Nonpeptide angiotensin II receptor antagonists. XI. Pharmacology of EXP3174: an active metabolite of DuP 753, an orally active antihypertensive agent

This report describes the pharmacology of (2-n-butyl-4-chloro-1- [(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazole-5-carboxylic acid (EXP3174). EXP3174 is a major metabolite generated after the oral dosing of 2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H- tetrazol-5-yl)biphenyl-4-yl)methyl]imidazole, potassium salt in rats. It displaced [3H]angiotensin II (AII) from its specific binding sites in rat adrenal cortical membranes with an IC50 of 3.7 x 10(-8) M. In the isolated rabbit aorta, EXP3174 caused nonparallel shifts to the right of the AII concentration-contractile response curves and reduced the maximal response by 30 to 40% with an apparent pA2 value of 10.09 and a KB value of 10(-10) M. At 10(-6) M, EXP3174 did not alter the contractile responses to norepinephrine and KCl. In the spinal pithed rat, EXP3174 at 0.03 to 0.3 mg/kg i.v. also inhibited the pressor responses to AII and angiotensin III noncompetitively and did not change the pressor responses to vasopressin and norepinephrine. When given i.v. and cumulatively to normotensive rats at 0.003 to 0.3 mg/kg, EXP3174 did not alter blood pressure but inhibited the pressor response to AII. In conscious renal artery-ligated rats, EXP3174 decreased blood pressure with an i.v. ED30 of 0.038 mg/kg and a p.o. ED30 of 0.66 mg/kg. These results demonstrate that EXP3174 is a selective and noncompetitive AII receptor antagonist and lacks agonistic effect. As EXP3174 is a potent antihypertensive agent, it may be responsible for part of the antihypertensive effect of DuP 753 in rats.     

Nonpeptide angiotensin II receptor antagonists. VIII. Characterization of functional antagonism displayed by DuP 753, an orally active antihypertensive agent

In the spinal pithed rat, DuP 753, 2-n-butyl-4-chloro-5-hydroxy-methyl-1-[(2'-(1H-tetrazol-5-yl)biphe nyl-4-yl) methyl] imidazole potassium salt, inhibited competitively the pressor response to angiotensin II (AII), whereas saralasin showed a noncompetitive pattern of interaction. It did not alter the pressor responses to vasopressin and norepinephrine as well as the heart rate response to isoproterenol. In the anesthetized rat, DuP 753 did not affect the vasodepressor response to bradykinin. Given p.o. or i.v., DuP 753 did not lower blood pressure in conscious normotensive rats, but it inhibited the pressor response to AII but not to vasopressin. It lowered blood pressure in furosemide-treated normotensive rats. Unlike saralasin, DuP 753 did not cause a transient increase in blood pressure even at 100 mg/kg i.v. DuP 753 at 3.5 micrograms i.c.v. inhibited the pressor response to i.c.v. AII, whereas DuP 753 at 10 mg/kg p.o. did not, suggesting that a single p.o. administration of DuP 753 does not affect brain AII receptors which are accessible by i.c.v. injection. Our study indicates that DuP 753 is a p.o. active, nonpeptide, selective, competitive AII receptor antagonist.     

