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• W2055364518 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. 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. The 5-lipoxygenase pathway is critical in inflammation, because it gives rise to a number of potent inflammatory mediators, including leukotriene (LT) 1The abbreviations used are: LT, leukotriene; BCNU, 1,2-bis[2-chloroethyl]-1-nitrosourea (carmustine); BSO, buthionine sulfoximine; DHEA, dehydroepiandrosterone; Me2SO, dimethyl sulfoxide; 5-HETE, 5S-hydroxy-6,8,11,14-eicosatetraenoic acid; 13-HODE, 13S-hydroxy-9Z,11E-octadecadienoic acid; 13-HpODE, 13S-hydroperoxy-9Z,11E-octadecadienoic acid; 5-HEDH, 5-hydroxyeicosanoid dehydrogenase; NEM, N-ethylmaleimide; 5-oxo-ETE, 5-oxo-6,8,11,14-eicosatetraenoic acid; PMA, phorbol 12-myristate 13-acetate; PMS, phenazine methosulfate; RP-HPLC, reversed-phase high performance liquid chromatography; tBuOOH, tert-butyl hydroperoxide; PBS, phosphate-buffered saline; PBMC, peripheral blood mononuclear cells. 1The abbreviations used are: LT, leukotriene; BCNU, 1,2-bis[2-chloroethyl]-1-nitrosourea (carmustine); BSO, buthionine sulfoximine; DHEA, dehydroepiandrosterone; Me2SO, dimethyl sulfoxide; 5-HETE, 5S-hydroxy-6,8,11,14-eicosatetraenoic acid; 13-HODE, 13S-hydroxy-9Z,11E-octadecadienoic acid; 13-HpODE, 13S-hydroperoxy-9Z,11E-octadecadienoic acid; 5-HEDH, 5-hydroxyeicosanoid dehydrogenase; NEM, N-ethylmaleimide; 5-oxo-ETE, 5-oxo-6,8,11,14-eicosatetraenoic acid; PMA, phorbol 12-myristate 13-acetate; PMS, phenazine methosulfate; RP-HPLC, reversed-phase high performance liquid chromatography; tBuOOH, tert-butyl hydroperoxide; PBS, phosphate-buffered saline; PBMC, peripheral blood mononuclear cells. B4, the cysteinyl-LTs, the lipoxins, and 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE) (1Funk C.D. Science. 2001; 294: 1871-1875Crossref PubMed Scopus (2966) Google Scholar, 2Levy B.D. De Sanctis G.T. Devchand P.R. Kim E. Ackerman K. Schmidt B.A. Szczeklik W. Drazen J.M. Serhan C.N. Nat. Med. 2002; 8: 1018-1023Crossref PubMed Scopus (326) Google Scholar). 5-Oxo-ETE, the most recently described member of this family, is the most active eosinophil chemoattractant among lipid mediators (3Powell W.S. Chung D. Gravel S. J. Immunol. 1995; 154: 4123-4132PubMed Google Scholar). It elicits a variety of responses in these cells, including actin polymerization, calcium mobilization, CD11b expression, degranulation, and migration (4O'Flaherty J.T. Kuroki M. Nixon A.B. Wijkander J. Yee E. Lee S.L. Smitherman P.K. Wykle R.L. Daniel L.W. J. Immunol. 1996; 157: 336-342PubMed Google Scholar, 5Czech W. Barbisch M. Tenscher K. Schopf E. Schröder J.M. Norgauer J. J. Investig. Dermatol. 1997; 108: 108-112Abstract Full Text PDF PubMed Scopus (36) Google Scholar, 6Powell W.S. Gravel S. Halwani F. Am. J. Respir. Cell Mol. Biol. 1999; 20: 163-170Crossref PubMed Scopus (51) Google Scholar) and induces eosinophil infiltration into rat lungs (7Stamatiou P. Hamid Q. Taha R. Yu W. Issekutz T.B. Rokach J. Khanapure S.P. Powell W.S. J. Clin. Investig. 1998; 102: 2165-2172Crossref PubMed Scopus (62) Google Scholar) and human skin (8Muro S. Hamid Q. Olivenstein R. Taha R. Rokach J. Powell W.S. J. Allergy Clin. Immunol. 2003; 112: 768-774Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) in vivo. 5-Oxo-ETE also has chemoattractant effects on neutrophils (9Powell W.S. Gravel S. MacLeod R.J. Mills E. Hashefi M. J. Biol. Chem. 1993; 268: 9280-9286Abstract Full Text PDF PubMed Google Scholar) and monocytes (10Sozzani S. Zhou D. Locati M. Bernasconi S. Luini W. Mantovani A. O'Flaherty J.T. J. Immunol. 