BMS‐180560, an insurmountable inhibitor of angiotensin II‐stimulated responses: comparison with losartan and EXP3174

Abstract

1 This study compares the activity of BMS-180560 (2-butyl-4-chloro-1-[[1-[2-(2H-tetrazol-5-yl)phenyl]-1H-indol-4-yl]methyl]-lH-imidazole-5-carboxylic acid), an insurmountable angiotensin II (All) receptor antagonist, with that of losartan and EXP3174 in functional and biochemical models of AII-receptor activation.2 BMS-180560 selectively inhibited ['25I]-Sar'Ile8AII (['25I]SI-AII) binding to rat aortic smooth muscle (RASM) cell and rat adrenal cortical AT, receptors (Ki = 7.6 ± 1.2 and 18.4 ± 3.9 nM respectively) compared to adrenal cortical AT2 receptors (Ki = 37.6 ± 1.3 JiM). The K, values of BMS-180560 and EXP3174, but not losartan, varied as a function of the BSA concentration used in the assays, indicating that the diacid drugs bound to albumin.3 BMS-180560 (3-300 nM) increased the KD of SI-AII for RASM cell AT, receptors. Only at high concentrations of BMS-180560 (300nM) were Bmax values decreased.4 BMS-1 80560 inhibited AII-stimulated contraction of rabbit aorta with a calculated KB = 0.068 ± 0.048 nM and decreased maximal AlI-stimulated contraction at 1 nM BMS-180560 by 75%. In the presence of 0.1% BSA, a higher KB value (5.2 ± 0.92 nM) was obtained. Losartan behaved as a competitive antagonist with a KB = 2.6 ± 0.13 nM. Contraction stimulated by endothelin-1, noradrenaline, KCl, or the TXA2 receptor agonist U-46619 were unaffected by BMS-180560 (1 nM).5 All stimulated the acidification rates of RASM cells as measured by a Cytosensor microphysiometer with an EC50 of 18 nM. Losartan (30 nM) shifted the All concentration-effect curves in a competitive manner whereas BMS-180560 (0.01 and 0.1 nM) decreased the maximum responses by 60 and 75% respectively. Inhibition by losartan and BMS-180560 could be reversed following washout although recovery took longer for BMS-180560. (17-55%). BMS-180560 (3 and 10 nM) increased the EC50 for All and decreased the maximum response by 30 and 80% respectively. The inhibition by EXP3174 and BMS-180560 could be reversed by inclusion of losartan (200 nM) indicating that the inhibition was not irreversible. 7 In conclusion, BMS-180560 is a potent, specific, predominantly competitive, reversible All receptor antagonist, which displays insurmountable receptor antagonism. At concentrations of BMS-180560 which have no effect on receptor number, BMS-180560 produced insurmountable antagonism of AII-stimulated second messenger formation, extracellular acidification, and smooth muscle contraction.

Distinction between surmountable and insurmountable selective AT1 receptor antagonists by use of CHO‐K1 cells expressing human angiotensin II AT1 receptors

“…This, in turn, triggers cell growth and constriction (Vincentini & Villereal, 1986). The initial AT 1 receptor-evoked responses can conveniently be measured in primary cell cultures and in cell lines (Dickinson et al, 1994;Koh et al, 1994;Panek et al, 1995;Perlman et al, 1995;Balmforth et al, 1997) and appear to be common to the biological actions of angiotensin II. In addition, it was also observed by Dickinson et al (1994) that angiotensin II increases the rate at which rabbit aortic smooth muscle cells acidify their environment with products of their energy metabolism.…”

“…The initial AT 1 receptor-evoked responses can conveniently be measured in primary cell cultures and in cell lines (Dickinson et al, 1994;Koh et al, 1994;Panek et al, 1995;Perlman et al, 1995;Balmforth et al, 1997) and appear to be common to the biological actions of angiotensin II. In addition, it was also observed by Dickinson et al (1994) that angiotensin II increases the rate at which rabbit aortic smooth muscle cells acidify their environment with products of their energy metabolism. This latter response is commonly observed for G-protein coupled receptors and it is claimed to occur irrespective of the initial cellular events (McConnell et al, 1992).…”

EXP3174: The Major Active Metabolite of Losartan

“…Based on these results, Sachinidis et al concluded that EXP3174 is a more potent angiotensin AT, receptor antagonist than is losartan in blocking angiotensin 11-induced cellular responses. Dickinson et al also reported that EXP3 174 noncompetitively inhibited angiotensin 11-stimulated phosphoinositide turnover in rat aorta smooth muscle cells (16).…”

Novel Dual Action AT1 and ETA Receptor Antagonists Reduce Blood Pressure in Experimental Hypertension

“…Human AT 1 receptor affinity was determined using a previously described standard binding assay (Dickinson et al, 1994). Human recombinant AT 1 receptor membranes (catalog no.…”

“…The membranes were incubated for 60 min at 37°C. Bound and free radioligand were separated and counted as described previously (Dickinson et al, 1994).…”