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• W2052258246 abstract "We previously showed that 6-trans isomers of leukotriene B4 but not leukotriene B4itself 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 Δ6- and Δ13-reductases could be separated by chromatography on DEAE-Sepharose. 5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is converted by the Δ6-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 Δ6-reductase and prostaglandin Δ13-reductase. Metabolism of 5-oxo-ETE by the Δ6-reductase results in loss of its biological activity. We previously showed that 6-trans isomers of leukotriene B4 but not leukotriene B4itself 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 Δ6- and Δ13-reductases could be separated by chromatography on DEAE-Sepharose. 5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is converted by the Δ6-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 Δ6-reductase and prostaglandin Δ13-reductase. Metabolism of 5-oxo-ETE by the Δ6-reductase results in loss of its biological activity. A major pathway in the metabolism of many eicosanoids is initiated by oxidation of one of the hydroxyl groups by an NAD+- or NADP+-dependent dehydrogenase. This is usually followed by reduction of an adjacent double bond by an olefin reductase in the presence of NADH or NADPH. A number of distinct cytosolic 15-hydroxyprostaglandin dehydrogenases oxo-dize various prostaglandins (PGs) 1The abbreviations used are: PGprostaglandinLTB4leukotriene B45-HETE(5S)-hydroxy-6,8,11,14-eicosatetraenoic acid5-oxo-ETE5-oxo-6,8,11,14-eicosatetraenoic acid5-oxo-15-HETE5-oxo-(15S)-hydroxy-6,8,11,13-eicosatetraenoic acid15-oxo-5-HETE(5S)-hydroxy-15-oxo-6,8,11,13-eicosatetraenoic acid515-diHETE, (5S,15S)-dihydroxy-6,8,11,13-eicosatetraenoic acidRP-HPLCreversed-phase high pressure liquid chromatography. to their biologically inactive 15-oxo metabolites (1Anggard E. Larsson C. Samuelsson B. Acta Physiol. Scand. 1971; 81: 396-404Crossref PubMed Scopus (196) Google Scholar, 2Okita R.T. Okita J.R. Crit. Rev. Biochem. Mol. Biol. 1996; 31: 101-126Crossref PubMed Scopus (66) Google Scholar, 3Braithwaite S.S. Jarabak J. J. Biol. Chem. 1975; 250: 2315-2318Abstract Full Text PDF PubMed Google Scholar). These products can then be reduced by cytosolic PG Δ13-reductases to biologically inactive 13,14-dihydro-15-oxo-PGs (1;4), which in turn can be further reduced to dihydro-PGs by ketoreductases (5Hamberg M. Samuelsson B. J. Biol. Chem. 1971; 246: 1073-1077Abstract Full Text PDF PubMed Google Scholar). prostaglandin leukotriene B4 (5S)-hydroxy-6,8,11,14-eicosatetraenoic acid 5-oxo-6,8,11,14-eicosatetraenoic acid 5-oxo-(15S)-hydroxy-6,8,11,13-eicosatetraenoic acid (5S)-hydroxy-15-oxo-6,8,11,13-eicosatetraenoic acid 15-diHETE, (5S,15S)-dihydroxy-6,8,11,13-eicosatetraenoic acid reversed-phase high pressure liquid chromatography. Lipoxygenase products can be metabolized by analogous pathways. We have shown that leukotriene B4 (LTB4) is converted to 12-oxo-LTB4 by an NAD+-dependent 12-hydroxyeicosanoid dehydrogenase in neutrophils (6Wainwright S.L. Powell W.S. J. Biol. Chem. 1991; 266: 20899-20906Abstract Full Text PDF PubMed Google Scholar). This is followed by reduction of the 10,11-double bond by a cytosolic NADH-dependent Δ10-reductase to give 10,11-dihydro-12-oxo-LTB4, which is then reduced to the corresponding dihydro compound by a ketoreductase (6Wainwright S.L. Powell W.S. J. Biol. Chem. 1991; 266: 20899-20906Abstract Full Text PDF PubMed Google Scholar). Metabolism of the potent neutrophil agonist (7Ford-Hutchinson A.W. Bray M.A. Doig M.V. Shipley M.E. Smith M.J. Nature. 