PHORBOL-MYRISTATE ACETATE STIMULATES THE FORMATION OF 5-OXO-6,8,11,14-EICOSATETRAENOIC ACID BY HUMAN NEUTROPHILS BY ACTIVATING NADPH OXIDASE

We have shown previously that human neutrophil microsomes contain a hi ghly specific dehydrogenase which, in the presence of NADP(+), convert s 5S-hydroxy-6,8,11,14-eicosatetraenoic acid (5S-HETE) to its 5-oxo me tabolite, 5-oxo-ETE, a potent agonist of these cells. However, intact neutrophils convert SS-HETE principally to its psi-oxidation product, 5,20-diHETE, and to only small amounts of 5-oxo-ETE. Phorbol myristate acetate (PMA) dramatically shifts the metabolism of 5S-HETE by intact cells so that 5-oxo-ETE is the major metabolite. The objective of thi s investigation was to determine the mechanism for the stimulatory eff ect of PMA on 5-oxo-ETE formation. The possibility that oxidants relea sed in response to PMA nonenzymatically oxidized 5S-HETE was ruled out , since PMA did not appreciably stimulate the formation of 5-oxo-ETE f rom 5R-HETE. On the other hand, inhibition of NADPH oxidase either by diphenylene iodonium or by mild heating nearly completely prevented th e stimulatory effect of PMA on the formation of 5-oxo-ETE. The possibi lity that this effect was mediated by superoxide seems unlikely, since it was still observed, although somewhat attenuated, in the presence of superoxide dismutase. Moreover, superoxide generated by another mec hanism (xanthine/xanthine oxidase) did not appreciably affect the form ation of 5-oxo-ETE by neutrophils, However, phenazine methosulfate, wh ich can nonenzymatically convert NADPH to NADP(+), mimicked the effect of PMA on 5-oxo-ETE formation by intact neutrophils. It is concluded that PMA acts by activating NADPH oxidase, resulting in conversion of NADPH to NADP(+), which enhances the formation of 5-oxo-ETE and reduce s the formation of 5,20-diHETE. Serum-treated zymosan has an effect on the metabolism of 5S-HETE similar to that of PMA in that it also stim ulates the formation of 5-oxo-ETE and inhibits that of 5,20 diHETE.