title: Ca2+-independent synergistic augmentation of O2- production by FMLP and PMA in HL-60 cells.
abstract: N-Formyl-Met-Leu-Phe (FMLP) and phorbol 12-myristate 13-acetate (PMA) caused a synergistic augmentation of superoxide anion (O2-) production in neutrophil-like HL-60 cells differentiated with dibutyryl cAMP. The present study was undertaken to investigate the mechanism of the synergistic augmentation of O2- production. FMLP increased intracellular free Ca2+ concentration ([Ca2+]i), which was slightly suppressed by PMA and completely inhibited by an intracellular Ca2+ chelating agent, O,O'-bis(2-aminophenyl)ethyleneglycol-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM). Although FMLP-induced O2- production was inhibited by BAPTA-AM, a major part of the synergistic augmentation of O2- production by FMLP and PMA remained after BAPTA-AM treatment, suggesting that a Ca2+-independent mechanism might be involved in the augmentation. FMLP and PMA caused an activation of phospholipase D (PLD) almost additively in a Ca2+-sensitive manner. The synergistic activation of mitogen-activated protein kinase (MAPK) was evoked by combined addition of PMA and FMLP in a BAPTA-AM resistant manner. However, PD98059, a MAPK kinase inhibitor, did not affect the synergistic augmentation of O2- production, although it potently inhibited the synergistic augmentation of tyrosine phosphorylation of MAPK. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, inhibited FMLP-induced O2- production, but it did not inhibit the synergistic augmentation of O2- production by PMA and FMLP. In contrast, staurosporine and GF109203X, protein kinase C inhibitors, reduced the synergistic augmentation induced by PMA and FMLP. In addition, pertussis toxin (PT) abolished the synergistic augmentation of O2- production. It is concluded that the synergistic augmentation of O2- production induced by PMA and FMLP is mediated through a PT-sensitive G protein and a protein kinase C in a Ca2+-independent manner.
authors: Department of Pharmaceutical Molecular Biology, Faculty of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.
authors: K
authors: K
authors: Tsukii
authors: N
authors: N
authors: Nakahata
authors: S
authors: S
authors: Tsurufuji
authors: Y
authors: Y
authors: Ohizumi
mesh_terms: D016718:Arachidonic Acid / Q000378:metabolism; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000187:drug effects / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D004357:Drug Synergism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D018922:HL-60 Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D037342:Pertussis Toxin; D010739:Phospholipase D / Q000187:drug effects / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D019311:Staurosporine / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013997:Time Factors; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D002614:Chelating Agents; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007211:Indoles; D008301:Maleimides; D010566:Virulence Factors, Bordetella; D013481:Superoxides; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D016718:Arachidonic Acid; D004533:Egtazic Acid; D009240:N-Formylmethionine Leucyl-Phenylalanine; D037342:Pertussis Toxin; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010739:Phospholipase D; D019311:Staurosporine; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D002118:Calcium