1980; 286: 264-265Crossref PubMed Scopus (1585) Google Scholar), LTB4, by this pathway results in considerable loss of biological activity (8Kumlin M. Falck J.R. Raud J. Harada Y. Dahlén S.E. Granström E. Biochem. Biophys. Res. Commun. 1990; 170: 23-29Crossref PubMed Scopus (22) Google Scholar, 9Kaever V. Damerau B. Wessel K. Resch K. FEBS Lett. 1988; 231: 385-388Crossref PubMed Scopus (21) Google Scholar, 10Powell W.S. Rokach J. Khanapure S.P. Manna S. Hashefi M. Gravel S. MacLeod R.J. Falck J.R. Bhatt R.K. J. Pharm. Exp. Ther. 1996; 276: 728-736PubMed Google Scholar). 12(S)-Hydroxy-5,8,10,14-eicosate-traenoic acid is metabolized in a similar manner by neutrophils (11Wainwright S. Falck J. Yadagiri P. Powell W.S. Biochemistry. 1990; 29: 10126-10135Crossref PubMed Scopus (30) Google Scholar). However, in this case, the 10,11-dihydro metabolite of 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid has been reported to be a potent proinflammatory agent (12Masferrer J.L. Rimarachin J.A. Gerritsen M.E. Falck J.R. Yadagiri P. Dunn M.W. Laniado-Schwartzman M. Exp. Eye Res. 1991; 52: 417-424Crossref PubMed Scopus (42) Google Scholar). LTB4 is metabolized by a similar pathway in monocytes (9Kaever V. Damerau B. Wessel K. Resch K. FEBS Lett. 1988; 231: 385-388Crossref PubMed Scopus (21) Google Scholar, 13Schönfeld W. Kasimir S. Knoller J. Jablonski K. König W. J. Leukocyte Biol. 1991; 50: 303-312Crossref PubMed Scopus (13) Google Scholar) and kidney (14Yokomizo T. Izumi T. Takahashi T. Kasama T. Kobayashi Y. Sato F. Taketani Y. Shimizu T. J. Biol. Chem. 1993; 268: 18128-18135Abstract Full Text PDF PubMed Google Scholar). However, in the latter case, the 12-hydroxy dehydrogenase is clearly distinct from the neutrophil enzyme (14Yokomizo T. Izumi T. Takahashi T. Kasama T. Kobayashi Y. Sato F. Taketani Y. Shimizu T. J. Biol. Chem. 1993; 268: 18128-18135Abstract Full Text PDF PubMed Google Scholar). We previously showed that neutrophils convert 6-trans isomers of LTB4, which are formed nonenzymatically from LTA4, to dihydro metabolites (15Powell W.S. Biochem. Biophys. Res. Commun. 1986; 136: 707-712Crossref PubMed Scopus (18) Google Scholar, 16Powell W.S. Gravelle F. J. Biol. Chem. 1988; 263: 2170-2177Abstract Full Text PDF PubMed Google Scholar). 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 (16Powell W.S. Gravelle F. J. Biol. Chem. 1988; 263: 2170-2177Abstract Full Text PDF PubMed Google Scholar). We initially speculated that the dihydro products of these reactions might have been 6,11-dihydro metabolites, due to migration of the two remaining double bonds. However, mass spectral evidence subsequently suggested that the products were 6,7-dihydro metabolites (17Wheelan P. Murphy R.C. J. Biol. Chem. 1995; 270: 19845-19852Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). The initial step in the formation of these substances is oxidation of the 5-hydroxyl group by a microsomal NADP+-dependent dehydrogenase that is highly specific for eicosanoids containing a (5S)-hydroxyl group followed by a 6-trans double bond (18Powell W.S. Gravelle F. Gravel S. J. Biol. Chem. 1992; 267: 19233-19241Abstract Full Text PDF PubMed Google Scholar). LTB4, which has a 6-cis double bond, is not metabolized by this pathway. The best substrate for 5-hydroxyeicosanoid dehydrogenase is (5S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), which is converted to 5-oxo-ETE (18Powell W.S. Gravelle F. Gravel S. J. Biol. Chem. 1992; 267: 19233-19241Abstract Full Text PDF PubMed Google Scholar), a potent activator of neutrophils (19Powell W.S. Gravel S. MacLeod R.J. Mills E. Hashefi M. J. Biol. Chem. 1993; 268: 9280-9286Abstract Full Text PDF PubMed Google Scholar, 20O'Flaherty J.T. Cordes J. Redman J. Thomas M.J. Biochem. Biophys. Res. Commun. 