The role of protein kinase-C (PKC) in control of the function of rat adrenal glomerulosa cells was studied. Phorbol 12-myristate 13-acetate (PMA), an activator of PKC, inhibited the stimulation of aldosterone production induced by K+(5.4 mM) or ACTH (5 pM) in a dose-dependent manner. Phorbol 12, 13-dibutyrate, another phorbol ester that activates PKC, also exerted an inhibitory effect, while the inactive 4a-phorbol 12, 13-didecanoate failed to affect aldosterone production. The inhibitory effect of PMA (5 nM) was reversed by preincubation of the cells with staurosporine (ST; 50 nM), an inhibitor of PKC. These data suggest that pharmacological activation of PKC initiates an inhibitory mechanism in rat glomerulosa cells. To elucidate whether PKC is activated by physiological stimuli, the effects of ST and down-regulation of PKC by prolonged pretreatment with PMA on stimulation of aldosterone production were studied. The effects of angiotensin-II (All) and K+, but not that of ACTH, were enhanced by ST pretreatment. This potentiation was prompt and transient in the case of All (2.5 nM), while it developed gradually when the cells were stimulated with K+(5.4 or 18 mM). Long term pretreatment (6 h) of glomerulosa cells with PMA also enhanced the stimulatory effect of All (300 pM) and K+(5.4 mM). These data together suggest that the actions of All and K+on aldosterone production involve a PKC-mediated inhibition. Activation of PKC by All is probably due to formation of diacylglycerol via receptor-mediated activation of phosphoino-sitide-specific phospholipase-C. Stimulation with K+caused a moderate accumulation of [3H] inositol phosphate in a concentration-dependent manner. Since this effect was abolished by nifedipine, activation of phospholipase-C may have been secondary to Ca2+entry. The concomitant formation of diacylglycerol may contribute to activation of PKC in K+stimulated cells. In conclusion, our data support the view that PKC participates in the physiological control of aldosterone production by rat adrenal glomerulosa cells. In addition to All, K+may activate PKC. Regardless of whether the enzyme is activated by phorbol esters or physiological stimuli, it exerts an inhibitory, rather than stimulatory, action on steroid production.
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