PHORBOL ESTER-INDUCED PRIMING OF SUPEROXIDE GENERATION BY PHOSPHATIDIC ACID-STIMULATED NEUTROPHILS AND GRANULE-FREE NEUTROPHIL CYTOPLASTS

This study was undertaken to examine the mechanisms involved in polymo rphonuclear leukocyte superoxide release stimulated by exogenous phosp hatidic acid (PA), Unlike the immediate burst of superoxide release af fected by membrane-permeable dioctanoyl-glycerol (DiC8-DAG), dioctanoy l phosphatidic acid (DiC8-PA) induced superoxide release after a lag p eriod of 5-20 min, This period was considerably reduced or eliminated when cells were primed by substimulatory levels of phorbol myristate a cetate (PMA), Granule-depleted neutrophil cytoplasts also responded to DiC8-PA with a burst of superoxide generation, Activation of the cyto plast superoxide generating system in response to DiC8-PA was also sig nificantly faster after cells had been preexposed to substimulatory le vels of PMA, indicating that at least a portion of the priming mechani sm was independent of PMA-induced degranulation. To further examine th e potential mechanism of PMA priming of responses to PA, we evaluated the activity of neutrophil ecto phosphatidic acid phosphohydrolase (ec to-PA phosphohydrolase), which generates diacylglycerol from exogenous PA, PIMA priming had no discernable effect on the activity of this en zyme, In addition, propranolol, an inhibitor of PA phosphohydrolase, d id not selectively inhibit PMA, priming of neutrophil responses to DiC 8-PA, indicating that priming did not result from acceleration of DiC8 -PA hydrolysis, We therefore investigated the possibility that activat ion of protein kinase C was the basis of the primed response, Several semiselective protein kinase C inhibitors (calphostin C, H-7, and acyl methylglycerol) inhibited DiC8-DAG- and DiC8-PA-induced superoxide rel ease as well as PMA-primed responses to approximately the same extent, These results are consistent with the hypothesis that neutrophil resp onses to phosphatidate are mediated by diglyceride generated by the ac tion of ecto-PA phosphohydrolase, PMA priming does not result from inc reased catalytic activity of ecto PA phosphohydrolase but rather seems to result from potentiation of an intermediate involved in the cells' response to multiple stimuli.