1993; 192: 129-134Crossref PubMed Scopus (50) Google Scholar) and eosinophils (21Powell W.S. Chung D. Gravel S. J. Immunol. 1995; 154: 4123-4132PubMed Google Scholar, 22O'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, 23Schwenk U. Schröder J.M. J. Biol. Chem. 1995; 270: 15029-15036Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Relatively little is known about the olefin reductase that converts 5-oxoeicosanoids to their dihydro metabolites. The objectives of this study were to investigate the regulation of this enzyme, its substrate specificity, and its subcellular localization. We also wanted to determine whether 5-oxo-ETE could be converted to a dihydro metabolite by this pathway and, if so, how this would affect its biological activity. Calmodulin was purchased from Biomol (Plymouth Meeting, PA), whereas the calmodulin inhibitor calmidazolium chloride (R24571) was obtained from Calbiochem. PGB2 was obtained from Sigma. 6-trans-LTB4, 12-epi-6-trans-LTB4, 15-oxo-PGF2α, and 13,14-dihydro-15-oxo-PGF2α were purchased from Cayman Chemical (Ann Arbor, MI). A number of the eicosanoids used in this study were prepared by total chemical synthesis. 5-Oxo-ETE, 8-trans-5-oxo-ETE, and 6,7-dihydro-5-oxo-ETE were prepared as described previously (24Khanapure S.P. Shi X.X. Powell W.S. Rokach J. J. Org. Chem. 1998; 63: 337-342Crossref Scopus (57) Google Scholar). 5-Oxo-[11,12,14,15-3H]ETE and 8-trans-5-oxo-[11,12,14,15-3H]ETE were prepared by reduction of an 11,14-diyne precursor with tritium gas (performed by American Radiolabeled Chemicals, St. Louis, MO) (25Khanapure S.P. Shi X.X. Powell W.S. Rokach J. J. Org. Chem. 1998; 63: 4098-4102Crossref Scopus (18) Google Scholar). Various eicosanoids were prepared biochemically. 5-Oxo-6-trans-LTB4 and 5-oxo-12-epi-6-trans-LTB4 were synthesized by incubation of 6-trans-LTB4 (2 μm) and 12-epi-6-trans-LTB4 (2 μm) (Cayman Chemical Co.), respectively, with a microsomal fraction from human neutrophils for 90 min in the presence of NADP+ (1 mm) (18Powell W.S. Gravelle F. Gravel S. J. Biol. Chem. 1992; 267: 19233-19241Abstract Full Text PDF PubMed Google Scholar). 5-Oxo-15-hydroxy-6E,8Z,11Z,13E-eicosatetraenoic acid (5-oxo-15-HETE) and 5-oxo-15-hydroxy-6E,8E,11Z,13E-eicosatetraenoic acid (8-trans-5-oxo-15-HETE) were prepared by incubation of 5-oxo-ETE and 8-trans-5-oxo-ETE, respectively, with soybean lipoxygenase (18Powell W.S. Gravelle F. Gravel S. J. Biol. Chem. 1992; 267: 19233-19241Abstract Full Text PDF PubMed Google Scholar). 5-HETE was synthesized by incubation of arachidonic acid (NuChek Prep, Inc., Elysian, MN) with potato 5-lipoxygenase (26Shimizu T. Honda Z. Miki I. Seyama Y. Izumi T. Radmark O. Samuelsson B. Methods Enzymol. 1990; 187: 296-306Crossref PubMed Scopus (31) Google Scholar). 5,15-Dihydroxy-6E,8Z,11Z,13E-eicosatetraenoic acid (5,15-diHETE) was prepared by incubation of 5-HETE with soybean lipoxygenase (27Maas R.L. Turk J. Oates J.A. Brash A.R. J. Biol. Chem. 1982; 257: 7056-7067Abstract Full Text PDF PubMed Google Scholar). 5-Hydroxy-15-oxo-6E,8Z,11Z,13E-eicosatetraenoic acid (15-oxo-5-HETE) was prepared by incubating 5,15-diHETE (2 μm) with the cytosolic fraction obtained from pregnant rabbit lungs in the presence of NAD+ (28Bergholte J.M. Soberman R.J. Hayes R. Murphy R.C. Okita R.T. Arch. Biochem. Biophys. 1987; 257: 444-450Crossref PubMed Scopus (34) Google Scholar, 29Sun F.F. Armour S.B. Prostaglandins. 