Characterization and Regulation of Angiotensin and Corticotropin Receptors on Cultured Bovine Adrenal Cells

Abstract

Cultured bovine adrenal fasciculata cells were used to characterize angiotensin II (A-II) and corticotropin (ACTH) receptors and to study their homologous and heterologous regulation. These cells contain one type of high affinity binding sites for A-II (K_D ∼ 2.4 ± 0.3 10⁻⁹M) and about 100000 sites/cell. Photoaffinity labeling followed by SDS-PAGE under reducing conditions revealed a single macromolecule of apparent MR 65000. Treatment of cells with increasing concentrations of A-II produced down-regulation of its own receptors and marked homologous and heterologous (ACTH) steroidogenic desensitization. However, the desensitization was not correlated with receptor loss and was mainly due to alterations of the steroidogenic pathway. Pretreatment of cells with ACTH also reduced A-II receptors, but this was not associated with steroidogenic desensitization. Bovine fasciculata cells contain two binding sites for ACTH: one of high affinity (K_D ∼ 2.6 ± 0.4 10⁻¹⁰M) and low capacity (2030 ± 390 sites/cell) and the other of low affinity and high capacity. Affinity cross-linking of ACTH to plasma membranes prepared from adrenal cells revealed a labeled macromolecule of apparent MR 43000. However, cross-linking experiments to intact cells revealed, both under reducing and non-reducing conditions, two labeled macromolecules of apparent MR of 123000 and 43000. Pretreatment of cells with ACTH enhanced its receptor and the cAMP and cortisol responses to further ACTH stimulation. These effects were time -and dose-dependent. The maximal effects were observed at 10⁻¹⁰ to 10⁻⁹M. A-II alone had no effect but it blocked partially the stimulatory action of ACTH.