interleukin-8 and 1-oleoyl-2-acetylglycerol

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).

Topics: Alkaloids; Aniline Compounds; Calcium; Cytoplasm; Diglycerides; Flow Cytometry; Fluorescent Dyes; Granulocytes; Humans; In Vitro Techniques; Interleukin-8; Kinetics; N-Formylmethionine Leucyl-Phenylalanine; Protein Kinase C; Receptors, Formyl Peptide; Receptors, Immunologic; Receptors, Peptide; Recombinant Proteins; Staurosporine; Tetradecanoylphorbol Acetate; Xanthenes