Binding of the antagonist [ [formula omitted]]candesartan to angiotensin II AT 1 receptor-tranfected Chinese hamster ovary cells

Abstract

Binding of the non-peptide angiotensin II AT 1 antagonist [ 3 H ](2-ethoxy-1-[(2′-(1 H-tetrazol-5-yl)biphenyl-4-yl)methyl]-1 H-benzimidazoline-7-carboxylic acid ([ 3 H ]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 [ 3 H ]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 II responses also recovered more rapidly in the presence of 2- n-butyl-4-chloro-5-hydroxymethyl-1-[(2′-(1 H-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazole (losartan), indicating that unlabelled ligands prevented reassociation. [ 3 H ]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.