Vol. 282, Issue 6, R1696-R1709, June 2002
Vasodilator responses to adenosine and hyperemia are
mediated by A1 and A2 receptors in the cat
vascular bed
Trinity J.
Bivalacqua,
Hunter C.
Champion,
David G.
Lambert, and
Philip J.
Kadowitz
Department of Pharmacology, Tulane University Health
Sciences Center, New Orleans, Louisiana 70112
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ABSTRACT |
Hemodynamic
responses to adenosine, the A1 receptor agonists
N6-cyclopentyladenosine (CPA) and adenosine
amine congener (ADAC), and the A2 receptor agonist
5'-(N-cyclopropyl)-carboxamido-adenosine (CPCA) were
investigated in the hindquarter vascular bed of the cat under
constant-flow conditions. Injections of adenosine, CPA, ADAC, CPCA,
ATP, and adenosine 5'-O-(3-thiotriphosphate) (ATP
S) into
the perfusion circuit induced dose-related decreases in perfusion pressure. Vasodilator responses to the A1 agonists were
reduced by the A1 receptor antagonists KW-3902 and
CGS-15943, whereas responses to CPCA were reduced by the
A2 antagonist KF-17837. Vasodilator responses to adenosine
were reduced by KW-3902, CGS-15943, and by KF-17837, suggesting a role
for both A1 and A2 receptors. Vasodilator
responses to ATP and the nonhydrolyzable ATP analog ATPS were
not attenuated by CGS-15943 or KF-17837. After treatment with the
nitric oxide synthase inhibitor
N
-nitro-L-arginine methyl ester,
the cyclooxygenase inhibitor sodium meclofenamate, or the ATP-dependent
K+ (K
) channel antagonists U-37883A or
glibenclamide, responses to adenosine and ATP were not altered. Responses to adenosine, CPA, and CPCA were increased in duration by
rolipram, a type 4 cAMP phosphodiesterase inhibitor, but were not
altered by zaprinast, a type 5 cGMP phosphodiesterase inhibitor. When
blood flow was interrupted for a 30-s period, the magnitude and
duration of the reactive vasodilator response were reduced by
A1 and A2 receptor antagonists. These data
suggest that vasodilator responses to adenosine and the A1
and A2 agonists studied are not dependent on the release of
cyclooxygenase products, nitric oxide, or the opening of
K channels in the regional vascular bed of the cat.
The present data suggest a role for cAMP in mediating responses
to adenosine and suggest that vasodilator responses to adenosine
and to reactive hyperemia are mediated in part by A1 and
A2 receptors in the hindquarter vascular bed of the cat.
purinergic responses; regional vascular bed; KF-17837; CGS-15943; reactive vasodilation
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INTRODUCTION |
ADENOSINE, AN ENDOGENOUS PURINE
nucleoside formed from the dephosphorylation of cAMP by the
ectoenzyme 5'-nucleotidase, mediates a variety of physiological
responses in mammalian tissues (11, 13, 14). Based on
molecular cloning studies (14), physiological responses to
extracellular adenosine are mediated by four adenosine receptor
subtypes (A1, A2a, A2b, and
A3). The A1 receptor is found in greatest
number in the brain, spinal cord, testis, and adipose tissue and is
coupled to several second messenger systems. In atrial and ventricular
myocytes, A1 receptor binding inhibits the activation of
adenylyl cyclase and increases an inwardly rectifying K+
current, leading to negative chronotropic and inotropic effects on the
heart (4). A2a receptor mRNA has been found in
highest concentration in the brain and thymus gland, whereas the
message for the A2b receptor subtype has been found in
human intestinal epithelium, and both receptor subtypes have been
identified in human cultured aortic endothelial cells (19, 32,
36). Activation of the A2 receptor results in
stimulation of adenylyl cyclase and may also involve stimulation of
nitric oxide (NO) formation and activation of ATP-dependent
K+ (K) channels (1, 21).
However, in isolated coronary arteries of the dog, inhibition of NO
synthesis did not affect the dilator properties of adenosine
(22). High levels of mRNA for the A3 receptor
subtype have been identified in human lung and liver, and there is
evidence that activation of this receptor subtype results in inhibition
of adenylyl cyclase and stimulation of phospholipase C
(40).
Physiological responses elicited by adenosine are varied, and
A2 receptor activation produces vasodilation in most
vascular beds in a variety of species (10, 24, 43). In the
rat renal artery, A1 receptor activation produces
constriction, whereas A2 receptor activation leads to
vasodilation (8, 16, 18, 21, 37). Adenosine has been shown
to have hypertensive activity in the pulmonary circulation in a number
of species (5, 24, 32), whereas in the feline pulmonary
vascular bed, responses to adenosine are tone dependent with a pressor
response mediated by A1 activation under low-tone
conditions and vasodilation mediated by A2 activation
observed under elevated-tone conditions (10). The
contribution of K channels to ischemic
vasodilation during reactive hyperemia has been well characterized
(2, 3, 28, 42). In the feline hindquarter vascular bed,
reactive hyperemia has been shown to be mediated in part by the opening
of K channels and the release of NO
(28). However, little is known about the contribution of
adenosine A1 and A2 receptors in the regulation of the peripheral vascular bed and on the reactive hyperemic response in the cat. Until recently, potent selective adenosine receptor antagonists have not been available to investigate the receptor subtypes involved in mediating responses to adenosine in physiological and pathophysiological conditions. It has been reported that KW-3902 and CGS-15943 are adenosine A1 receptor antagonists,
whereas KF-17837 is a selective A2 receptor antagonist
(16, 20, 38). Therefore, the present study was carried out
to determine the receptor subtype and the mechanisms involved in
mediating vasodilator responses to adenosine and reactive hyperemia in
the hindquarter vascular bed of the cat.
