Selective Al adenosine receptor antagonism augments

p-adrenergic-induced renin release in vivo

CYNTHIA A. PFEIFER, FUMIO SUZUKI, AND EDWIN K. JACKSON
Center for Clinical Pharmacology, University of Pittsburgh Medical Center,
Pittsburgh, Pennsylvania 15213-2582; and Kyowa Hakko Kogyo Company,
Shizuoka-ken 411, Japan

Pfeifer, Cynthia A., Fumio Suzuki, and Edwin K. whether an adenosine brake on renin release actually
Jackson. Selective Ai adenosine receptor antagonism aug- exists.
ments p-adrenergic-induced renin release in vivo. Am. J. To determine the in vivo existence of the adenosine
Physiol. 269 (Renal Fluid Electrolyte Physiol. 38): F469- brake on renin release, it is necessary to demonstrate
F479, 1995.-This study determines, in vivo, whether endog-
enous adenosine/Al receptor interactions at juxtaglomerular that in vivo inhibition of endogenous adenosine results
cells restrain the release of renin induced by receptor- in augmented renin release responses to appropriate
mediated activation of the adenosine 3’,5’-cyclic monophos- stimuli. In this regard, only a limited number of studies
phate pathway and whether endogenous adenosine/Az recep- have been performed (12, 22, 23, 27, 29, 31), and these
tor interactions diminish this restraining response. The studies employed high doses of nonselective, relatively
following four pharmacological probes were employed: 1) low-potency adenosine receptor antagonists [caffeine,
1,3-dipropyl&cyclopentylxanthine (DPCPX) and 2) FK-453, theophylline, and 1,3-dipropyl-8-p-sulfophenylxanthine
both selective A1 receptor antagonists; 3) FR-113452, a nearly (DPSPX)]. Therefore, an important caveat of all previ-
inactive enantiomer of FK-453; and 4) KF- 17837, a selective ous studies is that the pharmacological probes employed
AZ receptor blocker. Adult Sprague-Dawley rats were prepared
(adrenalectomized, renal denervated, uninephrectomized, and may have produced nonspecific effects; thus the exis-
treated with indomethacin, aldosterone, and hydrocortisone) tence of the adenosine brake on renin release is still in
to minimize endogenous stimulation of renin release and question.
received either vehicle (control group) or one of the four drugs. An additional serious caveat is that all previous
Intrarenal infusions of isoproterenol (3, 30, and 100 studies were performed with complex stimuli to renin
ngekg-lo min-l) caused dose-related increases in plasma renin release (sodium depletion, furosemide, hydralazine, and
activity (PRA). This PRA response was significantly aug- renal artery hypotension), which may have activated
mented in the groups receiving DPCPX (P = 0.0010) or multiple mechanistic pathways. If the stated rationale
FK-453 (P = 0.0001) but was not altered in the groups treated for the adenosine brake on renin release is correct, then
with FR-113452 (P = 0.3422) or KF-17837 (P = 0.2155). endogenous adenosine would restrain only renin release
Systemic and renal hemodynamics and renal electrolyte excre-
tions were monitored and could not account for the PRA responses that are mediated via the CAMP pathway.
augmentation caused by the Al antagonists. This study clearly Therefore, previous studies may have underestimated
demonstrates that endogenous adenosine acts on the A1 the quantitative importance of the adenosine brake by
receptor to restrain the renin release induced by activation of activating pathways of renin release not restrained by
intrarenal P-adrenoceptors and is not counteracted by endog- endogenous adenosine.
enous activation of the AZ receptor. Another issue not resolved by previous studies is
endogenous adenosine; adenosine receptor antagonists whether the A2 receptor diminishes the biological impor-
tance of the adenosine brake on renin release. It is well
known that adenosine can also stimulate adenylate
cyclase via a lower affinity AZ receptor (6). If endogenous
THE ADENOSINE BRAKE HYPOTHESIS asserts that endog- adenosine stimulates juxtaglomerular adenylate cyclase
enous adenosine restrains (brakes) the renin release via A2 receptors, then the biological impact of the
response to many pharmacological and physiological adenosine brake would be diminished because of the
stimuli (for review, see Ref. 17). The rationale for this counteracting influences of the A1 and A2 receptors.
hypothesis is that the renin release response to many Since all prior studies employed nonselective adenosine
stimuli may involve activation of adenylate cyclase in receptor antagonists, the relative importance of A1
the juxtaglomerular cell (JGC) (17), and adenosine can versus A2 receptors in the adenosine brake mechanism
inhibit adenylate cyclase via a high-affinity A1 adenosine remains unresolved.
receptor (32). Thus endogenous adenosine may be stra- In the past several years, remarkable advances have
tegically positioned to act as a molecular brake on renin been made in the medicinal chemistry of adenosine
release responses mediated via the adenosine 3’,5’-cyclic receptor antagonists, and this progress has made avail-
monophosphate (CAMP) pathway. able highly selective and potent adenosine receptor
Although the adenosine brake hypothesis has a logical blockers. Employing these state-of-the-art antagonists,
basis, it has received only limited experimental support. we have developed in vivo paradigms for determining,
Numerous studies have established that exogenous aden- with a high degree of certainty, the involvement of
osine and selective A1 receptor agonists inhibit renin endogenous adenosine/A1 receptor interactions (20, 21)
release responses both in vitro (10) and in vivo (2,9,11); and endogenous adenosine/A2 receptor interactions (18)
however, such studies indicate only that an adenosine in physiological processes. In addition, we have devel-
brake on renin release may exist but do not address oped recently a highly specific in vivo method for
036%6127/95 $3.00 Copyright o 1995 the American Physiological Society F469

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F470 CONFIRMATION OF THE ADENOSINE BRAKE HYPOTHESIS

receptor activation of the CAMP-mediated pathway of

renin release.
The overall aim of the present study was to employ
the aforementioned advances to more specifically ad-
dress the deficiencies of previous studies that examined
the adenosine brake hypothesis. In this regard, we had
two specific goals. First, we wished to determine defini-
tively and in vivo whether endogenous adenosine/A1
receptor interactions at JGCs restrain the release of
TERIAL
renin induced by activation of the CAMP pathway. A
positive result (renin response is augmented by Al
blockade) would clearly establish that an adenosine
brake on renin release exists, would suggest its quantita-
tive importance, and would establish that the adenosine
brake is mediated at least in part by Al receptors. [It is
now clear that adenosine can also inhibit adenylate
cyclase via A3 receptors (8); however, the present study
was not designed to address the role of A3 receptors in
the adenosine brake.] Our second specific goal was to
determine in vivo whether endogenous adenosine/A2
receptor interactions at JGCs diminish the biological
impact of the adenosine brake on renin release. A
negative result (renin response is unchanged by A2
blockade) would indicate that the biological impact of FL
the adenosine brake on renin release is not counteracted
by the adenylate cyclase-stimulating effects of A2 recep-
tors.

