Responses to angiotensin peptides are mediated by
AT1 receptors in the rat
Hunter C.
Champion,
Marc A.
Czapla, and
Philip J.
Kadowitz
Department of Pharmacology, Tulane University School of Medicine,
New Orleans, Louisiana 70112
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ABSTRACT |
The effects of the angiotensin
AT1 and
AT2 receptor antagonists
candesartan and PD-123,319 on hemodynamic responses to angiotensin peptides were investigated in the anesthetized rat. Injections of
angiotensin II and III caused dose-related increases in systemic arterial and in hindquarters perfusion pressure that were reduced in an
insurmountable manner by candesartan. Pressor responses to angiotensin
IV were also attenuated, and a vasodepressor or vasodilator response to
the angiotensin peptides was not unmasked by the
AT1 receptor antagonists
candesartan or losartan. The AT2 receptor antagonist PD-123,319 had no significant effect on increases in systemic arterial and hindquarters perfusion pressure in response to
the angiotensin peptides. Pressor responses to angiotensin peptides
were not altered by adrenergic nerve terminal and
-receptor blocking
agents or by the cyclooxygenase inhibitor sodium meclofenamate but were
increased by an inhibitor of nitric oxide synthase. The present results
suggest that pressor responses to the angiotensin peptides are mediated
by the activation of AT1 receptors
and that AT2 receptors, the
adrenergic system, or cyclooxygenase products do not appear to modulate
hemodynamic responses to the angiotensin peptides in the anesthetized
rat.
arterial pressure; candesartan; PD-123,319; angiotensin II, III,
IV; vasoconstrictor-vasodilator responses; hindquarters vascular bed; adrenergic system
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INTRODUCTION |
ANGIOTENSIN II is a potent vasoactive peptide formed
from angiotensin I that plays an important role in the regulation of vasomotor tone, sodium, and water homeostasis (13, 16-18, 21, 22,
27). Vasoconstrictor responses to angiotensin II are mediated by the
activation of angiotensin AT1
receptors in a number of vascular beds in a variety of species (1, 5,
6, 10, 12, 13, 18, 25, 30). Angiotensin II can be converted to
angiotensin III and IV by aminopeptidases, and angiotensin II and III
have similar AT1 receptor-mediated
pressor activity in the regional vascular bed of the cat (5, 9, 15, 16, 19). Angiotensin IV appears to have full agonist activity in the
regional vascular bed of the cat but has 100-fold less affinity for the
AT1 receptor (5, 8, 9, 13, 15,
16). Although angiotensin II and III bind to both
AT1 and
AT2 receptor subtypes, it appears
that most physiological responses are mediated by
AT1 receptor activation and
little, if anything, is known about the functional significance of
AT2 receptors in the
cardiovascular system (1, 2, 5-7, 11, 13, 20, 24, 25). It has been
reported that angiotensin II can produce biphasic changes in systemic
arterial pressure in the rabbit and rat (4, 14, 23, 24, 28). It has
also been reported that AT2
receptors mediate the depressor phase of a biphasic response to
angiotensin II and III in the anesthetized rat (24). In contrast, it
has been reported that the AT2
receptor antagonist PD-123,319 in intravenous doses up to 20 mg/kg did
not modify vasoconstrictor responses to angiotensin peptides in the
regional vascular bed of the cat (5). The difference in results in
regard to the biphasic character of the response to angiotensin II and
III and the effects of the AT2
receptor antagonist on hemodynamic responses to the angiotensin peptides may be due to differences in species (24). The present study
was, therefore, undertaken to investigate responses to angiotensin II,
III, and IV and the effects of the
AT1 and
AT2 receptor antagonists on
hemodynamic responses to angiotensin peptides in the systemic vascular
bed of the rat.
