THE SITE OF ACTION OF CA2+ IN THE ACTIVATION OF STEROIDOGENESIS - STUDIES IN CA2+-CLAMPED BOVINE ADRENAL ZONA-GLOMERULOSA CELLS

The Gaze-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%; EC(50) = 303 nM). By contrast, Ca2+ did not stimulate the conversion of II-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 Ca2+-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.