines of evidence [1,2] suggest that adenosine partly regulates coronary
vascular tone during myocardial ischemia via stimulation of A2 receptors,
which activates the adenylate cyclase system. Therefore, it is predictable that
ecto-5'-nucleotidase plays a role in controlling adenosine concentration of the
I The Department of Laboratory Medicine and

2The First Department of Internal Medicine, Okayama University Medical School,
Okayama, 700 Japan
160
5. The Role of Adenosine on Myocardial Reactive Hyperemia 161
interstitial space, and that adenyl ate cyclase is involved in myocardial reactive
hyperemia. It is generally accepted that forskolin is a potent activator of
adenylate cyclase through a mechanism not associated with receptor interaction
[3], and that a, ~-methylene adenosine 5-diphosphate (AOPCP) inhibits
ecto-5' -nucleotidase and decreases adenosine formation [4]. Thus, the first
purpose of the present study was to test the hypothesis that adenosine
participates in myocardial reactive hyperemia by activating adenylate cyclase
system. We also examined the possible involvement of ecto-5' -nucleotidase in
regulating adenosine concentration of the interstitial myocardial space.
Methods
Surgical Preparation
Healthy mongrel dogs, weighing 12-15 kg, were sedated with ketamine
hydrochloride, anesthetized with intravenous injection of pentobarbital
sodium, and artificially ventilated on a positive pressure respirator with a
mixture of room air and 100% oxygen in order to maintain arterial blood gases
within the physiological range. Thoracotomy through the left 5th intercostal
space exposed the heart for the implantation of an electromagnetic flow probe
and a plastic occlusive snare near the origin of the left anterior descending
coronary artery (LAD). A short polyethylene catheter was inserted
transepicardially into the coronary artery distal to the snare, and a Y-connector
linked this coronary catheter to two infusion syringes, permitting simultaneous
administration of two solutions at different rates. The blood pressure at the
aortic root and the left ventricular pressure were continuously monitored with
the coronary blood flow. A catheter in the great cardiac vein via the right atrial
appendage permitted sampling of the venous drainage of perfusion field under
study. After completion of the experiments, the animal was sacrificed with an
intracardiac injection of saturated KCI solution, and an intracoronary injection
of 0.5% Evans blue dye solution was given to delineate the myocardium under
study. The dye-stained myocardium was then excised and weighed for
normalizing coronary blood flow.
Experimental Protocol
The present study consisted of 3 series of experiments. The first experimental
series was designed to investigate the influence of forskolin upon reactive
hyperemia in response to coronary occlusions lasting for 5, 10, 15,20, and 30s.
Ten dogs were used for this study. For comparing coronary blood flow
response to each coronary occlusion, we calculated repayment of flow debt and
peak reactive hyperemic flow rate according to Coffman and Gregg [5]. The
order of occlusions was independently randomized, and each occlusion was
done with an interval of at least 1 min after the coronary flow returned to the
baseline level. The protocol of the first series of the experiment is illustrated in
162 D. Saito et al.
Experiment 1
Experiment 2
Experiment 3
..
RH
Saline
15min
-1
AOPCP
.. 15
H ..
Saline
FIG. 1. Experimental protocol. The inverted triangles represent the points for
observation of reactive hyperemia. FSK, Forskolin; 8-PT, 8-phenyltheophylline;
AOPCP, a, ~ -methylene adenosine 5-phosphate
Fig. 1. The first set of the experiment began with infusion of 5% ethanol
solution, the solvent of forskolin, for estimating the control coronary response.
The second set of observations was performed during forskolin infusion, and
the last set was started approximately 20 min after discontinuing the forskolin
infusion, during which the control solvent was administereQ to serve as the
post-control. The infusion was stopped during coronary occlusion in order to
avoid the accumulation of the infusate during the occlusions.
In the second experimental series, effects of 8-phenyltheophylline (8-PT) on
forskolin-induced changes in hyperemic response were investigated in 7 dogs.
This series of experiments consisted of six sets of observations (Fig. 1). The
first three sets were performed in the same way as the first experimental series.
After the third set was completed, intracoronary infusion of 8-PT at the
calculated coronary plasma concentration of 10 11M was started. The fourth set
of reactive hyperemia observations was then conducted 5 min after the start of
8-PT infusion. Subsequently, forskolin infusion was added at the same rate as
the second set with another syringe. Fifteen minutes later, during simultaneous
infusion of 8-PT and forskolin, the fifth set of reactive hyperemia was
conducted. Finally, the last set of reactive hyperemia response was observed
20 min after termination of the forskolin infusion, during which 8-PT alone was
continuously administered.
The last series of experiments was designed to investigate the influence of
AOPCP on reactive hyperemia in 8 dogs. At first, coronary occlusions were
started with infusion of physiological saline into the LAD. This served as the
control. Then, the second set of observations was conducted during
intracoronary infusion of AOPCP at a rate of a maximum dose which did not
TABLE 1. Hemodynamic effects of forskolin, 8-phenyltheophylline and AOPCP

BP HR CBF MV0 2
(mmHg) (beats/min) (mllmin per 100 g) (mllmin per 100 g)
Control 106 ± 7 151 ± 7 105 ± 10 11.0 ± 0.6
Forskolin 108 ± 6 150 ± 6 123 ± 12* 11.1 ± 0.7
Control 100 ± 6 141 ± 7 92 ± 10 11.2 ± 0.9
8-PT 104 ± 6 135 ± 6 84 ± 12* 11.1 ± 1.0
Control 128 ± 6 140 ± 6 101 ± 9 9.6 ± 0.5
AOPCP 122 ± 6 138 ± 5 110 ± 12 9.2 ± 0.6
'Significantly different from control, P < 0.05

BP, Blood pressure; HR, heart rate; CBF, coronary blood flow; MV0 2 , myocardial oxygen
consumption; 8-PT, 8-phenyltheophylline; AOPCP, a, ~ methylene adenosine 5-diphosphate
The data are expressed as mean ±standard error of the mean (SEM)
significantly affect the coronary blood flow rate. This set of occlusions was
begun 10 min after the start of AOPCP infusion. Finally, AOPCP was changed
to physiological saline, and the observations were repeated (Fig. 1).
In each experiment, arterial and coronary sinus bloods were sampled at a
steady state in each set in order to measure myocardial oxygen consumption.
An experiment was included in the data analysis if the control observations
met the following criteria: aortic blood pressure was over 80 mmHg, peak
reactive hyperemic flow rate following 15-s occlusion was more than 300%
above the baseline flow rate, and arterial oxyhemoglobin saturation was above
90%. Statistical analysis within groups was made by analysis of variance and
Bonferroni's (-test. Student's t-test for paired observations was used only to
assess the difference of % of increase of flow debt repayment between 8-PT
and forskolin infusion and simultaneous infusion of the two agents.
Results
Arterial P02 averaged 98 ± 2.1 mmHg, PC0 2 39 ± 0.7 mmHg, and pH 7.39 ±
0.02; none of these variables changed significantly during the course of the
experiment. Hemodynamic characteristics of three experimental series are
summarized in Table 1. Forskolin, 8-PT, and AOPCP did not change arterial
blood pressure, heart rate, or MV0 2 which remained stable during the course
of the experiment.
Effect of forskolin on reactive hyperemic responses of 10 dogs are
summarized in Table 2. Forskolin potentiated repayments of flow debt
significantly, compared with pre- and post-control responses, by 28%, 25%,
and 27% following coronary occlusions of 15, 20, and 30s, respectively.
Shorter occlusions did not provide significant changes in repayments of flow
debt. The peak reactive flow rate was not affected by forskolin after any
durations of coronary occlusions.
164 D. Saito et al.
TABLE 2. Hemodynamic effects of forskolin on myocardial reactive hyperemia

Duration Repayment of flow debt (%) Peak hyperemic flow rate (%)
(s)
Control Forskolin Post-control Control Forskolin Post-control
5 225 ± 24 237 ± 29 224 ± 31 245 ± 19 218 ± 20 237 ± 19
10 303 ± 49 318 ± 38 284 ± 40 360 ± 37 328 ± 33 348 ± 30
15 303 ± 35 400±41" 337 ± 33 393 ± 41 360 ± 33 402 ± 34
20 324 ± 41 405 ± 43" 322± 31 423 ± 45 388 ± 39 410 ± 35
30 318 ± 39 403 ± 39' 321 ± 40 462 ± 53 431 ± 45 451 ± 41
"Significantly different from control, and post control, P < 0.05

Duration, Occlusion duration
The data are expressed as mean ±standard error of the mean (SEM)
In the second experiment, blood pressure, heart rate, LVdP/dt, and MV02
were almost stable. Coronary blood flow increased after forskolin infusion, as
it did in the first experiment. 8-PT decreased the basal coronary flow, but when
forskolin was added, the basal flow again increased by about 18%. Results of
flow debt repayment and peak reactive hyperemic flow rate are shown in Fig.
2. Using forskolin alone, the results are essentially the same as those produced
in the first experiment. 8-PT infusion significantly reduced repayments of flow
debt following coronary occlusions of 15,20, and 30s by about 27%, compared
with those before 8-PT infusion. Peak reactive hyperemic flow rate was not
affected by combined infusion of forskolin and 8-PT.
* P<005
I*I I
15 sec.
l- I
NS
Ii
NS
I
l- I II :I: I
I I I
I
20 sec. I * I
l- T NS
Ii
NS
I
I I
l- 1
I
* II * I
I ! !
:I:
...
30see. I I
NS NS
L~ !
I II i
~
* I I ! I
Control FSK Post- 8-PT IWT 8-PT
con +FSK
FiG. 2. Effects of forskolin and 8-phenyltheophylline on flow debt repayment. Intra-
coronary forskolin enhances reactive hyperemic responses and 8-phenyltheophylline
diminished the forskolin-induced potentiation of reactive hyperemia. FSK, Forskolin;
8-PT, 8-phenyltheophylline; NS, not significant
20 sec
100
50 Con
.~~
0
~~'~III . til
c: 11 N4t11N"~rlt'''H{. .~ J:
E E
""- 0 E
E
i 50 ~~1 REPAYMENT 237 %
100
..
GI
:::I
..
II)
0 II)
u.. GI
0
'C 14
'\1/1 Q.
0 'C
.5! 0
co .5!
.
0 co
>-
CG 100
c:
...
0 50 Post-<:on
0
REPAYMENT 297 %
u ~I& 0
12 \\.,..
0
FIG. 3. Effects of AOPCP on reactive hyperemia following 20-s coronary occlusions.

The three panels show (from top to bottom) hyperemic responses before, during, and
20 min after termination of AOPCP infusion. Numbers refer to control and peak
hyperemic flow rates (mllmin). AOPCP, a, ~ -Methylene adenosine 5-phosphate
AOPCP in the coronary plasma concentration of 50 11M did not affect

coronary blood flow. Figure 3 shows a typical record of reactive hyperemia
following a 20-s coronary occlusion. Coronary blood flow increased slightly
after AOPCP started. The repayment of flow debt decreased from 328% to
237%, while peak hyperemic flow rate was not changed by AOPCP. After
termination of AOPCP infusion, the hyperemic response tended to return to
the control level. The effects of AOPCP on reactive hyperemia obtained from
8 dogs are summarized in Fig. 4. AOPCP attenuated repayment of flow debt
by 25% - 30% in response to coronary occlusion lasting for 15 s or longer.
Shorter occlusions did not change the repayment. Peak reactive hyperemic flow
rate was not affected by AOPCP infusion. The reactive hyperemia returned to
the control level after termination of AOPCP infusion.
Discussion
The present study demonstrated that forskolin potentiated and AOPCP
attenuated myocardial reactive hyperemia following brief coronary occlusions,
and that the forskolin-induced increase in hyperemic response was diminished
by 8-PT infusion.
166 D. Saito et al.
-+- saline
~ AOPCP
VS. saline
**p<O.Ol
* p<O.05
o
, , , 30 sec
5 10 15 20
Occlusion Time
FiG. 4. Effects of AOPCP on flow debt repayment and peak reactive hyperemia. Closed
circles, Control coronary responses during saline infusion; open circles, coronary
responses during AOPCP infusion; AOPCP, a, ~ -methylene adenosine 5-phosphate
Out of many metabolic factors which are proposed as regulating coronary

blood flow, adenosine is the most likely candidate to link the myocardial
metabolic state and the coronary vascular tone. The factors which promote
myocardial oxygen usage, such as LVdP/dt, heart rate, and blood pressure,
modify the volume of reactive hyperemic flow. Furthermore, coronary artery
pressure had a substantial influence on the characteristics of myocardial
reactive hyperemia after a brief coronary occlusion [6]. Therefore, when
observing the effect of an intervention on reactive hyperemia, it is important
that hemodynamic and metabolic conditions should remain stable, especially
coronary perfusion pressure and MV02 • In the present study, blood pressure
(i.e., coronary perfusion pressure), heart rate, LVdP/dt, and MV0 2 remained
constant throughout the experiments, so that these factors did not affect the
results.
Forskolin is widely accepted as a potent direct activator of adenylate cyclase
through a mechanism not associated with receptor interaction and, in low
concentrations, it acts synergistically to enhance receptor-mediated activation
of this enzyme [3].
It is known that the concentration of forskolin which decreased coronary
vascular resistance was 7- to 20-fold less than the concentration for positive
chronotropy and for positive inotropy in isolated guinea pig heart [7]. In a
lower dose, forskolin dilates directly, not through an indirect metabolic effect
secondary to increased myocardial oxygen demand. We observed that the
coronary plasma concentration of forskolin used in the present study (0.16-
0.34I1M) caused a slight increase in coronary blood flow in the absence of
changes in LVdP/dt, heart rate, blood pressure, and MV0 2 . This result is
consistent with the findings of Kusachi et al. [8], who used coronary plasma
concentrations between 0.16 and 0.48 11M of forskolin in the anesthetized dog.
It is clear that forskolin elicits physiological responses which have been shown
to be cyclic AMP dependent [9]. Vegesna and Diamond [10] indicated an
increased cAMP level with 0.1 11M of forskolin in the bovine coronary artery by
approximately 5.5-fold. Thus, we believe that the forskolin doses we employed
here were enough to increase the cAMP level in the coronary artery and
potentiated the effects of agonists which exert receptor-mediated stimulation of
adenylate cyclase. The present results, which demonstrated a potentiative
effect of forskolin on reactive hyperemic response, suggest that forskolin
directly enhanced receptor-mediated activation of adenyl ate cyclase. They also
showed that this enzyme contributes to myocardial reactive hyperemia,
because, if other substances which mediate coronary vascular tone through
receptor-mediated stimulation of adenylate cyclase during brief myocardial
ischemias did not appear at the time of reactive hyperemia, flow debt
repayments would not be potentiated.
The dose of 8-PT used here (plasma concentration 10 11M) did not affect
systemic hemodynamics or MV0 2 . Further more, the rate of forskolin- induced
increase in basal flow was 16%-18% regardless of 8-PT infusion, so that 8-PT
did not influence the coronary vasodilative effect of forskolin. In the presence
of 8-PT, the forskolin-induced increment in flow debt repayment diminished
significantly following 15-, 20- and 30-s occlusions. This result indicates that
adenosine is a major metabolic factor related to reactive hyperemia through
activation of adenylate cyclase.
In the last part of the present experiment, AOPCP blunted the reactive
hyperemic response following the coronary occlusions of longer than 10 s.
Schutz et al. [11] found that an intracoronary infusion of 50 11M AOPCP
reversibly inhibited dephosphorylation of AMP by 85% in the isolated
perfused guinea pig heart. However, they also observed that this dose of
AOPCP failed to attenuate the hypoxia-induced release of adenosine to
coronary effluent. Little contribution of ecto-5' -nucleotidase on adenosine
formation was also reported by Newby and Holmquist [12] in the intact
polymorphonuclear leukocytes in which the enzyme activity was inhibited by
coformycin. Contrarily, Imai et al. [13] reported contrasting data that, in the
isolated perfused guinea pig heart, reactive hyperemia was reduced under the
influence of AOPCP in association with the decrease in the amount of
adenosine emerging in the tissue fluid collected by the method of Deckere and
Ten Hoor, while the amount of adenosine in the coronary effluent remained
essentially the same with or without AOPCP. These results indicate that the
dose of AOPCP used in their experiments could inhibit ecto-5' -nucleotidase
168 D. Saito et al.
effectively and would attenuate myocardial reactive hyperemia, and also

suggest that the amount of adenosine released in coronary effluent was not a
satisfactory indicator of tissue adenosine concentration. Our findings on
AOPCP were consistent with Imai et al.'s results and suggested that ecto-5'-
nucleotidase is involved in adenosine production even by the in situ dog heart,
and that interstitial myocardial adenosine participates in reactive hyperemia
through regulating coronary adenylate cyclase system.
References
1. Saito D, Steihart CR, Nixon DG, Olsson RA (1981) Intracoronary adenosine
deaminase reduces canine myocardial reactive hyperemia. Am J Physiol 49:1262-
1267
2. Saito D, Hydo T, Takeda K, Abe Y, Tani H, Yamada N, Ueeda M, Nakatsu T
(1985) Intracoronary adenosine enhances myocardial reactive hyperemia after brief
coronary occlusion. Am J Physiol 248:H812- H817
3. Seamon KB, Daly JW (1981) Forskolin: A unique diterpene activator of cyclic
AMP-generating systems. J Cyclic Nucleotide Res 7:201-224
4. Olsson RA, Gentry MK, Townsend RS (1973) Adenosine metabolism: Properties
of dog heart microsomal 5'-nucleotidase. In: Current topics in coronary research.
Plenum, New York, pp 27-39
5. Coffman JD, Gregg DE (1960) Reactive hyperemia characteristics of the
myocardium. Am J Physiol 199:1143-1149
6. Dole WP, Montville WJ, Bishop VS (1981) Dependency of myocardial reactive
hyperemia on coronary artery pressure in the dog. Am J Physiol 240:H709- H715
7. Lindner E, Dohadwalla AN, Bhattacharya BK (1978) Positive inotropic and blood
pressure lowering activity of a diterpene derivative isolated from Coleus forskholi,
Forskolin. Arzneim-ittelforschung 28:284-289
8. Kusachi S, Bungi WJ, Olsson RA (1984) Forskolin potentiates the coronary
vasoactivity of adenosine in the open-chest dog. Circ Res 55:116-119
9. Ammon HPT, Muller AB (1985) Forskolin: From an ayurvedic remedy to a
modem agent. Plant a Med 6:473-477
10. Vegesna RVK, Diamond J (1983) Comparison of the effects of forskolin and
isoproterenol on cyclic AMP levels and tension in bovine coronary artery. Can J
Physiol Pharmacol 61: 1202-1205
11. Schutz W, Schrader J, Gerlach E (1981) Different sites of adenosine formation. Am
J Physiol 240:H963- H970
12. Newby AC, Holmquist CA (1981) Adenosine production inside rat
13. Imai S, Imai H, Jin H (1986) Myocardial tissue fluid adenosine and the reactive
hyperemic responses. Pflugers Arch 407 (Suppl 1):S17
6
Inhibition of PMN and Platelets in
the Coronary System by
Endothelium-Derived Adenosine,
PGE 1 and PGE 2
Stephan Nees and Andreas Dendorfer 1
Summary. With the aid of a novel blood filtration system into which
microbe ads coated with confluent endothelial cells can be incorporated,
cellular interactions can now be investigated in streaming whole blood in vitro
under conditions largely approximating those in vivo. Reductions in filtration
rate are elicited by f-MLP, ADP, and PAF in a concentration-dependent
manner, due to the activation and aggregation of neutrophils (PMN) and
platelets. These effects can be antagonized by exogenously applied adenosine,
PGE 1 and PGE2 in physiological concentrations. Cultured coronary endothelial
cells also inhibit the actions of the above-listed mediators of inflammation on
these blood cells, presumably by releasing the corresponding endogenously
produced adenosine and prostaglandins into the streaming blood. In the
physiological state, this inhibitory effect of the endothelium probably sup-
presses the manifestation of inflammatory processes in the intact parts of the
coronary system downstream of the inflammatory site.
Key words: Polymorphonuclear leukocytes-Platelets-Endothelial cells-
Adenosine-Prostaglandins PGE 1 and PGE2
Introduction
Recently, the tonicity of the myocardial resistance vessels, in connection with
the regulation of coronary blood flow, has been particularly at the focus of
interest. It is now well known that the tone of the corresponding smooth
muscle cells is subjected to mechanical, humoral, neural, and metabolical
factors [1]. A variety of factors are discussed as possible mediators of the
metabolical control of coronary flow [2]. Several contributions in this volume
address the question as to the importance of the role played by certain adenine
1 Department of Physiology, University of Munich, D-8000 Munich 2, Germany
169
170 s. Nees and A. Dendorfer
nucleotides or adenosine in such extraluminal control processes of the
myocardial blood supply. They may be produced and released from the
cardiomyocytes [3], from sympathetic nerve endings [4,5], from the vascular
endothelium [6,7], or from smooth muscle cells of the resistance vessels them-
selves [8].
But the perfusion of the myocardium in vivo also depends quite decisively on
the rheological properties of the blood itself, which are now known not to be
dependent solely upon the passive deformability of the red blood cells, as was
almost exclusively thought to be the case only a few years ago. Rather, the
rheological behavior of whole blood, in particular during its passage through
the microcirculation, also depends upon interactions of the platelets, PMN
(polymorphonuclear leukocytes), and the coagulation factors with the vascular
endothelium [9-12]. Apparently, these complex intraluminal processes are
under the influence of numerous activating and inhibiting substances [13,14],
the synthesis and release of which can be understood as a more and more
recognize able and fascinating level of intraluminal control of the blood supply
of all organs, including, of course, the heart.
H is difficult, if not impossible, to investigate the details of these molecular
control processes in the streaming blood in vivo, and to clarify their
biochemistry. For this reason, the findings thus far established in this area-
almost exclusively in intact microcirculation systems and beating hearts
[15,16]-have, unfortunately, only a qualitative character in this respect.
For corresponding quantitative studies, in contrast, an appropriate in vitro
system is required, in which whole blood or purified blood fractions can be
investigated in the presence or absence of endothelial cells, which
physiologically form the anti thrombogenic and antiinflammatory container of
the blood.
During recent years we have developed such a system, which is currently in
routine use in our laboratory. We now report on how several typical mediators
of acute inflammatory processes affect the rheological characteristics of flowing
whole blood, and how the effects of these substances can be effectively blocked,
interestingly enough, by typical prostanoids and purines that are constantly
being synthesized and released into the coronary circulation by its
endothelium.

Venous blood was freshly drawn from healthy donors, anticoagulated with
citrate, and stored in stoppered polyethylene tubes at 37°C. For the filtration
experiments at 37°C, one part of blood was diluted to a hematocrit of 6% with
a physiologically balanced salt solution (PBS), and adjusted to a final
concentration of free Ca2 + of 1,3mM. The filtration system itself, (developed
in cooperation with Gebr. Bosch, Jungingen, Germany) which enables a
quantitative assessment of cellular interactions in blood samples, is schemati-
cally outlined in Fig. 1. The cylindrical filtration chamber, equipped with an
6. Inhibition of PMN and Platelets in the Coronary System 171
electronically
regulated gas supply
pressure
device
pressured ai r
containing 5 % CO 2
gas outlet (p=12cm HP)
1,5 cylindrica l
diluted chamber
blood
suspended
' :T + - - microbeads
.. ,. ." . . . with
sl irrer - - - . l . -. L
confluent
endothelial
celis
nylon net
filter
back and fort h

movement
fi I t rate detector plotter
FIG. 1. Layout of the filtration system
axial stirrer, undergoes a continuous gentle back-and-forth movement around

its axis to prevent sedimentation of the blood cells. All parts of the chamber
consist of biologically inert materials. The bottom is a polycarbonate filter
(Nuc\eopore, Pleasanton, Calf.) with straight pores 51lm in diameter at a
density of 4000/cm2 . By means of an electronically controlled pressure regulator
and a feeder tube, the blood sample can be constantly equilibrated with
carbogen gas (95% O 2 , 5% CO 2 ) and the inside of the cylinder placed under
constant, low pressures at any desired level within 1-20 cm H 2 0. The filtration
rate established under a given condition is monitored with an electronic high
precision weighing scale (Bosch, Jungingen), the data being continuously
assimilated by a computer (Atari ST), differentiated , and plotted
logarythmically on the attached printer (NEC P6). Figure 2 illustrates a typical
set of curves, such as those attained for the filtration of blood samples in
the presence of various concentrations of the mediator of inflammation,
(f-MLP).
172 S. Nees and A. Dendorfer
••. 5
.......
.-.
(J
GI unstimulated
'"
;:;:r .4.0
----
........
5
~ .3.5 ··1 B-a/b Computer assisted analysis oC filtra·
..
GI
oW
CIS tion curves: definition oC set points:
C .3.0
.S: Start: at 90% of maximum flow
e
oW
rate (latest 10 sec after onset) .

.:: .2.5
fi: End: at 50% of maximum flow
• 2.0 rate (latest 50 sec after onset) .
Duration: minimum of 3 sec.

• 1.5
1
Filtration time [sec]
FIG. 2. Principle of the quantjtative evaluation of the filtration data. A few s after the
onset of filtration, the process is characterized by a certain phase in which a practically
constant fraction of the filter pores is being plugged by PMN and/or platelets per unit of
time. The duration of this phase depends primarily upon the rapidity of activation of
these cells, and the corresponding flow F through the filter can be described by the
relationship F = a x e- pt (a is a material constant of the filter; ~ is the plugging rate,
which is largely determined by the properties of the cell type mainly obstructing the
filter under the given conditions; t is the filtration time). Presented here schematically, ~
is derived from the decline of the logarythmically plotted curve of filtration rate vs.
time, the exact value being determined by the slope of the tangent drawn to the curve in
the described phase of filtration W = a/b). With the aid of a specially developed
computer program, the ~- values are automatically calculated and printed
To obtain pure red blood cell (RBC) suspensions, citrated whole blood had
to be freed of platelets and leukocytes. This was accomplished by running the
blood samples through microcrystalline cellulose, as described by Beutler et al.
[17]. Platelet rich plasma was obtained by centrifugation of citrated blood
(10 min 1500 rpm, Labofuge-Christ) in 15 ml Falcon tubes. Highly purified
PMNs were obtained by Percoll-density gradient centrifugation and repeated
washings in PBS. In order to be able to perform activation experiments in
these suspensions of platelets and PMNs under conditions imitating, as closely
as possible, ~hose in whole blood in PBS, they were supplemented with the
appropriate number of purified RBCs in Ca-containing plasma.
The influence of coronary endothelial cells on blood cell interactions was
investigated in special experiments, in which microbeads (Cytodex I,
Pharmacia, Uppsala) coated with confluent endothelial cells were added to the
blood samples. The isolation and cultivation of these cells, derived from guinea
pig hearts, have previously been described in detail [18].
-0.3
-0.1
10 9 8 7 6 -log [MJ
FiG. 3. Dose effect curves of (A) f-MLP, (8) PAF, (C) ADP on the plugging rate ~
during filtration of 1:5 diluted whole blood in the presence of physiological calcium
concentration
Results
As shown in Fig. 3, a concentration-dependent increase in the plugging rate B
(for definition see Fig. 2) of the filters ensued, when whole blood was filtrated
in the presence of f-MLP, adenosine 5'-diphosphate (ADP), and platelet
activating factor (PAF). Endotoxin from E.coli, (not shown in Fig. 2) on the
other hand, had no acute effect under these conditions. Comparative investi-
gations of the filters were performed by light and electron microscopy after
filtering whole blood, pure RBC suspensions, and mixed suspensions of PMNs
or platelets with RBCs, respectively, all in the presence of f-MLP. These
studies revealed that within the first 30 s of filtration, a selective activation of
the PMNs is incurred by f-MLP, which becomes especially manifested upon
contact of the PMNs with the filter, where they adhere in the vicinity of the
pores (Fig. 4a). Thereafter, an increasing interaction of the activated PMNs
with the platelets was noted, and, after about 2 min, the formation of fibrin
commenced. Finally, the pores of the filter became totally plugged by the
corresponding microthrombi.
ADP proved to be a specific stimulant of the platelets. Their primary
aggregation around the pore openings (Fig. 4b), however, led to a secondary
activation of more and more PMNs. In contrast, PAF stimulated PMNs and
platelets in whole blood simultaneously (Fig. 4c).
Of particular interest was the fact that all of these mediator-induced
interactions between PMN and platelets could be effectively inhibited by
supplementing whole blood with prostaglandin E, (PGE,) in increasing
concentrations. Figure 5 illustrates the corresponding decreases in the plugging
rate Binduced by this exogenously applied substance in whole blood containing
3 nM f-MLP. Further studies (not shown in Fig. 5) revealed that prostaglandin
E2 (PGE 2) was slightly less effective than PGE,. In contrast, the stable
a
174
B
-0.2
-0..1
9 8 7 6 5 4 3 -log [MJ
FiG. 5. Influence of (A) PGE j , (8) adenosine, (C) theophylline, and (D) SIN-Ion the
plugging rate ~ during filtration of whole blood supplemented with 3 nM f-MLP
derivative of prostacyclin PGI 2 , iloprost (purchased from Schering AG,

Berlin), selectively inhibited only the platelets.
In addition, adenosine proved to be a potent inhibitor of PMN and platelet
interactions already at physiological concentrations (Fig. 5). Interestingly,
A5MP developed its inhibitory action on both cell types only at concentrations
higher than 10-5 M (not shown in Fig. 5). Other naturally occurring purines
such as guanosine, inosine, hypoxanthine, xanthine, and uric acid proved to be
totally ineffective.
Another curve showing the inhibition of acute inflammatory responses of
cells in whole blood, by theophylline and in the absence of endothelial cells, is
also included in Fig. 5. It is immediately apparent that under such conditions,
inhibition by this methylxanthine only commences at > 100 11M, concentrations,
far above the therapeutically attainable levels of the drug. Surprisingly, SIN-l
(3-morpholino-sydnonimine), a rich source of endothelium-derived relaxing
factor (EDRF = NO) proved to be completely ineffective under the conditions
of our experiments.
Figure 6 illustrates the influence of coronary endothelial cells on the
inflammatory reactions brought about by f-MLP in whole blood. Whereas in
the absence of endothelial cells 3 nM f-MLP already induced a noticeable
increase in the plugging rate, ~ was considerably reduced in the presence of
these cells. This flow-improving effect was substantially diminished in the
presence of adenosine deaminase in the blood sample or after preincubation of
the endothelial cells with 10 11M acetylsalicylic acid (ASA) for 5 min. A
~~------------------------------------------------
FiG. 4. Scanning electron micrographs of polycarbonate filters through which 1:6
diluted whole blood has been passed for 30 s after the addition of a 2 nM F-MLP,
b 200nM ADP, c 2nM PAF. f-MLP primarily activates PMN, ADP platelets. PAF
activates PMN and platelets simultaneously
B FIG. 6. Plugging rate ~ in filtration

B F experiments with (A) whole blood
II" T = WB, (B) WB + 3nM FMLP, (C)
0 as in (B), but in the presence of
IT confluent endothelial cells, (D) as in
(C), but with endothelial cells
preincubated for 5 min with 10 ~M
-0.1 E ASA, (£) conditions as in (C), but
rr with adenosine deaminase added
C (llU/ml), (F) as in (D) and in the
rr presence of adenosine deaminase
A
...E1
combination of the enzyme and ASA almost abolished the influence of the
endothelial cells.
Discussion
The cell isolation and filtration methods developed by us now allow, for the
first time, acute inflammatory processes to be quantitatively studied in whole
blood under highly reproducible in vitro conditions. The filtration principle
employed takes into consideration recent insights concerning the activation of
PMNs. This refers to their more marked and, in many ways, more specific
activation encountered in contact with surfaces preincubated with plasma, as
opposed to the frequently investigated condition of rather dense suspensions of
purified PMNs [19]. In addition, comparative studies can now be performed in
whole blood and also in defined preparations of reduced blood samples which
contain only certain blood cell types in plasma. The greatest advantage,
however, would seem to be that even the vascular endothelium, which
represents the physiological container of blood, can be reliably and readily
introduced into the system. The importance of these measures is documented
by the excerpt of results presented in this chapter.
With the aid of exogenously added mediators of inflammatory reactions,
such as f-MLP and ADP, it is possible to induce a primary and selective
activation of PMNs or platelets, respectively, in whole blood. However, a rapid
coactivation of the other cell type always ensues, in acordance with the close
cooperation between PMNs and platelets already expressed in their common
evolutionary history. Mediators of acute inflammation like PAF, on the other
hand, simultaneously activate both PMNs and platelets.
The inhibitory effects of adenosine and of the prostaglandins PGE 1 and
PGE z on PMN and platelet activation in whole blood are of particular interest,
because these substances were already active at physiological concentrations.
All three substances are rapidly degraded in whole blood. The finding
presented here that cultured coronary endothelial cells can fully substitute for
added, exogenous adenosine, PGE 1 and PGE2 , therefore, probably indicates
that these compounds are continuously synthesized and released into the
streaming blood by the coronary endothelium. A new antiinflammatory, e.g.,
protective action of the intact coronary endothelium, can, therefore, be
proposed, which comes to bear whenever mediators of acute inflammatory
processes penetrate into the blood and threaten the blood supply of the
heart by eliciting aggregation of PMNs and platelets in the coronary
microvasculature (e.g., in the marginal zone of a myocardial infarction).
References
1. Feigl OE (1983) Coronary physiology. Physiol Rev 63:1-205
2. Olsson RA, Bunger R (1987) Metabolic control of coronary blood flow. Prog
Circ Res 47:807-813
4. Burnstock G (1972) Purinergic nerves. Pharmacol Rev 24:509-581
5. Fredholm BB, Hedquvist P, Lindstrom K, Wennmalm M (1982) Release of
nucleosides and nucleotides from the rabbit heart by sympathetic nerve stimulation.
Acta Physiol Scand 116:285-295
6. Nees S, Gerlach E (1983) Adenine nucleotide and adenosine metabolism in
cultured coronary endothelial cells: Formation and release of adenine compounds
and possible functional implications. In: Berne RM, Rail TW, Rubio R (eds)
Regulatory function of adenosine. Martinus Nijhoff, The Hague, pp 347-355
7. Nees S, Dendorfer A (to be published) New perspectives of myocardial adenine
nucleotide metabolism. In: Imai S, Nakazawa M (eds) Role of adenosine and
adenine nucleotides in the biological system. Elsevier, Amsterdam
8. Belloni FL, Bruttig SP, Rubio R, Berne RM (1986) Uptake and release of
adenosine by cultured rat aortic smooth muscle. Microvasc Res 32:200-210
9. Harlan JM (1985) Leukocyte-endothelial interactions. Blood 65:513-525
10. Stern DM, Carpenter B, Nawroth PP (1986) Endothelium and the regulation of
coagulation. Pathol Immun 5:29-36
11. Nees S, Stiegler H (1988) Neu erkannte physiologische und pathophysiologische
Merkmale des vaskuliiren Endothels. In: Peter K, Lawin P, Jensen U, Martin E
(eds) Schock: Strombahn, Mediatoren, Zelle. Georg Thieme, Stuttgart, pp 27-46
12. Henson PM (1990) Interaction between neutrophils and platelets. Lab Invest
62:391-393
13. Sandborg RR, Smolen JE (1988) Biology of disease. Early biochemical events in
leukocyte activation. Lab Invest 59:300-320
14. Gerlach E, Becker BF (1990) The vascular endothelium: Interactions with hemo-
static mechanisms (platelets, coagulation, fibrinolysis). In: Bleifeld W, Hamm CW,
Braunwald E (eds) Unstable angina. Springer-Verlag, Berlin Heidelberg, pp 3-15
15. Haljamiie H, Bagge K (1984) Leukocyte rheology in shock. Intensive Care News
1:4-8
16. Barroso-Arranda J, Schmid-Schonbein GW, Zweifach BW, Engler RL (1988)
Granulocytes and no-reflow phenomenon in irreversible hemorrhagic shock. Circ
Res 63:437-447
17. Beutler E, West C, Blume K-G (1976) The removal of leukocytes and platelets
from whole blood. J Lab Clin Med 88:328-333
18. Nees S, Gerbes AL, Gerlach E (1981) Isolation, identification, and continuous
culture of coronary endothelial cells from guinea-pig hearts. Eur J Cell Bioi 24:287-
297
19. Nathan CF (1987) Neutrophil activation on biological surfaces. J Clin Invest
80: 1550-1560
7
Effects of Exogenous Adenosine
on Human Coronary Circulation
Mario Marzilli, Gerald Klassen, Paolo Marraccini,
Maria Giovanna Trivella, Paolo Camici, and Antonio L'Abbate 1
Summary. A prominent role is attributed to adenosine in the regulation of

myocardial perfusion, but little information is available about the effects of
adenosine on coronary blood flow in conscious man. To investigate the
response of the human coronary circulation to adenosine, the great cardiac
vein flow and the coronary sinus flow were measured by the thermodilution
technique in a group of nine patients before and after the intracoronary bolus
injection of 0.1,0.5, 1, and 2.5mg of adenosine. In a subgroup of patients,
flow measurements were repeated after the intravenous bolus injection of 1,
2.5, and 5 mg of adenosine. The study was performed during cardiac catheter-
ization in subjects with normal coronary arteries and normal left ventricular
function.
Adenosine administration was free from significant side effects and was
followed by a dose-related coronary vasodilation; in the protocol we used,
maximal reduction of coronary resistance was achieved with 1 mg adenosine
injected either intracoronarily or intravenously. Following superselective
intracoronary adenosine injection, coronary vasodilation began immediately,
and persisted for 3-5 min; peak flow was reached with a two-step increment
and vascular regions not directly exposed to the drug also appeared to con-
tribute to the flow increment. Following intravenous adenosine injection,
coronary vasodilation began after 20-30 s, lasted several s, and reached a level
comparable to that achieved by the intracoronary administration of adenosine.
The observations of this study confirm that adenosine boluses can be safely
injected in human coronaries in order to achieve maximal coronary
vasodilation. The observations of this study suggest that, in addition to the
known receptor-agonist interaction, other mechanisms (endothelium-
dependent? neurally mediated?) contribute to the vasodilatory action of this
substance.
lIstituto di Fisiologia Clinica, CNR, University of Pisa, 56100 Pisa, Italy
179
180 M. Marzilli et al.
Key words: Adenosine-Coronary vasodilation-Coronary flow reserve
Introduction
Adenosine, an endogenous nucleoside formed mainly as a degradation product
of adenosine triphosphate (ATP) , is a powerful vasodilator, and may be an
important mediator of coronary autoregulation [1-3]. Adenosine is released
when oxygen demand increases and when myocardial blood supply is reduced,
and, by dilating resistance vessels, tends to restore the balance between oxygen
supply and demand [4-6].
When given intravenously to humans, adenosine stimulates respiration,
depresses sinus node automaticity and slows atrioventricular nodal conduction
[7-8]. In anesthetized patients, adenosine lowers blood pressure with minimal
changes of heart rate [9]. In conscious man, adenosine increases heart rate and
systolic blood pressure, with minimal effects on diastolic pressure [10-12].
Bolus injections of adenosine can reproduce angina pectoris-like chest pain in
healthy volunteers and epigastric pain in patients with duodenal ulcer [13,14].
Recently, adenosine has been shown to increase coronary blood flow during
reperfusion of ischemic myocardium, and intracoronary administration of
adenosine has been proposed to limit infarct size and to improve ventricular
function in patients undergoing pharmacological Or mechanical
revascularization [15,16].
The purpose of this study was to investigate the response of the human
coronary circulation to adenosine and to compare the effects of the
intracoronary administration with the effects of a peripheral injection of this
substance.
Methods
Study Population
The study was performed on nine patients (one male and eight female) ranging
in age from 40 to 61 years. These patients had been admitted to OUr ward for
the investigation of chest pain syndromes. The study was proposed to patients
that were found to have a normal EKG, negative exercise stress test, negative
echo-dipyridamole test, and negative ergonovine test; it was actually
performed on nine consecutive patients with these clinical features and who
were found to have normal Coronary vessels and normal left ventricular
function at cardiac catheterization. Written informed consent to the cardiac
catheterization and to the adenosine protocol was obtained from all
participants. Patients were studied in the fasting state, under mild sedation
(diazepam lOmg im.) after medications had been withdrawn for 72h.
7. Effects of Exogenous Adenosine on Human Coronary Circulation 181
Patient Instrumentation
After completion of diagnostic cardiac catheterization, heparin 10,000 U was
injected and a 5 F bipolar pacing catheter (Cordis) was positioned in the right
ventricular apex and set in the demand mode at approximately 10 beats/min
less than baseline rate. A three-thermistor thermodilution catheter (Webster)
was advanced into the great cardiac vein (GCV). The left coronary ostium was
reached with a 8 F PTCA guiding catheter (USCI) and an intracoronary
injection of isosorbide dinitrate (O.4mg) was given. A 0.014" very flexible
steerable guide wire (USCI) was introduced into the left anterior descending
(LAD) coronary artery and a 2.5F coronary infusion catheter (ACS) was
advanced over the wire and positioned in the proximal segment of the LAD
coronary artery. The wire was removed and both the guiding catheter and the
intracoronary catheter were connected to pressure transducers (Bell and
Howell). The catheters' positions were recorded on both tape and film, and
checked frequently during the study.
Flow Measurement
Great cardiac vein and coronary sinus (CS) blood flow were measured
according to the thermodilution method [17,18]. The following formula was
used to calculate blood flow:
Flow = [(Tb - Ti): (Tb - Tm) - 1] x Fi x C
where Tb is the blood temperature, Ti is the injectate temperature, Tm is the

temperature of the injectate/blood mixture, Fi is the pump infusion rate, and C
is a constant related to the thermal properties of the injectate (for normal
saline solution C = 1.19).
Adenosine Administration
Crystalline adenosine (Sigma Chemical Co.) was dissolved in normal saline
solution and concentrations of 0.1, 0.5, 1, and 2.5mg/ml were obtained.
Adenosine concentrations were confirmed by high pressure liquid
chromatography [19]. Adenosine solutions were prepared in sterile, pyrogen
free, 2ml vials by a pharmaceutical company (Laboratori Baldacci, Pisa, Italy).
Adenosine was injected as 1 ml boluses flushed with 1 ml of saline solution.
Study Protocol
Five to ten min after completion of patient instrumentation, flows were
measured in duplicate. The average of these two measurements was assumed
as basal values for CS and GCV flows. Flow measurements were then repeated
at each intracoronary injection of adenosine. The intracoronary adenosine
injections were done at 5 min intervals. After completion of this part of the
study, flow measurements were repeated after a peripheral (right antecubital

vein) injection of 5 mg adenosine (seven patients), 2.5 mg adenosine (five
patients), and 1 mg adenosine (three patients). Five-minute intervals were
interposed between consceutive measurements. In two patients a final
measurement of flows was performed after intravenous infusion of 0.56mg/kg
dypiridamole over 4 min. During this last flow measurement, the intracoronary
catheter and the coronary guiding catheter were separately pulled out of the
left coronary artery. The ECG lead 2, the aortic and intracoronary pressures,
and the flows were continuously displayed on a monitor and recorded on paper
when needed.
STATISTICAL ANALYSIS. Baseline and post-adenosine values of flow, pressure,
and heart rate were evaluated by analysis of variance. When a difference was
revealed, the residual mean square was applied in a Dunnett's test to
characterize which values were different from baseline. Differences were
analyzed by paired I-test. All null hypotheses were two-tailed, and the criterion
of significance was P < 0.05. All data are expressed as mean ± SEM.
Results
Patients' Tolerance and Side Effects