METHODS
Fig. 1. Surgical protocol. Adrenalectomy and uninephrectomy are
Experimental protocol. Male Sprague-Dawley rats, 427 t represented by the broken lines.
48 g (*SD, n = 35 experimental animals and 35 animals used
for blood donation) were obtained from Charles River (Wilm- average MABP and HR at 1-min or 5-min intervals (printer)
ington, MA). Animals were housed in the University of and 5-s intervals (digital display).
Pittsburgh animal care facility, and institutional guidelines for Next, the animal underwent bilateral adrenalectomy, right
animal welfare were followed at all times. Rats were kept in a uninephrectomy, and renal denervation (10% phenol). Supple-
12:12-h light-dark cycle (7 A.M. to 7 P.M.) at an ambient mentation of aldosterone (20 ng/min) and hydrocortisone (20
temperature of 22°C and relative humidity of 55% and were pg/min) was also provided to maintain hemodynamics for the
fed Wayne Rodent Blox 8604 (135 meq/kg sodium and 254 rest of the surgery and experiment. The left ureter was then
meq/kg potassium; Madison, WI) and water ad libitum. cannulated (PE-10) to continuously collect urine, and a transit-
Rats were anesthetized with pentobarbital sodium (45 time blood flow probe (model 1RB; Transonic Systems, Ithaca,
mg/kg ip), and body temperature was maintained at 37 t NY) was placed around the renal artery to continuously
0.5”C. Figure 1 illustrates the surgical protocol. After cannula- monitor renal blood flow (RBF). Intrarenal infusion of saline
tion of the trachea (PE-240), three polyethylene catheters or increasing step doses of isoproterenol (saline vehicle) at 20
(PE-50) were inserted into the left jugular vein. One catheter pl/min were obtained by carefully inserting a 32-gauge needle,
was used for both whole blood replacement (from a blood connected to a PE-10 catheter, into the renal artery proximal
donor rat) following arterial blood withdrawal and for supple- to the flow probe. (This needle was found to take up 5% or less
mental infusions of pentobarbital. The second was used for of the renal arterial lumen cross-sectional area.) Patency of
0.9% saline infusions (80 Fl/min). The third line (wrapped in this line was periodically checked, and animals that exhibited
foil to protect light-sensitive drugs) was used for infusions of any renal arterial bleeding at the needle insertion site were
adenosine receptor antagonists (DPCPX, FK-453, FR-113452, eliminated from the study. (This occurred infrequently but
or KF-17837)l dissolved in dimethyl sulfoxide (DMSO). A left our experience has been that any bleeding or trauma to the
carotid artery catheter (PE-50) was placed and connected to a renal artery induces greatly increased renin levels.) Each rat
digital blood pressure analyzer (Micro-Med, Louisville, KY) for then received a slow intravenous bolus of indomethacin (5
continuous measurement of mean arterial blood pressure mg/kg in 0.1 mol/l Na&O&
During the next l-h stabilization period, an intravenous
(MABP), heart rate (HR), and systolic/diastolic pressure. The
Micro-Med digital blood pressure analyzer was set to time infusion of [carbox$14C]inulin (0.75~FCi bolus followed by
0.05 pCi/min infusion) containing aldosterone (20 ng/min)
and hydrocortisone (20 kg/min) in saline at 80 pl/min was
commenced. Animals were randomly assigned to one of the
1 The chemical name for DPCPX is 1,3-dipropyl-%cyclopentylxan-
thine. The chemical name for FK-453 is (+)-(IX)-1-[(E)-3-(Z-phenylpyra- following five groups (n = 7lgroup): 1) DMSO only (control
zolol[ 1,5-alpyridin-3-yl)acryloyl]-2-piperidine ethanol. FR-113452 is group); 2) DPCPX (xanthine-based, selective A1 antagonist);
the S-enantiomer of FK-453. The chemical name for KF-17837 is 3) FK-453 (non-xanthine-based, selective Al antagonist);
(E)-1,3-dipropyl-7-methyl-8-(3,4-dimethoxyst~rl)xanthine. 4) FR-113452 (inactive enantiomer of FK-453); or 5) KF-

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 CONFIRMATION OF THE ADENOSINE BRAKE HYPOTHESIS F471

* PRA

/ Fig. 2. Experimental protocol time line.

- 60, min 60 Rats were randomized to 1-5 intra-
venous treatment infusions (control,
DPCPX, FK-453, FR-113452, and KF-
17837), which were initiated immedi-
0
ately after surgery. Continuous infu-
lntrarenal lsoproterenol Infusions (ng/kg/min) at 20 pl/min sions or collections are designated by
boxed areas. Experimental protocol was
divided into four 30-min intrarenal iso-
lnulin [carboxyl’4C] (0.75 PCi i.v. bolus + 0.05 pCi/80 pl/min, i.v.) proterenol infusion periods. Boxes UI-
U4, urine collections during the last 20
Aldosterone (20 ng/min) + hydrocortisone (20 pg/min) in saline min of each of the 4 isoproterenol infu-
sion periods. Small and large asterisks
CONTROL 1 DMSO vehicle, 0.65 pl/min, i.v. I represent midpoint and plasma renin
activity (PRA) arterial blood sampling,
DPCPX (A 1) 1 3 pg/kg/min, in DMSO at 0.65 pl/min, i.v. respectively.