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MATERIALS AND METHODS |
Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) of either
sex, weighing 305-490 g, were used in these experiments and were
housed in groups in a room maintained at 70°F with a 12:12-h
light-dark cycle. The rats were fed Laboratory Rodent Diet no. 5001 (PMI Feeds, St. Louis, MO), and water was available ad libitum. The
animals were anesthetized with pentobarbital sodium (Nembutal sodium
solution, 50 mg/kg ip; Abbott Laboratories, North Chicago, IL) or
thiobutabarbital (Inactin, 100 mg/kg ip; BYK-Gulden, Constance,
Germany). Supplemental doses of anesthetic were given as needed to
maintain a uniform level of anesthesia. The trachea was cannulated with
a polyethylene catheter (PE-240; Intramedic, Clay Adams, Sparks, MD),
and the rats either breathed room air or were ventilated with room air
enriched with 95% O2-5%
CO2 by a Harvard model 683 (South
Natick, MD) rodent ventilator at a tidal volume of 2.5 ml at a rate of
30 breaths/min. Polyethylene catheters (PE-50) were inserted into an
external jugular vein for the intravenous administration of drugs and
into the carotid artery for the measurement of systemic arterial
pressure. Systemic arterial pressure was measured with a
Viggo-Spectramed transducer (Oxnard, CA) and was recorded on a Grass
model 7 polygraph (Grass Instrument, Quincy, MA). Mean pressure was
derived by electronic averaging.
For studies in the hindquarters vascular bed, a 1.0- to 1.5-cm segment
of the distal aorta was exposed through a ventral midline incision and
cleared of surrounding connective tissue. After administration of
heparin sodium (1,000 U/kg iv), the aorta was ligated and catheters were inserted into the aorta proximal and distal to the ligature. Blood
was withdrawn from the proximal catheter and pumped at a constant-flow
rate with a Masterflex pump (Cole-Parmer Instrument, Chicago, IL) into
the distal aortic catheter. Perfusion pressure was measured from a
lateral tap in the perfusion circuit between the pump and the distal
aortic catheter. Hindquarters perfusion and systemic arterial pressures
were measured with Viggo-Spectramed transducers and were recorded on a
Grass model 7 polygraph. The pump flow rate was set so that perfusion
pressure was ~125 mmHg and was not changed during an experiment. The
perfusion pump flow rate was calibrated by timed collection of blood
from the outlet side of the perfusion pump, and the perfusion rate
ranged from 3 to 6 ml/min. Agonists were injected directly into the
hindquarters perfusion circuit distal to the pump in small volumes
(30-100 µl) in a random sequence. Responses to 3-4 agonists
could be investigated in the same animal, and systemic arterial or
hindquarters perfusion pressure was allowed to return to control value
before the next dose of an agonist was injected. The interval between
agonist injections was ~10 min. The hindquarters vascular bed was
denervated by ligating and cutting the lumbar sympathetic chain ganglia
bilaterally between L3 and L4. The extent of vascular isolation of the
hindquarters vascular bed was assessed by measuring pump-off occlusion
pressure, which usually approached small vein pressure (10-15
mmHg), indicating low collateral inflow and adequate vascular
isolation.
Candesartan (CV-11974,
2-ethoxy-1-{[2'-(1H-tetraxol-5-yl)biphenyl-4-yl]methyl}-1H-benzimadazole-7-carboxylic
acid; Astra-Hassle, Molndal, Sweden) was dissolved in a 1 N
Na2CO3/0.9%
NaCl solution (1:20). Losartan sodium (DUP-753; DuPont-Merck,
Wilmington, DE) was dissolved in a 5%
NaHCO3-dextrose (50:50) solution.
Sodium meclofenamate,
N
-nitro-L-arginine methyl ester
(L-NAME), angiotensin II, III, and IV, norepinephrine hydrochloride, tyramine hydrochloride,
,
-methylene ATP, calcitonin gene-related peptide (CGRP; Sigma Chemical, St. Louis, MO), PD-123,319 (Research Biochemicals, Natick, MA), reserpine phosphate (Serpasil), and phentolamine mesylate (CIBA-GEIGY, Summit, NJ) were dissolved in 0.9% NaCl. U-46619 (Upjohn,
Kalamazoo, MI) was dissolved in 100% ethanol at a concentration of 10 mg/ml and was diluted in 0.9% NaCl. BAY K 8644 (Miles, New Haven, CT)
was dissolved in a 1:4 solution of cremophor EL,
tris(hydroxymethyl)aminomethane (Tris), and
Tris · HCl (50 mM, pH 7.4). The resulting suspension was warmed, and polyethylene glycol and Tris (pH 7.4) were added to
make a stock solution that was stored in a brown bottle in a freezer.