Eight patients completed the intracoronary adenosine protocol. In the ninth
patient, the 2.5 mg dose was not injected because of the appearance of atrial
fibrillation after the intracoronary injection of 1 mg adenosine. The arrhythmia
was well tolerated and reverted spontaneously to sinus rhythm after 25 min.
The intracoronary administration of 0.1 and 0.5 mg adenosine went almost
unnoticed by the patients. Following 1 mg and 2.5 mg adenosine, six patients
complained of a variety of symptoms, including feelings of distress,
nervousness, head and neck flushing, an urge to breathe deeply, and chest
tightness. These symptoms were short-lasting and did not require medication.
No change in the QRS complex was noted and no modification of cardiac
iso-enzymes occurred (cardiac isoenzymes were measured in all patients prior
to the procedure and on the following morning). Overall, the intracoronary
and intravenous injections of adenosine appeared well tolerated and free from
clinically relevant complications and significant side effects.
Time Course of Coronary Flow Response to Adenosine

Following the intracoronary injection of adenosine, an immediate downward
displacement of the GCV and CS flow tracings was observed, indicating an
increase of coronary flow (Fig. 1). After 15-20s, a further displacement of the
flow tracings was observed, consistent with an additional flow increment. This
high flow was maintened for 20-30s and was eventually followed by a
progressive return to the basal levels. This "biphasic" response of coronary
.I,
.ECG
..Ll.L .I U ..L lL ..J.J.
II ADN 2.5 MG Ie I'-"'D,
II \
It
.\ORTIe PRESSURJ..:
~ 1l.
ftlAU J A ~ & jr---- /\
Ullill\ 1'1 l\L d\ fll Il
G:V~'LJ, ... " \ \JI\J
CS FLOW
1\J ~
. ~
.-
I
I
I
FIG. 1. Effects of the intracoronary injection of 2.5 mg adenosine (ADN 2.5 MG IC) on
the electrocardiogram (ECG), the aortic pressure and the intracoronary pressure
(superimposed with the same zero and gain), the great cardiac vein flow (GCV Flow)
and the coronary sinus flow (CS Flow). Vertical lines mark 1 s intervals. Adenosine
injection through the intracoronary catheter is indicated by the transient disappearance
of the intracoronary pressure, and is followed by a two-step flow increment and by a
transient increase in heart rate and aortic pressure
flow to intracoronary adenosine was already appreciable in some patients with

the low doses, and in all patients it was observed consistently after the highest
doses.
Time to peak flow, estimated from the end of the intracoronry bolus to the
moment of the maximal downward displacement of the flow tracing, increased
progressively with increasing adenosine dose. Peak flow was reached 19 ± 2, 23
± 3, 26 ± 3, and 28 ± 4s after the intracoronary injection of 0.1,0.5, 1, and
2.5 mg adenosine, respectively. Following the intravenous injection of
adenosine, the flow signal began to move from its basal level after 20-25 s,
reached the maximal displacement in 5-8 s, and returned to baseline in an
additional 10-15 s. The highest flow was observed 26 ± 4 and 31 ± 4 s after the
end of an intravenous injection of 2.5 and 5 mg adenosine, respectively
(Fig. 2).
Systemic Effects of Adenosine

Following the intracoronary injection of adenosine, we noted a transient and
minor increase in heart rate (10-12 bpm) and of systolic blood pressure (10-
15 mmHg). In some patients, phasic oscillations of aortic pressure were also
noted at a frequency of 4-8 cycle/min. These changes were more evident and
consistent after the highest doses and coincided in time with the patients'
symptoms, if present. A reduction of aortic pressure was noted as following the
JJ 1.1 ]. J j, J J J U U. J. J..lJJ'j .I U JJ j ~, Illi j J
II
KG
I I
_ AUNTie '-}(ESSUKE
I ~ ~
N /I II
" IA I~
- LA!) j)RESSliRE
r 1l1I1\ 111\ III
~ 1\11\11\J \ I\J \ ~ \ \ ~ \ ~
= I I l\J i'\ I~ I~ ~ \ \' I~ \\ ~ I~ ~
-
GCY FLOW
I
r- I'-- t--...
--
:::::: cs FLOW _ '- .....
FIG. 2. Effects of the intravenous injection of 2.5 mg adenosine on the elec-

trocardiogram (ECG), the aortic pressure, the intracoronary pressure (LAD pressure),
the great cardiac vein flow (GCV Flow) and the coronary sinus flow (CS Flow).
Intravenous adenosine injection causes a simultaneous and persistent increase of GCV
and CS flows. Aortic pressure decreases slightly following a transient incr.ease. Heart
rate initially slows and later increases
intravenous adenosine injection, associated with a minor increase in heart rate.

At the point when peak flow was observed, aortic pressure and heart rate were
similar to control values for both the intracoronary and intravenous adenosine
administration.
Coronary Blood Flow
Intracoronary Adenosine Administration

For each intracoronary adenosine dose, the basal GCV and CS flows, the
initial post-adenosine increments, and the maximal flow values were calculated.
By comparing the first increments of GCV and CS flows, it was readily
apparent that the initial CS flow increment was entirely due to the increment of
the GCV flow. Conversely, when maximal flows were compared, a progressive
enlargement of the CS-GCV difference was noted, thus demonstrating a flow
increment outside the GCV region that was statistically significant after 2.5 mg
adenosine.
The basal flow values, measured prior to each intracoronary adenosine
injection, were 58 ± 3 mllmin, 61 ± 6 mllmin, 62 ± 6 mllmin, and 60 ±
8 mllmin for GCV flow and 143 ± 15 mllmin, 147 ± 16 mllmin, 143 ± 19 mil
min, and 138 ± 14mllmin for CS flow. The injection of O.lmg adenosine
caused an initial increment of GCV flow from 58 ± 3 mllmin to 69 ± 10 ml/

min, followed by a further increase to a peak value of 93 ± 8 ml/min (P <
0.001). Simultaneously, CS flow increased from 143 ± 15 ml/min to a peak
value of 191 ± 23 mllmin (P < 0.05). The difference between GCV and CS
flows increased from 94 ± 12mllmin to 112 ± 22mllmin (P = NS).
The intracoronary injection of 0.5 mg adenosine caused an initial increase of
GCV flow to 67 ± 7mllmin and a peak rise to 115 ± 13mllmin (P < 0.001).
CS flow rose from 147 ± 18ml/min to 239 ± 28mllmin (P < 0.05). The
difference between GCV and CS flows was 145 ± 32mllmin (P = NS).
Following the intracoronary injection of 1 mg adenosine, GCV flow increased
to 72 ± 8mllmin and then to 139 ± 13mllmin (P < 0.001), and CS flow
reached a value of 248 ± 29mllmin (P < 0.01), the difference between GCV
and CS flows becoming 129 ± 27mllmin (P = NS). The intracoronary
injection of 2.5mg adenosine, the highest dose tested in this study, resulted in
an initial increase of GCV flow to 94 ± 15 ml/min and then to a peak value of
155 ± 17mllmin (P < 0.001). Peak CS flow after 2.5mg adenosine was 316 ±
38mllmin (P < 0.01). The difference between GCV and CS flows rose to 172
± 39mllmin (P < 0.02). However, maximal GCV and CS flows after 2.5mg
adenosine were not statistically different from maximal GCV and CS flows
following 1 mg adenosine.
As an index of coronary resistance, the ratio of mean aortic pressure and the
GCV flow was calculated. The "coronary resistance index" was calculated
under the basal condition and after each adenosine dose. The "coronary
resistance index" decreased from the initial value of 1.75 ± .10 mmHg/ml per
min to 1.04 ± .13mmHg/ml per min after O.lmg adenosine (P < 0.001), to
.89 ± 11mmHg/ml per min after 0.5mg adenosine (P < 0.001), to .76 ±
.WmmHg/ml per min after 1 mg adenosine (P < 0.01), and to .68 ±
.WmmHg/ml per min after 2.5mg adenosine (P < 0.001). Similar calculations
were performed using the CS flow values and showed that resistance to flow in
the CS was reduced by intracoronary adenosine from the initial value of 0.72 ±
0.09mmHg/mllmin to 0.49 ± .07mmHg/ml per min (P < 0.05), to 0.41 ±
0.06mmHg/ml per min (P < 0.05), to 0.40 ± 0.04mmHg/ml per min (P <
0.01), and to 0.35 ± 0.04mmHg/ml per min (P < 0.05). The values for "CS
and GCV resistance indexes" calculated after 1 mg adenosine were not
statistically different from the values obtained after 2.5 mg adenosine. The log
dose-response relationship was linear for an increase in flow in the GCV as
well as in the CS (P < 0.02).
Intravenous Adenosine Administration

The injection of adenosine in the right antecubital vein resulted in a dose-
related increase of coronary blood flow. After the 1 mg injection, GCV flow
rose from 69 ± 24 mllmin to 118 ± 19 mllmin and CS flow from 112 ±
11mllmin to 217 ± 36mllmin; with 2.5mg adenosine, GCV flow rose to 138 ±
Wmllmin (P < 0.001) and CS flow to 354 ± 73mllmin (P < 0.02). No further
200
150 .L
;r---
T ------ --i-
1····)----------------
-1
- - - -- - -
/1 / 1
100 lI' . /
" -0 I.C. ADN

50 .. LV. ADN
o+-----~----~----~----~----~--~
o 2 J 4 5 6
Adenosine (mg)
FiG. 3. Effect of intracoronary (IC) and intravenous (IV) injection of adenosine on the
great cardiac vein flow. Peak flow increment appears relatively independent from the
injection site
increase of GCV and CS flows was obtained with 5 mg adenosine. In fact, after
the 5 mg adenosine peak, GCV flow was 148 ± 8 mllmin and of CS flow was
338 ± 47 mllmin. When peak flow values obtained with the peripheral injection
of adenosine were compared with peak flow values obtained with the intra-
coronary administration of the same amounts of the drug, they were found
to be statistically similar (Fig. 3). In accordance with this observation, the
coronary resistance indexes were also very similar after the intracoronary and
intravenous injection of adenosine (Fig. 4).
Discussion
Safety Precautions
At the beginning of this study, little was known about the effects of the
intracoronary injection of adenosine in cOJ?scious humans. Therefore, the
possible risks contained in this experimental protocol were carefully analyzed
and preventive measures were taken. The intracoronary administration of
nitrates at the beginning of the study was performed in order to prevent a
possible vasoconstrictive response to the mechanical stimulation exerted by
catheters and guide wires. To prevent intracoronary clot formation and distal
coronary embolization, heparin was injected at the same does commonly used
2.0
1.5
1.0
Q)
u
C
<ll
+J
til 0.5
'M
til
Q)
0::
> o
U
l'J o 2 3 4 5 6
Adenosine (mg)
FIG. 4. Effect of adenosine on great cardiac vein resistance. A similar reduction of great
cardiac vein (GCV) resistance to flow is observed following intracoronary (open
squares) and intravenous (closed squares) injections
in our laboratory in PTCA procedures. A temporary pacing lead was placed in

the right ventricle in readiness to treat possible bradicardia or A-V block.
The super-selective injection of adenosine in the proximal left anterior
descending branch not only allowed a comparison of the flow response of the
area directly exposed to adenosine with the flow changes in the circumflex
territory not directly exposed to the substance [18], but also limited the amount
of myocardium affected by the drug, thus limiting the hemodynamic
consequences of possible negative inotropic action of adenosine.
Coronary Effects of Adenosine

Intracoronary and intravenous adenosine administration was followed by a
dose-related coronary vasodilation. The doses and the sequence of injections in
this protocol were arbitrary, and the indications are that a maximal coronary
vasodilation can be achieved with 1-2.5 mg adenosine, a dose similar to the
amount required to obtain maximal vasodilation in the forearm [20]. The
observations of this study suggest that the intracoronary injection of 1-2.5 mg
adenosine elicits a two-step vasodilatory response, the first wave of
vasodilation probably being limited to the vessels directly exposed to the
substance and the second wave involving the entire coronary circulation.
The occurrence of a vasodilatory response in the circumflex vascular bed
following the injection of adenosine into the LAD branch is suggested by (1)
the observation of a flow increment in the CS that is significantly larger than
the flow increment in the GCV, and (2) by the similarity of peak flows after
intracoronary and intravenous adenosine, when the entire coronary circulation

is obviously dilated. We realize, however, that these observations, as well as
the long time intervals between the injection of adenosine and peak flow
increments, seem to be in contrast with the short half-life of adenosine, which
is about lOs or less [21,22].
A backflow of adenosine from the LAD branch into the circumflex artery
could explain the vasodilation of this vascular bed, but this event seems
unlikely because the catheter tip was advanced 3-4cm downstream of the
LAD artery, because the injection rate was rather slow (due to the small lumen
of the 2.5F infusion catheter), and because the injectate volume was
intentionally limited to 1 ml. The similarity of the adenosine doses capable of
inducing maximal coronary vasodilation by the intravenous and the
intracoronary routes was unexpected and is difficult to understand.
Of 1 ml of any solution injected in aperipheral vein, approximately 0.05 ml is
estimated to reach the coronary circulation, if a coronary flow as high as 5% of
total cardiac output is assumed. Based on this hypothesis, following an iv.
injection of 2.5 mg of adenosine, 0.13 mg could reach the coronary circulation.
In the case of a substance with a short half-life such as adenosine, the amount
will be much less. Adenosine is avidly taken up by red blood cells and actively
removed by the pulmonary bed [23-25].
Consequently, some of the observations of this study remain difficult to
explain. These include the vasodilating response of the circumflex coronary
vascular bed following the superselective injection of adenosine in the LAD
coronary branch, the capability of the iv. injection of 2.5-5 mg adenosine of
inducing maximal vasodilation as the intracoronary injection, and the
persistance of a vasodilating effect 4-5 min after the administration.
Adenosine vasodilation is generally attributed to the interaction with
receptors present in the vascular smooth muscle cells [26-30]. However,
additional mechanisms could be activated by the injection of adenosine and
could contribute to the vasodilating response. These additional mechanisms
could be endothelial-mediated and neural-mediated.
In order to reach these receptors adenosine has to cross the endothelial
barrier. Endothelial cells are known to possess a system of ectonucleotidases
and efficiently take up extracellular adenosine by a carrier-mediated process
[31,32]. Liberation from the endothelial cells of the endothelial-derived
relaxing factor (EDRF) could paly a role in the initial wave of vasodilation;
smooth muscle receptor activation could be the mechanism of the second wave
of vasodilatation induced by intracoronary adenosine.
An alternative hypothesis to explain the two-step response and the long
duration of the vasodilatory effect of intracoronary adenosine despite its short
half-life could be that adenosine is rapidly transformed in or induces the
production of a second messenger with a longer half-life and/or is capable of
inducing cellular changes that persist longer than the receptor agonist
interaction. Neural reflexes may be elicited by the intracoronary injection of
adenosine, as suggested by the coincidence of the second phase of vasodilation
with a rise of aortic pressure. Similarly, intravenous administration may cause
reflexes originating in the lung causing indired coronary effects which result
from hemodynamic/sympathetic changes.
The dose of adenosine that induced maximal coronary vasodilation in this
study, both by the coronary and the venous routes, is markedly lower than the
maximal tolerated dose used by other researchers to investigate the systemic
and respiratory effects of this substance [11-15]. This is probably the reason
why this protocol was well tolerated by all patients and is consistent with the
observation that in other vascular regions the dose required to produce pain or
discomfort is greater than the vasodilating dose [20].
Maximal flow measured in this study was roughly 3 times greater than basal
flow. This ratio is rather low if compared with the "coronary reserve"
measured with intracoronary Doppler flow velocity probes [33]. The
thermodilution technique reflects, with sufficient accuracy, large changes in
volume flow in subjects with normal coronaries, as was the case in this study
[34]. The intracoronary Doppler flow velocity probes reflect changes in the
velocity of the blood in the vessel and may overestimate volume flow [34].
These methodological differences may account for the relatively low
"vasodilator reserve" estimated in this study.
To exclude artifactual limitations of flow due to the presence of a guiding
catheter in the left coronary ostium and of an infusion catheter in the LAD
branch, measurements of flows were repeated in two patients after the
intravenous injection of dypiridamole 0.56mg/kg. In both cases, dypiridamole
caused a flow increment comparable to that observed following 2.5 mg
adenosine, but the dypiridamole effect lasted much longer than that of
adenosine. This long-lasting vasodilation allowed us to repeat the flow
measurements after removing the infusion catheter from the LAD branch and
the guiding catheter from the coronary ostium. Neither GCV nor CS flows
increased after catheter removal, confirming that no artefactual limitation of
flow resulted from the experimental set-up of this study. An additional support
of this observation comes from the absence of a pressure gradient between the
guiding catheter and the infusion catheter, as would be expected in case a
significant resistance to flow were present.
Acknowledgment. This work was supported by a grant from the Ministero

Pubblica Istruzione.
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uptake of adenosine in canine skeletal muscle. Am J Physiol 250:H482- H489
33. Wilson RF, Laughlin DE, Ackell PH, Chilian WM, Holida MD, Hartley CJ,
Armstrong ML, Marcus ML, White CW (1985) Transluminal, subselective mea-
surement of coronary artery blood flow velocity and vasodilator reserve in man.
34. Marcus ML, Wilson RF, White CW (1987) Methods of measurements of
myocardial blood flow in patients: A critical review. Circulation 76:245-253
D
Role of Endothelial Cells
in Coronary Circulation
1
Endothelial Cell P2 Purinoceptors
Jeremy D. Pearson l and Thomas D. Carter
Summary. Endothelial cells possess P2 purinoceptors of the P2Y subclass.

These are coupled to the generation of inositol trisphosphate and hence the
mobilization of intracellular calcium stores. P2Y receptor-mediated changes in
intracellular ionized calcium have been followed in detail in cell populations
and in individual endothelial cells, and consist of a second phase of calcium
elevation requiring calcium entry, in addition to the first transient rise which is
due to discharge from internal stores. Synthesis of prostacyclin, a potent
vasodilator and inhibitor of platelet function, is driven by the first phase of the
calcium response. The properties of the second phase of the calcium response,
in contrast, are consistent with a causal role for this phase in the synthesis of
nitric oxide (endothelium-derived relaxing factor), although this has yet to be
established directly.
Key words: Endothelium-P2 Purinoceptors-Prostacyclin-EDRF-Nitric
oxide-Intracellular calcium
Introduction
Since the first proposal by Burnstock [1] that purinoceptors should be divided
into two types, PI (primarily recognizing adenosine) and P2 (primarily
recognizing adenosine 5 ' -triphosphate, ATP, or adenosine 5'-diphosphate,
ADP), there has been a dramatic increase in the interest in and recognition of
P2 purinoceptors and their possible pathophysiological roles [2]. With the
realization that ATP can be released from perivascular nerves, and the
knowledge that it is also present extracellularly in response to stimulation of
blood platelets or as a consequence of proteolytic or other damaging stimuli
iSection of Vascular Biology, Clinical Research Centre, Harrow, HAl 3UJ, UK

2 Divisonof Neurophysiology and Neuropharmacology, National Institute for Medical
Research, Mill Hill, London, UK
195
196 J.D. Pearson and T.D. Carter
acting on several cell types, notably endothelium [3,4], studies characterizing

the effects of adenine nucleotides acting via P2 receptors in the cardiovascular
system have multiplied (reviewed in [5]).
Interest has been promoted further by the possible contribution of ATP,
rather than or in addition to adenosine, as a mediator of the vasodilator
response to ischemia and hypoxia. The arguments in favor of this have been
well rehearsed elsewhere [6,7], but include the fact that ATP is at least as
potent as adenosine as a dilator in "the coronary bed [8], and describe the
difficulties in understanding how sufficient adenosine can be generated extra-
cellularly unless it is formed by the catabolism of extracellular adenine
nucleotides [9].
Extracellular ATP can act, usually as a vasoconstrictor, directly upon smooth
muscle P2 purinoceptors although its predominant action is normally
vasodilatation [5]. In this article we briefly review our work on the potent
endothelium-dependent vasodilator actions of ATP and their molecular
mechanisms.
Endothelial P2Y Purinoceptors

Endothelium-dependent vasodilatation in response to ATP was first described
by De Mey and Vanhoutte [10]. The phenomenon of endothelium-dependent
vasodilatation caused by a variety of agonists was originally characterised by
Furchgott and shown to be due to secretion by endothelium of a labile non-
prostanoid substance, now known to be nitric oxide [11,12]. The
pharmacological profile of a series of ATP analogues tested as endothelium-
dependent dilators [13] indicated that this response is due to action at P2Y
receptors, one of the proposed subclasses of P2 receptors [2,14]. At this
receptor, 2-purine substituted ATP analogues, such as 2-chloro-ATP, are more
potent than ATP, whereas analogues such as a,~-methylene-ATP are much
less potent.
A series of A TP analogues gave a similar profile of activity when endothelial
prostacyclin (PGIz) release was measured [15], indicating that the P2Y
purinoceptor is coupled to at least two effector pathways. In the absence of
selective antagonists, differences in the effectiveness of certain ATP analogues
at inducing PGI 2 release and endothelium-dependent vasodilatation [16] could
reflect actions at different receptor subtypes. However, there is no other
evidence to support this idea, and the results can be explained equally well in
terms of the different transduction mechanisms leading to PGIz and NO
synthesis (see below).
P2Y Purinoceptor Signal Transduction

In 1986, ATP was shown independently by 2 groups to elevate intracellular
ionized calcium concentrations, [Ca2 +]j, in endothelial cells [17,18], and shortly
afterwards to stimulate the transient production of inositol trisphosphate (IP3 )
1. Endothelial Cell P2 Purinoceptors 197
[19]. Therefore, it is likely that the endothelial P 2Y receptor is one of the

growing family of autacoid receptors known to be linked via a G protein to
phosphoinositidase C [20], and hence to the generation of IP 3 and the
mobilization of Ca 2 +j [21]. The P 2Y receptor has not yet been cloned from
any source, but it has been highly purified from avian erythrocytes, where it is
tightly linked to phosphoinositidase C in a G protein-dependent fashion [22].
Evidence that supports the coupling of a G protein to the endothelial P 2Y
receptor includes the effects of pertussis toxin and stable GTP analogues on
ATP-stimulated IP 3 production [23,24].
The pattern of the [Ca2 +]j changes in populations of endothelial cells has
been well defined using cells loaded with a calcium-sensitive fluorescent
indicator such as fura-2. There is an immediate transient dose-dependent
elevation of [Ca2 +]j in response to A TP, peaking within about 10 s from a
resting level of =O.II1M to a maximum of =2 11M. [Ca 2 +]j then declines
rapidly in the continued presence of the agonist (Fig. 1). This transient eleva-
tion is independent of the presence of extracellular Ca2 +, demonstrating that
the peak is due to mobilization of Ca2 + from internal stores [25]. When extra-
cellular Ca 2 + is present, [Ca 2 +]j falls to an elevated plateau level (again related
to the dose of ATP added: maximally =0.5IlM) that can be maintained for
many minutes. However, when extracellular CA 2 + is absent, [CA2+]j returns to
pre-stimulated levels within =2 min ([25], Fig. 1), suggesting that influx of
Ca 2 + is required to maintain the second phase of the response.
1+ 1mMEGTA 1
I ATP IEGTA I ATP ICa 2 +
t(100!!M) t(2mM) t (100!!M) t(2mM)
2 2
:?
~ 2:
2: .;:-'
~
C\J
+
C\J
ctJ
ctJ £
2.
a a
500 sec 500 sec
FIG. 1. ATP-stimulated changes in [Ca 2 +]j in populations of human umbilical vein

~ndothelial cells. Left panel shows initial transient peak followed by an elevated plateau
III ~he presence of 1 n:M extracellular Ca 2 +. On addition of 2mM EGTA, [Ca 2 +]j
rapidly falls to pre-stimulated levels. Right panel shows that in the absence of
extr2:cel!ular ~a2+ but in th~ presence of 1 mM EGTA, the initial transient peak of
rCa ]j IS eqUivalent to that III the left panel, but falls rapidly to pre-stimulated levels.
Wh.en extracellular Ca 2 + is introduced (in the continued presence of ATP) [Ca2 +l
rapidly rises and stabilises at an elevated plateau I
When [Caz+]j responses to A TP are studied in individual cells, a different

pattern is seen. Following an initial transient peak, the height of which is
apparently not related to the dose of ATP applied, a variable pattern of
sustained or oscillatory (spiking) elevations of [Caz+]j, with intermediate
returns to pre-stimulated levels, is found [26]. The dose-related response of the
initial transient peak in [Caz+]j detected in populations of cells, therefore,
seems to reflect an increasing likelihood that individual cells will respond as the
dose of ATP increases. The second phase of the [Caz+]j response, detected as a
plateau in populations of cells, reflects the summation of individual cell
oscillatory responses. Its dose relationship seems to be due to a dose
relationship at the single cell level between the concentration of A TP applied
and the frequency of the [Caz+]j oscillations or spikes [27]. Small groups of
endothelial cells in vitro can respond to ATP with synchronous [Caz+]j
oscillations [27], which raises the possibility that such coupled oscillations take
place in vivo, and hence that local endothelial responses dependent upon these
[Caz+]j changes (perhaps including NO synthesis - see below) occur in a
pulsatile fashion.
PGI2 Synthesis
Synthesis of PGIz (in response to thrombin) was first shown by Hallam et al.
[28] to require an elevation of [Caz+]j, as a consequence of IP3 production.
One of the main characteristics of receptor-stimulated PGIz production is its
transience: e.g., in the continued presence of ATP, release of PGIz ceased
within <5min [15,25]. A detailed study of ATP-stimulated PGIz synthesis
demonstrated that, like synthesis in response to thrombin, it required
mobilization of intracellular Caz+ [25]. The relationship between [Caz+]j and
PGIz release indicated (1) that a threshold [Ca2+]j of ;::::0.8 11M was required to
detect PGIz production, and (2) that this relationship (Fig. 2) was identical to
that obtained when PGIz production was driven exclusively by elevation of
[Caz+]j with an ionophore. These results imply that PGIz synthesis is driven
solely by an increase in [Caz+]j, and is short-lived because this increase is only
;::::0.8IlM for a short time. The most likely site of action for Caz+ is
phospholipase A z, the rate-limiting enzyme in the prostaglandin synthetic
pathway, which is known to be [Caz+ksensitive within the range of [Caz+]j
found in these experiments.
A second feature of PGIz synthesis stimulated by ATP (and other agonists)
is that repeated challenge with the agonist at short time intervals leads to
homologous desensitization of the response [29]. This is paralleled by a
decrease in the ability of ATP to mobilize CAz+ j, indicating diminished
receptor affinity or uncoupling of the receptor from phosphoinositidase C [26].
IP3 generation also leads to the production of a second potential messenger,
diacylglycerol (DAG), which can activate protein kinase C. Although there
was evidence from our earlier experiments (Fig. 2) that this pathway does not
contribute to the triggering of PGIz synthesis, it was plausible to consider that
20
15
o
Vi
a:;
u
'"......
0
10 ...
L-
0.>
0-
D'
• o
E
_N
(!)
a...
•
5 ... 0
o •
o
o~ 0 0
o ~ ...... ...o
I I I I I I
0.1 0.5 1 2 3 5
[Ca 2+1 i ()!M)
FiG. 2. Dose-response relationship between PGI2 production and [Ca 2+]j. PGI2 release
was measured after 5 min. These experiments were carried out in the absence of
extracellular Ca z+. Peak [Ca2 +]j values were measured in individual experiments after
adding increasing doses of either ATP (-), the more potent PZy agonist 2-chloro-ATP
(4), or ionomycin (0). (Redrawn with permission from data in [25])
activation of protein kinase C was involved in the desensitization process.

However, in a series of experiments in which we deliberately activated or
inhibited protein kinase C (with phorbol esters or staurosporine, respectively)
we demonstrated that desensitization is independent of protein kinase C
activation [26]. Thus, the mechanism leading to desensitization is not
established, but may involve activation of other protein kinases: it is known
that stimulation with A TP leads to the rapid phosphorylation of several
endothelial proteins via Ca2 + -dependent kinases [30].
Nitric Oxide Synthesis

As noted above, ATP acting at apparently the same endothelial P2Y receptor
also triggers the synthesis of nitric oxide (NO), which is responsible for
endothelium-dependent vasodilation in many vessels (although PGI2 is also a
potent vasodilator, in addition to its powerful inhibition of platelet
stimulation). The characteristics of NO production differ, however, in several
important respects from those of PGIz synthesis. First, endothelium-dependent
vasodilatation and NO release in response to ATP can be maintained for many

minutes, unlike PGI2 production [13,31]. Second, whereas PGIz synthesis is
affected relatively little by the absence of extracellular Ca2+, endothelium-
dependent vasodilatation is highly dependent upon extracellular Ca2+ [32].
NO synthase, which is the enzyme responsible for the synthesis of NO
from arginine, has been purified from endothelium and shown to be
Ca2+/calmodulin-dependent, and fully activated by [Ca2+] that are only
approximately at the threshold for PGI2 synthesis [33]. Taken together, these
results strongly suggest that NO synthesis is causally related to the second
phase of the [Ca2+]j response to ATP or other agonists.
Although direct proof of this has yet to be established, we have two further
pieces of data that support the concept. The first was obtained by using Mn2+
to monitor divalent cation influx into fura-2-loaded endothelial cells. Mn2+
quenches the fluorescence emission of fura-2, and there is good evidence that it
enters cells by a receptor-activated cation channel that also translocates Ca2+
[34]. Figure 3 demonstrates the use of this technique with endothelial cells. The
left panel shows that when Mn2+ is present extracellularly it is not translocated
to the cytoplasm until ATP is added, i.e., divalent cation entry is receptor-
IATP
t(100~M)
Mn 2
(100~M1f
+.1 I ATP
t(100~M)
r~, L_
_ _ _ Mn2+ (O.1mM)---
ATP I
(100~M)t tt t t
360
.",
.~
500 sec 500 sec
FIG. 3. Characteristics of ATP-stimulated divalent cation entry, monitored using Mn 2 +.

The left panel shows fluorescence at 500 nm measured as a consequence of excitation at
340nm (increases as [Ca 2 +]j rises, quenched by Mn2 +). Note that the addition of Mn 2 +
has no effect. When ATP is added, fluorescence due to excitation at 340 nm rapidly
increases as [Ca2 +]j increases, as in Fig. 1. After a delay of several seconds, fluorescence
due to excitation at both wavelengths drops as Mn2+ enters the cells. The right panel
shows a series of traces from parallel experiments. The top traces show [Ca2 +]i response
to ATP, in the absence of extracellular Ca2 + or Mn2 +. The bottom traces demonstrate
that Mn 2 + entry occurs when it is added after ATP via a channel that remains open for
>10 min
activated. Further, it shows that there is a detectable delay of several seconds

after internal Ca2+ stores have begun to discharge before Mn2+ quenches
fluorescence. P2 receptor-activated divalent cation entry in endothelial cells is,
therefore, controlled differently from in platelets, where Ca2+ entry precedes
discharge of Ca2+ from internal stores, or smooth muscle cells, where there is
electrophysiological evidence of direct coupling of the P2 receptor to cation
entry [35,36]. This result agrees with those of Jacob et al. [34,37]' who deduced
that the rate of cation entry into endothelial cells following agonist addition
was dependent upon the degree of emptying of the internal Ca2+ store, and
a consequence of Ca2+ mobilization rather than being coupled directly to
receptor occupation by the agonist.
The right panel of Fig. 3 demonstrates that divalent cation entry occurs
throughout the period for which [Ca2+]j is at an elevated plateau level. The
characteristics of this plateau, i.e., a [Ca2+]j of a few hundred nM maintained
by Ca2+ entry, thus agree closely with the time course of agonist-induced NO
release, its requirement for external Ca2+, and the [Ca2+]-dependence of
purified NO synthase.
A second feature of endothelium-dependent vasodilatation induced by
receptor stimulation, although not by calcium ionophores, is its ability to be
rapidly blocked by activation of protein kinase C [38]. Figure 4 demonstrates
that addition of the synthetic phorbol ester, PMA, which activates protein
kinase C, blocks the elevated plateau of [Ca2+]j induced by ATP in a similar
fashion, but does not inhibit the equivalent plateau phase induced by
i,onomYcin
PMA
(100nM)
i
1.0 B
ATP
(100~M) PMA
(10nM) 0.8
i i
~ 0.6
~
OJ
0
r:: +
OJ C\I
0 ell 0.4
III
~
Q.
0
:J
IT: 0.2
300 sec 300 sec
FiG. 4. Effects of activation of protein kinase C on steady-state elevation of [Ca 2+1i

induced by ATP (left panel) or increasing doses of ionomycin (right panel). 10nM PMA
rapidly blocks the plateau phase of the response induced by A TP, whereas 100 nM
PMA fails to alter ionomycin-induced sustained elevations in [Ca2 +1j
ionophore-mediated Caz+ entry. We have attempted to determine whether the

attenuation of elevated [Caz+]i by PMA is due to enhanced sequestration or
pumping out of Ca2 +i or to inhibited entry of external Ca2 +, both of which
have been reported to occur in neutrophils [39], but have as yet no clear data.
Finally, a further property of the plateau phase of [Ca 2 +]i is consistent with
it being tightly linked to NO synthesis, and more closely regulated by the state
of the Ca2 + store than by receptor occupation. When cells are challenged
sequentially with increasing doses of ATP at intervals of a few minutes, the
initial transient peak [Ca 2 +]i drops as desensitization occurs: in contrast, the
plateau phase of the response, like endothelium-dependent vasodilatation,
does not desensitize but increases cumulatively (Fig. 7 in [26]).
These results all strongly suggest that the second phase of [Ca2 +]i elevation is
tightly linked to the synthesis of NO. We are currently developing the on-line
technology required to measure NO release spectrophotometrically [31] in
order to resolve this question.
Discussion
Endothelial cell P2Y purinoceptors are coupled to the generation of IP3 and
hence the mobilization of Ca2+i from internal stores. Both of the physio-
logically important effector responses to P2Y receptor occupation, PGI 2 and
NO synthesis, are clearly consequent to these events. The former is driven
by the transient peak of [Ca2 +]i caused by the mobilization of Ca2+. In
contrast, NO synthesis seems to be regulated predominantly by the second
AlP ADP
\ I ~ Na+
1(1,4,5)P3
'~
Phosphopro1eins....--CaM kinase ..........- -
FIG. 5. Summary of PZy receptor-mediated transduction signals in endothelial cells

(detailed in [40]). Activation of protein kinase C (PKC) is believed to stimulate
phospholipase D (PLD) leading to choline synthesis. Release of Ca z+ from internal
stores activates calmodulin (CaM)-dependent kinases, efflux of K+, exchange of Na+
and H+ leading to cytoplasmic alkalinization, and (? directly) Ca 2 + influx
phase of the [Ca2 +]i response, which is not coupled directly to receptor
occupation but is related to Ca2+ entry. Other intracellular events known to
occur following activation of endothelial P2Y receptors are summarized in Fig.
5. Their relevance to endothelial pathophysiology is less well understood: the
current ideas have recently been discussed [40].
This review has focused upon the effects of A TP on endothelial Ca2 +
homeostasis. We believe that the insights obtained are applicable to a variety
of other agonists of endothelial functions, and expect that a better knowledge
of the differential regulation of PGI2 and NO synthesis will help in
understanding the nature of impaired endothelium-dependent responses in
atherosclerotic, hypertensive, or transplanted vessels.
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2
The Metabolic Barrier of
the Coronary Endothelium as a
Determinant of Flow Responses
Bernhard F. Becker, Birgit Leipert, Lisa Schwartz, and
Eckehart Gerlach 1
Summary. Numerous vasoactive substances are extensively degraded during

coronary passage in the isolated heart of the guinea pig, largely because of
enzymes located on or in the vascular endothelial cells. Examples of such
substances are adenosine, adenine nucleotides (ATP, ADP, AMP, a,B- and
B,y-methylene ATP) , bradykinin, and acetylcholine. Consequently, the
endothelium modulates the effective concentrations of these agonists at the cell
membrane receptors mediating coronary dilatation. This can be readily
evidenced for bradykinin by adding converting enzyme (kininase II) inhibitors,
such as ramiprilat, and for adenosine in the presence of cell uptake blockers,
e.g., dipyridamole. At least in the case of adenosine, the metabolic capacity of
the endothelium for adenosine phosphorylation or degradation is so great that,
at submicromolar concentrations, adenosine practically cannot pass from the
intra- to the extravascular space, or vice versa. As shown in experiments with
selectively prelabeled endothelial adenine nucleotides, even adenosine
escaping from endothelial cells is taken up and degraded downstream, still
within the coronary system. This finding supports the proposed role of the
endothelium in maintaining the homeostasis of adenosine. Due to endothelial
catabolism, it appears that coronary dilatation by many infused agonists must
be indirect, necessitating mediation by the endothelium itself. Moreover, the
enhanced dilatory efficacies of infused adenosine, acetylcholine, and
bradykinin in hearts where vascular permeability has been increased by
pretreatment with hydroxyl radicals presumably reflect facilitated access to
membrane receptors situated abluminally on the endothelial cells and/or on the
vascular smooth muscle cells (adenosine).
The coronary endothelium poses a powerful metabolic barrier, restricting the
passage of many vasoactive substances and determining the respective flow
responses. However, this barrier is not invariant, but can be modified by drugs
1 Physiologisches Institut der Universitat Munchen, 8000 Munich 2, Germany
206
2. The Metabolic Barrier of the Coronary Endothelium 207
or by enhanced vascular permeability, such as under inflammatory conditions

and in post-hypoxic myocardium.
Key words: Adenosine-Adenine nucleotides-Bradykinin-Acetylcholine-

Hydroxyl radicals
Introduction
Several potent vasoactive substances occurring in the blood or tissue fluids are
known to be metabolized during their passage through the coronary system.
This applies, for instance, to adenosine (Ado), for which uptake and
metabolism by the coronary endothelial cells are well-established processes
[1,2]. These cells also degrade the adenine nucleotides adenosine 5/-
triphosphate (ATP) , adenosine 5'-diphosphate (ADP) , and adenosine 5/-
monophosphate (AMP) by virtue of ecto-nucleotidases [2-4]. Similarly,
acetylcholine (Ach) infused into isolated guinea pig hearts is extensively
hydrolyzed and, thus, inactivated [5]. A substantial inactivation of infused
bradykinin (Bk) is also to be expected, since an angiotensin-converting
enzyme, identical to the Bk-degrading enzyme kininase II, is localized in the
coronary endothelium [6].
At the very least, the effective concentrations of the above-mentioned
vasodilators established at the smooth muscle or endothelial cell membrane
receptors mediating the respective vascular response will be modified by
endothelial metabolism. Indeed, if the coronary endothelium acts as a
sufficiently powerful metabolic barrier, then differences can be expected to
exist in the mechanisms by which vasodilation is elicited, depending upon
whether the agonists are acting from the intraluminal or interstitial side of the
vessel wall [4].
To quantify the importance of the endothelial barrier and to further explore
the active role of the endothelium in mediating flow increases in an intact
coronary vascular bed, experiments were performed on isolated perfused
hearts of guinea pigs. The agents tested included Ado, AMP, ATP and its
derivatives a,B- and B,y-methylene ATP (MeATP), Ach, Bk, and sodium
nitroprusside (SNP). Adenosine was generally infused at concentrations below
1 /lM, since at such levels the endothelium has been reported to completely
preclude access to the perivascular tissue [1,4]. Under this condition, dilatation
by infused Ado must, therefore, be mediated by the coronary endothelial cells
[4,7]. B,y-MeATP was of interest because it is not degraded intraluminally
by ecto-A TPases; however, there is recent evidence for dephosphorylation to
AMP by a pyrophosphohydrolase localized in the interstitial compartment [8].
In most vascular beds, dilatation by Ach and Bk was endothelium-dependent,
in contrast to the action of SNP [9], which served as a non-metabolizable
control agonist for directly inducing relaxation of the coronary smooth muscle
cells.
208 B.F. Becker et al.
Pretreatment of hearts with agents capable of generating hydroxyl radicals

created graded increases in paracellular leakage and, thus, in the access of
infused agonists to the abluminal vessel structures. It was, therefore, possible
to compare flow responses under conditions of "normal" and enhanced
permeability, such as may exist in inflamed or post-hypoxic tissue.

