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.     

Interaction between the partially insurmountable antagonist valsartan and human recombinant angiotensin II type 1 receptors

The interaction between the AT1 receptor-selective antagonist valsartan, and its human receptor, was investigated by direct radioligand binding as well as by its inhibition of angiotensin II induced inositol phosphate accumulation in CHO cells expressing human recombinant AT1 receptors. Specific binding of [3H]-valsartan rapidly reached equilibrium at 37 degrees C. It was saturable and occurred to a homogeneous class of sites with a K(D) of 0.88+/-0.076. It was inhibited by other AT1 receptor antagonists with the same potency order as previously described for the binding of [3H]-angiotensin II and [3H]-candesartan to human AT1 receptors (i.e. candesartan > or = EXP3174 > valsartan = irbesartan = angiotensin II > losartan). When valsartan and angiotensin II were applied simultaneously to the CHO-AT1 cells. the antagonist caused a rightward shift of the angiotensin II concentration response curve. Hence, valsartan interacts with the AT1 receptor in a manner that is competitive with angiotensin II. Pre-incubation of the cells with 0.5, 5 and 50 nM valsartan caused an additional, concentration-dependent, up to 55% decline of the maximal response. The partial nature of this insurmountable inhibition by valsartan was confirmed by biphasic antagonist concentration-inhibition curves. These data reflect the co-existence of a fast reversible/surmountable as well as a tight binding/insurmountable valsartan receptor complex. In agreement, pre-incubation of the CHO-AT1 cells with 5 and 50 nM valsartan produced a partial inhibition of the angiotensin II induced increase of the free intracellular calcium concentration. [3H]-Valsartan dissociated from its receptors with a half-life of 17 min. In functional recovery experiments with valsartan-pre-treated cells, the angiotensin II-mediated response was half-maximally restored within approximately 30 min. These kinetic data suggest that the insurmountable inhibition by valsartan is related to its relatively slow dissociation from the human AT1 receptors.     

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.     

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.     

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. 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)     

Comparative in vivo effects of irbesartan and losartan on angiotensin II receptor binding in the rat kidney following oral administration

We examined the ability of the new non-peptide angiotensin II receptor antagonist irbesartan to inhibit AT(1) receptors in vivo in the rat kidney following oral administration, compared with the prototype drug losartan. Male Sprague-Dawley rats (250-300 g) were gavaged with either irbesartan or losartan at doses of 1, 3, 10, 30 or 100 mg/kg, or with corresponding vehicle. Rats were killed at 0, 1, 2, 8, or 24 h after drug administration, trunk blood was collected and the kidneys were removed. The effects of irbesartan and losartan on angiotensin II receptor binding were determined by quantitative in vitro autoradiography using the specific radioligand (125)I-[Sar(1),Ile(8)]angiotensin II. High levels of angiotensin II receptor binding in the rat kidney were demonstrated in the glomeruli and inner stripe of the outer medulla, which was attributed to AT(1) receptors. At 1 h after dosing, irbesartan (1-100 mg/kg) and losartan (1-30 mg/kg) significantly inhibited AT(1) receptor binding in all anatomical areas of the kidney, in a dose-dependent manner, with a maximal effect at 100 mg/kg and 30 mg/kg respectively. For a 10 mg/kg dose, inhibition of AT(1) receptor binding was maximal around 1-2 h after oral administration of losartan, whereas maximal binding occurred between 2 and 8 h for irbesartan; both drugs produced persistent tissue blockade at 24h. In radioligand binding studies, irbesartan, losartan and EXP3174 (1x10(-10) to 1x10(-5) M) displaced (125)I-[Sar(1),Ile(8)]angiotensin II binding from renal AT(1) receptors in a concentration-dependent manner, with a rank order of potency of irbesartan>EXP3174>losartan. The concentration required to displace 50% of radioligand binding (IC(50)) by irbesartan, EXP3174 and losartan was 1.00+/-0.2 nM, 3.5+/-0.4 nM and 8.9+/-1.1 nM respectively. In conclusion, the findings of the present study suggest that irbesartan and losartan produce effective and sustained inhibition of AT(1) receptors in vivo in the kidney following oral administration. However, irbesartan appears less potent, with respect to dosage, than losartan in vivo, despite having a higher affinity for AT(1) receptors in vitro. The reason for this apparent discrepancy is unclear, but it may reflect the slower onset of action of irbesartan and its rate of tissue accessibility. Inhibition of angiotensin II receptors in target tissues such as the kidney may represent an important action of AT(1) receptor antagonists, which may contribute to the beneficial effects of these agents in the clinical setting.     

Nonpeptide angiotensin II receptor antagonists: insurmountable angiotensin II antagonism of EXP3892 is reversed by the surmountable antagonist DuP 753

Effects of 2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)biphen yl-4- yl)methyl]imidazole, potassium salt (DuP 753), a surmountable angiotensin II (AII) receptor antagonist, on the insurmountable AII antagonism induced by 2-n-propyl-4-trifluoromethyl-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4- yl)methyl]imidazole-5-carboxylic acid (EXP3892) were examined. In the rabbit aorta, EXP3892 exhibited selective and insurmountable AII antagonism. DuP 753 at 10(-6) M, added before or after EXP3892, reversed partially the depressed AII maximal response caused by 10(-9) M EXP3892. Repeated washing of the rabbit aorta created with DuP 753 at 10(-6) M or EXP3892 at 10(-9) M did not restore completely the sensitivity to AII for at least 2 hr. In the pithed rat, EXP3892 showed selective and insurmountable AII antagonism. DuP 753 at 0.1 to 3 mg/kg i.v., given before or after EXP3892, reversed the reduced AII-maximal response induced by EXP3892 at 0.1 mg/kg i.v. We propose that DuP 753 by binding to the AII receptor induces conformational changes resulting in a reduction of the affinity of the receptor for coupling factors/transducer proteins, which causes surmountable antagonism. EXP3892 would diminish the binding capacity for coupling factors accounting for insurmountable antagonism. As DuP 753 and EXP3892 compete for the same AII receptor, the reduced AII-maximal response by EXP3892 may be reversed by DuP 753.     

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.     

Comparative effects of angiotensin II and angiotensin III in rabbit adrenal and aortic tissues

The addition of angiotensin II (AII) and angiotensin III (AIII) to isolated tissue baths produced the same maximal contractile response of rabbit aortic strips. AIII was about 10 times less potent, the slope of its concentration-response curve was less steep and its rate of onset slower than that of AII. The responses of both AII and AIII were inhibited with equal potency by the surmountable AII antagonist Phe4, Tyr8-AII and its unsurmountable analog Sar1, Leu8-AII but the kinetic patterns of inhibition by both were less well defined with the agonist AIII than with AII. The addition of AIII to tissues which had exhibited a maximal response to AII did not increase the level of contraction, in contrast to the case when norepinephrine was added to tissues contracted by AII. Both AII and AIII displaced [125I]AII binding from rabbit adrenal membranes; AIII was 6 times less potent than AII but displayed competitive kinetics as an inhibitor of [125I]AII binding. In further studies two binding sites for [125I]AII were identified in adrenal membranes, having KD values of 2.0 +/- 0.2 and 19.6 +/- 2.3 nM, respectively. Each site was inhibited by both AII and AIII and the ratio of the apparent Ki values for the two hormones was not significantly different. The Hill coefficient for the high affinity site was, however, lower for AIII than AII. We interpret our data to suggest that AII and AIII act on the same receptors. AIII apparently binds less efficiently than does AII in both rabbit adrenal membranes and rabbit aortic strips.     

Hepatic binding sites for angiotensen II in the rat.

1. 125I-labelled (Asn1,Val5)-angiotensin II (125I-labelled AII) incubated with purified rat liver membranes was degraded with time, as estimated by three techniques: binding to an excess of specific antibody, polyacrylamide-gel electrophoresis and rebinding to fresh membranes. Degradation was inhibited in the presence of an excess of beta 1-24-corticotrophin but still very marked. 2. 125 I-labelled AII became bound to purified rat liver membranes. Association and dissociation rates were slow. Binding was competitively inhibited by (Asn1,Val5)-AII, (Asp1,Ile5)-AII and (Des,Asp1, Ile5)-AII. Apparent KD was approximately 0.1 nmol/l. 3. Bound hormone was also partly degraded independently of time. 4. Angiotensinases inhibitors had different effects on 125I-labelled AII binding. A clear increase was observed in the presence of beta 1-24-corticotrophin and phenylmethylsulphonylfluoride whereas binding was decreased in the presence of EDTA or 8-hydroxyquinoline. 5. These results demonstrate the presence of high-affinity binding sites for AII and of angiotensinases in hepatic membranes.     

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.     

Angiotensin-converting enzyme-independent angiotensin formation in a human model of myocardial ischemia: modulation of norepinephrine release by angiotensin type 1 and angiotensin type 2 receptors

Angiotensin II (Ang II) promotes norepinephrine (NE) release from cardiac sympathetic nerve endings. We assessed in a human model in vitro whether locally formed Ang II contributes to NE release in myocardial ischemia. Surgical specimens of human right atrium were incubated in anoxic conditions. After 70 min of anoxia, NE release (carrier-mediated; caused by NE transporter reversal) was 8-fold greater than normoxic release. Angiotensin-converting enzyme inhibition with enalaprilat failed to reduce anoxic NE release. In contrast, prevention of chymase-dependent Ang II formation with chymostatin, Bowman-Birk inhibitor, or alpha(1)-antitrypsin significantly inhibited anoxic, but not exocytotic, NE release. Two mast-cell stabilizers, cromolyn and lodoxamide, markedly reduced NE release, implicating cardiac mast cells as a major source of chymase. Angiotensin type 1 receptor (AT(1)R) blockade with EXP3174 inhibited NE release, whereas angiotensin type 2 receptor (AT(2)R) blockade with PD123319 did not. Interestingly, PD123319 reversed the inhibitory effect of EXP3174. Furthermore, synergisms were uncovered between EXP3174 and an AT(2)R agonist, and between EXP3174 and a Na(+)/H(+) exchanger inhibitor. Thus, angiotensin-converting enzyme-independent Ang II formation via chymase is important for carrier-mediated ischemic NE release in the human heart. Locally generated Ang II promotes NE release by acting predominantly at AT(1)Rs, which are likely coupled to the Na(+)/H(+) exchanger. Effects of Ang II at AT(2)Rs, seemingly opposite to those resulting from AT(1)R activation, are uncovered when AT(1)Rs are blocked. Because NE release is associated with coronary vasoconstriction and arrhythmias, and mast-cell density and chymase content increase in the ischemic heart, the notion that chymase-generated Ang II plays a major role in carrier-mediated NE release may have important clinical implications.     

Pharmacological characterization of the nonpeptide angiotensin II receptor antagonist, SK&F 108566.

The angiotensin II (AII) antagonist activity of (E)-alpha-[[2-butyl-1-[(4-carboxyphenyl)methyl]-1H-imidazol-5- yl]methylene]-2-thiophenepropanoic acid (SK&F 108566), was examined in a number of in vitro and in vivo assays. In rat and human adrenal cortical membranes, SK&F 108566 displaced specifically bound [125I]AII with IC50 of 9.2 and 3.9 nM, respectively. SK&F 108566 also inhibited [125I]AII binding to human liver membranes (IC50 = 1.7 nM) and to rat mesenteric artery membranes (IC50 = 1.5 nM). In rabbit aortic smooth muscle cells, SK&F 108566 caused a concentration-dependent inhibition of AII-induced increases in intracellular Ca++ levels. In rabbit aortic rings, SK&F 108566 produced parallel rightward shifts in the AII concentration-response curve without affecting the maximal contractile response. Schild analysis of the data yielded a KB value of 0.26 nM and a slope not different from 1, indicative of competition antagonism. SK&F 108566 had no effect on the contractile responses to KCl, norepinephrine or endothelin in rabbit aorta. In conscious normotensive rats, i.v. administration of SK&F 108566 (0.01-0.3 mg/kg) produced dose-dependent parallel shifts in the AII pressor dose-response curve. Administration of SK&F 108566 (3-10 mg/kg) intraduodenally or intragastrically to conscious normotensive rats resulted in a dose-dependent inhibition of the pressor response to AII (250 ng/kg, i.v.). At 10 mg/kg, i.d., significant inhibition of the pressor response to AII was observed for 3 hr. In this same rat model, SK&F 108566 had no effect on base-line pressure or on the pressor response to norepinephrine or vasopressin. The data demonstrate that SK&F 108566 is a potent, highly selective, competitive nonpeptide AII antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Pharmacological profile of TH-142177, a novel orally active AT1-receptor antagonist

