Calcium/Calmodulin-dependent Conversion of 5-Oxoeicosanoids to 6,7-Dihydro Metabolites by a Cytosolic Olefin Reductase in Human Neutrophils

Kiflu Berhane,Andrew A Ray,Subhash P Khanapure,Joshua Rokach,William S Powell

doi:10.1074/jbc.273.33.20951

Kiflu Berhane, Andrew A Ray + Show 3 more

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https://doi.org/10.1074/jbc.273.33.20951

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Abstract

We previously showed that 6-trans isomers of leukotriene B4 but not leukotriene B4 itself are converted to dihydro metabolites by human neutrophils. The first step in the formation of these metabolites is oxidation of the 5-hydroxyl group by 5-hydroxyeicosanoid dehydrogenase. The objective of the present investigation was to characterize the second step in the formation of the dihydro metabolites, reduction of an olefinic double bond. We found that the olefin reductase reduces the 6,7-double bond of 5-oxoeicosanoids, is localized in the cytosolic fraction of neutrophils, and requires NADPH as a cofactor. Neutrophil cytosol converts a variety of both 5-oxo- and 15-oxoeicosanoids to dihydro products. However, conversion of 5-oxoeicosanoids to their 6,7-dihydro metabolites is inhibited by EGTA and a calmodulin antagonist and stimulated by the addition of calcium and calmodulin, whereas the reduction of 15-oxoeicosanoids to their 13,14-dihydro metabolites is slightly inhibited by calcium. Furthermore, eicosanoid Delta6- and Delta13-reductases could be separated by chromatography on DEAE-Sepharose. 5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is converted by the Delta6-reductase to 6,7-dihydro-5-oxo-ETE, which is 1000 times less potent than 5-oxo-ETE in mobilizing calcium in neutrophils. We conclude that neutrophils contain both 5-oxoeicosanoid Delta6-reductase and prostaglandin Delta13-reductase. Metabolism of 5-oxo-ETE by the Delta6-reductase results in loss of its biological activity.

Highlights

We previously showed that 6-trans isomers of leukotriene B4 but not leukotriene B4 itself are converted to dihydro metabolites by human neutrophils
To investigate the subcellular localization of the olefin reductase required for the formation of these products, 5-oxo-12-epi-6-trans-leukotriene B4 (LTB4) was incubated with subcellular fractions from neutrophils in the presence of different cofactors
6-trans-LTB4 was incubated with the cytosolic fraction from neutrophils in the presence of NADPH was its dihydro metabolite (Fig. 1B)

Summary

Introduction

We previously showed that 6-trans isomers of leukotriene B4 but not leukotriene B4 itself are converted to dihydro metabolites by human neutrophils. A major pathway in the metabolism of many eicosanoids is initiated by oxidation of one of the hydroxyl groups by an NADϩ- or NADPϩ-dependent dehydrogenase This is usually followed by reduction of an adjacent double bond by an olefin reductase in the presence of NADH or NADPH. We previously showed that neutrophils convert 6-trans isomers of LTB4, which are formed nonenzymatically from LTA4, to dihydro metabolites [15, 16] This reaction proceeds by a sequence analogous to that described above for LTB4, the initial step being oxidation of the 5-hydroxyl group, followed by reduction of one of the double bonds and the oxo group [16]. The best substrate for 5-hydroxyeicosanoid dehydrogenase is (5S)-hydroxy-6,8,11,14eicosatetraenoic acid (5-HETE), which is converted to 5-oxoETE [18], a potent activator of neutrophils [19, 20] and eosinophils [21,22,23]

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