Pharmacology of DuP 532, a selective and noncompetitive AT1 receptor antagonist

DuP 532 (2-propyl-4-pentafluoroethyl-1-[(2'-(1H-tetrazol-5-yl)bip hen yl- 4-yl)methyl]imidazole-5-carboxylic acid) inhibited the specific binding of [125I]angiotensin II (AII) for the subtype receptor AT1 in rat adrenal cortical membranes with an IC50 of 3.1 X 10(-9) M, but not the [125I]AII binding for the subtype AT2 sites in rat adrenal medulla tissues. It inhibited the contractile response to AII selectively and noncompetitively in the isolated rabbit aorta with a KB value of 1.1 X 10(-10) M. The selective AII antagonism was confirmed in the guinea pig ileum and the pithed rat. In conscious rats, DuP 532 inhibited the AII-induced pressor effect, aldosterone secretion, and water drinking induced by AII. In conscious renal hypertensive rats, DuP 532 decreased blood pressure with i.v. and p.o. ED30 of 0.02 and 0.21 mg/kg, respectively. The antihypertensive effect of DuP 532 at 0.3 to 3 mg/kg p.o. lasted for at least 24 hr. In conscious spontaneously hypertensive rats, DuP 532 given i.v. or p.o. at 0.3 to 3 mg/kg reduced blood pressure dose-dependently. DuP 532, at doses up to 100 mg/kg i.v., did not cause a pressor response in conscious normotensive rats, suggesting lack of agonism. DuP 532 exerted selective AII antagonism in conscious dogs. In conscious furosemide-treated dogs, DuP 532 given either at 0.3 and 1 mg/kg i.v. or at 1 to 10 mg/kg p.o. decreased blood pressure. As the AT1 receptors are responsible for AII-induced vasoconstriction, aldosterone secretion, and water drinking, our study indicates that DuP 532 is a potent, orally active, selective, and noncompetitive AT1 receptor antagonist and antihypertensive agent.     

Nonpeptide angiotensin II receptor antagonists. VII. Cellular and biochemical pharmacology of DuP 753, an orally active antihypertensive agent

2-n-Butyl-4-chloro-5-hydroxymethyl-1-[2'-(1H-tetrazole-5-yl)biphenyl-4-y l) methyl]imidazole, potassium salt (DuP 753) is a potent, p.o. active antihypertensive agent exerting its action by specific blockade of angiotensin II receptors. It inhibited the specific binding of labeled angiotensin II to its receptor sites in rat adrenal cortical membranes and in cultured rat smooth muscle cells with IC50 values of 19 and 20 X 10(-9) M, respectively. Functional antagonism was demonstrated by its blockage of angiotensin II (3 X 10(-8) M)-induced 45Ca++ efflux in rat aortic smooth muscle cells with an IC50 of 2 X 10(-8) M. In rabbit aorta, DuP 753 antagonized the contractile response to angiotensin II competitively with a pA2 value of 8.48 but had no effect on the responses induced by norepinephrine or KCl. In both in vitro and in vivo assays, no partial agonistic effect was detected even with concentrations of up to 10(-5) M. In addition, this agent (10(-5) or 10(-4) M) exhibited no direct effect on converting enzyme (rabbit lung) or renin (rat plasma). These data demonstrate that DuP 753, is a potent and highly specific angiotensin II receptor antagonist. This agent may be a useful experimental or therapeutic tool for interference with the renin-angiotensin system in health and diseases.     

Functional studies of nonpeptide angiotensin II receptor subtype-specific ligands: DuP 753 (AII-1) and PD123177 (AII-2)

DuP 753 and PD123177 are two nonpeptide angiotensin II (AII)-specific ligands, which show high affinities for two respective and distinct subtypes of AII binding sites, i.e., AII-1 and AII-2 sites, respectively, in the rat adrenal gland, brain and uterus. The objective of this study is to identify the functions of these subtype binding sites in the adrenal, sympathetic ganglia, brain and vascular smooth muscle. In conscious rats, DuP 753 at 1, 3 and 10 mg/kg i.v. but not PD123177 at 30 and 100 mg/kg i.v. inhibited the AII-induced aldosterone increase. In the isolated perfused rat adrenal gland, DuP 753 at 10(-6) and 10(-4) M but not PD123177 at 10(-3) M blocked the AII-induced epinephrine secretion. In control and chemically sympathectomized pithed rats, the pressor and tachycardiac responses to AII were blocked by DuP 753 at 10 mg/kg i.v. but not by PD123177 at 100 mg/kg i.v. In conscious rats, DuP 753 at 10 mg/kg s.c. but not PD123177 at 100 mg/kg s.c. inhibited the AII-induced water drinking. In the rabbit aorta, DuP 753 at 10(-6) M but not PD123177 at 10(-4) M inhibited the contractile effect of AII. In conscious renal artery-ligated hypertensive rats, DuP 753 but not PD123177 at 0.1 to 10 mg/kg i.v. lowered blood pressure. In summary, a function of the PD123177-sensitive AII binding site (AII-2) has not yet been identified. However, the DuP 753-sensitive site (AII-1) appears to mediate the AII-induced responses such as adrenal aldosterone and catecholamine secretion, release of catecholamine from sympathetic ganglia, drinking and vasoconstriction.     

