PHOSPHATIDIC-ACID AND DIACYLGLYCEROL SYNERGIZE IN A CELL-FREE SYSTEM FOR ACTIVATION OF NADPH OXIDASE FROM HUMAN NEUTROPHILS

NADPH oxidase, the respiratory burst enzyme of human neutrophils, is a multi-component complex that is assembled and activated during stimul ation of the cells by inflammatory or phagocytic stimuli. The signal m echanisms leading to activation of the enzyme are unclear, but it is l ikely that phospholipases are involved. Recent work has shown that pho sphatidic acid, the initial product of phospholipase D activation, is a weak activator of NADPH oxidase in a cell-free system. We now show t hat diacylglycerol enhances the cell-free activation of NADPH oxidase activation by phosphatidic acid. 1,2-Didecanoyl phosphatidic acid (10: 0-PA) and 1,2-dioctanoylglycerol (8:0-DG) each increased levels of NAD PH oxidase activity in mixtures of membrane and cytosolic fractions ab out 2-fold. The combination of both lipids increased NADPH oxidase act ivity approximately 12-fold, indicative of a synergistic response. Fat ty acid and neutral lipid metabolites of 10:0-PA or 8:0-DG were ineffe ctive, suggesting activation is directly mediated by phosphatidic acid and diacylglycerol. Activation was time- and concentration-dependent with maximum activation at 30-60 min and a sharp peak of maximal activ ity at 10 muM 10:0-PA and 30 muM 8:0-DG. In lipid specificity studies, activity of PA or DG decreased with increasing acyl chain length but was restored by introducing unsaturation in the acyl chain. Natural fo rms of PA stimulated levels of activity comparable to that seen with 1 0:0-PA. Synthetic and natural phosphatidylserines, but not other phosp holipids, could replace phosphatidic acid in the synergistic response. These studies provide direct evidence for a synergistic interaction b etween phosphatidic acid and diacylglycerol in mediating a cellular fu nction: the assembly and activation of NADPH oxidase. Our results supp ort the concept that the generation of second messenger lipids by phos pholipase D is a key step in activation of the respiratory burst enzym e.