Role of AT1 receptors and autonomic nervous system in mediating acute pressor responses to ANG II in anesthetized mice

Trinity J. Bivalacqua, Ajay Dalal, Hunter C. Champion, and Philip J. Kadowitz

Department of Pharmacology, Tulane University School of Medicine, New Orleans, Louisiana 70112


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Hemodynamic responses to angiotensin II and the role of AT1 and AT2 receptors and the autonomic nervous system in mediating acute responses to angiotensin II were investigated in anesthetized CD1 mice. Injections of angiotensin II caused dose-related increases in systemic arterial pressure that were antagonized by candesartan. Pressor responses to angiotensin II were not altered by PD-123,319 in doses up to 25 mg/kg iv. At the lowest dose studied (20 µg/kg iv), the inhibitory effects of candesartan were competitive, whereas at the highest dose (100 µg/kg iv) the dose-response curve for angiotensin II was shifted to the right in a nonparallel manner with inhibitory effects that could not be surmounted. The inhibitory effects of candesartan were selective and were similar in animals pretreated with enalaprilat (1 mg/kg iv) to reduce endogenous angiotensin II production. Acute pressor responses to angiotensin II were not altered by propranolol (200 µg/kg iv), phentolamine (200 µg/kg iv), or atropine (1 mg/kg iv) but were enhanced by hexamethonium (5 mg/kg iv). Increases in total peripheral resistance induced by angiotensin II were inhibited by the AT1-receptor antagonist but were not altered by AT2-, alpha -, or beta -receptor antagonists. These results suggest that acute pressor responses to angiotensin II are mediated by AT1 receptors, are buffered by the baroreceptors, and are not modulated by effects on AT2 receptors and that activation of the sympathetic nervous system plays little if any role in mediating rapid hemodynamic responses to the peptide in anesthetized CD1 mice.

angiotensin II; mouse; systemic arterial pressure; AT2 receptors; heart rate; cardiac output


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ANGIOTENSIN II plays an important role in regulating arterial pressure, sodium, and water balance (7, 19, 24, 25, 29). Angiotensin II binds with high affinity to AT1 and AT2 receptors (1, 2, 5-8, 29). Angiotensin II-induced increases in systemic arterial pressure and the release of aldosterone from the adrenal cortex are mediated by AT1 receptors, whereas less is known about the role of the AT2 receptor in the regulation of cardiovascular function (3-6, 8, 12, 14, 24-27, 31, 33). AT1 receptors are widely distributed, and AT1-receptor antagonists are effective in the treatment of essential hypertension and congestive heart failure (1, 5, 7, 20, 22, 32). AT2 receptors are highly expressed in the fetus and are downregulated with time, suggesting that they are involved in growth and development (6, 16, 28). The AT2 receptor has been shown to have an inhibitory effect on cell growth mediated through an effect on the mitogen-activated protein kinase pathway in vascular smooth muscle cells (16, 18, 28). A new approach to the study of the role of AT1 and AT2 receptors in mediating hemodynamic responses to angiotensin II has been the development of models in which AT1 and AT2 receptors have been deleted and AT2 receptors have been overexpressed (10, 11, 15, 23, 30). The results of studies in transgenic mice overexpressing the AT2 receptor and in mice in which AT2 receptors are knocked out suggest a role for the AT2 receptor in the regulation of blood pressure and responses to angiotensin II (10, 11, 15, 23, 30). However, the results of pharmacological studies in the anesthetized cat and rat and in the pithed and sodium-depleted rat do not support the concept that AT2 receptors are involved in mediating or modulating acute hemodynamic responses to angiotensin II (3, 4, 14, 33). In an attempt to characterize acute hemodynamic responses to angiotensin II and to define the role of AT1 and AT2 receptors and the autonomic nervous system in mediating pressor responses to angiotensin II in the mouse, a species in which phenotype changes have been reported in AT1- and AT2-receptor knockouts, the effects of candesartan and of PD-123,319, AT1- and AT2-receptor antagonists, and of autonomic blocking agents on rapid increases in systemic arterial pressure and total peripheral resistance in response to angiotensin II were investigated in anesthetized CD1 mice (10, 11, 15, 23).


