Kinetic studies on the interaction of nonlabeled antagonists with the angiotensin II receptor

Abstract

Angiotensin AT 1 receptor antagonists are divided into two types, surmountable, based on the way they inhibit angiotensin II-induced vasoconstriction. To elucidate what causes the difference, we studied how antagonists associate with and dissociate from AT 1 receptor sites in rat liver membranes. Three antagonists, 6-propyl-7-oxo-4[[2′-(1 H-tetrazol-5-yl)biphenyl-4 -yl]methyl]-4,7-dihydropyrazolo[1,5- a]pyrimidine-3-carboxylic ac acid (SRL1080227), 2-ethoxy-1-[[2′(1 H-tetrazol-5-yl)biphenyl-4-yl]methyl]-3 H-benzimidazole-7-carboxylic a acid (CV-11974) and 2-butyl-3-[[2′-(1 H-tetrazol-5-yl)biphenyl-4-yl]-3 H-imidazo-[4,5- b]pyridine (FK739), showed competitive antagonism when they were added simultaneously with [ 125I]angiotensin II, but CV11974 and SRL1080227 showed apparently noncompetitive antagonism when membranes were preincubated with each antagonist. The longer the preincubation time with CV11974 or SRL1080227 was, the more effectively the antagonist inhibited [ 125I]angiotensin II binding, while the inhibition by FK739 did not change with the preincubation time. To estimate their dissociation rate from the receptor binding site, we studied [ 125I]angiotensin II binding to membranes which had been preincubated with each antagonist and washed twice. Membranes pretreated with FK739 completely recovered the ability to bind [ 125I]angiotensin II with a period of 60 min, while menbranes preincubated with CV11974 did not read this level of recovery. [ 125I]angiotensin II binding to membranes preincubated with SRL1080227 increased gradually, but did not reach the control level during the experiment. The kinetic properties of SRL1080227, CV11974 and FK739 were consistent with their characteristic modes in inhibiting angiotensin II-induced contraction of isolated rabbit aorta and decreasing blood pressure of spontaneously hypertensive rats. The present results support the hypothesis that slow dissociation of an antagonist from the receptor binding site is involved in its apparently noncompetitive behavior in receptor binding and its insurmountable property in functional studies.