Characterization of Bovine Ovary Angiotensin II Receptors Using Subtype-Selective Antagonists

Angiotensin-II (AII) receptors have been classified as AT1 and AT2 subtypes based on selective antagonists. AII binding sites in bovine ovary membranes were characterized using the radiolabeled AII antagonist, [125I]Sar1,Ile8-AII ([125I]SIA). The binding was specific and saturable with dissociation constant (Kd) and maximum binding (Bmax) of 0.18 +/- 0.08 nmol/l and 32.5 +/- 1.3 fmol/mg, respectively. Pretreatment of ovarian membranes with dithiothreitol (10 mumol/l) doubled the specific binding of [125I]SIA twofold to 63.5 +/- 2.8 fmol/mg. Guanine nucleotide had no significant effect on the affinity of agonist (AII) to compete for [125I]SIA binding. AII and a series of AII-related analogs were used in competition binding experiments, and the data were compared with those obtained with membranes prepared from bovine adrenal cortex and bovine cerebellum. The membranes from ovary and cerebellum showed similar binding characteristics, but they differed from those of adrenal cortex. CGP42112A and WL-19, AT2-subtype selective antagonists, inhibited [125I]SIA binding to ovarian membrane with IC50 values of 28 +/- 4 and 26.7 +/- 2.8 nmol/l, respectively. SK&F 108566 and DuP 753, AT1-subtype-selective antagonists, had very little effect on [125I]SIA binding to ovarian membranes. These data directly demonstrate that bovine ovary membranes have predominantly AT2-subtype AII receptors.