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Adult CD1 mice (Harlan Sprague Dawley, Indianapolis, IN) weighing 20-35 g were anesthetized with thiobutobarbital sodium (90 mg/kg ip; Inactin, BYK-Gulden, Constance, Germany) and ketamine hydrochloride (3 mg/kg ip; Ketaset, Fort Dodge Labs, Fort Dodge, IA). Supplemental doses of thiobutobarbital were given intraperitoneally as needed to maintain a uniform level of anesthesia. The trachea was cannulated with PE-90 tubing (Intramedic, Clay Adams, Parsippany, NJ), and the mice spontaneously breathed room air enriched with 95% O2-5% CO2. PE-10 catheters were inserted into an external jugular vein for the intravenous administration of drugs and into a carotid artery for the measurement of systemic arterial pressure. Systemic arterial pressure was measured with a Viggo-Spectramed transducer (Viggo-Spectramed, Oxnard, CA) and was recorded on a Grass model 7 polygraph (Grass Instrument, Quincy, MA). Mean pressure was derived by electronic averaging, and heart rate was determined from the interval between arterial pressure pulses with a tachograph (Grass model P44).

In the first series of experiments in the anesthetized mouse, acute changes in systemic arterial pressure in response to intravenous injections of angiotensin II, norepinephrine, and the thromboxane A2 mimic U-46619 were compared and agonist doses were expressed on a nanomoles per kilogram basis to take molecular weight into account.

In the second set of experiments, the effects of candesartan in doses of 20, 40, and 100 µg/kg iv on responses to angiotensin II were investigated. The effects of candesartan on responses to norepinephrine, U-46619, vasopressin, and acetylcholine were investigated to assess the selectivity of the AT1-blocking effect.

In the third set of experiments, the effects of PD-123,319 in doses of 10 and 25 mg/kg iv on responses to angiotensin II were investigated to determine if activation of AT2 receptors had a role in mediating or modulating acute hemodynamic responses of angiotensin II in the anesthetized mouse.

In the fourth set of experiments, the effects of candesartan and PD-123,319 on responses to angiotensin II were investigated in mice pretreated with enalaprilat (1 mg/kg iv) to inhibit angiotensin-converting enzyme and decrease endogenous angiotensin II production.

In the next set of experiments, the effects of propranolol (200 µg/kg iv), phentolamine (200 µg/kg iv), atropine (1 mg/kg iv), and hexamethonium (5 mg/kg iv) on responses to angiotensin II were investigated to determine the role of the autonomic nervous system in mediating or modulating rapid hemodynamic responses to the peptide in the anesthetized mouse.

In the last set of experiments, increases in total peripheral resistance induced by intravenous injections of angiotensin II were evaluated and cardiac output was measured by the thermodilution technique. A known volume (20 µl plus catheter dead space) of 0.9% NaCl solution at 23°C was injected into the right atrium, and changes in blood temperature were measured in the root of the aorta by a cardiac output computer (Cardiotherm 500, Columbus Instruments, Columbus, OH) equipped with a small animal interface. The thermistor microprobe catheter (Columbus Instruments, Fr-1) was inserted into the right carotid artery and advanced to the aortic arch where aortic blood temperature was measured. A catheter placed in the right jugular vein was advanced to the right atrium for rapid bolus administration of the saline injectate. The saline solution was injected with a constant-rate syringe (Hamilton, Reno, NV) to ensure rapid and repeatable injection of the saline indicator solution. Thermodilution curves, which were inscribed within a 10- to 12-s period, were recorded on a chart recorder (Western Graphtec, Irvine, CA), and systemic arterial pressure was monitored from a catheter in a femoral artery. Catheter placement was verified at postmortem examination. In these experiments, cardiac output was measured during the control period and when systemic arterial pressure attained a peak value after injection of angiotensin II in a dose of 1 µg/kg iv. Total peripheral resistance was calculated by dividing mean arterial pressure by the cardiac output, and the effects of candesartan, PD-123,319, phentolamine, and propranolol were investigated.