FK453 (Al) 1 10 pg/kg/min, in DMSO at 0.65 pl/min, i.v. I

FR113452 ] 10 pg/kg/min, in DMSO at 0.65 pl/min, i.v. I

KFl7837 (A2) 1 30 dkdmin, in DMSO at 4.0 ut/min. ix

17837 (selective AZ antagonist). All drugs were dissolved in artery were cannulated. Rats then underwent identical bilat-
DMSO. These were given intravenously as follows: DMSO eral adrenalectomy, uninephrectomy, renal denervation with
vehicle (0.65 kl/min), FR-11342 or FK-453 (10 pg*kg-lamin phenol (10% in ethanol), and administration of indomethacin
at 0.65 kl/min), DPCPX (3 kg* kg-l min-l at 0.65 kllmin), or l (5 mg/kg iv bolus). After a l-h stabilization period, arterial
KF-17837 (30 pg*kg-l*min- l at 4.0 pl/min> and maintained blood was collected into a heparinized syringe at room tempera-
for the duration of the experiment. All of these adenosine ture and used as needed for intravenous whole blood replace-
antagonist infusions were administered for at least 30 min ment in the experimental animal.
before the experimental protocol was initiated, and FR- Plasma renin activity analysis and validation. Arterial
113452, FK-453, and KF-17837 were prepared and infused in blood samples collected for PRA measurement were gently
darkness to prevent light-sensitive degradation. The doses, mixed with disodium EDTA (final disodium EDTA concentra-
route of administration, and duration of infusion of DPCPX, tion was 1 mg/ml) and centrifuged at 3,000 g (Eppendorf
FK-453, FR-113452, and KF-17837 were determined previ- centrifuge 5415 C; Brinkmann Instruments, Westbury, NY)
ously by our laboratory. The doses were selected such that for 15 min at room temperature. Plasma was immediately
FK-453 and DPCPX would provide a high and comparable stored at -40°C until analyses. Five different conditions of
degree of Ai receptor blockade (20, 21). The same dose of collecting, processing, and storing blood samples were investi-
FR-113452 does not antagonize Ai receptors (20, 21) but gated to determine whether cryoactivation or alterations in
would share any nonspecific effects with FK-453, thereby renin activity occurred. We found that combinations of prompt
acting as a second control for FK-453. The dose of 30 or delayed addition of disodium EDTA, freezing, and/or
pg. kg-l min- l for KF-17837
l was the maximal dose that centrifugation did not significantly alter PRA values. Also,
specifically antagonizes the A2 receptor without crossover cryoactivation was not evident in blood samples stored 6 h on
antagonism at the Ai receptor (18). ice or in plasma stored 4 wk at 4°C. Rat PRA appeared to be
The experimental protocol (Fig. 2) was composed of four very stable under a variety of collection and storage conditions;
30-min periods. During period 1, only saline was infused into however, to remain consistent, we always collected plasma in
the renal artery, whereas isoproterenol was infused during the above fashion.
periods 2-4 at 3, 30, and 100 ng* kg-l emin, respectively. PRA was estimated by using the Rainen Angiotensin I 1251
During each period, the last 20 min of urine was collected, and Radioimmunoassay Kit (DuPont-NEN Research Products,
a 0.5-ml midpoint arterial plasma sample was obtained for Boston, MA) with the following modifications. First, in sample
calculation of hematocrit and glomerular filtration rate (GFR). processing before radioimmunoassay, plasma sample pH was
At the end of each period, a second 0.5-ml arterial sample was brought to and maintained at 5.5-6.0 with a small volume (5%
withdrawn for plasma renin activity (PRA) determination, and of plasma volume) of concentrated maleic acid (276 mmol/l)
blood was replaced 1: 1 with prepared rat donor whole blood. At instead of the large volume (200% of plasma volume) of
the end of the experiment, animals were terminated with an maleate buffer (200 mmol/l) provided with the kit. This
overdose of pentobarbital. prevented possible dilutional effects on renin kinetics de-
Donor whoZe Hood replacement. To maintain stable hemody- scribed by Osmond et al. (26) in the subsequent l-h heat
namics despite multiple blood sampling during the experi- incubation step [angiotensin I (ANG I) generation]. Second,
ment, the experimental rat blood was replaced 1:l with whole after heat incubation, all samples were diluted 40-fold with
blood obtained from a second blood donor rat prepared simi- assay buffer, and both samples and assay standards were
larly to minimize renin production. Briefly, the trachea, right brought to a composition of 10% plasma matrix by the addition
jugular (with a saline infusion of 80 Fl/min) and left carotid of prepared renin-free rat plasma. This was done to correct for

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F472 CONFIRMATION OF THE ADENOSINE BRAKE HYPOTHESIS

a prominent plasma matrix interference effect that was seen RESULTS

for rat plasma samples (interference was proportional to the
percentage of plasma in a diluted sample). Because all stan- PRA, sodium excretion rate (U,$>, RBF, and MABP
dards and samples were at a fixed plasma matrix percentage of are illustrated for the DPCPX (Fig. 3), FR-113452 and
lo%, this essentially eliminated the interference effect, and FK-453 (Fig. 4), and KF-17837 (Fig. 5) treatment
accuracy was greatly improved. After matrix correction and groups vs. the control group. The U&, RBF, and
40-fold dilution, we prepared plasma samples according to the MABP parameters were chosen to highlight the involve-
Rainen kit protocol without further modification. ment (or lack thereof) of any macula densa- or barorecep-
Once matrix correction was established, further extensive
validations were performed on identical plasma samples (with tor-induced changes in PRA caused by the adenosine
high or low PRA values) to confirm repeatability of PRA receptor antagonists or the experimental protocol. Six
measurements after undergoing different l-h heat incuba- additional renal parameters that can also influence
tions, repeated freeze/thawing, and prolonged frozen storage PRA, i.e., GFR, urinary flow (UV), filtration fraction
(up to 4 mo at - 40°C). Intra-assay precision (coefficient of (FF), potassium excretion (U&), and fractional excre-
variation, CV) was below 10% for all conditions tested. Interas- tions of sodium (FEN,) and potassium (FE& are listed
say CV was less than 15% (calculated from the means of for each treatment group over baseline and all three
identical samples processed by 3 different batches of kits). isoproterenol dose periods (Table 1).
Sample PRA values were also found to be stable after at least 4
mo of freezing. These validation studies demonstrated that, Selective augmentation of renin responsesby AI recep-
despite different conditions (freezing, heat incubation, and tor antagonists. Both the DPCPX (A1 antagonist) and
different kits), PRA values were highly reproducible. Alto- control groups demonstrated a consistent dose-depen-
gether, we have demonstrated that this modified assay and dent increase in PRA with increasing isoproterenol
sample preparation and collection were rigorous and stable, doses (Fig. 3). DPCPX treatment (3 kg* kg-l min-l) l

making comparisons between different rat plasma samples caused no PRA augmentation at baseline but caused a
under different conditions consistent and resilient to change. nearly twofold increase in PRA at the 3, 30, and 100
These qualities were essential to verify because of the pivotal
ngv kg-l min-l isoproterenol doses. This augmentation
role of PRA in this study.
l