Effect of captopril and the nonpeptide angiotensin II antagonists, SK&F 108566 and EXP3174, on renal function in dogs with a renal artery stenosis.

Treatment with an angiotensin converting enzyme (ACE) inhibitor can result in acute renal failure in patients with a renal artery stenosis. In the present study the effects of the selective nonpeptide angiotensin II antagonists, SK&F 108566 ((E)-alpha-[[2-Butyl-1-[(4-carboxyphenyl)methyl]-1H-imidazol-5-yl] methylene]-2-thiophenepropanoic acid) and EXP3174 (2-n-Butyl-4-chloro-1-[(2'-(1H-tetrazol-5-yl)-biphenyl-4-yl) methyl]imidazole-5-carboxylic acid hydrochloride) (the active metabolite of DuP 753, losartan) were compared with the ACE inhibitor, captopril, in the anesthetized dog which had been uninephrectomized and the remaining renal artery clamped to reduce renal blood flow (RBF) by approximately 50%. All three agents resulted in dose-dependent reductions in mean arterial pressure (MAP), glomerular filtration rate (GFR) and RBF. The maximum responses to captopril and SK&F 108566 were similar with MAP, RBF and GFR all decreasing approximately 30%. EXP3174 also resulted in decreases in GFR and RBF of approximately 30%; however, there was a smaller (approximately 17%) decrease in MAP. The data indicate that the possible bradykinin enhancing activity of ACE inhibitors may not provide any moderating activity of ACE inhibitor-induced reduction in GFR observed in dogs with a renal artery stenosis.     

In vivo pharmacology of L-158,809, a new highly potent and selective nonpeptide angiotensin II receptor antagonist.

L-158,809 (5,7-dimethyl-2-ethyl-3-[[2'-(1H-tetrazol-5yl)[1,1']-bi- phenyl-4-yl]-methyl]-3H-imidazo[4,5-b]pyridine) is a potent, competitive and specific antagonist of AT1 subtype of angiotensin II (AII) receptors in in vitro radioligand binding and functional isolated tissue assays. The present study was carried out to characterize the in vivo pharmacology of this potent AII receptor antagonist. In conscious, normotensive and anesthetized pithed rats, L-158,809 inhibits AII (0.1 microgram/kg i.v.) elevations in blood pressure without altering pressor responses to methoxamine or arginine vasopressin. In conscious rats, the relative potencies (ED50) were 29 micrograms/kg i.v. and 23 micrograms/kg p.o. Duration of action with single i.v. or p.o. doses exceeded 6 hr in rats. In similar experiments using rhesus monkeys, the potencies of L-158,809 were 10 micrograms/kg i.v. and approximately 100 micrograms/kg p.o. In these rats and monkeys, L-158,809 was 10 to 100 times more potent than DuP-753 (losartan) and approximately 3 times more potent than the metabolite, EXP3174. AII-induced elevation of plasma aldosterone in rats was also inhibited by L-158,809. Unlike angiotensin converting enzyme inhibitors, L-158,809 did not potentiate the hypotensive responses to i.v. bradykinin. L-158,809 was antihypertensive in high renin hypertensive rats (aortic coarction) and volume-depleted rhesus monkeys. The maximum hypotensive responses with acute doses of L-158,809 were equal to those with an angiotensin converting enzyme inhibitor in these renin-dependent animal models. From these in vivo data, L-158,809 is a selective AII receptor antagonist with high potency, good p.o. absorption, long duration and antihypertensive efficacy equal to angiotensin converting enzyme inhibition after single doses.     

