Primary cultures of bovine adrenal glomerulosa cells will be used to study the mechanisms by which angiotensin II affects steroidogenesis. Attention will focus on the role of calcium in the actions of the peptide, and studies performed to test the hypotheses: (1) that an increase in cytosolic free calcium is needed for angiotensin-stimulated cholesterol side chain cleavage activity; (2) that the increase in enzyme activity is accompanied by an increase in the phosphorylation state of one or more of the components of the hydroxylase system; and (3) that the phosphorylation of cell protein(s) is catalyzed by calcium dependent kinases. Effects of angiotensin on cytosolic free calcium will be measured spectrofluorometrically using Quin 2 accetoxymethyl ester and correlated with those on pregnenolone production and protein phosphorylations. The role of calcium in the activation of pregnenolone production and the phosphorylation of mitochondrial cytosolic proteins will also be studied by (1) comparing the actions of the peptide in the presence and absence of calcium, (2) comparing the actions of angiotensin and those of ionophore, and (3) examining the effects of agents (verapamil and TMB-8) that prevent calcium translocation/mobilization on the actions of the hormone. Measurements of pregnenolone production from cholesterol and 25-hydroxycholesterol, mitochondrial cholesterol content and cholesterol ester hydrolase activity will be used to delineate actions of angiotensin and calcium on enzyme activation and substrate mobilization. Phosphoproteins will be resolved by 1- and 2-dimensional electrophoresis and visualized by autoradiography. Changes in the phosphorylation state of cell proteins will be quantitated by scanning densitometry and correlated with those in free calcium levels, pregnenolone production and calcium-dependent kinase activity. The role of calcium-, calcium/calmodulin- and calcium/phospholipiddependent protein kinases will be examined by comparing phosphorylation patterns induced by angiotensin with those produced in vitro in the presence of specific promotors of protein kinases. Changes in the phosphorylation state of a component(s) to the cholesterol side chain cleavage system will be examined by immunoisolating [32p] phosphate-labeled cytochrome P-450, adrenodoxin and adrenodoxin reductase from total radiolabeled proteins. These studies should provide important insight into the mechanism by which angiotensin affects glomerulosa cell function.