Renin-free rat plasma used to correct for matrix interfer- of PRA by the Al antagonist, DPCPX, was found to be
ence was prepared by bilaterally nephrectomizing (under highly significant as indicated by both the significant
aseptic conditions) an anesthetized rat (pentobarbital, 45 group (P = 0.0333) and interaction terms (P = 0.0010).
mg/kg ip). After 24 h of recovery, the animal was reanesthe- Interestingly, PRA was found to be statistically elevated
tized, the carotid artery was cannulated, and blood was from control at the low isoproterenol dose of 3 ng. kg-l l

collected into tubes containing disodium EDTA. The blood was min-l, an isoproterenol dose which did not stimulate
centrifuged, and plasma was stored at -40 to -70°C. PRA control group PRA levels.
levels of this plasma (used to fix plasma matrix at 10% for Treatment with the Al antagonist, FK-453 (10
matrix correction) were undetectable by the Rainen radioim-
munoassay. Pde min-l),
l caused a statistically significant aug-
PRA was recorded as nanograms of ANG I generated at mentation of PRA at baseline and at the 30 and 100 ng*
37°C per milliliter of plasma per hour (ng ANG I-ml-l lh-l) kg-l min-l doses of isoproterenol (Fig. 4). The inactive
l

and was calculated by the RiaSmart Software package (Pack- enantiomer, FR-113452 (10 pg. kg-l min-l), however,
l

ard Instrument, Meridan, CT), using a weighted logit curve- caused absolutely no augmentation of PRA release over
fitting program. The Rainen Angiotensin I Radioimmunoassay any isoproterenol dose and was nearly indistinguishable
Kit has a standard curve from 0.1 to 5.0 rig/ml and was found from the control PRA response.
to be accurate and linear for diluted samples up to at least 12.0
rig/ml. KF-17837 (30 kg. kg-l min-l) did not significantly
l

Statistics. All data in Figs. 1-5 and Table 1 are presented as reduce the PRA response in basal or isoproterenol-
means 2 SE, and statistical analyses were calculated using the stimulated conditions (Fig. 5).
Number Cruncher Statistical System (version 5.1; NCSS, Hemodynamic and renal responses to Al and A2
Kaysville, UT). Statistical analyses compared parameters of receptor antagonism. No statistically significant differ-
each adenosine antagonist treatment group with the corre- ences were found between the DPCPX group and
sponding control group across the range of different doses of control group values for UN& RBF, or MABP, thus
isoproterenol. A general linear model two-factor analysis of excluding these parameters from influencing PM aug-
variance (ANOVA) for repeated measures was used at a
significance level of P < 0.05. The P values for comparisons mentation (Fig. 3). GFR, FF, and U& were also un-
between treatment groups (factor l), doses (factor Z), and the changed compared with the control group; only a tran-
interaction between treatment group and dose (G x D) are sient increased diuresis (UV) was noted in the DPCPX
listed for all graphs. If a significant interaction between group during the first experimental period, which re-
treatment and dose (G x D) or a significance among treat- turned to control levels for the next three isoproterenol
ments or doses was detected, comparative tests (Fisher’s doses (Table 1). FEN, and FEx were statistically elevated
least significant difference) were initiated to identify the signifi- for most of the experiment in the DPCPX group compared
cant differences. with the control group (Table 1).
DPCPX was purchased from Research Biochemicals (Natick,
MABP and RBF were also unchanged compared with
MA). FK-453 and FR-113452 were kindly provided by the
Fujisawa Pharmaceutical (Osaka, Japan), and KF-17837 was the control group for both FK-453 and FR-113452;
kindly provided by Kyowa Hakko Kogyo (Shizuoka-ken, Ja- however, FK-453 induced a transient and statistically
pan). DMSO used as drug vehicle and isoproterenol were significant elevation of U& (Fig. 4). Five of the six
purchased from Sigma Chemical (St. Louis, MO). renal parameters for the FR-113452 group were similar

