External calcium is required for activation of phospholipase C by angiotensin II in adrenal glomerulosa cells

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Abstract
Previous studies have shown that external calcium (Ca2+) is required for the effects of angiotensin II (AII) on aldosterone secretion in adrenal glomerulosa zone. Using bovine adrenal glomerulosa cells prepared by collagenase dispersion, we examined whether external Ca2+ is required for the activation of phospholipase C by AII. Adrenal glomerulosa cells were exposed to Ca-EGTA buffered media to provide accurate estimates of external free Ca2+ concentrations. Phospholipase C activation was evaluated by measurement of inositol phosphates production. At 0.1 μM Ca2+ and less, sustained AII effects on inositol monophosphate (IP), inositol bisphosphate (IP2) and inositol trisphosphate (IP3) were markedly inhibited. Increasing the Ca2+ concentration to 50μkM or greater fully restored All-induced inositol phosphates production. AII-induced increases in cytosolic Ca2+ measured by Quin-2 fluorescence, were diminished at lower external Ca2+ concentrations. Treating adrenal glomerulosa cells with Chelex-100, a strong Ca2+ binding resin, blocked early activation of phospholipase C by AII. Inhibition of IP3 production was also observed when inhibitors of Ca2+ movement across the plasma membrane were used, viz., La2+, TMB-8 and nifedipine. The requirement for Ca2+ during AII-induced activation of phospholipase C may be explained, at least partly by a requirement for Ca2+ at a site between the AII receptor and Phospholipase C.