The site of action of Ca2+ in the activation of steroidogenesis: studies in Ca2+-clamped bovine adrenal zona-glomerulosa cells

The Ca(2+)-messenger system plays a crucial role in the regulation of steroid production in adrenal zona-glomerulosa cells, as it is known to mediate the action of both angiotensin II and K+. In the present study we used intact isolated glomerulosa cells in which the cytosolic free Ca2+ concentration ([Ca2+]c) was clamped at various levels with the Ca2+ ionophore ionomycin in order to locate the site(s) of action of Ca2+. By measuring in parallel steroid synthesis and [Ca2+]c, we show that Ca2+ levels (50-860 nM) regulate the production of both pregnenolone (up to 669 +/- 71.1% of the basal production) and aldosterone (up to 301 +/- 42.2%; EC50 = 303 nM). By contrast, Ca2+ did not stimulate the conversion of 11-deoxycorticosterone into aldosterone. Ca2+ modulation did not affect the formation of pregnenolone from freely diffusible analogues of cholesterol, indicating that Ca2+ acts at a step upstream of cholesterol side-chain cleavage. Moreover cycloheximide, an inhibitor of protein translation and of adrenocorticotropin-induced facilitation of intramitochondrial cholesterol transport, the rate-limiting step in steroidogenesis, also blocked Ca(2+)-triggered pregnenolone formation. This is consistent with a model in which Ca2+ promotes cholesterol transfer between mitochondrial membranes. In addition, agents using the cyclic AMP pathway as well as angiotensin II potentiated the steroidogenic response to increases in [Ca2+]c by augmenting both the efficacy and the potency of Ca2+. This effect of angiotensin II did not involve protein kinase C. These results establish a direct link between agonist-induced [Ca2+]c rises and a specific step of the steroidogenic pathway.