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 CONFIRMATION OF THE ADENOSINE BRAKE HYPOTHESIS F473

80.0 2-Factor ANQVA (Ml)

~~~
Group p - 0.0333
Dose p = 0.0001
60.0 GxD p - 0.0010
*+

40.0

20.0

00.

25.000 1
+ Group
Dose
p - NSD
p - 0.0199
Fig. 3. Four different
(Ai antagonist,
groups are illustrated.
parameters
A) and control
Parameters
for DPCPX
(0) treatment
(from top Jo

15.000

5.000'
1:-+=+ + GxD p = NSD bottom) are PRA, sodium excretion
renal blood flow (RBF),
blood pressure
the intrarenal
points represent
Two-factor
(MABP)
infusions

ANOVA
sures for each parameter
rate (&V),
and mean arterial
and are plotted against
of isoproterenol.
means 2 SE; n = 7/group.
tables for repeated
are listed, G x D is
Data

mea-

the interaction term between group and dose.

NSD, no significant difference (P > 0.05).
*P < 0.05, statistically significant for DPCPX
group vs. corresponding control group. +P <
25.0 Group p = NSD
0.05, statistically significant change from basal
Dose p = 0.0009 measurement (0 ng. kg-l. min-1 isoproter-
20.0 GxD p = NSD enol) within the same group.

15.0
10.0

160 Group p - NSD

Dose p = NSD
GxD p = NSD

80 J , I I I
0 3 30 100

Dose of lsoproterenol (ng/kg/min)

to the control group, and only GFR was slightly but
significantly decreased during some periods (data not
shown). Altered renal parameters for FK-453 included a
significant but transient elevation in UV and FEN,
compared with the control group.
KF-17837 did not alter RBF but did cause a sustained
elevation of U& and MABP (Fig. 5). Other altered
renal parameters are shown in Table 1 and include a
sustained elevation of UV, FEN,, and FEx compared
with the control group.
HR and hematocrit (Hct) remained statistically the
same among all treatment groups across all four isopro-
terenol periods (data not shown). Average values for all
treatment groups combined (means t SE, n = 35), for
the first (0 ng. kg-l 0min-l isoproterenol) or last period
(100 ngekg-l*rnin-l isoproterenol), respectively, were
363 t 6 and 382 t 7 beats/min for HR and 47.2 t 0.4
and 45.7 t 0.5% for Hct. PRA levels of blood donor rats
used for the control, DPCPX, FR-113452, FK-453, and
KF-17837 treatment groups were 18.8 t 4.8,36.6 t 7.6,
14.9 t 3.7, 17.3 t 6.7, and 20.3 t 7.4 ng ANG
Iml-l-h-l (*SE, n = 7/group), respectively, and were
not statistically different compared with the control
blood donor group. Poststabilization measurements were
also made for PRA, MABP, and RBF parameters (data
not shown) just after the conclusion of the l-h surgical
recovery period (but prior to initiating the first experi-
mental period). These baseline parameters for all treat-
ment groups were found to be similar to the control
parameters except for a statistically elevated MABP for
the KF-17837 group. Because these three parameters
remained stable and were the same as the values
obtained for the first experimental period (baseline),
this demonstrated that, by the end of the l-h recovery

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F474 CONFIRMATION OF THE ADENOSINE BRAKE HYPOTHESIS

2-Factor ANOVA (FM)

Group p - 0.0115
Dose p = 0.0001
60.0 - GxD p - 0.0001
[ l and 0 I

Group p = NSD
Dose p = 0.0001
GxD p = NSD
[ n and 0 1

Group p - 0.0229
Fig. 4. Four different parameters for FK-453 F 25.000 1* * Dose p = 0.0002
(Ai antagonist, l ), FR-113452 (less active --
E GxD p = 0.0014
enantiomer of FK-453, n ), and control (0) z
treatment groups. Parameters (from top to E 1 l and 0 I
15.000
bottom) are PRA, UNJ, RBF, and MABP and s Group p -NSD
are plotted against intrarenal infusions of l> 11 Dose p= NSD
isoproterenol. Data points are means + SE; a 5.000 J GxD p = NSD
n = 7/group. Two-factor ANOVA tables for Z?
repeated measures for each parameter are 1 a and 0 I
listed. G x D, interaction term between group
and dose. ANOVA tables of comparisons be-
tween FK-453 or FR-113452 against control
are designated by solid circle or solid square
symbols, respectively. * P < 0.05, statistically Group p = NSD
significant for either the FK-453 or FR- z Dose p = 0.0001
113452 group vs. corresponding control group. ‘g GxD p = NSD
+P < 0.05, statistically significant change $j [ l and 0 I
from basal measurement (0 ngskg-l *rnin-l 2
isoproterenol) within the same group. Group p = NSD
Dose p = 0.0009
ii GxD p = NSD
u
[ m and 0 I

Group p = NSD
160 Dose p = NSD
GxD p - NSD
[ l and 0 1

Group P = NSD
Dose p = NSD
80 J I I 1 1 GxD p = NSD
[ m and o 1
0 3 30 100

Dose of lsoproterenol (ng/kg/min)

period, MABP, RBF, and PRA responses were fully (16), it is likely th at en dogenous adenosine/Al receptor
stabilized and reproducible in the absence of p-adrener- interactions restrain the renin release response to all
gic stimulation. receptor-mediated physiological or pharmacological
stimuli that utilize the adenylate cyclase pathway to
DISCUSSION
activate renin secretion. However, the lack of PRA
These results provide the first strong evidence that reduction after A2 blockade suggests that endogenous
endogenous adenosine interacts with Al receptors to adenosine/Ag receptor interactions do not significantly
attenuate (brake) P-adrenoceptor-mediated renin re- affect renin release. Thus this study also provides strong
lease in vivo. Thus this study clearly establishes that the evidence that interactions of endogenous adenosine
adenosine brake on renin release exists and that it is with A2 receptors do not diminish the biological impact
mediated at least in part by the AI receptor. Because of the adenosine brake on renin release.
P-adrenoceptors stimulate renin release in vitro (30) The highly selective AI receptor antagonist, DPCPX
and in vivo (11, 25) via activation of adenylate cyclase (inhibition constant, Ki = 0.46 and 340 nmol/l for the AI

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 CONFIRMATION OF THE ADENOSINE BRAKE HYPOTHESIS F475

80.0 2-Factor ANOVA (RIM)

Group p - NSD
E- Dose p = 0.0001
3 60.0 GxD p - NSD
E
E
z 40.0
P
5 20.0
CL

Group P = 0.0014
Fig. 5. Four different parameters for KF-
Dose p - 0.0001
17837 (AZ antagonist, +) and control (0)
GxD P = 0.0252 treatment groups. Parameters (from top to
bottom) are PM, UNJ, RBF, and h4ABP and
are plotted against intrarenal infusions of
isoproterenol. Data points are means + SE;
= 7/group (n = 5 for RBF parameter for
h- 17837 group). Two-factor ANOVA tables
for repeated measures for each parameter are
listed. *P < 0.05, statistically significant for
KF- 17837 group vs. corresponding control
group. +P < 0.05, statistically significant
25.0 1 Group P = NSD change from basal measurement (0 ngekg-l.
Dose P = NSD min-l isoproterenol) within the same group.
20.0 GxD P = NSD

15.0 ip
10.0 1

Group p= 0.0001
Dose p = NSD
GxD p - NSD

80 J , I I 1

0 3 30 100

Dose of lsoproterenol (ng/kg/min)