Ado, AMP, and ATP were supplied by Boehringer-Mannheim, Germany. Bk,
a,~- and ~,y-methylene adenosine 5f-triphosphate (MeATP), blue dextran
(mol. weight approx. 2 x 106 ) and theophylline came from Sigma, Munich,
Germany. Ach, SNP, and the components of the Krebs-Henseleit buffer and of
the system used to generate hydroxyl radicals were purchased from Merck,
Darmstadt, Germany. [18- 14C]-adenosine (specific activity 55 Ci/mol) was
obtained from Amersham Buchler, Braunschweig, Germany. The drug
ramiprilat was generously provided by Hoechst AG, Frankfurt, Germany.
Hearts of guinea pigs (male, 250-300 g) were isolated and perfused with
modified Krebs-Henseleit buffer, as previously described [10,11], in a non-
working mode (Langendorff preparation) at 37°C and pH7.4. Perfusion was
generally performed at constant pressure (80cmH20); in some experiments
constant volume perfusion was employed. To collect the so-called
"transudate", the fluid forming on the epicardial surface of the ventricles (c.f.,
8,12], all the truncated vessels on both the left and the right atrium were
ligated. Drops of transudate were sampled from the apex of the heart for
periods of 2min. Coronary effluent perfusate was sampled from the canulated
pulmonary artery (I-min collection periods).
The vasoactive agents were infused into the coronary system via a mixing
chamber fitted with a magnetic stirrer and located in the aortic feedline just
proximal to the heart. Flow responses were always assessed in the 5th -6th min
of application, i.e., after a steady-state had been reached. To selectively label
the adenine nucleotide pool of the endothelial cells in guinea pig hearts,
[8- 14C]-adenosine was infused for 30min at submicromolar concentrations
[4,7,10]. Radiactivity was measured by scintillation counting (Packard Tri-carb
4000, counting efficiency 80% ). Separation and analyses of adenine
nucleotides, purine nucleosides, and bases in samples of perfusate were
achieved by high pressure liquid chromatography techniques [10]. Dextran blue
was determined photometrically at 610nm in 1:5 diluted samples of perfusate
and transudate [12], collected in the 10th-15th min of perfusion with Krebs-
Henseleit buffer containing 0.25% by weight of the macromolecular substance.
To generate hydroxyl radicals (OH) intravascularly, H2 0 2 (final
concentration 10 IlM) and a solution containing FeS04, ethylenediamine
tetraacetate and ascorbic acid (final concentrations 10 IlM, 100 IlM, and 10 IlM,
respectively) were infused from two separate syringes. These components
catalytically form OH at a rate of approximately 0.004fmole/min, as
determined via a chemiluminescence assay [11,13].
Results
Assessment of Intracoronary Degradation

In the case of adenosine, uptake and degradation during coronary passage are
readily demonstrable with the aid of [8- 14C]-Ado. Table 1 lists the proportion
of the label emerging again unchanged in the venous effluent during infusion of
10-7 M-Iabeled Ado, as well as the total radioactivity being released from the
heart at that moment. The latter represents the sum of 14C-Ado and
degradatives (inosine, hypoxanthine, xanthine, and uric acid) reappearing from
the myocardial tissue. From the data, it is obvious that about 80% of the
infused Ado was being taken up, most of this being incorporated
intracellularly. About 20% of the infused Ado emerged again as vasoinactive
degradation products. Appropriately, the extent of uptake depended upon the
duration of contact with the vascular tissue, proportionally less Ado
disappearing from the perfusate as the rate of coronary flow increased (Table 1).
In hearts where the adenine nucleotide pool of the endothelial cells had been
selectively prelabeled with 14C-Ado, radioactive Ado and degradatives were
still released into the coronary effluent perfusate long after cessation of Ado-
infusion. As shown in Fig. 1, 30min after labeling, about twice as much
radioactivity was present as inosine than as Ado. However, when the
adenosine uptake blocker, dipyridamole, was added to the perfusate, the
amount of 14C-inosine dropped markedly, while the Ado counts rose by a
corresponding amount. This result indicates that endogenously formed and
released Ado also undergoes reuptake and degradation in the coronary system.
Nucleoside transport blockers, such as dipyridamole, are well known to
enhance the dilatory action of infused Ado. An analogous potentiation of the
dilatory efficacy of bradykinin in the presence of ramiprilat, a fast-acting
converting enzyme inhibitor, is evidenced in Fig. 2. The drug had no effect on
either basal coronary flow or on the action of SNP (10-5 M).
The comparative metabolic activities of the endothelial ecto-ATPase,
-ADPase and -5 ' -nucleotidase were established by infusing ATP or AMP into
TABLE 1. Uptake and degradation of adenosine in the coronary system. 14C-adenosine

(10- 7 M) was infused at various rates of coronary flow and the amounts of radioactive
adenosine (Ado) and of total radioactive material emerging in the coronary effluent
perfusate were determined in the 5-6th min of infusion. Effluent values are expressed
as % of the infusion level. Means ±SD, 3 hearts each
Venous recovery [% of infusion level]
Ado Total radioactivity
4 15 ±6 37 ± 10
5 19 ± 4 38 ± 7
6 23 ± 4 40 ± 6
Radioactivity Coronary Flow

Bq Imin ml/min
9000
10
I ~
6000 + Ramiprilat, 10mg It
5
Bradykinin,lO- lO M
3000
o 1
o 5 10 15 min
FIG. 2. Potentiation of the dilatory effect of
bradykinin by co-infusion of ramiprilat, a fast-acting
Adenosine /':, Inosine inhibitor of the converting enzyme (kininase II)
FIG. 1. Release of radioactive adenosine and inosine from guinea pig hearts, 30-35 min
after pre labeling with [8-1 4C]-adenosine (3 x 1O- 7 M, 30 min). Rates of release under
control conditions (~) and in the presence of 5 x 1O- 7 M dipyridamole (0) are given ±
standard error of the mean (SEM) for 6 and 5 hearts, respectively. Dipyridamole was
applied for 10 min, starting 25 min after labeling
guinea pig hearts and analyzing some of the metabolites appearing in the
coronary effluent perfusate under steady-state conditions (Table 2). While the
amount of ATP emerging unchanged was very small (3%-5%) and ADP
comprised some 15%, AMP seemed to be more resistant to
dephosphorylation. Indeed, when AMP itself was infused, about 70%
reappeared as such at the high flow rates (23-25 mllmin per g) pertaining
under these conditions. Interestingly, infused ~,'Y-methylene ATP, a Pz-
purinoceptor agonist, was also degraded in the heart, about 30% emerging as
TABLE 2. Degradation of infused ATP and AMP in isolated guinea pig hearts. The
values (±SD) represent the recovery of vasoactive metabolites in the coronary effluent
perfusate, expressed as percentage of the infusion concentration (5 x 1O-7 M). All
hearts were perfused at constant pressure. Coronary flow in the two groups averaged
25.0 and 23.2 ml/min/g, respectively. n, Number of hearts; ATP, ADP, AMP, adenosine
5'-tri-, di- and monophosphate; Ado, adenosine
Metabolites recovered [%]
Nucleotide infused ATP ADP AMP Ado
ATP (n = 4) 4±3 13 ± 6 49 ± 12 6±2
AMP (n = 5) 71 ± 5 4±2
AMP and Ado under low flow conditions. These metabolites may, in fact, have
been vasoactively important, because the dilatory action of ~,y-MeATP could
be substantially inhibited by the Prreceptor antagonist theophylline (results not
shown). Dephosphorylation of a,~-methylene ATP to the vasoinactive ADP
analogue only amounted to 7% and, thus, cannot account for the absence of
dilatation in response to infusions of this particular triphosphate (5 x 10- 7 to
10- 5 M) in the guinea pig heart.
Effect of Increased Vascular Permeabilty

Pretreatment of hearts for 40 min with a system of reagents capable of
generating hydroxyl radicals tripled the rate of formation of transudate on the
epicardial surface (Fig. 3). Furthermore, the concentration gradient for infused
dextran blue existing between the intravascular perfusate and the transudate
was substantially lowered (Fig. 3). Both findings are indicative of an increase in
vascular permeability, with the facilitated passage of the macromolecular
dextran suggesting this leak to be paracellular in character.
The data in Table 3 reflect that this also allows substances such as Ado to
more easily overcome the metabolic barrier posed by the endothelial cell
lining. The observed doubling in the transudate concentration of Ado during
infusion, considered together with the threefold rise in transudate flow (c.f.,
Fig. 3), signifies an estimated 6 times higher rate of passage into the interstitial
space. Since the concentration of Ado in the coronary effluent stayed low,
pretreatment with OR had not detectably decreased endothelial uptake, the
mechanism responsible for the removal of Ado.
Enhancing the passage of infused vasoactive agents into the perivascular
space caused marked alterations in some of the vascular responses. As shown
in Fig. 4, the dilatory efficacies of Ach, Bk and Ado were improved, while that
of ~,y-methylene ATP was reduced. Dilatation by SNP remained uninfluenced.
In the case of Ado, it was established that the phenomenon corresponded to a
shift to the left of the concentration-response curve for coronary flow (Fig. 5).
Discussion
The extensive degradation and incorporation of infused ATP, ADP, AMP, and
adenosine within the coronary system results from the selective endowment of
the endothelial cells with specific enzymes and membrane transport systems
[2,4,14]. The ensuing ability of the coronary vascular endothelium to act as a
metabolic barrier probably also restricts passage of adenine nucleotides and
Ado from the interstitial to the intravascular space. This conclusion is
supported by previous studies performed on hearts in which the endothelial
adenine nucleotide pool had been selectively prelabeled by infusing radioactive
Ado [10]. In the presence of the transport inhibitor dipyridamole, there was a
substantial rise in the release of unlabeled Ado-stemming from the
Transudate Flow Interstitial Dextran Blue

(ml.min-l~ 9- 1) (0'0 of perfusate cone.)
***
(5)
0:10 100
**
(5)
~
0.05 50
(4) (4)
-
~
:g
:g
0
Control OH Control OH
FiG. 3. Enhancement of vascular permeability by pretreatment of hearts with a chemical
system capable of generating hydroxyl radicals (OB). Transudate formation and
permeability towards dextran blue, as assessed by the relative concentrations of dextran
blue in the transudate and the coronary perfusate, were determined 10-15 min after
cessation of OR production. Mean values ±SD, number of hearts in parentheses, ** P
< 0.01, ***P < 0.001 vs control (t-test for unpaired samples)
TABLE 3. Metabolism of adenosine in control hearts and hearts pretreated for 40 min
with hydroxyl radicals. Ado (100 nM) was infused 5 min after cessation of OR genera-
tion and the concentrations in the coronary effluent and transudate determined under
steady-state conditions of flow (range 14-18mllmin per g). Mean values ±SD, number
of hearts in parentheses
Adenosine concentrations [nM]
Coronary effluent Transudate
Control 35.6 ± 9.9 (6) 8.4 ± 6.6 (5)
OR-Pretreated 32.4 ± 9.8 (7) 15.5 ± 7.7 (5)
D Control ~ OH
Flow Increase
(x+SD)
ml * *
(11) (6)
minxg (5)
(7)
10 * (5)
(6) T~
(14) *
(7)
T
5 (5) (13)
T I
o ~
Bk Ado MeATP SNP
0.1 nM 0.1 ~M 0.5 ~M 10 ~M
FIG. 4. Effect of pretreatment of hearts with hydroxyl radicals (OR) on coronary flow
responses. Vasodilators were tested 5-lOmin after washout of the OR-generating
system (see Methods). Basal coronary flow ranged from 7 to 9mllmin per g in all
groups. Numbers of hearts in parentheses, *P < 0.05 vs control (t-test for unpaired
samples), MeATP = p,y-methylene ATP
l1CF
17)
20
---'JlL 15)
min·g
OH- Pretreated
15
10
/
/
/
5 /
/ 114)
/
o i
0.1 OS i
5 ~M
Adenosine Concentration
FIG. 5. Concentration dependence of adenosine-induced increases of coronary flow

(L\CF), as influenced by pretreatment of hearts with hydroxyl radicals. For details, see
legends to Figs. 3 and 4; - 0 - , control hearts; -e-, OR-pretreated hearts; mean
values ±SD; numbers of hearts in parentheses
213
cardiomyocyte nucelotide pool-whereas the chief degradative, uric acid,

showed a corresponding drop. Since the enzyme responsible for uric acid
formation in the heart, xanthine dehydrogenase, only occurs in the
microvascular endothelial cells [4,10,14], it was clear that endogenously formed
adenosine is also largely taken up and metabolized by the coronary
endothelium. Moreover, the finding that endothelial cells take up and
metabolize Ado released from endothelial cells upstream in the coronary
system (Fig. 1) confirms the postulated role of the vascular endothelium in
establishing the homeostasis of Ado [15].
Because practically no infused Ado could be found in the transudate, a fluid
considered to represent in its composition the ventricular interstitial fluid
[8,12], it seems that Ado infused at low concentrations mediates coronary
dilatation via the endothelium. However, at concentrations exceeding the
metabolic capacity of the endothelium, a direct effect of Ado on the vascular
smooth muscle cells, also leading to relaxation, will come to bear. Other
observations have led to the same conclusion [1,4,16,17].
The a,~- and ~,y-methylene derivatives of ATP also seem subject to
metabolic degradation in the guinea pig heart. Whereas a,~-MeATP could be
degraded to a,~-MeADP by the endothelial ecto-ATPase, in the case of ~,y
MeATP the barrier may be a function of perivascular cells rather than of the
endothelium [8]. Catabolism may explain the respective lack of vasodilatory
activity and the inhibition of flow increase by theophylline. The latter should,
according to the pharmacology of purinoceptors, be without influence on the
endothelial P2y-receptor-mediated effect of ~,y-MeATP [18,19]. Alternatively,
the absence of dilatation could reflect an extremely low sensitivity of P2y-
receptors in the guinea pig heart towards methylene analogues of A TP.
Notwithstanding, the endothelium strongly modifies the concentrations of
endogenously occurring adenine nucleotides, Ado, and Bk at the vessel wall
and, thus, at the respective receptors mediating changes in vascular tone. This
is indirectly evidenced by the potentiating effect of adenosine uptake blockers
on the action of Ado [19,20] and that of the converting enzyme inhibitor
ramiprilat on dilatation by Bk.
It is of particular interest that enhancing vascular permeability markedly
stimulated not only dilatation by Ado and Bk, but also that by Ach, another
compound readily degraded intravascularly [5]. Due to rapid paracellular
transit in such hearts, more Ado and, by corollary, more Bk and Ach were
gaining access to the perivascular space, i.e., to the abluminal side of the
endothelial cells and to the vascular smooth muscle cells. With respect to Ado,
the stronger dilatory response of "leaky" hearts could result from an enhanced
or additional action of this nucleoside on smooth muscle adenosine receptors,
since these are known to also induce relaxation [16]. The potentiated responses
of "leaky" hearts to Ach and Bk must be explained differently, mainly because
smooth muscle cells of the coronary resistance vessels do not appear to possess
receptors mediating dilatation by these substances; Ach and Bk effects are
totally endothelium-dependent in the guinea pig heart [17]. Hence, it is
tempting to assume that receptors mediating dilatation by Ach and Bk are not
only present at the luminal, but also at the abluminal side of the endothelial
cell membrane.
There may also be abluminal endothelial receptors for Ado and adenine
nucleotides, because the reduced dilatory propensity of ~,y-MeATP in
permeabilized hearts could reflect an overriding P2x-receptor-mediated
contraction of the vascular smooth muscle. Although such effects of adenine
nucleotides have been characterized in hearts of other species and in other
vessel preparations [18], there is as yet no real proof for the existence of
P2x-purinoceptors in the guinea pig coronary system. In the case of Ach, no
strong vascular constriction was to be expected in the wake of increased
permeability, since the contraction of coronaries via muscarinic receptors on
the vascular smooth muscle cells seems to be far weaker in the guinea pig than,
e.g., in the rat [21].
Finally, the hearts pretreated with hydroxyl radicals showed no electron-
microscopically detectable vascular damage. This is the same situation as that
encountered in posthypoxic heart tissue [22], in which increased vascular
permeability is a well-known disturbance. Thus, the endothelial metabolic
barrier is most probably infringed in reperfused and in inflamed myocardium,
and alterations of vascular responsiveness must be expected under such
pathological conditions. The changes, however, will probably not be uniform,
depending upon the vasoactive substance and, most likely, on the species.
References
1. Nees S, Gerlach E (1983) Adenine nucleotide and adenosine metabolism in
cultured coronary endothelial cells: Formation and release of adenine compounds
and possible functional implications. In: Berne RM, Rail TW, Rubio R (eds)
Regulatory function of adenosine. Martinus Nijhoff, The Hague, pp 347-360
2. Pearson JD, Gordon JL (1985) Nucleotide metabolism by endothelium. Ann Rev
PhysioI47:617-627
3. Baer HP, Drummond GI (1969) Catabolism of adenine nucleotides by the isolated
perfused rat heart. Proc Soc Exp Bioi Med 127:33-36
4. Nees S, Herzog V, Becker BF, Bock M, Des Rosiers Ch, Gerlach E (1985) The
coronary endothelium: A highly active metabolic barrier for adenosine. Basic Res
Cardiol 80:515-529
5. Dieterich HA, Loffelholz K (1977) Effect of coronary perfusion rate on the
hydrolysis of exogenous and endogenous acetylcholine in the isolated heart.
Naunyn Schmiedebergs Arch Pharmacol 296:143-148
6. Seitz RJ, Neuen E, Henrich M, Schrader J, Wechsler W (1987) Angiotensin
I-converting-enzyme (ACE): A marker for vascular endothelium. In: Cervos-
Navarro J, Ferszt (eds) Stroke and microcirculation. Raven, New York, pp 111-
115
7. Newman WH, Becker BF, Heier M, Nees S, Gerlach E (1988) Endothelium-
mediated coronary dilatation by adenosine does not depend on endothelial
adenylate cyclase activation: Studies in isolated guinea pig hearts. Pflugers Arch
413:1-7
8. Imai S, Chin W-P, Jin H, Nakazawa M (1989) Production of AMP and adenosine in
heart. Pflugers Arch 414:443-449
9. Furchgott RF (1990) Studies on endothelium-dependent vasodilation and the
endothelium-derived relaxing factor. Acta Physiol Scand 139:257-270
10. Becker BF, Gerlach E (1987) Uric acid, the major catabolite of cardiac adenine
nucleotides and adenosine, originates in the coronary endothelium. In: Gerlach E,
Becker BF (eds) Topics and perspectives in adenosine research. Springer-Verlag,
Berlin, pp 209-222
11. Becker BF, Reinholz N, 6zc;elik T, Leipert B, Gerlach E (1989) Uric acid as
radical scavenger and antioxidant in the heart. Pflugers Arch 415:127-135
12. Wienen W, Kammermeier H (1988) Intra- and extracellular markers in interstitial
transudate of perfused rat hearts. Am J Physiol 254:H785-H794
13. Becker BF, Reinholz N, Leipert B, Raschke P, Permanetter B, Gerlach E (to be
published) The role of uric acid as an endogenous radical scavenger and
antioxidant. Chest
14. Nees S, Dendorfer A (1990) Catabolism of adenine nucleotides and adenosine by
the coronary microvasculature: Physiological consequences. Jap J Pharmacol 52
(Suppl II):P33
15. Gerlach E, Becker BF, Nees S (1987) Formation of adenosine by vascular
endothelium: A homeostatic and antithrombogenic mechanism? In: Gerlach E,
Becker BF (eds) Topics and perspectives in adenosine research. Springer-Verlag,
Berlin, pp 309-320
16. Headrick JP, Berne RM (1990) Endothelium-dependent and -independent
relaxations to adenosine in guinea pig aorta. Am J Physiol 259:H62-H67
17. Becker BF, Leipert B, Gerlach E (to be published) Alterations of microvascular
flow responses in the heart by oxygen-derived radicals, HOCI and the K+-channel
blocker glibenclamide. In: Niimi H, Hori M, Naritomi H (eds) Microcirculatory
disorders in the heart and brain. Gordon and Breach, New York
18. Burnstock G (1988) Local purinergic regulation of blood pressure. In: Vanhoutte
PM (ed) Vasodilatation: Vascular smooth muscle, peptides, autonomic nerves, and
endothelium. Raven, New York, pp 1-14
19. Olsson RA, Pearson JD (1990) Cardiovascular purinoceptors. Physiol Rev 70:761-
845
20. Becker BF, Bardenheuer H, Overhage de Reyes I, Gerlach E (1985) Effects of
theophylline on dipyridamole-induced coronary venous adenosine release and
coronary dilation. In: Stefanovich V, Rudolphi K, Schubert P (eds) Adenosine:
Receptors and modulation of cell function. IRL, Oxford, pp 441-449
21. Kitamura K, Kuriyama H (1979) Effects of acetylcholine on the smooth muscle cell
of isolated main coronary artery of the guinea pig. J Physiol (Lond) 293:357-363
22. Ward BJ, Firth JA (1989) Effect of hypoxia on endothelial morphology and
interendothelial junctions in the isolated perfused rat heart. J Mol Cell Cardiol
21:1337-1347
3
Regulation of Vascular Tone
by Endothelium-Derived
Contracting Factor (EDCF)
Takayuki Ito, Toshio Kato, Yoshio Iwama, Masahito Muramatsu,
Kiyokazu Shimizu, Hiroshi Asano, Kenji Okumura,
Hidekazu Hashimoto, and Tatsuo Satake
Summary. Our purpose was to identify endothelium-derived contracting factor

produced by acetylcholine stimulation in the aorta of spontaneously hyperten-
sive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. The rings of the
thoracic aorta were obtained from age-matched SHR and WKY rats, and
changes in isometric tension were recorded. The relaxant responses to
acetylcholine in the aortic rings from SHR were significantly weaker than those
from WKY rats. The relaxant responses to acetylcholine were significantly
enhanced by pretreatment with a cyclooxygenase inhibitor (indomethacin) or
the thromboxane A 2iprostaglandin H2 receptor antagonist (ONO-3708) in
aortic rings from both SHR and WKY rats. A thromboxane A2 synthetase
inhibitor (OKY-046) did not affect the acetylcholine-induced relaxation in the
aortic rings from either SHR or WKY rats. When these rings were pretreated
with an inhibitor of nitric oxide production (N-nitroarginine methylester), the
relaxant response induced by acetylcholine was completely inhibitied. In the
organ bath solution, prostaglandin E2 and 6-keto-prostaglandin F la con-
centrations increased after acetylcholine stimulation, but prostaglandin F 2a and
thromboxane B2 concentrations did not. Exogenous prostaglandin H 2, a stable
analogue of thromboxane A 2, and prostaglandin F2a induced contractions of
the SHR rings at a lower concentration than prostaglandin E 2, prostaglandin
D 2, and prostaglandin 12, These contractile responses to various prostaglandins
were markedly inhibited by pretreatment with ONO-3708. A prostacyclin
sinthetase inhibitor did not affect the relaxant responses to acetylcholine in the
SHR rings. These results show that endothelium-derived contracting factor is
produced and released by acetylcholine stimulation the aorta of not only SHR
but also in WKY rats, and suggest that prostaglandin H 2, a precursor of the
released prostaglandins, is a strong candidate for being endothelium-derived
contracting factor produced by acetylcholine stimulation. It was also suggested
The Second Department of Internal Medicine, Nagoya University School of Medicine,

Nagoya, 466 Japan
217
218 T. Ito et al.
that in the rat aorta treated by acetylcholine, the vascular tonus is regulated by
two factors, prostaglandin H2 (EDCF) and nitric oxide (endothelium-derived
relaxing factor).
Key words: Endothelium-Acetylcholine-Prostaglandin H2-Spontaneously
hypertensive rats-Aorta
Introduction
In 1980, Furchgott and Zawadzki found that acetylcholine (ACh) induced
endothelium-dependent relaxation in the rabbit aorta [1]. Since then, various
substances have been reported to induce endothelium-dependent relaxation in
most of the blood vessels in mammals [2]. Several pharmacological
observations have strongly suggested that there is more than one endothelium-
derived relaxing factor (EDRF) [3] and that nitric oxide is one of them [4].
After the discovery of EDRF, studies by Vanhoutte and coworkers [5-8]
have shown that endothelial cells produce not only EDRF but also
endothelium-derived contracting factors (EDCF) after various stimulations.
Several substances have been suggested as EDCF. In canine basilar arteries,
calcium ionophore A23187, arachidonic acid, and ACh caused endothelium-
dependent contractions. In the case of A23187 and arachidonic acid, TXA2
contributed to the endothelium-dependent contractions [9]. A recent
publication has suggested that one of the EDCF may be the super oxide anion
[10].
In 1988, endothelin was identified as an EDCF. The endothelin-induced
contraction is resistant to cyclooxygenase inhibitors, lipoxygenase inhibitors,
and p-adrenergic, histaminergic, and serotonergic antagonists [11].
In the aorta of spontaneously hypertensive rats (SHR), ACh causes a
simultaneous release of EDRF and EDCF that differs from endothelin, which
is a peptide and is considered to be a cyclooxygenase product [12]. However,
the details remain obscure. We conducted the present study to identify the
EDCF which is produced by ACh stimulation in the SHR aorta.

The thoracic aorta from male SHR and normotensive Wistar-Kyoto (WKY)
rats matched for age (30-32 weeks) and body weight (330-400 g) was used.
Blood pressure measured by the tail cuff method in the unanesthetized state
was 191.9 ± 3.4mmHg in the SHR (n = 8) and 140.6 ± 1.5mmHg in the
WKY rats (n = 8).
The rats were decapitated and the thorax was opened. The thoracic aorta
was immediately removed and placed in cold Krebs-Henseleit solution. The fat
and connective tissues on the vascular surface were removed, and the vessel
was cut by scissors into rings (5 mm in length). After two stainless steel wires
3. Regulation of Vascular Tone 219
were inserted into the vascular lumen, the rings were suspended in an organ
chamber which contained 30 ml buffer bubbled with a mixture of 95% O 2 and
5% CO2, maintained at 37°C, and connected to a force-transducer. The resting
tension was adjusted to 1.0 g. The intimal surface was removed by gentle
rubbing with a swab inserted into the vascular lumen. Removal of the intima
was confirmed by the absence of responses to 10- 7 MACh.
Responses to Acetylcholine and Effects of Inhibitors

and an Antagonist
The aortic rings with and without endothelium from SHR and WKY rats were
contracted with 1O- 7 M norepinephrine (NE). After the contraction reached a
plateau, ACh was added at cumulative concentrations of 10- 8 to 10- 5 M to
relax the rings. Fifteen minutes before the induction of contractions by NE,
indomethacin [13], which is a cyclooxygenase inhibitor (1O- 5 M), OKY-046
[14], which is a TXA2 synthetase inhibitor (10- 5 M), and ONO-3708 [15],
which is a TXA2/prostaglandin H2 (PGH2) receptor antagonist (1O- 6 M) were
added separately to the bath solution, and each effect on relaxant responses to
ACh was evaluated. The rates of relaxation were expressed as percentages to
the contraction induced by 10- 7 M NE.
Concentrations of Prostaglandins in the Bath Solution

This and the following experiments were done in the aortic rings from only the
SHR.
In the SHR rings, changes in the concentrations of various prostaglandins
(PGs) in the bath solution were evaluated before and after addition of ACh.
Immediately before the induction of contractions by NE, at the peak response
to 1O- 7 M NE, and at the peak response to 1O- 5 M ACh after the cumulative
addition of 10- 8 to 10- 5 MACh, the organ bath solution (1 ml) was obtained,
and the concentrations of PGE2, 6-keto-PGF1'H and TXB 2 (using an RIA kit
prepared by New England Nuclear) and PGF2a (using an RIA kit prepared by
Clinical Assays) were determined by the radioimmunoassay methods of Jaffe
[16] and Powell [17].
Responses to Exogenous Prostaglandins

PGH2> PGF2a , a stable analogue of TXA 2 (STA 2) [15], PGE2, and PGD 2 were
added at cumulative concentrations of 10-9 -10- 6 M and PGI2 at 10-9 -10- 5 M
to the bath solution for the SHR aortic rings with and without endothelium at
the basal state and at the precontracted (by NE 1O- 7 M) state. The vascular
responses and the effects of ONO-3708 (1O- 6 M) on these responses were
evaluated. The rate of contraction was expressed as percentages to the
contraction induced by 1O-7 M NE.
220 T. Ito et al.
Effects of a Prostacyclin Synthetase Inhibitor

Tranylcypromine [18], a prostacyclin synthetase inhibitor, was added to the
bath solution at a concentration of 10-4 M 15 min before the induction of
contraction by NE, and its effects upon relaxant responses to ACh in the SHR
rings were evaluated.
Effects of an Inhibitor of Nitric Oxide

The SHR aortic rings were contracted with NE (10- 7 M), and, after observing
a plateau of contraction, relaxed with ACh (10- 5 M). Indomethacin, ONO-
3708, and an inhibitor of nitric oxide N-nitroarginine methylester [19] (NNM,
10- 3 M) were added to the bath 15 min before induction of contraction with
NE.
Drugs
The following drugs from Sigma Chemical Co. (St. Louis, Mo.) were used:
I-norepinephrine bitartrate, acetylcholine chloride, indomethacin,
tranylcypromine, sodium nitroprusside, PGI2 , and NNM. Ono Pharmaceutical
Company (Osaka, Japan) provided the PGF2a , PGE2 , PGD 2 , PGH2 , OKY-
046, and ONO-3708. Results were expressed as the means ± -SEM. For
statistical analysis, Student's (-test for paired or unpaired observations and the
Wilcoxon-Man-Whitney test were used. Values of P < 0.05 were considered to
be significant.
Results
Responses to Acetylcholine
In the aortic rings from both SHR and WKY rats, the maximum relaxation was
observed with 1O- 7 M ACh. At higher concentrations, ACh induced
contractions (Figs. 1a,2). In the rings without endothelium from SHR and
WKY rats, responses to ACh were negligible (Fig. 1b). The ACh-induced
relaxation in rings from SHR rats was significantly weaker than that in rings
from WKY rats at ACh concentrations of 10- 7 to 1O- 5 M (Fig. 2).
Effects of Inhibitors and an Antagonist

In the SHR rings, relaxant responses were enhanced by pretreatment with
indomethacin (10- 5 M) at ACh concentrations of 10- 7 -1O- 5M, and the
contractile responses observed at high ACh concentrations disappeared
(Fig. 2). Similarly, in rings from the WKY rats, relaxant responses were
significantly enhanced at ACh concentrations of 10- 6 _10- 5 M, resulting in
similar responses in rings from both SHR and WKY rats (Fig. 2).
OKY-046 (1O- 5 M) did not affect the relaxant responses to ACh in rings
from either SHR or WKY rats.
a b
ACh 10-810-7
ACh 10-8 1D-6 10- 5MWO
I 10-7 I I I I +
j
I
O.le
I
NE 10-7M ~ NE 1ri-7M
FiG. 1. Typical records of acetylcholine (ACh)-induced responses in spontaneously

hypertensive rat aortic rings a with and b without endothelium. Rings were contracted
with 1O- 7M norepinephrine (NE) , and ACh was cumulatively added at 1O- 8 -1O- 5 M.
Wo, wash out. (From [25] with permission)
o - SHR control
.. -- .. SHR indomethacin 10-'M
1>-1>. WKY control
.---. WKY indomethacin 10-'M
(n = 6) **
20
~
= 40
-..,..,
C>
><
II)
a::
60
80
100~~------~----~----~-
10- 1 10- 6 10- 5
Acetylcholine (M)
FIG. 2. Cumulative concentration-response curves for acetylcholine in spontaneously

hypertensive rat (SHR) and Wistar-Kyoto (WKY) rat aortic rings with and without
endothelium and showing effects of indomethacin (lO-5M) on the curves. Rate of
relaxation was expressed as percentages to contraction induced by 10-7 M
norepinephrine. Results are shown as the means ± SEM. **P < 0.01 between the
presence and absence of indomethacin. (From [25] with permission)
222 T. Ito et al.
ACh
0.1 g
I
NE 10-7M
L min
10
FiG. 3. Effects of ONO-3708 (1O- 6 M) on acetylcholine (ACh)-induced responses in
spontaneously hypertensive rat aortic rings with endothelium. N, Norepinephrine; Wo,
wash out. (From [25] with permission)
To determine whether TXA2 is involved, the effects of ONO-3708 (1O- 6 M)

were evaluated. In the SHR aorta rings, relaxant responses were enhanced by
pretreatment with ONO-3708 at ACh concentrations of 10- 7 _10- 5 M, and the
contractile responses observed at high ACh concentrations disappeared (Figs.
3,4). Similarly, in the rings from the WKY rats, relaxant responses were
enhanced at ACh concentrations of 10- 6 _10- 5 M, resulting in relaxation
similar in degree to that of rings from SHR and WKY rats (Fig. 4). The degree
of the enhancement of relaxation was comparable with that after pretreatment
with indomethacin.
Concentrations of Prostaglandins in the Bath Solution

In the SHR rings, the concentrations of PGE2 and 6-keto-PGF la increased
about three- to fourfold after ACh stimulation. The concentration of PGE2 in
the bath solution after ACh stimulation was about lO- lO M, and that of 6-keto-
PGF la was about 1O-9 M. The concentrations of PGF2a and TXB 2 did not
change significantly (Fig. 5).
Responses to Exogenous Prostaglandins

In the SHR rings with endothelium, PGF2a , STA2 , PGH 2 , PGE2 , PGD 2 , and
PGI 2 induced only contractions at the basal state. STA2 , PGF2a and PGH2
induced contractions at 1O-7 M or more, while PGE2 and PGD 2 induced
contractions only at a high concentration of 10- 6 M and PGI2 at 10- 5 M (Fig.
6). ONO-3708, at a concentration of 1O- 6 M, inhibited all contractile responses
to the various prostaglandins (Fig. 6). In the SHR rings without endothelium at
the basal state, results similar to those in the SHR rings with endothelium were
obtained (data not shown).
In the SHR rings with and without endothelium, PGF2a , STA2 , PGH2 ,
PGE2 , and PGD 2 also induced only contractions at the precontracted state.
0 - - - 0 SHR control
.. -- .. SHR ONO-3JOB IO-6M
t.-t.WKY control
A---A WKY ONO-3JOB IO-6M
(n =6)
**
20
-......
c 40
co
><
Q)
co:: 60
80
~'.-.,
.......
".,......
,,~:-:--------
~---------i:
100~~------~----~------~
10- 7 10- 6
Acetylcholine (M)
FIG. 4. Effects of aNa-3708 (10- 6 M) on acetylcholine-induced responses in

spontaneously hypertensive rat (SHR) and Wistar-Kyoto (WKY) rat rings with
endothelium. Rate of relaxation was expressed as percentages to contraction induced by
1O- 7 M norepinephrine. Results are shown as means ± standard error of the mean
(SEM). * *P < 0.01 between the presence and absence of aNa-3708. (From [25] with
permission)
Results similar to those at the basal state were obtained, although the
percentage of contraction was reduced (data not shown).
Effects of a Prostacyclin Synthetase Inhibitor

The contractions in the SHR aortic rings induced by PGI 2 at high
concentrations presented the possibility that PGI 2 is EDCF. Therefore, the
effects of tranylcypromine (10- 4 M) on relaxant responses to ACh in SHR
rings were evaluated. Because tranylcypromine reduced the NE-induced
contractions, the rings were contracted with 10- 6 M NE to obtain responses
comparable with those in the control group. Tranylcypromine had no
significant effect on ACh-induced relaxation in SHR rings.
PGI 2 is known to have potent vasodilating effects. In the SHR rings with and
without endothelium at the basal state, contractions were induced with 10- 5 M
PGIz, but there were no significant changes at 10- 9 _10- 6 M. At the
precontracted state, negligible or only slight relaxations were observed at PGI 2
224 T. Ito et al.
c:::J control
~NE10-1M
~ NE 10- 1M + ACh 10- 5M

(n = 6)
PG [2 6-keto-PG F1,. PG F2,. TX 8 2
pg/ml ~ ** pg/ml ~ **,---,

pg/ml pg/ml
30 300
10 10
20 200
5 5
10 100
0 0 0 0
FiG. 5. Changes in concentrations of various prostaglandins (PG) in an organ bath
solution in spontaneously hypertensive rat aortic rings. The solution was obtained and
analyzed by radioimmunoassay immediately before the induction of contractions by
norepinephrine (NE) (control), when contraction induced by 1O- 7 M NE was stabilized,
and when response induced by 10- 5 M acetylcholine (A Ch) reached a peak after
cumulative ACh addition at 10- 8 _10- 5 M. Results are expressed as means ± standard
error of the mean (SEM). TX, thromboxane. ** P < 0.01 between the concentration
before and after ACh addition (From [25] with permission)
concentrations of 10- 9 _10- 6 M. Contractions were induced at 10- 5 M (data not

shown).
Effects of an Inhibitor of Nitric Oxide

When ACh (10- 5 M) was administered to the organ bath contracted with NE, a
rapid relaxant response followed by a contractile response were observed.
When ONO-3708 or indomethacin was added before ACh administration, the
contractile response was completely inhibited, and only the relaxant response
was observed (Fig. 7). By pretreatment with NNM (10- 3 M) in the presence of
ONO-3708, this relaxant response induced by ACh was completely inhibited,
and tension was sustained at a plateau (Fig. 7).
Discussion
The present study confirmed significantly weaker relaxant responses to ACh at
concentrations of 10- 7 _10- 5 M in the SHR aortic rings than in the WKY rat
aortic rings. Indomethacin and ONO-3708 enhanced the relaxations at ACh
concentrations of 10- 7 _10- 5 M in the SHR rings and at 10- 6 _10- 5 M in the
WKY rat aortic rings, resulting in similar responses in both the SHR and WKY
0-----0 ST A,
0-----. . ST A, +ONO-3108 lO-'M **
~ PGH, 1
.-----~ PG H, +ONO-3J08 lO-'M l'
0-----<0 PG F, •
• ----.. PG F1.+ONO-3108 lO-'M

