125I]EXP985: A highly potent and specific nonpeptide radioligand antagonist for the AT 1 angiotensin receptor

Abstract

[ 125I]EXP985 is the first nonpeptide radioligand with high specific activity for the AT 1 angiotensin receptor. The biochemical and pharmacological profiles of this ligand were determined using either ligand-receptor binding techniques in rat adrenal cortical microsomes or cellular Ca 2+ mobilization in rat smooth muscle cells. Specific binding with 0.1 nM [ 125I]EXP985 increased slowly with time reaching an equilibrium at 60 min of incubation (22°C). Scatchard analysis of the inhibition / binding data revealed a single class of binding sites having a K d of 1.49 ± 0.06 nM and a Bmax of 3.6 ± 0.1 pmol/mg protein. These sites were saturable and the ligand-receptor complex dissociated with a t 1/2 of 58 min. The binding was inhibited by Ang peptides with the following order of potency and IC 50 (nM) : Ang II (3.7) > Ang III (69) > Ang I (3650), and by the nonpeptide AT 1 receptor antagonist, losartan, with an IC 50 of 3.2 nM, PD123177, an AT 2 selective antagonist, showed minimal inhibitory effect. Specific binding of [ 125I]EXP985 was found on rat aortic smooth cells. Ang II-induced Ca 2+ mobilization in these cells was blocked by EXP985 in a noncompetitive manner. These data show that [ 125I]EXP985 (or its unlabeled) is a potent and highly specific radioligand or noncompetitive antagonist which represents a novel tool to further our understanding of the biochemistry of AT 1 receptors.