and A2 receptors, respectively; see Ref. 5), augments
PRA release over control levels for all three doses of
isoproterenol (Fig. 3) but not for basal levels of PRA (0
ng* kg-l min- l dose of isoproterenol). Augmentation
l tubule, as has been recently reported (21).
occurred in a nonlinear fashion, as shown by the greater
PRA differential between DPCPX-treated rats com-
pared with the control rats at increasing doses of
isoproterenol. The nonparallel nature of PRA augmenta-
tion was also reflected in the statistically significant
interaction term (P = 0.0010). This augmentation of
PRA release was clearly not influenced by changes in
hemodynamics, because RBF, MABP, HR, and Hct were
not statistically different compared with control rats.
DPCPX also did not cause a significant increase in U&?,
although it did cause an early transient increase in FEN,
and UV, which returned to control values during later
periods (Table 1). Because hemodynamics, GFR, and FF
were unaltered, the increased FEN, and UV was prob-
ably due to a transient Al-antagonist-induced inhibition
of sodium and water reabsorption by the proximal
To further verify that endogenous adenosine/Al inter-
actions inhibit renin release responses to p-adrenergic
stimulation, the chemically dissimilar (nonxanthine)
but also highly selective Al receptor antagonist, FK-453,
was used and compared with both its inactive enanti-
omer (FR-113452) and to the control group. A dose of 10
pg*kg-l*min-l for both FK-453 and FR-113452 was
chosen because of the equivalent Al blocking ability of
this dose of FK-453 compared with 3 pg. kg-l emin-l
DPCPX (20, 21). FR-113452 is a much less active Al
receptor antagonist (573.fold less potent in receptor
binding studies) compared with FK-453 and at 10
kg* kg-l emin-l shows no Al (or AZ) blocking action.

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F476 CONFIRMATION OF THE ADENOSINE BRAKE HYPOTHESIS

Infusion Rate, ng. kg-l - min- l

Treatment Group 0 (saline) 3 100

GFR, ml/min
Control 2.98 + 0.18 2.80 f 0.14 2.92 2 0.13 2.72 k 0.10
DPCPX 2.612 0.09 2.58 2 0.13 2.63 2 0.13 2.64 + 0.08
FK-453 2.63 + 0.13 2.63 + 0.17 2.52 + 0.17 2.37 2 0.18
KF-17837 2.56 iz 0.13 2.70 + 0.16 2.69 + 0.20 2.57 AI 0.14
UV, pl/min
Control 52.0 + 4.8 60.9 + 5.0 63.7 + 6.1 52.6 + 6.5
DPCPX 80.3 2 9.9* 65.3 2 6.6 80.4 AI 6.7 58.5 + 7.6
FK-453 93.3 t 10.3* 95.7 t 10.1* 77.4 t 10.8 56.6 + 12.0
KF-17837 116 + 16* 1312 10” 123 + 7* 104* 1O”“r
FF, %
Control 4328 3927 4127 4327
DPCPX 29+5 30*6 3327 33+55
FK-453 2822 2923 30+3 2924
KF-17837 3325 3526 3526 3325
U& kmol/min
Control 3.17 + 0.25 2.79 2 0.22t 2.78 + 0.28t 2.31+ 0.21-f
DPCPX 3.82 t 0.38 3.22 t 0.36-f= 3.23 + 0.32t 2.46 IL 0.27t
FK-453 3.42 2 0.14 3.22 + 0.14 2.53 + 0.19.f 2.12 2 0.33-f
KF-17837 4.08 k 0.26* 3.95 ,+ 0.25* 3.37 ,+ 0.117 2.74 k 0.30-t-
FEK, %
Control 27.84 + 2.75 26.80 2 1.70 25.75 2 2.76 23.65 k 2.30
DPCPX 37.77 2 2.69* 34.07 + 2.96* 34.52 + 2.76” 27.18 + 2.88.f
FK-453 32.86 + 3.36 32.75 2 2.36 26.22 2 2.06t 23.32 + 2.lO”r
KF-17837 39.59 2 3.19* 39.29 2 3.55* 35.0 t 2.8*? 29.4 2 l.S”-t
FENa, %
Control 3.09 f 0.37 3.28 + 0.35 3.59 * 0.37 3.26 k 0.42
DPCPX 4.75 in 0.46* 4.05 * 0.33 4.78 k 0.31* 3.44 2 0.40
FK-453 5.62 2 0.42* 5.81 it 0.65* 4.78 * 0.53 3.84 + 0.44.f
KF-17837 5.94 2 0.70* 6.19 + 0.51* 5.51+ 0.46* 4.58 + 0.37*

Values are means 2 SE; n = 7lgroup (except n = 5 for the FF parameter for KF-17837). Renal parameters are listed for control and adenosine
antagonist treatment groups for all 4 isoproterenol infusions. (Parameter data for FR- 113452 treatment group are statistically indistinguishable
from control group and thus are not presented.) *P < 0.05, statistically significant vs. corresponding control treatment value. tP < 0.05,
statistically significant vs. corresponding basal measurement (0 ng. kg- l. min-l isoproterenol) within a treatment group. GFR, glomerular
filtration rate; UV, urine flow rate; FF, filtration fraction; U& potassium excretion rate; FE K, fractional excretion of potassium, and FENa,
fractional excretion of sodium; DPCPX and FK-453, Al adenosine receptor antagonists. FR-113452, S-enantiomer of FK-453; and KF-17837, A2
adenosine receptor antagonist.

Although sharing no Al-blocking effects at this dose, documented in rats (21). Human normotensive patients
being enantiomers, these two agents would likely show (3), as well as essential hypertensive patients (34),
similar “nonspecific” effects on renin release. In this receiving FK-453 also showed early transient natriure-
regard, FR-113452 is an ideal second negative control sis and diuresis without associated changes in renal
for FK-453. FK-453 augmented baseline PRA as well as hemodynamics. This phenomenon, which occurs for two
the PRA response to the 30 and 100 ng. kg-l emin-l structurally distinct A1 blockers, DPCPX and FK-453,
doses of isoproterenol. As with DPCPX, the augmenta- suggests a common tubular action mediated by Al
tion by FK-453 was nonlinear. In contrast to FK-453, blockade. FR-113452 did not increase sodium excretion
FR-113452 did not augment PRA at any dose of isopro- or cause diuresis, confirming that these events are A1
terenol. The augmentation of PRA in the present study receptor mediated.
by FK-453 concurs well with recent studies in humans, Endogenous adenosine can also act on the A2 receptor,
which found that oral FK-453 caused a sustained two- but the affinity of the A2 receptor for adenosine is 10 (Aza
fold increase in PRA in hypertensive patients (34) and a receptor) to 1,000 (Agb receptor) times lower than the
threefold increase in plasma renin concentrations in affinity of the Al receptor for adenosine (6). Stimulation
normotensive patients (3). of the A2 receptor by micromolar concentrations of
Hemodynamic (RBF and MABP) and renal param- exogenous adenosine and A2 agonists has been demon-
eters (GFR, FF, FEx, and UxV) for both FK-453 and strated in vitro (10) and in vivo (9); however, it is
FR-113452 were also similar to corresponding param- questionable as to whether endogenous levels of adeno-
eters in the control group. An early significant increase sine in the kidney are sufficiently high to activate
in UV, U&, and FEN, for the FK-453 group was found stimulatory A2 receptors. Nonetheless, if A2 receptors
for the first two isoproterenol doses (0 and 3 are functionally activated by endogenous adenosine, this
ng= kg-l min-l) but returned to control and FR-113452
l would theoretically stimulate renin release and dimin-
group levels during subsequent periods. This pattern of ish the biological impact of the adenosine brake on renin
FK-453-induced transient diuresis and natriuresis with release. Importantly, A2 receptor blockade by KF-17837
no change in hemodynamics has also been previously did not reduce renin release induced by P-adrenoceptor

