Inhibition of rabbit aortic angiotensin II (AII) receptor by CV-11974, a new nonpeptide AII antagonist

Abstract

The angiotensin II (AII) antagonistic action of CV-11974 (2-ethoxy-1-[[2'-(1 H-tetrazol-5-yl)biphenyl-4-yl]methyl]benzimidazole-7-carboxylic acid) was investigated in an AII-receptor binding assay using rabbit aortic membranes and an AII-induced contraction assay using rabbit aortic strips. A single class of [ 125I]AII-(Sar 1,Ile 8) binding sites was found in the membranes with a dissociation constant ( K d ) of 0.15 nM and a receptor concentration ( B max) of 86.9 fmol/mg protein. CV-11974 markedly reduced K d without affecting B max. The specific binding of [ 125I]AII-(Sar 1,Ile 8) in this preparation was inhibited competely by CV-11974 [the inhibition constant ( K i ) = 0.64 nM], DuP 753 [an angiotensin II type I (at 1) receptor-selective antagonist] ( K i = 51 nM) and EXP3174 (an active metabolite of DuP 753) ( K i = 6.8 nM), but was not affected by PD123177 (an AT 2 receptor-selective antagonist). These results suggest that the single binding site in rabbit aortic membranes is an AT 1 receptor subtype. The affinity of CV-11974 to these AT 1 receptors was approximately 80 and 10 times higher than that of DuP 753 and EXP3174, respectively. CV-11974 showed no appreciable affinity for the AT 2 receptors found in bovine cerebellum. In the in vitro functional study, CV-11974 markedly reduced the AII-induced maximal contractile response of rabbit aortic strips (pD' 2 = 9.97). In contrast. Compound 7-H, which lacks the carboxyl group at the benzimidazole ring of CV-11974, inhibited the contraction in a competitive manner. The inhibition by CV-11974 was long lasting. These results suggest that CV-11974 is a potent and long-acting AT 1 receptor-selective, competitive antagonist. The carboxyl group at the benzimidazole ring plays an important role in the interaction between CV-11974 and the AT 1 receptor.