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

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha -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

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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, Nomega -nitro-L-arginine methyl ester (L-NAME), angiotensin II, III, and IV, norepinephrine hydrochloride, tyramine hydrochloride, alpha ,beta -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, alpha ,beta -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 alpha -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.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha -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 1.   Influence of reserpine (1.5 mg/kg ip) and phentolamine (1 mg/kg iv) on pressor responses in the rat

                              
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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

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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Table 3.   Effects of candesartan and PD-123,319 on mean vascular pressures in the rat


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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).

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).

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).

Selectivity studies. The effects of candesartan on vasoconstrictor responses to U-46619, alpha ,beta -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, alpha ,beta -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 alpha ,beta -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).

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).

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).

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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, alpha ,beta -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 alpha -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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Bauer, P. H., A. T. Chiu, and J. C. Garrison. DuP 753 can antagonize the effects of angiotensin II in rat liver. Mol. Pharmacol. 39: 579-585, 1991[Abstract].

2.   Blankley, J. C., J. C. Hodges, S. R. Klutchko, R. J. Himmelsbach, A. Chucholoski, C. J. Connolly, S. J. Neergaard, M. S. van Nieuwenhze, A. Sebastian, J. Quin, A. D. Essenburg, and D. M. Cohen. Synthesis and structure-activity relationships of a novel series of nonpeptide angiotensin II receptor binding inhibitors specific for the AT2 subtype. J. Med. Chem. 34: 3248-3260, 1991[Medline].

3.   Bottari, S. P., M. DeGasparo, U. M. Stackelings, and N. R. Levens. Angiotensin II receptor subtypes: characterization, signalling mechanisms, and possible physiological implications. Front. Neuroendocrinol. 14: 134-171, 1993.

4.   Campbell, W. J., Jr., J. A. Donohue, and L. H. Duket. Alterations in responses to bradykinin, angiotensin I, and angiotensin II during the induction phase of one-kidney, one-wrapped hypertension and associated arterial disease in rabbits. Am. J. Pathol. 98: 457-484, 1980[Medline].

5.   Champion, H. C., E. A. Garrison, L. S. Estrada, J. M. Potter, and P. J. Kadowitz. Analysis of responses to angiotensin I and angiotensin I-(3-10) in the mesenteric vascular bed of the cat. Eur. J. Pharmacol. 309: 251-259, 1996[Medline].

6.   Champion, H. C., and P. J. Kadowitz. Analysis of effects of candesartan in the mesenteric vascular bed of the cat: evidence for the presence of spare angiotensin AT1 receptors. Hypertension 30: 1060-1066, 1997.

7.   Chang, R. S. L., and V. J. Lotti. Two distinct angiotensin II receptor binding sites in rat adrenal revealed by new selective nonpeptide ligands. Mol. Pharmacol. 37: 347-351, 1990[Abstract].

8.   Cheng, D. Y., B. J. DeWitt, E. L. Dent, B. D. Nossaman, and P. J. Kadowitz. Analysis of responses to angiotensin IV in the pulmonary vascular bed of the cat. Eur. J. Pharmacol. 261: 223-227, 1994[Medline].

9.   Cheng, D. Y., B. J. DeWitt, T. J. McMahon, and P. J. Kadowitz. Comparison of pressor responses to angiotensin I, II, and III in pulmonary vascular bed of cats. Am. J. Physiol. 266 (Heart Circ. Physiol. 35): H2247-H2255, 1994[Abstract/Free Full Text].

10.   Chiu, A. T., W. F. Herblin, D. E. McCall, R. J. Ardecky, D. J. Carini, J. V. Duncia, L. J. Pease, P. C. Wong, R. R. Wexler, A. L. Johnson, and P. M. B. W. M. Timmermans. Identification of angiotensin II receptor subtypes. Biochem. Biophys. Res. Commun. 165: 196-203, 1989[Medline].

11.   De Gasparo, M., S. Whitebread, M. Mele, A. S. Motani, P. J. Whitcombe, H. Ramjoue, and B. Kamber. Biochemical characterization of two angiotensin II receptor subtypes in the rat. J. Cardiovasc. Pharmacol. 16: S31-S35, 1990[Medline].

12.   DiSalvo, J., and C. B. Montefusco. Conversion of angiotensin I to angiotensin II in the canine mesenteric circulation. Am. J. Physiol. 221: 1576-1579, 1971[Medline].

13.   Dudley, D. T., R. L. Panek, T. C. Major, G. H. Lu, R. F. Burns, B. A. Klinkefus, J. C. Hodges, and R. E. Weishaar. Subclasses of angiotensin II binding sites and their functional significance. Mol. Pharmacol. 38: 370-377, 1990[Abstract].

14.   Fleming, J. T., and I. G. Joshua. Mechanism of biphasic arteriolar response to angiotensin II. Am. J. Physiol. 247 (Heart Circ. Physiol. 16): H88-H94, 1984[Abstract/Free Full Text].

15.   Garrison, E. A., and P. J. Kadowitz. Analysis of responses to angiotensin I-(3---10) in the hindlimb vascular bed of the cat. Am. J. Physiol. 270 (Heart Circ. Physiol. 39): H1172-H1177, 1996[Abstract/Free Full Text].