Candesartan (Astra Hässle, Mölndal, Sweden) was dissolved in a 1 N Na2CO3-0.9% NaCl solution (1:20). PD-123,319 (Research Biochemical, Natick, MA) was dissolved in 0.9% NaCl. Candesartan and PD-123,319 solutions were made just before use and were injected into the external jugular vein. Angiotensin II, acetylcholine chloride, propranolol hydrochloride, bradykinin, norepinephrine hydrochloride, atropine sulfate, hexamethonium bromide, vasopressin (Sigma, St. Louis, MO), enalaprilat (Merck, Rahway, NJ), diethylamine/nitric oxide (DEA/NO; Research Biochemical, Natick, MA), and phentolamine mesylate (Ciba-Geigy, Summit, NJ) were dissolved in 0.9% NaCl, and the solutions were divided into aliquots and stored in 1-ml plastic tubes in a freezer. U-46619 (Upjohn, Kalamazoo, MI) was dissolved in 100% ethanol at a concentration of 10 mg/ml and was diluted in 0.9% NaCl, and the solutions were divided into aliquots and stored in 1-ml plastic tubes in a freezer. The aliquots were thawed and used on the day of the experiment. During an experiment the agonist solutions were kept on crushed ice. The agonists were administered intravenously in small volumes (10-30 µl) over a period of 10-15 s in a random sequence. In experiments when the effects of antagonists on responses to the angiotensin II were investigated, increases in mean systemic arterial pressure were compared before and beginning 10-20 min after administration of the antagonist. Control experiments with the injection of equal volumes of the saline vehicle or candesartan vehicle had no consistent effect on systemic arterial pressure or on responses to angiotensin II. Increases or decreases in systemic arterial pressure were expressed as peak change from baseline in millimeters of mercury. Changes in cardiac output were expressed in milliliters per minute, and changes in heart rate were measured as percent change from baseline.

The hemodynamic data were analyzed with a one-way ANOVA with a Scheffé's post hoc test or a paired t-test. A P value of less than 0.05 was used as the criterion for statistical significance.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of antagonists and inhibitors. The effects of the antagonists and inhibitors used in this study on baseline values for mean systemic arterial pressure and heart rate are summarized in Table 1. In regard to agents that interfere with the renin-angiotensin system, candesartan in doses up to 100 µg/kg iv had a significant effect on baseline mean arterial pressure or heart rate when values were compared before and 10-20 min after administration of the AT1-receptor antagonist. PD-123,319, when administered in a dose of 10 mg/kg iv, had no significant effect on arterial pressure or heart rate, whereas when administered in a dose of 25 mg/kg iv, there was a significant reduction in mean arterial pressure (Table 1). In regard to administration of the angiotensin-converting enzyme inhibitor enalaprilat (1 mg/kg iv), there was a significant reduction in mean arterial pressure but no significant change in heart rate (Table 1).

                              
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Table 1.   Mean systemic arterial pressure and heart rate before and after administration of antagonists or inhibitors

The effects of the autonomic blocking agents on mean systemic arterial pressure were evaluated, and arterial pressure was decreased significantly after administration of phentolamine (200 µg/kg iv), propranolol (200 µg/kg iv), or hexamethonium (5 mg/kg iv; Table 1). After administration of propranolol, there was a significant reduction in heart rate, whereas atropine (1 mg/kg iv) produced a significant increase in heart rate but no change in mean systemic arterial pressure (Table 1). The greater reduction in systemic arterial pressure and decrease in heart rate after administration of propranolol when compared with hexamethonium may be related to unopposed parasympathetic actions on the heart and/or by the membrane-stabilizing properties of the beta -receptor antagonist.

Pressor responses to angiotensin II. Injections of angiotensin II in doses of 0.1-3 nmol/kg iv in anesthetized CD1 mice caused rapidly developing dose-related increases in systemic arterial pressure (Fig. 1). Angiotensin II was ~10-fold more potent than norepinephrine and the thromboxane A2 mimic U-46619 in increasing systemic arterial pressure in the mouse when doses are expressed on a nanomole basis to take molecular weight into account (Fig. 1).


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Fig. 1.   Dose-response curves comparing acute increases in systemic arterial pressure in response to iv injections of angiotensin II, norepinephrine, and thromboxane A2-receptor agonist U-46619. Doses are expressed on a nmol basis to take molecular weight into account; n = no. of animals.

