ACTIVATION OF NADPH OXIDASE AND PHOSPHOLIPASE-D IN PERMEABILIZED HUMAN NEUTROPHILS - CORRELATION BETWEEN OXIDASE ACTIVATION AND PHOSPHATIDIC-ACID PRODUCTION

A major function of human neutrophils (PMN) during inflammation is formation of oxygen radicals through activation of the respiratory burst enzyme, NADPH oxidase. Stimulus-induced production of both phosphatidic acid (PA) and diglyceride (DG) has been suggested to mediate oxidase activity; however, transductional mechanisms and cofactor requirements necessary for activation are poorly defined. We have utilized PMN permeabilized with Staphylococcus aureus alpha-toxin to elucidate the signal pathway involved in eliciting oxidase activity and to investigate whether PA or DG act as second messengers. PMN were permeabilized in cytoplasmic buffer supplemented with ATP and EGTA for 15 min before addition of NADPH and various cofactors. Oxidase activation was assessed by superoxide dismutase inhibitable reduction of ferricytochrome C; PA and DG levels were measured by radiolabeled product formation or by metabolite mass formation. Both superoxide (O2-) and PA formation were initiated by 10 muM GTPgammaS; addition of cytosolic levels of calcium ions (Ca2+, 120 nM) enhanced O2- and PA formation 1.5-2 fold. DG levels showed little change during these treatments. PA formation preceded O2- production and varying GTPgammaS levels had parallel effects on O2- and PA formation. However, while PA formation and oxidase activation occurred in tandem at Ca2+ levels of <1 muM, higher calcium enhanced PA formation but inhibited O2- production. Removal of ATP completely blocked O2- production but had little effect on PA formation; in contrast, if ATP was replaced with ATPgammaS, parallel production of PA and O2- occurred in the absence of other cofactors. Finally, while inhibition of PA production by ethanol pretreatment led to inhibition of O2- formation in PMN treated with GTPgammaS alone, in cells stimulated with a combination of GTPgammaS and Ca2+, ethanol continued to inhibit PA formation but had no effect on O2- production. Our results do not support a role for DG in the signal transduction path leading to oxidase activation and, while we show a close correlation between oxidase activation and PA production under many physiologic conditions, we also demonstrate that PA is not sufficient to induce oxidase activation and O2- formation can occur when PA production is inhibited.