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 protein kinase C (PKC) in adrenal glomerulosa cells stimulated by angiotensin II (ANG II). PKC activity in cells was monitored by measuring phosphorylation of a synthetic KRTLRR peptide, a specific substrate for PKC, immediately after the permeabilization of the cells with digitonin [Heasley and Johnson J. Biol. Chem. (1989) 264, 8646-8652]. Addition of 1 nM ANG II induced a gradual increase in KRTLRR peptide phosphorylation, which reached a peak at 30 min, and phosphorylation was sustained thereafter. When the action of ANG II was terminated by adding [Sar1,Ala8]ANG II, a competitive antagonist, both Ca2+ entry and KRTLRR phosphorylation ceased rapidly, whereas diacylglyercol (DAG) content was not changed significantly within 10 min. Similarly, when blockade of Ca2+ entry was achieved by decreasing extracellular Ca2+ to 1 microM or by adding 1 microM nitrendipine, KRTLRR peptide phosphorylation was decreased within 5 min. In addition, restoration of Ca2+ entry was accompanied by an immediate increase in KRTLRR peptide phosphorylation. Under the same condition, DAG content did not change significantly. We then examined the role of the PKC pathway in ANG II-induced aldosterone production. Ro 31-8220 inhibited ANG II-induced KRTLRR phosphorylation without affecting the activity of calmodulin-dependent protein kinase II. In the presence of Ro 31-8220, ANG II-mediated aldosterone production was decreased to approx. 50%. Likewise, intracellular administration of PKC19-36, a sequence corresponding to residues 19-36 of the regulatory domain of PKC known to inhibit PKC activity, attenuated ANG II-mediated activation of PKC and aldosterone output. These results indicate a critical role of Ca2+ entry in the regulation of PKC activity by ANG II.
Kojima
I
I
Cell Biology Research Unit, Gunma University, Maebashi, Japan.
Kawamura
N
N
Shibata
H
H
D000450:Aldosterone / Q000378:metabolism; D000595:Amino Acid Sequence; D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D004075:Diglycerides / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015384:Zona Glomerulosa / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism
D002147:Calmodulin; D004075:Diglycerides; D000804:Angiotensin II; D000450:Aldosterone; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium