The transport of the angiotensin II receptor antagonist losartan and its interaction with organic anion transport were examined in the isolated perfused rabbit proximal tubule. Losartan reversibly inhibited the secretion of para-aminohippurate (PAH) in a concentration-dependent manner (IC50 = 15 +/- 0.5 microM). Other angiotensin II receptor antagonists also inhibited PAH secretion with similar potencies: eprosartan, 11 +/- 2.3 microM; irbesartan, 17 +/- 2.2 microM; and valsartan 3 +/- 0.6 microM. [3H]Losartan was secreted by the proximal tubule by a saturable and probenecid-sensitive mechanism. The affinity of losartan for the organic anion transporter (Km = 12.3 +/-1.8 microM) was significantly greater than that of PAH (Km = 88.5 +/- 10.7 microM). [3H]Losartan secretion was stimulated in the presence of alpha-ketoglutarate, suggesting that losartan, like PAH, enters the cell in exchange for a dicarboxylate. These results demonstrate that losartan and probably other nonpeptide angiotensin II receptor antagonists are secreted by an organic anion transporter that is similar to, if not identical with, the classic PAH transporter.

The dibasic amino acid, L-arginine, is a substrate for both nitric oxide synthase (NOS) and arginase and therefore, plays an important role in cell signaling and cell growth. We examined the effects of various NOS inhibitors on L-arginine transport into rat renal brush border membrane (BBM) vesicles. L-Arginine uptake was stimulated in the presence of an inwardly directed Na+ gradient and an imposed inside negative potential in BBM but not basolateral membrane vesicles. In BBM vesicles, the L-arginine analogs, N-iminoethyl-L-orinithine and Nw-monomethyl-L-arginine (L-NMMA) were potent inhibitors of L-arginine uptake (IC50 of 0.48 and 0.82 mM, respectively), while Nw-nitro-L-arginine was less active (IC50 = 10 mM) and Nw-nitro-L-arginine methyl ester (L-NAME) was inactive. The inhibition of L-arginine transport by L-NMMA was competitive in nature. L-NIO, L-NMMA as well as L-arginine and L-lysine but not Nw-nitro-L-arginine methyl ester, trans-stimulated L-arginine uptake when preloaded into BBM vesicles. The L-arginine analogs had no effect on the transport of the neutral amino acid, L-leucine, in the same preparations. The data suggest that in addition to inhibiting NOS, the L-arginine analogs, N-iminoethyl-L-orinithine, L-NMMA and to a lesser extent L-NA, also inhibit L-arginine transport across the BBM of proximal tubules.

[125I]Endothelin-1 bound with high affinity to a single site on both brush border membranes (Kd=192±26 pM, Bmax=314±49 fmol/mg) and basolateral membranes (Kd=94.7±3.4 pM, Bmax=612±107 fmol/mg) isolated from rat renal cortex. Competition binding experiments using subtype selective ligands revealed that the proportion of ETB to ETA receptors was 80:20 and 60:40 in the brush border membrane and the basolateral membrane, respectively. The results demonstrate that endothelin-1 binds to brush border membranes, and that endothelin ETB receptors may be involved in the previously described effects of endothelin-1 on brush border membrane Na+ transport.

The angiotensin II (AII) antagonist, losartan, increases uric acid excretion when administered to humans. However, the active metabolite of losartan, EXP 3174, and other nonpeptide AII antagonists such as eprosartan and SB 203220 are devoid of uricosuric activity. To investigate the mechanism of losartan-induced uricosuria, we examined the effects of losartan, EXP 3174, eprosartan and SB 203220 on OH- -dependent [14C]urate uptake into rat proximal tubule brush-border membrane vesicles. Losartan (10 microM) inhibited [14C]urate uptake at all time points examined, except at equilibrium (2 hr). Losartan had no effect on urate uptake in the absence of an OH- gradient. The inhibitory effect of losartan on urate uptake was concentration dependent (IC50 = 9.5 +/- 1.4 microM) and competitive in nature. The other AII antagonists also inhibited urate uptake but were 6-8-fold less potent than losartan with IC50 values of EXP 3174 (65 +/- 13 microM), eprosartan (60 +/- 7.0 microM) and SB 203220 (74 +/- 12.5 microM). In contrast to the effects of the nonpeptide AII antagonists, the peptide antagonist, Sar1,Ile8-AII, as well as AII itself had no effect on urate uptake. These results suggest that the uricosuric activity of losartan is, at least in part, due to inhibition of urate reabsorption in the proximal tubule and is unrelated to AII receptor activity. Furthermore, losartan has a greater affinity for the urate/anion exchanger than the other AII antagonists tested. These results are in direct agreement with observations made after administration of these compounds to humans.