1996; 157: 4664-4671PubMed Google Scholar). Its biological effects are mediated by a highly selective Gi protein-coupled receptor that has recently been cloned (11Hosoi T. Koguchi Y. Sugikawa E. Chikada A. Ogawa K. Tsuda N. Suto N. Tsunoda S. Taniguchi T. Ohnuki T. J. Biol. Chem. 2002; 277: 31459-31465Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 12Jones C.E. Holden S. Tenaillon L. Bhatia U. Seuwen K. Tranter P. Turner J. Kettle R. Bouhelal R. Charlton S. Nirmala N.R. Jarai G. Finan P. Mol. Pharmacol. 2003; 63: 471-477Crossref PubMed Scopus (93) Google Scholar). This receptor is quite distinct from all other eicosanoid receptors and is highly expressed on eosinophils, neutrophils, and monocytes, in that order (11Hosoi T. Koguchi Y. Sugikawa E. Chikada A. Ogawa K. Tsuda N. Suto N. Tsunoda S. Taniguchi T. Ohnuki T. J. Biol. Chem. 2002; 277: 31459-31465Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 12Jones C.E. Holden S. Tenaillon L. Bhatia U. Seuwen K. Tranter P. Turner J. Kettle R. Bouhelal R. Charlton S. Nirmala N.R. Jarai G. Finan P. Mol. Pharmacol. 2003; 63: 471-477Crossref PubMed Scopus (93) Google Scholar). The regulation of 5-oxo-ETE biosynthesis is not well understood, and it can be formed both enzymatically (13Powell W.S. Gravelle F. Gravel S. J. Biol. Chem. 1992; 267: 19233-19241Abstract Full Text PDF PubMed Google Scholar) and non-enzymatically via lipid peroxidation (14Hall L.M. Murphy R.C. Chem. Res. Toxicol. 1998; 11: 1024-1031Crossref PubMed Scopus (17) Google Scholar). It is formed enzymatically by the oxidation of 5S-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-HETE) by 5-hydroxyeicosanoid dehydrogenase (5-HEDH), a highly selective NADP+-dependent microsomal enzyme that is present in human neutrophils, eosinophils, monocytes, lymphocytes, and platelets (13Powell W.S. Gravelle F. Gravel S. J. Biol. Chem. 1992; 267: 19233-19241Abstract Full Text PDF PubMed Google Scholar, 15Zhang Y. Styhler A. Powell W.S. J. Leukoc. Biol. 1996; 59: 847-854Crossref PubMed Scopus (40) Google Scholar, 16Powell W.S. Gravel S. Khanapure S.P. Rokach J. Blood. 1999; 93: 1086-1096Crossref PubMed Google Scholar). Alternatively, 5-oxo-ETE can be formed directly from 5-hydroperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-HpETE) by murine macrophages, but not human neutrophils, in the presence of a cytosolic protein (17Zarini S. Murphy R.C. J. Biol. Chem. 2003; 278: 11190-11196Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The mechanism for this reaction may be akin to that previously reported for the decomposition of hydroperoxy fatty acids in the presence of heme-containing proteins (18Hamberg M. Lipids. 1975; 10: 87-92Crossref PubMed Scopus (144) Google Scholar). Unlike 5-HEDH, the pathway in murine macrophages is not stereoselective for the S configuration at C5 (17Zarini S. Murphy R.C. J. Biol. Chem. 2003; 278: 11190-11196Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Despite the presence of very high 5-HEDH activity in microsomal fractions from inflammatory cells, unstimulated intact cells form only very small amounts of 5-oxo-ETE when incubated with 5-HETE. This may be because of limited availability of NADP+, which is present at very low levels in the cytosol, as cells maintain this cofactor in its reduced state (NADPH) as a protective mechanism against oxidative stress (19Schafer F.Q. Buettner G.R. Free Radic. Biol. Med. 2001; 30: 1191-1212Crossref PubMed Scopus (3551) Google Scholar). In neutrophils, 5-oxo-ETE synthesis is dramatically increased by stimulation of the respiratory burst, which would increase the levels of NADP+ in the cytosol (20Powell W.S. Gravelle F. Gravel S. J. Biol. Chem. 1994; 269: 25373-25380Abstract Full Text PDF PubMed Google Scholar). This raised the possibility that other factors that affect the NADP+ levels in cells could also regulate 5-oxo-ETE synthesis. Reactive oxygen species, such as superoxide and H2O2, are formed at inflammatory sites and in pathological states associated with leukocyte infiltration (21Bowler R.P. Crapo J.D. J. Allergy Clin. Immunol. 2002; 110: 349-356Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, 22Nedeljkovic Z.S. Gokce N. Loscalzo J. Postgrad. Med. J. 2003; 79: 195-199Crossref PubMed Scopus (140) Google Scholar). H2O2, which is formed from superoxide by superoxide dismutase, can cause cellular damage and oxidize protein thiol groups, thereby affecting the activities of various intracellular signaling molecules (23Dröge W. Physiol. Rev. 2002; 82: 47-95Crossref PubMed Scopus (7276) Google Scholar). It can also affect the cellular redox status, as a major pathway for its inactivation is its GSH-dependent reduction to H2O by glutathione peroxidase (19Schafer F.Q. Buettner G.R. Free Radic. Biol. Med. 2001; 30: 1191-1212Crossref PubMed Scopus (3551) Google Scholar). This results in the formation of GSSG, which is recycled to GSH by glutathione reductase, which concomitantly oxidizes NADPH to NADP+. The latter is in turn reduced back to NADPH by the pentose phosphate pathway (PPP), which uses glucose 6-phosphate as its initial substrate, and is a major defense mechanism against oxidative stress. It seemed possible that the impact of oxidative stress on NADP+ levels could be sufficient to enhance 5-oxo-ETE synthesis. Although our preliminary experiments with neutrophils did not appear to support this hypothesis, further studies using U-937 monocytic cells indicated that in these cells oxidative stress is indeed a very strong stimulus for 5-oxo-ETE formation. This also proved to be true for blood monocytes, lymphocytes, and platelets, with neutrophils being the only exception. The effects of oxidative stress and the respiratory burst on 5-oxo-ETE synthesis could potentially be opposed by the PPP, depending on its ability to reduce NADP+ compared with the rate of formation of NADP+ by glutathione reductase or NADPH oxidase. This proved to be the case for U-937 cells, neutrophils, and monocytes, but not for lymphocytes and platelets. Materials—5-HETE was prepared by total organic synthesis (24Zamboni R. Rokach J. Tetrahedron Lett. 1983; 24: 999-1002Crossref Scopus (44) Google Scholar). 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) (25Hamberg M. Samuelsson B. J. Biol. Chem. 1967; 242: 5329-5335Abstract Full Text PDF PubMed Google Scholar). 5S-HpETE was purchased from Cayman Chemical, Ann Arbor, MI. Phorbol 12-myristate 13-acetate (PMA), phenazine methosulfate (PMS), dehydroepiandrosterone (DHEA), 3-amino-1,2,4-triazole, N-ethylmaleimide (NEM), tert-butyl hydroperoxide (tBuOOH), H2O2, 1,2-bis[2-chloroethyl]-1-nitrosourea (BCNU; carmustine), buthionine sulfoximine (BSO), dimethyl sulfoxide (Me2SO), and diamide were purchased from Sigma. Auranofin was obtained from ALEXIS Corporation (Lausen, Switzerland). Sodium azide and glucose were purchased from Fisher Scientific. RPMI 1640 and other products for cell culture were purchased from Invitrogen. U937 Cells—U937 cells, obtained from ATCC (Manassas, VA), were cultured in 10% fetal bovine serum in modified RPMI 1640 medium containing l-glutamine (2 mm), sodium bicarbonate (1.5 g/liter), glucose (4.5 g/liter), HEPES (25 mm), and sodium pyruvate (1.0 mm). Cells were maintained at a density of 0.1–1.5 × 106 cells/ml. Preparation of Blood Cells—Neutrophils were prepared by treatment of whole blood with Dextran T-500 (Amersham Biosciences) for 45 min, followed by centrifugation over Ficoll-Paque (Amersham Biosciences) and hypotonic lysis of any remaining red blood cells in the pellet (13Powell W.S. Gravelle F. Gravel S. J. Biol. Chem. 1992; 267: 19233-19241Abstract Full Text PDF PubMed Google Scholar). The neutrophils were suspended in phosphate-buffered saline (PBS–, 137 mm NaCl, 2.7 mm KCl, 1.5 mm KH2PO4, and 8.1 mm Na2HPO4, pH 7.4). Human monocytes were prepared as described in the literature (26Fischer D.G. Hubbard W.J. Koren H.S. Cell. Immunol. 