Summary— The pharmacological properties of TH-142177 (N-n-butyl-N-[2'-(1-H-tetrazole-5-yl)biphenyl-4-yl]-methyl-(N-carboxymethyl-benzylamino)-acetamide), a novel antagonist of the angiotensin II (AII) AT₁ receptor, were studied in vitro and in vivo, and compared to those of losartan. In the rat isolated aorta, TH-142177 produced parallel shifts to the right of the concentration-response curves for AII-induced contractions without affecting the maximal response (pA₂ = 9.07). The inhibitory potency of TH-142177 in the aorta was about three times greater than that of losartan. TH-142177 completely inhibited the specific binding of [¹²⁵I]AII to AT₁ receptor in rat aortic membranes (K_i = 1.6 × 10⁻⁸ M), whereas specific [¹²⁵I]AH binding to AT₂ receptor in bovine cerebellum and human myocardium was not affected by concentrations of TH-142177 up to 10⁻⁵ M. Losartan also inhibited the [¹²⁵I] AII binding to rat aortic membranes ( K _i = 2.2 × 10⁻⁸ M). Following the intravenous administration to anesthetized normotensive rats, TH-142177 dose-dependently inhibited the increase in systolic blood pressure induced by an intravenous bolus injection of AII that was 1.5 times less potent than losartan. Furthermore, the oral administration of TH-142177 to conscious renal hypertensive rats exerted a dose-dependent reduction of systolic blood pressure without significantly effecting the heart rate. TH-142177 was at least three times more potent than losartan. These results demonstrate that TH-142177 is a potent and selective antagonist of AT₁ receptors and by oral administration has a long-lasting antihypertensive activity.     

In vitro pharmacological characterization of UP 269-6, a novel nonpeptide angiotensin II receptor antagonist

Summary— The in vitro pharmacology of UP 269-6, a novel nonpeptide angiotensin II antagonist, was examined in radioligand binding and functional isolated tissue assays. UP 269-6 bound selectively to AT₁ receptors as evidenced by the inhibition of specific [¹²⁵I] Sar¹, Ile⁸-AII binding in rat adrenal membranes (IC₅₀ = 35.8 nM) and in cultured vascular smooth muscle cells (IC₅₀ = 23.8 nM). UP 269-6 displayed a very high selectivity for the AT₁ compared to the AT₂ receptor subtype (IC₅₀ > 10,000 nM). UP 269-6 inhibited the AII-induced contraction of isolated rabbit aortic strips. The pattern of AII antagonism suggested competitive antagonism at low concentrations (10⁻¹⁰, 3 × 10⁻¹⁰, 10⁻⁹ M) of UP 269-6 and insurmountable antagonism at higher concentrations (3 × 10⁻⁹, 10⁻⁸, 3 × 10⁻⁸ M). Based on the calculated p A₂ values, UP 269-6 (9.86 ± 0.25) was an angiotensin II receptor antagonist as potent as L-158,809 (9.82 ± 0.37) and much more potent than losartan (7.96 ± 0.38). UP 269-6 was devoid of affinity (IC₅₀ > 10,000 nM) for many other receptors, ion channels and uptake sites, demonstrating its high specificity for AII receptors. Furthermore, this compound did not affect the contractile response to KCl or phenylephrine in rabbit aorta and exhibited no effect on angiotensin converting enzyme activity. These data demonstrate that UP 269-6 is a highly potent, selective and specific AT₁ receptor antagonist.     