Nonpeptide angiotensin II receptor antagonists. III. Structure-function studies

A series of 1-benzylimidazole-5-acetate derivatives defining the critical substituents on the phenyl ring was synthesized in order to improve on the affinity of 2-butyl-4-chloro-1-(2-nitrobenzyl)imidazole-5-acetate, sodium (S-8308) for the angiotensin II (AII) receptor. The analogs, substituted with -1-(4-carboxybenzyl) (EXP6155),-1-[4-(2-carboxybenzamido)benzyl] (EXP6159) and the 5-methylacetate of EXP6159 (EXP6803), were found to inhibit the binding of [3H]AII to AII receptors in rat adrenal cortical microsomes with 9-, 35- and 107-fold higher affinity, respectively, than that of S-8308 (IC50, 15 X 10(-6) microM). Scatchard analysis of the [3H]AII binding revealed that in the presence of EXP6155 (10(-6) M), the dissociation constant for AII was increased from 1.2 to 3.9 X 10(-9) M, whereas the total number of binding sites remained unchanged, suggesting a competitive nature of antagonism. A similar order of affinity or potency (saralasin much greater than EXP6803 greater than EXP6159 greater than EXP6155 greater than S8308) was observed in various in vitro and in vivo assays: rat smooth muscle cells AII binding, 45Ca++ influx in rat aortic rings, contractile response in isolated rabbit aorta and AII-induced pressor response in anesthetized rats. Responses (45Ca++ and contraction) elicited by norepinephrine or by KCl were unaltered by these agents at concentrations of up to 10(-4) M. In addition, they exerted no direct effect on the activity of rabbit angiotensin converting enzyme and rat renin. In conscious renal artery-ligated rats, EXP6155, EXP6159 and EXP6803 were p.o. inactive, but caused a rapid decrease in mean arterial pressure when administered i.v.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cardiac angiotensin receptors: effects of selective angiotensin II receptor antagonists, DUP 753 and PD 121981, in rabbit heart.

Angiotensin II (AII) elicits a positive inotropic response in cardiac muscle preparations from several species including humans. The purpose of this study was to characterize the AII binding sites and inotropic responses in rabbit ventricle using the selective AII receptor antagonists/ligands, DuP 753 (AT1) and PD 121981 (AT2). Biphasic displacement of specific 125I-Sar1,Ile8-AII binding was observed with both DuP 753 and PD 121981, suggesting the presence of two AII binding sites. The high affinity site for DuP 753 (29 nM) was a low affinity site for PD 121981 (91 microM), and the high affinity site for PD 121981 (78 nM) was a low affinity site for DuP 753 (81 microM). Of the specific AII binding, 70% was DuP 753 (AT1)-sensitive sites. Positive inotropic responses to AII in isolated papillary muscles from rabbit heart were antagonized competitively by both DuP 753 and PD 121981. The potencies of DuP 753 (pA2 = 7.99) and PD 121981 (pA2 = 4.28) to antagonize AII inotropic responses were similar to their potencies to displace 125I-Sar1,Ile8-AII from AT1 sites. There was no apparent functional consequence of AII interaction with AT2 site. Inotropic responses to isoproterenol were unaffected by DuP 753 and PD 121981. Therefore, there are two binding sites for AII in rabbit ventricle; however, only one site, AT1, participates in the inotropic response to AII. The roles of these receptor subtypes in other cardiac responses to AII have yet to be determined. Also, DuP 753 and PD 121981 are useful tools to study these two AII binding sites in cardiac preparations.     

The antihypertensive effect of the angiotensin II receptor antagonist DuP 753 may not be due solely to angiotensin II receptor antagonism.