0-----0 PG E,
200 ..-----+ PG E1 +ONO-3J08 10-'M

----- PG D1
,..-----x PG D1 +ONO-3J08 lO-'M
",-"PGI 1
..-----* PG 11
(n ~ 6)
tfe.. 150
-...
0:::
C>
-
c..>
~
0:::
~ 100
50
10 -9 10- 8 10- 7 10- 6 10- 5

Concentration (M)
FIG. 6. Responses of spontaneously hypertensive rat aortic rings with endothelium to
various prostaglandins (PG) and effects of ONO-3708 (10- 0 M). Rate of contraction
was expressed as percentages to contraction induced by 10- 7 M norepinephrine. Results
are shown as means ± standard error of the mean (SEM). PG, Prostaglandin; STAb a
stable analogue of thromboxane A 2 . ** P < 0.01 between presence and absence of
ONO-3708. (From [25] with permission)
rat aortic rings. ONO-3708 did not affect the relaxations to sodium
nitroprusside nor the contractions induced by NE. Therefore, the relaxant
responses to ACh would not be enhanced by the direct effect of ONO-3708 on
the vascular smooth muscle. These results suggest that a substance that is
inhibited by indomethacin and ONO-3708 is produced and released by ACh
stimulation in the endothelium. This substance (EDCF), produced and
released simultaneously with EDRF, seems to weaken ACh-induced
relaxations. EDCF has been considered to be present only in SHR [12].
However, our results suggest that EDCF is also produced and released by ACh
stimulation in the WKY rat aorta. Endothelium-dependent relaxation have
226 T. Ito et al.
Ach 10- 5M
~
0
0--0 control
20 t!r--6 NNM +ONO-3708
~ >+-+< Indomethacin
-
c:
<:> 40 - ONO-3708
~
(n = 7)
><
...
~
a::
60
80
100
0 10 20 30 min
FiG. 7. Effects of ONO-3708, indomethacin, and ONO-3708 plus N-nitroarginine
methylester (NNM) on acetylcholine-induced responses in spontaneously hypertensive
rat aortic rings with endothelium. Rings were contracted with 10- 7 M norepinephrine
and 10-5 M acetylcholine was added. The rate of relaxation was expressed as the
percentage of contraction induced by 1O- 7 M norepinephrine
been reduced in vascular smooth muscle of the rat with increasing age [20].
The existence of EDCF in WKY rats suggests that EDCF may participate in
the reduced endothelium-dependent relaxations with increasing age.
Therefore, age and blood pressure may promote endothelium-dependent
contractions in the aorta of the rat.
Reports have suggested that a substance in the cyclooxygenase system [12] is
the EDCF produced and released from the aorta of SHR. This was confirmed by
the present study. To identify EDCF, the possibility of TXA2 involvement was
evaluated first. This EDCF was not inhibited by OKY-046, a TXA 2 synthetase
inhibitor, but was inhibited by ONO-3708, a TXA 2 /PGH 2 receptor antagonist.
ONO-3708, which inhibits the actions of TXA2 and PGH2 [15], also inhibited
contractions induced by PGF2a , PGE2, PGD 2, and PGI2. This result shows that
ONO-3708 is not a selective antagonist to TXA2 and PGH2. Consequently,
EDCF seems to be a cyclooxygenase product(s) other than TXA 2.
Next, the concentrations of various prostaglandins in the organ bath solution
were determined. After ACh stimulation, the concentrations of PGE2 and
6-keto-PGF1a increased about three-to fourfold. The concentration of PGE2 in
the bath solution after ACh stimulation was about lO-lOM, and that of 6-keto-
PGF1a , (i.e., PGh) was about 1O- 9 M. Luscher et al. [21] have obtained similar
results. The effects of various exogenous prostaglandins on the blood vessel
were than evaluated. PGF2a , STA2, PGH2, PGE2, PGD 2, and PGI2 caused
contractions. PGE2 and PGD 2 induced contractions only at a high

concentration of 1O- 6 M and PGI 2 at 1O- 5 M. The concentrations of PGE2 and
PGI2 measured in the bath solution were very low, and the concentrations that
induced contractions were about 104 times higher. Although the degree of
transfer of a substance produced in the tissue to the solution is not known, the
local concentrations of the prostaglandins released from the endothelium
within the blood vessel wall would be much higher than those measured in the
organ chamber. Tranylcypromine, a prostacyclin synthetase inhibitor of ACh-
induced relaxations, had no effect, which suggests that PGIz is not increased in
the tissue to the degree that induces vascular contractions. Therefore, there is
only a slight possibility that PGI 2 or PGE 2 is EDCF. In addition, that there was
no increase in the concentrations of PGF2a and TXB 2 in the solution excludes
the possibility that PGF2a or TXA 2 is EDCF. These results suggest a
cyclooxygenase product or products other than TXA2 , PGE2 , PGI2 , or PGF2a
as the EDCF produced and released by ACh stimulation in the SHR and WKY
rat aorta.
PGI 2 is produced in endothelial cells and has potent vasodilating effects.
When administered exogenously, it is known to induce biphasic responses in
some types of blood vessels: relaxation is observed at low concentrations and
contraction at high concentrations [22-24]. After ACh stimulation, the
concentration of PGI2 in the solution increased. The degree of the involvement
of this increased PGI2 in relaxations in the rat aorta was evaluated. The aortic
rings at the basal state showed no changes in tension at PGI 2 concentrations of
10- 9 _10- 6 M. Contractions were induced at 10- 5 M. The rings which
contracted with 1O- 7 M NE showed negligible change or only slight relaxation
at PGI 2 concentrations of 10- 9 -10- 6 M. At 10- 5 M, contractions were
induced. Therefore, the PGI 2 which was increased by ACh stimulation is not
likely to be involved in either contractions or relaxations in the rat aorta.
In the present experiment, we could not identify EDCF produced by ACh
stimulation in the rat aorta but we did find that: (1) there is a very low
possibility that the final product in the cyclooxygenase system is an EDCF, (2)
exogenous PGH 2 induced contractions at 1O- 7 M or more, (3) the contractions
induced by PGH2 were inhibited by ONO-3708, (4) the concentrations of PGIz
and PGE2 increased after ACh stimulation in the organ bath solution (the
concentration of PGI 2 in the bath solution after ACh stimulation was about
1O- 9 M and that of PGF2 was about 1O- 1O M, and (5) the volume of the aortic
ring was about 1/104 of that of the organ bath solution. Taking the
concentrations of PGs measured in the organ bath solution and the volume
ratio of the vascular tissue to the organ bath solution into consideration, the
concentration of PGH2 , a precursor of the released prostaglandins, would be at
least 1O- 6 M or more in the vascular tissue. Therefore, it seems that the
concentration of PGH2 produced in the tissue is sufficient to induce vascular
contractions. These observations suggest that PGH2 is a strong candidate for
EDCF produced by ACh stimulation in the rat aorta [25], and that both EDCF
(PGH2) and EDRF (nitric oxide) play important roles in the regulation of the
vascular tonus in hypertensive blood vessels.
228 T. Ito et al.
References
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relaxation of arterial smooth muscle by acetylcholine. Nature 288:373-376
2. Peach MJ, Loeb AL, Singer HA, Saye JA (1985) Endothelium-derived vascular
relaxing factor. Hypertension 7 (Suppl 1):194-1100
3. Rubanyi GM, Vanhoutte PM (1987) Nature of endothelium-derived relaxing factor:
Are there two relaxing mediators? Circ Res 61 (Supplll):1161-1167
4. Palmer RMJ, Ferrige AG, Moncada S (1987) Nitric oxide release accounts for the
5. De Mey JG, Vanhoutte PM (1983) Anoxia and endothelium-dependent reactivity
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6. Rubanyi GM, Vanhoutte PM (1985) Hypoxia releases a vasoconstrictor substance
from the canine vascular endothelium. J Physiol 364:45-56
7. Miller VM, Vanhoutte PM (1985) Endothelium-dependent contractions to
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248:H432- H437
8. Katusic ZS, Shepherd JT, Vanhoutte PM (1987) Endothelium-dependent
contraction to stretch in canine basilar arteries. Am J Physiol 252:H671- H673
9. Katusic ZS, Shepherd JT, Vanhoutte PM (1988) Endothelium-dependent
contractions to calcium ionophore A23187, arachidonic acid, and acetylcholine in
canine basilar arteries. Stroke 19:476-479
10. Vanhoutte PM, Katusic ZS (1988) Endothelium-derived contracting factor:
Endothelin and/or superoxide anion? Trends Pharmacol 9:229-230
11. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki
Y, Goto K, Masaki T (1988) A novel potent vasoconstrictor peptide produced by
vascular endothelial cells. Nature 332:411-415
12. Luscher TF, Vanhoutte PM (1986) Endothelium-dependent contractions to
acetylcholine in the aorta of the spontaneously hypertensive rat. Hypertension
8:344-348
13. Vane JR (1971) Inhibition of prostaglandin synthesis as a mechanism of action for
aspirin-like drugs. Nature 231:232-235
14. Iizuka K, Akahane K, Momose D, Nakazawa M (1981) Highly selective inhibitors
of thromboxane synthetase. 1. Imidazole derivatives. J Med Chern 24:1139-1148
15. Katsura M, Miyamoto T, Hamanaka N, Knodo K, Terada T, Ohgaki Y, Kawasaki
A, Tsuboshima M (1983) In vitro and in vivo effects of new powerful thromboxane
antagonists (3-alkylamino pinane derivatives). Adv Prostaglandin Thromboxane
Leukotriene Res 11:351-357
16. Jaffe BM, Behrman HR, Parker CW (1973) Radioimmunoassay measurement of
prostaglandins E, A, and F in human plasma. J Clin Invest 52:398-405
17. Powell WS (1980) Rapid extraction of oxygenated metabolites of arachidonic acid
from biological samples using octadecylsilyl silica. Prostaglandins 20:947-957
18. Johnson AR (1980) Human pulmonary endothelial cells in culture: Activities of
cells from arteries and cells from veins. J Clin Invest 65:841-850
19. Griffith T, Randall M (1989) Nitric oxide comes of age. Lancet 11:875-876
20. Soltis EE (1987) Effect of age on blood pressure and membrane-dependent vascular
responses in the rat. Circ Res 61:889-897
21. Luscher TF, Romero JC, Vanhoutte PM (1986) Bioassay of endothelium-derived
vasoactive substances in the aorta of normotensive and spontaneously hypertensive
rats. J Hypertension 4 (Suppl 6):S81-S83
22. Pomerantz K, Sintetos A, Ramwell P (1978) The effect of porstacyclin on the

human umbilical artery. Prostaglandins 15: 1035-1044
23. Chapleau CE, White RP (1979) Effects of prostacyclin on the canine isolated
basilar artery. Prostaglandins 17: 573 - 580
24. Van Dam J, Maddox YT, Ramwell PW, Kot PA (1986) Role of the vascular
endothelium in the contractile response to prostacyclin in the isolated rat aorta. J
25. Kato T, Iwama Y, Okumura K, Hashimoto H, Ito T, Satake T (1990) Prostaglandin
H2 may be the endothelium-derived contracting factor released by acetylcholine in
the aorta of the rat. Hypertension 15:475-481
4
Flow-Induced Calcium Response in
Cultured Vascular Endothelial Cells
Joji Ando\ Shigenobu Araya, Youichi Katayama, Akira Ohtsuka2 ,
and Akira Kamiya 3
Summary. Hemodynamic forces influence vascular endothelial cell functions.

However, the mechanism involved is not well understood. We have studied
how endothelial cells perceive blood flow and the substance(s) that may
mediate such flow-induced changes in cell functions. In this paper we report
that intracellular free Ca2 +, a major component of an internal signalling
system, is a mediator of the endothelial cell response to flow. Cultured
mono layers of bovine fetal aortic endothelial cells loaded with the highly
fluorescent Ca2 + sensitive dye Fura-2 were subjected to fluid flow in a flow-
loading chamber, and simultaneous changes in intracellular free Ca2+
concentration ([Ca2 +]j) were measured using photometric fluorescence
microscopy. Application of medium flow to cells led to an immediate increase
in [Ca2 +]j, following by a rapid decline, and then a sustained increase
somewhat higher than control levels during the entire period of flow
application. Image analysis of Fura-2 fluorescence obtained before and after
the initiation of flow application showed that all the cells exposed to flow
underwent an increase in [Ca2 +]j, although the increase in individual cells
varied in degree.
Key words: Vascular endothelial cells-Shear stress-Intracellular calcium-
Hemodynamic force-Fura-2
Introduction
Vascular tone plays an important role in regulating blood flow and is
modulated by alterations in nervous activity, by circulating vasoactive agents,
I Department of Cardiovascular Biomechanics, Faculty of Medicine, University of

Tokyo, Tokyo 113, Japan
2Research Institute of Applied Electricity, Hokkaido University, Sapporo, 060 Japan
3 Institute of Medical Electronics, Faculty of Medicine, University of Tokyo, Tokyo, 113
Japan
230
4. Flow-Induced Calcium Response in Cultured Vascular Endothelial Cells 231
U
Blood flow
Shear stress
dU Cell morphology
• r = wdI Cell alignment
Histamine t
Prostacyclin t
EDRF t Nucleus
Endothelin t
~
~
DNA synthesis t
Cell cytoskeleton
Microfilament t
Stress fiber t
Cell motility t
Migration t
Endothelial cell
FIG. 1. A schematic model of blood flow and endothelial cells. Endothelial cells are
constantly exposed to shear stress which is generated by blood flow. If the velocity of
blood flow is U, the intensity of shear stress (t) can be calculated by the equation: , = Jl
dU/dl where Jl is blood viscosity and I is the radius of vessels. Flow modulates various
endothelial cell functions. EDRF, Endothelium derived relaxing factor
and by metabolic changes. Recently, it has been elucidated that vascular

endothelial cells influence vascular tone by producing various potent
vasodilative and vasoconstrictive mediators, termed endothelium-derived
relaxing factor (EDRF) and endothelium-derived contracting factor (EDCF) ,
respectively [1-4]. As shown in Fig. 1, vascular endothelial cells covering the
inner surface of vessels are continually exposed to hemodynamic shear stress
generated by blood flow . Recent evidence has shown that endothelial cells
change their functions in response not only to chemical agents but also to such
physical force as hemodynamic shear stress . For instance, fluid shear stress
enhances the synthesis of histamine [5), prostacyclin [6] and endothelin [7), and
stimulates endocytosis [8] and cytoskeleton formation [9] in these cells. We
recently showed that shear stress also enhances the migration and proliferation
of cultured endothelial cells during the repair of mechanical denudation [10] .
However, the manner in which endothelial cells sense blood flow or shear
stress and modulate their functions has not been clearly defined.
Figure 2 shows a schematic diagram of the internal signalling system which is
now generally accepted as existing in a wide variety of cell types. When
external signals or stimuli arrive at the cell membrane , they are recognized by
232 J. Ando et al.
External Stimuli
Cell membrane
Ca++ 1
Ca++ PI cAMP ATP
1 I \
~/~Ky~
/
Ca + + Phosphorylation
\ / Cellular
responses
FIG. 2. A schematic diagram of the internal signalling system. R, Receptor; G, GTP

binding protein; AC, adenyl ate cyclase; ATP, adenosine 5'-triphosphate; cAMP,
adenosine 3',5'-cyclicphosphate; PLC, phopholipase C; PI, phosphatidylinositol; DAG,
diacylglycerol, IP3 , inositol 1,4,5-triphosphate
specific surface receptors on the plasma membrane and are transformed into
such internal signals as cyclic adenosine 5'-monophosphate (cAMP),
diacylglycerol (DAG), inositol 1,4,5-triphosphate (IP 3 ), and Ca2 +. These
internal signals, i.e., second messengers, are known to regulate cellular
response to external stimuli [11,12]. In this study, we focused on the role of
cytoplasmic Ca2+ in the response of endothelial cell to fluid flow. Using the
intracellularly trapped fluorescent Ca2+ indicator, Fura-2 [13], we examined
changes in intracellular free Ca2 + when cultured endothelial cells are subjected
to medium flow.
Methods and Materials
Cell Culture
Bovine fetal aortic endothelial cells were isolated and cultured as described
previously [10]. Cells were cultured in tissue-culture flasks in medium 199
supplemented with L-glutamine (2mM), 20% fetal bovine serum, penicillin

(lOOU/ml) and streptomycin (100Ilg/ml). Cells were grown at 37°C in a 95%
air and 5% CO2 atmosphere. Cultures were subcultured weekly by brief
treatment with 0.05% trypsin and 2mM EDTA. Cell density was determined
with a Coulter Counter (Coulter Electronics Ltd., Luton, UK). The cells were
then diluted with fresh medium and inoculated into culture flasks at 1 x 104
cells/cm2 . "Culture age" was defined as the number of cumulative population
doublings (CPDs) calculated at each subculture. Cells used in the present
experiments were in a state of less than 20 CPDs. All cultures satisfied multiple
criteria of endothelial origin including (1) characteristic cobblestone monolayer
morphology, (2) immunofluorescent staining for Factor VIII-related antigen
[14], (3) uptake of fluorescent acetylated low density lipoprotein [15], and (4)
expression of angiotensin-I converting enzyme [16]. Endothelial cells were then
cultured on 0.3-mm-thick quartz coverslips.
Measuring Intracellular Free Calcium Concentrations

with Fura-2
Cells were incubated at 37°C in medium 199 containing 61lM Fura-2/AM
(the acetoxymethyl ester of the fluorescent tetracarboxylated chelator Fura-2,
Dojin Co., Kumamoto, Japan). Thirty minutes later, cell monolayers were
washed several times with Fura-2-free medium, and maintained thereafter in
fresh medium. The coverslips were placed in a specially designed flow chamber
(Fig. 3) which was mounted on the stage of a photometric fluorescence
microscope (MICROPHOT FX-Pl, Nikon, Tokyo). A group of 30-40 cells
was chosen and centered in the measuring field of the microscope. Changes in
free intracellular Ca2 + concentration ([Ca2 +]j) were determined by the method
of Williams et al. [17] with a slight modification. Fura-2 was excited alternately
with light at 340 nm and at 380 nm, and the intensity of emitted light was
measured with a photomultiplier tube through a 500nm bandpass filter.
Inasmuch as Fura-2 exhibits a spectral shift upon binging to calcium, an
increase in [Ca2 +]j increased the fluorescence excited by 340-nm light (F340)
and decreased the fluorescence from 380-nm excitation (F380). The ratio of
these two values (F340:F380) reflects changes only in Ca2 + concentrations,
whereas dye concentration, cell thickness, and absolute optical efficiency of the
instrument are constant in these ratios [13]. After subtracting the
autofluorescence at the two wave lengths, F340:380 ratios were calculated.
Time course of such parameters as F340, F380, and F340:F380 ratios following
flow application were shown on a cathode-ray tube (CRT) display.
Video images of F340 and F380 were recorded on a magnetic floppy disk of a
TV-photo player (R-3000, Fujix, Tokyo, Japan) through a silicon-intensified
target camera (SIT-tube c-2400, Hamamatsu-Photonics, Hammamatsu, Japan)
before and after the initiation of flow application. They were then converted
into a digital image of 256 graduations (8 bits) at 512 x 512 points and stored
in a frame memory (Edec Image PC). Using a NEC microcomputer (PC9801)
connected to the frame memory, the ratio (F340/F380) was calculated at every
234 J. Ando et al.
Flow-
C~ a
Flow chamber
3Q (1-4
u =2ab
(Ya )2)
u =tlow velocity (em/sec)
Q =volume flow (ee/see)
a, b = cross-sectional dimensions (em)
y = lateral distance from tube axis (em)
r =shear stress (dyn/em')
/1 =fluid viscosity (poise)
FIG. 3. Flow-loading chamber. Intensity of shear stress (T) to the cells was calculated by
the equation shown in this figure.
point in each image and subjected to data processing in order to construct a

three-dimensional display of a spatial F340/F380 distribution.
Flow-Loading Apparatus
For applying flow on cultured endothelial cells, we used the same flow device
as described earlier [18]. The flow chamber consisted of 0.2 x 4.0 x 50-cm
polymethacrylate plates (Fig. 3). Both ends of the chamber were connected via
silicone tubing to the reservoirs and to a centrifugal pump through which the
medium flows. A depression was made in the upper surface of the chamber to
secure the coverslip upon which endothelial cells were cultured, so that the cell
layer would face a nonturbulent stream of medium circulating through the
chamber. After the coverslip was fixed in the chamber, the entire circuit was
filled with medium and maintained at 37°C by an automatic temperature
controller; pH was adjusted to 7.3-7.4 by bubbling the medium with a mixture
of 95% air and 5% CO 2 , Flow rate was monitored by an electromagnetic
flowmeter (Nihon Koden, Tokyo) inserted into the outflow tube, and internal
pressure was measured through a nylon catheter by a Statham P231D pressure

transducer (Statham Instruments, Oxnard, Calif.). Pulsation of the medium
flow caused by the pump was absorbed by a silicone rubber depulsator (Cole-
Parmer, Chicago, 111.).
Intensity of shear stress (t, dyne/cm2) to the endothelial cell layer was
calculated as shown in Fig. 3. The maximum Reynolds number corresponding
to the highest flow rate used in this study (23mlls) was 912. Under these
conditions we assumed that the flow was laminar with negligible effects of
secondary velocity, because the viscous force was predominantly greater than
the inertial force.
Results
Effect of Hydrostatic Pressure on [Ca 2 +Ji

When flow is applied to the cell layer by medium perfusion in the chamber, it
results in the application of hydrostatic pressure load to the cells. In order to
determine the effect of hydrostatic pressure in rCa 2+]j, we applied only a
hydrostatic pressure load to the cell layer by elevating the position of the
reservoir so that the pressure in the chamber might increase from 50 to
95 mmHg in a stepwise manner in the absence of medium flow. As shown in
Fig. 4, the pressure load alone had no effect on [Ca2+]j.
2
Pressure Load
----~-----~~--~--------~~-----
(mmHg)
o
o 3 6
time (minutes)
FIG. 4. Effect of hydrostatic pressure on [Ca 2+1i in the absence of flow
236 J. Ando et al.
F340/F380
- - F340/F380
--- F340
_.- F380
500 2
.~
-
(/)
~ 300
-
"-
c
''---------
..c
OJ
:.::i
\
\
\" ............ --.- .--._.- . --.,...-__ 0_.-
100 o
o
- Medium Flow
Shear Stress:6.3dynes/cm 2
3
FIG. 5. Time course of F340, F380, and F340/F380 following the application of medium
6 (min)
flow
Flow-Induced ccl+ Response

When the medium was perfused at a flow rate of 14.2 mIls to yield a shear
stress load of 6.3 dynes/cm2, [Ca 2 +]j increased immediately, reaching a peak
within 30 s. After peaking, it reached a new steady state, which remained
slightly higher than the control level throughout the period of flow application.
When flow stopped, [Ca 2 +]j declined slowly to the control level (Fig. 5).
Image Analysis of the Flow-Induced Ca 2 + Response

Since Fura-2 fluorescence measured with a photomultiplier tube reflects total
fluorescence from 30-40 cells existing in a measuring field, it is not known
whether or not all the cells respond to flow. To clarify this point, video images
of cellular Fura-2 fluorescence were recorded, using a SIT-tube, and analyzed.
Figure 6 shows three-dimensional images of the Fura-2 fluorescence ratio
(F340/F380) before and 15 s after the initiation of flow application. The Z axis
represents F340/F380 which reflects intracellular calcium concentration. All
cells exposed to flow underwent an increase in [Ca2+]j, although the increase in
individual cells varied in degree.
control
flow
FiG. 6. Flow-induced Ca2+ response illustrated by three-dimensional Fura-2 image. The

lower part of the panel shows the image obtained 15 s after the initiation of flow
application (shear stress: 6.3 dynes/cm2)
Ca 2 + Response to Flow in Ca 2 + Free Medium

The Ca2+ response to flow was examined using M199 with 2 mM EDTA added.
The perfusion of such Ca 2 +-free medium also induced [Ca 2 +]j responses in
endothelial cells. In the absence of extracellular Ca2 +, the early peak rise in
[Ca 2 +]j occurred, but the sustained rise in [Ca 2 +]j seen in the presence of
extracellular Ca 2 + disappeared (Fig. 7). These data suggested that the flow-
induced sustained rise in [Ca2+]j is caused by influx of extracellular Ca2 + across
the plasma membrane.
Effect of Calcium Blockers on the Ca 2 + Response

Similar experiments were carried out with a calcium channel blocker,
nicardipine. In the presence of 2 x 10-5 M nicardipine, apparent inhibition was
noted in the flow-induced sustained rise in [Ca2 +k This inhibitory effect was
not clear at lower concentrations (2 x 1O-6 M, data not shown). These
observations suggest that calcium antagonist-sensitive calcium channels are
involved in the flow-induced influx of extracellular Ca2 + across the plasma
membrane.
238 J. Ando et al.
- - Control
- - - EGTA
(2mM)
2
,
\
\
"-\
\
"- "-
"-
"-
'--~--_/----------
Medium Flow
o Shear Stress:6.3dynes/cm 2
o 3 6 (min)
FIG. 7. Flow-induced [Ca2 +]j response in the absence of extracellular Ca2+
Discussion
The present data demonstrate that application of flow to endothelial cells by
fluid perfusion leads to an early peak and sustained rise in [Ca2 +]j. In the
absence of extracellular calcium, the early peak rise in [Ca2 +]j occurred, but
the sustained rise in [Caz+] diminished. This finding indicates that influx of
extracellular Ca2 + across the plasma membrane is a major mechanism effecting
the sustained rise in [Ca2 +]j. Nicardipine also prevented the sustained rise in
[Ca2 +]j seen in its absence, indicating that the influx of extracellular Ca2 +
occurs through calcium antagonist-sensitive calcium ion channels. The fact that
the early rise in [Ca2 +]j occurs even in the absence of extracellular Ca2 +
suggests that other mechanisms, besides influx of extracellular Ca2 +, is also
involved in the flow-induced Ca2+ response. It is natural to suppose that the
early rise in [Ca2+]j is due to release of Ca2+ from intracellular stores (e.g.,
mitochondria, endoplasmic reticulum, and plasma membrane). Thus, there are
two mechanisms by which Ca2 + can be made available during the response to
flow: Ca2 + mobilization from intracellular stores accounts for the early rise,
whereas influx of extracellular Ca2+ is required for sustained elevation of
[Ca2 +k
Because cytoplasmic free Ca2 +, a second messenger in the internal signalling
system of the cell, responds to fluid flow, it would seem likely that a sensing
mechanism exists in endothelial cells in order to perceive blood flow as a
stimulus, and to mediate the signal to intracellular organelles. Lansman et al.
[19] have recently shown that the opening frequency of ion channels gating
calcium influx into cells increases when the vascular endothelial cell membrane
is mechanically· stretched. Based on these findings, they hypothesized the
presence of a "mechanotransducer" regulating the ion channels. As noted
above, calcium ion channels are also involved in the endothelial cell response
to flow. However, our data demonstrated that application of flow produces
the Ca2+ response even in the absence of extracellular Ca2 + or in the pres-
ence of calcium channel blockers. Thus, a flow-sensing mechanism can not
be explained by calcium ion channels alone. It is therefore possible that a
flow-sensing mechanism of endothelial cell differs from a stretch-sensing
mechanism. On the other hand, Nakache and Gaube [20] reported that
application of flow to bovine pulmonary artery endothelial cells leads to a
membrane hyperpolarization. Using a patch-clamp method. Olesen et al. [21]
demonstrated a K+ selective, shear-stress-activated ionic current in bovine
aortic endothelial cells, and suggested that the K+ current or the resulting
membrane hyperpolarization could play the role of a transducer between the
flow and the endothelial cell membrane. Although it is not clear at present
what these K+ fluxes have to do with the flow-induced Ca2 + response reported
here, the clarification of their relationship seems to be of great importance in
understanding any flow-sensing mechanism of endothelial cells.
It is well known that vascular endothelial cells respond to a variety of
chemical stimuli and modulate their functions. For example, prostacyclin,
labile and potent vasodilators, are released from endothelial cells in response
to histamine, bradykinin, thrombin, and adenosine 5 ' -triphosphate (ATP) [22-
25]. Recent studies have revealed that such chemical stimuli induce rapid
alterations in intracellular Ca2+ concentrations in endothelial cells [26,27]. The
pattern of Ca2 + response to chemical stimuli is very similar to that of Ca2 +
response to flow. In general, an interaction of an agonist and a membrane
surface receptor induces inositol phospholipid turnover [28]. This turnover of
membrane phospholipids is paralleled by an increase in intracellular
concentration of Ca2+. For example, using cultured porcine aortic endothelial
cells, Lambert et al. [29] showed that when bradykinin stimulates
phospholipase C metabolism of phosphatidylinositol 4, 5-biphosphate to form
inositol trisphosphate (IP3 ), Ca2 + is mobilized simultaneously, and suggest that
IP3 production initiates Ca2 + mobilization. A recent study [30] has shown that
IP3 increases Ca2+ levels within the cells by triggering the passage of Ca2 +
through membranes of intracellular Ca2+ stores. Thus, with regard to chemical
signals such as hormones and neurotransmitters, the mode of signal
transduction has been studied in detail. However, the signalling system
mediating hemodynamic forces to intracellular organelles is, for the most part,
unknown. The biochemical approach including the measurements of guanine
nucleotide binding proteins, inosital phospholipids, and diacylglycerol should
prove useful in this regard.
Acknowledgements. This work was partly supported by a Grant-in-Aid for

Scientific Research (A), no. 01440085, (B), no. 02454251, from the Japanese
240 J. Ando et al.
Ministry of Education, Science and Culture, and a research fund from the
Atherosclerosis Study Association.
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18. Ando J, Komatsuda T, Kamiya A (1988) Cytoplasmic calcium response to fluid
shear stress in cultured vascular endothelial cells. In Vitro Cell Dev Bioi 24:871-
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19. Lansman JB, Hallman TJ, Rink TJ (1987) Single stretch-activated ion channels in
vascular endothelial cells as mechanotransducers. Nature 352:811-813
20. Nakache M, Gaub HE (1988). Hydrodynamic hyperpolarization of endothelial

cells. Proc Nat! Acad Sci USA 85:1841-1843
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24. Stanfield MC, Schilling WPI, Ritchie AL, Eskin, SG, Navaro LT, Kunze DL (1987)
Bradykinin-induced increases in cytosolic calcium and ionic currents in cultured
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5
Endothelin and Vasoconstriction
Hisashi Kai, Mayuko Kodama, Hiromichi Yamamoto,
and Hideo Kanaide
Summary. We investigated the effect of endothelin-1 (ET) on cytosolic free

Ca2+ concentration ([Ca2+]j) and intracellular Ca2+ store in vascular smooth
muscle cells (VSMCs). Using quin2 microfluorometry, effects of ET on [Ca2+]j
were investigated in rat aortic VSMCs in primary culture. In Ca2+-containing
solution, ET induced a rapid and sustained [Ca2+]j elevation. The sustained
component of [Ca2+]j elevation was inhibited by diltiazem. In Ca2+-free
solution, ET induced only a rapid and transient component of elevation, which
was not inhibited by diltiazem. When the caffeine-sensitive intracellular Ca2+
store was depleted in Ca2+-free solution, ET did not increase [Ca2+]j. 45Ca2+
flux study showed that ET released Ca2+ from intracellular store in VSMCs.
Front-surface fluorometry with fura-2-loaded strips of porcine coronary artery
were used to simultaneously measure the effects of ET on [Ca2+]j and tension
development. In the Ca2+ -containing solution, ET induced rapid and sustained
increases in [Ca2+]j and tension. In the Ca2+-free solution, ET induced rapid
and transient increases in [Ca2+]j and tension. Pretreatment for depletion of
histamine-sensitive intracellular Ca2+ store did not affect ET-induced transient
increases in [Ca2+]j and tension in the Ca2+-free solution. Conversely, when
the caffeine-sensitive store or both caffeine- and histamine-sensitive stores
were depleted, ET induced a contraction with no change in [Ca2+]j. This
Ca2+-independent contraction was markedly inhibited by H-7, a protein kinase
C inhibitor. Thus, we conclude that ET-sensitive intracellular Ca2+ store
overlaps with the caffeine-sensitive one, and that the ET-induced contraction
depends on (1) Ca2+ release from intracellular store, (2) extracellular Ca2+-
dependent mechanism in the sustained phase, and (3) Ca2+-independent
mechanisms mediated by protein kinase C-related phosphorylation of
contractile elements.
Division of Molecular Cardiology, Research Institute of Angiocardiology, Faculty of

Medicine, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812, Japan
242
5. Endothelin and Vasoconstriction 243
Key words: Endothelin-1-Vascular smooth muscle-Muscle contraction-

Intracellular calcium concentration
Introduction
Endothelin-l (ET) is a potent vasoconstrictor peptide which was isolated form
the conditioned medium of cultured porcine aortic endothelial cells by
Yanagisawa et a1. [1]. Earlier studies suggested that ET may be an endogenous
voltage-dependent Ca2+ channel agonist because of its properties of action and
the structural homologies with neurotoxins which affect ion channels [1,2].
Recent studies have shown that ET has multiple sites of action in vascular
smooth muscle [3-5]. We reported studies focusing upon the effects of ET on
Ca2+ homeostasis in vascular smooth muscle [6-8]. We now summarize our
findings on the effects of ET on cytosolic free Ca2+ concentration ([Ca2+]i) and
on Ca2+ mobilization from extracellular space and/or intracellular Ca2+ store
in rat aortic vascular smooth muscle cells (VSMCs) in primary culture, and the
effects of ET on [Ca2+]i and tension development of strips of the porcine
coronary artery.

1. Studies in the Rat Aortic VSM Cs in Primary Culture
VSMCs were obtained from aortic media of male Wistar rats, and then
cultured as previously described [9]. Only primary cultures were used the for
45Ca2+ flux study and for the quin2 microfluorometry.
45CA2+ FLUX STUDY. The 45Ca2+ efflux study was done as follows: after rinsing
with physiological salt solution (PSS), the cultured VSMCs were equilibrated
with 45Ca2+ (15 IlCi/mt) in 1 ml of PSS for 3 h at 37°C. The cells were then
incubated in 1.4 ml PSS or Ca2+-free PSS containing 2 mM ethylene glycol bis
(p-aminoethyl ether) N, N, N', N'-tetraacetic acid (EGTA) at 37°C, and
changes of the solution were made every min for 25 min. ET was added at
10 min of the efflux time. The amount of 45Ca2+ released from the VSMCs at
each time interval was measured by liquid scintillation counting with 10 ml
ACS II (Amersham Co., Arlington Heights, 111.). Fraction lost or 45Ca2+
released per min per 106 cells was calculated. In order to measure the 45Ca2+
influx, the cultured VSMCs were rinsed with PSS and incubated for 15 min in
PSS at 37°C. The cells were preincubated with ET for 0,2, 5, and 1Omin, and
then incubated in 1 ml PSS containing ET and 45Ca2+ (1IlCi/ml) for 2 min. In
order to terminate 45Ca2+ uptake and remove external 45Ca2+, the cells were
rinsed with Ca2+-free PSS containing 2mM EGTA at 4°C for 10min. Miasiro
et a1. describe this process in detail [6].
QUlN2 MICROFLUOROMETRY. VSMCs cultured on chamber slides were loaded
with quin2 by incubating with growth medium containing 50 11M quin2/AM at
244 H. Kai et a1.
37°C for 60min. Optic measurements were performed in PSS at 25°C. Details
of the quin2 microfluorometry can be found in the report by Kanaide et al.
[10]. Briefly, the fluorescence intensity in a small spot «1Ilm2) in the cytosol
31lm apart from the nucleus was measured using a fluorescence microscope
(Model Standard 18, Zeiss) equipped with a water immersion objective system
(Plan-Neofluor 63, Zeiss), an appropriate combination of filters (Zeiss and
Toshiba) in which cells were excited at wavelengths between 350 and 360nm
and analyzed at wavelengths between 470 and 560 nm and a pinhole diaphragm
(Zeiss) in the light axis. For optical measurement, each cell was exposed to the
excitation light only once for no longer than 2 s, to avoid the photo bleaching
effect on quin2. The estimate of [Ca2 +]j was made as previously described [10].
2. Studies in the Medial Strips of Porcine Coronary Artery

Left coronary circumflex arteries were isolated from porcine hearts
immediately after the animals were sacrificed. Segments located 2-3 cm from
the origin were excised. After the endothelial cells had been removed by
rubbing the luminal surface with a cotton web, the preparations were cut into 1
X 5 mm circular strips of 0.1 mm thickness and were incubated in the gassed
(95% 0 2 /5% CO 2 ) Krebs-Henseleit solution (KHS) with 251lM fura-2/AM and
5% fetal bovine serum for 3-4 h at 37°C. The strip was then sustained in 10 ml
organ bath filled with gassed KHS at 37°C. The contractile force and the
fura-2'Ca 2 + fluorescence of the strip were monitored simultaneously, using
front-surface fluorometry described elsewhere [8,11]. The contractile tension
was monitored using a force transducer (TB-612T, Nihon Koden, Tokyo), and
the fluorescence was monitored using a spectrofluorometer especially designed
for fura-2 fluorometry (CAM-OF-1, Japan Spectroscopic Co., Tokyo), in
which the strip was alternatively excited at wavelengths of 340 and 380 nm and
was analyzed at a wavelength of 500 nm. The absolute value of [Ca2 +)z was
calculated from the ratio (R) of the fluorescence at 340nm excitation (F340) to
that at 380nm excitation (F380), as described by Kodama et al. [8].
CHEMICALS. ET was purchased from Peptide Institute (Osaka, Japan), and
quin2/AM and fura-2/AM were purchased from Dotite (Kumamoto, Japan)
and Molecular Probe Inc. (Eugene, Ore.), respectively. N-(6-aminohexyl)-5-
chloro-1-naphtalene sulfonamide (W-7) and 1-(5-isoquinolinesulfontyl)-2-
methylpiperazine dihydrochloride (H-7) were purchased from Seikagaku
Kogyo Co. Ltd. (Tokyo). All other reagents were of the highest commercially
available grade.
Results
Effects of ET on the Ca 2 + Homeostasis of Cultured VSMCs