Working solutions of all agonists were prepared on a frequent basis,
stored in brown, stoppered glass bottles, and kept on crushed ice
throughout the duration of the experiment. The agonists used in these
studies were injected intravenously or directly into the hindquarters
perfusion circuit. Vehicles for the agonists and antagonists did not
alter systemic arterial or hindquarters perfusion pressure or responses
to the vasoactive agents.
In the first series of experiments, responses to angiotensin II, III,
and IV and the effects of candesartan and PD-123,319 on changes in
systemic arterial pressure in response to the angiotensin peptides were
investigated. The doses of candesartan used were based on doses used in
previous studies and on pilot studies in which it was observed that
responses to angiotensin II and III were greatly diminished or
abolished by candesartan in intravenous doses of 100-1,000 µg/kg
(6, 17, 19, 26). Candesartan has been reported to be potent and highly
selective for the AT1 receptor (6,
17, 19, 26). The selectivity of the inhibitory effect of the compound
was assessed by investigating the effects of the
AT1 receptor antagonist on
responses to norepinephrine, U-46619,
,
-methylene ATP, BAY K
8644, and CGRP, and the duration of the angiotensin receptor blockade
was assessed by following responses to angiotensin II for a period of 3 h after administration of the antagonist. The inhibitory effects of
larger (10 mg/kg iv) and smaller (10 µg/kg iv) doses of candesartan
on responses to the angiotensin peptides were also investigated. The
effects of the AT2 receptor
antagonist PD-123,319 in an intravenous dose of 10 mg/kg on responses
to the angiotensin peptides were investigated (24). In binding studies
PD-123,319 has been shown to have high affinity for the
AT2 receptor, and the dose of
PD-123,319 used was based on previous studies in the literature (2, 3,
10, 11, 24). It should be pointed out, however, that, since a selective
AT2 receptor agonist is not
available, the efficacy of the AT2
receptor blockade cannot be assessed in in vivo experiments (5).
Because changes in total peripheral resistance and in cardiac output
contribute to the changes in systemic arterial pressure in response to
the angiotensin peptides, vascular responses were further analyzed
under conditions of controlled blood flow in the hindlimb vascular bed
of the rat, where changes in perfusion pressure directly reflect
changes in regional vascular resistance and denervation prevented
reflex changes in perfusion pressure.
In experiments in which the role of the adrenergic nervous system in
mediating increases in systemic arterial pressure was assessed, the
effects of pretreatment with the adrenergic neuronal blocking agent
reserpine and treatment with the
-receptor-blocking agent
phentolamine were investigated. Pretreatment with reserpine (1.5 mg/kg
ip) 24 h before the experiment resulted in a decrease in systemic
arterial pressure from 125 ± 4 to 95 ± 5 mmHg compared with
values in control animals. Administration of phentolamine in an
intravenous dose of 1 mg/kg decreased systemic arterial pressure from
131 ± 6 to 107 ± 5 mmHg. The administration of the cyclooxygenase inhibitor sodium meclofenamate in an intravenous dose of
2.5 mg/kg had no consistent effect on systemic arterial pressure. The
nitric oxide synthase inhibitor
L-NAME increased systemic
arterial pressure from a baseline value of 122 ± 9 to 209 ± 10 mmHg when administered in an intravenous dose of 50 mg/kg.
The hemodynamic data are expressed in absolute millimeters Hg change
from baseline and were analyzed using a one-way analysis of variance
and Scheffé's F-test with a
Bonferroni-Dunn procedure or a paired
t-test. A
P value of <0.05 was used as the
criterion for statistical significance.
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RESULTS |
Analysis of responses to angiotensin II: role of the adrenergic
nervous system, the cyclooxygenase pathway, and nitric oxide.
Intravenous injections of angiotensin II and of angiotensin III in
doses of 0.1-3 µg/kg caused dose-related increases in systemic arterial pressure in the anesthetized rat (Fig.
