ROLE OF PROTEIN KINASE-C ON THE STEROIDOGENIC EFFECT OF ANGIOTENSIN-II IN THE RAT ADRENAL GLOMERULOSA CELL

The role of protein kinase C (PKC) in the ster-oidogenic action of angiotensin II (All) was investigated by depletion of endogenous PKC using prolonged incubation with phorbol ester and direct measurement of PKC in isolated rat adrenal glomerulosa cells. PKC activity was measured by incor-poration of32P from [γ32P]ATP into histone in the presence of cytosolic and detergent-solubilized membrane fractions purified by diethylaminoethyl cellulose chromatography. Basal PKC ac-tivity was higher in cytosol than in membranes (1, 000 ± 57 and 413 ± 14 pmol P incorporated/mg-min, respectively). After incubation of the cells with All for 5, 15, 30, and 60 min, PKC activity in the cytosol decreased by 5, 18, 25, and 27%, respec-tively, while in the membrane there was a transient increase of 15% at 15 min returning to basal by 60 min. Incubation of the cells with 100 nM 12-0-tetradecanoylphorbol-13-acetate (TPA) resulted in transient translocation of PKC activity to the mem-brane (15 min) which was followed by a 64% decrease in total cellular enzyme activity after 3 h. In PKC-depleted cells, the aldosterone response to ACTH was increased by 25% but All-stimulated steroidogenesis was unchanged. In contrast, in cells in which PKC was translocated to the membrane by a 15 min preincubation with TPA, aldosterone response to All was en-hanced by 40%, while the response to ACTH was reduced by 30%; under these conditions membrane PKC levels rapidly returned to basal. However, the changes in aldosterone response were still evident when addition of All or ACTH was delayed for up to 30 min after removal of TPA, indicating a persistent modification in the cell membrane secondary to PKC activation. Aldosterone responses to potassium were not altered by prein-cubation of the cells with TPA. The inactive phorbol ester analog, 4a-hydroxyphorbol-12, 13-dibutyrate, had no effect on the steroid responses to either stimulus. The small but significant translocation of PKC activity from cytosol to membrane after treatment of rat adrenal glomerulosa cells with All suggests that All activates PKC. However, the fact that aldosterone responses to All are potentiated during TPA-induced PKC translocation to the membrane suggests that All and phorbol esters do not share the same mechanism of action in the regulation of steroidogenesis. In addition, the full aldosterone response to All despite marked cellular PKC depletion after prolonged preincubation with TPA argues against the involvement of PKC in the stimulation of aldosterone production by AIL We propose that PKC activation by All may mediate effects other than steroidogenesis, such as trophic maintenance of the adrenal glomerulosa or adrenal growth. (Endocrinology 126: 125-133, 1990). © 1990 by The Endocrine Society.