As shown in Fig. la, in VSMCs in primary culture, the application of ET
markedly increased the Ca2 + efflux within 1 min in a dose-dependent manner
FIG. 1. Effects of ET on 45Ca2+ efflux from flit a

aortic VSMCs in primary culture in a normal 0.2
PSS and in b Ca 2+-free PSS containing 2 mM tl
EGTA. ET of 10-7 M was added at 10 min as ..Q
c
indicated by the arrow (e, subject; 0, control). :;:::;
o
(From [6) with permission) :6.... 0.1
LL
10 15 20 25
Efflux time (min)
c:
,
ET
~
.!!!
Qi
0.2
u
"b
"-
<5
E
-S
"0
Q) 0.1
en
<t1
Q)
~
<t1
U
'"
0.0
0 5 10 15 20 25
Efflux time (min)
(10-9 -10-7 M). Then, the 45Ca2+ efflux rate gradually reverted to control
levels. In the absence of extracellular Ca2+, ET also increased the 45Ca2+ efflux
with a similar time course and concentration-dependent relationship as
observed in the presence of extracellular Ca2+ (Fig. 1b). On the other hand,
there was no apparent increase in the 45Ca2+ influx into the cultured VSMCs
observed 2-lOmin after treatment with ET in normal PSS, compared with
findings in the control cells. These results suggested that ET induces a
mobilization of Ca2+ in the cultured VSMCs and that the release of Ca2+ from
the intracellular store has an important role in the mechanism of the ET-
induced elevation of [Ca2+k
In quin2 microfluorometry, it was shown that in normal PSS, ET induced a
rapid and concentration-dependent (10-9 -10-7 M) increase in [Ca2+]j of quin2-
loaded VSMCs within 30 s, and that this elevated level remained unchanged for
at least 20 min (Fig. 2a). The effect of ET was slightly altered by washout with
PSS. Effects of ET on [Ca2+]j in the absence of the extracellular Ca 2 + are
shown in Fig. 2b. When VSMCs were exposed to Ca 2 +-free PSS containing
2mM EGTA, [Ca2+]j rapidly decreased to reach a lower steady level. The
246 H. Kai et al.
10 20 30 40 50 60
DURATION OF INCUBATION
IN 2 MM EGTA PSS (MIN)
b'@z
::::J
>- 35
a: 2min
ri
Iii
~ 30
w
()
z
w
&l
~ 25 EGTA
o
w
~ ()
z
w
&lw
a:
o
~
FIG. 2. Effects of ET on [Ca 2 +]i in quin2-loaded VSMCs in primary culture from rat
aorta. a Time courses of the fluorescence change of cytosolic spots, [Ca2+]i' observed
when 1O- IO M (\7), 1O- 9M (D), 1O- 8M (/0), and 1O- 7 M (0) ET were applied to VSMCs
in normal PSS, as indicated by the arrow. The estimated levels of [Ca2+]i observed just
before and 5min after application with 1O- 7 M ET in normal PSS were 109nM and
225nM, respect'ively. b Time courses of [Ca 2+]i when 1O- 10 M (".), 1O- 9 M (_), 1O- 8 M
(.~), and 1O- 7 M (e) ET were applied to VSMCs in Ca 2 +-free PSS containing 2mM
EGTA, as indicated by the arrow. The estimated [Ca 2 +]i levels just before and 30s after
application with 1O- 7 M ET in Ca 2 +-free PSS were 55nM and 135nM, respectively.
c Effects of the duration of incubation in Ca 2 +-free PSS on the levels in response to the
first applications with 1O- 7 M ET (D), 1O- 2 M caffeine (~), and 1O- 6 M norepinephrine
(WJ). d Effects of the repetitive caffeine treatments on ET-induced [Ca 2 +]i changes in
Ca 2 +-free PSS. After 10 min incubation in Ca 2 +-free PSS, the following procedures
were carried out. a VSMCs were not exposed to caffeine, b VSMCs were exposed to
10- 2 M caffeine (CF) once for 2 min., VSMCs were exposed to caffeine for 2 min c twice
and d 5 times, each with 2 min interval. Data are mean ± S. D. of 5 experiments in Fig.
2a, b, and d or 4 experiments in Fig. 2c, and the number of the cells counted in each
experiment was 8. (From [7] with permission)
subsequent application with ET induced a rapid and concentration-dependent

(10-9 -10-7 M) elevation of [Ca2 +k The elevation of [Ca 2 +]j in VSMCs in
Ca2 +-free solution was transient with a peak level observed at 30 s. There was a
gradual reversion to the pre-exposed level, within 3 min, thereby suggesting
that ET increased [Ca2 +]j, in part, by releasing Ca2 + from the intracellular Ca2 +
store in the cultured VSMCs. Pretreatment with diltiazem for 15 min inhibited
the ET-induced elevation of [Ca2+]j in a concentration-dependent manner in
normal PSS, especially the sustained component, while the peak level and the
time course of ET-induced elevation of [Caz+]j in Caz+-free PSS were not
influenced by pretreatment with diltiazem.
In order to determine the characteristics of the ET-sensitive intracellular
Caz+ store, the following studies were preformed. Effects of the duration of
incubation in Ca2+ -free PSS on [Caz+]j change induced by the first application
with 1O- 7 M ET were compared with those with 1O- 2 M caffeine and 1O-5 M
norepinephrine (Fig. 2c). Duration of the preincubation period of VSMCs in
Ca2+ -free PSS (10-60 min) had no effect on the peak level of [Caz+]j elevation
induced by subsequent treatment with ET. As previously reported [12], the
longer the duration of incubation in Caz+-free PSS, the lesser was the extent of
norepinephrine-induced [Caz+]j elevation. The caffeine-induced [Caz+]j
elevation was little affected by the duration of exposure to Caz+-free PSS, as in
the case of ET. As shown in Fig. 2d, repetitive applications with lO- z M
caffeine in Caz+-free PSS for 2 min at 2 min intervals revealed a series of
transient [Caz+]j elevations. The peak levels of the response progressively
decreased with each treatment, and the fifth application with caffeine led to no
cellular response. After pretreatment with caffeine in Ca2 +-free PSS, the
transient [Caz+]j elevation induced by the subsequent application with ET was
diminished. The greater the number of treatments with caffeine the lesser the
response of [Caz+]j induced by ET. Finally, under the condition where the
caffeine-sensitive intracellular Caz+ store was practically depleted by five
different treatments with caffeine in Caz+-free PSS, the subsequent application
with ET induced no significant elevation of [Caz+]j (Fig. 2d.d).
Effects of ET on [Ca 2 +Ji and Tension Development of

Porcine Coronary Arterial Strips
Figure 3 shows a representation of typical recordings of effects of ET on
[Caz+]j and tension development of fura-2-loaded porcine coronary arterial
medial strips recorded simultaneously using front-surface fluorometry. In
normal KHS, ET induced a rapid and sustained increase in [Caz+]j and tension
in a concentration-dependent manner (Figs. 3,4). The contraction and [Ca2+]j
elevation remained unchanged for as long as ET was applied, and these effects
were not readily eliminated with washes in PSS. Although the maximum level
of [Caz+]j elevation induced by 10- 7 M ET was estimated to be 740 nM and
was about 90% of that observed with the depolarization in 118 mM K+ KHS
(970nM), the maximum level of tension reached 125% of that observed in
118mM K+ KHS. When a strip was exposed to Caz+-free KHS, the level of
[Caz+]j gradually decreased to reach a lower steady state within 10min, with no
change in the tension. The subsequent application of ET in the absence of
extracellular Caz+ induced a rapid and transient increase in [Caz+]j and tension
(Fig. 3b). The increased [Caz+]j and tension reverted to pre-exposure levels
within lOmin, despite the continuous presence of ET. The maximum tension
induced by 10-7 M ET in Caz+-free KHS reached 105% of that observed in
118mM K+ KHS, while the maximum level of [Ca2 +]j induced by 10- 7 M ET
in Ca2+-free KHS was as high as 58% (430nM) of that in 118mM K+ KHS.
248 H. Kai et al.
a b
F340
F380
r
L
r
'I
F340
r~
nM
L r--y--
§[r- r-
F380
Ratio n~ 970
Ratio ~[
150 r~~
Tension
J250 m9
5min
Tension
JLlJ'OO~
CJ
118mM K fET-
5mln
r
I
2mM EGTA
F340
F380 "-
Rot,o'i['70f
150
'----
~200mg
5min
Tension
=
118mM K
CF His
2mM EGTA
FIG. 3. Effects of ET on [Ca2 +]i and the tension of porcine coronary arterial strips
loaded with fura-2. a Representative time course of the effects of 10- 7 M ET on the
fluorescence intensity and tension of porcine coronary arterial strip in normal KHS and
bin Ca 2 +-free KHS containing 2 mM EGTA. The estimated levels of [Ca 2 +]i of strips in
normal and Ca 2 +-free KHS were 150 nM and 70 nM, respectively. Peak levels of
fluorescence and tension boserved when strip was exposed to 1O- 7 M ET in normal KHS
were about 90% (the estimated [Ca 2 +k 740nM) and 125%, respectively, of the peak
levels observed in 118mM K+ KHS (100% (970nM), 100%). In Ca2 +-free solution,
peak levels of fluorescence and tension induced by 1O- 7 M ET were about 58%
(430nM) and 105%, respectively, of those observed with strips in 118mM K+ KHS. c A
representative time course of the effects of 10- 7 M ET on [Ca 2 +]i and tension of a
porcine coronary arterial strip after depletion of intracellular Ca2 + stores in Ca2 +-free
KHS. The strip was exposed to 2 x 10- 2 M caffeine (CF) 7 times for 1 min with an
a
0 % nM
F 100
« 970
a:
UJ
~~
()
z ,....,
UJ 50 ()
() III
(f) ~
UJ
a:
0
::>
...J 0 0 - 150
U.
d4 + +- +-
b 'J6
150
100
z
o
Ci5
z
~ 50
10 9 8 7 6 (-log M)
ENDOTHELIN
FIG. 4. Concentration-dependent effect of ET on a [Caz+]i and b tension of porcine
coronary arterial strips. The fluorescence intensity and tension at normal and 118 mM
K+ KHS were assumed to be 0% and 100%, respectively, [Caz+]i and tension were
measured at peak levels from recordings such as those shown in Fig. 3. Data are means
± S.D. of 4 experiments. a 0, In normal KHS (EC so = 3.2 X 10- 10 M);~, in Ca2+-free
KHS (EC50 = 6.6 X 10- 9 M);-after depletion of intracellular Ca z+ store in Ca z+-free
KHS. b 0, in normal KHS (EC so = 6.0 x 10- 10 M); ., in Ca 2 +-free KHS (EC 50 = 9.0
X 10- 9 M); .. , after depletion of intracellular Ca z+ store in Ca 2 +-free KHS (EC 50 = 2.0
x 1O- x M). (From [9], with permission)
~~---------------------------------------------
interval of 2 min and subsequently to 10- 5 M histamine (His) 4 times for 1 min with an
interval of 3min. Thereafter, 1O- 7 M ET was applied to the strip in which the intra-
cellular Ca 2 + stores were practically depleted. F340 and F380 traces are the fluorescence
changes emitted at 500nm when the strip was alternately (400Hz) exposed to the
excitation light at 340 and 380 nm, respectively. The ratio of the fluorescence excited at
340 nm to that at 380 nm was calculated and referred to as Ratio. Tension trace shows
the tension development monitored simultaneously. Before starting all of the experi-
ments, the fura-2-loaded strip was suspenqed in an organ bath filled with normal KHS
for 60-90 min and was then exposed to 118 mM K+ KHS for 5 min and washed with
normal KHS. (From [8] with permission)
250 H. Kai et al.
Therefore, irrespective of the presence and absence of extracellular Caz+, ET

induced a greater contraction for the given [Caz+]i elevation than that
expected, compared with the relationship between [Ca2+]i and tension seen
with contraction in 118mM K+ KHS.
The peak levels and time course of increases in the fluorescence and the
tension induced by ET were not affected by the duration of the exposure to
Caz+-free KHS (10-120 min). In Caz+-free KHS, when the strip was exposed
to 10- 5 M histamine 4 times for 2 min with an interval of 3 min, a series of
increases in [Caz+]i and tension was observed and the peak levels of [Caz+]i
and tension were progressively reduced with each application of histamine. No
increase in [Ca2+]i and tension was observed with the fourth application of
histamine. Under the condition in which the histamine-sensitive intracellular
Caz+ store was then practically depleted, ET induced a transient contraction
and elevation of [Caz+k The levels were similar to those observed when ET
was applied to the strip in Ca z+-free KHS, without repeated treatment with
histamine. When the strip was exposed to 2 x lO- z M caffeine 7 times for
1 min with an interval of 2 min in Caz+-free KHS, a series of increases in
[Caz+]i and tension was also observed, and the peak levels of [Ca2+]i and
tension were progressively reduced with each application. The fourth or fifth
application with caffeine induced no response of contraction, while the
elevation of [Caz+]i was evident for up to the sixth application. When the
caffeine-sensitive intracellular Ca z+ store was depleted with repetitive
applications of caffeine, the subsequent application of ET induced a transient
contraction but no increase in [Caz+k A similar contraction with no change of
[Ca2+]i was observed when ET was subsequently applied to the strip in which
both caffeine-sensitive and histamine-sensitive intracellular Ca z+ stores were
practically depleted by repetitive treatments with caffeine and histamine in
Caz+-free KHS (Fig. 3b). This effect depended upon the concentration of ET
(Fig. 4). In the strip in which the intracellular Ca2+ stores were depleted, the
tension induced by 10- 7 M ET was about 28% of that observed with strips
exposed to 118 mM K+ KHS. When the strips were exposed to 5 x 10- 5 M of
W-7, a relatively specific calmodulin inhibitor [13], for 15 min after the
procedure in which the intracellular Caz+ stores were depleted, the contraction
induced by the subsequent application with ET had little effect (97% of the
peak tension observed without W-7). Conversely, when the strips were treated
for 15 min with 10- 5 M of H-7, a relatively selective protein kinase C inhibitor
[14], the contraction induced by the subsequent application with ET after
depletion of the intracellular Caz+ stores was markedly inhibited (32% of the
peak tension observed without H-7).
Discussion
It has been suggested that ET functions as an endogenous modulator of the
voltage-dependent Caz+ channel, and that the mechanism of action of ET on
vasoconstriction strongly depends upon the presence of extracellular Caz+
[1,2]. However, the 45Ca2+ flux study suggested that extracellular Ca2+-
independent mechanisms may be involved in the action of ET on rat aortic
VSMCs in primary culture [6]. In addition, the evaluation using quin2
microfluorometry study revealed that ET induced a sustained elevation of
[Ca2+]j in the presence of extracellular Ca2+, and that a transient elevation of
[Ca2+]j was evoked by ET not only in the presence but also in the absence of
extracellular Ca2+, thereby suggesting that a release of Ca2+ from the
intracellular store is also involved in the mechanism of action of ET on [Ca2+]j
elevation in cultured VSMCs from the rat aorta [7]. Since the sustained
component of the ET-induced [Ca2+]j elevation in Ca2+-containing solution
was not observed in Ca2+ -free solution, and was markedly inhibited by the
Ca2+ antagonist diltiazem, this component may be mediated by an extracellular
Ca2+ -dependent mechanism such as the Ca2+ influx via the voltage-dependent
Ca2+ channel. The maximum response of the [Ca2+]j increase was observed
with 1O- 7 M ET, and the level was estimated to be 225nM. This level was
similar to that observed when VSMCs were exposed to 16mM K+ PSS, and it
was much less than that observed when VSMCs were exposed to lOOmM K+
PSS or 1O- 5M norepinephrine (543nM or 368nM, respectively). This may
explain why a significant net increase in 45Ca2+ accumulation into VSMCs was
not observed when cells were exposed to ET, in the 45Ca2+ influx study [6].
We reported that rat aortic VSMCs in primary culture apparently possess
two distinguishable types of intracellular Ca2+ stores: a caffeine- and K+
depolarization-sensitive store and a histamine- and norepinephrine-sensitive
one [12,15]. Since the ET-sensitive intracellular Ca2+ store was resistant to
[Ca2+]j depletion in Ca2+-free PSS, as in the case of the caffeine-sensitive one,
the ET-sensitive store is distinguishable from the norepinephrine- and
histamine-sensitive one in which Ca2+ is readily depleted by the decrease in
[Ca2+]j in Ca2+ -free PSS in rat aortic VSMCs in primary culture [12,15]. When
the caffeine-sensitive Ca2+ store was practically depleted by repeated
treatments with caffeine, the subsequent exposure to ET led to no increase in
[Ca2+]j. Therefore, it was suggested that the ET-sensitive store overlaps with
the caffeine-sensitive one in VSMCs in primary culture.
Front-surface fluorometry is a newly developed method for simultaneous
monitoring of tension and [Ca2+]j in fura-2-loaded muscle strip, and presents
high sensitivity and specificity for detecting [Ca2+]j change [8,11]. We have
shown that, as in the case of rat aortic VSMCs in primary culture, ET increases
[Ca2+]j by means of both a release of Ca2+ from the intracellular store and
extracellular Ca2+ -dependent mechanisms. Irrespective of the presence and the
absence of extracellular Ca2+, the rapid increase in [Ca2+]j mainly relates to a
release of Ca2+ from the intracellular store, which practically determines the
extent of tension development [8]. The sustained phase of [Ca2+]j increase and
tension development observed in the presence of extracellular Ca2+ probably
depends upon extracellular Ca2+ [8]. As in the case of rat aortic VSMCs in
primary culture, the ET-sensitive intracellular Ca2+ store overlaps with the
caffeine-sensitive one but not with the histamine-sensitive one in the smooth
muscle cell of the porcine coronary artery, since ET induced a release of Ca2+
252 H. Kai et al.
from the intracellular store when the histamine-sensitive store was depleted,
but not after depletion of the caffeine-sensitive one [8].
In addition to these two Ca2+ -dependent components of the contraction, a
Ca2 +-independent component is probably involved in ET-induced contraction
since ET evoked contraction accompanied with no apparent change in [Ca2 +]j
in strips in which the intracellular Ca2+ stores had been depleted. The findings
that the Ca2+ -independent component of the ET-induced contraction was
markedly inhibited by the protein kinase C inhibitor, H-7, but not by the
calmodulin inhibitor, W-7, suggested that this component of contraction is
probably mediated by a protein kinase C-related phosphorylation of contractile
elements, in a manner different from the usual Ca2 +-calmodulin-mediated
myosin light chain phosphorylation.
The three components of the ET-induced contraction showed differences in
the values in ECsos. As shown in the legend for Fig. 4, ECsos in normal KHS
and Ca2 +-free KHS were 6.0 x 10- lO M and 9.0 x 1O- 9 M, respectively, and
that observed when the intracellular Ca2+ stores were depleted in Ca2 +-free
KHS was 2.0 x 10- 8 M. These findings suggested that ET induces contraction
mainly due to Ca2+ -dependent mechanism, with low ECso values.
The ET-induced tension development in relation to an increase in [Ca2 +]j in
normal and Ca2+ -free KHS was much greater than that expected from the
relationship between [Ca2+]j and tension observed with K+ -depolarization-
induced contraction. Since these findings were apparent when the
concentration of ET was below the levels in which the Ca2+ -independent
contraction was negligible (e.g., 10- 9 M), it was implied that ET may increase
the sensitivity for Ca2 + in the Ca2 +-mediated contractile system, or may
amplify the Ca2 +-mediated tension development.
Thus, ET has multiple sites of action on vascular smooth muscle cells, and
the maintenance and regulation of tension in the sustained phase of the ET-
induced contraction in the presence of the extracellular Ca2 + is no doubt
mediated by complicated and diversified systems, probably including
extracellular Ca2+ -dependent mechanisms and the Ca2 +-independent
component.
Acknowledgments. We thank Ms. M. Ohara for critical comments. This work

was supported in part by Grants-in-Aid for Scientific Research on Priority
Areas (No. 01641532, 02223107 and 02257207) and for General Scientific
Research (No. 01480250 and 02670399) from the Ministry of Education,
Science, and Culture, Japan and Grants for the "Research Program on Cell
Calcium Signals in the Cardiovascular System" from Suzuken Memorial
Foundation, the Tokyo Biochemical Research Foundation, Uehara Memorial
Foundation, Casio Science Promotion Foundation, and Ciba-Geigy Foundation
(Japan) for the Promotion of Science.
References
1. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui M,
Yazaki Y, Goto K, Masaki T (1988) A novel potent vasoconstrictor peptide
produced by vascular endothelial cells. Nature 332:373-376
2. Goto K, Kasuya Y, Matsuki N, Takuwa Y, Kurihara H, Ishikawa T, Kimura S,
Yanagisawa M, Masaki T (1989) Endothelin activates the dihydrophyridine-
sensitive, voltage-dependent Ca2 + channel in vascular smooth muscle. Proc Natl
Acad Sci USA 86:3915-3918
3. Hirata Y, Yoshimi H, Takaichi S, Yanagisawa M, Masaki T (1988) Binding and
receptor of down-regulation of a novel vasoconstrictor endothelin in cultured rat
vascular smooth muscle cells. FEBS Lett 239:13-17
4. Resink TJ, Scott-Burden T, Buhler FR (1988) Endothelin stimulates phospholipase
C in cultured vascular smooth muscle cells.Biochem Biophys Res Comm 157:1360-
1368
5. Komuro I, Kurihara H, Sugiyama T, Yazaki Y (1988) Endothelin stimulates c-fos
and c-myc expression and proliferation of vascular smooth muscle cells. FEBS Lett
238:249-252
6. Miasiro N, Yamamoto H, Kanaide H, Nakamura M (1988) Does endothelin
mobilize calcium from intracellular store sites in rat aortic vascular smooth muscle
cells in primary culture? Biochem Biophys Res Comm 156:312-317
7. Kai H, Kanaide H, Nakamura M (1989) Endothelin-sensitive intracellular Ca2 +
store overlaps with caffeine-sensitive one in rat aortic smooth muscle cells in
primary culture. Biochem Biophys Res Comm 158:235-243
8. Kodama M, Kanaide H, Ade S, Hirano K, Kai H, Nakamura M (1989) Endothelin-
induced Ca-independent contraction of the porcine coronary artery. Biochem
Biophys Res Comm 160: 1302-1308
9. Yamamoto H, Kanaide H, Nakamura M (1983) Metabolism of glycosaminoglycans
of cultured rat aortic smooth muscle cells altered during subculture. Br J Exp
Pathol64:156-165
10. Kanaide H, Kobayashi S, Nishimura J, Hasegawa M, Shogakiuchi Y, Matsumoto
T, Nakamura M (1988) Quin2 microfluorometry and effects of verapamil and
diltiazem on calcium release from rat aortic smooth muscle cells in primary culture.
Circ Res 63:16-26
11. Hirano K, Kanaide H, Nakamura M (1989) Effects of okadaic acid on cytosolic
calcium concentrations and on contractions of the porcine coronary artery. Br J
Pharmacol 98: 1261-1266
12. Kanaide H, Shogakiuchi Y, Nakamura M (1987) The norepinephrine-sensitive Ca2+
storage site differs from the caffeine-sensitive site in vascular smooth muscle cells of
the rat aorta. FEBS Lett 214:130-134
13. Hidaka H, Asano M, Iwadare S, Matsumoto M, Tatsuka T, Aoki N (1978) A novel
vascular releasing agent, N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide
which affects vascular muscle actomyosin. J Pharmacol Exp Ther 207:8-15
14. Hidaka H, Inagaki M, Kawamoto S, Sasaki Y (1984) Isoquinolinesulfonamides,
novel and potent inhibitors of cyclic nucleotide-dependent protein kinase and
protein kinase C. Biochemistry 23:5036-5041
15. Matsumoto T, Kanaide H, Shogakiuchi Y, Nakamura M (1989) Characteristics of
the histamine-sensitive calcium store in vascular smooth muscle; comparison with
norepinephrine- or caffeine-sensitive stores. J BioI Chern 265:5610-5616
6
Cascade of Pathophysiological Events
Leading to Spasm of Coronary
Arteries *
Hitonobu Tomoike i , Kensuke Egashira, Yusuke Yamamoto,
Hiroaki Shimokawa, Yasuo Hayashi, Akira Yamada,
Kazushige Nagasawa, Wataru Mitsuoka, Shogo Egashira,
Takeshi Kuga, Hirofumi Tagawa, and Motoomi Nakamura 2
Introduction
Coronary spasm plays an important role in variant angina, effort angina, acute
myocardial infarction, and/or sudden death [1-4]. Augmented responses of the
coronary artery to vasotonic agents have been documented angiographically in
patients with variant angina; however, the mechanisms of enhanced luminal
narrowing remain unclarified, both clinically and experimentally. In order to
elucidate factors involved in the enhanced responses of the coronary artery, we
developed an animal model with the following features [5,6]: (1) transient
changes in coronary diameter can be assessed angiographically, (2) coronary
spasm can be repeatedly provoked, and (3) myocardial ischemia at the area
distal to the site of the stenosed coronary artery can be documented. We chose
G6ttingen miniature swine as an animal model of coronary spasm [5], because
(1) repeated examinations of coronary angiography and endothelial balloon-
denudation were feasible using a catheterization technique and (2) pigs seem to
be the most appropriate animal model for inducing atherosclerosis by changes
which occur that closely resemble those seen in humans. We used mainly
coronary arteriography for documentation of spastic events, because this
technique is the only available tool for determining regional differences in
vascular responsiveness to vasoactive substances in situ [6]. Regional intimal
thickening along the left coronary artery was produced to mimic the diseased
state of humans, this being the area in which the endothelial denudation had
been one of procedures for inducing atherosclerotic lesions in experimental
animals [7,8].
Present address: IThe First Department of Internal Medicine, Yamagata University

School of Medicine, Yamagata, 990-23 Japan
z"Nakamura Gakuen" Graduate School of Nutritional Science, Fukuoka, 814 Japan
* Experiments were done in the Research Institute of Angiocardiology and
Cardiovascular Clinic, Faculty of Medicine, Kyushu University, Fukuoka, 812 Japan
254
6. Coronary Spasm 255
Agonist Specificity of the Segments Previously Denuded

The segment of the left coronary artery in 4- to 5-month-old Gottingen
miniature pigs were denuded by a 2F Fogarty embolectomy catheter under the
guidance of fluoroscopy. Then, the pigs were fed either a 2% cholesterol diet
or low-cholesterol laboratory chow for 3-6 months. The cholesterol level
increased from 57 ± 6 to 222 ± 27 mg/dl in pigs fed the cholesterol diet but
remained unchanged in pigs fed the low-cholesterol diet (48 ± 5 to 55 ±
6mg/dl).
Three to six months after denudation of the left coronary artery,
angiography revealed no significant stenosis at the basal state in either group of
pigs [9]. Responses of the coronary artery to vasoactive stimuli were examined
angiographically and the degree of luminal narrowing was derived as a
percentage change compared with the diameter after administration of
nitroglycerin (20/lg/kg iv.). The ECG was monitored to document ischemic
changes associated with coronary artery spasm. Intracoronary administration of
histamine or serotonin but not phenylephrine, STA2 , or leukotrienes (LTC4
and LTD 4 ) enhanced the constriction of the previously denuded area [6,9-12]
(Fig. 1). ECG-ST changes were occasionally noted in cases with severe
SWINE MODEL
Specific & Non-specific Vasoconstriction to Agonists
DENUDED SITE INTACT SITE
100
* P<OOl between
Denuded & Intact
-
c:
o
-u
....
en
c: 50 1* SEROTONIN
o
p<ot
CIM~IDINE
g (
HISTAMINE
en ~ ~ STAI P<O~I.!1
{, SEROTONIN
~ ~~STAI
Ii It P<QOS
~-----Q Q_ _ _ _Q PHENYLEPHRINE
la! PHENYLEPHRINE
OL--------
Before 3M After Before 3M After
Denudation Derudation
FIG. 1. Vasoconstrictive responses before and 3 months after balloon-denudation in a

swine model. Augmented luminal narrowing in response to histamine and serotonin was
noted at the denuded site. Data area presented as the mean ± SE. (Modified with
permission from [6])
256 H. Tomoike et al.
FIG. 2. Comparison of observed and pre-

(!) 100 • 0
Z dicted percentage of luminal narrowing.
o (Detailed in [9])
~
o
a:
a: • 0
~
z • 0
';I.. SO
o
LU
>
a:
LU
(f)
CD
o
o so 100
PREDICTED" NARROWING
narrowing. Diphenhydramine and ketanserin totally attenuated histamine- and

serotonin-induced hyperconstrictions of the coronary artery in vivo,
respectively. Such enhanced responses were events topologically related to the
segments with the intimal thickening. The geometric effects of the intimal
thickening on luminal constriction did not play a significant role in the further
augmentation of spasm (Fig. 2). These lines of evidence suggest that receptors
related to HI and S2 were altered during the process of intimal hyperplasia.
The mechanisms of agonist specificity for enhanced responsiveness remain
unelucidated.
Structural Characteristics Responsible for Coronary Spasm

Blood components and neurohumoral factors have been examined as some of
the possible agents for inducing the augmented transient constriction of the
coronary artery. In order to determine the pharmacological characteristics of
the spastic coronary artery in the absence of both blood and neurohumoral
influences, coronary hemodynamics were examined in electrolyte-perfused
isolated porcine heart [13]. Following in situ provocation of coronary spasm by
histamine, the heart was isolated and perfused with oxygenated Krebs-
Henseleit solution under a constant perfusion pressure of 90 mmHg. After
90 min of equilibration, coronary angiograms of the arrested heart were taken
before and after the infusion of vasoactive agents. Histamine 10-5 M narrowed
the luminal diameter by 29 ± 4% and 67 ± 3% (P < 0.001) in non-spastic and
spastic segments, respectively. The site of augmented constriction was the same
in vivo as well as in isolated hearts. KCI 40 mM or phenylephrine reduced the
coronary diameters to a similar degree for denuded and for non-denuded
coronary arteries. Accordingly, agonist specificity of the spastic vessel also was
confirmed in the isolated heart.
6. Coronary Spasm 257
The dose-response relation to histamine (10-7 - 10-5 M) of the spastic

segment was not altered by pretreatment with guanethidine, atropine, or
tetrodotoxin. Ca2+-free solution with EGTA 2mM completely blocked the
luminal narrowing due to 118 mM KCl, and also attenuated the extent of
narrowing at the spastic segment to 19 ± 1%. Mepyramine, an HI blocker,
abolished the constrictive response to histamine [13]. Thus, the functional state
of Hrreceptor may playa pivotal roJe in the expression of augmented luminal
narrowing along the spastic vessel. Problems which remain include the
mechanisms of changes in receptor function, including signal transduction
during the process of intimal hyperplasia.
Isometric tension studies demonstrated both a reduced level of endothelium-
dependent relaxation to serotonin and an increased response of the medial
smooth muscles to histamine at the spastic site [14].
Conclusions
A cascade of structural and functional changes following mechanical injury of
the intima is involved in the appearance of coronary spasm, along with
arteriosclerosis. Chronological studies on receptor function and/or signal
transduction in both endothelium and vascular smooth muscle may elucidate
the mechanisms related to the enhanced responses of the vessel to autacoids.
Acknowledgments. Our gratitude is extended to Dr. Y. Kikuchi and Professors

K. Tanaka and K. T. Lee for advice and discussion, and to Ms. M. Sasaki for
secretarial services.
This work was partly supported by grants for Scientific Research Nos.
02454259, 02404045 from the Ministry of Education, Science and Culture,
Japan, a grant for Research on Cardiovascular Disease (1s-1) from the Ministry
of Health and Welfare, Japan, a grant from the Japan Medical Association,
and a grant from the Uehara Memorial Foundation.
References
1. Maseri A, Chierchia S (1982) Coronary artery spasm. Demonstration, diagnosis and
consequences. Prog Cardiovasc Dis 25:169-181
2. Oliva P, Potts D, Pluss R (1973) Coronary artery spasm in Prinzmetal angina:
Documentation by coronary arteriography. N Engl J Med 288:745-751
3. Miller DD, Waters DD, Szlachcic J (1982) Clinical characteristics associated with
sudden death in patients with variant angina. Circulation 66:588-592
4. Maseri A, L'Abbate A, Baroldi G, Chierchia S, Marzilli M, Ballestra AM, Severi
A, Parodi 0, Biagini A, Distante A, Pesola A (1978) Coronary vasospasm as a
possible cause of myocardial infarction. A conclusion derived from the study of
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E
Ischemia and Reperfusion Injury in the
Experimental and Clinical Studies
1
Coronary Blood Flow
in Reperfused Myocardium
Thomas A versano 1
Summary. The factors which determine coronary blood flow in myocardium

subjected to ischemia and reperfusion are numerous, complex, and
interrelated. They can be conveniently, if arbitrarily, divided into three broad
categories. First, cellular and protein elements of the coronary perfustate, such
as the leukocyte and fibrinogen, may affect coronary blood flow through effects
on the microvasculature and on blood rheological properties, respectively.
Second, the coronary conduit itself, the blood vessel, can also be affected by
ischemia and reperfusion, particularly in its endothelial component and the
associated vascular responses. Finally, the environment in which the coronary
vessel finds itself, determined by characteristics of the myocyte and the
interstitial space, can affect coronary flow by itself being profoundly changed
after ischemia and reperfusion. While ischemia and reperfusion affects all three
of these major determinants of coronary blood flow, whether or not coronary
blood flow responses following reperfusion have a role in producing
myonecrosis is unclear at this time.
Introduction
The factors influencing coronary blood flow in normal myocardium are
numerous, complex, interactive, and incompletely understood. In the more
complex setting of ischemia and reperfusion, still less is known about the
regulation of coronary flow. Nevertheless, studies of coronary flow in
reperfused myocardium and in other settings that mimic some characteristic of
reperfused myocardium do make it possible to at least enumerate, if not order
in quantitative importance, the myriad determinants of coronary flow in this
setting.
1 Johns Hopkins Medical Insititutions, MD, Baltimore, 21205 USA
261
262 T. Aversano
The purpose of this brief review is to highlight those factors that may affect
coronary blood flow in myocardium subjected to periods of ischemia and
reperfusion. No attempt was made to be exhaustive in this space, but rather to
suggest a framework in which coronary flow responses may be viewed. Such a
framework is important because it reveals the complexities involved in
interpreting coronary flow reponses and because it provokes awareness of
potential confounding influences.
Interest in coronary blood flow regulation in ischemia and reperfusion
centers mainly on its potential to influence myonecrosis. In addition to
potentially influencing the extent of myocardial cell necrosis, however,
coronary blood flow in reperfused myocardium may influence infarct expansion
[1] and possibly post-infarction arrythmias [2].
The determinants of coronary blood flow may be conveniently, if artificially,
divided into factors that influence characteristics of the coronary vessel itself
(i.e., its endothelial and smooth muscle components), the vessel's physical
environment (e.g., the myocytes and interstitial space) and the nature of the
coronary perfusate (its cellular and protein components). Ischemia and
reperfusion may affect all these properties simultaneously and to a varying
degree. It is important to note that most animal models of ischemia and
reperfusion not only vary among themselves, but they differ markedly from the
clinical situation of acute coronary artery thrombosis superimposed on chronic,
atherosclerotic coronary artery disease in which thrombolytic agents are
utilized to effect reperfusion. While the simpler animal models are necessary to
advance our know lege of the pathophysiology of ischemia and reperfusion, the
application of the results of such studies to the clinical setting must be made
with the usual caution.
The Coronary Perfusate
The cellular and protein components of the coronary perfusate, particularly the
leukocyte and fibrinogen, can importantly determine coronary blood flow
following ischemia and reperfusion. Evidence for an association between
leukocyte accumulation and abnormal flow responses in reperfused
myocardium has led to the notion that the former causes the latter, either by
directly plugging the coronary vasculature (as microspheres do [3]) or indirectly
via a variety of vasoactive substances elaborated by the leukocyte [4].
Peak coronary blood flow induced by endothelium-independent vasodilators
has been shown to be normal following 10 min of ischemia and reperfusion
[5]-a period sufficient to produce stunning-while it is clearly reduced
following 1 h of ischemia followed by reperfusion [6]. Interestingly, indium-
labeled leukocyte accumulation is undetectable after 12 min of ischemia
followed by reperfusion, and is quite marked after 40 or 90 min of ischemia
followed by reperfusion [7]. These data suggest a link between leukocyte
accumulation and the reduction in maximum coronary conductance.
1. Coronary Blood Flow in Reperfused Myocardium 263
Further evidence of a link between granulocyte accumulation and abnormal

flow responses has been provided by Martin et al. [8]. These investigators have
demonstrated that even when the epicardial coronary artery is not occluded
and its perfusion pressure is kept constant, granulocyte activation by
intracoronary administration of complement component CSa causes
granulocyte accumulation which is associated with coronary flow reduction and
deterioration of regional function [8]. These effects were reversible. Rather
than physical obstruction of the vasculature by leukocytes causing the
reduction in flow, this group suggests that the flow response is more likely due
to production of thromboxane A2 and leukotrienes, which are potent
vasoconstrictors [4].
In addition to impeding antegrade coronary flow following ischemia and
reperfusion, the granulocyte also appears to influence coronary collateral blood
flow as shown by Engler et al. [9]. When perfused with normal whole blood,
both endocardial and epicardial collateral flow decrease over a 1-h period of
ischemia, while perfusion with leukocyte-poor blood is attended by an increase
in endocardial flow and no change in epicardial collateral flow [9]. These
observations suggest that neutrophils may obfuscate what would otherwise be a
rise in collateral conductance during a 1-h period of ischemia [9]. This could be
due to microvascular plugging or elaboration of vasoconstrictor substances [9].
Another property of the coronary perfusate that may importantly determine
coronary blood flow following ischemia and reperfusion is blood viscosity. In
the setting of human acute myocardial infarction, blood viscosity is higher than
in a control population and may increase in the days following infarction [10].
In addition, a major protein component of blood viscosity, fibrinogen
concentration, is reduced to a varying degree by the various thrombolytic
agents available for use in acute myocardial infarction [11]. Thus, blood
viscosity can be pathologically altered in acute coronary thrombosis and can be
manipulated in the course of thrombolytic therapy. What effect can blood
viscosity have on coronary blood flow?
At a shear rate of S.2 s-l, blood viscosity is approximately 9.S cP in control
populations and rises to 12.S cP in patients with acute myocardial infarction
[10]. By Poiseuille's law, this increase in viscosity of 33% can be expected to
translate to a 33% reduction in maximum coronary conductance. Since, in the
clinical setting, reperfusion most often occurs into an artery with a residual,
high-grade coronary stenosis, it is likely that the coronary circulation distal to
this reperfused but stenosed coronary artery is near maximal vasodilation.
Thus, changes in viscosity may be important determinants of coronary flow in
this setting since autoregulation is essentially exhausted.
The effect of perfusate viscosity on the relationship between coronary
pressure and flow in the fully vasodilated canine circulation has been recently
reported by Drossner and Aversano [12] and illustrates the magnitude of the
flow response that occurs in association with changes in blood viscosity within
this range. At a coronary perfusion pressure of 60mmHg, there is a 40%
reduction in coronary blood flow when a viscosity change of this magnitude is
effected by hemodilution [12].
264 T. Aversano
Interestingly, if all cellular and protein components of the perfusate are

removed, not only does maximum coronary conductance increase, but the zero
flow pressure is reduced to a level equal to right and left atrial pressures [12].
Which perfusate components determine the zero-flow pressure is unknown, but
it is interesting to recall that, while outnumbered by the red cell, the leukocyte
is the most viscous, as well as the most vicious, cell.
Properties of the Conduit