Increases in systemic arterial pressure in response to angiotensin II injections were associated with small but significant increases in heart rate (Fig. 2; Table 2). Group data illustrating the time course of the acute increase in systemic arterial pressure and in heart rate in response to an intravenous injection of angiotensin II (1 µg/kg iv) are shown in Fig. 2A. The peak increase in arterial pressure occurred within 30 s, whereas the peak increase in heart rate occurred within 50 s (Fig. 2A).


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Fig. 2.   Time course showing means ± SE of grouped data for increase in systemic arterial pressure and heart rate in response to iv injections of angiotensin II (1 µg/kg) during control period (A), after administration of candesartan (100 µg/kg iv; B), and after administration of candesartan and PD-123,319 (10 mg/kg iv) (C). bts/min, Beats per minute.


                              
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Table 2.   Influence of candesartan, PD-123,319, propranolol, and atropine on increases in heart rate in response to intravenous injections of angiotensin II in mice

Effects of the AT1-receptor antagonist. After administration of the AT1-receptor antagonist candesartan in a dose of 20 µg/kg iv, increases in systemic arterial pressure in response to angiotensin II were reduced significantly and the dose-response curve for angiotensin II was shifted to the right in a parallel manner (Fig. 3A). However, in two other groups of mice when the inhibitory effects of candesartan in doses of 40 and 100 µg/kg iv were investigated, pressor responses to angiotensin II were markedly reduced or abolished and the dose-response curves were shifted to the right in a nonparallel manner (Fig. 3, B and C). Group data illustrating the effect of candesartan (100 µg/kg iv) on the increase in systemic arterial pressure and heart rate measured over a 5-min period are shown in Fig. 2, where it can be seen that the increases in pressure and heart rate are markedly reduced or abolished and a secondary decrease in arterial pressure is not observed after administration of the AT1-receptor antagonist (Fig. 2, A and B).


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Fig. 3.   Influence of candesartan on increases in systemic arterial pressure in response to iv injections of angiotensin II. Responses to angiotensin II were determined before and beginning 10 min after administration of candesartan in 3 groups of animals in doses of 20 µg/kg iv (A), 40 µg/kg iv (B), and 100 µg/kg iv (C). Values are means ± SE; n = no. of animals. * Significantly different from control (P <0.05, ANOVA).

Although candesartan antagonized pressor responses to angiotensin II, increases in systemic arterial pressure in response to norepinephrine, U-46619, and vasopressin and decreases in systemic arterial pressure in response to acetylcholine were not altered by the AT1-receptor antagonist candesartan (100 µg/kg iv; Fig. 4). In other experiments, depressor responses to the nitric oxide donor DEA/NO, albuterol, and bradykinin were not altered by candesartan (100 µg/kg iv; data not shown). The increases in heart rate in response to intravenous injections of angiotensin II were reduced significantly after administration of candesartan (100 µg/kg iv), PD-123,319 (25 mg/kg iv), propranolol (200 µg/kg iv), and atropine (1 mg/kg iv; Table 2). Increases in heart rate in response to angiotensin II were not altered after administration of the alpha -receptor antagonist phentolamine (200 µg/kg iv; data not shown).


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Fig. 4.   Influence of candesartan (100 µg/kg iv) on increases in systemic arterial pressure in responses to iv injections of norepinephrine, vasopressin, and thromboxane A2-receptor agonist U-46619 and decreases in systemic arterial pressure in responses to iv injections of acetylcholine; n = no. of animals.

Influence of PD-123,319. The effect of the AT2-receptor antagonist PD-123,319 on increases in systemic arterial pressure in response to angiotensin II were investigated, and these data are summarized in Fig. 5. After administration of PD-123,319 in doses of 10 and 25 mg/kg iv in two groups of mice, increases in systemic arterial pressure in response to angiotensin II were not changed (Fig. 5). PD-123,319 (10 mg/kg iv) did not alter pressor responses to norepinephrine and U-46619 or depressor responses to acetylcholine, DEA/NO, or albuterol (data not shown). The administration of PD-123,319 (10 mg/kg iv) to mice treated with candesartan (100 µg/kg iv) had no effect on the reduction in the response to angiotensin II induced by candesartan (Fig. 2).