1). Intravenous injections of angiotensin
IV in doses of 10-100 µg/kg induced dose-related increases in
systemic arterial pressure, and this fragment was ~100-fold less
potent than angiotensin II or III in increasing systemic arterial
pressure in the rat (Fig. 1). The role of the adrenergic system in
mediating or modulating responses to angiotensin II and III was
investigated, and increases in systemic arterial pressure in response
to intravenous injections of angiotensin II and III were not altered in
animals pretreated with reserpine (1.5 mg/kg ip) 24 h before the
experiment or in animals that were treated with the
-receptor-blocking agent phentolamine in an intravenous dose of 1 mg/kg (Table 1). Reserpine pretreatment significantly enhanced the pressor response to intravenous injections of norepinephrine and significantly reduced the pressor response to
intravenous injections of tyramine (Table 1). Phentolamine significantly reduced the pressor response to intravenous injections of
norepinephrine (Table 1). Increases in systemic arterial pressure in
response to angiotensin II and III were not altered after treatment with the cyclooxygenase inhibitor sodium meclofenamate (2.5 mg/kg iv;
Table 2). The role of nitric oxide release in modulating pressor responses to angiotensin II and III was investigated, and,
after treatment with the nitric oxide synthase inhibitor L-NAME (50 mg/kg iv), pressor
responses to angiotensin peptides and norepinephrine were increased
significantly (Table 2).

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Fig. 1.
Effect of candesartan (1 mg/kg iv) on increases in systemic arterial
pressure in response to iv injections of angiotensin II, III, and IV.
Responses were determined before and beginning 10 min after
administration of the AT1 receptor
antagonist, and responses to larger doses of angiotensin II were
determined after administration of candesartan.
n, Number of animals. Values are
expressed as means ± SE. * Significantly different from
control [P <0.05, analysis of
variance (ANOVA)].
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Table 2.
Influence of L-NAME (50 mg/kg iv) and
sodium meclofenamate (2.5 mg/kg iv) on pressor
responses in the rat
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Role of AT1 and
AT2 receptors.
The role of AT1 and
AT2 receptors in mediating or
modulating pressor responses to angiotensin II and III was
investigated, and candesartan and PD-123,319 in the doses used had no
significant effect on baseline systemic arterial pressure when values
were compared before and 10-20 min after administration of
candesartan in intravenous doses of 1 and 10 mg/kg and PD-123,319 in a
dose of 10 mg/kg (Table 3). The increases in systemic
arterial pressure in response to intravenous injections of angiotensin
II and III were reduced significantly after administration of the
AT1 receptor antagonist
candesartan in an intravenous dose of 1 mg/kg, and there was little
tendency for the blockade to be surmounted or overcome when doses of
angiotensin II up to 1,000 µg/kg were injected intravenously after
administration of the AT1 receptor
antagonist (Fig. 1). The AT1
receptor blockade induced by candesartan was long in duration, and
pressor responses to angiotensin II were markedly attenuated for
periods up to 3 h after administration of the
AT1 receptor antagonist (Fig.
2). Although pressor responses to angiotensin II were
markedly suppressed for periods up to 3 h after administration of the
AT1 receptor antagonist, increases in systemic arterial pressure in response to intravenous injections of
norepinephrine were not altered during this same time period (Fig. 2).

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Fig. 2.
Time course of effect of candesartan on responses to injections of
angiotensin II (0.3 µg/kg iv; top)
and norepinephrine (1 µg/kg iv;
bottom). Responses to angiotensin II
and norepinephrine were determined before and at intervals up to 180 min after administration of AT1
receptor antagonist in a dose of 1 mg/kg iv. * Significantly
different from control (P <0.05,
ANOVA).
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The role of the AT2 receptor in
mediating or modulating pressor responses to angiotensin II and III was
investigated, and these data are summarized in Fig. 3.