The vascular conduit-the coronary vessel-is a complex structure composed
of endothelial, smooth muscle, and other cells, and of a variety of proteins
including collagen and elastin. Early studies, such as those by Crystal et al.
[13], showed that ischemia and reperfusion resulted in loss of certain flow
responses, in this case autoregulatory responses. More recently, most
published reports on the effect of ischemia and reperfusion concern changes
induced in the endothelial component's structure and function.
In an elegant study by Dauber et al. [14], even very brief periods (e.g.,
15 min) of ischemia followed by reperfusion lead to changes in both vascular
permeability and to endothelium-dependent relaxation, while histologically the
distal microvasculature appeared normal, suggesting that endothelial cell
function is abnormal even before structural injury is apparent. Longer periods
of ischemia (e.g., 60min) followed by reperfusion result in more pronounced
functional abnormalities and to clear histologic abnomalities of the distal
microvasculature characterized by intracellular vacuolization and loss of tight
junctions, perivascular edema, and surrounding myocardial cell injury [14).
Furthermore, the degree of protein leak was inversely related to the degree of
ischemia, the latter dependent upon collateral flow during the ischemia period
[14].
Even the proximal vasculature is affected by periods of ischemia and
reperfusion. Ouyang et al. [15] studied canine epicardial coronary arteries after
90 min of ischemia and 2 h of reflow. In these studies, the epicardial coronary
artery of an intact dog was occluded and reperfused in vivo; it was then
removed and its responses to endothelium-dependent and-independent agents
were studied in vitro [15). The non-occluded artery served as a control. While
the response to endothelium-independent vasoconstrictors, such as potassium,
and vasodilators, such as nitroglycerin, was not abnormal, the response to
acetylcholine, an endothelium-depentent vasodilator, was markedly reduced
following ischemia and reperfusion [15). Interestingly, the endothelium
appeared histologically normal.
These and other studies suggest that endothelial cell dysfunction can occur in
the absence of apparent structural injury, and that the degree of dysfunction
and loss of structural integrity depend upon the degree and duration of
coronary occlusion.
What causes endothelial cell functional and structural alterations? In the
study of epicardial coronary arteries by Ouyang et al. [15] noted above,
another set of experiments demonstrated that at least in the epicardial

coronary artery, coronary occlusion alone-even without reperfusion-is
sufficient to result in loss of endothelium-dependent vasodilation. In this
system, the epicardial coronary artery is removed after occlusion and is never
reperfused with blood. It is, of course, reperfused with oxygenated HEPES
solution but not with the protein and cellular components of the coronary
perfusate. Thus, reperfusion in the presence of leukocytes does not appear to
be necessary to induce the injury to endothelium of the epicardial coronary
artery. Indeed, the endothelium of the epicardial vessel is never even
hypoxic, raising the possibility that factors other than ischemia, such as
altered shear forces, may affect endothelial cell function following coronary
occlusion.
Another possible pathophysiological mechanism of such injury involves the
generation of free radicals by the endothelial cells themselves. Zweier et al.
[16] have shown that cultured endothelial cells subjected to periods of hypoxia
followed by reoxygenation generate free radicals and die. Endothelial cells
treated in the same way in the presence of superoxide dismutase, catalase, or
xanthine oxidase reduced both the generation of free radicals and preserved
cell viability [16]. Such a mechanism of endothelial cell injury might have more
importance in the distal microvasculature where coronary occlusion would be
expected to result in severe hypoxia.
Jackson et al. [17] added electrolysis-generated free radicals to the perfusate
of isolated rat hearts and demonstrated evidence for both vascular and
myocardial effects in the absence of cellular or protein elements of blood. In
particular, perfusion with free radical-containing perfusate resulted in a
decrease in coronary conductance. Therefore, free radicals, per se, can inflict
at least functional changes in the coronary circulation.
Thus, ischemia and reperfusion lead to varying degrees of endothelial cell
structural and functional derangements, primarily dependent upon the degree
and duration of ischemia. Free radical generation, perhaps by both leukocytes
and endothelial cells, may be an important pathophysiological mechanism of
these derangements, particularly in the microvasculature. In the epicardial
vessel, coronary occlusion per se, with its associated stop-flow condition and
altered shear forces, appears to lead to abnormal responses to endothelial-
dependent vasodilators in the absence of hypoxia.
The Conduit Environment
Relatively little attention has been paid to how changes in the mechanical
properties of the blood vessel's environment that attend periods of ischemia
and reperfusion influence coronary blood flow. In order to detect such effects,
the coronary circulation must usually be fully vasodilated, either
pharmacologically following reactive hyperemia or because of the presence of a
critical epicardial coronary artery stenosis. This is so because autoregulatory
266 T. Aversano
mechanisms can offset changes in coronary hemodynamics. Since reperfusion

usually occurs into a critically stenosed coronary artery, the mechanical
properties of the blood vessel's environment may importantly determine
coronary flow.
As noted previously, even relatively brief periods of ischemia and
reperfusion are attended by changes in microvascular permeability [14]. The
formation of intersitial edema can, in turn, adversely affect coronary blood
flow, particularly when the vasodilator reserve is exhausted.
The influence of interstitial edema on coronary blood flow is described in
a study by Van Dijk et al. [18]. These investigators shows that increasingly
longer periods of crystalloid perfusion in isolated cat hearts resulted in a
progressive increase in ventricular wall water content [18]. As intersitial edema
increased, maximum coronary .conductance decreased, and the zero-flow
pressure increased, resulting in a detectable fall in coronary blood flow when
ventricular wall volume increased as little as 2% [18]. While not an ischemia-
reperfusion experiment, this study points out the magnitude and nature of the
changes that interstitial edema can effect.
Ischemia and reperfusion can also be attended by changes in left ventricular
diastolic pressure, which itself can dramatically alter coronary blood flow in the
fully vasodilated coronary circulation [19]. In a study by Aversano et al. [19],
even small changes in preload, particularly at low perfusion pressure (as may
occur distal to a critical coronary stenosis), reduced coronary blood flow
markedly. For example, at a coronary perfusion pressure of 35 mmHg, a rise in
left ventricular (LV) diastolic pressure from 8 to 18 mmHg reduce coronary
flow by 50% [19].
Even more subtle effects of ischemia and reperfusion can affect coronary
blood flow. After even very brief (1 min) periods of ischemia and reperfusion,
Hori et al. [20] could detect changes in ventricular relaxation characterized by
T, the time constant for isovolumic LV pressure dacay. Following reperfusion
after even brief ischemia, relaxation is impaired with T increasing to nearly
twice its preischemic value [20]. Since most coronary blood flow occurs in
diastole, can such impaired ventricular relaxation affect coronary flow?
In a study by Domalik-Wawrzynski et al. [21] 5 min of ischemia followed by
2 min of reperfusion were attended by impairment of relaxation. Associated
with this impairment was a decrease in early diastolic coronary blood flow [21].
Treatment with verapamil, which prevented the relaxation abnormality, also
prevented the early diastolic flow reduction [21]. Thus, even the subtle
impairment of relaxation that follows a very brief period of ischemia and
reperfusion can, indeed, have potentially deleterious effects on coronary blood
flow.
Thus, the mechanical influences of the blood vessel's environment on
coronary blood flow can be quantitatively important, particularly in the circula-
tion whose vasodilator reserve is exhausted. Recognition of such influences
further complicates the interpretation of coronary hemodynamic changes in a
setting of ischemia and reperfusion.
No-reftow
Perhaps the most dramatic flow response that follows ischemia and reperfusion
involves the so-called no-reflow phenomenon. This term refers to the
observation that following periods of ischemia and reperfusion, a variety of
techniques have shown that there are areas within the ischemic zone which fail
to reperfuse despite release of the epicardial coronary occlusion. That the
coronary conduit perfusate, environment, and the conduit itself are involved in
the pathophysiology of no-reflow is underscored by the associated histology
which includes damaged microvascular structures filled with leukocytes and red
cells and surrounded by evidence of severe myocyte necrosis and interstitial
edema.
It now appears that such no-reflow zones actually do have flow at the onset
of reperfusion, but the flow falls to zero in these regions over time. In a study
by Ambrosio et al. [22], dogs were subjected to 90 min of ischemia followed by
either 2min or 3.5h of reflow. The area of no-reflow was defined by injection
of the fluorescent marker, thioflavin S, into the left atrium at the termination
of the reflow period [22]. Areas identified as no-reflow zones at 3.5 h had high
flows (measured using radio labeled microspheres) at 2min after reflow and a
reduced, but substantial, flow at 30 min after reflow, although they had near
zero flow by 3.5h [22]. No reflow, then, is not present at the onset of occlusion
release, but rather appears to develop over time. Observation of leukocytes
trapped in the microvasculature supposrts the hypothesis that leukocyte
plugging is the cause of the no-reflow phenomenon.
Furthermore, it appears that removal of leukocytes from the blood
reperfusing the ischemic region reduces the extent of no-reflow [23]. Reduction
in no-reflow zones was associated with reduced infarct size [23]. That free
radical-induced damage to the microvasculature, perhaps effected by
leukocytes or their products, was also involved in this phenomenon is
supported by the observation that free radical scavengers reduce the extent of
no-reflow [24).
A study by Przyklenk and Kloner [24] has shown that application of
superoxide dismutase (SOD) and catalase in an ischemia-reperfusion model in
dogs reduced microvascular damage and the no-reflow phenomenon compared
with control animals. Interestingly, myonecrosis still occured to the same
degree in control animals and those treated with SOD and catalase despite
preservation of microvascular structural integrity and limitation of no- or
low-reflow. Others have shown reduction of the extent of myonecrosis in
similar models with these same free-radical scavangers [25). The potential
reasons for this difference are beyond the scope of this paper. However, the
level of coronary collateral flow during the ischemic period may itself
determine whether or not free-radical scavangers work to reduce myonecrosis:
Przyklenk suggests that animals with higher than average collateral flow are
most helped, while Ambrosio suggests the opposite [24,25]. The resolution of
these divergent observations awaits further study.
268 T. Aversano
Relation Between Reperfusion Injury and

Coronary Blood Flow
While a multitude of studies suggest a variety of actual and potential abnormal
flow responses in myocardium subjected to periods of ischemia and
reperfusion, the relationship between these conditions and myonecrosis is
unclear.
In the study by Przyklenk and Kloner [24], a dissociation between coronary
flow and myonecrosis is suggested. That is, even though free radical scavengers
were able to preserve microvascular integrity and although the degree of no- or
low-reflow was reduced, the extent of myocardial necrosis was not different
from control animals [24]. The situation is more complex than it might first
appear, however, because if animals with lower than average collateral flow
« 0.06ml/min per gm) are excluded, then SOD and catalase did reduce the
infarct size in treated animals [24]. As noted above, these data have been
interpreted as showing that the level of coronary blood flow determines the
effect of the free-radical scavanger on infarct size. This study does not,
therefore, rule out a possible link between preserved microvascular integrity,
reduction of no- or low-reflow zones and infarct size reduction.
The study by Ambrosio et al. [22] mentioned above also suggests a
relationship between coronary flow responses and myonecrosis. After 90 min of
occlusion and 2 min of reflow, the endocardium in the ischemic region had
patchy filling of endocardial capillaries and a Swiss cheese appearance, the
latter representing areas of no-reflow [22]. After 90 min of occlusion and 3.5 h
of reflow, the endocardium showed extensive confluent areas of non-filling
involving large segments of the ischemic zone's endocardial wall and abrupt
truncation of arterioles, suggesting obstruction [22]. There was evidence of
severe histologic damage and leukocyte infiltration in the no-reflow areas [22].
These data support the idea that the progressive impairment of flow
associated with leukocyte accumulation and vascular obstruction at least
contributes to post-reperfusion myonecrosis. Nevertheless, a clear-cut cause
and effect association cannot be made.
Conclusions
Coronary blood flow in reperfused myocardium can be affected by a variety of
factors, only a very few of which have been highlighted in this brief overview.
Teasing out what factors are most important in any given situation is quite
difficult in a setting so complex as this. In addition, the question of whether
derangements in coronary blood flow per se contribute to the extent of
myocyte injury following reperfusion seems unsettled at the present time.
In the clinical setting of acute myocardial infarction, coronary occlusion is
usually superimposed on a chronic atherosclerotic plaque. The vascular
reponses of this plaque are themselves abnormal, and the contents of the
occlusive thrombus include a variety of substances and formed elements that
can affect both the distal and proximal coronary vasculature. Furthermore, the
thrombolytic agents used to effect reperfusion in the clinical setting have
varying abilities to affect factors which determine coronary blood flow, such as
blood viscosity through fibrinogen depletion. Other methods of reperfusion,
such as angioplasty, may themselves introduce changes in coronary flow. We
have not considered any of these factors in our discussion, and most
experimental models understandably do not account for them. They are import-
ant to recognize, however, so that our threshold for applying knowledge gained
in the simpler setting of animal models to the clinical setting is raised. We are
at the beginning of our understanding.
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coronary pressure-flow relationship. Am J Physiol 259:H603-H609
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flow and volume during reperfusion in canine hearts. Clin Exp Pharmacol Physiol
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14. Dauber 1M, VanBenthuysen KM, McMurtry IF, Wheeler GS, Lesnefsky EJ,
Horwitz LD, Weil JV (1990) Functional coronary microvascular injury evident as
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pressure-flow relation after transition from blood to Tyrode perfusion. Am J
PhysioI255:H476-H482
19. Aversano T, Klock FJ, Mates RE, Canty JM (1984) Preload-induced alterations in
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2
Continuity of Myocardial Stunning-
Latent Myocardial Damage
After Coronary Occlusion
Mamoru Miura, Takashi Saito, and Tomohiro Kanazawa 1
Summary. In order to investigate the contrinuity of myocardial stunning, the

pre-stage of manifest myocardial stunning or latent myocardial damage was
studied in 20 anesthetized, open-chest dogs. Myocardial contractile function
(percent of systolic shortening: %SS) was evaluated by sonocrystals.
Myocardial tissue PC0 2 , pH, and extracellular K+ concentration (K+ e) were
measured in the middle layer of myocardium. Epicardial surface ECG (AC-
and DC-coupled) were recorded. A 2-min occlusion of the left anterior
descending coronary artery (LAD) was used as a tool for analyzing latent
myocardial damage after reperfusion. During occlusion, %SS, PC02 , pH, and
K+ e were observed in ischemic myocardium. There were no differences in these
variables between the first 2-min occlusion (T-1) and the second 2-min
occlusion (T-2) which was done 110 min after T-l. Therefore, these two
occlusions affected the ischemic myocardium almost identically in this
situation.
For investigating the effects of brief ischemia on IPyocardium, 5-min LAD
occlusion was introduced 15 min after T-l. Although %SS seemed to be
completely recovered 90 min after reperfusion, early contractile failure in %SS
was observed during T-2. The R wave amplitude during T-2 showed earlier
progression of the first decrease phase and reduced the rise of the second
increase phase. Also, during T-2, ST deviation in the ECG was reduced, and
PC0 2 , pH, and K+ e were decreased. Then, metabolic damage was latently
continued for 90 min after reperfusion.
It is concluded that a 5-min coronary occlusion causes latent myocardial
dysfunction and latent metabolic damage even after 90 min of reperfusion. This
latent myocardial damage of myocardial function and metabolism may be quite
important as a pre-stage of manifest myocardial stunning.
1 The Second Department of Internal Medicine, Akita University School of Medicine,

Akita, 010 Japan
271
272 M. Miura et al.
Key words: Latent myocardial damage-Myocardial stunning-Myocardial

contractile function-Myocardial metabolism-Brief ischemia
Introduction
Myocardium reperfused after brief ischemia exhibits prolonged and reversible
depression of contractile function, the so called myocardial stunning. The
severity and the time course of myocardial dysfunction after reperfusion
depend upon the duration of myocardial ischemia. This phenomenon is very
important for the indication of coronary revascularization therapy and the
prognosis of patients with coronary heart disease. In order to study the
pathophysiological states of myocardial stunning, the reversibility,
accumulation, and the continuity of the ischemic myocardial damage [1,2]
should be analyzed.
We present an analysis of the continuity of myocardial stunning, especially
the pre-stage of manifest myocardial stunning or latent myocardial damage.
Methods
Twenty adult mongrel dogs of either sex were anesthetized with intravenous
sodium pentobarbital (25 mg/kg) and ventilated with a Harvard respirator.
After thoracotomy, the heart was exposed in a pericardial cradle and the left
anterior descending coronary artery (LAD) was dissected between the first and
second diagonal branches and fitted with a pneumatic occluder in order to
produce ischemic and reperfused myocardium. In order to study regional
myocardial function, a pair of sonocrystals was implanted in the middle layer of
the LAD-perfusing region. The segmental length of enddiastole (EDL) ,
endsystole (ESL) and at the beginning of ejection (SLej) was measured in a
short axis. The regional myocardial contractility was expressed as percent of
systolic shortening (%SS: (EDL-ESL)/EDL x 100), percent of shortening of
ejection phase (%EJ: (SLej-ESL) EDL x 100) and percent of shortening of
isometric contraction phase (%IS: (EDL-SLej)/EDL x 100) calculated from
the segmental length of each phase.
Myocardial tissue PCO z and pH were measured in the middle layer of the
same region. Metabolic rate (MR) was calculated by the following equation
MR(PCO z) = PCOz(t) - PCOz(! - 10) (mmHg), MR (H+ concentration) =
lO-pH(I) _ lO-pH(1 - 10) (nM/L), X!; value of X at tmin after occlusion.
Extracellular K+ concentration (K+ e) was also measured in the same region.

K+ equilibrium potential (Ek) [3] was calculated with the following equation:
Ek = 61.5 mV x log (Ko/ (Ki - 0.33 x delta Ko), 61.5 mV; Nernst slope
(37°C), Ko; extracellular K+, Ki; intracellular K+ (100 mM).
We also recorded AC- and DC-coupled epicardial surface ECG and
measured the R wave, ST segment, and TO shift, as well as the activation
2. Continuity of Myocardial Stunning 273
time. Left ventricular and aortic pressures were measured by catheter-tip

manometers.
In order to reveal the influence of brief ischemia, i.e., 5-min coronary
occlusion in this experiment, 2-min coronary occlusion was used as a tool for
analyzing latent myocardial damage after reperfusion. At first, LAD was
occluded for 2 min and reperfused for 15 min. This 2-min occlusion was called
trial 1. The dogs were divided into 2 groups. In the control group (n=lO),
subsequent 95-min reperfusion was continued. In the 5-min occlusion group
(5-min group; n=lO), LAD was occluded for 5 min and reperfused for 90 min.
Then, LAD was occluded for 2 min as trial 2 in both groups. Two trials were
compared in each group and between the two groups in order to evaluate
myocardial damage caused by 5-min occlusion.
Statistical Analysis
Data were presented as mean ± standard error of the mean (SEM). Student's
t-test was used and differences were considered significant when P < 0.05.
Results
Myocardial contractile function expressed as %SS, %EJ, and %IS was
completely recovered after 90 min of reperfusion. %SS and %EJ during two
trials in the 2 groups are shown in Fig. 1a,b. In the control group, no significant
differences were found between trials 1 and 2. In the 5-min group, the earlier
deterioration of myocardial contractile function in trial 2 was apparently shown
while transient post-ischemic hyperfunction occurring immediately after reper-
fusion disappeared in trial 2. However, %IS during trials 1 and 2 was not
significantly different between the control and 5-min groups.
In Fig. 2a, R wave amplitude in the epicardial surface ECG showed a
biphasic response (first a decrease phase and second an increase phase) during
trials 1 and 2 in the control group. In the 5-min group, the first decrease phase
of R wave appeared earlier in trial 2 than in trial 1, and the second increase
phase was significantly reduced in trial 2. The response of activation time was
similar to that of R wave (Fig. 2b). There was negative correlation between R
wave amplitude and EDL in both the two trials and the two groups:
Control: trial 1: y = -23.6X - 1.0, r = -0.80, P < 0.01
trial 2: y = -19.6X - 0.4, r = -0.77, P < 0.01
5-min: trial 1: y = -20.4X - 1.1, r = -0.76, P < 0.01
trial 2: y = -24.4X - 0.6, r = -0.79, P < 0.01
(y, delta R wave amplitude; X, delta EDL)
On the epicardial surface ECG (AC-coupled), ST elevation was not different
between trials 1 and 2 in the control group but was significantly reduced in trial
2 in the 5-min group (Fig. 3, Table 1). TO shift on DC-coupled ECG in trial 2
was also reduced in the 5-min group.
274 M. Miura et al.
a Control Group
%Systolic Shortening
%
120
-4
ot 15 30 4560 90 12015 30 45 60 90 120 sec 5 10 15 min
t
occlusion reperfusion
%Ejection Shortening
%
120
o Trial 1
• Trial2
(mean±SEM)
• : p<O.05
•• : p<O.01
----------------------------------~~~---
o 15 30 45 60 90 ,20 15 30 45 60 90 120sec 5 10 15 min
t
occlus reperfusion
FIG. 1. a Time course of % systolic shortening and % ejection shortening during trials 1
and 2 (2-min occlusion) in the control group. b Time course of % systolic shortening
and % ejection shortening during trials 1 and 2 in the 5-min occlusion group
b 5min Occlusion Group
%Systolic Shortening
-4
o 15 30 4560 90 12015 30 45 60 90 120 sec 5 10 15 min
t 1
%Ejection Shortening
%
120
60
o Trial 1
• Trial 2
40
(mean±SEM)
2 * : p<O.05
** : p<O.01
o
----------------------------------~~-------
~ 15 30 4560 90 ~20 15 30 45 60 90 120 sec 5 1Q 15 min
r
FiG. 1. cont.
276 M. Miura et al.
a R wave Amplitude
control group 5min occlusion group
O(
10
%
140 140
120 120
*
100 100
80 80
60 0-") Trial 1
60
e-e Tfial 2
meanoSEM
O T,-~,~~-r--T------,•sec o ...
T * P<005
..,.--..,.--.,..-.,..-.,..---~sec
befcre 15 30 45 60 120 befcre 15 30 45 60 120
b Activation Time
% control group % 5min occlusion group
120 120
100 100
80 80
0-") Trial 1
• • TriaJ2
60 60 meanoSEM
* P<O.05
o Tf-r-~~_~--~' sec o I,--,--~~~~---~, sec
befCfe 15 30 45 60 120 befcre 15 30 45 60 120
FIG. 2. a Time course of R wave amplitude during trial 1 and 2 in control (2-min)
occlusion) and 5-min occlusion groups. b Time course of activation time during trial 1
and 2 (2-min occlusion) in control and 5-min occlusion groups
K+ c was significantly increased from 45 S after coronary occlusion. Ek is

shown in Table 1. The K+ c was positively correlated to TO shift in both the
two trials and the two groups:
Control: trial 1: y = 5.90X + 0.31, r = 0.92, P < 0.001
trial 2: y = 6.08X + 0.40, r = 0.90, P < 0.001
FIG. 3. ST deviation and TQ shift during ST Deviation

trials 1 and 2 (2-min occlusion) in control mV
and 5-min occlusion groups. Results are 10
NS P<0.05
mean ± standard error of the mean
(SEM). NS, Not significant.
OL-~~~~~--~--L-~
Trial 1 Trial 2
control group 5 min occlusion
group
O~--~~-m~~~r--r-m~~
-5
NS P<0.05
mV
TO Shift
TABLE 1. ST deviation, TQ shift, extracellular K+ concentration, tissue PC0 2 , CO 2

metabolic rate, tissue pH, and proton metabolic rate during trials 1 and 2 (2-min
occlusion) in the control and 5-min occlusion groups
Control group 5-min Occlusion group
Trial r Trial2 a Triall a Trial2 a
ST Deviation (mV) 6.5 ± 1.0 6.0 ± 0.7 6.5 ± 0.6 3.6 ± 0.8-
TO Shift (mV) 5.4 ± 0.9 5.5 ± 1.0 5.8 ± 0.7 3.3 ± 0.5-
Extracellular K+ concentration
(mmoIlL) 4.61 ± 0.11 4.67 ± 0.10 4.71 ± 0.22 3.99 ± 0.09*-
Tissue pC02 (mmHg) 60.9 ± 3.6 59.3 ± 3.7 59.3 ± 4.0 48.0 ± 3.5-
CO2 Metabolic rate (mmHg/min) 11.9 ± 3.9 12.0 ± 4.1 11.6 ± 3.5 5.5 ± 3.8*-
Tissue pH (pH unit) 7.12 ± 0.03 7.12 ± 0.03 7.12 ± 0.03 7.20 ± 0.02-
Proton metabolic rate (nmoI/L) 17.4 ± 6.6 16.8 ± 3.7 17.6 ± 5.6 8.5 ± 4.8**
*P < 0.05; *-P < 0.01 (Compared to trial!: intragroup comparison)

a2-min Occlusion
Results are mean ±SEM
278 M. Miura et al.
5-min: trial 1: y = 7.57X + 0.42, r = 0.93, P < 0.001

trial 2: y = 6.50X + 0.23, r = 0.91, P < 0.001
(y, TO shift; X, delta extracellular K+ concentration)
As shown in Table 1, the changes of myocardial tissue PC0 2 , pH, and K+ e
were not significantly different between the two trials in the control group, but
were markedly depressed in trial 2 compated to trial 1 in the 5-min group. The
MR calculated from PC02 and pH (time differential of CO 2 and proton) in the
two trials showed no significant differences in the control group. On the
contrary, a marked depression of these variables in trial 2 was shown in the
5-min group.
Discussion
Although myocardial contractile function seemed to recover completely after
90 min of reperfusion, earlier deterioration of %SS on the ischemic segmental
length appeared faster during trial 2 than trial 1 in the 5-min group, and
transient post-ischemic hyperfunction appeared immediately after reperfusion
disappeared during trial 2 in the 5-min group. %EJ showed the same responses
as %SS. Therefore, myocardium having reperfused after 5 min of ischemia
exhibits latent depression of contractile function that is prolonged more than
90 min after reperfusion.
On epicardial surface ECG (AC-coupled), R wave amplitude showed the
same biphasic response during trial 2 as in trial 1 in the control group. On the
other hand, the first decrease phase of R wave amplitude was earlier during
trial 2 than during trial 1, and the second increase phase of R wave amplitude
was significantly decreased during trial 2 in the 5-min group. The response of
activation time showed a similar biphasic pattern to that of R wave amplitude.
As already reported [4], intramyocardial conduction time is the major deter-
minant factor of R wave amplitude. Consequently, the altered response of R
wave is closely related to the earlier appearance of the first phase of decrease
and the diminished peak of increase in the activation time.
R wave amplitude is affected by EDL and K+ c in brief myocardial ischemia.
There was negative correlation between R wave amplitude and EDL in both
the two trials and the two groups. The higher R wave amplitude was the
shorter EDL was.
Dominguez and Fozzard [5] reported that the elevation of K+ c caused a
biphasic change of intra-myocardial conduction time. Less than 4.0 MEq/L of
K + c increased the velocity and more than 4.0 MEq/L decreased it. In our
study, however, K+ c did not significantly increase within 45 S after occlusion.
Therefore, we postulate that the rapid ventricular wall thinning and stretching
caused by ischemia produce the first decrease phase of R wave amplitude. The
decrease of R wave amplitude correlates to the stretching of the wall accom-
panied by dyskinesis. K+ c significantly increased more than 4S S after occlusion.
Therefore, K + c increase only affects the second increase phase of R wave
amplitude.
ST elevation during trial 2 was significantly reduced in the 5-min group
compared to trial 1. ST elevation on AC-coupled ECG is the sum of TO shift,

reflecting the diastolic injury current and true ST elevation during electrical
systole. In our study, reduced ST elevation during trial 2 in the 5-min group
was accompanied by the reduction of TO shift on DC-coupled ECG.
K+ loss from myocardial cells is the major determinant factor of ischemic ST
change. The change in K+ c was consistent with reduced ST elevation during
trial 2 of the 5-min group. The reduction of Ek was also consistent with the
reduction of K + c in trial 2 of the 5-min group. Moreover, there was close
positive correlation between K + c and TO shift in both the two trials and the
two groups. Therefore, the reduction of ST elevation may be introduced by the
reduction of intracellular K+ loss during trial 2 of the 5-min group. It is
clinically important that the reduction of ST elevation due to brief ischemia
may cause an error in judgment of the degree of the next ischemia to appear
on ECG.
Maximal PC0 2 and pH showed no significant differences between trials 1
and 2 in the control group. CO 2 and proton are the end products of myocardial
metabolism. These variables calculated from myocardial tissue PC02 and pH
indicate the MR of ischemic myocardium and suggest its metabolic viability.
The MR showed no significant differences during two trials in the control
group but showed marked depression during trial 2 in the 5-min group.
Therefore metabolic damage due to 5-min ischemia was latently continued for
90 min after reperfusion.
The mechanism of K+ c elevation in brief ischemia is supposed to enhance
the permeability of cell membranes. Therefore, these reductions in CO 2 and
proton may partly explain the diminished K+ loss from ischemic myocardium
and the consequently reduced ST elevation during trial 2 in the 5-min group.
From these findings, latent myocardial damage, i.e., latent myocardial
dysfunction and latent metabolic damage, may certainly exist before manifest
myocardial stunning and is quite important for clarifying the continuity of
myocardial stunning.
References
1. Miura M, Saito T, Tajika T, Kanazawa T (1987) The experimental study of the
coronary reperfusion in the acute myocardial ischemic: The feasibility of the myo-
cardial salvage. Jpn Circ J 51:1082-1090
2. Miura M, Matsu-oka H, Kanazawa T (1989) The protection of the acute ischemic
myocardium: Merits and demerits of the coronary circulation. Jpn Circ J 53:1084-
1091
3. Kleber AG (1983) Resting membrane potential, extracellular potassium activity and
intracellular sodium activity during acute global ischemia in isolated perfused guinea
pig hearts. Circ Res 52:442-450
4. Miura M, Kadowaki K, Saito T, Hosoya K, Yoshida K, Tajika T, Ono Y, Ikeda S,
Shozawa T, Kanazawa T (1982) Transient decrease of R wave amplitude during
acute myocardial ischemia. Jpn Heart J 23 (suppl 1): 456-458
5. Dominguez G, Fozzard HA (1970) Influence of extracellular K+ concentration on
cable properties and excitability of sheep cardiac Purkinje fibers. Circ Res 26:565-
574
3
Complement-Induced Myocardial
Ischemia: Neutrophil
and Vascular Mechanisms
Robert L. Engler, Ughetta del Baiza, and Bruce R. It0 1
Introduction
Medical therapy for acute myocardial infarction is currently directed at
preventing acute arrhythmia, limiting the extent of necrosis, and preventing
ventricular dilation. Experimental studies dealing with constraining the extent
of necrosis in acute coronary occlusion have had limited success in the absence
of reperfusion. Restoration of blood flow was found to be by far the most
effective mechanism of reducing the degree of necrosis during acute myocardial
infarction. Studies in experimental animals indicate that reperfusion within
2-3 h of coronary occlusion in canine hearts results in significant salvage of
tissue, defined as the restoration of contractile function to tissue which would
have otherwise developed into necrosis. Thus, in the 1980s, attempts to limit
injury during acute myocardial infarction are primarily directed at reperfusion.
It should be noted, however, that other measures, such as the administration of
beta-adrenergic blocking agents, are effective in reducing mortality and
perhaps morbidity and should not be neglected.
With the advent of reperfusion therapy using thrombolytic agents,
angioplasty, or surgery it became evident that the degree of irreversible injury
was increased by reperfusion; thus, for the last 10 years, pharmacologic
adjuncts to reperfusion have been sought. Reperfusion therapy during acute
myocardial ischemia might either accelerate or increase the degree of
irreversible injury present at the moment of reperfusion. This phenomenon has
been termed "reperfusion injury" and denotes that component of lethal tissue
injury actually induced by consequences of acute reperfusion. Despite the
presence of reperfusion injury, restoring blood flow to the tissue will still
salvage myocardium when compared to the alternative of no reperfusion at all.
The reperfusion injury might be reduced or prevented by pharmacological
interventions.
1 VA Medical Center, Research Service (151), San Diego, CA 92161, USA
280
3. Complement-Induced Myocardial Ischemia 281
The three lines of evidence that supported the concept of reperfusion injury
in cardiac muscle were (1) the discovery of superoxide dismutase and xanthine
oxidase hypothesis, (2) the description of leukocyte capillary plugging leading
to a capillary no-reflow phenomena, and (3) the demonstration that
neutropenia could reduce infarct size. The discovery of superoxide dis mutase
[1] led to the hypothesis by McCord et al. that endothelial cell xanthine
dehydrogenase could be converted to the oxidase form during ischemia which,
upon the reintroduction of molecular oxygen with reperfusion, resulted in the
production of superoxide from the metabolism of hypoxanthine to xanthine
and uric acid [2,3]. McCord proposed that superoxide initiated reperfusion
injury to tissue [4]. Investigation of the involvement of neutrophils stemmed
from the fundamental observations that leukocytes move slowly through
capillaries and upon reduction in perfusion pressure can cause a complete
stoppage of flow [5,6]. Neutrophils were shown to plug myocardial capillaries
during ischemia and reperfusion and result in a capillary no-reflow phenomena
[7]. Furthermore, reduction in the number of neutrophils or inhibition of
arachidonic acid metabolism have been shown to reduce infarct size in models
of ischemia and reperfusion [8-13].
A proposed scheme whereby reperfusion might trigger additional
inflammatory or free radical mediated injury is illustrated in Fig. 1. Xanthine
dehydrogenase is present in endothelial cells in many organs. During ischemia,
this enzyme, which normally requires nicotinamide adenine dinucleotide
(NADH) to metabolize hypoxanthine, is converted to the enzyme xanthine
oxidase by a calcium-dependent protease. Xanthine oxidase uses molecular
oxygen in the conversion of hypoxanthine to uric acid and thus produces
superoxide. Superoxide and its radical products (hydroxyl radical and hydrogen
peroxide) may cause tissue injury by direct oxidation, such as by initiating
chain lipid peroxidation of biologic membranes, or indirectly, by activating or
MYOCARDIAL ISCHEMIA & REPERFUSION
-----~
: XANTHINEt-- 02- -
/
PMN ACTIVATION •
~ COMPLEMENT
U~.X.!.D~~_: ~ 1 ~ ACTIVATION
"
CAPILLARY PLUGGING VENOUS SECRETAGOG
CAPILLARY NO REFLOW MARGINATION ACTIVATION
?
/
SUPEROXIDE DEGRANULATION
/
INCREASED VASCULAR RESISTANCE
Y
MYOCARDIAL DYSFUNCTION
FiG. 1. Possible mechanisms of interaction of inflammatory and ischemic injury

282 R. L. Engler et al.
initiating chemotactic factors which recruit neutrophils which magnify the in-
jurious inflammatory response [14]. This mechanism appears active in intestinal
ischemia and reperfusion, and perhaps in cardiac ischemia and reperfusion of
some species, but is believed to be inactive in human myocardium due to the
natural absence of xanthine oxidase [1S].
Mechanisms other than xanthine oxidase for the activation of neutrophils
during ischemia and reperfusion have been identified. The most prominent of
these is serum complement. Early studies demonstrated that complement
depletion using cobra venom factor reduced both the extent of myocardial
infarction and inflammatory reaction [16-18]. Complement activation and
binding of C1q to ischemic myocardium has been shown to begin at about
IS min after coronary occlusion and to be significant by 4S min [19]. Thus,
complement activation appears to be a major mechanism of inflammatory
activation during ischemia and reperfusion.
Decreased perfusion pressure per se is also a mechanism for neutrophil
recruitment. Neutrophils accumulate in tissue during decreased perfusion
pressure and lead to a capillary plugging phenomenon [S-7]. It appears that
with reperfusion during reversible ischemia, there is actually a net wash-out of
neutrophils from the stunned myocardium [20]. One of the mechanisms of
neutrophil release from this reversibly injured tissue may be adenosine release.
Adenosine has been shown to inhibit neutrophil adherence, superoxide
production, and endothelial cell killing [21,22]. Augmented adenosine
production from adenosine triphosphate (ATP) catabolism using S amino 4
imidazole carboxamine riboside (AICA-riboside) reduces neutrophil
accumulation and increases collateral blood flow [23]. Following more
prolonged ischemia, reperfusion dramatically increases neutrophil
accumulation [24]. Finally, arachidonic acid metabolism is initiated during
ischemia and may be part of a positive feedback loop for neutrophil activation
[12,2S].
Model System for Studying Complement

Neutrophils contain specific cell surface receptors for the complement
component CSa which result in adherence, activation, directed chemotaxis, and
superoxide production. Accordingly, we developed a porcine model whereby
CSa could be injected directly into the coronary artery under controlled
conditions wherein coronary vascular resistance and regional fUl)ction could be
monitored. Systemic injection of activated complement as either purified CSa
or zymosan-activated plasma results in diffuse activation of circulating
neutrophils which accumulate in the lungs, and are associated with
thromboxane-mediated acute pulmonary hypertension as well as acute right
heart failure which is often lethal [26-30]. We chose a pig model because
collateral blood flow was minimal (which would enable us to prevent systemic
recirculation of the CSa-activated neutrophils) and pig CSa has close homology
with human CSa. Both human and porcine CSa bind to and activate porcine
neutrophils.
Under general anesthesia with alpha-chloralose, the heart was exposed and
the left anterior descending coronary artery cannulated and perfused with
carotid artery blood. The extracorporeal perfusion circuit contained a low-
impulse servo-controlled perfusion pump specially designed for coronary
perfusion. The extracorporeal circuit can be used for insertion of filters to
remove neutrophils and for the injection of CSa or other pharmacologic agents.
The coronary vein adjacent to the cannulated left anterior descending coronary
artery (LAD) was catheterized, tied proximally, and drained into a beaker.
The coronary venous blood was reinfused into the jugular vein except during
CSa injections, when it was collected and discarded in order to avoid
recirculation of CSa. Regional function was monitored using ultrasonic
dimension crystals in both the LAD perfused area (treatment area) and a
circumflex (control) area, to be certain that systemic circulating factors were
not altering myocardial function.
When CSa is injected into the coronary artery under servo-controlled
constant coronary perfusion pressure, a highly reproducible effect of neutrophil
trapping, increased coronary vascular resistance, and regional myocardial
dysfunction occurs [31] (Fig. 2). A I-min injection of SOO nanograms CSa
results in the trapping of 1.6 X 106 cells per gram perfused myocardium [32].
This extent of neutrophil trapping is close to the estimated number of
neutrophils accumulating during 3 h of acute myocardial ischemia without
reperfusion [20,24]. Reperfusion for S min after 3 h of ischemia doubles the
neutrophil accumulation [24]. One hour of reperfusion following 40 or 90min
of ischemia results in 3.3 x 106 and S.7 X 106 cells per gram, respectively [20].
Thus, the number of polymorphonuclear neutrophils (PMN) trapped during
CSa injection is between 28% and 49% of the number that accumulate in
commonly used canine myocardial ischemia and reperfusion models. The
increase in coronary vascular resistance during CSa injection was promptly
followed by reactive hyperemia. The time course of this effect is compatible
with the known dynamics of CSa-induced neutrophil activation which is quite
brief as the CSa bound to the neutrophil receptor is rapidly internalized and
processed [33,34]. The decline in regional function reaches a nadir of
approximately 2S%-SO% of the baseline. When flow was reduced by reducing
the perfusion pressure in the servo-controlled pump to achieve flow equivalent
to that seen during CSa, function decreased to an equivalent level. Thus, the
degree of dysfunction observed was compatible with that caused by the
ischemia and did not necessarily implicate a direct negative inotropic effect of
CSa, although the latter is certainly not excluded by these experiments.
Injection of CSa during constant flow perfusion results in an increase in
coronary vascular resistance, but only mild regional dysfunction. This regional
dysfunction occurring at constant flow could be due to direct negative inotropic
effects of CSa, or regional mal distribution of flow causing ischemia during CSa
injection (e.g., subendocardial ischemia with lUXury flow to the epicardium).
Possible mechanisms of increased coronary vascular resistance during CSa
injection include microvascular obstruction by activated neutrophils (leukocyte
capillary plugging), arteriolar obstruction by aggregating neutrophils, in
vivo production of a vasoconstrictor substance, or chemical neutralization
284 R.L. Engler et al.
Coronary Blood Flow (ml/min/gm)
Mean Coronary Blood Flow (ml/min/gm)

\lO-
064 c.+
.
. ~
~
,
f ;1:' ::tl bf.
Left-ventricular Pressure (mmHg)
FIG. 2. Response to intracoronary (LAD) injection of 500 ng of C5a over 1 min in a pig.
Coronary blood flow (phasic and mean, top and second panel) decreases promptly
during the injection and shows reactive hyperemia after injection stops. Regional
shortening in the left anterior descending (LAD) perfusion bed decreases by about
50%. Segment shortening in the circumflex (eire) perfusion bed and the left ventricular
systolic pressure are minimally affected
of an intrinsic vasodilator (e.g. , endothelial-derived relaxing factor [EDRF]

neutralized by superoxide).
Arachidonic Acid Metabolites

Several metabolites of arachidonic acid are known potent coronary
vasoconstrictors. Both thromboxane A2 and the peptido-leukotrienes, LTC 4
and 0 4 , are coronary constrictors [35-37]. Furthermore, combined cycio-
oxygenase and lipoxygenase blockade reduced neutrophil infiltration and
injury during myocardial ischemia and reperfusion [38]. Studies in
complement-mediated lung injury had implicated neutrophils and thromboxane
production as etiologic in the severe pulmonary hypertension and edema that
accompanies systemic complement activation [27,28,30]. More recent data
have suggested that in the lung, cell types other than neutrophils may also be
involved in thromboxane production. We hypothesized that TxA2 was a
mediator of C5a-induced myocardial ischemia.
First, we determined that serial injections (30 min apart) of C5a resulted in
equivalent vasoconstriction, regional dysfunction, neutrophil trapping, and
thromboxane production. The administration of aspirin or indomethacin
resulted in no detectable change in any of these parameters except for
complete blockade of thromboxane production [32]. Ibuprofen had a small but
significant effect upon reducing the ischemia, neutrophil trapping, and
dysfunction. Ibuprofen is known to have inhibitory effects on neutrophil
function independent of cyclooxygenase blocking activity [39,40]. Next, the
administration of the thromboxane receptor antagonist, BM13505, decreased
the ischemic response by about 50% without effecting neutrophil accumulation
[41]. The apparent paradoxical difference between cyclooxygenase blockade
and TxA2 receptor antagonism might be explained by shunting. In vitro studies
have found shunting of arachidonic acid metabolism through an alternate
pathway (leukotriene production) in the presence of blockade in the
cyclooxygenase pathway [42]. Thus, an increased production of leukotrienes
might give an equivalent vasoconstriction reducing the effect of thromboxane
blockage (Fig. 3).
Administration of the leukotriene C41D4 receptor antagonist, LYl71883,
also diminished the myocardial ischemic response to C5a. Combined receptor
antagonism with both agents completely prevented the reduction in flow and
regional function accompanying C5a injection, but did not diminish the
neutrophil accumulation [41]. This evidence strongly supports the concept that
C I
yc ooxygena~~o ~
,
Phospholipids
Arachidonic Acid
BIOCk~ """"
PGG2+ PGH2 5-HPETE
~ "LTA4
TXA2 Others PG12 ~
~ 1
I
LTB4 Ly4l
TXB 2 6-KETO-PGF-1 a LTD 41
Antagonist Antagonist
Receptor Receptor Receptor
FiG. 3. Arachidonic acid metabolism results in production of two potent coronary

vasoconstrictors, thromboxane A2 (TxA 2 ) and the peptido-leukotrienes (LTC4, LTD4).
Blockade of cyc100xygenase results in enhanced leukotriene production. Dual receptor
antagonism completely prevented the ischemic response to intracoronary C5a without
effecting neutrophil trapping
vasoconstrictor production is the mechanism producing myocardial ischemia in

response to C5a, and that microvascular obstruction by the accumulated or
trapped neutrophils is not the direct mechanism.
Further confirmation that this extent of neutrophil trapping (1.6 X 106
PMN/g) does not per se induce myocardial ischemia was obtained from
preliminary studies using intracoronary injection of LTB4 in the same model.
Accumulation of neutrophils was slightly greater during LTB4 administration
then during C5a administration. However, there was no increase in vascular
resistance and no change in regional myocardial function in response to LTB4
[43] (Fig. 4). From the studies involving the TxA2 and LTC4 1D 4 receptor
antagonists and LTB4, it could be concluded that arachidonic acid metabolism
is critical for inducing the ischemic response to complement, and that there is
an association between neutrophil accumulation and the production of myo-
cardial ischemia, but that vascular obstruction by neutrophils is not the
mechanism of ischemia.
Preliminary studies have been performed to assess the question of whether
neutophils are necessarily involved, and what is the relative role of free radical
production. Studies performed with two different types of filters to remove
neutrophils in the extracorporeal coronary perfusion circuit have shown
complete prevention of the ischemic response to C5a. However, following
removal of these filters, reinjection of C5a produced equivalent neutrophil
trapping but without myocardial ischemia. Furthermore, neutropenia and
thrombocytopenia failed to prevent the C5a-induced ischemic response [44,45].
These studies suggest that a cell type other than the neutrophil is responsible
for the thromboxane and/or leukotriene production. Investigations are
currently under way to confirm these findings, and to ascertain which cell
Effect of PMN activation on pig heart

Adherence vs secretagogue activation without ischemia
200.----------------------------------------,
150~--------------------------------------~
100 f-------v0.;
50 f--r-----v~
Flow(%) Function (%)
DC5a E2I LTB-4

FIG. 4. Comparison of intracoronary CSa (white) and leukotriene B4 (LTB4) (shaded)
effects on myocardial function, coronary blood flow, and neutrophil trapping. Despite
equivalent neutrophil trapping, LTB4 did not alter regional functions (% of preinjection
value) or coronary flow (% of preinjection value)
type(s) are responsible for arachidonic acid metabolism in response to C5a.