A possible gender difference in the antidiuretic activity of vasopressin was studied in male and female Sprague-Dawley rats. Infusion of vasopressin (3-100 pg.kg-1.min) into conscious, chronically instrumented water-loaded rats resulted in a dose-dependent antidiuresis in both male and female rats. Male rats, however, were more than three times more sensitive to vasopressin than female rats. Thus the effective doses of vasopressin (pg.kg-1.min-1) to decrease urine flow to 30 microliters.min-1.100 g-1 (18 +/- 5 in males; 58 +/- 12 in females), to increase urine osmolality to 600 mosmol/kgH2O (35 +/- 5 in males; 119 +/- 15 in females), and to decrease free water clearance to 30 microliters.min-1.100 g-1 (8 +/- 3 in males; 28 +/- 7 in females) were significantly (P < 0.05) lower in males. Furthermore, in vitro studies in papillary collecting duct cells demonstrated a significantly higher density of vasopressin V2 receptors and a greater ability of vasopressin to stimulate adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in males than in females. Vasopressin V2-receptor density (maximum binding) was 359 +/- 47 and 238 +/- 22 fmol/mg in male and female rats, respectively (P < 0.05). There was no difference in apparent dissociation constants (Kd). Vasopressin resulted in a dose-dependent increase in cAMP accumulation in papillary collecting duct cells, and at the highest concentration of vasopressin used (10(-8) M) cAMP increased from 44 +/- 10 to 182 +/- 51 fmol/micrograms protein in males and from 30 +/- 4 to 91 +/- 18 fmol/micrograms protein in females (P < 0.05). (ABSTRACT TRUNCATED AT 250 WORDS)

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We examined the role of angiotensin II (AII) receptor subtypes in the regulation of hormone-stimulated cyclic AMP (cAMP) accumulation in isolated rat glomeruli. All inhibited cAMP formation induced by histamine, serotonin and parathyroid hormone, but not by prostaglandin E2 or calcitonin gene-related peptide. Angiotensin III but not the angiotensin fragments (1-7) and (3-8) also showed inhibitory activity. The inhibition of histamine-induced cAMP accumulation by AII was concentration-dependent and was absent in glomeruli isolated from pertussis toxin-treated rats. The effect of AII on histamine-induced cAMP levels was not mimicked by the protein kinase C activator, phorbol-12-myristate-13-acetate, nor was the effect of AII inhibited by the protein kinase C inhibitors, staurosporine and H-7. The angiotensin II receptor subtype 1 (AT1) antagonists, SK&F 108566 and losartan, attenuated the inhibitory effect of AII on histamine-stimulated cAMP accumulation, whereas the AT2 selective antagonists, CGP 42112A, WL-19 and PD 123319, had no effect. Displacement of [125I]AII from glomerular membrane using the subtype-selective antagonists confirmed that the glomerular AII receptor has characteristics of an AT1 subtype. The results suggest that AII, through activation of the AT1 receptor, may act to maintain the contractile state of glomerular mesangial cells by attenuating the increase in cAMP levels induced by some hormones.

We characterized the endothelin (ET) receptor subtype responsible for the inhibition of vasopressin (AVP)-induced increases in osmotic water permeability (Pf) and cAMP accumulation in rat inner medullary collecting ducts (IMCD). ET-1 (10 nM) produced a rapid and transient decrease in AVP-stimulated Pf from 1241 +/- 112 to 224 +/- 38 microns/sec. At the same concentration (10 nM), the selective ETB receptor agonist sarafotoxin 6c (S6c) produced the same degree of inhibition with a time course identical to that of ET-1. Exposure of IMCDs to the ETA-selective antagonist BQ123 (100 nM) had no effect on ET-1-induced inhibition of AVP-dependent Pf. In suspensions of IMCD cells, ET-1, ET-3 or S6c produced concentration-dependent inhibition of AVP-stimulated cAMP accumulation to the same extent and with similar potencies (IC50 = 10-30 nM). BQ123 (1 nM to 10 microM) had no effect on ET-1-induced inhibition of AVP-stimulated cAMP formation. Saturation binding experiments with radiolabeled ET-1 and the selective ETB agonist IRL1620 and competition binding studies with selective ETA and ETB receptor ligands demonstrated that > or = 80% of the ET-1 binding sites in IMCD membranes were of the ETB subtype. Therefore, results from functional, biochemical and binding studies suggest that the ETB receptor is the ET receptor subtype that inhibits AVP action in the rat IMCD.