Increases in systemic arterial pressure in response to angiotensin II
and III were not changed after administration of the
AT2 receptor antagonist PD-123,319 in an intravenous dose of 10 mg/kg (Fig. 3). The subsequent
administration of candesartan (1 mg/kg iv) significantly attenuated
pressor responses to angiotensin II and III (Fig. 3). PD-123,319, in an
intravenous dose of 10 mg/kg, had no significant effect on the increase
in systemic arterial pressure in response to intravenous injections of
angiotensin IV (Fig. 3). However, subsequent administration of
candesartan (1 mg/kg iv) significantly attenuated the pressor response
to angiotensin IV (Fig. 3). Increases in systemic arterial pressure in
response to norepinephrine were not altered by PD-123,319 or by
PD-123,319 and candesartan (Fig.
3).

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Fig. 3.
Influence of PD-123,319 (10 mg/kg iv) and subsequent administration of
candesartan (1 mg/kg iv) in the same group of animals on increases in
systemic arterial pressure in response to iv injections of angiotensin
II, III, and IV, and norepinephrine. * Significantly different
from control (P <0.05, ANOVA).
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Analysis of responses to angiotensin peptides in the hindquarters
vascular bed.
Under constant-flow conditions, injections of angiotensin II, III, and
IV into the perfusion circuit induced dose-related increases in
hindquarters perfusion pressure (Fig. 4).
The effects of candesartan on hindquarters vasoconstrictor responses to
the angiotensin peptides were investigated, and these data are also summarized in Fig. 4. After the administration of candesartan in an
intravenous dose of 1 mg/kg, increases in hindquarters perfusion pressure in response to angiotensin II, III, and IV were significantly decreased (Fig. 4). Responses to higher doses of angiotensin II were
evaluated after administration of candesartan, and the
AT1 receptor blockade was not
surmounted or overcome when doses of angiotensin II up to 100 µg were
injected into the hindquarters perfusion circuit (Fig. 4).
Vasoconstrictor responses to norepinephrine were not altered after
administration of candesartan in an intravenous dose of 1 mg/kg (Fig.
4). The administration of the AT2
receptor antagonist PD-123,319 in an intravenous dose of 10 mg/kg had
no significant effect on increases in hindquarters perfusion pressure in response to angiotensin II or III (Fig.
5). However, the subsequent administration
of candesartan in these animals in a dose of 1 mg/kg significantly
decreased hindquarters vasoconstrictor responses to angiotensin II and
III (Fig. 5).

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Fig. 4.
Influence of candesartan on increases in hindquarters perfusion
pressure in response to injections of angiotensin II, III, and IV, and
norepinephrine. Responses to angiotensin peptides were determined
before and beginning 10 min after administration of candesartan (1 mg/kg iv). Peptides were injected directly into hindquarters perfusion
circuit, and larger doses of angiotensin II were injected after
candesartan was administered. * Significantly different from
control (P <0.05, ANOVA).
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Fig. 5.
Influence of PD-123,319 (10 mg/kg iv) and subsequent administration of
candesartan (1 mg/kg iv) in the same group of animals on increases in
hindquarters perfusion pressure in response to injections of
angiotensin II (top) and III
(bottom). * Significantly
different from control (P <0.05,
ANOVA).
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Selectivity studies.
The effects of candesartan on vasoconstrictor responses to U-46619,
,
-methylene ATP, and BAY K 8644 and on vasodilator responses to
CGRP were investigated in the hindquarters vascular bed of the rat, and
these data are summarized in Fig. 6. After
administration of candesartan in an intravenous dose of 1 mg/kg,
increases in hindquarters perfusion pressure in response to U-46619,
,
-methylene ATP, and BAY K 8644 and decreases in perfusion
pressure in response to CGRP were not altered (Fig. 6).

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Fig. 6.
Effect of candesartan on increases in hindquarters perfusion pressure
in response to the thromboxane A2
mimic U-46619, the purinergic P2x
receptor agonist , -methylene ATP, and the calcium channel opener
BAY K 8644 and on decreases in perfusion pressure in response to
calcitonin gene-related peptide (CGRP). Responses were determined
before and beginning 10 min after administration of candesartan in a
dose of 1 mg/kg iv. n, Number of
experiments. * Significantly different than control
(P <0.05, ANOVA).
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Effects of a larger dose of candesartan and of losartan.