16.   Garrison, E. A., J. A. Santiago, and P. J. Kadowitz. Analysis of responses to angiotensin peptides in the hindquarters vascular bed of the cat. Am. J. Physiol. 268 (Heart Circ. Physiol. 37): H2418-H2425, 1995[Abstract/Free Full Text].

17.   Mizuno, K., S. Nimura, M. Tani, I. Saito, H. Sanada, M. Takahashi, K. Skazaki, M. Yamaguchi, and S. Fukuchi. Hypotensive activity of TCV-116, a newly developed angiotensin II receptor antagonist, in spontaneously hypertensive rats. Life Sci. 51: PL183-PL187, 1992[Medline].

18.   Navar, L. G., E. W. Inscho, D. S. A. Majid, J. D. Imig, L. M. Harrison-Bernard, and K. D. Mitchell. Paracrine regulation of the renal microcirculation. Physiol. Rev. 76: 425-536, 1996[Abstract/Free Full Text].

19.   Noda, M., Y. Shibouta, Y. Inada, M. Ojima, T. Wada, T. Sanada, J. Kubo, Y. Kohara, T. Naka, and K. Nishikawa. Inhibition of rabbit aortic angiotensin II (AII) receptor by CV-11974, a new nonpeptide AII antagonist. Biochem. Pharmacol. 46: 311-318, 1993[Medline].

20.   Osei, S. Y., R. K. Minkes, J. A. Bellan, and P. J. Kadowitz. Analysis of the inhibitory effects of DuP753 and EXP 3174 on responses to angiotensin II in the feline hindquarters vascular bed. J. Pharmacol. Exp. Ther. 264: 1104-1112, 1993[Abstract].

21.   Peach, M. J. Renin-angiotensin system: biochemistry and mechanisms of action. Physiol. Rev. 57: 313-370, 1977[Free Full Text].

22.   Regoli, D., W. K. Park, and N. D. Rioux. Pharmacology of angiotensin. Pharmacol. Rev. 26: 69-123, 1974[Medline].

23.   Rowe, B. P., and A. Nasjeletti. Biphasic blood pressure response to angiotensin II in the conscious rabbit: relation to prostaglandins. J. Pharmacol. Exp. Ther. 255: 559-563, 1983.

24.   Scheuer, D. A., and M. H. Perrone. Angiotensin type 2 receptors mediate depressor phase of biphasic pressure response to angiotensin. Am. J. Physiol. 264 (Regulatory Integrative Comp. Physiol. 33): R917-R923, 1993[Abstract/Free Full Text].

25.   Sechi, L. A., E. F. Grady, C. A. Griffin, J. E. Kalikyak, and M. Schambelan. Distribution of angiotensin II receptor subtypes in a rat and human kidney. Am. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F236-F240, 1992[Abstract/Free Full Text].

26.   Shibouta, Y., Y. Inada, M. Ojima, T. Wada, M. Noda, T. Sanada, K. Kubo, Y. Kohara, T. Naka, and K. Nishikawa. Pharmacological profile of a highly potent and long-lasting angiotensin II receptor antagonist, 2-ethoxy-1-{[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl}-1H-benzimidazole-7-carboxylic acid (CV-11974), and its prodrug, (+)-1-(cyclohexyloxycarbonyloxy)-ethyl-2-ethoxy-1-{[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl}-1H-benzimidazole-7-carboxylate (TCV-116). J. Pharmacol. Exp. Ther. 266: 114-120, 1993[Abstract].

27.   Timmermans, P. B. M. W. M., P. C. Wong, A. T. Chiu, W. F. Herblin, P. Benfield, D. J. Carini, R. J. Lee, R. R. Wexler, J. M. Saye, and R. D. Smith. Angiotensin II receptor and angiotensin II receptor antagonists. Pharmacol. Rev. 45: 205-251, 1993[Medline].

28.   Tofovic, S. P., A. S. Pong, and E. K. Jackson. Effect of angiotensin subtype 1 and subtype 2 receptor antagonists in normotensive versus hypertensive rats. Hypertension 18: 774-782, 1991[Abstract].

29.   Wada, T., Y. Inada, T. Sanada, M. Ojima, Y. Shibouta, M. Noda, and K. Nishikawa. Effect of an angiotensin II receptor antagonist, CV-11974, and its prodrug, TCV-116, on production of aldosterone. Eur. J. Pharmacol. 253: 27-34, 1994[Medline].

30.   Wong, P. C., S. D. Hart, J. V. Duncia, and P. B. M. W. M. Timmermans. Nonpeptide angiotensin II receptor antagonists: studies with DuP 753 and EXP 3174 in dogs. Eur. J. Pharmacol. 202: 323-330, 1991[Medline].

31.   Wong, P. C., S. D. Hart, A. M. Zaspel, A. T. Chiu, R. J. Ardecky, R. D. Smith, and P. B. M. W. M. Timmermans. Functional studies of nonpeptide angiotensin II receptor subtype-specific ligands: DuP 753(AII-1) and PD123177(AII-2). J. Pharmacol. Exp. Ther. 255: 584-592, 1990[Abstract].


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