The angiotensin II (AII) receptor antagonist, DuP 753 (10 mg/kg intraduodenal), produced a sustained and long-lasting antihypertensive effect in conscious renin-dependent hypertensive rats. Blood pressures were still reduced markedly 24 to 72 hr after administration of a single dose of DuP 753. However, pressor responses elicited by either angiotensin I or AII were not blocked at these times despite the continued antihypertensive effect of DuP 753. In a model of orthostatic hypotension, DuP 753 and the selective alpha-1 adrenoceptor antagonist prazosin produced a marked orthostatic hypotension response in renin-dependent hypertensive rats as demonstrated by potentiation of the decrease in blood pressure induced by a 90 degrees tilt. The nonpeptide AII receptor antagonist SK&F 108566 (10 mg/kg intraduodenal) did not produce orthostatic hypotension and the angiotensin converting enzyme inhibitor enalapril produced only a slight orthostatic response to tilting. In conscious spontaneously hypertensive rats (SHR), allowed 3 to 4 days to recover from surgery, administration of either enalapril (1 mg/kg i.v.) or SK&F 108566 (10 mg/kg i.v.) did not significantly effect blood pressure. In SHR tested within 24 hr of surgery, enalapril was effective in lowering blood pressure. In contrast, in surgically recovered SHR, DuP 753 (10 mg/kg i.v.) produced an antihypertensive effect that was slow in onset, sustained and extremely long in duration. Blood pressures did not return to predrug levels until 48 hr after administration of DuP 753. Stimulation of the thoracolumbar sympathetic outflow in pithed rats produced frequency-dependent pressor responses that were significantly potentiated by continuous infusion of a subpressor dose of AII.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nonpeptide angiotensin II receptor antagonists. IX. Antihypertensive activity in rats of DuP 753, an orally active antihypertensive agent

In conscious renal artery-ligated rats, a high renin hypertensive rat model, DuP 753, a p.o. active nonpeptide angiotensin II (AII) receptor antagonist, decreased blood pressure at 0.1 to 3 mg/kg given i.v. or at 0.3 to 10 mg/kg given p.o. with an i.v. ED30 of given i.v. or at 0.3 to 10 mg/kg given p.o. with an i.v. ED30 of 0.78 mg/kg and a p.o. ED30 of 0.59 mg/kg. The antihypertensive efficacy of DuP 753 was similar to that of captopril. Unlike the peptide AII antagonist saralasin, DuP 753 did not cause a transient increase in blood pressure, suggesting absence of agonistic activity. At 3 mg/kg p.o., DuP 753 lowered blood pressure for at least 24 hr and did not change heart rate, suggesting a long duration of antihypertensive effect. At 3 mg/kg i.v., DuP 753 inhibited the pressor response to AII but not to norepinephrine or vasopressin. Pretreatment of renal artery-ligated rats with captopril, saralasin or propranolol, but not with prazosin, hydralazine or indomethacin, abolished or reduced the antihypertensive effect of DuP 753. In the deoxycorticosterone acetate hypertensive rat, a low renin model, DuP 753 did not lower blood pressure. These results suggest that DuP 753 is a p.o. active, antihypertensive agent in renal artery-ligated rats with a similar antihypertensive efficacy as captopril. The antihypertensive effect of DuP 753 is most likely related to the blockade of the vasoconstrictor effect of AII. Unlike saralasin, DuP 753 does not have agonistic activity.     

Characterization of renal angiotensin II receptors using subtype selective antagonists.

Two angiotensin II (AII) receptor subtypes, AT1 and AT2, have recently been identified based on their relative affinities for selective peptide and nonpeptide antagonists. In the present study we used various AII peptide analogs, the AT1 subtype selective antagonists, DuP 753 and SK&F 108566, and the AT2 subtype selective antagonists, WL-19 and CGP 42112A, to determine whether AII receptor subtypes exist in the kidney. In agreement with previous studies, octapeptide (Sar1,Ile8-AII) and heptapeptide (AIII and Ile8-AIII) AII analogs displaced [125I]AII bound to rat glomerular membranes with similar affinities. However, in membranes derived from cortical tubules and the outer medulla, the heptapeptide analogs were 20-fold less potent in competing with [125I]AII binding than octapeptide analogs. The AT1 subtype selective nonpeptide AII antagonists, DuP 753 and SK&F 108566, totally displaced [125I]AII binding from all three membrane preparations in a monophasic manner with IC50 values in the 5 to 30 nM range. The AT2 selective peptide antagonist, CGP 42112A, had a low affinity in AII three membranes (IC50 = 450-1050 nM), whereas the nonpeptide AT2 selective antagonist, WL-19, had no activity at concentrations up to 10 microM. Dithiothreitol and the nonhydrolyzable GTP analog, 5'-guanylyl-imidodiphosphate, inhibited AII binding to all three membrane preparations. Based on these results, we conclude that the AII receptors located on glomeruli, tubules and in the outer medulla belong to the AT1 subtype, and that the physiologically important renal actions of AII are mediated through activation of AT1 receptors.     

