Binding of the antagonist [H-3]candesartan to angiotensin II AT(1) 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-carboxyl ic acid ([H-3]candesartan) to human angiotensin II AT(1) receptor-transfect ed 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 bin ding 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 i ncreased 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 angiote nsin 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]imid azole (losartan), indicating that unlabelled ligands prevented reassociatio n. [H-3]candesartan saturation binding experiments required a long time to reach equilibrium. Therefore, the equilibrium dissociation constant(K-d = 5 1 +/- 8 pM) was calculated from the association and dissociation rate const ants. Our findings indicate that the insurmountable nature of candesartan i n functional studies is related to its slow dissociation from the receptor. (C) 1999 Elsevier Science B.V. All rights reserved.