1981; 58: 426-435Crossref PubMed Scopus (87) Google Scholar) with some modifications. Peripheral blood mononuclear cells (PBMC), present at the interface after centrifugation of leukocytes over Ficoll-Paque as described above, were washed by centrifugation and resuspended in ice-cold RPMI 1640 (4 °C). The washed cells (2 × 107 cells in 10 ml per dish) were then added to Corning tissue culture dishes (Fisher, Nepean, Ontario, Canada) that had been pretreated with 1.5 ml of autologous plasma for 20 min at 37 °C. After incubation for 30 min at 37 °C the loosely adherent lymphocytes were removed with gentle streams of the culture medium from a 10-ml pipette. The lymphocytes were aspirated and the wash-aspiration procedure was repeated twice more with RPMI at 37 °C and then with PBS– at 4 °C. The monocyte-enriched adherent cells were detached from the plastic by incubation for 30 min with ice-cold 2 mm EDTA in PBS– in the cold room, followed by removal with a rubber policeman. For preparation of platelets, whole blood (20 ml) was collected in medium (2.8 ml) containing citric acid (15.5 mm), sodium citrate (90 mm), NaH2PO4 (16 mm), dextrose (161 mm), and adenine (2 mm) (16Powell W.S. Gravel S. Khanapure S.P. Rokach J. Blood. 1999; 93: 1086-1096Crossref PubMed Google Scholar). After centrifugation at 200 × g for 15 min, the supernatant was diluted with an equal volume of medium containing 94 mm citrate and 140 mm dextrose, pH 6.5. After centrifugation at 1000 × g for 10 min, the pellet was suspended in PBS– to give a platelet concentration of 3 × 108 cells/ml. Preparation of Microsomal Fractions from U937 Cells—Cells were washed by centrifugation, resuspended in 20 ml of PBS– 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 × 6 s with 30-s intervals for cooling. The disruptate was successively centrifuged at 4 °C at 1,500 × g for 10 min, 10,000 × g for 10 min, and 150,000 × g for 80 min and the final pellet resuspended in PBS–. Protein was measured with the Bio-Rad DC (detergent compatible) protein assay kit and adjusted to a concentration of 150 μg/ml. Incubation Conditions—Unless otherwise indicated, incubations were performed in PBS– supplemented with calcium (1.8 mm) and magnesium (1 mm) (PBS+) in the absence of glucose. Suspensions (1 ml) of U937 cells, lymphocytes, monocytes, neutrophils (2 × 106 cells/ml), platelets (108 cells/ml), or U937 cell microsomes (150 μg of protein/ml) were incubated with 5-HETE (1 μm) for 5 min unless otherwise indicated. Cells or microsomes were preincubated with various substances, none of which affected 5-HEDH activity directly, as indicated in Table I.Table IEffects of various agents on microsomal 5-HEDH activityCompoundAction5-Oxo-ETE% control3-Amino-1,2,4-triazole (5 mm, 30 min)Inhibits catalase (48Margoliash E. Novogrodsky A. Biochem. J. 1957; 68: 468-475Crossref Scopus (179) Google Scholar)106.6 ± 7.9Auranofin (10 μm, 30 min)Inhibits thioredoxin reductase (31Gromer S. Arscott L.D. Williams Jr., C.H. Schirmer R.H. Becker K. J. Biol. Chem. 1998; 273: 20096-20101Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar)—aDid not affect H2O2-stimulated 5-oxo-ETE synthesis in U937 cells.Azide (1 mm, 30 min)Inhibits heme enzymes (myeloperoxidase, catalase) (28Kalyanaraman B. Janzen E.G. Mason R.P. J. Biol. Chem. 1985; 260: 4003-4006Abstract