Acute inhibition of corticosteroidogenesis by inhibitors of calmodulin action

To identify the possible role of calmodulin in ACTH function, we tested the ability of chlorpromazine (CP) and other calmodulin antagonists to inhibit steroidogenesis of isolated adrenocortical cells of the rat. CP reversibly inhibited maximal ACTH-induced corticosterone (B) production. The presence of the drug did not alter the ED₅₀ of ACTH stimulation (3.2 × 103 pg/ml), suggesting that it inhibited ACTH-induced steroidogenesis in a noncompetitive manner. The CP concentration required for half-maximal inhibition was 8.2 μM, a value close to the dissocation constant of the CP-calmodulin complex (5.3μM). Concentrations greater than 40 μM resulted in complete inhibition. Similar concentrations of CP inhibited ACTH-induced cAMP accumulation in a dose-dependent manner, indicating an effect of the drug on early events in ACTH action. In addition, CP also apparently acted at a site distal to the point of cAMP formation, as shown by the finding that it inhibited cAMP-induced B production. CP inhibition of ACTH-induced B production was independent of the Ca²⁺ concentration, suggesting that the drug did not compete with Ca²⁺ directly. Concentrations of CP greater than 20 μM inhibited protein synthesis as measured by leucine incorporation into cellular proteins. Thus, although the inhibitory effect of high concentrations of CP on steroidogenesis might be explained by an effect on protein synthesis, the inhibition seen at 10 μM appeared to be independent of protein synthesis. Other antagonists of calmodulin action inhibited maximal ACTH-induced B production with the following relative potencies: trifluoperazine < CP < haloperidol < chlordiazepoxide. This order is similar to that reported for inhibition of calmodulin-activated phosphodiesterase and for binding to calmodulin. These findings suggest that calmodulin may modulate the effect of ACTH on steroidogenesis at multiple sites.