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MATERIALS AND METHODS |
One hundred and seventeen adult mongrel cats of either sex,
weighing 2.4-4.6 kg, were sedated with ketamine hydrochloride (10-15 mg/kg im) and were anesthetized with pentobarbital sodium (30 mg/kg iv). Supplemental doses of pentobarbital sodium were given as
needed to maintain a uniform level of anesthesia. The trachea was
cannulated, and the animals breathed spontaneously or were ventilated
with a Harvard model 607 ventilator at a volume of 40-60 ml at
15-22 breaths/min. An external jugular vein was catheterized for
intravenous administration of drugs, and a carotid artery was
catheterized for the measurement of systemic arterial pressure. For
constant-flow perfusion of the hindquarter vascular bed, a 3- to 4-cm
segment of the distal abdominal aorta was exposed through a ventral
midline incision and was cleared of surrounding connective tissue by
blunt dissection. After administration of heparin sodium (1,500 U/kg
iv), the aorta was ligated, and catheters were inserted proximal and
distal to the ligature. Branches of the aorta distal to the origin of
the external iliac arteries were ligated to restrict blood flow to the
hindlimb. The hindquarter vascular bed was denervated by ligating and
cutting the lumbar sympathetic chain ganglia between L3 and
L4. Blood was withdrawn from the proximal catheter and
pumped at a constant rate with a Sigmamotor model T-8 pump into the
distal aortic catheter. For the reactive vasodilator experiments, blood
flow to the hindquarter vascular bed was interrupted by stopping the
perfusion pump for a 30-s period. When the pump was started and blood
flow was restored, a reactive vasodilator response was observed
(28). Perfusion pressure was monitored from a lateral tap
in the perfusion circuit located between the pump and the distal aortic
catheter. Hindquarter perfusion pressure and systemic arterial pressure
were measured with Statham P23 transducers and were recorded on a Grass
model 7 polygraph. Mean pressures were derived by electronic averaging, and the flow rate was set so that hindquarter perfusion pressure approximated systemic arterial pressure and was not changed during the
experiment. The flow rate, determined by timed collection, ranged from
24 to 36 ml/min. Agonists were injected directly in the hindlimb
perfusion circuit distal to the pump in small volumes (30 and 100 µl)
in a random sequence, and antagonists were injected intravenously.
These procedures have been described previously (9, 28,
33).
For constant-flow perfusion of the mesenteric vascular bed, the
superior mesenteric artery was approached through a midline abdominal
incision and cleared of surrounding connective tissue. The mesenteric
vascular bed was denervated by ligating and cutting the perivascular
nerves to the small intestine as they course along the superior
mesenteric artery. After the administration of heparin sodium (1,000 U/kg), the femoral artery was cannulated and connected to the inlet
side of the perfusion circuit. The outlet side of the perfusion circuit
was connected to a catheter inserted in the superior mesenteric artery.
Blood flow to the small intestine was maintained constant with a
Sigmamotor model T-8 perfusion pump. Superior mesenteric perfusion
pressure was measured using a lateral tap in the perfusion circuit
located between the pump and the outlet side of the perfusion circuit. Superior mesenteric perfusion pressure and systemic arterial pressure were measured with Statham P23 pressure transducers and were recorded on a Grass model 7 polygraph. Mean pressures were derived by electronic averaging, and the perfusion rate was set so that superior mesenteric arterial perfusion pressure approximated systemic arterial pressure and
was not changed during the experiment. The flow rate was determined by
timed collection and ranged from 24 to 34 ml/min. The agonists used in
these experiments were injected directly in the superior mesenteric
arterial perfusion circuit distal to the pump in small volumes in a
random sequence.
In the first set of experiments, hindquarter responses to injections in
the perfusion circuit of the adenosine A1 receptor agonist
N6-cyclopentyladenosine (CPA), the
A2 receptor agonist
5'-(N-cyclopropyl)-carboxamido-adenosine (CPCA), adenosine,
ATP, and adenosine 5'-O-(3-thiotriphosphate) (ATPS) were
investigated under constant-flow conditions, with doses expressed on a
nanomole basis to take into account differences in molecular weight. In
the second set of experiments, the effects of selective adenosine
A1 receptor antagonists CGS-15943 and KW-3902 and the
A2 receptor antagonist KF-17837 on vasodilator responses were investigated, with agonist responses being compared before and
after the administration of CGS-15943 (0.5 mg/kg iv), KW-3902 (2 mg/kg
iv), and KF-17837 (2-3 mg/kg iv). The doses of CGS-15943, KW-3902,
and KF-17837 were determined in pilot experiments. In the third set of
experiments, the selectivity of the blockade induced by the
A1 and A2 antagonists was investigated, and, in the fourth set of experiments, the mechanism by which adenosine produces vasodilation in the hindquarter vascular bed was investigated. To investigate the role of NO,
N-nitro-L-arginine methyl ester
hydrochloride (L-NAME; 100 mg/kg iv) was administered, and
responses to adenosine were evaluated beginning 20 min after completion
of L-NAME administration. Vasodilator responses to ACh were
compared before and after L-NAME to assess the degree of NO
synthase inhibition. To investigate the role of vasodilator
prostaglandins in mediating responses to adenosine, the cyclooxygenase
inhibitor sodium meclofenamate was injected in a dose of 2.5 mg/kg iv
over a 10-min period, and responses were evaluated beginning 20 min
after completion of the injection. Vasodilator responses to arachidonic
acid were compared before and after sodium meclofenamate to assess the
degree of cyclooxygenase inhibition. The role of K
channel activation was investigated, and responses to adenosine were
compared before and after administration of the K channel antagonist U-37883A or glibenclamide (5 mg/kg iv). Responses to
the K channel opener levcromakalim were compared
before and after administration of U-37883A or glibenclamide to assess
the K channel blockade. In the fifth set of
experiments, the role of cAMP and cGMP in mediating responses to
adenosine, CPA, and CPCA was evaluated. Rolipram, a type 4 cAMP
phosphodiesterase inhibitor, was injected in a dose of 0.5 mg/kg iv,
and zaprinast, a type 5 cGMP phosphodiesterase inhibitor, was injected
in a dose of 1 mg/kg iv. Responses to adenosine, CPA, and CPCA were
compared before and beginning 20 min after administration of rolipram
or zaprinast. In the final set of experiments, the role of
A1 and A2 receptors in mediating the reactive
hyperemic response was studied under constant-flow conditions in the
hindquarter vascular bed of the cat. The effect of a 30-s period of
interruption of hindquarter blood flow induced by stopping the
perfusion pump was assessed in terms of total area under the perfusion
pressure curve over time, the duration of the reactive hyperemic
response, and the percent decrease in hindquarter perfusion pressure
(28). In this set of experiments, the effects of
CGS-15943, KF-17837, the combination of CGS-15943 and KF-17837, and of
the passage of time on the hindquarter reactive vasodilator response
were investigated.