Full Text PDF PubMed Google Scholar)110.5 ± 6.3BCNU (100 μm, 30 min)Inhibits glutathione and thioredoxin reductases (29Schallreuter K.U. Gleason F.K. Wood J.M. Biochim. Biophys. Acta. 1990; 1054: 14-20Crossref PubMed Scopus (104) Google Scholar)106.8 ± 4.7BSO (1 mm, 24 h)Inhibits γ-glutamylcysteine synthetase (depletes GSH) (32Troyano A. Fernandez C. Sancho P. de Blas E. Aller P. J. Biol. Chem. 2001; 276: 47107-47115Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar)—bDid not affect PMS-stimulated 5-oxo-ETE synthesis in U937 cells.DHEA (100 μm, 30 min)Inhibits Glu-6-P dehydrogenase (blocks PPP) (34Tsutsui E.A. Marks P.A. Reich P. J. Biol. Chem. 1962; 237: 3009-3013Abstract Full Text PDF PubMed Google Scholar)114.5 ± 0.2Diamide (250 μm)Converts GSH → GSSG (33Kosower N.S. Kosower E.M. Wertheim B. Correa W.S. Biochem. Biophys. Res. Commun. 1969; 37: 593-596Crossref PubMed Scopus (404) Google Scholar)84.8 ± 8.6Me2SO (1%, 30 min)Vehicle110.3 ± 5.1Glucose (1 mg/ml, 30 min)Gluc → Gluc-6-P → PPP103.9 ± 7.9H2O2 (100 μm)Induces oxidative stress93.2 ± 1.5NEM (100 μm, 10 min)Alkylates SH groups (49Friedmann E. Biochim. Biophys. Acta. 1952; 9: 65-75Crossref PubMed Scopus (29) Google Scholar)92.4 ± 12.1PMA (30 nm, 6 min)Stimulates protein kinase C and the respiratory burst (50Nauseef W.M. Volpp B.D. McCormick S. Leidal K.G. Clark R.A. J. Biol. Chem. 1991; 266: 5911-5917Abstract Full Text PDF PubMed Google Scholar)102.1 ± 1.1PMS (100 μm, 6 min)Converts NADPH → NADP+ (51Smith P.J.C. Nature. 1961; 190: 84-85Crossref Scopus (10) Google Scholar)88.2 ± 3.1tBuOOH (100 μm)Induces oxidative stress84.9 ± 5.1a Did not affect H2O2-stimulated 5-oxo-ETE synthesis in U937 cells.b Did not affect PMS-stimulated 5-oxo-ETE synthesis in U937 cells. Open table in a new tab Analysis of Eicosanoids by Precolumn Extraction/Reversed-phase (RP)-HPLC—All incubations were terminated by addition of methanol (0.65 ml) and cooling to 0 °C. The concentration of methanol in each sample was adjusted to 30% by addition of water and either 13-HODE (120 ng) or prostaglandin B2 (130 ng) were added as internal standards. Eicosanoids were analyzed by precolumn extraction/RP-HPLC (27Powell W.S. Anal. Biochem. 1987; 164: 117-131Crossref PubMed Scopus (93) Google Scholar) using a Waters Millenium system (Waters Associates, Milford, MA). Products were quantitated by comparing the areas of their peaks of UV absorbance at their λmax with that of the internal standard. The extinction coefficients used were: 5-HETE (235 nm; 27,000), 5-oxo-ETE (280 nm, 20,500), 5-oxo-20-HETE (280 nm, 20,5000), LTB4 (270 nm, 39,500), prostaglandin B2 (280 nm, 28,680), and 13-HODE (235 nm, 23,000). The identities of the products measured were confirmed by examination of their complete UV spectra. Data Analysis—The results are presented as mean ± S.E. Statistical significance was assessed using one-way repeated measures analysis of variance followed by the Bonferroni test. A p value of less than 0.05 was considered significant. “n” refers to the number of separate experiments performed. In the case of blood cells, a different donor was used for each experiment. Oxidant Stress Enhances the Synthesis of 5-Oxo-ETE—As U937 cells possess high 5-HEDH activity, they were used as a model to investigate the effects of oxidative stress induced by H2O2 on 5-oxo-ETE synthesis. Following incubation of these cells with 5-HETE (1 μm) in the presence or absence of H2O2 (100 μm), the amounts of 5-oxo-ETE were measured by HPLC using 13-HODE as an internal standard. In the absence of H2O2 very little 5-oxo-ETE was detected (Fig. 1A, left side), whereas in its presence, a large amount was formed (Fig. 1A, right side). There was no evidence for the formation of ω-oxidation products of either 5-HETE or 5-oxo-ETE by these cells. The time courses for the effects of H2O2 (100 μm) and tBuOOH (100 μm) on 5-oxo-ETE formation are shown in Fig. 1B. Both hydroperoxides strongly stimulated 5-oxo-ETE synthesis to a similar extent over the first 5 min. However, the amount of 5-oxo-ETE formed in the presence of H2O2 subsequently declined, whereas it continued to increase in the presence of tBuOOH to reach maximal levels between 10 and 15 min. 