The in vitro pharmacological profile of KR31080, a nonpeptide AT1 receptor antagonist

Summary— KR31080 (2-butyl-5-methyl-6-(1-oxopyridin-2-yl)-3-[[2'-(1H-tetrazol-5-yl) biphenyl-4-yl]methyl]-3H-imidazo[4,5-b] pyridine) is a potent inhibitor of angiotensin type 1 (AT₁) receptors in rabbit aorta and human recombinant AT₁ receptors. In the isolated rabbit thoracic aorta, KR31080 caused a nonparallel shift to the right of the concentration-response curves to angiotensin II (All) with decreased maximal response (pD'₂ = 10.1 ± 0.1), but had no effect on the contractile response induced by norepinephrine. KR31080 inhibited specific [¹²⁵I]AII binding to rabbit aortic membranes (AT, receptors) and [¹²⁵I][Sar¹, Ile⁸]AII binding to human recombinant AT₁ receptors in a concentration-dependent manner with IC₅₀ values of 0.84 ± 0.08 nM and 1.92 ± 0.15 nM, respectively, but did not inhibit specific [¹²⁵I)AII binding to bovine cerebellum membranes (ÀT₂ receptors). In the Scatchard analysis, KR31080 interacted with rabbit aortic AT₁ receptors in a competitive manner, similar to losartan. These results demonstrate that KR31080 is a potent and AT₁ selective angiotensin receptor antagonist which exerts a competitive antagonism in the [¹²⁵I]AII binding assay and insurmountable AT₁ receptor antagonism in the functional study.     

Binding of KRH-594, an antagonist of the angiotensin II type 1 receptor,to cloned human and rat angiotensin II receptors

We studied the binding properties of KRH-594, a new selective antagonist of angiotensin II (AII) type 1 (AT1) receptors, to rat liver membranes and to recombinant AT1 and AT2 receptors. Preincubation of rat liver membranes with KRH-594 produced maximal inhibition of [125I]-AII binding when the preincubation time was 1-2 h. Preincubation with KRH-594 for 2 h decreased the B(max) value and increased the Kd value. For human AT1, human AT2, rat AT1A and rat AT1B receptors, the Ki values for KRH-594 were 1.24, 9360, 0.67, and 1.02 nm, respectively. The rank order of K1 values for human AT1 receptors was KRH-594 > EXP3174 > candesartan = AII. The order of specificities for human AT1 and AT2 receptors was candesartan > EXP3174 > KRH-594. Although a 2-h preincubation of human AT2 receptors with KRH-594 (30 microM) or CGP 42112 (a selective AT2 receptor antagonist; 0.3 nM) inhibited binding of [125I]-AII, the suppression by KRH-594 was not significant. These results indicate that KRH-594 binds potently to AT1 receptors in an insurmountable manner, and that at a very high dose (30 microM) it may also bind to AT2 receptors, but in a surmountable manner.     

Antihypertensive mechanism of captopril in renal hypertensive rats: studies with a nonpeptide angiotensin II receptor antagonist and an angiotensin II monoclonal antibody

The validity of using EXP6803, a nonpeptide angiotensin II (AII) receptor antagonist, and KAA8, an AII monoclonal antibody, as specific tools for studying the physiology of AII has been established previously. In this study, we used these specific probes to examine the role of blocking AII formation in the antihypertensive effect of captopril in conscious renal artery-ligated rats (RALRs), a high renin, renal hypertensive model. Mean arterial pressure and plasma renin activity in a typical group of RALRs averaged 175 +/- 5 mm Hg and 28.2 +/- 6.2 ng of angiotensin 1 per ml/hr (n = 6), respectively. The antihypertensive effect of captopril (3 mg/kg i.v.) was determined in RALRs given either EXP6803 (30 mg/kg + 2 mg/kg/min i.v.) or KAA8 (10 mg + 1 mg/min i.v. per rat) with the corresponding vehicle-treated RALRs. These doses of EXP6803 and KAA8 were very effective in blocking the pressor response to AII but not to norepinephrine or vasopressin in RALRs. Captopril decreased mean arterial pressure by 44 +/- 2 and 53 +/- 8 mm Hg in the groups treated with the vehicles of EXP6803 (n = 5) and KAA8 (n = 5), respectively. In the presence of EXP6803 (n = 5) or KAA8 (n = 5), the antihypertensive effect of captopril was almost or totally abolished. Indomethacin did not alter the antihypertensive effect of captopril. These results suggest that the antihypertensive effect of captopril in conscious RALRs is due mainly to the blockade of AII formation. Furthermore, circulating AII rather than locally formed AII appears to play a major role in maintaining hypertension in hypertension in RALRs.     

Cytoskeleton-dependent endocytosis is required for apical type 1 angiotensin II receptor-mediated phospholipase C activation in cultured rat proximal tubule cells.

Renal proximal tubule sodium reabsorption is enhanced by apical or basolateral angiotensin II (AII). Although AII activates phospholipase C (PLC) in other tissues, AII coupling to PLC on either apical or basolateral surfaces of proximal tubule cells is unclear. To determine if AII causes PLC activation, and the differences between apical and basolateral AII receptor function, receptors were unilaterally activated in rat proximal tubule cells cultured on permeable, collagen-coated supports. Apical AII incubation resulted in concentration- and time-dependent inositol trisphosphate (IP3) formation. Basolateral AII caused greater IP3 responses. Apical AII-induced IP3 generation was inhibited by DuP 753, suggesting that the type 1 AII receptor subtype mediated proximal tubule PLC activation. Apical AII signaling did not result from paracellular ligand leak to basolateral receptors since AII-induced PLC activation occurred when basolateral AII receptors were occupied by Sar-Leu AII or DuP 753. Inhibition of endocytosis with phenylarsine oxide prevented apical (but not basolateral) AII-induced IP3 formation. Cytoskeletal disruption with colchicine or cytochalasin D also prevented apical AII-induced IP3 generation. These results demonstrate that in cultured rat proximal tubule cells, AII is coupled to PLC via type 1 AII receptors and cytoskeleton-dependent endocytosis is required for apical (but not basolateral) AII receptor-mediated PLC activation.     

Nitric oxide and angiotensin II. Glomerular and tubular interaction in the rat.

Nitric oxide (NO) has been proposed to modulate the renal response to protein as well as basal renal hemodynamics. We investigated whether NO and angiotensin II (AII) interact to control glomerular hemodynamics and absolute proximal tubular reabsorption (APR) during glycine infusion and in unstimulated conditions. In control rats, glycine increased single nephron GFR and plasma flow with no change in APR. The NO synthase blocker, NG-monomethyl L-arginine (LNMMA), abolished the vasodilatory response to glycine, possibly through activation of tubuloglomerular feedback due to a decrease in APR produced by LNMMA + glycine. Pretreatment with an AII receptor antagonist, DuP 753, normalized the response to glycine at both glomerular and tubular levels. In unstimulated conditions, LNMMA produced glomerular arteriolar vasoconstriction, decreased the glomerular ultrafiltration coefficient, and reduced single nephron GFR. These changes were associated with a striking decrease in APR. DuP 753 prevented both glomerular and tubular changes induced by LNMMA. In conclusion, NO represents a physiological antagonist of AII at both the glomerulus and tubule in both the basal state and during glycine infusion; and inhibition of NO apparently enhances or uncovers the inhibitory effect of AII on proximal reabsorption.     