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Fig. 5.   Influence of PD-123,319 in doses of 10 (A) and 25 (B) mg/kg iv on increases in systemic arterial pressure in response to iv injections of angiotensin II. Responses to angiotensin II were determined before and beginning 10 min after administration of PD-123,319; n = no. of animals.

Changes in total peripheral resistance. Changes in cardiac output and in total peripheral resistance in response to angiotensin II injections were evaluated in anesthetized CD1 mice, and these data are summarized in Table 3. Cardiac output measured at the peak of the increase in systemic arterial pressure in response to injections of angiotensin II (1 µg/kg iv) was unchanged or increased slightly, whereas calculated total peripheral resistance was increased significantly (Table 3). The increase in total peripheral resistance in response to angiotensin II was not changed significantly after administration of PD-123,319 (10 mg/kg iv), phentolamine (200 µg/kg iv), or propranolol (200 µg/kg iv; Table 3). The increase in total peripheral resistance in response to intravenous injections of angiotensin II was decreased significantly after administration of candesartan (100 µg/kg iv; Table 3). The increase in total peripheral resistance in response to an intravenous injection of angiotensin II was not altered after administration of the candesartan vehicle (1 N Na2CO3 diluted 10:1 in 0.9% NaCl) in a volume of 0.25 ml/kg iv (data not shown).

                              
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Table 3.   Changes in MAP, CO, and TPR in response to angiotensin II after PD-123,319, candesartan, phentolamine, and propranolol in anesthetized mice

Responses in enalaprilat-treated animals. In mice pretreated with enalaprilat in a dose of 1 mg/kg iv to reduce endogenous angiotensin II production, increases in systemic arterial pressure in response to intravenous injections of angiotensin II were decreased significantly by candesartan (100 µg/kg iv; Fig. 6). In mice pretreated with enalaprilat, increases in systemic arterial pressure in response to angiotensin II were not changed by PD-123,319 (25 mg/kg iv; Fig. 6). Treatment with enalaprilat significantly attenuated the increase in systemic arterial pressure in response to angiotensin I and enhanced the decrease in systemic arterial pressure in response to bradykinin but did not alter pressor responses to angiotensin II or to norepinephrine (Fig. 6). Administration of PD-123,319 or candesartan did not significantly decrease arterial pressure in enalaprilat-pretreated mice.


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Fig. 6.   Effects of AT1- and AT2-receptor antagonists on increases in systemic arterial pressure in response to angiotensin I, angiotensin II, norepinephrine (NE), and bradykinin (BK) in mice pretreated with enalaprilat (1 mg/kg iv). Responses to agonists were determined before and beginning 10 min after administration of candesartan (100 µg/kg iv) and PD-123,319 (25 mg/kg iv) in animals pretreated with enalaprilat; n = no. of animals. * Significantly different from control (P < 0.05).

Effects of adrenergic- and cholinergic-receptor antagonists. The effects of phentolamine, propranolol, and atropine on increases in systemic arterial pressure in response to angiotensin II were investigated, and these data are summarized in Fig. 7. After administration of phentolamine in a dose of 200 µg/kg iv, increases in systemic arterial pressure in response to intravenous injections of angiotensin II were not changed, whereas the pressor response to norepinephrine was significantly reduced (Fig. 7A). After administration of propranolol in a dose of 200 µg/kg iv, increases in systemic arterial pressure in response to intravenous injections of angiotensin II were not changed, whereas decreases in systemic arterial pressure in response to isoproterenol were significantly reduced (Fig. 7B). After administration of atropine in a dose of 1 mg/kg iv, pressor responses to angiotensin II were not altered, whereas vasodepressor responses to acetylcholine were significantly decreased (Fig. 7C).


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Fig. 7.   Influence of phentolamine (200 µg/kg iv; A), propranolol (200 µg/kg iv; B), and atropine (1 mg/kg iv; C) on increases in systemic arterial pressure in response to iv injections of angiotensin II. Responses to angiotensin II were determined before and beginning 10 min after administration of phentolamine, propranolol, and atropine; n = no. of animals. * Significantly different from control (P < 0.05).