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 myeloperoxidase (28Kalyanaraman B. Janzen E.G. Mason R.P. J. Biol. Chem. 1985; 260: 4003-4006Abstract Full Text PDF PubMed Google Scholar), two key enzymes involved in H2O2 metabolism. Azide did not significantly alter the response to H2O2 over the first 5 min, but thereafter strongly enhanced its effect on 5-oxo-ETE synthesis (Fig. 1B). Because of the short-lived effect of H2O2, all subsequent incubations with 5-HETE were performed for a period of 5 min. No detectable oxidation of 5-HETE occurred when it was incubated with H2O2 or tBuOOH in the absence of cells (data not shown). The effects of various concentrations of different hydroperoxides on 5-oxo-ETE formation by U937 cells are shown in Fig. 1C. tBuOOH (EC50, 3.5 ± 0.9 μm) is the most potent stimulator of 5-oxo-ETE formation, followed by H2O2 (EC50, 12.1 ± 2.6 μm). Both hydroperoxides elicited an ∼7.5-fold increase in 5-oxo-ETE synthesis when compared with controls. The linoleic acid metabolite 13-HpODE also stimulated 5-oxo-ETE formation, but was somewhat less potent. Because of the limited availability of this substance we were unable to determine the maximal response. In contrast to hydroperoxides, PMA, which is a potent stimulator of 5-oxo-ETE formation by neutrophils (20Powell W.S. Gravelle F. Gravel S. J. Biol. Chem. 1994; 269: 25373-25380Abstract Full Text PDF PubMed Google Scholar), had no detectable effect on its formation by U937 cells (Fig. 1C). The Glutathione Redox Cycle Mediates the Effect of Oxidant Stress on 5-Oxo-ETE Formation—An important means of cellular inactivation of hydroperoxides is their GSH-dependent reduction by glutathione peroxidase, followed by the reduction of the resulting GSSG back to GSH by glutathione reductase, which is accompanied by the formation of NADP+. To determine whether the stimulatory effect of hydroperoxides on 5-oxo-ETE formation could be mediated by the glutathione redox cycle, we tested the effects of various reagents that affect this cycle. The glutathione reductase inhibitor BCNU (carmustine) (29Schallreuter K.U. Gleason F.K. Wood J.M. Biochim. Biophys. Acta. 1990; 1054: 14-20Crossref PubMed Scopus (104) Google Scholar) completely blocked the stimulatory effect of H2O2 on 5-oxo-ETE formation (Fig. 2A) and had a similar effect on the response to tBuOOH (data not shown). For comparison, baseline conversion of 5-HETE to 5-oxo-ETE (±S.E.) in the absence of H2O2 or inhibitors is shown by the horizontal lines in Fig. 2A. The effect of BCNU (30 μm) on the concentration-response to H2O2 is shown in Fig. 2B. In contrast to its strong inhibitory effect on H2O2-induced 5-oxo-ETE formation, BCNU had little effect on the response to PMS (Fig. 2B, inset), which acts by a different mechanism by non-enzymatically converting intracellular NADPH to NADP+ (30Davis G. Thornalley P.J. Biochim. Biophys. Acta. 1983; 724: 456-464Crossref PubMed Scopus (53) Google Scholar). Nor did it affect the synthesis of 5-oxo-ETE by microsomal fractions from U937 cells (Table I). BCNU also inhibits thioredoxin reductase (29Schallreuter K.U. Gleason F.K. Wood J.M. Biochim. Biophys. Acta. 1990; 1054: 14-20Crossref PubMed Scopus (104) Google Scholar), which