Protein kinase C regulates IL-8 and fMLP induced cytoplasmic Ca2+ increase in human granulocytes by receptor modulation measurements by flow cytometry.
Changes in cytosolic free Ca2+ influence important granulocyte functions like chemotactic behavior, adherence to endothelia, and phagocytosis. In the following study we used a simple reproducible procedure involving flow cytometry in combination with the fluorescent dye Fluo-3 to measure Ca2+ changes in human granulocytes. The aim of our study was to investigate the involvement of protein kinase C in regulating cytosolic free Ca2+ concentrations after stimulation of cells with IL-8 and fMLP. Both reagents induced a 5-6 fold increase in cytosolic Ca2+. Experiments conducted in Ca(2+)-free media showed a minor 18-29% decrease in cytosolic Ca2+ response, suggesting that intracellular Ca(2+)-stores are the main source for Ca2+ release after fMLP or IL-8 stimulation. Activators of protein kinase C, phorbol myristate acetate (PMA) and 1-oleyl-2-acetyl-sn-glycerol (OAG), inhibited cytosolic Ca(2+)-increase completely when induced by IL-8 and by 68-82% in the case of fMLP. Staurosporine, an inhibitor of protein kinase C, was able to attenuate or even abolish the PMA/OAG-effect. Our results show that changes in cytosolic Ca2+ due to IL-8 and fMLP signalling can be regulated by protein kinase C in human granulocytes. This regulatory role of protein kinase C involves some form of receptor modulation (i.e. phosphorylation, internalization, shedding).
Schöndorf
M
M
Department of Clinical Biochemistry, University of Bonn, Germany.
Bidlingmaier
F
F
von Ruecker
A A
AA
D000470:Alkaloids / Q000494:pharmacology; D000814:Aniline Compounds; D002118:Calcium / Q000097:blood*; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D004075:Diglycerides / Q000494:pharmacology; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006098:Granulocytes / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D016209:Interleukin-8 / Q000494:pharmacology*; D007700:Kinetics; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000187:drug effects / Q000502:physiology*; D018000:Receptors, Peptide / Q000187:drug effects / Q000502:physiology*; D011994:Recombinant Proteins / Q000494:pharmacology; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014966:Xanthenes
D000470:Alkaloids; D000814:Aniline Compounds; D004075:Diglycerides; D005456:Fluorescent Dyes; D016209:Interleukin-8; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D018000:Receptors, Peptide; D011994:Recombinant Proteins; D014966:Xanthenes; C059715:Fluo-3; D009240:N-Formylmethionine Leucyl-Phenylalanine; C038237:1-oleoyl-2-acetylglycerol; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium