151

Distribution and Metabolism of Angiotensin I

and II in the Blood Vessel Wall
Peter Gohlke, Peter Bunning, and Thomas Unger

Hie demonstration of all components of the renin-angiotensin system in vascular tissue has raised questions
as to the precise location of the local angiotensin II generation within the vascular wall. We investigated the
metabolism of angiotensin I to angiotensin II in the vascular wall in the isolated rabbit thoracic aorta.
Angiotensin I ( 3 x 1 0 ' M) applied into the aortic lumen was partially converted to angiotensin II (14% after
60 minutes), but most of the luminal angiotensin I was degraded to peptide fragments or diffused as intact
angiotensin I, peptide fragments, or both, into the vessel wall. Incubation studies with [3H] angiotensin I
revealed that angiotensin I or angiotensin I fragments mainly diffused into the medial layer of the aorta and
to a lesser degree into the adventitia and the endothelium. After removal of the endothelium, angiotensin II
generation could no longer be detected. Addition of the angiotensin converting enzyme inhibitor ramiprilat
(10~7 M) to the incubation medium led to a complete blockade of angiotensin II generation by endothelial
angiotensin converting enzyme. Our results underline the importance of the endothelium for conversion of
angiotensin I to angiotensin II and provide evidence that conversion of angiotensin I to angiotensin II is
predominantly achieved by endothelial cells. They also support the concept of an endocrine versus
autocrine/paracrine renin-angiotensin system where the endothelium of the vasculature is the critical target
site for angiotensin II production by both systems and, thus, the most important site for the actions of
angiotensin converting enzyme inhibitors. (Hypertension 1992^20:151-157)
KEY WORDS • angiotensin I • angiotensin II • kininase II • angiotensin converting enzyme
inhibitors • endothelium, vascular • renin-angiotensin system • rabbit studies
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B iochemical, immunohistochemical, and molecu-

lar biological studies have provided evidence
for the presence of all the components of the
renin-angiotensin system (RAS) (renin, angiotensino-
gen, angiotensin converting enzyme (ACE), angiotensin
peptides, and angiotensin receptors) in a variety of
tissues, suggesting the existence of local tissue RAS.1-3
In addition, local production of angiotensin I (Ang I)
and angiotensin II (Ang II) has been demonstrated in
cell culture and in organ preparations,4"6 and evidence
that such a local Ang II production may also occur in
humans has been found in human subjects in vivo.7 A
local Ang II synthesis in the vascular wall is of special
interest in view of the multiple vascular actions of Ang
II. Ang II not only exerts a direct vasoconstrictor effect,4
but was also reported to enhance sympathetic norad-
renergic transmission8-9 and to have mitogenic and
trophic actions in the vasculature via stimulation of
platelet-derived relaxing factor A-chain or the proto-
oncogenes c-myc and c-fos.ia~13
However, a precise characterization of a local vascu-
lar RAS is complicated by the lack of information about
the origin and location of each component of the
vascular RAS. Ang I may be synthesized locally within
From the Department of Pharmacology and the German Insti-
tute for High Blood Pressure Research, University of Heidelberg
(P.G., T.U.), and Hoechst AG (P.B.), Frankfurt, FRG.
Address for correspondence: Dr. Peter Gohlke, Department of
Pharmacology, University of Heidelberg, Im Neuenheimer Feld
366, Heidelberg, FRG.
Received September 10, 1991; accepted in revised form March
27, 1992.
the vascular wall (e.g., medial and adventitial layer) by
the action of vascular renin on vascular angiotensino-
gen, both derived from either local production or sys-
temic uptake.13-16 However, the last step in the RAS
cascade, i.e., the conversion of Ang I to Ang II, still
remains controversial with regard to the site of Ang II
production as well as the enzymes involved.
Theoretically, several Ang II-generating pathways
can participate in vascular Ang II production. Ang I
may be converted to Ang II by endothelial ACE,
extraendothelial ACE, or by other enzymes in the
vascular wall like the chymostatin-sensitive Ang II gen-
erating enzyme.17-18 Furthermore, alternative pathways
for Ang I conversion to Ang II, for example, a consec-
utive action of carboxypeptidases to generate Ang II,
may be involved in vascular Ang II production. More-
over, Ang I is not only metabolized to Ang II but also to
peptide fragments by the action of aminopeptidases,
carboxypeptidases, and endopeptidases localized on
vascular endothelial and smooth muscle cells.19-21
In the present study we used an in vitro model, the
isolated thoracic rabbit aorta, to investigate the vascular
metabolism of Ang I to Ang II and to further analyze
the distribution of these peptides and their fragments in
the vascular wall.
Methods
Aortic Preparation
Male Chinchilla rabbits (Mollegard, Skensved, Den-
mark) weighing 2-3 kg were killed by cervical disloca-
tion. The thoracic aorta was removed and rinsed with a
modified Krebs-Henseleit buffer, pH 7.4 (mM: NaCl
 152 Hypertension Vol 20, No 2 August 1992
37t-
FIGURE 1. Schematic diagram shows in vitro model of
isolated rabbit thoracic aorta. The isolated rabbit thoracic
aorta was cannulated at both ends and suspended in an organ antibody is 100% for Ang I-(2-10) and less than 0.1%
chamber containing a modified Krebs-Henseleit buffer, pH for Ang II.
7.4, gassed with 95% 0-2-5% CO2. The incubation bath was
kept at 37°C. For aortic incubation the aortic lumen is filled Distribution of Angiotensin I and Angiotensin II in
(1), the tubes are clamped (2), and incubation starts. At the
end of the incubation period the clamps are opened and the
aortic eluate is collected (4) by gently pushing air through the in the vascular wall was investigated by instilling tritium-
system (3).
118.0, KC1 4.75, KH2PO4 1.2, CaCl2 1.7, MgSO4 1.2,
NaHCO 3 25.0, HEPES 10.0, glucose 11.0) that was
gassed with a 95% C>2-5% CO2 mixture. After dissec-
tion of perivascular tissue all side branches were tied
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off. The aorta was cannulated at both ends and sus-
pended in an organ chamber containing the modified
Krebs-Henseleit buffer (pH 7.4) gassed with 95%
C>2-5% CO 2 and 10~5 M verapamil. The incubation bath
was kept at 37°C (Figure 1). The aorta was instilled with
200-300 (il of the modified Krebs-Henseleit buffer,
closed at both ends, and preincubated for 1 hour before
angiotensin incubations were started.
In a separate experiment aorta preparations were
examined histologically by silver nitrate staining to 22
verify the integrity of the endothelium. The aorta was
pinned on a styropore block and rinsed three times with
isotonic glucose solution. After incubation with silver
nitrate (0.25%) for 30 seconds the aorta was rinsed
again with isotonic glucose, fixed overnight in 4% for-
malin, transferred into xylol via increasing ethanol
concentrations (10-96%), and embedded in Entellan
(Merck, Darmstadt, FRG) for microscopic examination.
Aortic preparations were also stained after denudation
of the endothelium to demonstrate the completeness of
endothelium removal. Routinely, silver nitrate staining
was carried out at the end of incubation experiments to
examine the status of the aortic preparation.
Angiotensin Incubation
In a first experiment the aortic vessels were filled with
3x10"' M Ang I and incubated for 5,10,15, 30,45, and
60 minutes (n=3). In a second series, vessels (n=3)
were incubated in reverse order for 60, 45, 30, 15, 10,
and 5 minutes. Each incubation period was followed by
a washout period of 10 minutes with buffer. One aorta
was used for an additional incubation period of 90
minutes. In a second experiment, aortic vessels (n=3)
were incubated with 3 x 10"' M Ang I together with the
ACE inhibitor ramiprilat (10~7 M) and incubated for 5,
10, 15, 30, 45, and 60 minutes. Each incubation period
was followed by a washout period of 10 minutes with
buffer containing 10"7 M ramiprilat. In a third experi-
ment, endothelium-denuded aortas (n=3) were incu-
bated with 3x10"' M Ang I for periods of 5,10, 15, 30,
45, and 60 minutes. In a fourth set of experiments, aortic
vessels (n=3) were incubated with 10~8 M Ang II (n=3)
for the same incubation periods. Incubations were ter-
minated by sampling the luminal fluid.
Aliquots of 200 /A were added to 50 fil of 0.5 M HC1
and stored at -20°C. The concentrations of Ang I and
immunoreactive Ang II (irAng II) were determined by
radioimmunoassay (RIA) as described previously.23
Cross-reactivity of the Ang II antibody is 100% for
angiotensin III, Ang II-(3-8), and Ang II-(4-8) and
0.5% for Ang I. The cross-reactivity of the Ang I
the Vascular Wall
The time-dependent distribution of Ang I and Ang II
labeled Ang I or Ang II into the aortic lumen.
[Tyr3^,3H(AT)]-Ang I (0.54 mCi/ml; 33.67 Ci/mmol; cus-
tom synthesis) and [Tyr3-5,3H(A')]-Ang II (1 mCi/ml;
86.3 Ci/mmol) were obtained from New England Nu-
clear, Du Pont de Nemour, Boston, Mass. [3H]Ang I or
pHJAng II at a concentration of 10~8 M was instilled
into the aortic lumen and incubated for 5,10,15, 30, 45,
60, and 90 minutes. One aorta was used for each time
period of incubation. At the end of the incubation
period, radioactivity was measured in the luminal fluid;
the endothelial, medial, and adventitial layer; and in the
incubation bath. The vascular layers were separated
mechanically and transferred into a porcelain vessel for
catalytic combustion in the oxygen stream of a combus-
tion furnace (OX 300, Zinsser Analytics, Frankfurt,
FRG). This procedure resulted in the generation of
tritium, which was rinsed together with scintillator fluid
(Quickscint 212, Zinsser Analytics) into a vial (Zinsser
Analytics). The recovery was determined using a known
amount of radioactivity with and without catalytic com-
bustion. Radioactivity was measured in a ^-scintillation
counter (2000 CA Tricarb, Packard Instruments Co.,
Inc., Meriden, Conn.) and expressed as disintegrations
per minute (dpm).
Results
Angiotensin Incubation
Ang I decreased time-dependentry in the aortic lu-
men, whereas irAng II appeared after 5 minutes in the
lumen and increased with time throughout the incuba-
tion period (Table 1, Figure 2A). After 60 minutes of
incubation the intraluminal Ang I concentration had
decreased by 73%. At this time point, the irAng II
concentration in the lumen reached 14% of the initial
concentration of Ang I. Addition of the ACE inhibitor
ramiprilat (10~7 M) into the incubation medium re-
duced intraluminal irAng II concentration to virtually
zero (Figure 2B). The decrease of the intraluminal Ang
 Gohlke et al Vascular Angjotensin Metabolism 153

TABLE 1. Intralumlnal Incubation of Angiotcnsln I in

Rabbit Aorta
Incubation AngI Angll
period (min) (pg/50#d) (pg/50^)
0 239.2+40.7
5 219.3±26.7 7.7±1.6
10 204.2+56.0 12.4±3.2
15 152.3±31.4 18.4±2.6
30 111J+23.4 2O.5±3.6
45 98.2+24.6 28.8±9.2 3t «t
60 60.3+15.8 31J±5.6 TIME (MWUIE)
Intraluminal angiotensin I (Ang I) and angiotensin II (Ang II) FIGURE 3. Line plot shows percentage amount of intralu-
were measured by radioimmunoassay after different incubation minal angiotensin II after intraluminal angiotensin II incuba-
periods. Values are expressed as mean±SEM (n«=6). Time zero
represents the initial intraluminal administered concentration of tion (10~* M) in rabbit thoracic aorta with intact endothelium
AngI. (n=3).

similar to Ang I (71% decrease after 60 minutes of

I concentration was initially delayed, but after 60 min-
utes of incubation a reduction similar to that in un-
treated aortas was observed (69% with ramiprilat versus
73% without ramiprilat). Removal of the endothelium
prevented the irAng II generation without affecting Ang
I decrease in the aortic lumen (Figure 2C). Administra-
tion of Ang II into the aortic lumen revealed kinetics
100
incubation) (Figure 3).
The histological examination of the aortic luminal
surface demonstrated the integrity of the endothelium
before (Figure 4A) and after (Figure 4B) consecutive
angiotensin incubations. The endothelium remained
intact with some small scattered lesions after consecu-
tive angiotensin incubations. The completeness of the
removal of the endothelium was also verified histologi-
calry (Figure 4C). Only smooth muscle cells of the
vascular media could be detected in the denuded aortic
vessels.

50
Distribution of Angiotensin I and Angiotensin II in
the Vascular Wall
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The time-dependent distribution of radioactivity in

