Characterization of Distinct Angiotensin II Binding Sites in Rat Adrenal Gland and Bovine Cerebellum Using Selective Nonpeptide Antagonists

We investigated the characteristics of 125I-AII binding to rat adrenal and bovine cerebellar membranes in the presence and absence of new nonpeptide angiotensin II (All) receptor ligands. The imidazole All ligands, DUP753 and WL19, both produced biphasic competition curves to 125I-All binding in rat adrenal glomerulosa and adrenal medulla particles, suggesting the existence of two distinct All binding sites. Antagonist affinity (Ki) and binding capacity (Bmax) for each binding site was determined using nonlinear analysis of competition data fit to a two-site model. The high capacity site (68% of total specific 125I-All bound) in glomerulosa had high affinity for DUP753 (4.6 ± 0.8 nM) and low affinity for WL19 (29 ± 3 μM), and the low capacity site had high affinity for WL 19 (3.3 ± 1.4 nM) and low affinity for DUP753 (51 ± 9 μM). Conversely, in medulla, the high capacity site (77% total binding) had high affinity for WL 19 (19 ± 6 nM) and low affinity for DUP753 (29 ± 8 μM), and the low capacity site had low affinity for WL19 (25 ± 7 μM) but a high affinity for DUP753 (2.8 ± 2.0 nM). In glomerulosa, binding parameters for the nonpeptide ligands at each site derived from monophasic competition curves obtained in the presence of either 0.3 μM DUP753 or WL 19 to selectively block the high or low capacity binding site, respectively, were similar to values determined from the biphasic competition curves. Unlike the nonpeptide inhibitors, unlabeled All yielded monophasic inhibition curves. WL19 and DUP753 also blocked 125I-All binding to bovine cerebellar membranes (Ki = 0.14 ± 0.03 and 170 ± 21 μM, respectively), and both displayed monophasic competition curves. All-induced contraction of rabbit aorta was potently and competitively blocked by DUP753 (K = 3.9 ± 0.4 nM) but not by WL 19. Thus, the Ki values for DUP753 at the DUP753-sensitive binding site in rat adrenal glomerulosa and medulla are similar to the affinity of DUP753 for All receptors in rabbit aorta, suggesting a similarity among these sites. The Ki values for WL19 at the WL19-sensitive site in rat adrenal gland and bovine cerebellum differ from 7–40-fold and represent binding site(s) distinct from the DUP753-sensitive site.