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

The Gaze-messenger system plays a crucial role in the regulation of st eroid 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 sho w that Ca2+ levels (50-860 nM) regulate the production of both pregnen olone (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+ modula tion did not affect the formation of pregnenolone from freely diffusib le analogues of cholesterol, indicating that Ca2+ acts at a step upstr eam of cholesterol side-chain cleavage. Moreover cycloheximide, an inh ibitor of protein translation and of adrenocorticotropin-induced facil itation of intramitochondrial cholesterol transport, the rate-limiting step in steroidogenesis, also blocked Ca2+-triggered pregnenolone for mation. This is consistent with a model in which Ca2+ promotes cholest erol transfer between mitochondrial membranes. In addition, agents usi ng the cyclic AMP pathway as well as angiotensin II potentiated the st eroidogenic response to increases in [Ca2+](c) by augmenting both the efficacy and the potency of Ca2+. This effect of angiotensin II did no t involve protein kinase C. These results establish a direct link betw een agonist-induced [Ca2+](c) rises and a specific step of the steroid ogenic pathway.