Abstract
5-Oxo-ETE (5-oxo-6,8,11,14-eicosatetraenoic acid) is a highly potent granulocyte chemoattractant that acts through a selective G-protein coupled receptor. It is formed by oxidation of the 5-lipoxygenase product 5-HETE (5S-hydroxy-6,8,11,14-eicosatetraenoic acid) by 5-hydroxyeicosanoid dehydrogenase (5-HEDH). Although leukocytes and platelets display high microsomal 5-HEDH activity, unstimulated intact cells do not convert 5-HETE to appreciable amounts of 5-oxo-ETE. To attempt to resolve this dilemma we explored the possibility that 5-oxo-ETE synthesis could be enhanced by oxidative stress. We found that hydrogen peroxide and t-butyl hydroperoxide strongly stimulate 5-oxo-ETE formation by U937 monocytic cells. This was dependent on the GSH redox cycle, as it was blocked by depletion of GSH or inhibition of glutathione reductase and mimicked by oxidation of GSH to GSSG by diamide. Glucose inhibited the response to H2O2 through its metabolism by the pentose phosphate pathway, as its effect was reversed by the glucose-6-phosphate dehydrogenase inhibitor dehydroepiandrosterone. 5-Oxo-ETE synthesis was also strongly stimulated by hydroperoxides in blood monocytes, lymphocytes, and platelets, but not neutrophils. Unlike monocytic cells, lymphocytes and platelets were resistant to the inhibitory effects of glucose. 5-Oxo-ETE synthesis following incubation of peripheral blood mononuclear cells with arachidonic acid and calcium ionophore was also strongly enhanced by t-butyl hydroperoxide. Oxidative stress could act by depleting NADPH, resulting in the formation NADP+, the cofactor for 5-HEDH. This is opposed by the pentose phosphate pathway, which converts NADP+ back to NADPH. Oxidative stress could be an important mechanism for stimulating 5-oxo-ETE production in inflammation, promoting further infiltration of granulocytes into inflammatory sites.
Highlights
5-Oxo-ETE (5-oxo-6,8,11,14-eicosatetraenoic acid) is a highly potent granulocyte chemoattractant that acts through a selective G-protein coupled receptor
Glucose inhibited the response to H2O2 through its metabolism by the pentose phosphate pathway, as its effect was reversed by the glucose-6-phosphate dehydrogenase inhibitor dehydroepiandrosterone. 5-Oxo-ETE synthesis was strongly stimulated by hydroperoxides in blood monocytes, lymphocytes, and platelets, but not neutrophils
To determine whether the transient effect of H2O2 could be because of its metabolism, incubations were performed in the presence of sodium azide, which inhibits both catalase and Oxidative Stress and 5-Oxo-ETE Synthesis
Summary
Materials—5-HETE was prepared by total organic synthesis [24]. 13S-Hydroxy-9Z,11E-octadecadienoic acid (13-HODE) and 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13-HpODE) were prepared by oxidation of linoleic acid with soybean lipoxygenase Type 1B (Sigma) [25]. 5S-HpETE was purchased from Cayman Chemical, Ann Arbor, MI. RPMI 1640 and other products for cell culture were purchased from Invitrogen. After centrifugation at 1000 ϫ g for 10 min, the pellet was suspended in PBSϪ to give a platelet concentration of 3 ϫ 108 cells/ml. Preparation of Microsomal Fractions from U937 Cells—Cells were washed by centrifugation, resuspended in 20 ml of PBSϪ supplemented with 1 mM phenylmethylsulfonyl fluoride, and disrupted by sonication at a setting of 40 cycles/s (model 4710 ultrasonic homogenizer, Sonics and Materials, Newtown, CT) on ice for 5 ϫ 6 s with 30-s intervals for cooling. Suspensions (1 ml) of U937 cells, lymphocytes, monocytes, neutrophils (2 ϫ 106 cells/ml), platelets (108 cells/ml), or U937 cell microsomes (150 g of protein/ml) were incubated with 5-HETE (1 M) for 5 min unless otherwise indicated. In the case of blood cells, a different donor was used for each experiment
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