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.     

Ischemia promotes renin activation and angiotensin formation in sympathetic nerve terminals isolated from the human heart: contribution to carrier-mediated norepinephrine release

We recently reported that in the ischemic human heart, locally formed angiotensin II activates angiotensin II type 1 (AT(1)) receptors on sympathetic nerve terminals, promoting reversal of the norepinephrine transporter in an outward direction (i.e., carrier-mediated norepinephrine release). The purpose of this study was to assess whether cardiac sympathetic nerve endings contribute to local angiotensin II formation, in addition to being a target of angiotensin II. To this end, we isolated sympathetic nerve endings (cardiac synaptosomes) from surgical specimens of human right atrium and incubated them in ischemic conditions (95% N(2,) sodium dithionite, and no glucose for 70 min). These synaptosomes released large amounts of endogenous norepinephrine via a carrier-mediated mechanism, as evidenced by the inhibitory effect of desipramine on this process. Norepinephrine release was further enhanced by preincubation of synaptosomes with angiotensinogen and was prevented by two renin inhibitors, pepstatin-A and BILA 2157BS, as well as by the angiotensin-converting enzyme inhibitor enalaprilat and the AT(1) receptor antagonist EXP 3174 [2-N-butyl-4-chloro-1-[2'-(1H-tetrazol-5-yl)biphenyl-4-yl] methyl]imidazole-5-carboxylic acid]. Western blot analysis revealed the presence of renin in cardiac sympathetic nerve terminals; renin abundance increased ~3-fold during ischemia. Thus, renin is rapidly activated during ischemia in cardiac sympathetic nerve terminals, and this process eventually culminates in angiotensin II formation, stimulation of AT(1) receptors, and carrier-mediated norepinephrine release. Our findings uncover a novel autocrine/paracrine mechanism whereby angiotensin II, formed at adrenergic nerve endings in myocardial ischemia, elicits carrier-mediated norepinephrine release by activating adjacent AT(1) receptors.     

In vitro and in vivo characterization of the activity of telmisartan: an insurmountable angiotensin II receptor antagonist

In vitro studies have shown that telmisartan is an insurmountable angiotensin II subtype-1 (AT1) receptor antagonist. Herein, the molecular basis of this insurmountable antagonism has been investigated in vitro, and the effect of telmisartan has been compared in vivo with that of irbesartan and candesartan. Association and dissociation kinetics of telmisartan to AT1 receptors have been characterized in vitro on rat vascular smooth muscle cells (RVSMC) expressing solely the AT1 receptor subtype. In a second set of experiments, the antagonistic efficacy of single intravenous doses (0.1, 0.3, and 1 mg/kg) of telmisartan was compared with that of irbesartan (0.3, 1.0, 3.0, and 10.0 mg/kg) and candesartan (0.3 and 1 mg/kg) in conscious, normotensive, male Wistar rats. The results show that the specific binding of [(3)H]telmisartan to the surface of living RVSMC is saturable and increases quickly to reach equilibrium within 1 h. Telmisartan dissociates very slowly from the receptor with a dissociation half-life (t(1/2)) of 75 min, which is comparable with candesartan and almost 5 times slower than angiotensin II (AngII). In vivo, telmisartan blunts the blood pressure response to exogenous AngII dose dependently. The blockade is long lasting and remains significant at 24 h at doses >0.1 mg/kg. Ex vivo assessment of the AT1 receptor blockade using an in vitro AngII receptor binding assay shows similar results. When administered intravenously in rats, telmisartan is 10-fold more potent than irbesartan and comparable to candesartan. Taken together, our in vitro data show that the insurmountable antagonism of telmisartan is due at least in part to its very slow dissociation from AT1 receptors.