A NOVEL ECTO-PHOSPHATIDIC ACID PHOSPHOHYDROLASE ACTIVITY MEDIATES ACTIVATION OF NEUTROPHIL SUPEROXIDE GENERATION BY EXOGENOUS PHOSPHATIDIC-ACID

Phosphatidic acid (PA) added to intact cells activates a variety of pr ocesses including mitogenesis in fibroblasts and superoxide generation in neutrophils. We have investigated the mechanism of activation of s uperoxide generation in intact human neutrophils by a short-chain (dio ctanoyl) PA (diC8PA). After a lag, diC8PA caused a high rate of supero xide production (19.6 nmol of cytochrome c reduced/min/10(6) cells). A ctivation did not require extracellular Ca2+ and coincided with near q uantitative conversion of diC8PA to dioctanoylglycerol (diC8-glycerol) . diC8PA also activated cellular phospholipase D with release of long- chain PA and secondary production of long-chain diradylglycerol (sn-1, 2-diacylglycerol and 1-O-alkyl-2-acylglycerol). The metabolism of diC8 PA to diC8-glycerol was catalyzed by a novel PA phosphohydrolase on th e outer leaflet of the plasma membrane as demonstrated by the exclusiv e release of P(i) into the extracellular medium. This enzyme also show ed activity toward PA containing long-chain unsaturated fatty acids. T he ecto-PA phosphohydrolase differed from the intracellular PA phospho hydrolase based on its relative insensitivity to desipramine and N-eth ylmaleimide. The enzyme was also present in Chinese hamster ovary (CHO ) cells and its activity did not change in transfected CHO cells expre ssing the two membrane-associated isoforms of alkaline phosphatase, in dicating that the PA phosphohydrolase was not alkaline phosphatase. No n-hydrolyzable phosphonate analogs of diC8PA poorly stimulated superox ide production. Activation of superoxide generation by diC8PA was inhi bited by staurosporine, suggesting a protein kinase C-dependent mechan ism. We suggest that the action of a novel ecto-PA phosphohydrolase pe rmits exogenously added short-chain PA to serve as ''timed-release dia cylglycerol'' and that its biological effects in neutrophils are secon dary to diacylglycerol-mediated protein kinase C activation.