Preparation of drugs.
ATP, ATPS, adenosine, acetylcholine bromide, L-NAME
(Sigma Chemical, St. Louis, MO), and albuterol sulfate (Schering,
Kenilworth, NJ) were dissolved in 0.9% NaCl. U-37883A (Upjohn,
Kalamazoo, MI) was dissolved in 0.9% NaCl with sonication.
Glibenclamide (Sigma) was dissolved in a 10% ethanol/saline solution
at a concentration of 10 mg/ml and was diluted with 0.9% NaCl.
Levcromakalim (SmithKline Beecham, Sussex, UK) was dissolved in 20%
ethanol-saline solution at a concentration of 1 mg/ml and was diluted
with 0.9% NaCl. Zaprinast
(2-O-propoxyphenyl-8-azapurin-6-one; Rhone-Poulenc, Degenham, Essex, UK) was dissolved in 0.15 N NaOH in normal saline in a
concentration of 3 mg/ml. Rolipram (SmithKline Beecham) was dissolved
in 20% dimethyl sulfoxide (DMSO) and diluted with normal saline.
CGS-15943 (RBI, Natick, MA) was dissolved in DMSO with sonication.
KF-17837 and KW-3902 (provided by Dr. Fumio Suzuki, Pharmaceutical
Research Laboratories, Kyowa Hakko Kogyo, Schizuoka, Japan) were
dissolved in propylene glycol. CPA, adenosine amine congener (ADAC),
and CPCA (RBI) were dissolved in 1 N acetic acid and diluted with
normal saline. The vehicles for these agents had no consistent effect
on baseline vascular pressure or responses to the vasoactive agonists.
The drug solutions were stored in dark bottles in a freezer, and
working solutions prepared on a frequent basis were kept on crushed ice
during an experiment.
The hemodynamic data are expressed in absolute units as means ± SE, except in experiments with L-NAME in which baseline
tone was markedly increased, and responses are expressed as percent decrease to take into account changes in baseline perfusion pressure. In experiments carried out to determine the role of A1 and
A2 receptors on the response to reactive hyperemia, the
area under the curve was measured with a planimeter or using a Bruning
model 4849 area graph grid. The data were analyzed using a one-way
ANOVA with repeated measures and Scheffé's F-test
with a Bonferonni/Dunn procedure or a paired t-test. A
P value <0.05 was used as the criterion for statistical significance.
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RESULTS |
Responses to purinergic agonists.
Responses to purinergic agonists were investigated in the hindquarter
vascular bed of the cat under constant-flow conditions, and
dose-response curves are shown in Fig. 1.
Injections of the purinergic agonists into the hindquarter perfusion
circuit produced dose-related decreases in perfusion pressure (Fig. 1).
When doses are expressed on a nanomole basis, adenosine and the
A2 receptor agonist CPCA were the most potent vasodilators,
with dose-response curves 2 log units to the left of the curve for the
A1 agonist CPA and 1 log unit to the left of the
dose-response curves for ATP and the degradation-resistant ATP analog
ATPS (Fig. 1).
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Fig. 1.
Dose-response curves comparing decreases in hindquarter
perfusion pressure in response to injections of the purinergic agonists
in the hindquarter vascular bed. The order of potency in dilating the
hindquarter vascular bed was adenosine = 5'-(N-cyclopropyl)-carboxamido-adenosine (CPCA) > ATP = adenosine 5'-O-(3-thiotriphosphate)
(ATPS) > N6-cyclopentyladenosine (CPA)
when doses are expressed in nmol to take molecular weight into account.
n, No. of experiments.
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Role of A1 and A2 receptors.
The role of A1 and A2 receptors in mediating
vasodilator responses to adenosine was investigated, and these data are
summarized in Figs.
2-4.
Decreases in hindquarter perfusion pressure in response to adenosine
were decreased significantly [51 ± 4 to 21 ± 4 mmHg (59%
decrease) at the 30-µg dose] after administration of the A2 receptor antagonist KF-17837 (2-3 mg/kg iv; Fig.
2). Treatment with KF-17837 significantly attenuated vasodilator
responses to the A2 receptor agonist CPCA [47 ± 3 to
14 ± 2 mmHg (70% decrease) at the 1-µg dose] without altering
responses to the A1 agonists CPA or ADAC or to ATP or
ATPS (Fig. 2). KF-17837 had no significant effect on vasodilator
responses to ACh, levcromakalim, or albuterol (data not shown).
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Fig. 2.
Influence of the adenosine A2 receptor antagonist
KF-17837 (2-3 mg/kg iv) on vasodilator responses to adenosine,
CPCA, CPA, adenosine amine congener (ADAC), ATP, and ATPS in the
hindquarter vascular bed. Responses to the purinergic agonists were
compared beginning 10-20 min after administration of the
A2 receptor antagonist. n, No. of experiments.