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 CONFIRMATION OF THE ADENOSINE BRAKE HYPOTHESIS F477

stimulation, which indicates that endogenous adenosine tinct A1 receptor antagonists, augmented renin release
does not influence renin release via A2 receptors. Inter- responses, whereas FR-113453 did not, it is virtually
estingly, KF-17837 did increase MABP, which likely certain that the augmentation produced by DPCPX and
accounts for the increase in sodium excretion induced by FK-453 was mediated via blockade of the Al receptor.
KF-17837 (pressure natriuresis). We do not know to A third strength of the study design was the use of
what extent the pressor response to KF-17837 observed KF-17837. Until very recently, truly selective A2 recep-
in the present study is mediated by blocking vasodila- tor antagonists for in vivo studies were unavailable.
tory AZ receptors. KF-17837 represents a prototype for a new class of
The animal model of uninephrectomy, renal denerva- selective AZ receptor antagonists (28), and we previously
tion, and adrenalectomy (or P-adrenoceptor blockade) performed extensive studies to determine the dose and
has been extremely useful and versatile in investigating route of KF-17837 to provide selective A2 receptor
endogenous renal mechanisms of renin release in the blockade (18). The lack of reduction of renin release
past (14). All of the features of the current study were responses by KF-17837 clearly indicates that the adeno-
crucial to permit a strong inference regarding the role of sine brake on renin release is not diminished by interac-
endogenous adenosine/A1 receptor interactions in the tions of endogenous adenosine with AZ receptors.
regulation of p-adrenoceptor-induced renin release. First, An important issue in the current study was the
several key steps were taken to minimize basal renin possible confounding influences of changes in hemody-
release and to assure that the renin release response was namics and renal excretory function induced by the
mediated exclusively by the direct effects of the intrare- treatments or animal model. It is noted that all experi-
nally infused isoproterenol on intrarenal P-adrenocep- mental groups, including the control group, had ele-
tors, as follows. 1) To diminish macula densa-induced vated sodium excretion (FEN, values of 3% or more),
renin release, rats were fed a salt-replete diet and were diuresis, and high RBF (15 ml/min), which were rela-
maintained on a supplementary infusion of hydrocorti-
tively maintained and constant over the entire experi-
sone and aldosterone. 2) Indomethacin pretreatment ment. These alterations appear to stem from complete
should have blocked prostacyclin production (7), a puta- renal denervation, which causes the well-documented
tive mediator of both baroreceptor- and macula densa-
“denervation natriuresis and diuresis” phenomenon
induced renin release via adenylate cyclase. 3) Unilat-
(13). Second, when we characterized these adenosine A1
eral nephrectomy was performed so that renin release
antagonists on the cardiovascular and renal systems
came only from the kidney receiving intrarenal infu-
using a rat model without denervation, we found that
sions of isoproterenol. 4) Activation of renin release by
FEN, was much lower (-0.29 t 0.08%) (21). We were
endogenous catecholamines was prevented by bilateral
adrenalectomy and renal denervation. 5) The intrarenal concerned that the higher sodium excretion could poten-
delivery of isoproterenol prevented systemic changes in tially induce the tubuloglomerular feedback (TGF) mech-
arterial blood pressure that would have been produced anism of the macula densa, which could, in turn, inhibit
by intravenous administration of isoproterenol. 6) Iso- renin release. If the TGF mechanism plays a significant
proterenol was chosen over norepinephrine or renal role, then it may mask augmentation or accentuate
nerve stimulation to exclude any a-adrenoceptor effects reduction of the renin release response to Al or A2
on sodium excretion, renin release, and renal hemody- blockade respectively. However, all treatment groups,
namics (30). Finally, 7) the doses of isoproterenol were including control, experienced similar sodium excretion,
selected to provide a wide range of stimulus intensities and basal PRA levels for these groups were nearly
to the JGCs without inducing systemic or renal hemody- identical as well. Importantly, this suggests that the
namic or renal excretory changes. Therefore, the above TGF mechanism, which may have been activated, was at
precautions, by minimizing basal renin release and least not differentially activated among the five groups.
restricting the renin release response to that induced Possible clinical relevance of the adenosine brake
directly by isoproterenol, permitted a precise assess- mechanism. The clinical relevance of adenosine regula-
ment of the role of endogenous adenosine as a negative tion of p-adrenergic-induced renin release in humans
modulator of P-adrenoceptor-induced renin release. may be enormous. Caffeine, widely consumed in bever-
A second important feature of the current study was ages across the world, is a nonselective Al/AZ xanthine
the selection of A1 antagonists and dosing regimens. The antagonist and thus has the capacity to block adeno-
A1 antagonists used in this study were chosen because of sine’s natural braking effect on renin release. In general,
their high A1 selectivity and different chemical struc- moderate caffeine consumption (250 mg/day) has little
tures and chiral properties, and the doses and route of effect on normal renal physiology. However, conditions
administration were chosen after an extensive in vivo of uncompensated congestive heart failure and liver
evaluation of their antagonistic properties (20,21>. Our cirrhosis with ascites are associated with abnormally
previous studies have shown that the doses of DPCPX elevated plasma renin levels, sodium retention, and
and FK-453 used in the current study provide a high and sympathetic nerve activity and set the stage for poten-
comparable blockade of the A1 receptor without antago- tial detrimental effects caused by caffeine. Caffeine may
nizing the A2 receptor (20,21). Furthermore, the match- cause further augmentation of renin release, which, in
ing dose of FR-113452 (the less active enantiomer of turn, increases sodium and water retention, further
FK-453) was shown previously not to block Al receptors. exacerbating these conditions. Because of the decreased
Since both DPCPX and FK-453, two structurally dis- metabolizing ability of the cirrhotic liver, even low

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F478 CONFIRMATION OF THE ADENOSINE BRAKE HYPOTHESIS