the luminal fluid, the vascular wall, and the incubation
oi-^ bath after instillation of [3H]Ang I into the aortic lumen
60 is presented in Figure 5. Radioactivity in the vessel
100 lumen, representing Ang I, Ang I fragments, or both,
decreased with time in a linear fashion. After 60 min-
utes' and 90 minutes' incubation, 37% and 46% of the
initial amount of radioactivity had diffused into the
vascular wall, mainly into the medial layer (25% after 60
minutes and 31% after 90 minutes). Only small amounts
of radioactivity were measured in the endothelial and
• * ••*••»
adventitial layers (Figure 5).
60
Radioactivity in the incubation bath, reflecting pen-
etration of Ang I or its fragments, or both, through all
100 vascular layers, remained low up to 30 minutes and then
increased to 8.2% and 8.6% after 60 minutes and 90
minutes, respectively.
50 The distribution of [3H]Ang II in the vascular wall is
shown in Figure 6. The results were comparable to those
for [3H]Ang I, except for higher levels of radioactivity in
the incubation bath after 60 and 90 minutes (14% and
16.5%, respectively).
30 60 The fate of Ang I in the aortic wall was summarized
TIME (MINUTE)
from both experiments. For each incubation time we
added the percentage amount of Ang I left in the aortic
FIGURE 2. Line graphs show percentage amount of intralu- lumen, the percentage amount of irAng II generated
minal angiotensin I (Ang I) and angiotensin II (Ang II) after within the lumen, and the percentage amount of radio-
intraluminal Ang I incubation (3xlO~9 M) in rabbit thoracic activity that had diffused out of the lumen. The differ-
aorta. Panel a: With intact endothelium (n=6). Panel b: ence between the sum of these three parameters and
After addition of the angiotensin converting enzyme inhibitor the initial amount of Ang I (100%) provides a measure
ramiprilat (10~7 M) (n=3). Panel c: After removal of the for Ang I degradation products that were not detected
endothelium (n=3). in the Ang I or Ang II RIA. The results demonstrate
 154 Hypertension Vol 20, No 2 August 1992

t f

i V.
FIGURE 4. Photomicrographs show his-
tological examination of rabbit aorta after
silver nitrate staining of cell borders of
endothelial and smooth muscle cells. Mag-
nification, xl50. Panel A: Before incuba-
i tions. Panel B: After consecutive incuba-
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tions. Panel C: After removal of the

endothelium.