*Response is significantly different from control.
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Fig. 3.
Influence of the A1 receptor agonist CGS-15943 (0.5 mg/kg iv) on vasodilator responses to adenosine, ADAC, CPCA, ATP, and
ATPS in the hindquarter vascular bed. Responses to the purinergic
agonists were compared before and beginning 10-20 min after
administration of the A1 receptor antagonist. n,
No. of experiments. *Response is significantly different from
control.
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Fig. 4.
Effect of adenosine A1 and A2
receptor antagonists CGS-15943 and KF-17837 on the vasodilator response
to adenosine in the hindquarter vascular bed. The response to adenosine
was determined after administration of CGS-15943 (0.5 mg/kg iv) and
again after administration of KF-17837 (2-3 mg/kg iv) in 7 animals. *Response is different from control. **Response is different
from that obtained after administration of CGS-15943.
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The role of A1 receptors in mediating hindquarter
responses to adenosine was investigated, and, after administration of
the A1 receptor antagonist CGS-15943 (0.5 mg/kg iv),
vasodilator responses to adenosine were reduced significantly [55 ± 7 to 35 ± 4 mmHg (36% decrease) at the 30-µg dose; Fig.
3]. After administration of CGS-15943, vasodilator responses to the
A1 agonists CPA and ADAC were reduced significantly
[33 ± 3 to 14 ± 4 mmHg (58% decrease) at 30 µg for CPA
and from 42 ± 5 to 19 ± 4 mmHg (55% decrease) for ADAC at
the 30-µg dose] without altering responses to the A2
agonist CPCA (Fig. 3). CGS-15943 had no significant effect on
vasodilator responses to ATP and ATPS (Fig. 3) or on responses to
ACh, levcromakalim, or albuterol (data not shown). In an additional set
of experiments, the role of A1 receptors in mediating
hindquarter responses to adenosine, CPA, and CPCA was investigated
using the selective A1 receptor antagonist KW-3902 (2 mg/kg
iv), and these data are summarized in Table
1. Vasodilator responses to adenosine and
the A1 receptor agonist CPA were significantly decreased
after treatment with KW-3902, whereas vasodilator responses to the
A2 receptor agonist CPCA were not changed significantly
(Table 1). Treatment with KW-3902 had no significant effect on
vasodilator responses to ATP, ACh, or albuterol (data not shown).
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Table 1.
Effect of the adenosine A1 receptor antagonist KW-3902 on
vasodilator responses to adenosine, CPA, and CPCA in the
hindquarters vascular bed of the cat
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The combined effect of CGS-15943 and KF-17837 on the vasodilator
response to adenosine was assessed and, after administration of
CGS-15943 (0.5 mg/kg iv), the vasodilator response to adenosine was
reduced significantly (46% decrease); the administration of KF-17837
(2-3 mg/kg iv) to the same animals produced a significantly greater (73%) decrease in the response to adenosine than did CGS-15943 alone (Fig. 4).
Influence of NO synthase and cyclooxygenase inhibitors and a
K
channel antagonist.
To determine if hindquarter vasodilator responses to adenosine are
mediated or modulated by the release of NO, responses were compared
before and after administration of the NO synthase inhibitor L-NAME, and these data are summarized in Fig.
5A. After administration of
L-NAME in a dose of 100 mg/kg iv, vasodilator responses to adenosine were not reduced at a time when responses to ACh were significantly decreased (Fig. 5A). The NO synthase inhibitor
did alter vasodilator responses to albuterol or levcromakalim (data not
shown).
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Fig. 5.
A: influence of the nitric oxide synthase inhibitor
N-nitro-L-arginine methyl ester
hydrochloride (L-NAME; 100 mg/kg iv) on responses to
adenosine and ACh. B: influence of the cyclooxygenase
inhibitor sodium meclofenamate (2.5 mg/kg iv) on responses to adenosine
and arachidonic acid (AA). Responses to agonists were compared before
and beginning 10-20 min after administration of the inhibitors.
n, No. of experiments. *Response is significantly different
from control.
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To ascertain if responses to adenosine are modulated by the release of
vasodilator products in the cyclooxygenase pathway, responses were
compared before and after administration of the cyclooxygenase
inhibitor sodium meclofenamate in a dose of 2.5 mg/kg iv, and these
data are summarized in Fig. 5B. After administration of
sodium meclofenamate, responses to adenosine were not significantly different from control, whereas vasodilator responses to the
prostaglandin precursor arachidonic acid were reduced significantly
(Fig. 5B).
The role of K channels in mediating the response to
adenosine was investigated, and, after administration of the
K channel antagonist U-37883A or glibenclamide (5 mg/kg iv), vasodilator responses to adenosine were not significantly
reduced at a time when vasodilator responses to the
K channel opener levcromakalim were reduced
significantly (Fig. 6, A and
B).
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Fig. 6.
A: influence of the K channel
antagonist glibenclamide (5 mg/kg iv) on vasodilator responses to
adenosine and the K channel opener levcromakalim
(LK) in the hindquarter vascular bed of the cat. B:
influence of the K channel antagonist U-37883A (5 mg/kg iv) on vasodilator responses to adenosine and levcromakalim in
the hindquarter vascular bed of the cat. C: influence of
U-37883A on vasodilator responses to adenosine and levcromakalim in the
mesenteric vascular bed of the cat. Responses to agonists were compared
before and beginning 10-20 min after administration of the
inhibitor. n, No. of experiments; *Response is significantly
different from control.
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In another set of experiments in the mesenteric vascular bed of the cat
under constant-flow conditions, vasodilator responses to adenosine were
compared before and after administration of U-37883A (5 mg/kg iv) to
determine if K channel activation plays a role in
mediating responses to adenosine in another regional vascular bed in
the cat. Vasodilator responses to adenosine were not significantly
reduced at a time when responses to the K channel
opener levcromakalim were decreased after administration of U-37883A in
the mesenteric vascular bed (Fig. 6C).