Effects of hexamethonium. The effects of the ganglionic blocking agent hexamethonium on the increase in systemic arterial pressure in response to intravenous injections of angiotensin II are summarized in Fig. 8. After administration of hexamethonium in a dose of 5 mg/kg iv, the increases in systemic arterial pressure in response to intravenous injection of angiotensin II in doses of 0.3 µg/kg and 1 µg/kg were larger in amplitude and longer in duration as measured by the recovery half-time (t1/2) of the pressor response (Fig. 8).


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Fig. 8.   Influence of hexamethonium (5 mg/kg iv) on increases in systemic arterial pressure in response to iv injections of angiotensin II (0.1 µg/kg-1 µg/kg). Increase in arterial pressure and duration of pressor response as measured by recovery half-time (t1/2) of response were compared before and beginning 10 min after administration of ganglionic blocking agent; n = no. of mice. * Significantly different from control (P < 0.05).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

New findings from these studies in anesthetized CD1 mice are that angiotensin II induced rapid increases in arterial pressure that 1) are associated with a beta -adrenergic receptor-mediated increase in heart rate; 2) are, along with rapidly developing increases in total peripheral resistance, mediated by the activation of AT1 receptors with little if any contribution from the autonomic nervous system; 3) are enhanced by hexamethonium, suggesting that acute pressor responses are buffered by the baroreceptors; and 4) are, along with rapid increases in total peripheral resistance, not modulated by counteracting AT2-receptor-mediated effects.

The results of the present investigation showing that rapid increases in systemic arterial pressure and total peripheral resistance in response to angiotensin II in CD1 mice are antagonized by candesartan, whereas PD-123,319 was without effect, and that the inhibitory effects of candesartan were selective in the mouse are consistent with the hypothesis that acute pressor responses to angiotensin are mediated by the activation of AT1 receptors (3, 4, 7, 12, 14, 33).

In contrast to results with candesartan, the AT2-receptor antagonist PD-123,319 in doses up to 25 mg/kg iv had no significant effect on increases in systemic arterial pressure in response to angiotensin II, suggesting that pressor responses are not mediated or modulated by the actions of the peptide on the AT2 receptor and are consistent with previous results (3, 4, 14, 33). The results of the present study based on the use of an AT2-receptor antagonist and the acute effects of PD-123,319 are not consistent with the results of studies in genetically altered mice in which the AT2 receptor is either deficient or is overexpressed in the heart (10, 11, 15). It has been reported that the targeted disruption of the mouse AT2-receptor gene resulted in an increase in baseline systemic arterial pressure and increased sensitivity to the pressor actions of angiotensin II (11). There is, however, some disagreement about the effects of AT2-receptor deletion on baseline arterial pressure, and the enhanced pressor response to angiotensin II can only be observed after treatment with an angiotensin-converting enzyme inhibitor (10, 11). The results of experiments in the AT2-receptor knockout suggest that AT1 and AT2 receptors mediate mutually counteracting blood pressure responses to angiotensin II (10, 11). However, in the present study systemic arterial pressure was not increased after administration of PD-123,319 in doses of 10-25 mg/kg iv. In addition, the AT2-receptor antagonist did not enhance pressor responses to intravenous injections of angiotensin II in control or enalaprilat-treated mice. Moreover, in experiments with the AT1-receptor antagonist candesartan, pressor responses to angiotensin II were reduced at the lowest dose of the antagonist studied and were abolished after treatment with the highest dose. Although pressor responses to angiotensin II were abolished, a depressor response to angiotensin II was not uncovered. The present results with larger doses of candesartan are consistent with results in the AT1A-AT1B receptor double-knockout mouse in which pressor responses to angiotensin II were abolished and a depressor response was not observed (23). The observation that pressor responses to angiotensin II are not reversed after AT1-receptor blockade is not in agreement with results of a study in the anesthetized rat (26). The reason for the difference in results in the studies of Scheuer and Perrone (26) in the rat and in the present study in the mouse or in previous studies in the regional vascular bed of the rat and cat is uncertain but may involve species difference (3, 4, 11, 33).

