Roles of phosphatidylinositol 3-kinase and phospholipase D in temporal activation of superoxide production in FMLP-stimulated human neutrophils.

To determine the temporal roles of phosphatidylinositol 3-kinase (PI3-kinase) and phospholipase D (PLD) during human neutrophil activation stimulated by a chemotactic peptide, we examined the kinetics of these enzymes and related them to a neutrophil function (superoxide production). Both wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), potent and specific inhibitors of PI3-kinase, inhibit PI3-kinase activity in human neutrophils and significantly inhibit superoxide production from the early phase. Ethanol has no effect on PI3-kinase and markedly inhibits superoxide production at the late phase. Although these agents are inhibitory to different degrees, when neutrophils are simultaneously treated with ethanol and PI3-kinase inhibitors, superoxide is not produced. These results suggest that PI3-kinase and PLD play a pivotal role in the signal transduction pathway of the chemo-attractant-receptor involved neutrophil activation. These enzymes produce second messengers which are required for subsequent superoxide production in human neutrophils. NADPH oxidase is activated in a PI3-kinase-dependent manner at the early phase, and PLD activity follows it and is related to superoxide production at the late phase in human neutrophils by stimulation with FMLP.