The effects of a larger dose of candesartan and of losartan on
responses to angiotensin II and III were investigated to determine if a
vasodepressor or vasodilator response could be unmasked, and these
results are summarized in Figs. 7 and
8. After administration of candesartan in
an intravenous dose of 10 mg/kg, increases in systemic arterial
pressure in response to intravenous injections of angiotensin II and
III and increases in hindquarters perfusion pressure in response to
intra-arterial injections of angiotensin II and III were abolished and
a vasodepressor or vasodilator response was not unmasked (Fig. 7).
Candesartan in an intravenous dose of 10 mg/kg had no significant
effect on pressor responses to intravenous or intra-arterial injections
of norepinephrine (data not shown).

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Fig. 7.
Effect of a large dose of candesartan (10 mg/kg iv) on increases in
systemic arterial pressure in response to iv injections of angiotensin
II and III in 1 group of animals (left
panels) and on increases in hindquarters perfusion pressure in response
to ia injections of angiotensin II and III in a second group of animals
(right panels). Responses to
angiotensin peptides were determined before and beginning 10 min after
administration of candesartan in a dose of 10 mg/kg iv.
n, Number of animals.
* Significantly different from control
(P <0.05, ANOVA).
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Fig. 8.
Top: influence of losartan (DUP-753;
10 mg/kg iv) on increases in systemic arterial pressure in response to
iv injections of angiotensin II and angiotensin III in 1 group of
animals. Bottom: influence of
PD-123,319 (10 mg/kg iv) and subsequent administration of losartan (10 mg/kg iv) on increases in systemic arterial pressure in response to iv
injections of angiotensin II and III in a second group of animals.
* Significantly different from control
(P <0.05, ANOVA).
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The administration of PD-123,319 (10 mg/kg iv) had no significant
effect on the pressor response to angiotensin II and III, whereas
responses to the angiotensin peptides were significantly reduced after
the subsequent administration of losartan (10 mg/kg iv) in these same
animals (Fig. 8).
The effects of a lower dose of candesartan on responses to the
angiotensin peptides were investigated, and these data are summarized
in Fig. 9. After administration of
candesartan in an intravenous dose of 10 µg/kg, increases in systemic
arterial pressure in response to intravenous injections of angiotensin
II and III were reduced significantly (Fig. 9). Pressor responses to
intravenous injections of norepinephrine were not changed after
administration of candesartan (10 µg/kg iv) (data not shown).

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Fig. 9.
Effect of a low dose of candesartan (10 µg/kg iv) on increases in
systemic arterial pressure in response to iv injections of angiotensin
II, III, and IV. Responses to angiotensin peptides were determined
before and beginning 10 min after administration of
AT1 receptor antagonist.
* Significantly different from control
(P <0.05, paired
t-test).
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DISCUSSION |
The present results show that angiotensin II and III increase systemic
arterial and hindquarters perfusion pressures in a dose-related manner
in the rat. The increases in systemic arterial and hindquarters
perfusion pressure in response to angiotensin II and III were markedly
inhibited or abolished by candesartan, an angiotensin
AT1 receptor antagonist. The
AT1 receptor blockade was not
surmounted or overcome when larger doses of angiotensin II were
injected after administration of candesartan, and the dose-response
curves for angiotensin II and III were shifted to the right in a
nonparallel manner and exhibited very little positive slope. Although
responses to angiotensin II and III were markedly attenuated,
candesartan had no significant effect on increases in systemic arterial
or hindquarters perfusion pressure in response to norepinephrine. The
selectivity of the inhibitory effects of candesartan was further
studied in the hindquarters vascular bed; and, in addition to not
altering responses to norepinephrine, the
AT1 receptor antagonist in a dose
(1 mg/kg iv) that markedly inhibited responses to the angiotensin
peptides had no effect on vasoconstrictor responses to U-46619,
,
-methylene ATP, and BAY K 8644 or on vasodilator responses to
CGRP. The inhibitory effect of candesartan on pressor responses to the
angiotensin peptides was long in duration with little tendency for
responses to return toward control value at periods up to 3 h after
administration of the AT1 receptor
antagonist. Increases in systemic arterial pressure in response to
norepinephrine did not change over this 3-h period after administration
of the AT1 receptor antagonist, suggesting that vascular responsiveness was not altered. The present data suggest that candesartan is a selective, potent, long-acting angiotensin AT1 receptor
antagonist and that the AT1
receptor blockade induced by candesartan in intravenous doses of 1 and 10 mg/kg is noncompetitive in nature. The inhibitory effects of candesartan on increases in systemic arterial and hindquarters perfusion pressure in response to angiotensin II, III, and IV were
similar. These data indicate that increases in systemic arterial pressure and hindquarters vascular resistance in response to
angiotensin II, III, and IV are mediated by an
AT1 receptor mechanism and are
consistent with previous studies (3, 5-7, 9, 15-17).
