RATE OF CALCIUM-ENTRY DETERMINES THE RAPID CHANGES IN PROTEIN-KINASE-C ACTIVITY IN ANGIOTENSIN II-STIMULATED ADRENAL GLOMERULOSA CELLS

The present study was conducted to monitor precisely the activity of p rotein kinase C (PKC) in adrenal glomerulosa cells stimulated by angio tensin II (ANG II). PKC activity in cells was monitored by measuring p hosphorylation of a synthetic KRTLRR peptide, a specific substrate for PKC, immediately after the permeabilization of the cells with digiton in [Heasley and Johnson J. Biol. Chem. (1989) 264, 8646-8652]. Additio n of 1 nM ANG II induced a gradual increase in KRTLRR peptide phosphor ylation, which reached a peak at 30 min, and phosphorylation was susta ined thereafter. When the action of ANG II was terminated by adding [S ar(1),Ala(8)]ANG II, a competitive antagonist, both Ca2+ entry and KRT LRR phosphorylation ceased rapidly, whereas diacylglyercol (DAG) conte nt was not changed significantly within 10 min. Similarly, when blocka de of Ca2+ entry was achieved by decreasing extracellular Ca2+ to 1 mu M or by adding 1 mu M nitrendipine, KRTLRR peptide phosphorylation wa s decreased within 5 min. In addition, restoration of Ca2+ entry was a ccompanied by an immediate increase in KRTLRR peptide phosphorylation. Under the same condition, DAG content did not change significantly. W e then examined the role of the PKC pathway in ANG II-induced aldoster one production. Ro 31-8220 inhibited ANG II-induced KRTLRR phosphoryla tion without affecting the activity of calmodulin-dependent protein ki nase II. In the presence of Ro 31-8220, ANG II-mediated aldosterone pr oduction was decreased to approx. 50%. Likewise, intracellular adminis tration of PKC19-36, a sequence corresponding to residues 19-36 of the regulatory domain of PKC known to inhibit PKC activity, attenuated AN G II-mediated activation of PKC and aldosterone output. These results indicate a critical role of Ca2+ entry in the regulation of PKC activi ty by ANG II.