Activation of human neutrophil NADPH oxidase by phosphatidic acid or diacylglycerol in a cell-free system - Activity of diacylglycerol is dependent on its conversion to phosphatidic acid

The superoxide-generating neutrophil NADPH oxidase can be activated in cell -free reconstitution systems by several agonists, most notably arachidonic acid and the detergent sodium dodecyl sulfate. In this study, we show that both phosphatidic acids and diacylglycerols can serve separately as potent, physiologic activators of NADPH oxidase in a cell-free system. Stimulation of superoxide generation by these lipids was dependent upon both Mg2+ and agonist concentration. Activation of NADPH oxidase by phosphatidic acids di d not appear to require their conversion to corresponding diacylglycerols b y phosphatidate phosphohydrolase, since diacylglycerols were much slower th an phosphatidic acids to activate the system and required the presence of A TP. Stimulation of the oxidase by dioctanoylglycerol proved to be by a mean s other than the activation of protein kinase C. Instead, dioctanoylglycero l was converted to dioctanoylphosphatidic acid by an endogenous diacylglyce rol kinase present in the cell-free reaction system. This conversion was se nsitive to the diacylglycerol kinase inhibitor R59949 and explains the mark edly slower kinetics of activation and the novel ATP requirement seen with dioctanoylglycerol. The level of dioctanoylphosphatidic acid formed was sub optimal for NADPH oxidase activation but could synergize with the unmetabol ized dioctanoylglycerol to activate superoxide generation.