Although increases in systemic arterial and hindquarters perfusion
pressure in response to angiotensin II, III, and IV were attenuated or
abolished by candesartan, a hypotensive or vasodilator response was not
observed after administration of the
AT1 receptor antagonist. These
data suggest that vasoconstriction is the predominant response to the
angiotensin peptides, and that this response is mediated by the
activation of AT1 receptors in the
rat. The present data indicate that blockade of the
AT1 receptor with candesartan or
losartan does not unmask a vasodilator response. Moreover, when the
dose of candesartan was increased to 10 mg/kg iv, increases in systemic
arterial and hindquarters perfusion pressures in response to
angiotensin II and III were abolished, and a vasodepressor or
vasodilator response was not observed. It is possible that at high
doses candesartan may also block
AT2 receptors; however, in a
manner similar to that observed with candesartan, responses to
angiotensin II or III were not reversed by losartan in the present
study. The increases in systemic arterial pressure in response to
angiotensin II, III, and IV were not altered by the AT2 receptor antagonist
PD-123,319, whereas administration of candesartan to the same animals
inhibited pressor responses to the angiotensin peptides. Increases in
hindquarters perfusion pressure in response to angiotensin II and III
were not altered by PD-123,319. These data are consistent with previous
studies in the regional vascular bed of the cat and provide support for the hypothesis that increases in systemic arterial and hindquarters perfusion pressure in response to the angiotensin peptides are mediated
by the activation of AT1 receptors
and that AT2 receptors play
little, if any, role in modulating these responses in the rat (5, 6).
The present data with PD-123,319 are consistent with results of studies
with the AT2 receptor antagonists
PD-123,177 in the rat and PD-123,319 in the regional vascular bed of
the cat (5, 6, 8, 9, 16, 29, 31). However, the efficacy of the
AT2 receptor blockade induced by
PD-123,317 or PD-123,319 cannot be confirmed in in vivo studies, since
a selective AT2 receptor agonist
is not available at the present time.
It has been reported that angiotensin II and III induce biphasic
changes in systemic arterial pressure in the anesthetized rat, and that
losartan eliminated the pressor response and enhanced the depressor
response (24). In the present studies, however, biphasic changes in
pressure in response to the angiotensin peptides were almost never
observed and responses to angiotensin II and III were not enhanced by
the AT2 receptor antagonist
PD-123,319. These data indicate that angiotensin peptides do not induce
vasodilation in the hindquarters vascular bed of the rat, that
increases in systemic arterial and hindquarters perfusion pressure in
response to the peptides were attenuated or abolished but were not
reversed by candesartan in doses of 1 and 10 mg/kg iv, and that similar results were obtained with the competitive
AT1 antagonist losartan. These
data suggest that AT2 receptors do
not mediate a vasodilator response or modulate pressor responses to the
angiotensin peptides in the systemic and hindquarters vascular beds in
the rat and are in agreement with previous studies (5, 6, 15, 16, 20).
However, the efficacy and selectivity of the
AT2 receptor blockade must be
clarified in future studies when a selective AT2 receptor agonist is developed.
