Ca2+-independent synergistic augmentation of O-2(-) production by FMLP andPMA in HL-60 cells

N-Formyl-Met-Leu-Phe (FMLP) and phorbol 12-myristate 13-acetate (PMA) cause d a synergistic augmentation of superoxide anion (O-2(-)) production in neu trophil-like HL-60 cells differentiated with dibutyryl cAMP. The present st udy was undertaken to investigate the mechanism of the synergistic augmenta tion of O-2(-) production. FMLP increased intracellular free Ca2+ concentra tion ([Ca2+](i)), which was slightly suppressed by PMA and completely inhib ited by an intracellular Ca2+ chelating agent, O,O'bis(2-aminophenyl)ethyle neglycol-N,N,N'N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM). Alt hough FMLP-induced O-2(-) production was inhibited by BAPTA-AM, a major par t of the synergistic augmentation of O-2(-) production by FMLP and PMA rema ined after BAPTA-AM treatment, suggesting that a Ca2+-independent mechanism might be involved in the augmentation. FMLP and PMA caused an activation o f phospholipase D (PLD) almost additively in a Ca2+-sensitive manner. The s ynergistic activation of mitogen-activated protein kinase (MAPK) was evoked by combined addition of PMA and FMLP in a BAPTA-AM resistant manner. Howev er, PD98059, a MAPK kinase inhibitor, did not affect the synergistic augmen tation of O-2(-) production, although it potently inhibited the synergistic augmentation of tyrosine phosphorylation of MAPK. Wortmannin, a phosphatid ylinositol 3-kinase inhibitor, inhibited FMLP-induced O-2(-) production, bu t it did not inhibit the synergistic augmentation of O-2(-) production by P MA and FMLP. In contrast, staurosporine and GF109203X, protein kinase C inh ibitors, reduced the synergistic augmentation induced by PMA and FMLP. In a ddition, pertussis toxin (PT) abolished the synergistic augmentation of O-2 (-) production. It is concluded that the synergistic augmentation of O-2(-) production induced by PMA and FMLP is mediated through a PT-sensitive G pr otein and a protein kinase C in a Ca2+-independent manner.