The role of tyrosine kinases in capacitative calcium influx-mediated aldosterone production in bovine adrenal zona glomerulosa cells

In adrenal glomerulosa cells, the stimulation of aldosterone biosynthesis by angiotensin II (Ang II) involves the activation of a capacitative Ca2+ influx through calcium release-activated calcium (CRAC) channels. In various mammalian cell. systems, it has been shown that CRAC channel activation and Ca2+ entry require tyrosine kinase activity. We have therefore examined in this work whether similar mechanisms contribute to Ang II-induced mineralocorticoid biosynthesis. In fluo-3-loaded isolated bovine glomerulosa cells, two inhibitors of tyrosine kinases, genistein. and methyl-2,5-dihydroxycinnamate (MDHC) (100 mu M) prevented capacitative Ca2+ entry elicited by Ang II (by 54 and 62% respectively), while the inhibitor of epidermal growth factor (EGF) receptor tyrosine kinase, lavendustin A, was without effect. Similar results were observed on Ca2+ influx triggered by thapsigargin, an inhibitor of microsomal Ca2+ pumps. The inhibitors blocked Ang II-stimulated pregnenolone and aldosterone production in the same rank order. In addition to its specific effect on capacitative Ca2+ influx, genistein also affected the late steps of the steroidogenic pathway, as shown by experiments in which the rate-limiting step (intramitochondrial cholesterol transfer) was bypassed with 25-OH-cholesterol (25-OH-Chol), cytosolic calcium was clamped at stimulated levels or precursors of the late enzymatic steps were supplied. In contrast, genistin, a structural analogue of genistein devoid of tyrosine kinase inhibitory activity, was almost without effect on pregnenolone or 11-deoxycorticosterone (DOC) conversion to aldosterone. These results suggest that, in bovine adrenal glomerulosa cells, Ang II promotes capacitative Ca2+ influx and aldosterone biosynthesis through tyrosine kinase activation.