Influence of rolipram and zaprinast.
The role of changes in cAMP and cGMP levels in mediating responses to
adenosine in the hindquarter vascular bed was assessed by investigating
the effects of type 4 cAMP and type 5 cGMP phosphodiesterase inhibitors
on the duration of the vasodilator response as measured by the recovery
half-time (T1/2) of the response. The
T1/2 is defined as the time required for the
pressure to return to 50% of the maximal decrease in perfusion
pressure. The time course of the decrease in hindquarter perfusion
pressure in response to adenosine (3 µg) and albuterol and the
effects of rolipram are shown in Fig. 7.
After administration of the type 4 phosphodiesterase inhibitor rolipram
(0.5 mg/kg iv), the T1/2 of the vasodilator responses to adenosine, CPA, CPCA, and albuterol was increased significantly, whereas the T1/2 of the response
to the NO donor 2-(N,N-diethylamino)-diazenolate
2-oxide (DEA/NO) was not altered (Figs. 7 and
8). The T1/2 (s)
of the response to adenosine was increased from 15 ± 1 to 23 ± 1 s after treatment with rolipram, and the
T1/2 (s) of the response to the 
-agonist
albuterol was increased from 52 ± 7 to 220 ± 25 s
after treatment with rolipram (Figs. 7 and 8). The time course of the
decreases in hindquarter perfusion pressure in response to adenosine
and DEA/NO and the effect of zaprinast are shown in Fig.
9. The T1/2 (s) of
the vasodilator response to adenosine (3 µg) was not significantly
different after treatment with zaprinast (15 ± 2 to 14 ± 1 s; Figs. 9 and 10). The
T1/2 of the vasodilator response to DEA/NO (3 µg) was increased from 71 ± 18 to 104 ± 16 s after
treatment with zaprinast (Figs. 9 and 10). After administration of the
type 5 cGMP phosphodiesterase inhibitor zaprinast (1 mg/kg iv),
vasodilator responses to adenosine, CPA, CPCA, and albuterol were not
altered, whereas the T1/2 of the vasodilator
response to DEA/NO was increased significantly (Figs. 9 and 10).
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Fig. 7.
Records from an experiment showing the time course of the
decrease in hindquarter perfusion pressure in response to adenosine and
albuterol before and after administration of the type 4 cAMP
phosphodiesterase inhibitor rolipram (0.5 mg/kg iv). Responses to the
vasodilator agonists were compared before and beginning 10-20 min
after administration of the phosphodiesterase inhibitor. n,
No. of experiments.
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Fig. 8.
Bar graphs summarizing the effects of the type 4 cAMP
phosphodiesterase inhibitor rolipram (0.5 mg/kg iv) on the duration of
responses to adenosine, CPCA, CPA, the adrenergic
2-receptor agonist albuterol, and the nitric oxide (NO)
donor DEA/NO as measured by the half-time
(T1/2) of the vasodilator response.
Responses to the vasodilator agonists were compared before and
beginning 10-20 min after administration of the phosphodiesterase
inhibitors. n, No. of experiments. *Response is
significantly different from control.
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Fig. 9.
Records from an experiment showing the time course of the decrease
in hindquarter perfusion pressure in response to adenosine and DEA/NO
before and after administration of the type 5 phosphodiesterase
inhibitor zaprinast (1 mg/kg iv). Responses to the vasodilator agonists
were compared before and beginning 10-20 min after administration
of the phosphodiesterase inhibitor. n, No. of experiments.
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Fig. 10.
Bar graphs summarizing the effect of the type 5 cGMP
phosphodiesterase inhibitor zaprinast (1 mg/kg iv) on response duration
as measured by the T1/2 of vasodilator responses
to adenosine, CPCA, CPA, albuterol, and DEA/NO. Responses to the
vasodilator agonists were compared before and beginning 10-20 min
after administration of the phosphodiesterase inhibitors. n,
No. of experiments. *Response is significantly different from
control.
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Role of A1 and A2 receptors in the reactive
hyperemic response.
The effects of CGS-15943 (0.5 mg/kg iv) and of KF-17837 (3 mg/kg iv) on
the reactive vasodilator response to a brief period of ischemia
were investigated; these data are summarized in Fig. 11. When blood flow was restored after
a 30-s period of ischemia, a significant decrease in
hindquarter perfusion pressure (vasodilator response) lasting 125 ± 5 s was observed (Fig. 11). The hyperemic response to a 30-s
period of arterial inflow occlusion was reduced after administration of
CGS-15943, KF-17837, and the combination of CGS-15943 and KF-17837
(Fig. 11, A-C). The area under the curve, the duration of
the reactive vasodilation, and the percent decrease in hindquarter
perfusion pressure after a 30-s period of ischemia were
reduced significantly after administration of CGS-15943, KF-17837, and the combination of CGS-l5943 and KF-17837 (Fig. 11,
A-C). The percent decrease in hindquarter perfusion pressure in response to a 30-s occlusion was not significant after
administration of the A1 receptor antagonist CGS-15943
(Fig. 11A). The reactive hyperemic response was reproducible
with respect to time, and the area under the curve, duration of the
reactive vasodilation, and the percent decrease in hindquarter
perfusion pressure after a 30-s period of arterial occlusion were not
reduced after treatment with vehicle (Fig. 11D).
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Fig. 11.
Bar graphs showing the effect of CGS-15943 (0.5 mg/kg iv;
A), KF-17837 (3 mg/kg iv; B), CGS-15943 (0.5 mg/kg iv) and KF-17837 (3 mg/kg iv; C), and saline
(D) on the area under the curve (AUC, in
mmHg · min), the duration of the reactive vasodilator response,
and the percent decrease in hindquarter perfusion pressure in response
to a 30-s period of arterial inflow occlusion. The reactive hyperemic
responses were compared before and beginning 10 min after injection of
CGS-15943 or KF-17837 and after administration of CGS-15943 and
KF-17837 or saline. n, No. of experiments. *Response is
significantly different from control.
|
|
Effects on baseline tone.
