Abstract
Phosphatidic acid (PA) added to intact cells activates a variety of processes including mitogenesis in fibroblasts and superoxide generation in neutrophils. We have investigated the mechanism of activation of superoxide generation in intact human neutrophils by a short-chain (dioctanoyl) PA (diC8PA). After a lag, diC8PA caused a high rate of superoxide production (19.6 nmol of cytochrome c reduced/min/10(6) cells). Activation did not require extracellular Ca2+ and coincided with near quantitative 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 diC8PA to diC8-glycerol was catalyzed by a novel PA phosphohydrolase on the outer leaflet of the plasma membrane as demonstrated by the exclusive release of Pi into the extracellular medium. This enzyme also showed activity toward PA containing long-chain unsaturated fatty acids. The ecto-PA phosphohydrolase differed from the intracellular PA phosphohydrolase based on its relative insensitivity to desipramine and N-ethylmaleimide. The enzyme was also present in Chinese hamster ovary (CHO) cells and its activity did not change in transfected CHO cells expressing the two membrane-associated isoforms of alkaline phosphatase, indicating that the PA phosphohydrolase was not alkaline phosphatase. Non-hydrolyzable phosphonate analogs of diC8PA poorly stimulated superoxide production. Activation of superoxide generation by diC8PA was inhibited by staurosporine, suggesting a protein kinase C-dependent mechanism. We suggest that the action of a novel ecto-PA phosphohydrolase permits exogenously added short-chain PA to serve as "timed-release diacylglycerol" and that its biological effects in neutrophils are secondary to diacylglycerol-mediated protein kinase C activation.
Highlights
Phosphatidic acid(PA) added to intacctells activates a variety of processes including mitogenesis in fibroblasts and superoxide generation in neutrophils
PA generated via agonist activation incided with near quantitative conversionof diC8PA of phospholipase D in Balb/c 3T3 cells [13] and vascular to dioctanoylglycerol. diC8PA ac-smooth muscle [3] correlates with mitogenesis and increased tivated cellular phospholipase D with releaseof long- DNA synthesis
Themetabolism of diC8PA todiC8-glycerol was catalyzebdy a novel PA phosphohydrolase on the outer leaflet of the plasma membrane as demonstrated by the exclusive releaseof Pi into the extracelphospholipase D or P A phosphohydrolase have implicated both P A and diacylglycerol in cell activation, including stimulation of superoxide generation [14, 15]
Summary
Recent studies have focused attention on possible roles of (“flip-flop”)or a carrier mechanism where it could exert its phosphatidic acid (PA)’ as asecond messenger Calcium ionophores activate a variety of cellular processes, including superoxide generation in phagocytic cells [26,27].PA extracts Ca2+from an aqueous into an organic phase [28] and has a Ca2+ionophoretic effect in model membranes [29]. Following lipid extraction as above, the CHC1, phase was analzyed for diC8glycerol and long-chain diradylglycerol by the enzymatic conversion to [32P]PAusing E. coli diglyceride kinase and [Y-~'P]ATP[37]. Were resuspended in 100 mM aminomethylpropanol, pH 10.5, Upon treatment of diacetone mannitol with periodate, the resulting and alkaline phosphatase activity was determined by measuring p aldehyde derivative was treated with the lithium saltof bisphosphon- nitrophenylphosphate hydrolysis as described by Nair et al [44].
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