Some type of transcellular metabolism may be involved [46].
Preliminary evidence also suggests that hydroxyl radical production is
necessary, at least in part, for the full ischemic response. Prior administration
of the hydroxyl radical scavenger dimethythiourea diminishes the ischemic
response and TxA2 release without altering neutrophil trapping [47].
Conclusion
Our results to date suggest an important role for arachidonic acid metabolism
in response to activated complement in acute myocardial ischemia. Neutrophil
trapping induced by purified C5a to the extent found in these experiments is
insufficient to explain the increased vascular resistance that is seen. More
extensive neutrophil trapping during more prolonged ischemia with reperfusion
could indeed lead to elevated vascular resistance by mechanical obstruction
alone. Preliminary evidence suggests that cells other than polymorphonuclear
leukocytes are involved in the production of eicosanoid coronary
vasoconstrictors.
References
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erythrocuprein (hemcuprein). J Bioi Chern 244:6049-6055
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LF, Downey 1M (1985) Xanthine oxidase as a source of free radical damage in
myocardial ischemia. J Mol Cell CardioI17:145-152
3. Granger DN, Rutili G, McCord 1M (1981) Superoxide radicals in feline intestinal
ischemia. Gastroenterology 81:22-29
4. McCord 1M (1985) Oxygen derived free radicals in postischemic tissue injury. N
Engl1 Med 312:159-163
5. Bagge U, Amundson B, Lauritzen C (1980) White blood cell deformability and
plugging of skeletal muscle capillaries in hemorrhagic shock. Acta Physiol Scand
180:159-163
6. Braide M, Amundson B, Chien S, Bagge U (1984) Quantitative studies on the
influence of leukocytes on the vascular resistance in a skeletal muscle preparation.
Microvasc Res 27:331-352
7. Engler R, Schmid-Schoenbein GW, Pavelec R (1983) Leukocyte capillary plugging
in myocardial ischemia and reperfusion in the dog. Am J Path 111:93-111
8. Bendar M, Smith B, Pinto A, Mullane KM (1985) Nafazatrom-induced salvage of
ischemic myocardium in anesthetized dogs is mediated through inhibition of
neutrophil function. Circ Res 57: 131-141
9. Litt MR, Jeremy RW, Weisman HF, Winkelstein JA, Becker LC (1989) Neutrophil
depletion limited to reperfusion reduces myocardial infarct size after 90 minutes of
ischemia: Evidence for neutrophil-mediated reperfusion injury. Circulation
80: 1816-1827
10. Mullane KM, Fornabaio D (1988) Thromboxane synthetase inhibitors reduce

infarct size by a platelet dependent, aspirin sensitive mechanism. Circ Res 62:668-
678
11. Mullane KM, Read N, Salmon JA, Moncada S (1984) Role of leukocytes in acute
myocardial infarction in anesthestized dogs. J Pharmacol Exp Ther 228:510-522
12. Mullane KM, Smith CW (1990) The role of leukocytes in ischemic damage,
reperfusion injury and repair of the myocardium. In: Piper HM (ed)
Pathophysiology of severe ischemic myocardial injury. Kluwer, pp 239-267
13. Romson JL, Hook BG, Kunkel SL, Abrams GD, Schork MA, Lucchesi BR (1983)
Reduction of the extent of ischemic myocardial injury by neutrophil depletion in
the dog. Circulation 67:1016-1023
14. Granger DN (1988) Role of xanthine oxidase and granulocytes in ischemia-
reperfusion injury. Am J Physiol 255:H1269-H1275
15. Eddy U, Stewart JR, Jones HP, Engerson TD, McCord JM, Downey JM (1987)
Free radical-producing enzyme, xanthine oxidase, is undetectable in human hearts.
Am J PhysioI253:H709-H711
16. Hill JH, Ward PA (1970) The phlogistic role of C3 leukotactic fragments in
myocardial infarcts of rats. J Exp Med 885-900
17. Maroko PR, Carpenter CB, Chiariello M, Fishbein MC, Radvany P, Knostman JD,
Hale SL (1978) Reduction by cobra venom factor of myocardial necrosis after
coronary artery occlusion. JCI 61:661-670
18. Pinckard RN, O'Rourke RA, Crawford MH, Grover FS, McManus LM, Ghidoni
JJ, Storrs SB, Olson MS (1980) Complement localization and mediation of ischemic
injury in baboon myocardium. J Clin Invest 66:1050-1056
19. Rossen RD, Swain JL, Michael LH, Weakey S, Giannini E, Entman ML (1985)
Selective accumulation of the first component if complement and leukocytes in
ischemic canine heart muscle. A possible initiator of an extra myocardial
mechanism of ischemic injury. Circ Res 57:119-130
20. Go LO, Murry CE, Richard VJ, Weischedel GR, Jennings RB, Reimer KA (1988)
Myocardial neutrophil accumulation during reperfusion after reversible or
irreversible ischemic injury. Am J Physiol 255:H1188-H1198
21. Cronstein BN, Kramer SB, Weissmann G, Hirschhorn R (1983) Adenosine: A
physiological modulator of superoxide anion generation by human neutrophils. J
Exp Med 158:1160-1177
22. Roberts PA, Newby AC, Hallett MB, Campbell AK (1985) Inhibition by adenosine
of reactive oxygen metabolite production by human polymorphonuclear leucocytes.
Biochem J 227:669-679
23. Gruber HE, Hoffer ME, McAllister DR, Laikind PK, Lane TA, Schmid-
Schoenbein GW, Engler RL (1989) Increased adenosine concentration in blood
from ischemic myocardium by AICA riboside: Effects on flow, granulocytes and
injury. Circulation 80:1400-1411
24. Engler RL, Dahlgren M, Petersen M, Dobbs A, Schmid-Schoenbein GW (1986)
Accumulation of polymorphonuclear leukocytes during 3 hour experimental
myocardial ischemia. Am J PhysioI251:H93-H100
25. Sen A, Miller JC, Reynolds R, Willerson JT, Buja LM, Chien KR (1988) Inhibition
of the release of arachidonic acid prevents the development of sarcolemmal
membrane defects in cultured rat myocardial cells during adenosine triphosphate
depletion. J Clin Invest 82: 1333-1338
26. Cooper JD, McDonald JWD, Ali M, Menkes E, Masterson J, Klement P (1980)
Prostaglandin production associated with the pulmonary vascular response to
complement activation. Surgery 88:215-221
27. Gee M, Perkowski SZ, Havill A, Flynn T (1983) Role of prostaglandins and
leukotrienes in complement initiated lung vascular injury. Chest 83:82s-85s
28. Kaslovsky RA, Horgan MJ, Lum H, McCandless BK, Gilboa N, Wright SD, Malik
AB (1990) Pulmonary edema induced by phagocytosing neutrophils. Protective
effect of monoclonal antibody against phagocyte CD18 integrin. Circ Res 67:795-802
29. McDonald JWD, Ali M, Morgan E, Townsend ER, Cooper JD (1983)
Thromboxane synthesis by sources other than platelets in association with
complement induced pulmonary leukostasis and pulmonary hypertension in sheep.
Circ Res 52:1-6
30. Perkowski SZ, Havill AM, Flynn JT, Gee MH (1983) Role of intrapulmonary
release of eicosanoids and superoxide anion as mediators of pulmonary dysfunction
and endothelial injury in sheep with intermittent complement activation. Circ Res
53:574-583
31. Martin SE, Chenoweth DE, Engler RL, Roth DM, Longhurst JC (1988)
Complement C5a decreases regional coronary blood flow and myocardial function
in pigs: Implications for a granulocyte mechanism. Circ Res 63:483-491
32. Ito BR, Roth DM, Chenoweth DE, Lefer AM, Engler RL (1989) Thromboxane is
produced in response to intracoronary infusion of complement C5a in pigs.
Cyclooxygenase blockade does not reduce the myocardial ischemia and leukocyte
accumulation. Circ Res 65:1220-1232
33. Chenoweth DE (1987) In: Ross GD (ed) Immunobiology of the complement
system. Academic, New York, pp 88-119
34. Hugli TE (1981) In: Atssi MZ (ed) Critical reviews in immunology. CRC, Boca
Raton, Florida, pp 321-380
35. Boyd ED, Feuerstein G, Goldstein RE (1983) Coronary constriction by leukotriene
C4, and E4 in the intact pig heart. Am J CardioI51:1451-1454
36. Laurindo FRM, Finton CK, Ezra D, Czaja JF, Feuerstein GZ, Goldstein RE
(1988) Inhibition of eicosanoid-mediated coronary constriction during myocardial
ischemia. FASEB J 2:2479-2486
37. Michelassi F, Landa L, Hill RD, Lowenstein E, Watkins WD, Petkau AJ, Zapol
WM (1982) Leukotriene D4: A potent coronary artery vasoconstrictor associated
with impaired ventricular contraction. Science 217:841-843
38. Ernst E, Hammerschmidt DE, Bagge U, Matrai A, Dormandy JA (1987)
Leukocytes and the risk of ischemic disease. JAMA 257:2318-2324
39. Nielsen VG, Webster RO (1987) Inhibition of human polymorphonuclear leukocyte
function by ibuprofen. Innumopharmacology 13:61-71
40. Minta JO, Williams MD (1985) Some nonsteroidal antiinflammatory drugs inhibit
the generation of superoxide anions by activated polymorphs by blockade of ligand-
receptor interactions. J Rheum 12:751-757
41. Ito BR, Roth DM, Engler RL (1990) Thromboxane A2 and peptidoleukotrienes
contribute to the myocardial ischemia and contractile dysfunction in response to
intracoronary infusion of complement c5a in pigs. Circ Res 66:596-607
42. Docherty JC, Wilson TW (1987) Indomethacin increases the formation of
lipoxgenase products in calcium inophore stimulated human neutrophils. Biochem
Biophys Res Commun 148:534-538
43. Engler RL, Ito BR, Roth DL, Chenoweth D (1988) Dissociation of leukocyte
accumulation from myocardial dysfunction: LTB4 versus C5a induced PMN
activation (abstract). Circ 78 (Suppl 11):11-651
44. delBalzo U, Ito BR, Nork SE, Engler RL (1990) Leukocyte depletion does not
attenuate the myocardial ischemia and thromboxane production in response to
complement C5a. Circ 82: III-1096
45. Engler RL, delBalzo U, Nork SE, Ito BR (1990) Leukocyte filtered blood at-
tenuates the complement C5a induced myocardial ischemia and thromboxane
production: Leukocyte dependent or independent? Circ 82:III-2789
46. Feinmark SJ, Cannon PJ (1986) Endothelial cell leukotriene C4 synthesis results
from intercellular transfer of Leukotriene A4 synthesized by polymorphonuclear
leukocytes. JBC 261:16466-16472
47. Ito BR, Libraty D, Engler RL (1990) Oxygen-derived free-radicals are responsible
for the release of thromboxane A2 induced by the intracoronary infusion of
complement C5a in swine (abstract). FASEB J 4:A945
4
Reoxygenation-Induced Heart
Microvasculature Endothelial Cell
Injury and Neutrophil Hyperreaction:
Role of Arachidonate Lipoxygenase
Metabolism
Tsunehiko Kuzuya, Youngjoon Kim, Shiro Hoshida, Masashi Nishida,
Hisakazu Fuji, Masatsugu Hori, Akira Kitabatake,
and Michihiko Tada 1
Summary. In order to clarify the mechanism by which neutrophils are

activated in the reperfused coronary vasculature, we investigated intercellular
actions between endothelial cells and neutrophils under hypoxia followed by
reoxygenation. We cultured canine coronary endothelial cells and neutrophils
under hypoxia «1 torr:45min)-reoxygenation (100 torr:5h). Adhesion and
diapedesis of neutrophils was measured by counting the cells which adhered on
the endothelial cells and infiltrated under the endothelial cell monolayer.
Arachidonate lipoxygenase metabolites, hydroxyeicosatetraenoic acids (HETE),
and oxygen-free radicals were assayed by high pressure liquid chromatography
(HPLC) and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) spin trapping,
respectively. Under sustained hypoxia, there was no increase in neutrophil
function, free radical generation, or HETE production. After reoxygenation,
diapedesis and chemotaxis of the leukocytes were markedly enhanced. Under
this condition, the production of 12-, 5-, and 15-HETE was markedly in-
creased. Potent signals of the DMPO radical adducts were also detected after
reoxygenation. DMPO-OOH generation was peaked at 1 h after reoxygenation
and DMPO-OH production was peaked at 3 h after reoxygenation. The pre-
treatment of endothelial cells with AA-861 (a lipoxygenase inhibitor) or
CV-3611 (a radical scavenger) markedly attenuated the re-oxygenation-induced
cell injury and free radical generation, resulting in the suppression of neutrophil
hyperreaction. There was a close correlation between HETE production and
neutrophil function. These results indicate that augmented lip oxygenase
metabolism in the reoxygenated endothelial cells may serve to enhance
neutrophil function, and that augmented free-radical generation from the co-
1 The First Department of Medicine, Osaka University, School of Medicine, Osaka, 553
Japan
291
292 T. Kuzuya et al.
cultured system may enhance microvasculature endothelial cell injury under

hypoxia-reoxygenation.
Key words: Endothelial cell injury-Hypoxia-reoxygenation-Neutrophil
function- Arachidonate lip oxygenase metabolism-Oxygen-derived free
radicals
Introduction
Several studies point to a detrimental effect of reperfusion on endothelial
function and coronary vasodilator reserve. In particular, extensive capillary
plugging by neutrophils can reduce the microvascular bed resulting in impaired
coronary blood flow response, the "no reflow" phenomenon. These
microcirculatory disorders can interfere with the functional recovery of the
postischemic myocardium [1] and, furthermore, lead to the propagation of
ultimate infarct size resulting from coronary occlusion-reperfusion [2].
However, it is not understood how circulating neutrophils are functionally
activated in the reperfused ischemic myocardial tissue. In order to clarify the
mechanism by which neutrophils are activated in the reperfused vasculature,
we investigated endothelial dysfunction and intercellular actions between
endothelial cells and neutrophils under hypoxia followed by reoxygenation.

Endothelial cells were isolated from canine heart microvasculature by
enzymatic and mechanical treatment. After removing the epicardial large
vessels and endocardial endothelium, the left ventricular myocardium was cut,
gently homogenized, and washed with minimum essential medium (MEM).
The endothelial cells were enzymatically dissociated with 0.05% trypsin, 0.05%
collagenase, and 0.05 mM DETAPAC in calcium-free phosphate-buffered
saline (PBS) for 30 min at 37°C. After the dissociation reaction was stopped by
adding MEM with 10% fetal bovine serum (PBS), 10 mM glucose, and 1 mM
calcium (pH 7.4), the suspension was incubated in culture dishes (3001 Falcon)
in order to remove contaminated fibroblasts. After the endothelial cell-rich
suspension was subsequently collected and washed, the final pellet was
resuspended in Medium 199 with 20% FBS, buffered by 5 g NaHC0 3 and
25 mM HEPES. The cells were cultured in 35 mm or 60 mm dishes (3801, 3802
Falcon) and used for experiments at 7-14 days after the cells had grown to
confluence. The purity was confirmed by their morphology on inverted
microscope and by being immune-positive for coagulant factor 8-related
antigen. Polymorphonuclear leukocytes (PMN) were isolated from canine
peripheral blood by Percoll gradient [3]. Endothelial cells were incubated
under hypoxia (95% nitrogen, 5% carbon dioxide, and oxygen concentration
of less than 1 mmHg, for 45 min) followed by reoxygenation (95% air, 5%
4. Reoxygenation-Induced Heart Microvasculature Endothelial Cell Injury 293
carbon dioxide, and oxygen concentration of 100mmHg, for 5h). Under these
conditions, we measured the generation of oxygen-derived free radicals
and hydroxyeicosatetraenoic acids (HETEs), arachidonate lipoxygenase
metabolites, by the electron paramagnetic resonance (ESR) spin-trapping
technique [4] and by high performance liquid chromatography, respectively.
For investigating the interaction between leukocytes and endothelial cells,
we added 1 x 105 PMN on the endothelial cell monolayer and incubated it for
30 min. Leukocytic adhesion to endothelial cells was assessed by counting the
number of cells in the washed medium. Leukocytic diapedesis was assayed by
counting the number of infiltrated cells under the endothelial cells after
washing out the medium, using inverted microscopy. The data of adhesion and
diapedesis were represented by a percentage of control obtained under
normoxic incubation. In some experiments, cultured dishes were pre incubated
in the presence of drugs before co-culture and then washed out.
Results
Free Radical Generation and Lipoxygenase Metabolism

in the Reoxygenated Endothelial Cells
Figure 1 shows representative spectra of DMPO radical adducts generated
from monocultured endothelial cells. After 45 min of hypoxia subtle signals of
DMPO radical adducts were detected. These radical signals were markedly
enhanced after reoxygenation, which consisted of hydroperoxyl DMPO and
hydroxyl DMPO. Under normoxia, no signal was detected. Thus, it is clear
that oxygen-free radicals are generated from the reoxygenated endothelial
cells.
Figure 2 shows RETE production in endothelial cells under hypoxia-
reoxygenation: three kinds of HETEs were produced with the maximum
production occurring about 3 h after reoxygenation. These findings indicate
that 15-, 5-, and 12-lipoxygenase metabolism in endothelial cells were potently
augmented by hypoxia followed by reoxygenation. We also found that the
augmented lipoxygenase metabolism is closely correlated with the free-radical
production mentioned above. Figure 3 shows the data, examining the effects of
radical scavengers and enzyme inhibitors on free radical generation from
reoxygenated endothelial cells. At 1 h after reoxygenation, DMPO radical
adducts were inhibited by CV-3611 [5], a free-radical scavenger, SOD, and
allopurinol, a xanthine oxidase inhibitor. On the other hand, at 3 h after
reoxygenation, DMPO radical adducts were inhibited by CV-3611,
dimethylsulfoxide (DMSO), hydroxyl-radical scavengers, and nordihydro-
guaiaretic acid (NDGA), a lipoxygenase inhibitor. Thus, these results suggest
that oxygen-free radicals generated in endothelial cells are originated not only
from xanthine oxidase but also from the lipoxygenase system. It is interesting
Normoxia
Hypoxia 45 min
5 sec after reoxygenation
~\lI~
1 hr after reoxygenation
FiG. 1. EPR spectra of DMPO radical adducts formed in heart microvasculature endo-
thelial cells
how these reoxygenation-induced metabolic reactions are coupled with endo-

thelial cell function, especially in view of the endothelial-leukocyte interaction.
Interaction Between Endothelial Cells and Leukocytes

When the functional alteration of leukocytes co-cultured with endothelial cells
were investigated under hypoxia-reoxygenation, the leukocytic adhesion and
diapedesis were markedly enhanced after reoxygenation (Fig. 4). It should be
noted that these leukocytic hyperreactions are peaked at 1-3 h after
reoxygenation, when the production of free radicals and HETEs are markedly
enhanced.
In order to investigate the role of arachidonate lipoxygenase reactions in the
enhancement of leukocytic function, we examined the effects of inhibitors of
T ___O
T
° ...
10
15-HETE
T/'"
0
u.
\
~
Qj
u
"'
0
:::.
w
W
J:
'00
.s 5-HETE
c: 5
T~ ti..--'£
0
.+i
u
::::J
/-~
"C
0
L.
11. T
6
W
I-
o ,.., T
T _ _6 - -....
:/:/1~~
W
I
~o ~C_ _ _f 12-HETE
....-~~
C ...
0 o45 min-~
0 1 2 3 4 5
Hypoxia REOXYGENATION (hr)
FIG.2. HETEs production in cardiac microvasculature endothelial cells under hypoxia-
reoxygenation
arachidonate metabolism on leukocytic adhesion and diapedesis Cfable 1). All

drugs were added to endothelial cells and pre incubated for 30 min and washed
out from the medium before hypoxia. The leukocytic adhesion and diapedesis
were significantly inhibited by the lipoxygenase inhibitors, AA-861 and
nordihydroguaiaretic acid (NDGA), whereas these leukocytic activities were
enhanced by a cyclooxygenase inhibitor, indomethacin. Under these
conditions, there was a close correlation between HETE production and
activities of the leukocytes. Therefore, it is clear that augmented lipoxygenase
metabolism in endothelial cells at least contributes to the enhancement of
leukocytic chemotactic activity.
Discussion
We assayed chemotaxis and free radical generation in leukocytes co-cultured
with endothelial cells. The chemotaxis of leukocytes and free-radical
generation were markedly enhanced by reoxygenation. The enhanced
3
?
'c:::J
to2
f
+'
1i
!1
.l!lo
~ O~~~~~~--~~~~~~--~
~ 3 3 hr after reoxygenation
"ii
o
~
0:::2
o
D..
~1
FIG. 3. Effect of radical scavengers and enzyme inhibitors on free radical generation
from endothelial cells under hypoxia-reoxygenation
chemotaxis was decreased by the inhibition of lipoxygenase metabolism and

augmented by the inhibition of cyclooxygenase metabolism in endothelial cells.
These results suggest that augmented lipoxygenase metabolism in endothelial
cells promotes chemotaxis of leukocytes and free-radical generation, leading to
endothelial cell injury.
The mechanism of these results can be considered as follows. Lipoxygenase
and cyclooxygenase metabolism in endothelial cells are supposed to regulate
chemotaxis of leukocytes and free-radical generation in coronary vessels.
Lipoxygenase metabolites, 12-HETE and 5-HETE, stimulate leukocytic
chemotaxis. In addition, free radicals are produced through the enzymatic
lipoxygenation of unsaturated fatty acids. A cyclooxygenase metabolite,
prostacyclin, has strong chemorepellant activities. The production of HETE
is increased in ischemic myocardium under reperfusion [6,7]. Thus,
reoxygenation-induced augmentation of lipoxygenase metabolism would
stimulate leukocytic chemotaxis [1] and free-radical generation [8,9]. Free
radicals are also supposed to inactivate prostacyclin synthetase. The
chemotactic regulative mechanism of lipoxygenase versus cyclooxygenase might
200
0-
......
•
(5
(5
........c:
....
....c: 0
U
U
0
....0
.....0
150~
*'
~
Z
~
Q) a..
0 ......
co 150
0
.!:2
Q) VI
.I:. Q)
+>
0
"0
Q)
"0 a.
c: m
W (5
c:
0
c:
0
.iii
Q)
.I:.
"0
<t:
100
Hypoxia 1 2 3 4 5
REOXYGENATION TIME (hr)
FIG. 4. Chemoattractive activity of PMN
TABLE 1. Chemotaxis and HETE production in PMN-endothelial cell co-culture: pre-

treatment of endotherial cells with inhibitors of arachidonate metabolism
Control AA-861 NDGA Indomethacin
(2J1M) (10J1M) (10 J1M)
PMN Adhesion 20S.3 144.1 ' IS7 .6' 321.8'
(% of control) ±1O.6 ±S.9 ±S.8 ±11.2
PMN Diapedesis 177.S 119.1' 127.3' 263.2'
('Yo of control) ±4.2 ±6.9 ±4.7 ±lOo4
HETE Production 44.6 27.7' 32.9' SOo4'
(ng/10 7 cells) ±2.8 ±204 ±3.3 ±3.1
' P < 0.01 vs Control, n = 6

HETE, Hydroxyeicosatetraenoic acid; NDGA, nordihydroguaiaretic acid
Results are mean ±standard error of the mean (SEM)
be collapsed under hypoxia-reoxygenation. Once this happens, activated

leukocytes produce the strong chemotactic factors, leukotrienes and lactoferin.
The accumulation of leukocytes adhering to myocardium induces further injury
of coronary microvasculature and myocardial edema.
References
1. Hoshida S, Kuzuya T, Nishida M, Kim Y, Kitabatake A, Kamada T, Tada M (1989)
Attenuation of neutrophil function by inhibitors of arachidonate metabolism reduces
the extent of canine myocardial infarction. Am J Cardiol 63:24E-28E
2. Kuzuya T, Hoshida S, Nishida M, Kim Y, Kamada T, Tada M (1987) Altered
lipoxygenase metabolites and leukocyte involvement in an acute occlusion-
reperfusion model of canine myocardial infarction. Jpn Circ J 51:465-470
3. Kuzuya T, Hoshida S, Suzuki K, Sasaki T, Kitabatake A, Kamada T, Minamino T,
Tada M (1988) Polymorphonuclear leukocyte activity and ventricular arrhythmia in
acute myocardial infarction. Am J Cardiol 62:868-872
4. Zweier JL, Kuppusamy P, Lutty G (1988) Measurement of endothelial cell free
radical generation: Evidence for a central mechanism of free radical injury in
postischemic tissue. Proc Nat! Acad Sci USA 85:4046-4050
5. Kuzuya T, Hoshida S, Nishida M, Kim Y, Fuji H, Kitabatake A, Kamada T, Tada
M (1989) Role of free radicals and neutrophils in canine myocardial reperfusion
injury: Myocardial salvage by a novel free radical scavenger, 2-octadecylascorbic
acid. Cardiovasc Res 23:323-330
6. Kuzuya T, Hoshida S, Nishida M, Kim Y, Kamada T, Tada M (1987) Increased
production of arachidonate metabolism in an occlusion-reperfusion model of canine
myocardial infarction. Cardiovasc Res 21:551-558
7. Tada M, Kuzuya T, Hoshida S, Nishida M (1988) Arachidonate metabolism in
myocardial ischemia and reperfusion. J Mol Cell CardioI20:135-141
8. Zweier JL, Flasherty JT, Weisfeldt ML (1987) Direct measurement of free radical
generation following reperfusion of ischemic myocardium. Proc Nat! Acad Sci USA
84:1404-1407
9. Kuzuya T, Hoshida S, Kim Y, Nishida M, Fuji H, Kitabatake A, Kamada T, Tada
M (1990) Detection of oxygen-derived free radical generation in the canine
postischemic heart during late phase of reperfusion. Circ Res 66:1160-1165
5
Possible Mechanisms of the Beneficial
Effects of Nitroglycerin in Patients with
Effort Angina: Potential Roles of
Collateral Circulation
Kazuhisa Kodama, Yasushi Okazaki, Shinsuke Nanto,
Masayoshi Mishima, At~ushi Hirayama!, Hiroshi Sato,
Masafumi Kitakaze, Masatsugu Hori,2 and Michitoshi Inoue 3
Summary. Although nitrates are very effective for the treatment of effort
angina, the precise mechanisms of the beneficial effects are still unclear. In
order to clarify what the mechanisms of the beneficial effects of one of these
nitrates, nitroglycerin, we performed exercise stress tests on patients with
effort angina and examined the effects of nitroglycerin on coronary and
systemic hemodynamics using the following two exercise protocols. In the first,
a noninvasive study, 105 patients with stable effort angina were evaluated, and
in the second, an invasive study, 50 patients with stable effort angina were
employed. In the noninvasive study, the rough correlation between the severity
of coronary stenosis and the exercise tolerance time was shown, and those
patients with collaterals showed a greater increase in exercise time after
sublingual nitroglycerin administration compared to those without collaterals.
In the invasive study, the reduction in pulmonary arterial end-diastolic pressure
during 3 min of exercise correlated well with the increase in exercise time after
pretreatment with sublingual nitroglycerin. Furthermore, the coronary
angiogram during exercise-induced angina showed the more enhanced
collateral opacification after pretreatment with nitroglycerin. Thus, the effects
of nitroglycerin on the exercise tolerance time are considered to be due to: (1)
relaxation of the stenotic coronary artery and the improvements of coronary
circulation, (2) reduction of preload due to decreae in the systemic peripheral
vascular resistance, (3) improvement of myocardial energy efficiency possibly
due to reduction of ventricular volume, and (4) increase in coronary blood flow
to the ischemic area through collaterals.
It is well known that nitrates, such as nitroglycerin, are very effective for the
treatment of effort angina. However, the precise mechanisms of the beneficial
effects of nitroglycerin are still unclear. We will discuss how nitrates affect
1 Cardiovascular Division of Osaka Police Hos~ital, Osaka, 543 Japan

2The First Department of Medicine and Medical Information Science, Osaka
University School of Medicine, Osaka, 553 Japan
299
300 K. Kodama et al.
. ormal FIG. 1. Relationship between

myocardial oxygen consump-
t
Se~mental
tion and coronary flow
coronar arteries
_ - - - Diseased
~ Normal zone
Myocardial oxygen consumption
pathophysiologic aspects of effort angina and how they improve myocardial

ischemia during exercise.
Key words: Exercise tests-Nitroglycerin-Effort angina-Collateral
The Mechanisms of Occurrence of Myocardial Ischemia

in Patients with Effort Angina During Exercise
The Responses of Coronary Arteries

Braunwald and Sobel [1] proposed the following mechanisms concerning the
occurrence of myocardial ischemia in patients with severe organic coronary
stenosis (Fig. 1). In normal coronary arteries, coronary blood flow is
predominantly increased according to the increased myocardial oxygen
consumption; however, in stenotic coronary arteries, coronary blood supply
does not increase as much as the myocardium demands. This imbalance
between myocardial oxygen supply and demand is thought to be the primary
cause of acute myocardial ischemia in patients with effort angina during
exercise, and is considered to be an important element in the occurrence of
exertional angina. Figure 2 supports this idea. We measured coronary blood
flow in a patient who had a severe stenosis in the proximal left anterior
descending coronary artery by the thermodilution method proposed by Ganz et
al. [2], and also assessed myocardial lactate metabolism by measurements of
lactate concentrations in arterial and great cardiac vein blood. When the
cardiac pacing rate was increased, coronary blood flow, i.e . , both coronary
sinus flow and great cardiac vein flow, increased due to increased myocardial
oxygen consumption. Six min after the onset of pacing, lactate production was
observed although coronary blood flow was further increased . The fact that
5. Possible Mechanisms of the Beneficial Effects 301
CSF
GCVF
7.0
.3.0
I
~~:~~]
o .3 6 9 (min)
FIG .2. Coronary blood flow, lactate extraction, and heart rate during atrial pacing
stress test in a patient with effort angina. Ao, Blood from aorta; CS, blood from
coronary sinus; GCV, blood from great cardiac vein ; CSF, coronary sinus flow; GCVF,
great cardiac vein flow; HR, heart rate
coronary blood flow was able to increase despite the existence of myocardial
ischemia suggests that myocardial oxygen supply can not catch up with the
increase in oxygen consumption, leading to the imbalance between myocardial
oxygen supply and demand.
Recently, there have been several reports in support of the idea that the
changes in coronary vasomotion in patients with effort angina may modify its
clinical features. The lack of the reproducibility in some patients with effort
angina hints at this hypothesis. Gage et al. [3] showed that in patients with
effort angina, the diameter of the stenotic proximal coronary artery even
decreases during exercise although the diameter of the non-stenotic coronary
artery conversely increases. Thus, we can clearly state that the causes of effort
angina consist of two components of abnormalities in coronary arteries:
coronary stenosis due to organic changes and changes in coronary vasomotion.
It has also been reported that collateral flow largely influences myocardial
ischemia. In addition, several investigators have suggested that coronary
microcirculatory disturbances may be involved in the pathophysiology of
angina; however, we will not discuss this aspect here.
The Responses of Myocardium

When myocardial oxygen consumption is increased, an imbalance between
myocardial oxygen supply and demand may easily occur in patients with effort
angina during exercise because myocardial oxygen supply through the stenotic
coronary artery is severely limited. The increases in myocardial oxygen
demand during exercise are predominantly determined by the heart rate,
myocardial contractility, preload, and afterload. Augmentation of any of these
factors may act as a cause of the onset of effort angina if any factor contributes
to making myocardial oxygen demand greater than oxygen supply.
Furthermore, an external compressive force to intramyocardial small coronary
arteries may reduce coronary blood flow. When preload is increased during
exercise, coronary blood flow during diastole is depressed because myocardium
is well perfused by small coronary arteries, predominantly during the diastolic
phase.
Clinical Assessments of the Severity of Effort Angina

The functional severity of effort angina has been estimated by comparing the
exercise tolerance time to the onset of angina during exercise. Indeed, this
index is also clinically important for assessing the exercise capability of the
patients with effort angina. Figure 3 shows the relationship between the
exercise tolerance time and pressure-rate products in 50 patients with classical
effort angina during exercise. The exercise tolerance time and pressure-rate
products varied 2-10min and 10-30 x 103 mmHg/min, respectively. There
seems to be no relationship between these two indexes when compared inter-
individually. However, the exercise tolerance time and maximal exercise work
load were highly reproducible when two exercise tests were performed in each
patient. (Fig. 4). This observation indicates that assessment of the exercise
tolerance time is very suitable to evaluate the effects of drugs in patients with
stable effort angina.
Possible Effects of Nitroglycerin in

Effort Angina During Exercise
Pharmacological Effects of Nitroglycerin

Nitroglycerin is believed to have several myocardial and coronary effects. First,
nitroglycerin potently relaxes coronary arterial smooth muscles. Nitric oxide
(NO) produced during the degradation process of nitroglycerin was recently
FIG. 3. Relationship between

30 •
exercise tolerance time and
rate-pressure product III 50 •
patients with effort angina. I •
RPP, Rate pressure product
c:
• ••
25
'E
"":I:
0()
• •••
E
E
20
• •
•
§
"-
• • • •
• • • •
• •
•• •• • •
il.
il.
c:<: 15 • ••
•
• •
••
10
T
234 567 8 9 10
Exercise tolerance time (min)
C,(kpm)
(" (min' 1
3.000
,"
\0
• •
•
2.000
., ~
•
•
• •
r=O.961
1.000
r=0.959
y =0.9H5x +0.369 .v=O.996x+ 104.62
L-______ ~ ________ ~ ________ ~~
10 1.000 2.000 3.000

a C,lmin) b (",Ikpm)
FIG. 4. a Reproducibility in exercise tolerance time and b maximal exercise work load
during the two exercise stress tests in 50 patients with effort angina
reported to increase cyclic GMP concentrations in coronary smooth muscle

cells [4]. The increased cyclic GMP concentrations activate cyclic GMP kinase;
this activation is reported to decrease Ca2+ concentrations and enhance
dephosphorylation of the myosin light chain in coronary smooth muscle cells.
Both of these actions of cyclic GMP are thought to relax coronary smooth
muscles. Intriguingly, one of the endothelial-dependent relaxant factors is
recently identified NO which is believed to relax coronary arterial smooth
muscle through the common pathway of the effects of nitroglycerin.
In the clinical setting, nitroglycerin is thought to reduce the severity of
myocardial ischemia in patients with effort angina in three different ways:
nitroglycerin may (1) decrease the tones of the stenotic coronary arteries, (2)
increase the coronary collateral flow, and (3) decrease the preload due to the
decreases in systemic vascular resistance. When the coronary artery has severe
orgainc lesions, nitroglycerin no longer affects any decease in the tones of
stenotic lesions. However, when the abnormalities of coronary vasomotion
overlap the coronary stenotic lesions, nitroglycerin may improve the
myocardial perfusion because of its potent coronary vasodilatory actions. Gage
et al. (3) also reported that nitroglycerin can affect vasomotor tones of stenotic
coronary arteries during exercise in patients with effort angina.
Effects of Nitroglycerin on Collateral Circulations

In order to te<;t these three possibilities concerning the beneficial effects of
nitroglycerin on effort angina, we performed the exercise stress test on patients
with effort angina and examined the effects of nitroglycerin on coronary and
systemic hemodynamics using the following two exercise protocols. The first
study was performed using the noninvasive ergometer exercise test, and the
second study was done using the invasive ergometer exercise test under cardiac
catheterization. In the first noninvasive study, 105 patients with stable effort
angina were evaluated, and in the second invasive study 50 patients with stable
effort angina were employed (Table 1). The measured parameters were blood
pressure, heart rate, pressure rate products, and the severity of coronary artery
in the noninvasive study (Table 2). In the invasive study, the measured
parameters were aortic and pulmonary arterial pressures, cardiac output,
TABLE 1. Subjects
Noninvasive study
105 Patients with effort angina
Without previous MI: 47
With previous MI: 58
Invasive study
50 Patients with effort angina
Without previous MI: 30
With previous MI: 20
MI, Myocardial infarction

TABLE 2. Measured parameters

Noninvasive study
Clinical profile (history, NYHA classification, etc.)
Hemodynamics (BP, HR)
Severity of coronary stenosis
Invasive study
Clinical profile
Hemodynamics (AoP, CO, and PAP during exercise)
Coronary blood flow
Myocardial oxygen consumption
Coronary angiography during exercise
AoP, Aortic pressure; BP, hlood pressure; CO, cardiac output;