The effects of the antagonists and inhibitors used in the present study
on mean systemic arterial and hindquarter perfusion pressures are
summarized in Table 2. Because cardiac
output was not measured, changes in total peripheral resistance could
not be analyzed. However, because blood flow to the hindquarter
vascular bed was maintained constant, changes in perfusion pressure
reflect changes in vascular resistance in the bed. The largest increase in vascular resistance observed was in experiments with
L-NAME, suggesting that NO release plays an important role
in regulating vascular tone. A large increase in baseline tone, by
changing initial value, will enhance vasodilator responses; therefore, in experiments with the NO synthesis inhibitor, vasodilator responses are expressed in terms of the percent decrease in perfusion pressure to
take changes in initial tone into account. In these experiments, both
negative (albuterol and levcromakalim) and positive control agonist
responses (ACh) are used to assess changes in vascular responsiveness.
The cyclooxygenase inhibitor sodium meclofenamate and the
K channel antagonists had only small effects on
perfusion pressure, and the efficacy of the pharmacological probes was
assessed using the prostaglandin precursor arachidonic acid and the
K channel opener levcromakalim.
View this table:
[in this window]
[in a new window]
|
Table 2.
Effect of antagonists and inhibitors used in this study on systemic
arterial pressure and hindquarter perfusion pressure in the cat
|
|
The phosphodiesterase inhibitors rolipram and zaprinast caused
significant decreases in hindquarter vascular resistance, suggesting a
role for cAMP and cGMP turnover in regulating baseline tone in this
bed. The effects of these agents on response duration, as shown in
Figs. 7-10, were evaluated using appropriate negative and positive
control agonist injections to assess changes on response duration.
Both KF-17837 and CGS-15943 caused small significant increases in
perfusion pressure, suggesting a role for tonic activation of adenosine
receptors in the regulation of baseline tone in the vascular bed. The
efficacy and selectivity of the blockade were assessed using selective
A1 and A2 receptor agonists. Neither KF-17837
nor CGS-15943 altered vasodilator responses to ACh, albuterol, or
levcromakalim, and KW-3902 did not alter responses to ATP, ACh,
albuterol, or levcromakalim. In addition to assessing the selectivity
and efficacy of the inhibitory effects of the pharmacological probes
used, control experiments were carried out to assess the effects of
time and the vehicles used in this study on vascular responses.
| |
DISCUSSION |
New findings from this study are that vasodilator responses to
adenosine in the hindquarter vascular bed of the cat are mediated by
A1 and A2 receptors, whereas the release of NO,
vasodilator prostaglandins, and the opening of K channels appear to have no important role. The present data suggest a
role for cAMP in mediating vasodilator responses, and, in addition, these results show that A1 and A2 receptor
antagonists reduce the reactive hyperemic response after a 30-s period
of ischemia in the hindquarter vascular bed of the cat.
The results of the present investigation show that adenosine, the
A1 receptor agonists CPA and ADAC, and the A2
receptor agonist CPCA produce dose-related decreases in hindquarter
perfusion pressure in the cat (Fig. 1). Inasmuch as blood flow to the
hindquarter vascular bed was maintained constant, the decreases in
perfusion pressure reflect decreases in hindquarter vascular
resistance. In terms of relative vasodilator activity, adenosine and
CPCA were approximately equivalent and were about 100-fold more potent than the A1 receptor agonist CPA. ATP and the
degradation-resistant ATP analog ATPS have significant vasodilator
activity and were halfway between the A1 and A2
receptor agonists in vasodilator potency (Fig. 1). The results of
experiments with the A1 and A2 receptor
agonists suggest that A1 and A2 receptors
mediating vasodilation are present in the hindquarter vascular bed of
the cat (Figs. 1-3). To further test the hypothesis that
A1 and A2 receptors are present and to
determine the role of these receptor subtypes in mediating the response
to adenosine, the effects of the A2 receptor antagonist
KF-17837 and the A1 antagonists CGS-15943 and KW-3902 were
investigated. KF-17837 attenuated responses to the A2
agonist CPCA without altering responses to the A1 agonists
CPA and ADAC, and the A2 receptor antagonist reduced the
vasodilator response to adenosine (Fig. 2). These data indicate that
A2 receptors mediating vasodilation are present and that
vasodilator responses to adenosine are mediated in part by the
activation of A2 receptors, a finding consistent with
results from a number of studies (11, 14, 23, 43). The
A1 receptor antagonists CGS-15943 and KW-3902 decreased
vasodilator responses to the A1 agonist CPA without altering responses to the A2 agonist CPCA (Fig. 3 and Table
1). These results provide support for the hypothesis that
A1 receptors mediating in part the vasodilator response to
adenosine are present in the hindquarter vascular bed. This finding is
at variance with results from a number of studies indicating that
A2 receptors mediate vasorelaxant responses to adenosine
and that A1 receptors have no significant role (11,
18, 43). Earlier studies have shown that the hypotensive
response to CPA may be the result of bradycardia and a reduction in
cardiac output after A1 receptor activation
(34). However, in the present study under constant-flow conditions, vasodilator responses to direct local injections of the
A1 receptor agonists CPA and ADAC into the perfusion
circuit were rapid in onset, suggesting that responses to CPA and ADAC were the result of a direct effect on A1 receptors on
resistance elements in the hindquarter vascular bed. These results are
consistent with the results of studies in the hindlimb and
diaphragmatic vascular beds in the rat (6, 12). The
observation that higher doses of the A1 agonist relative to
doses of the A2 agonist were required to induce
vasodilation suggests that the A1 receptor mediating
vasodilation in the hindquarter vascular bed of the cat may be of an
unusual low-affinity type and is in agreement with a previous study in
which high concentrations of CPA were required to relax isolated bovine
coronary arterial rings (27).