The further evolution of the imidazole-5-acrylic acid series of nonpeptide angiotensin II receptor antagonists is detailed (for Part 1, see: J. Med. Chem. 1992, 35, 3858). Modifications of the N-benzyl ring substitution were undertaken in an effort to mimic the Tyr4 residue of angiotensin II. Introduction of a p-carboxylic acid on the N-benzyl ring resulted in the discovery of compounds with nanomolar affinity for the receptor and good oral activity. SAR studies of these potent antagonists revealed that the thienyl ring, the (E)-acrylic acid, and the imidazole ring in addition to the two acid groups were important for high potency. Also, overlay comparisons of the parent diacid with both angiotensin II and a representative biphenylyltetrazole nonpeptide angiotensin II receptor antagonist are presented. The parent diacid analog, SK&F 108566 or (E)-3-[2-butyl-1-(4-carboxybenzyl)-1H-imidazole-5-yl]-2-[(2- thienyl)methyl]propenoic acid, is currently in clinical development for the treatment of hypertension.

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 ability of alpha(2)-adrenoceptor agonists to inhibit vasopressin (VP)-stimulated cAMP accumulation in collecting tubules and to inhibit the antidiuretic effect of VP in rats is clearly established. However, in other species, such as the dog, alpha(2)-adrenoceptor-induced inhibition of VP action has not been convincingly demonstrated. In the present study, we examined the effects of epinephrine and other alpha(2)-adrenoceptor agonists on VP-stimulated cAMP accumulation in inner medullary collecting tubule cells and/or cortical collecting tubules from a number of species. Epinephrine, oxymetazoline, clonidine, and guanabenz inhibited VP-induced cAMP formation in rat inner medullary collecting tubule cells with IC50s ranging from 10 to 30 nM. However, epinephrine or guanabenz had no effect on VP-stimulated cAMP formation in cells from dog, pig, rhesus monkey, or human inner medulla. Similarly, epinephrine inhibited VP-induced cAMP accumulation in cortical collecting tubules dissected from rat kidneys but not from dog or rabbit kidneys. We conclude that there is a marked species difference in the ability of alpha(2)-adrenoceptor agonists to inhibit VP-induced cAMP formation at the tubular level. This may explain the difficulty in demonstrating an alpha(2)-adrenoceptor agonist-induced inhibition of VP action in other species such as dog and man.

The effects of calcitonin gene-related peptide (CGRP) on lumen diameter and adenylate cyclase activity in isolated intracerebral arterioles were examined. CGRP produced a concentration-dependent relaxation of spontaneous tone developed by the arterioles with an EC50 value of 3.9 x 10(-9) M. Calcitonin, as well as substance P, which is frequently colocalized with CGRP, had no effect on arteriolar tone. CGRP also relaxed arterioles contracted with the thromboxane mimetic, U-44619, yielding an EC50 value of 3 x 10(-9) M. The CGRP fragment, human CGRP(8-37), antagonized CGRP-induced relaxation in a noncompetitive manner. Adenylate cyclase activity in single arterioles was stimulated by CGRP, but not by substance P, in a concentration-dependent fashion. Half maximal stimulation occurred at 8 x 10(-9) M, whereas maximum stimulation (2.5-fold over basal) occurred at 10(-7) M. CGRP(8-37) inhibited CGRP-stimulated adenylate cyclase activity over a concentration range of 10(-9) to 10(-5) M. The results demonstrate that CGRP stimulates adenylate cyclase activity and is a potent vasodilator of small parenchymal cerebral arterioles in vitro and may play an important role in the regulation of cerebral blood flow.