In vitro pharmacology of a nonpeptidic angiotensin II receptor antagonist, SC-51316.

The properties of a novel nonpeptidic angiotensin II (AII) receptor antagonist, 2,5-dibutyl-2,4-dihydro-4-([2-(1H-tetrazol-5-yl)(1,1'-biphenyl) -4'-yl]methyl)-3H-1,2,4-triazol-3-one (SC-51316), are described. SC-51316 inhibited [125I]AII binding selectively to the AT1 receptor with IC50 values of 3.6 and 5.1 nM in rat adrenal cortical and rat uterine membrane preparations, respectively. The compound was a competitive and reversible antagonist of AII-mediated contraction of rabbit aortic rings with a pA2 of 8.86. In addition, SC-51316 inhibited AII-induced aldosterone release from rat adrenal zona glomerulosa cells and blocked inhibition of renin release by AII from rat kidney slices with pA2 values of 8.62 and 8.9, respectively. The agent (0.1 mM) did not inhibit angiotensin-converting enzyme or plasma renin activity. These data demonstrate that SC-51316 is a potent AII receptor antagonist which may prove to be useful as a pharmacologic tool for studying the role of the renin-angiotensin system in cardiovascular diseases.     

Renal hemodynamic and excretory responses to PD 123319 and losartan, nonpeptide AT1 and AT2 subtype-specific angiotensin II ligands.

Angiotensin receptor subtypes have been described and pharmacologically characterized. DuP 753 (losartan) selectively antagonizes the angiotensin type 1 receptor, whereas PD 123319 selectively binds to an angiotensin type 2 receptor. These studies compared the renal response to treatment with the nonpeptides, DuP 753 and PD 123319, and the peptide antagonist, saralasin, in anesthetized mongrel dogs. Saralasin and DuP 753 increased renal blood flow and were mildly natriuretic. DuP 753 was roughly 10-fold less potent than saralasin. PD 123319 had no effect on renal hemodynamics, but produced dose-related increases in urine volume and free water clearance. PD 123319 had no effect on circulating vasopressin levels, suggesting the change in water handling by the kidney was not due to inhibition of vasopressin release. A direct effect of PD 123319 at the level of the renal tubule has not been ruled out. This is the first report of a renal functional response to an angiotensin type 2 receptor ligand and suggests that the angiotensin type 2 receptor may be related to water handling by the kidney.     

In vitro pharmacology of L-158,809, a new highly potent and selective angiotensin II receptor antagonist.

L-158,809 interacted in a competitive manner with rabbit aortic angiotensin II (AII) receptors as determined by Scatchard analysis of the specific binding of [125I]Sar1Ile8-AII. The affinity of L-158,809 (IC50 = 0.3 nM) for AII receptors in this tissue was appreciably greater than that of other reported nonpeptide AII antagonists such as DuP-753 (IC50 = 54 nM) and EXP3174 (IC50 = 6 nM) and similar to the natural ligand, AII. L-158,809 also exhibited a high potency at AII receptors in several other tissues from different animal species (IC50 = 0.2-0.8 nM). In vitro functional assays utilizing AII-induced aldosterone release in rat adrenal cortical cells demonstrated further that L-158,809 acts as a competitive, high affinity antagonist of AII (pA2 = 10.5) and lacks agonist activity. L-158,809 also potently inhibited AII-induced inositol phosphate accumulation in vascular smooth muscle cells and contractile responses to AII in isolated blood vessels. The specificity of L-158,809 for AII receptors was demonstrated by its lack of activity (IC50 greater than 1 microM) in several other receptor binding assays and its inability to affect in vitro functional responses produced by other agonists. L-158,809 demonstrated a very high selectivity for the AT1 compared to the AT2 receptor subtype (AT2 IC50 greater than or equal to 10 microM). The high affinity and selectivity makes L-158,809 a valuable new tool for investigating the physiological and pharmacological actions of AII.