The reason for the difference in results in the present study and in
previous studies in the rat is uncertain and does not involve
differences in anesthetic, since similar data were obtained with
pentobarbital or thiobutabarbital anesthesia, but may involve differences in experimental procedure employed (24). The results of the
present study showing that candesartan inhibits angiotensin II-induced
pressor responses in an insurmountable or noncompetitive manner are
consistent with results in isolated rabbit aorta and in the mesenteric
vascular bed of the cat (6, 19, 26). The results of the present study
extend previous work by showing that candesartan attenuates
hindquarters vasoconstrictor responses to angiotensin II, III, and IV
in a selective noncompetitive manner in the rat and that a depressor
response is not observed under constant-flow conditions after
administration of the AT1 receptor antagonist.
The role of the adrenergic nervous system in mediating pressor
responses to the angiotensin peptides in the rat was investigated and,
after treatment with the adrenergic neuronal blocking agent reserpine,
responses to intravenous injections of the angiotensin peptides were
not changed. Pretreatment with reserpine decreased the pressor response
to the indirect-acting agonist tyramine and enhanced the response to
norepinephrine, indicating that adrenergic nerve terminal function was
impaired. The effect of the
-receptor blocking agent phentolamine
and reserpine on responses to the angiotensin peptides was similar.
Treatment with phentolamine in a dose that reduced the pressor response
to norepinephrine was without effect on pressor responses to
angiotensin II and III. The results with reserpine and with
phentolamine indicate that, under the conditions of the present
experiments, the increases in systemic arterial pressure in response to
intravenous injections of angiotensin II and III, which are mediated by
the activation of AT1 receptors,
are not dependent on an interaction with the adrenergic nervous system.
In addition to having an interaction with the adrenergic nervous
system, it has been postulated that angiotensin peptides act on the
endothelium and that responses are modulated by the release of
endothelial factors (27). To ascertain the role of endothelial factors,
the effects of the cyclooxygenase inhibitor sodium meclofenamate and of
the nitric oxide synthase inhibitor L-NAME on responses to the
angiotensin peptides were investigated. The results with meclofenamate
in a dose that attenuates responses to the prostaglandin precursor,
arachidonic acid, suggest that increases in systemic arterial pressure
in response to intravenous injections of angiotensin II and III are not
modulated by the release of vasodilator products in the cyclooxygenase
pathway. Nitric oxide is released from the endothelium and could
modulate pressor responses to the angiotensin peptides. The results of the present study with L-NAME
show that pressor responses to angiotensin II and III are increased
significantly. However, this effect is not specific for angiotensin
peptides in that pressor responses to norepinephrine were also
increased to a similar extent. The results of these experiments when
taken together suggest that responses to the angiotensin peptides are
mediated by the activation of AT1
receptors on vascular smooth muscle and that the release of
norepinephrine, vasodilator prostaglandins, or the activation of
AT2 receptors does not play an
important role in mediating or modulating the pressor response. It
should, however, be pointed out that the in vivo
AT2 receptor blocking properties
of PD-123,319 require further study. The results with
L-NAME suggest, however, that
pressor responsiveness may be modulated by the release of nitric oxide
from the endothelium.
In conclusion, the results of the present study show that
AT1 receptor antagonists inhibit
responses to angiotensin II, III, and IV in the systemic and
hindquarters vascular bed in the rat. These data suggest that the
antagonism induced by candesartan in intravenous doses of 1 and 10 mg/kg is noncompetitive in nature and selective and that pressor
responses to the angiotensin peptides are not reversed by
AT1 receptor antagonists in the
rat. The AT2 receptor antagonist
PD-123,319 did not alter responses to the angiotensin peptides in the
systemic or hindquarters vascular bed of the rat. The present data
suggest that the predominant response to the angiotensin peptides is an
AT1 receptor-mediated pressor
response that is not dependent on an interaction with the adrenergic
nervous system and that AT2
receptors or the release of cyclooxygenase products does not appear to
play an important role in modulating pressor responses to the
angiotensin peptides in the anesthetized rat.
 |
FOOTNOTES |
Address for reprint requests: P. J. Kadowitz, Dept. of Pharmacology,
Tulane Univ. School of Medicine, 1430 Tulane Ave., New Orleans, LA
70112.
Received 17 July 1997; accepted in final form 29 September 1997.
 |
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