Calcium Modulation of Acth and Cholera Toxin Stimulated Adrenal Steroid and Cyclic-AMP Biosynthesis

Abstract

AbstractMonolayer cultures of mouse adrenal cortex tumors have been used to investigate the effects of Ca2+ on the binding of 125I-labelled ß1-24-ACTH peptide to the cell, ACTH-stimulated cyclic-AMP and steroid biosynthesis. Stimulation with ACTH results in increased intracellular cyclic-AMP levels and release of cyclic-AMP into the medium. The specific activities of intracellular and extracellular cyclic-AMP produced by cells incubated with 3H-adenine are the same, suggesting that they are products of a common pool of ATP. ACTH-stimulated cyclic-AMP release continues after intracellular cyclic-AMP levels have decreased and reflects continuing cyclic-AMP biosynthesis. In incubations of two hours or less with 5.0 mM Ca2+, maximum steroidogenesis occurs with 0.1 milliunit/ml ACTH. Further increases in cyclic-AMP production are elicited with higher concentrations of ACTH. In the absence of Ca2+, both cyclic-AMP and steroid syntheses are attenuated, maximum steroidogenesis requiring > 10 milliunits/ml ACTH.The proportion of 125I-ßI-24-ACTH bound to the intact cell to ACTH added is constant over a range of concentrations of ACTH from 7 pg to 17 ng/ml. Although binding of ACTH is decreased approximately 30% in the absence of exogenous Ca2+, Ca2+ has no effect on this binding at Ca2+ concentrations from 0.1-5.0 mM. However, the quantity of cyclic-AMP released at each concentration of ACTH is increased as the concentration of Ca2+ is elevated. Steroidogenesis is also increased until a level of cyclic-AMP production corresponding to maximal steroidogenesis is reached. In contrast, the steroidogenic response to added cyclic-AMP or dibutryl-Cyclic-AMP is unaffected by Ca2+. The specific activity of 3H-cyclic-AMP produced in cultures incubated with 3H-adenine is lower in Ca2+-deprived cells.These data suggest that the amount of cyclic-AMP produced in response to the quantity of ACTH bound is dependent on the concentration of Ca2+ in the system. Steroidogenic activity is related primarily to the level of cyclic-AMP production and is uninfluenced by Ca2+. The lower levels of cyclic-AMP found in Ca2+ deprived cells are not due to enhanced cyclic-AMP degradation.V. cholerae enterotoxin (choleragen) is also a potent stimulator of cyclic-AMP and steroid synthesis in this system, but its mode of action differs from that of ACTH. Ca2+ has the same qualitative effects on cyclic-AMP and steroid synthesis in choleragen-stimulated cells as those observed in ACTH-stimulated cells. The data presented in this report provide direct evidence that Ca2+ alters some property of the plasma cell membrane in the intact cell to produce an enhancement in the adenylate cyclase response to a stimulatory peptide.