Binding of the antagonist [3H]candesartan to angiotensin II AT1 receptor-tranfected Chinese hamster ovary cells

Binding of the non-peptide angiotensin II AT(1) antagonist [H-3](2-ethoxy-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]-1H-benzimidazoline-7-carboxylic acid ([H-3]candesartan) to human angiotensin II AT(1) receptor-transfected Chinese hamster ovary (CHO-AT(1)) cells was inhibited to the same extent by angiotensin ii and non-peptide angiotensin ii AT(1) antagonists. No binding was observed in control CHO-K-1 cells. Dissociation was slow (k(-1) = 0.0010 +/- 0.0001 min(-1)) after removal of the free [H-3]candesartan but increased 5-fold upon addition of supramaximal concentrations of angiotensin II AT(1) antagonists. Angiotensin II responses recovered equally slow from candesartan-pretreatment. When washed and further incubated, these angiotensin LT responses also recovered more rapidly in the presence of 2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazole (losartan), indicating that unlabelled ligands prevented reassociation. [H-3]candesartan saturation binding experiments required a long time to reach equilibrium. Therefore, the equilibrium dissociation constant(K-d = 51 +/- 8 pM) was calculated from the association and dissociation rate constants. Our findings indicate that the insurmountable nature of candesartan in functional studies is related to its slow dissociation from the receptor. (C) 1999 Elsevier Science B.V. All rights reserved.