let
75
* • ANG n
A • -* AJl«mliH»
25
UciWtita-
t
• 30 6t 9t
TOR (Mixirn)
FIGURE 5. Line plot shows distribution of radioactivity in
the rabbit aorta after intraluminal [3H]'angiotensin I incuba-
tion. One aorta was used for each time period DPM,
disintegrations per minute.
that Ang I degradation products increased up to 30
minutes of incubation and remained constant thereaf-
ter, indicating a steady state between degradation and
diffusion (Figure 7).
Discussion
The isolated thoracic rabbit aorta represents a valu-
able model for studies on the metabolism and distribu-
tion of angiotensin peptides in the vascular wall. Incu-
bation of intact aortic vessels with Ang I may be
particularly suited to mimicking the fate of circulating
Ang I in vivo, and after removal of the endothelium,
metabolic processes in deeper layers of the vascular wall
can be studied. The lack of a pulsatile perfusion inher-
ent to this model has to be acknowledged. However, this
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drawback is outweighed by the stability and reproduc-
ibility of the system and can be accepted if one limits the
investigation to general features of peptide handling by
the vascular wall.
Incubation of aortic vessels with Ang I for 60 minutes
resulted in a 14% conversion of Ang I to Ang II by
endothelial ACE. Since an unknown proportion of
newly generated Ang II might have been enzymaticalh/
degraded or could have penetrated into the vascular
wall, or both, the actual generation of Ang II might have
been even greater in view of the fact that the incubation
of aortic vessels with Ang II revealed a similar "escape"
of Ang II, its fragments, or both, into the vascular wall
as observed with Ang I.
1M
75
| nique. The authors demonstrated that 42% of the ACE
5«
25
-*- - IKIWIIH-
3#
TDfS (MNVTD
FIGURE 6. Line plot shows distribution of radioactivity in
the rabbit aorta after intraluminal [sHj'angiotensin II incuba-
tion. One aorta was used for each time period. DPM,
disintegrations per minute.
Gohlke et al Vascular Angiotensin Metabolism 155
100T
80 B DEGRADA-
TION
D DIFFUSION
• ANG I
5 10 15 30 45 60 SO
FIGURE 7. Bar graph shows fate of intraluminal angiotensin
I (Ang I). For each incubation time we added the percentage
amount of intraluminal Ang I left in the aortic lumen (see
Figure 1) (black columns), the percentage amount of angio-
tensin II (Ang II) generated within the lumen (see Figure 1)
(dotted columns), and the percentage amount of radioactivity
that had diffused out of the lumen (see Figure 6) (white
columns). The difference between the sum of these three
parameters and the initial amount of Ang I (100%) provides
a measure for intraluminal Ang I degradation products
(hatched columns).
Our results confirm previous results by Bunning et
al,24 who determined a conversion rate of Ang I to Ang
II of 10-20% after incubation with a high concentration
of 5xlO" 7 M Ang I using two in vitro systems, the
isolated thoracic rabbit aorta and cultured endothelial
cells from rabbit aorta. Similar conversion rates were
reported in dog kidney (4-19%),25"27 in dog mesenteric
arteries (25%),M and in the isolated perfused rat heart
)
Our data further demonstrate that the major portion
of luminal Ang I diffused into the vessel wall or was
degraded to peptide fragments. Incubation studies with
radioactively labeled Ang I revealed that Ang I, Ang I
fragments, or both, accumulated mainly in the medial
layer of the vascular wall. This raises the question of a
possible conversion of Ang I to Ang II within the medial
layer. Generation of Ang I in the vascular media is
possible since renin and angiotensinogen both were
shown to be present in this layer of the vascular wall and
were either synthesized locally or taken up from plas-
ma.1316 However, it is still controversial whether the last
step in the RAS cascade, the generation of Ang II,
occurs in the vascular media, since the precise location
of ACE within the vascular wall is by no means clear. In
a study by Velletri and Bean30 ACE activity was mea-
sured in the vascular wall using a radioenzymatic tech-
activity was localized in the medial layer while 58% was
found in the adventitial layer. A major drawback of this
study, however, is the lack of ACE activity in the intimal
layer. In another study by Pipili et al31 ACE activity was
measured in intact as well as in endothelium-denuded
aortic rings. Although this study suggested that ACE
was present in extraendothelial sites, the exact localiza-
tion of ACE, e.g., medial or adventitial layer, or both,
still remains obscure. Indirect evidence for an extraen-
dothelial conversion of Ang I to Ang II in the vascular
wall was provided by studies measuring the contraction
 156 Hypertension Vol 20, No 2 August 1992
of endothelium-denuded aortic rings after addition of
Ang I in rabbit aorta32 and in rat aorta.33-34 In these
studies aortic rings, suspended in an organ bath, were
used for the measurement of isometric contraction after
exogenous addition of Ang I. With this model, the
medial as well as the adventitial site of the vascular wall
is exposed to the peptide. Thus, it is possible that
conversion of Ang I to Ang II occurs primarily in the
adventitial layer (e.g., vasa vasorum). In contrast, in our
model of the isolated thoracic rabbit aorta, Ang I was
applied intraluminally and thus was exposed to endo-
thelial enzymes or, after removal of the endothelium, to
enzymes on vascular smooth muscle cells.
In the present study, removal of the aortic endothe-
lium completely abolished the conversion of Ang I to
Ang II. Thus, there was no evidence for an extraendo-
thelial generation of Ang II in the vascular wall. Our
results are supported by a number of studies investigat-
ing the localization of ACE in the vascular wall by
means of biochemical, immunocytochemical, and auto-
radiographic methods.18J5-36 Generally, these studies
failed to detect any ACE activity in the vascular medial
layer, thus pointing to the endothelium as the major site
of Ang II generation.
The decrease of luminal Ang I after 1 hour was not
different between intact or endothelially denuded aor-
tas, despite the fact that conversion of Ang I to Ang II
was abolished in the latter. This finding may be ex-
plained either by an increased diffusion rate into the
vascular wall due to the absence of the endothelium as
diffusion barrier or by an increased degradation of Ang
I by peptidases on vascular smooth muscle cells of the
Downloaded from http://ahajournals.org by on April 20, 2020
medial layer.
Inhibition of the endothelial ACE by addition of the
ACE inhibitor ramiprilat (10~7 M) to the incubation
medium resulted in a complete blockade of Ang II
generation. However, despite the lack of Ang I conver-
sion to Ang II, the ACE inhibitor did not affect the Ang
I decrease in the aortic lumen during an incubation of 1
hour. A compensatorily increased degradation of Ang I
by other endothelial peptidases may account for this
finding. Similar results were obtained by Luckhoff et
al,37 who investigated the effects of enalapril (10~7 M)
on the metabolism of Ang I (5 x 10~7 M) in the isolated
rabbit aorta. In contrast, Whalley38 observed a signifi-
cant attenuation of the Ang I breakdown by rabbit aorta
and human basilar artery in the presence of captopril
(5xlO~ 6 M), although in that study the Ang II produc-
tion remained unaffected by the ACE inhibitor. These
contradictory results may be explained by the very high
concentration of Ang I (10~4 M) used in their study.
In conclusion, our results emphasize the importance
of an intact endothelium for vascular Ang II generation
and provide evidence that conversion of Ang I to Ang II
is predominantly achieved by endothelial cells. They
also support the concept of an endocrine versus auto-
crine/paracrine RAS, where the vascular endothelium is
the strategic site for Ang II production for both systems
and thus the most important target site for the action of
ACE inhibitors.
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