Inhibitory Actions of Calmodulin Antagonists on Steroidogenesis in Zona Glomerulosa Cells

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Abstract
The stimulation of aldosterone (Aldo) production in the [rat] adrenal glomerulosa cell by angiotensin II (AII) and ACTH is highly dependent on Ca. To determine the role of calmodulin (CM) as a regulator of intracellular Ca during hormonal activation of steroidogenesis, the actions of a series of CM inhibitors on the stimulation of Aldo biosynthesis by AII, the Ca ionophore A23187, ACTH and cAMP were examined in isolated adrenal glomerulosa cells. The CM antagonists, pimozide, W7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide] and W5 [N-(6-aminohexyl)-1-naphthalenesulfonamide] caused dose-dependent inhibition of hormone-stimulated Aldo production, in proportion to their reported binding affinities for CM. The maximum inhibitory concentration (5 .mu.M) of the most potent CM antagonist, pimozide, had no effect on the conversion of progesterone to Aldo. Submaximal inhibitory concentrations of W7 and W5 also reduced conversion of progesterone to Aldo, indicating that their inhibitory action is not only due to their CM antagonist properties. The relative sensitivities of the stimuli of Aldo release to inhibition by all 3 CM inhibitors were A23187 = AII > ACTH > 8-bromo-cAMP (8-Br-cAMP). Aldo responses to ACTH were more sensitive to inhibition pimozide than those to 8-Br-cAMP, suggesting that ACTH-induced cAMP formation is also dependent on CM. Pimozide (5 .mu.M) completely inhibited the Aldo responses to AII and A23187; the Aldo responses to ACTH and 8-Br-cAMP were only partially inhibited. At submaximal inhibitory concentrations, pimozide caused a decrease in the maximum response to AII without changing the concentration of the peptide required for half-maximum stimulation of Aldo production. The inhibition of Aldo production by pimozide was accompanied by parallel inhibition of pregnenolone accumulation measured in the presence of cyanoketone. No effect of pimozide (5 .mu.M) was observed on the binding of 125I-AII to adrenal glomerulosa cells, placing the CM-dependent site between the receptor and the side-chain cleavage enzyme. CM-mediated regulation is essential for the steroidogenic response to Ca ionophore and AII, but is only partially required for the responses to ACTH and 8-Br-cAMP.