Activation of protein kinase C in human neutrophils attenuates agonist-stimulated rises in cytosolic free Ca2+ concentration by inhibiting bivalent-cation influx and intracellular Ca2+ release in addition to stimulating Ca2+ efflux.
Stimulation of fura-2-loaded human neutrophils with formylmethionyl-leucyl-phenylalanine (FMLP) or ionomycin elevated the cytosolic free Ca2+ concentration, [Ca2+], to a maintained elevated level. Activation of protein kinase C (C-kinase) with phorbol 12-myristate 13-acetate, 4 beta-phorbol 12,13-didecanoate or dioctanoylglycerol caused decreases in [Ca2+]i from this level. 4 alpha-Phorbol didecanoate, which does not activate C-kinase, had no effect. These results confirm previous reports that C-kinase activation decreases neutrophil [Ca2+]i by stimulating removal of Ca2+ from the cytosol. Further experiments showed that activation of C-kinase attenuated the component of the FMLP-stimulated [Ca2+]i rise that was dependent on external Ca2+. C-kinase activation also inhibited FMLP-stimulated entry of the quenching cation, Mn2+, used as an indicator of bivalent-cation entry. In contrast, C-kinase activation caused only a partial inhibition of FMLP-stimulated release of Ca2+ from intracellular stores. 4 alpha-Phorbol didecanoate was ineffective in inhibiting Ca2+ entry, Mn2+ entry and intracellular Ca2+ release. Addition of FMLP also stimulated a decrease in the ionomycin-elevated [Ca2+]i, and this effect was blocked by staurosporine, a protein kinase inhibitor. These results show that, in addition to stimulating Ca2+ efflux, C-kinase activation in neutrophils inhibits FMLP-stimulated entry of bivalent cations, and partially inhibits intracellular release of Ca2+. Further, FMLP itself can modulate [Ca2+]i by activation of C-kinase.
McCarthy
S A
SA
Department of Cellular Pharmacology, Smith Kline and French Research Ltd., Welwyn, Herts., U.K.
Hallam
T J
TJ
Merritt
J E
JE
D001572:Benzofurans; D001692:Biological Transport; D002118:Calcium / Q000097:blood*; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation; D005456:Fluorescent Dyes; D016257:Fura-2; D006801:Humans; D066298:In Vitro Techniques; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D008345:Manganese / Q000097:blood; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D011493:Protein Kinase C / Q000097:blood*; D015290:Second Messenger Systems; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D001572:Benzofurans; D005456:Fluorescent Dyes; D008345:Manganese; D015759:Ionomycin; D009240:N-Formylmethionine Leucyl-Phenylalanine; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D016257:Fura-2