Another possible explanation for the vasodilation observed in response
to injections of the A1 receptor agonists may be that CPA
and ADAC are interacting with A2 receptors. However, the
high selectivity of CPA (26) and ADAC (44)
for the A1 receptor and the absence of an inhibitory effect
of the A2 antagonist KF-17837 on responses to CPA and ADAC
suggest that activation of the A2 receptor would not
account for the observed results. Furthermore, the A1
receptor antagonist KW-3902 also attenuated vasodilator responses to
CPA at a time when responses to CPCA were not altered in the
hindquarter vascular bed of the cat (Table 1; see Refs. 10
and 34). These results suggest that A2 receptor activation does not contribute to the vasodilator response to CPA or ADAC in the
present study. Previous reports have shown that CGS-15943 acts as a
nonselective A1 and A2 receptor antagonist;
however, the present data indicate that CGS-15943 in the dose used is
selective for A1 receptors in the hindquarter vascular bed
of the cat (16, 44). The selectivity of an antagonist for
a receptor could depend on the species, experimental preparation, or
vascular bed studied, and, in pilot studies, high doses of CGS-15943
also attenuated responses to the A2 agonist CPCA. The
results of the present study showing that vasodilator responses to ATP
and ATPS are similar, that these agents are less potent than
adenosine, and that responses are not altered by A1 or
A2 receptor antagonists suggest that ATP breakdown and
adenosine formation do not contribute to vasodilator responses to ATP
in the hindquarter vascular bed and are in agreement with studies in
the pulmonary vascular bed in this species (10, 32).
It has been reported that vasorelaxant responses to adenosine are
endothelium dependent, involving the release of NO, and are dependent
on the release of cyclooxygenase products or the opening of
K channels (1, 20, 23, 43). The
mechanism underlying vasodilator responses to adenosine was
investigated, and responses to the purinergic agonist were not altered
after administration of the NO synthase inhibitor L-NAME or
the cyclooxygenase inhibitor sodium meclofenamate in doses that
decreased responses to ACh and arachidonic acid (Fig. 5). These data
suggest that adenosine does not induce vasodilation by releasing NO or
products in the cyclooxygenase pathway in the hindquarter vascular bed.
The role of K channel activation was investigated
in the hindquarter and mesenteric vascular beds of the cat, and, after
treatment with the K channel antagonist U-37883A or
glibenclamide in doses that reduced vasodilator responses to the
K channel opener levcromakalim, responses to
adenosine were not altered (Fig. 6), suggesting that the opening of
K channels is not involved in mediating the
vasodilator response to adenosine in the hindquarter and mesenteric
vascular beds of this species.
Adenosine A2 receptors are reported to be coupled to
adenylyl cyclase and increase cAMP levels (23); in studies
on the mechanism by which adenosine dilates the hindquarter vascular
bed, the duration of the vasodilator response before and after
administration of the type 4 cAMP and type 5 cGMP phosphodiesterase
inhibitors was measured. After treatment with the type 4 inhibitor
rolipram in a dose that significantly increased the duration of the
response to the 2-receptor agonist albuterol, the
T1/2 of the vasodilator response to adenosine,
CPA, and CPCA was increased significantly, whereas the duration of the
response to DEA/NO was not altered (Figs. 7 and 8). There are numerous
reports in the literature that associate the A1 receptor
with inhibition, not stimulation, of adenylyl cyclase (13, 38,
43). However, in the hindquarter vascular bed of the cat,
A1 receptor activation does not appear to decrease cAMP
formation, which could evoke a vasoconstrictor response. The type 5 cGMP phosphodiesterase inhibitor zaprinast, in a dose that increased
the duration of the vasodilator response to DEA/NO, did not alter the
T1/2 of the response to adenosine, CPA, CPCA, or
albuterol (Figs. 9 and 10). These data suggest that vasodilator
responses to adenosine, CPA, and CPCA are not associated with an
increase in cGMP levels in the hindquarter vascular bed of the cat and
that increases in cAMP levels not mediated by the release of a
vasodilator prostaglandin may be involved.
The signal transduction mechanism for A1 receptors has been
extensively studied, and the original definition of A1
receptor activation was based on inhibition of adenylyl cyclase
(17, 20). The inference that A1 receptor
activation may be associated with increased cAMP formation goes against
a great deal of biochemical evidence in the literature (17, 20,
23, 43). A decrease in cAMP levels in vascular smooth muscle is
usually associated with vascular smooth muscle contraction and
vasoconstriction. The results of the present study and previous results
in the literature show that A1 receptor activation results
in vasodilation (6, 7, 12). The present data differ from
data in several studies in which A1 receptor-induced
vasodilation has been associated with the activation of
K channels or the release of NO (1, 20, 23,
25, 43). Although there is disagreement about the role of
K channels or NO in A1
receptor-mediated responses, the present study is the only known data
suggesting that vasodilator responses to A1 activation may
be associated with increased cAMP levels in resistance vessel elements
in the hindlimb circulation of the cat. We therefore wish to be very
cautious in suggesting a relationship between A1 receptor
activation and increased cAMP levels. The doses of the A1
agonists CPA and ADAC required to induce vasodilation were much higher
than doses of the A2 agonist CPCA or adenosine. It is known
in isolated tissue studies that the affinity of CPA or ADAC for the
A1 receptor is as high as CPA for the A2
receptor (11, 27). Moreover, neither CPA nor ADAC is
specific and, at high concentrations, can activate A1
receptors. The present data are unusual in that the A1
receptor, which in most studies mediates a contractile response,
appears to be of the low-affinity type (11, 27). In the
present study, both CPA and ADAC are full agonists capable of inducing
a maximal vasodilator response at high doses, and responses to CPA and
ADAC are not blocked by KF-17837 in doses that block responses to CPCA.
Moreover, responses to CPA and ADAC are blocked by CGS-15943 in a dose
that does not block the response to CPCA. In experiments with the
selective A1 antagonist KW-3501, responses to CPA were
decreased significantly at a time responses to CPCA are not changed.
In regard to the question of receptor affinity, results of experiments
in isolated porcine coronary arterial rings show that CPA causes
contraction at low concentrations (10
8 to
106 M) and relaxation at high concentrations
(106 to 105 M), whereas the A2
agonist
N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]- adenosine (DPMA) caused relaxation at low concentrations
(27). The results of this study in isolated
coronary arterial rings are similar to the present data in that high
concentrations of the A1 agonist CPA were required to
induce vasorelaxation. It is therefore postulated that the
A1 receptor mediating vasodilation in some organ systems may be an usual low-affinity-type receptor.
Reactive hyperemia is the increase in blood flow observed after a
period of arterial occlusion and has been described in a number of
vascular beds from a variety of species (2-5, 8, 28, 35, 39,
42). In the rat mesenteric circulation, adenosine receptor
antagonists reduced the reactive hyperemic response (35). Furthermore, the coronary reactive hyperemic response in the dog was
decreased after treatment with adenosine deaminase, suggesting that
adenosine mediates the increase in blood flow after a brief period of
ischemia (39). These results suggest that
adenosine accounts for approximately one-third of the blood flow
increase after a period of ischemia and suggest that other
vasoactive factors play a role in mediating the hyperemic response. In
the present study under constant-flow conditions in the hindquarter
vascular bed of the cat, the A1 receptor antagonist
CGS-15943 and the A2 receptor antagonist KF-17837
attenuated the maximal observed decrease in hindquarter vascular
resistance, the duration of the reactive hyperemic response, and the
area under the curve after a 30-s period of ischemia (Fig. 11).
The reactive hyperemic response was reproducible with respect to time
and was not dependent on the presence of an intact sympathetic
innervation. The reduction in the response to a 30-s period of
ischemia after treatment with A1 and A2
receptor antagonists suggests that the reactive hyperemic response in
the hindlimb vascular bed is mediated in part by the activation of
adenosine receptors (Fig. 11). The inhibitory effect of treatment with
both CGS-15943 and KF-17837 on the duration of the response to a 30-s
period of ischemia and the percent decrease in hindquarter
perfusion pressure were not significantly greater than the inhibitory
effect of CGS-15943 or KF-17837 alone. The reason that the inhibitory
effect of combined treatment is not significantly greater than
treatment with CGS-15943 or KF-17837 alone is uncertain but may be
related to the complex nature of the reactive hyperemic response. In
previous studies in the hindlimb vascular bed of the cat, the reactive
hyperemic response was in part attenuated by K
antagonists and to a lesser extent by an inhibitor of NO synthesis but
was not altered by a cyclooxygenase inhibitor (28). When
taken together with the present data, these results suggest that, in
the hindlimb circulation of the cat, activation of adenosine receptors
and K channels and the release of NO may all play a
role in mediating the reactive hyperemic response. The results of the
present investigation and of previous studies in the literature provide
support for the hypothesis that the reactive hyperemic response in
several vascular beds in different species may in part involve the
activation of adenosine receptors (35, 39).
In summary, the results of the present investigation suggest that
A1 and A2 receptors mediating vasodilation are
present in the hindquarter vascular bed of the cat and that adenosine
acts on both receptor subtypes to induce vasodilation. Moreover, the present data suggest that vasodilator responses to ATP are not mediated
by adenosine formed from ATP breakdown and that the response to
adenosine is increased in duration by a cAMP phosphodiesterase inhibitor but is not altered by inhibitors of NO synthase or cGMP phosphodiesterase. Furthermore, vasodilator responses to adenosine are
not altered by inhibitors of K channel activation
or the cyclooxygenase pathway. In addition, the reactive hyperemic
response after a brief period of arterial occlusion is dependent in
part on the activation of adenosine receptors. These data suggest that
vasodilator responses to adenosine and the reactive hyperemic response
are mediated at least in part by the activation of A1 and
A2 receptors. These results also suggest that responses to
adenosine do not involve the release of NO, vasodilator prostaglandins,
or the opening of K channels in the hindquarter
vascular bed of the cat and suggest that increases in cAMP levels may
be involved.
Perspectives
The broad implications of the present study are that, since
vasodilator responses to adenosine are reduced by A1 and
A2 selective receptor antagonists, these results suggest
that both receptor subtypes are present and mediate responses to
adenosine. The observation that responses to adenosine are increased in
duration by a cAMP phosphodiesterase inhibitor but are not altered by
inhibitors of K channels, NO synthase, or
cyclooxygenase suggests that the response is mediated in part by an
increase in cAMP levels but that K channel opening,
the release of NO, or vasodilator prostaglandins are not involved. The
observation that the response to reactive hyperemia is reduced by
A1 and A2 receptor antagonists suggests that
the release of adenosine from an endogenous source mediates in part the
reactive hyperemic response by activating A1 and
A2 receptors in the hindlimb circulation.
| |
ACKNOWLEDGEMENTS |
This study was supported by a grant from the American Heart
Association-Louisiana and National Heart, Lung, and Blood Institute Grant HL-62000.
| |
FOOTNOTES |
Address for reprint requests and other correspondence:
P. J. Kadowitz, Dept. of Pharmacology SL83, Tulane Univ.
School of Medicine, 1430 Tulane Ave., New Orleans, LA 70112. (E-mail: pkadowi{at}tulane.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpregu.00394.2001
Received 10 July 2001; accepted in final form 28 January 2002.
| |
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