HR, heart rate; PAP, pulmonary arterial pressure
coronary blood flow by the thermodilution method, and regional myocardial

oxygen consumption. In addition a coronary angiogram during exercise was
performed. The exercise protocol of the noninvasive study consisted of
stepwise increments of the work load. The endpoint of the exercise was
determined to be the time of the onset of angina of grade II in all patients.
Premedication of sublingual nitroglycerin increased the exercise time
significantly compared to the results without nitroglycerin. In all 105 patients,
the exercise time was increased significantly, however, each value greatly
differed inter-individually, and the extent of increases in exercise time was
different in each individual (Fig. 5). In order to investigate the reason for these
findings, we hypothesized that differences in capability to increase coronary
blood flow by administration of nitroglycerin may depend upon the severity of
the coronary sclerosis. This is because atherosclerosis is reported to attenuate
vasodilatory actions caused by endogenous vasoactive substances, such as
acetylcholine. Thus, we determined the relationship between the increase in
exercise time by nitroglycerin and the coronary sclerotic index proposed by
Knobel et al. [5] (Fig. 6). Unfortunately, these two parameters do not seem to
be correlated significantly. On the other hand, when we divided the patients
into 2 groups, with and without collateral circulation, the patients with
collaterals had a tendency to have greater increments in exercise time with
nitroglycerin treatment (Fig. 7a). Excluding the patients with previous
myocardial infarction, the tendency becomes more apparent (Fig. 7b), and
there seems to be a close relation between the response to nitroglycerin and
the existence of collaterals. These results could lead us to support the idea that
nitroglycerin potently increases coronary collateral flow to the ischemic regions
in patients with effort angina.
The protocol of the invasive study is composed of two successive bicycle
ergometer exercise tests in the supine position (Fig. 8). Between the first
(control) and second exercises, the patients were allowed a 30- min recovery
period. During this interval, sublingual nitroglycerin was administered for the
second exercise. Again, in this invasive exercise test, we demonstrated direct
p<O.OI
FiG. 5. Exercise tolerance time

before (Control) and after (NTG)
pretreatment with sublingual
nitroglycerin in 105 patients with
10 effort angina
II
6
c
'e -H-
~
~
LIJ
4
('onlrol NTG
JOO
0
0 0 • 00
t 177
0
t
•
200
00
•
0 0 0 0 .1\00
00 0 1::..
0
+ 1<;0
.,
)(
",
0
I::.
0 0 8 0
.125 0
c 0 0
.~
0... 0
0
«fD
•
0 0 0
oF 1200
., 0 0 0
•
..
•
0
u
'"
t-
•o 0
8
<II
c • I 0
0
::;
u
100 • ~ 0
• • Collaleral ( - )
•
•• ••
o Collaleral (+ )
• • • I::. Collaleral poor
•
I
I
10 20 30 40 50 60 70 80 90 100 110
NTG response (,1%)
FiG. 6. Relationship between increase in exercise tolerance time after pretreatment with
sublingual nitroglycerin (NTG response) and coronary sclerotic index
306
a p<O.OI b
72.112 ± 6.44 36.113 ± 6.21
p<O.OI
200 00 •
00
150
81.9H 13.57 31.47 ±4.49

o
-
~ o
•
-
c o
o
0-
6-0 100 o
~
~ 100
...
-
U
f-
o c'"
Z
• o
,
00 0-
I
t~ ~
- •••
V
cW
00
I f-
Z 50 000
50 cxx&
fb 00
&,
o
o
00 I t
oL-------------~~__
CollalCral (+ ) Collateral ( -- ) Collatcral ( -t-) Collateral ( -)
FIG. 7. a Comparison of increase in exercise tolerance time after pretreatment with

sublingual nitroglycerin (NTG response) between patients with (open circles) and
without (closed circles) collaterals. b The results in a excluding the patients with
previous myocardial infarction
Ergometer stress te:.t Ergometer ,tress test

Control study NTG study
450 kpm
300 kpm,>",~,,:
150 kpm -'?\:_';;':4,-,______3~es~,_in----"==
.~ / r
oI 2 3 4 ,'i 6 7!1 I 2 J 4 5 ,-- () I 2 3 4 5/)7 !I I 2 3 4 '; --
_-LL'..l1--11-L1..ll-L.,I....L,.';' / I I I I I I _ _~--1-L..lI-LI~I-LI..lI~I/~/~~I-LI..lI-L1LI
• Webstcr
• Swan-(iam • lIemodynamic 'parameters • Ilemodynamic parameters
catheteri/ation measurement measurement
• Arterial canulation • 12-lead FCC; • 12-lead ECG
FIG. 8. Exercise protocol of the invasive study. NTG, Nitroglycerine

FIG. 9. Coronary angiograms of a representative case with collaterals during an

exercise-induced anginal attack. Cant., Coronary angiogram during control study; Ex. ,
at the onset of angina during exercise ; NTG, coronary angiogram during NTG study
evidence that nitroglycerin may relax the collaterals. Figure 9 shows the
coronary angiogram of a representative case with good collaterals during an
exercise-induced anginal attack. The left half shows the coronary angiograms
during the control exercise , and the right half shows those during the exercise
after administration of sublingual nitroglycerin. The upper panels show the
coronary angiograms at rest, and the lower two panels during the exercise-
induced anginal attack. As shown in the left lower panel, collateral flow was
visible or even more enhanced compared with that in the control rest. These
tendencies were common findings in patients with good collaterals. These
results suggest that patients with good response to nitroglycerin have good
collaterals and increase the exercise time through increase in coronary flow by
the administration of nitroglycerin .
Effects of Nitroglycerin on Ventricular Volume

The differences in increments in the exercise time before and after
pretreatment with sublingual nitroglycerin are shown in Fig. 10. Figure 11
shows the changes in stroke work index during exercise. The extent of the
15 p<O.OOI .,
FIG. 10. Exercise tolerance time
(ETT) before (Control) and after
(NTG) pretreatment with sublingual
nitroglycerin in 50 patients with
effort angina T
I
I
I
10 I
I
-t-
I
C I
'e I
I
l- I
I- ...L
1.0.1
Control NTG
('ontrol study
a
100
NE XO
.......
;;
...
.D
----
E nO
ell
~
Vl
40
~ Mean value
1 20 30 10 20 30
10
PAED!' (mmHg)
FIG, 11. Relationship between pulmonary arterial end-diastolic pressure (PAEDP) and
stroke work index (SW/) in a control study and b NTG study
309
•• 7.0
120 •• ••
Ne 5.0
....c
'e
~
c; .1.0
•
1.0
T
CN 6 9 eN 6 9
lOTt" (min)
... 120
:r::
OIl
:r::
e e
e e 20
~
~
ow
~ lOll
«
~ ~ 10
e
r .~CN 6 9
••
CN 9
lOTT (minI
• p<O.OOS C>--<> : ("ontrol study
•• P<O.OOI _ _ _ : NT(j study
1(" vs. N. mean ~ S/)o
paired II
FiG. 12. Hemodynamic parameters during exercise stress tests in control and NTG
studies, C, at the resting condition of control study; C/, cardiac index; HR, heart rate;
mean BP, mean aortic pressure; N, at the resting condition of NTG study; ETT,
exercise tolerance time
increments of stroke work index was smaller in the control exercise of patients
with effort angina than that in normal subjects (Fig. lla), suggesting that the
contraction efficiency may become worse due to ischemia. In contrast, after
pretreatment with nitroglycerin, the slope between the pulmonary arterial
end-diastolic pressure vs stroke work index became steeper and almost
returned to the levels of the normal subjects (Fig. llb), suggesting that the
contraction efficiency had been greatly improved by the administration of
sublingual nitroglycerin. We carefully analyzed the systemic hemodynamics
(Fig. 12). Both the aortic and pulmonary arterial pressures were shifted to the
lower levels after administration of nitroglycerin, suggesting that the preload
reductions by the systemic vasodilator effect of nitroglycerin were clearly
NTG response in ETT (%)

50 100 150 200
O~O~.~------~-----------'-----------'-----------;
o
o MI(+)
o
•
- 10
.
• MI(-)
-20
_.
---.... --
0-
o
o
UJ
-30 •
o
0
<: 0- • 0
0-
_ • TOlal cases
. --'-'----
I
·e
-0 -40 ::-i- ___________ y::.:. -0.14x-24.S
c:
• o. • 0 •
• ...... _ _ n.:::35, r= -0.39 (p<0.05)
~ -50 o
c:
o
o
c..
~ -60
o o MI(-)
I- y= -0.26x-16.5
Z o
-70 n=19, r=-O.IS (p<O.OI)
FIG. 13. Relationship between the increase in exercise tolerance time after pretreatment
with nitroglycerin (NTG response in ETT) and the reduction in pulmonary arterial
end-diastolic pressure at 3 min during exercise. ETT, Exercise tolerance time; MI,
myocardial infarction; PAEDP, pulmonary arterial end-diastolic pressure
observed at the same work load. The relation between the reduction in
pulmonary arterial end-diastolic pressure and the increase in exercise time is
shown in Fig. 13. There is a significant relation between both the larger
reduction of preload and the greater increase in exercise tolerance time in all
patients (dotted line). Excluding the patients who suffered from previous
myocardial infarctions, this relation becomes more prominent (Fig. 13 (closed
line)); the reduction in pulmonary arterial end-diastolic pressure at the same
work load is well correlated with the increase in exercise tolerance time. These
results indicate that nitroglycerin significantly reduces the preload, leading to
the lengthening of the exercise time and attenuation of ischemia in patients
with effort angina. However, we could not exclude the possibility that the
beneficial effects of the reduction of preload may be attributed to the reduction
of the external compressive force against the intramyocardial small coronary
arteries.
Effects of Nitroglycerin on Stenotic Coronary Arteries

We investigated coronary circulation and myocardial energy metabolism. The
rate-pressure product in patients with premedication with nitroglycerin was
shifted to the same extent compared to that without nitroglycerin. However,
the rate-pressure product showed a greater value at the maximal exercise work
a b
R P P ( x 10" mmHg/min) MV02 (mf/min)
20
20
**
10
10
5
'" p<0.05
** p<O.OOI
I I
C N 3 5 7 9 C N 3 5 7 9
ETT (min) ETT (min)
cr---o : COnlrol siudy. ------ : NTG siudy
FIG. 14. a Rate pressure product and b myocardial oxygen consumption during exercise
in control and NTG study. C, at the resting condition of control study; N, at the resting
condition of NTG study; ETT, exercise tolerance time; MV02> myocardial oxygen
consumption; RPP, rate pressure product
load (Fig. 14a). The regional myocardial oxygen consumption in patients with
premedication of sublingual nitroglycerin was also shifted to lower levels
compared to that without nitroglycerin. However, at the onset of angina, the
regional myocardial oxygen consumption reached an even higher level than
that without nitroglycerin (Fig. 14b). These obserVations may suggest two
different mechanisms of the effects of nitroglycerin in patients with effort
angina: nitroglycerin has the effect of reducing the myocardial oxygen
consumption and of increasing coronary flow. We evaluated the relation
between the increase in exercise time and the reduction in regional myocardial
oxygen consumption at the maximal work load (Fig. 15). The increase in
exercise time and the reduction in regional myocardial oxygen consumption
have a positive correlation (dotted line). Excluding the patients with previous
infarctions, the tendency becomes more prominent (Fig. 15). These results
indicate that nitroglycerin has the potential to reduce the myocardial oxygen
consumption and to increase coronary blood flow; the former may be mediated
through the reduction of preload and the latter may be mediated through
coronary vasodilation of the stenotic lesions during exercise.
Conclusions
Our findings are as follows:
1. The exercise tolerance time in patients with effort angina was easily
reproducible, but the values differed within each individual
MI(-l
y=0.47x·-19.9
40
Total n
MI(-) n
=
~
23
14
• r=0.79 (p<O.OI)
MI(+)n~9 Total cases
.
D [ ~·:-0.19x-10.0
r=0.48 (p<0.05)
, , ... ...' ........

.......'"0
• • •
20
,' ~
c " ,......................
..................... .. o
,"'"
~
" ~
o O~------~~~------~~-------'--------~
I- 50 /100 150 200
Z ,- 0 ~ ..
,- .........,'~ NTG response in ETT (%)
•. 0
............... 0 MI(t)
.. 0 0 y=0.2Ix- 21.9
• r=0.67 (p<O.OI I •
o
MI(-l
MII+1
•
FIG. 15. Relationship between the increase in exercise time after pretreatment with
sublingual nitroglycerin (NTG response in ETT) and the increase in myocardial oxygen
consumption at the maximal exercise work load (NTG response in MV0 2). ETT,
Exercise tolerance time, MI, myocardial infarction, MV02 , myocardial oxygen
consumption
2. The differences in exercise tolerance time in each individual in the group

with effort angina seemed to be related with differences in coronary artery
sclerosis, existence of collaterals, and differences in hemodynamic responses
3. Thus, the effects of nitroglycerin on exercise tolerance time are considered
to be due to:
(a) Relaxation of the stenotic coronary artery and improvements of the
coronary circulation
(b) Reduction of preload due to the decrease in systemic peripheral
vascular resistance
(c) Improvement of myocardial energy efficiency possibly due to the
reduction of ventricular volume
(d) Increase in coronary blood flow to the ischemic area through collaterals
References
1. Braunwald E, Sobel BE (1988) Coronary blood flow and myocardial ischemia. In:
Heart disease, a textbook of cardiovascular medicine. Saunders, Philadelphia, pp
1191-1221
2. Ganz W, Tamura K, Marcus HS, Donosa R, Yoshida S, Swan HJC (1971)

Measurement of coronary sinus blood flow by continuous thermodilution in man.
Vasoconstriction of stenotic coronary arteries during the dynamic exercise in patients
with classic angina pectoris: Reversibility by nitroglycerin. Circulation 73:865-876
4. Armstrong PW, Moffat JA (1983) Tolerance to organic nitrates: Clinical and
experimental perspectives. Am J Med 74 (Suppl):73
5. Knobel SB, Elliott WC, McHenry PL, Ross E (1971) Myocardial blood flow in
coronary artery disease. Am J Cardiol 27:51
6
Importance of Collateral Circulation
in Acute Myocardial Infarction
Shigetake Sasayama, Masatoshi Fujita, and Tadakazu Hirai!
Abstract. Although a functional role of coronary collaterals has been

continuously debated, we observed the following facts in our studies during
intracoronary thrombolytic therapy: (1) myocardial ischemia is important for
the development of collateral circulation, (2) collaterals can perfuse the
infarcted myocardium, and (3) the presence of collaterals prevents the left
ventricular aneurysm formation in acute myocardial infarction even when the
amount of the salvaged tissue is small. Thus, coronary collaterals are not
merely markers of severe ischemia but help to preserve the functional integrity
of the myocardium in the presence of coronary obstruction.
Key words: Myocardial infarction-Coronary collateral circulation-Anginal
pain-Creatine Kinase-Ventricular aneurysm
Introduction: Collateral Circulation in Coronary Artery

Disease
The functional roles of coronary collaterals in humans qave been disputed for
many years. Some investigators have drawn the pessimistic conclusion that
coronary collaterals in humans are more an indication of severe regional
ischemia rather than a sign of biological compensation for a perfusion deficit
[1,2]. On the other hand, many other clinical investigators have offered
evidence that coronary collaterals play a salutary functional role and help to
preserve ventricular performance in hearts with coronary obstruction [3].
Earlier studies on the evaluation of collateral circulation have been done on
post-mortem hearts in order to correlate the extent of the collateral network
with clinical findings during life. These studies demonstrated a better
1 The Second Department of Internal Medicine, Toyama Medical and Pharmaceutical

University, Toyama, 930-01 Japan
315
316 S. Sasayama et al.
developed collateral circulation in patients with a longer duration of ischemic

symptoms. The concept of a functional significance of coronary collaterals to
prevent ischemic events was evidenced by an example of infarction at a
distance where, after earlier occlusion or critical subtotal stenosis of the artery,
collaterals successfully maintain perfusion of the myocardium originally
supplied by the diseased vessel while a second occlusion of the collateral
source vessel resulted in infarction of the distant collateral-dependent area [4].
Similarly, the interventricular septum has a dual blood supply from the anterior
and posterior descending arteries, with many collaterals traversing between
them, and occlusion of one of these vessels can be often compensated with
maintenance of tissue viability of the septum [5].
Chest Pain and Angiographic Visualization of Collaterals

Coronary arteriography has permitted the correlation of the anatomic
appearance of coronary collateral vessels to the underlying coronary arterial
disease. Particularly, coronary thrombolysis, which has been currently used in
selected patients for restoring myocardial perfusion early after acute
myocardial infarction, offers the greatest opportunity to evaluate the role of
coronary collaterals in maximizing myocardial salvage. We studied 37 patients
with acute myocardial infarction who had intracoronary thrombolysis in the
first 6 h after the onset of symptoms. All these patients showed a total
occlusion of one of the major branches of the coronary arteries, and none had
a previous history of myocardial infarction. Collateral channels were visualized
in 9 of the 18 patients (50%) who had anginal attacks for more than 1 week
before infarction, whereas they were seen in only 2 of the 19 patients (11%)
without pre-infarction angina [6] (Fig. 1). The development of coronary
collaterals is a dynamic process extending over several months. The extent of
collateral circulation after myocardial infarction is related to the length of time
between myocardial infarction and coronary angiographic study [7]. Although
the histologic transformation of the collateral vessel starts during the first day
with a rapid increase in luminal dimensions, cellular proliferation actually
occurs during the next 3 or 4 days [8]. Thus, our study, conducted as early as
within 6 h of the onset of myocardial infarction, might reasonably account for
the substantial effect of myocardial ischemia before the onset of acute
myocardial infarction in promoting visualization of coronary collaterals during
coronary angiography.
Post-infarction angina was also noted in patients with well-collateralized
hearts. Forty-one patients who had undergone intracoronary thrombolytic
therapy were divided into 4 groups, depending upon the results of
intracoronary thrombolytic therapy and the presence or absence of pre-
infarction angina [9]. Group I (n = 12): successful thrombolysis with pre-
infarction angina; group II (n = 6): unsuccessful thrombolysis with pre-
infarction angina; group III (n = 15): successful recanalization without pre-
infarction angina, and group IV (n = 8): unsuccessful recanalization in the
absence of pre-infarction angina.
6. Importance of Collateral Circulation in Acute Myocardial Infarction 317
I I I
50- I .0- I
50"10
I I
I I
I I
40 I I
z I
x 0.8 I
o I w
f0- I o I
e[
I z I
N
30 I I
-J I I
e[
:::>
(/)
I '5- -
I
> I
20 I
-J
e[
I
0:: I
W I
f0-
e[ 10 11%
-J 0.2
-J
o
U
0 o
Angina (+) Anginal-) Angina 1+) Anginal-)
group I group IT g ro up I group II
n = 18 n = 19 n = 18 n = 19
FIG. 1. Angiographic evidence of collateral development in patients with pre-infarction

angina. There are significant differences in collateral visualization and collateral index
between patients with and without pre-infarction angina. (From [6] with permission)
Post-infarction angina developed within 10 days after acute infarction was

observed in 7 out of 12 patients in group I (58%), 4 out of 6 patients in group
II (67%), 6 out of 15 patients in group III (40%), and in 1 out of 8 patients in
group IV (13%) (Fig. 2). From these observations, the presence of pre-
infarction angina appears to be an important factor predisposing the
development of post-infarction angina even when intracoronary thrombolysis
was unsuccessful. The post-infarction angina signifies the presence of a viable
myocardial tissue in the perfusion territory of the ischemia-related coronary
artery and a limited coronary perfusion reserve. Collateral circulation was
probably responsible for preserving myocardial integrity in the region at risk,
but this tissue could be rendered ischemic when myocardial metabolic activity
was augmented. In patients who developed post-infarction angina, neither the
thrombolysis nor the collaterals was sufficient to prevent recurrent symptoms.
However, the presence of post-infarction angina may not be merely a marker
of severe ischemia but it may indicate the presence of viable tissue which might
be salvaged by a more effective procedure for recanalization.
Perfusion of Collateral Circulation

Creatine kinase (CK) is an enzyme contained in myocardial cells which leaks
out of damaged cells. Dynamic changes in serum CK activity offer a useful
measure for estimating the extent of infarction. The CK time-activity curve
70,-
r--------- P <O,05---------.,
I
67%
60 -
........
-*''"
58%
50 -
C
011
C
-
'"
C
0
40
40%
+l
u
L-
30 -
.....'"
C
.....II)
I
20 -
0
0..
10 - 13%
0
Group I Groupn Groupm GrouplV'
Pre-infarction Pre-infarction Pre-infarction Pre-infarction
anllina(+) anllina(+) anllina(-) anllina(-)
RecanaJlization( + ) RecanaJlization( - ) RecanaJlization( + ) RecanaJlization( - )
(n=12) (n=6) (n=15) (n=8)
FiG. 2. Prevalence of post-infarction angina in patient groups subdivided according to

the extent of recanalization and presence or absence of the pre-infarction angina.
Despite unsuccessful recanalization, four out of six patients with pre-infarction angina
(group II) developed post-infarction angina, indicating a limited coronary perfusion
reserve and a viable myocardial tissue. (From [9] with permission)
during acute myocardial infarction can be characteristically modified by

recanalization of the occluded coronary artery. We assessed the kinetics of CK
in 32 patients undergoing intracoronary thrombolysis within 6 h of acute
myocardial infarction [10]. The time to peak CK level was 11 ± 1 h (standard
error of the mean) after acute myocardial infarction in 19 patients in whom the
thrombolysis was successful, 16 ± 1 h in 6 patients who had a significant
collateral circulation but the thrombolysis was unsuccessful, and 21 ± 1 h in 7
patients who had inadequate collaterals and the recanalization was unsuccessful
(Fig. 3). There were no significant differences in CK peak value and CK
cumulative release-hence infarct size-among these 3 groups. These data
imply that collateral blood flow supply appears to be inadequate compared
with the ante grade flow through the recanalized coronary artery, but that the
collateral channels visualized by arteriography definitely contribute to the
washout of CK produced in the myocardium at jeopardy and that these vessels
provide significant blood supply to the myocardium at risk.
hrs
30
25 * P < 0.05 •
~ 20
U • f •••
~
• ••
0
Cl>
a. 15
•
•
••
1•••
0
+-
••• •
•
Cl>
E 10
2• ••
•••
f- ••••
•
5 (n = 19) (n= 6) (n= 7l
I I I 1
"* "* 1
0
*
groupA groupB groupe
recanalization(+ ) recanalization(-) recanalization(-)
collaterals(+) collaterals(-)
FIG. 3. Time to peak creatine kinase (CK) in 3 patient groups subdivided according to
whether thrombolysis was successful and whether collateral development was adequate.
The time to peak CK was significantly shorter in patients with adequate collateral
perfusion than in those without collaterals. (From [10] with permission)
Coronary Collateral Circulation and

Left Ventricular Function
A number of investigations have convincingly demonstrated the salutary effect
of early recanalization of an infarct-related coronary artery in the presence of a
residual flow on the regional and global left ventricular function in the later
stages. However, attempts to correlate cardiac function with the presence
of collateral channels have failed to draw a definite conclusion [11 ,12].
Recently, we assessed the relation of the coronary collateral circulation to
regional myocardial function and, later, the formation of ventricular aneurysm
in 47 patients with the first acute anterior myocardial infarction [13]. All these
patients had a complete occlusion of the proximal part of the left anterior
descending coronary artery and were treated with intracoronary thrombolysis
during the first 6h after the onset of symptoms. When the infarct-related
coronary artery was recanalized with less than 90% residual narrowing in
diameter, left ventriculography was performed in a 30° right anterior oblique
projection. The hemodynamic study was repeated within 28-40 days (average
35 days) following acute myocardial infarction, and a left ventriculogram was
obtained in the same manner.
The boundaries of end-diastolic and end-systolic left ventricular silhouettes
were delineated by manual tracings using a sonic digitizing device. The left
ventricular volumes were calculated by the area-length method. The two
ventricular silhouettes were superimposed with external reference markers.
Regional shortening was quantified by a radial coordinate system originating at
the center of gravity of the end-diastolic silhouette. Thirty-two radial grids
were drawn around the center of gravity, and the lengths of each radial grid at
end diastole and end systole were measured to characterize the centripetal
motion of a given surface point.
A left ventricular aneurysm was diagnosed when all the following three
criteria were met: (1) systolic bulging of the involved segment, (2) absence of
%
70
60
*p<0.05
!:
50
~0
~
!Z. 40
!:
~0 30
G)
·n
IZI
>
..:I 20
10
0
groupA groupB groupe
recanalization~) recanalization(-) recanalizationH
collateralsHi collateralsH
FiG. 4. Effect of the recanalization and development of collaterals on left ventricular
(LV) ejection fraction in the acute stage (open bars), and in the chronic stage of the
infarction (hatched bars). (From [13] with permission)
trabeculation in the involved segment, and (3) well-defined demarcation of the

infarcted segment from normally contracting myocardium.
Of the 47 patients, 25 had successful thrombolysis (group A), 10 had
significant collateral circulation but unsuccessful reperfusion (group B), and 12
had unsuccessful recanalization in the absence of significant collateral perfusion
(group C).
In the chronic phase, the heart rate decreased in groups A and B. The left
ventricular end-diastolic pressure fell in group A. End-diastolic volume
remained unchanged in groups A and B but increased significantly in group C.
The ejection fraction was augmented in groups A and B, but reached a
statistical significance only in the former (Fig. 4).
The percentage of segmental shortening in the infarcted area was significantly
improved only in group A. There were no significant changes in regional
segmental shortening of the non-infarcted areas during the convalescent stage
in all groups (Fig. 5). These findings suggest, but do not prove, that collaterals
are able to prevent necrosis and increase the likelihood that reperfusion
procedures will have functionally beneficial effects. However, even a good
collateral network may not be equivalent to the effects of a reperfusion attempt
by intracoronary thrombolysis.
The development of a post-infarction aneurysm has been related to the
absence of residual flow to the myocardium at risk. In our attempt to analyze
the prevalence of left ventricular aneurysm formation by cineventriculography
in the patients mentioned above, we found an aneurysm in 1 out of 25 patients
(4%) in group A, 1 out of 10 patients (10%) in group B, and 7 out of 12
patients (58%) in group C (Fig. 6). The incidence of aneurysm formation was
30%
*p<0.05
20
~
Q)
I-<
<
+-'
0 10
~
H
I:::
·n 0
..:I
."
0°
-10 groupA groupB groupe
recanalizationH1 recana.lizationf-) recanalizationH
collateruslt) collateralsH
FIG. 5. Effect of the recanalization and development of collaterals on the percent of
regional segment shortening (%AL) in acute stage (open bars) and chronic stage of the
infarction (hatched bars). (From [13] with permission)
25
20
* p<0.05
*
~CD 15
'.;:1
&! 5890
Io-t
0 10%
M 10
CD
,Q
§
:z;
group! groupB
recanaJiza:tionH-) recanalizationH recanalizationH
collatera.ls(-+i collateraLsH
FiG. 6. The prevalence of left ventricular aneurysm formation in patients with acute
myocardial infarction who were treated with intracoronary thrombolysis. Open bars,
Patients without left ventricular aneurysm; hatched bars, patients with left ventricular
aneurysm; numbers on the top of the bars, percentage of the left ventricular aneurysm
formation in each group. (From [13] with permission)
much higher in patients with poor collateral circulation than in those with
significant collateral development.
Earlier observations by others have suggested that the paucity of collaterals
could be the cause of aneurysm formation [7,14]. Forman et al. [14] analyzed
factors involved in left ventricular aneurysm formation after a transmural
anterior myocardial infarction. They demonstrated that an aneurysm is
generally associated with total occlusion of a poorly collateralized left anterior
descending artery, and that it is rarely seen in multivessel disease with either
extensive collaterals or a nonoccluded left anterior descending artery.
These retrospective studies could not differentiate whether collateral vessels
were present before the infarction or if they developed afterwards. Our study
provided, for the first time, an insight into this question, and presented the
evidence that pre-existing collateral vessels play an important role in protecting

against aneurysm formation despite the lack of evidence that they provided
myocardial salvage.
The mechanism by which infarct expansion and aneurysm formation was
reduced is unknown, but as long as collaterals can perfuse the infarcted
myocardium [10], islands of residual subepicardial cells could limit the extent
of transmurality [15].
In conclusion, our findings substantiate the importance of residual blood
supply to the infarcted myocardium. Thus, intervention to re-establish flow
either by ante grade flow or by way of collateral circulation would prevent left
ventricular aneurysm formation even when the amount of the preservation of
regional myocardial function is minimal.
References
1. Helfant RH, Gorlin R (1972) The coronary collateral circulation. Ann Intern Med
77:995-997
2. Berger BC, Watson DD, Taylor GJ, Burwell LR, Martin RP, Beller GA (1980)
Effect of coronary collateral circulation on regional myocardial perfusion assessed
with quantitative thallium-201 scintigraphy. Am J Cardiol 46:365-370
3. Cohen MV (1985) Functional significance of coronary collaterals in man. In:
Coronary Collaterals: Clinical and experimental observations. Futura, New York,
pp 93-185
4. Fulton WFM (1964) Anastomotic enlargement and ischaemic myocardial damage.
Br Heart J 26:1-15
5. James TN (1971) Coronary circulation in acute myocardial infarction. Br Heart J 33
(Suppl): 138-144
6. Fujita M, Sasayama S, Ohno A, Nakajima H, Asanoi H (1987) Importance of
angina for development of collateral circulation. Br Heart J 57:139-143
7. Aygen M (1977) Collateral circulation and regional myocardial function. Bibl
Cardiol 36: 136-140
8. Schaper W (1971) The collateral circulation of the heart. North Holland,
Amsterdam
9. Fujita M, Sasayama S, Araie E, Ohno A, Yamanishi K, Hirai T (1988) Significance
of pre-infarction angina for occurrence of post-infarction angina. Eur Heart J
9:159-164
10. Hirai T, Fujita M, Sasayama S, Ohno A, Yamanishi K, Nakajima H, Asanoi H
(1987) Importance of coronary collateral circulation for kinetics of serum creatine
kinase in acute myocardial infarction. Am J Cardiol 60:446-450
11. Schwartz H, Leiboff RL, Katz RJ, Wasserman AG, Bren GB, Varghese PJ, Ross
AM (1985) Arteriographic predictors of spontaneous improvement in left
ventricular function after myocardial infarction. Circulation 71:466-472
12. Saito Y, Yasuno M, Ishida M, Suzuki K, Matoba Y, Emura M, Takahashi M (1985)
Importance of coronary collaterals for restoration of left ventricular function after
intracoronary thrombolysis. Am J CardioI55:1259-1263
13. Hirai T, Fujita M, Nakajima H, Asanoi H, Yamanishi K, Ohno A, Sasayama S
(1989) Importance of collateral circulation for prevention of left ventricular
aneurysm formation in acute myocardial infarction. Circulation 79:791-796, 1989
14. Forman MB, Collins HW, Kopelman HA, Vaughn WK, Perry JM, Virmani R,
Friesinger GC (1986) Determinants of left ventricular aneurysm formation after
anterior myocardial infarction: A clinical and angiographic study. J Am Coll
Cardiol8:1256-1262
15. Hochman JS, Choo H (1987) Limitation of myocardial infarction expansion by
reperfusion independent of myocardial salvage. Circulation 75:299-306
Index
A) adenosine receptors 123, 147 Blood-flow velocity 10

A2 adenosine receptors 123, 160 Bradykinin 206
Acetylcholine 43, 206, 217 Brief ischemia 271
Acetylsalicylic acid (ASA) 169
Adenine nucleotides 206 45Ca 2+ 242
Adenosine 56,133,147,160,169,179, Calcium-dependent protease 280
206 cAMP 56
deaminase 109 Carotid body chemoreceptor 43
receptor 56 Catalase 261
Adenylate cyclase 123 Catheter-tipped Doppler probe 3
Alpha receptors 43, 56 C4/D4 receptor antagonist 280
Alpha-l receptor 43 Cellular energy state 123
Alpha-2 receptor 43 Chronotropic 147
a, f)-methylene adenosine 5-diphosphate Collateral 299
(AOPCP) 160 Complement component C5a 261, 280
Alpha-receptor-mediated vasoconstriction Coronary arterial disease 31, 91
43 blood flow 56, 261
5 amino 4 imidazole carboxamine riboside collateral circulation 315
(AICA-riboside) 280 flow 123, 133
AMP 133 reserve 179
Anginal pain 315 velocity 3
"Anti-slosh" hypothesis 43 microcirculation 22
AOPCP 133 resistance 109
Aorta 217 sinus flow (CS flow) 179
Aortic regurgitation 3 stenosis 78
Arachidonate lipoxygenase metabolism vasodilation 179
291 vasomotion 78, 91
Atherosclerotic vessels 78 C)Q 280
ATP-sensitive K+ channel 123 Creatine kinase 147, 315
Atropine 254 Cytosolie 5' -nucleotidase 123, 147
Autoregulation 109
AV node 123 D4 280
1, 2-diacyl glycerol (DG) 56
Beta receptors 43, 56 Diacylglycerol (DAG) 230
Beta-l receptor 43 Dilated cardiomyopathy 3
Beta-2 receptor 43 Dilazep 22
Bezold-larisch reflex 43 5, 5-dimethyl-l-pyrroline-N-oxide
325
326 Index
(DMPO) 291 Inosine monophosphate (IMP) 147

Dipyridamole 109 Inositol phosphates (IP) 56
Dromotropic effects 147 1, 4, 5-triphosphate (IP3) 230
Dynamic exercise 91 Interstitial fluid 109
Intracellular calcium 195, 230
Ecto-5'-nucleotidase 123, 133, 160 concentration 242
EDCF 217, 230 Intraluminal applications 133
Effort angina 299 control 169
EHNA 109 Intramyocardial coronary artery and vien
Endocardial adenosine concentrations 10
109 small vein velocity 10
Endothelial cell injury 291 Intravital microscope 22
cells 169 Ischemia 56, 109
Enbdothelin-l 242
Endothelium 133, 195, 217 K+ Channels 123
Endothlium-dependent relaxation facter Kininase II 206
(EDFR) 43,78,169,179,195,217,
230, 254, 280 Laser Doppler velocimeter 10
Energy charge 147 Latent myocardial damage 271
Epicardial adenosine concentrations 109 Leukocyte capillary plugging 280
Equilibrium constants 147 Leukotriene 261, 280
Exercise tests 299 LTC4 280
Exercise-induced vasoconstriction 91
Extracellular K + concentration (K +e) 271 Mepyramine 254
Extraluminal applications 133 Metabolic vasodilation 109
Extravascular compression 91 Methoxamine 56
Mg2+ ion 133
F340 : F380 ratios 230 Microcirculation 280
FFf system 3 Monoamines 78
Flow debt repayment 160 Muscarinic K+ channel 123
FMLP 169 Muscle contraction 242
Forskolin 160 Myocardial contractile function 271
Fourier transform signal processor infarction 315
(FFf) 3 metabolism 271
Free radicals 261 oxygen consumption 43
Fura-2 230 perfusion 31
stunning 56, 271
Geometry of stenosis 91 Myokinase 147
Granulocyte accumulation 261
Great cardiac vein flow (GCV flow) 179 N-13 ammonia 31
Guanethidine 254 Negative chronotropic 123
dromotropic 123
8-7 56 inotropic 123
Hemodynamic force 230 Neutrophil function 291
High-speed motion picture camera 22 NG-nitro-L-arginine 123
video camera 22 Nicardipine 230
Histamine 78, 254 Nicotine 43
HPLC 291 Nitric oxide 195
Hydroxyeicosatetraenoic acids (HETE) Nitroglycerin 78, 299
291 Noninvasive techniques 31
Hydroxyl radicals 206 No-reflow phenomenon 261, 280
Hypoxia 109, 133 Norepinephrine 56
Hypoxia-reoxygenation 291 5'-nucleotidase 147
Inner/outer flow ratio 43 0-15 Water 31

Index 327
ONO-3708 217 Rubidium-82 31

Optical fiber 10
Oxygen supply/demand 109 SA node 123
tension 109 S-adenosylhomocysteine hydrolase 109
Oxygen-derived free radicals 291 Septal artery velocity 10
Serotonin 78
PI 195 Shear stress 230
P2 195 SHR 217
Purinoceptors 195 SIT camera 22
PAF 169 Small arterioles 22
Parasympathetic 43 epicardial coronary artery and vein 10
Patch-clamp method 230 venules 22
Peptido-leukotrienes 280 Spontaneously hypertensive rats 217
PGH 2 217 Stenotic vasoconstriction 91
Phasic myocardial perfusion 10 Stress-induced ischemia 31
Phentolamine 22 Sublingual nitroglycerin 91
Phenylephrine 254 Superoxide dismutase (SOD) 261,280
8-phenyl-theophylline 56 Supine bicycle exercise 91
Phosphoinositide turnover 78 exercise test 91
Phospholipase C 78 Sympathetic 43
Phosphorylation potential 147
Platelet aggregation 56 T 261
Platelets 169 Tetrodotoxin 254
PMA 56 Thioflavin S 261
3IP-NMR 147 Thromboxane 280
Polymorphonuclear leukocytes 169 A2 261
Positron emission tomography 31 receptor antagonist, BM13505 280
Prazosin 56 Transmural adenosine gradient 109
Propranolol 22 TXA 2 217
Prostacyclin 195
Prostaglandin H2 217 Upright exercise test 91
Prostaglandins PGE 1 and PGE 2 169
Protein kinase C 56 Vascular contraction 78
PTCA 31 endothelial' cells 230
smooth muscle 242
Quantitative coronary arteriography 91 cells (VSMCs) 242
Quin2 242 Ventricular aneurysm 315
Verapamil 261
Reactive hyperemia 160 Vessel capacitance 91
Red cell velocity 22
Reflex 43 Xanthine oxidase 261, 280
Reperfusion injury 56, 261, 280
Reynolds number 230 Zero-cross system 3

Regulation of Coronary Blood Flow PDF

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M. Inoue· M. Hori· S. Imai· R.M.

With 149 Illustrations

ISBN 978-4-431-68369-8 ISBN 978-4-431-68367-4 (eBook)

©Springer Japan 1991

The importance of the physiology and pathophysiology of coronary circulation

I am most grateful to the contributors for their efforts in providing the

A New Approaches for Coronary Flow Measurement

B Neural Control of Coronary Blood Flow

1. Autonomic Control of Coronary Blood Flow

3. Vasoacative Monoamines in the Regulation of Arterial Tone

C Role of Adenosine in Regulation of Coronary Blood Flow

1. The Role of Adenosine in the Metabolic Regulation of Coronary

D Role of Endothelial Cells in Coronary Circulation

1. Endothelial Cell P 2 Purinoceptors

4. Flow-Induced Calcium Response in Cultured Vascular Endothelial

E Ischemia and Reperfusion Injury in the Experimental and

1. Coronary Blood Flow in Reperfused Myocardium

Akita, H. 78 Hirayama, A. 299 Leipert, B. 206

Shimokawa, H. 254 Tamaki, N. 34 Uematsu, M. 3

Summary. The recent development of a catheter-tipped Doppler probe has

Effects of the Range Gate on

Sample posit ion from the catheter tip

Zero- :---1- 1--.--1- f----1_ -'---1-

Influence of Catheter Insertion Direction on Coronary Flow

Distance from 2mm 4mm

I•• ~ • I :, 8", .' ••'.:: 5.Bm

along the ultrasound beam simultaneously within the depth of 15 mm , thereby

o 100 (em / see)

Doppler velocimeter. The velocity profile was parabolic in the retrograde

Comparison of the Volumetric Data Obtained by the

DCM T.T. GOM

In patients with dilated cardiomyopathy, coronary flow acceleration in early

6. Doi Y, Tanouchi J, Uematsu M, Ishihara K, Yoshida Y, Inoue M (1990) Abnormal

Summary. Our laser Doppler velocimeter (LDV) with an optical fiber is a

I Department of Medical Engineering and Systems Cardiology, Kawasaki Medical

Principle of Fiber Optic Laser Doppler Velocimetry

He-Ne laser P.B.S. Shifter P.B.S.

The back-scattered light signal has a Doppler-shift frequency Ai from 4 MHz

Four Different Routes of Access of the Fiber Probe to

(1) epicardial large (2) epicardial small

Opt ical fibe r

Blood Flow Velocity Waveforms and Profiles in Proximal

Blood Velocity Waveforms for the Stenotic Coronary

Blood Velocity Waveforms in the Epicardial Small Artery

Blood Velocity Waveforms in Intramyocardial Arteries and

FIG. 5. An example of velocity waveforms in a small epicardial artery of the left

waveforms are measured in small arteries and veins. Therefore, measurements

Blood-Flow Velocity Waveforms in the Coronary Vein:

FIG. 9. A schematic drawing of possible blood flow directions in intramyocardial

venous flow by access route 4 may be effective for clinical monitoring of

20. Hiramatsu 0, Mito K, Kajiya, F (1990) Evaluation of the velocity waveform in

Summary. Direct and continuous visualization of the coronary microcircula-

1 The First Department of Internal Medicine, Tohoku University School of Medicine,

<art Stabilizing Needles

purpose, we have developed a new microscope system, the "floating objective

Partial Restraint of Card;iac Motion

mercury~ standard microscope

FIG. 2. A method for epiilumination of epimyocardium and the image acquisition

Image Analysis and Velocity Measurement

TABLE 1. Phasic change of internal diameter in small arterioles and venules

Photonics) or on prints produced by a video graphic printer (model UP-811 ,

Change of Microvascular Diameter During the Cardiac Cycle

Phasic Change of Blood Flow in Coronary Microcirculation