Zhang Y, Styhler A, Powell W S
Meakins-Christie Laboratories, McGill University, Montreal, Quebec, Canada.
J Leukoc Biol. 1996 Jun;59(6):847-54. doi: 10.1002/jlb.59.6.847.
We recently demonstrated that the arachidonate metabolite 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) is converted by a highly specific dehydrogenase in human neutrophils to 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), which is a potent stimulator of these cells. The objective of this study was to determine whether 5-oxo-ETE is also formed by monocytes and lymphocytes. Human monocytes (74 +/- 2% pure) and lymphocytes (86 +/- 1% pure) were prepared by successive centrifugations of leukocytes over Ficoll-Paque and Percoll. Both cell types converted 5-HETE to a single major product, which was identified as 5-oxo-ETE. The formation of 5-oxo-ETE was stimulated about twofold by phorbol myristate acetate (PMA; 30 nM). Dehydrogenase activity in monocyte fractions did not appear to be due to platelet contamination, since depletion of platelets did not reduce enzyme activity. The dehydrogenase was localized in membrane fractions from monocytes and required NADP+ as a cofactor. It was specific for eicosanoids containing a 5S-hydroxyl group followed by a 6-trans double bond. We also investigated the formation of 5-oxo-ETE from endogenous arachidonic acid by monocytes. 5-Oxo-ETE, 5-HETE, and leukotriene B4 (LTB4) were present in comparable amounts after incubation of these cells with A23187. PMA (EC50 approximately 4 nM) stimulated the formation of 5-oxo-ETE and 5-HETE and, to a lesser extent, LTB4. Although monocytes released considerably less 5-HETE and LTB4 than neutrophils, they released comparable amounts of 5-oxo-ETE. Unlike neutrophils, monocytes did not convert any of these substances to detectable amounts of omega-oxidation products. Although lymphocytes were capable of converting 5-HETE to 5-oxo-ETE, they released little or no 5-lipoxygenase products in response to A23187. We conclude that monocytes have a high capacity to synthesize 5-oxo-ETE and that its formation is stimulated by activation of protein kinase C.
我们最近证明，花生四烯酸代谢物5(S)-羟基-6,8,11,14-二十碳四烯酸（5-HETE）在人类中性粒细胞中被一种高度特异性的脱氢酶转化为5-氧代-6,8,11,14-二十碳四烯酸（5-氧代-ETE），后者是这些细胞的一种强效刺激物。本研究的目的是确定单核细胞和淋巴细胞是否也能生成5-氧代-ETE。通过在Ficoll-Paque和Percoll上对白细胞进行连续离心制备了人类单核细胞（纯度为74±2%）和淋巴细胞（纯度为86±1%）。两种细胞类型都将5-HETE转化为单一主要产物，该产物被鉴定为5-氧代-ETE。佛波醇肉豆蔻酸酯乙酸盐（PMA；30 nM）使5-氧代-ETE的生成增加约两倍。单核细胞组分中的脱氢酶活性似乎并非由于血小板污染，因为去除血小板并未降低酶活性。该脱氢酶定位于单核细胞膜组分中，需要NADP⁺作为辅因子。它对含有5S-羟基且随后有一个6-反式双键的类花生酸具有特异性。我们还研究了单核细胞从内源性花生四烯酸生成5-氧代-ETE的情况。在用A23187孵育这些细胞后，5-氧代-ETE、5-HETE和白三烯B4（LTB4）的含量相当。PMA（半数有效浓度约为4 nM）刺激了5-氧代-ETE和5-HETE的生成，对LTB4的刺激程度较小。尽管单核细胞释放的5-HETE和LTB4比中性粒细胞少得多，但它们释放的5-氧代-ETE量相当。与中性粒细胞不同，单核细胞不会将这些物质中的任何一种转化为可检测量的ω-氧化产物。尽管淋巴细胞能够将5-HETE转化为5-氧代-ETE，但它们在对A23187的反应中释放很少或不释放5-脂氧合酶产物。我们得出结论，单核细胞具有合成5-氧代-ETE的高能力，并且其生成受到蛋白激酶C激活的刺激。