consumption of caffeine (90 mg/day) has been reported 2. Arend, L. J., A. Haramati, C. I. Thompson, and W. S.
to cause elevated fasting levels of caffeine, which in- Spielman. Adenosine-induced decrease in renin release: dissocia-
tion from hemodynamic effects. Am. J. Physiol. 247 (RenaZ FZuid
creased PRA eightfold in 28 of 44 cirrhotic patients Electrolyte Physiol. 16): F447-F452, 1984.
tested (15). 3. Balakrishnan, V. S., G. A. Coles, and J. D. Williams. A
Renovascular hypertension is another high-renin con- potential role for endogenous adenosine in control of human
dition that may worsen with caffeine disruption of the glomerular and tubular function. Am. J. Physiol. 265 (RenaZ
FZuid Electrolyte PhysioZ. 34): F504-F510, 1993.
adenosine brake. In two-kidney, one-clip rats (Goldblatt
4. Brown, N. J., 3. Porter, D. Ryder, and R. A. Branch. Caffeine
model of renovascular hypertension) given caffeine potentiates the renin response to d&oxide in man. Evidence for a
(equivalent to 4-5 cups of coffee/day in humans), regulatory role of endogenous adenosine. J. PharmacoZ. Exp.
development and severity of hypertension was markedly Ther. 256: 56-61,199l.
accelerated (19, 24) with corresponding elevations in 5. Bruns, R. F., J. H. Fergus, E. W. Badger, J. A. Bristol, L. A.
Santay, J. P. Hartman, S. J. Hays, and C. C. Huang. Binding
plasma renin levels (24). High-renin hypertensive pa- of the Al-selective adenosine antagonist 8-cyclopentyl-1,3-dipropyl-
tients that consume caffeine may also be at higher risk xanthine to rat brain membranes. Naunyn-Schmiedeberg’s Arch.
for heart attack or stroke. Laragh and colleagues (1) Pharmacol. 335: 59-63,1987.
have found compelling evidence that a high-renin profile 6. Bruns, R. F., G. H. Lu, and T. A. Pugsley. Adenosine receptor
is an independent and significant cardiovascular risk subtypes: binding studies. In: Topics and Perspectives in Adeno-
sine Research, edited by E. Gerlach and B. F. Becker. Berlin:
factor. Springer-Verlag, 1987, p. 59-73.
Disruption of the adenosine brake may be of conse- 7. Campbell, W. B., R. M. Graham, and E. K. Jackson. Role of
quence in essential and borderline hypertension. Essen- renal prostaglandins in sympathetically mediated renin release in
tial hypertensive patients are sometimes placed on one the rat. J. Clin. Invest. 64: 448-456, 1979.
8. Carruthers, A. M., and J. R. Fozard. Effect of pertussis toxin
or more antihypertensive therapies (low-sodium diets,
treatment on the putative adenosine A3 receptor-mediated hypo-
diuretics, and/or vasodilators) that inherently stimulate tensive response in the rat. Eur. J. PharmacoZ. 250: 185-188,
renin release. Low-sodium diets have clearly been found 1993.
to increase renal sympathetic overflow and PRA in 9. Churchill, P. C., and A. Bidani. Renal effects of selective
humans. A low-sodium diet significantly elevates PRA in adenosine receptor agonists in anesthetized rats. Am. J. PhysioZ.
252 (RenaZ FZuid Electrolyte Physiol. 21): F299-F303, 1987.
rats partially through a p-adrenergic-mediated compo- 10. Churchill, P. C., and M. C. Churchill. Ai and A2 adenosine
nent, and PRA levels increase further with caffeine (33). receptor activation inhibits and stimulates renin secretion of rat
Vasodilators, diazoxide and hydralazine, in combination renal cortical slices. J. Pharmacol. Exp. Ther. 232: 589-594,
with caffeine, have also been shown to increase PM in 1985.
11. Deray, G., R. A. Branch, W. A. Herzer, A. Ohnishi, and E. K.
humans (4) and rats (31), respectively, also with sympa-
Jackson. Adenosine inhibits P-adrenoceptor but not DBcAMP-
thetic neural involvement. induced renin release. Am. J. Physiol. 252 (Renal Fluid Electro-
In summary, the present study firmly establishes that Zyte Physiol. 21): F46-F52, 1987.
there exists in vivo an adenosine brake on renin release, 12. Deray, G., R. A. Branch, and E. K. Jackson. Methylxanthines
mediated in part by the A1 receptor, that restrains augment the renin response to suprarenal-aortic constriction.
Naunyn-Schmiedeberg’s Arch Pharmacol. 339: 690-696,1989.
physiological and pharmacological receptor-mediated 13. DiBona, G. F., and L. L. Rios. Renal nerves in compensatory
stimuli that act via the CAMP pathway. Furthermore, renal response to contralateral renal denervation. Am. J. PhysioZ.
the adenosine brake on renin release is not opposed by 238 (Renal Fluid Electrolyte Physiol. 7): F26-F30, 1980.
interactions of endogenous adenosine with A2 receptors. 14. Gerber, J. G., A. S. Nies, and R. D. Olsen. Control of canine
renin release: macula densa requires prostaglandin synthesis. J.
We thank Richard A. Conn and William A. Herzer for excellent Physiol. Lond. 319: 419-429, 1981.
technical assistance and support, as well as Stevan P. Tofovic, 15. Hasegawa, M., S. Yamada, and C. Hirayama. Fasting plasma
Subhash J. Vyas, and Curtis K. Kost, Jr., for critical reading of the caffeine level in cirrhotic patients: relation to plasma levels of
manuscript. We thank Fujisawa Pharmaceutical (Osaka, Japan) for catecholamines and renin activity. Hepatology 10: 973-977,1989.
generous donations of FK-453 and FR-113452 compounds. We also 16. Henrich, W. L., and W. B. Campbell. Importance of calcium in
thank the Kyowa Hakko Kogyo Company (Shizuoka-ken, Japan) for renal renin release. Am. J. Physiol. 251 (Endocrinol. Metab. 14):
generous donation of the KF-17837 compound. E98-E103,1986.
This work was supported by National Heart, Lung, and Blood 17. Jackson, E. K. Adenosine: a physiological brake on renin release.
Institute Grants HL-40319, HL-35909, and HL-14192 and by the Annu. Rev. Pharmacol. Toxicol. 31: l-35, 1991.
American Heart Association. 18. Jackson, E. K., W. A. Herzer, and F. Suzuki. KF’17837 is an
Portions of this work were presented at the Fifth International AZ adenosine receptor antagonist in vivo. J. Pharmacol. Exp.
Symposium on Adenosine and Adenine Nucleotides (From MoZecuZar Ther. 267: 1304-1310,1993.
Biology to Integrative Physiology), May 9-13, 1994, in Philadelphia, 19. Kost, C. K., P. Li, C. A. Pfeifer, and E. K. Jackson. Telemetric
PA. blood pressure monitoring in benign 2-kidney, l-clip renovascular
Address for reprint requests: E. K. Jackson, Center for Clinical hypertension: effect of chronic caffeine ingestion. J. PharmacoZ.
Pharmacology, 623 Scaife Hall, Univ. of Pittsburgh Medical Center, Exp. Ther. 270: 1063-1070,1994.
20. Kuan, C. J., W. A. Herzer, and E. K. Jackson. An experimen-
200 Lothrop St., Pittsburgh, PA 15213-2582.
tal paradigm for investigating the role of endogenous adeno-
Received 10 February 1995; accepted in final form 5 April 1995. sine/Al receptor interactions in vivo. J. PharmacoZ. Exp. Ther.
263: 657-662,1992.
21. Kuan, C. J., W. A. Herzer, and E. K. Jackson. Cardiovascular
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