The effects of candesartan and of PD-123,319 were similar in control mice and in mice pretreated with enalaprilat to inhibit endogenous angiotensin II production. In enalaprilat-treated animals, pressor responses to angiotensin I were inhibited and depressor responses to bradykinin were enhanced. In animals pretreated with the angiotensin-converting enzyme inhibitor, candesartan in a dose of 100 µg/kg iv completely blocked pressor responses to angiotensin II, and in enalaprilat-pretreated animals, PD-123,319 in a dose of 25 mg/kg iv had no significant effect on the pressor response to angiotensin II.

The results of the present study with acute AT2-receptor blockade in CD1 mice are not in agreement with results of studies in mice lacking the AT2 receptor. Support for the concept that angiotensin II activates AT1 and AT2 receptors with mutually counteracting hemodynamic actions was not obtained in the present experiments in mice or in previous studies in the rat or cat and may involve obvious species differences (3, 4, 14, 33). The difference between earlier studies in rats or cats vs. transgenic mice in addition to species difference may involve chronic manipulation of the AT2 receptor, as opposed to acute administration of an AT2 antagonist. The hypothesis that acute and chronic effects of an AT2-receptor antagonist may be different is suggested by studies showing that chronic administration of PD-123,319 inhibits angiogenesis and vasodilation induced by angiotensin II (17). The reason for the difference in results in the present study and in the study of Ichiki et al. (11) is uncertain but may be due to differences in the strain of mice used or the use of thiobutobarbital anesthesia in our studies. It is also possible that unidentified compensatory mechanisms may develop in AT2-receptor-deficient mice. It is also possible that acute pharmacological blockade of the AT2 receptor does not mimic the phenotype of mice deficient in AT2-receptor expression since birth or the effects of chronic AT2-receptor blockade (11, 17).

In the present experiments in the anesthetized CD1 mouse, intravenous injections of angiotensin II caused small but significant increases in heart rate. The increases in heart rate were attenuated by candesartan, which also antagonized pressor responses to the peptide. The increase in heart rate in response to angiotensin II was antagonized by propranolol, whereas the beta -receptor antagonist in the dose used did not change the increase in arterial pressure in response to the peptide. These data in the anesthetized mouse suggest that increases in heart rate in response to angiotensin II are mediated by an adrenergic mechanism but that the heart rate increase does not in any large measure contribute to the increase in systemic arterial pressure induced by the peptide. The administration of atropine in a dose of 1 mg/kg iv increased baseline heart rate and perhaps, because of the increase in baseline heart rate, attenuated the increase in heart rate but did not alter the pressor response to angiotensin II in the mouse. The results with the muscarinic antagonist provide additional support for the concept that changes in heart rate play a minor role in mediating the acute arterial pressure increase in response to intravenous injections of angiotensin II in the anesthetized CD1 mouse. Moreover, acute increases in systemic arterial pressure in response to angiotensin II were not altered by phentolamine in a dose that attenuated pressor responses to norepinephrine. These data suggest that increases in alpha -adrenergic receptor activity do not play an important role in mediating acute pressor responses to angiotensin II in the mouse. The increase in heart rate in response to angiotensin II was attenuated by PD-123,319 in a dose of 25 mg/kg iv. However, increases in systemic arterial pressure in response to angiotensin II were not altered by the AT2-receptor antagonist. It is, however, uncertain if the inhibitory effect of the high dose of PD-123,319 on the chronotropic response to angiotensin II or on baseline blood pressure is due to AT2-receptor blockade or a nonspecific effect of the high dose of the antagonist.

In a number of studies, the pressor response to angiotensin II is opposed to the baroreceptor reflex, and, as a result, after ganglionic blockade the angiotensin II response is enhanced (9). Moreover, the angiotensin II-mediated increase in sympathetic activity may not be revealed unless the baroreceptors are denervated (9). Therefore, one interpretation of the lack of effect of phentolamine or propranolol is that autonomic blockade may have attenuated the inhibitory effect of the baroreflex while blunting the indirect actions due to sympathoexcitation (9). The observation that increases in arterial pressure in response to angiotensin II are enhanced and prolonged by hexamethonium provides support for the concept that the baroreflex is buffering the increase in arterial pressure in response to angiotensin II in the mouse (9). Furthermore, it is possible that an indirect sympathoexcitatory action of angiotensin II may be revealed by baroreceptor deafferentation in the mouse. However, these experiments are beyond the technical capability of our laboratory at the present time. The measurement of cardiac output and the calculation of angiotensin II-induced increases in total peripheral resistance in animals treated with phentolamine or propranolol provide support for the conclusion that the autonomic nervous system is not involved in mediating the acute hemodynamic response to the peptide. The observation that increases in arterial pressure and total peripheral resistance are blocked by candesartan but are not changed by PD-123,319 provides support for the concept that acute vasoconstrictor responses to the peptide are mediated by actions at the AT1 receptor and that acute responses are not counterbalanced by opposing effects on the AT2 receptor in anesthetized mice.

