Powell W S, Gravelle F, Gravel S
Meakins-Christie Laboratories, Department of Medicine, McGill University, Montreal, Quebec, Canada.
J Biol Chem. 1994 Oct 14;269(41):25373-80.
We have shown previously that human neutrophil microsomes contain a highly specific dehydrogenase which, in the presence of NADP+, converts 5S-hydroxy-6,8,11,14-eicosatetraenoic acid (5S-HETE) to its 5-oxo metabolite, 5-oxo-ETE, a potent agonist of these cells. However, intact neutrophils convert 5S-HETE principally to its omega-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 this investigation was to determine the mechanism for the stimulatory effect of PMA on 5-oxo-ETE formation. The possibility that oxidants released in response to PMA nonenzymatically oxidized 5S-HETE was ruled out, since PMA did not appreciably stimulate the formation of 5-oxo-ETE from 5R-HETE. On the other hand, inhibition of NADPH oxidase either by diphenylene iodonium or by mild heating nearly completely prevented the stimulatory effect of PMA on the formation of 5-oxo-ETE. The possibility 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 mechanism (xanthine/xanthine oxidase) did not appreciably affect the formation of 5-oxo-ETE by neutrophils. However, phenazine methosulfate, which 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 reduces 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 stimulates the formation of 5-oxo-ETE and inhibits that of 5,20-diHETE.
我们之前已经表明，人中性粒细胞微粒体含有一种高度特异性的脱氢酶，在NADP⁺存在的情况下，该酶可将5S-羟基-6,8,11,14-二十碳四烯酸（5S-HETE）转化为其5-氧代代谢产物5-氧代-ETE，这是这些细胞的一种强效激动剂。然而，完整的中性粒细胞主要将5S-HETE转化为其ω-氧化产物5,20-二氢二十碳四烯酸（5,20-diHETE），仅产生少量的5-氧代-ETE。佛波酯（PMA）可显著改变完整细胞对5S-HETE的代谢，使5-氧代-ETE成为主要代谢产物。本研究的目的是确定PMA对5-氧代-ETE形成的刺激作用机制。由于PMA不会明显刺激5R-HETE生成5-氧代-ETE，因此排除了PMA诱导释放的氧化剂非酶氧化5S-HETE的可能性。另一方面，二亚苯基碘鎓或轻度加热对NADPH氧化酶的抑制几乎完全阻断了PMA对5-氧代-ETE形成的刺激作用。这种作用由超氧化物介导的可能性似乎不大，因为在超氧化物歧化酶存在的情况下，尽管作用有所减弱，但仍可观察到该作用。此外，通过另一种机制（黄嘌呤/黄嘌呤氧化酶）产生的超氧化物对中性粒细胞形成5-氧代-ETE没有明显影响。然而，吩嗪硫酸甲酯可非酶地将NADPH转化为NADP⁺，它模拟了PMA对完整中性粒细胞形成5-氧代-ETE的作用。得出的结论是，PMA通过激活NADPH氧化酶起作用，导致NADPH转化为NADP⁺，从而增强5-氧代-ETE的形成并减少5,20-diHETE的形成。血清处理的酵母聚糖对5S-HETE代谢的影响与PMA类似，即它也刺激5-氧代-ETE的形成并抑制5,20-diHETE的形成。