could also potentially explain its inhibitory effect on the response to H2O2. To determine whether thioredoxin redox cycling is involved in this response we investigated the effects of the thioredoxin reductase inhibitor auranofin on H2O2-induced 5-oxo-ETE formation. Auranofin, which is a very potent inhibitor of this enzyme (IC50, 4 nm) (31Gromer S. Arscott L.D. Williams Jr., C.H. Schirmer R.H. Becker K. J. Biol. Chem. 1998; 273: 20096-20101Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar), had no effect on the formation of 5-oxo-ETE in response to H2O2 at concentrations as high as 10 μm (Table II). This inhibitor was also without effect on PMS-induced 5-oxo-ETE formation.Table IIEffects of inhibition of thioredoxin reductase on 5-oxo-ETE formation by U937 cellsTreatment5-Oxo-ETE (pmol/ml)ControlH2O2PMSVehicle23 ± 2142 ± 9137 ± 21Auranofin (1 μm)21 ± 1152 ± 11140 ± 12Auranofin (10 μm)20 ± 3133 ± 6137 ± 10 Open table in a new tab To provide further evidence for the role of GSH in the response to H2O2 we used two approaches to deplete cellular GSH. NEM, which is a very efficient alkylator of SH groups, thus preventing the formation of GSSG, completely suppressed H2O2-induced 5-oxo-ETE synthesis to levels below baseline (Fig. 2A). Like BCNU, NEM did not have a direct effect on 5-HEDH (Table I). The response to H2O2 was also inhibited by about 50% following culture of U937 cells for 24 h in the presence of BSO (Fig. 2C), which depletes cells of GSH by inhibiting γ-glutamylcysteine synthetase (32Troyano A. Fernandez C. Sancho P. de Blas E. Aller P. J. Biol. Chem. 2001; 276: 47107-47115Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). In contrast, BSO had no effect on the stimulatory effect of PMS on 5-oxo-ETE formation. We also examined the effects of diamide, which acts by non-enzymatically oxidizing intracellular GSH to GSSH (33Kosower N.S. Kosower E.M. Wertheim B. Correa W.S. Biochem. Biophys. Res. Commun. 1969; 37: 593-596Crossref PubMed Scopus (404) Google Scholar) (Fig. 2D). Diamide (EC50, 15.5 ± 2.5 μm) strongly stimulated the formation of 5-oxo-ETE to an extent similar to that observed with H2O2. The time course for 5-oxo-ETE synthesis in the presence of diamide, shown in the inset to Fig. 2D, is very similar to that observed with tBuOOH (Fig. 1B), except that the response appears to be somewhat more sustained. In contrast, diamide had no effect on the conversion of 5-HETE to 5-oxo-ETE by U937 cell microsomes (Table I). Activation of the Pentose Phosphate Pathway Inhibits 5-Oxo-ETE Formation—The PPP is the major mechanism whereby cells maintain a high ratio of NADPH to NADP+. To determine whether this pathway could affect the formation of 5-oxo-ETE, we investigated the effects of glucose, which is metabolized by the PPP following its phosphorylation to glucose 6-phosphate, and galactose, which is not a substrate for this pathway. Glucose (IC50, 0.42 ± 0.08 mm) strongly inhibited the formation of 5-oxo-ETE in the presence of H2O2, whereas galactose had no effect (Fig. 3A). To further substantiate that the inhibitory effect of glucose is mediated by its metabolism by the PPP, we examined the effect of DHEA, which is an inhibitor of glucose-6-phosphate dehydrogenase, the initial enzyme in this pathway (34Tsutsui E.A. Marks P.A. Reich P. J. Biol. Chem. 1962; 237: 3009-3013Abstract Full Text PDF PubMed Google Scholar). The inhibitory effect of 5.6 mm glucose on H2O2-in" @default.
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• W2055364518 title "Oxidative Stress Stimulates the Synthesis of the Eosinophil Chemoattractant 5-Oxo-6,8,11,14-eicosatetraenoic Acid by Inflammatory Cells" @default.
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