Cardiac AT2-receptor expression is low in adult mice; however, cardiac-specific overexpression of the AT2 receptor resulted in decreased pressor and chronotropic responses to angiotensin II (15). The change in response to angiotensin II in the transgenic mice overexpressing the AT2 receptor was blocked by PD-123,319 in a dose of 10 mg/kg iv (15). Studies in mice overexpressing the cardiac AT2 receptor suggest that stimulation of cardiac AT2 receptors exerts an inhibitory effect on the pressor action of angiotensin II by attenuating the AT1 receptor-mediated positive chronotropic effects of the peptide (15). These data suggest that, when overexpressed, the AT2 receptor may play a role in modulating the actions of angiotensin II on pacemaker function and, in this manner, alter pressor responses to the peptide (15). However, in the present study in CD1 mice, PD-123,319 in doses up to 25 mg/kg iv did not alter pressor responses to angiotensin II, suggesting that AT2 receptors do not play an important role in modulating rapid blood pressure responses to angiotensin II in the adult mouse. The results of the present study are not surprising in view of studies showing that AT2-receptor expression is very low in the heart of adult mice (15). Although the present data, based on the acute effects of PD-123,319, suggest that AT2 receptors do not play an important role in modulating acute pressor responses to angiotensin II, it is possible that these receptors may assume a more prominent role in pathophysiological conditions, such as heart failure and left ventricular hypertrophy, in which the expression of the AT2-receptor phenotype is upregulated (13, 15, 20).

The results of experiments with the higher dose of candesartan show that pressor responses to angiotensin II are abolished and that the inhibitory effect is not surmounted when larger doses of the peptide up to 100 nmol/kg iv are injected. These data suggest that the inhibitory effect of the high dose of candesartan on the pressor response to angiotensin II is noncompetitive in nature. The mechanism mediating the noncompetitive blockade of pressor responses to angiotensin II is uncertain but a nonspecific inhibitory effect of the AT1-receptor antagonist is unlikely, because pressor responses to norepinephrine, vasopressin, and the thromboxane A2 receptor mimic U-46619 were not altered. It is also unknown if noncompetitive blockade of pressor responses to angiotensin II induced by candesartan will translate into greater antihypertensive efficacy when compared with the actions of agents that are competitive AT1-receptor antagonists.

In summary, the results of the present investigation show that acute increases in systemic arterial pressure and total peripheral resistance in response to angiotensin II are inhibited by the AT1-receptor antagonist candesartan but are not altered by PD-123,319. These data suggest that acute vasoconstrictor responses to angiotensin are mediated by AT1 receptors and that AT2 receptors have little if any role in mediating or modulating rapid hemodynamic responses to angiotensin II. Angiotensin II increased heart rate through a beta -adrenergic receptor mechanism; however, increases in heart rate do not appear to play an important role in mediating pressor responses to angiotensin II in CD1 mice. Pressor responses to angiotensin II are enhanced and prolonged by hexamethonium but are not altered by propranolol or phentolamine, suggesting that the baroreflex buffers responses to the peptide but that increased sympathetic nervous system activity does not contribute to the acute pressor response. These data suggest that acute vasoconstrictor responses to angiotensin II are mediated by AT1 receptors and are not modulated by AT2 receptors and that rapid hemodynamic responses are not dependent on increases in sympathetic nervous system activity in anesthetized CD1 mice.


    FOOTNOTES

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. §1734 solely to indicate this fact.

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.

Received 6 January 1999; accepted in final form 10 June 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Endocrinol Metab 277(5):E838-E847
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