Properties of Angiotensin II Receptors in the Bovine and Rat Adrenal Cortex

Abstract

Abstract Receptors for angiotensin II have been identified and characterized in bovine and rat adrenal cortex by binding studies with tritiated and monoiodinated angiotensin II. The angiotensin II binding sites of bovine adrenal cortex homogenate were enriched severalfold in a microsomal membrane fraction together with alkaline phosphatase, adenylate cyclase, 5'-nucleotidase and 21β-hydroxylase. Uptake of angiotensin II by adrenal cortex particles and adrenal cells was rapid, reaching equilibrium at 15 to 30 min in the presence of 0.2 to 1.5 nm angiotensin II. The equilibrium association constant of angiotensin II for bovine adrenal cortex receptors was 0.5 nm-1 at 22°, and that for rat adrenal particles was significantly higher. Dissociation of bound angiotensin II from adrenal particles and cells was also rapid, with initial half-time of 13 to 23 min. Angiotensin II released from adrenal cortex binding sites at low pH retained activity in subsequent binding studies, whereas angiotensin II in the incubation medium was rapidly inactivated. Degradation of angiotensin II by adrenal cortex particles was partially inhibited by addition of unrelated peptides including glucagon and insulin, by reducing agents such as dithiothreitol, and by reduced temperature. Fragments and analogues of angiotensin II showed binding-inhibition potencies which correlated with their biological activities in vivo. In particular, competitive antagonists, such as the (Sar1, Ala8) derivative of angiotensin II, inhibited angiotensin II binding in proportion to their antagonistic activity in vivo. This system provides a simple and rapid method for evaluation of the competitive binding activity of angiotensin agonists and antagonists in vitro. In contrast to angiotensin II, the decapeptide angiotensin I exhibited relatively low affinity for angiotensin II binding sites in competitive studies, and direct binding studies with monoiodinated 125I-angiotensin I showed considerably lower uptake than that of angiotensin II. The uptake of labeled angiotensin I and II by adrenal medulla homogenates was much lower than that of adrenal cortex particles, and again angiotensin II showed higher binding affinity than angiotensin I. These findings illustrate the presence of high affinity specific binding sites for angiotensin II in bovine and rat adrenal cortex and suggest a plasma membrane location for the angiotensin II receptors.