1974; 7: 327-338Crossref PubMed Scopus (58) Google Scholar). Similarly, 15-oxo-[5,6,8,9,11,12,14,15-3H]PGF2α was synthesized by incubation of [5,6,8,9,11,12,14,15-3H]PGF2α (180 Ci/mmol (NEN Life Science Products) with pregnant rabbit lung cytosol (1 ml, equivalent to 20 mg of tissue) for 10 min at 37 °C (29Sun F.F. Armour S.B. Prostaglandins. 1974; 7: 327-338Crossref PubMed Scopus (58) Google Scholar). This product cochromatographed with authentic unlabeled 15-oxo-PGF2α. Human neutrophils were purified by treatment of blood with Dextran T-500, centrifugation over Ficoll-Paque, and hypotonic lysis of the remaining red cells (30Böyum A. Scand. J. Clin. Lab. Invest. 1968; 21: 77-89PubMed Google Scholar). The cells (25 × 106/ml) were suspended in 20 mm phosphate buffer, pH 7.4, containing 0.3m sucrose, phenylmethylsulfonyl fluoride (1 mm), leupeptin (2 μg/ml), and aprotinin (2 μg/ml). The neutrophils were then disrupted by sonication (model 4710 Ultrasonic Homogenizer; Sonics & Materials, Danbury, CT) in an ice bath for 2 × 5 s at a power setting of 1 and for a further 5 s at a power setting of 2. The sonicate was centrifuged successively at 1500 × g for 10 min, 10,000 × g for 10 min, and 200,000 × g for 60 min. The 10,000 ×g and 200,000 × g pellets were suspended in phosphate-buffered saline (1.25 times the original volume) containing calcium and magnesium. The 200,000 × gsupernatant fraction (10 ml) was incubated with 1 ml of DEAE-Sepharose for 30 min at 4 °C with constant mixing. The gel suspension was packed into a column that was eluted with 1) 20 mmphosphate buffer, pH 7.4 (10 ml); 2) 50 mm NaCl in 20 mm phosphate (10 ml); and 3) 250 mm NaCl in 20 mm phosphate (10 ml). The initial unretained fraction contained the Δ13-reductase, which was monitored using 15-oxo-5-HETE as substrate, whereas the 250 mm NaCl fraction contained the Δ6-reductase, which was monitored using 8-trans-5-oxo-[3H]ETE as substrate. Fractions obtained as described above were incubated with various substrates, and the reactions were terminated by the addition of methanol (0.6 ml) and stored at −80 °C until analysis by RP-HPLC. After the samples were thawed, water was added to give a final volume of 4 ml (i.e.15% methanol). Eicosanoids were analyzed by automated precolumn extraction/RP-HPLC as described previously (31Powell W.S. Anal. Biochem. 1987; 164: 117-131Crossref PubMed Scopus (93) Google Scholar). Products were detected and UV spectra recorded using a Waters model M991 diode array detector. Dihydro products were quantitated on the basis of UV absorbance or (when 5-oxo-[3H]ETE, 8-trans-5-oxo-[3H]ETE, or 15-oxo-[3H]PGF2α were the substrates) measurement of radioactivity. Different conditions were used for the analysis of metabolites of oxoeicosanoids (i.e. 5-oxo-ETE and 8-trans-5-oxo-ETE), hydroxyoxoeicosanoids, and 15-oxo-PGF2αa. All mobile phases contained 0.02% acetic acid. Conditions for oxoeicosanoids were as follows: Spherisorb ODS-2 column (3.2 × 250 mm; 5-μm particle size; Phenomenex); 60% acetonitrile in water, isocratic for 40 min at 0.5 ml/min. Conditions for hydroxyoxoeicosanoids were as follows: Novapak C18column (3.9 × 150 mm; Waters); linear gradient between 37 and 45% acetonitrile over 30 min at 1 ml/min. Conditions for 15-Oxo-PGF2αa were as follows: Novapak C18column; 31% acetonitrile, isocratic at 1 ml/min. PGB2 (250 ng/sample) was used as an internal standard. 12-epi-6-trans-LTB4(2 μm) was incubated with the 1500 × gsupernatant fraction from human neutrophils for 90 min at 37 °C in the presence of NADP+ (1 mm). The reaction was terminated by the addition of methanol (0.5 volumes). Water was added to give a final concentration of methanol of 15%, and the mixture was centrifuged at 1000 × g for 10 min. The supernatant was extracted without acidification on a C18 Sep-Pak (Waters-Millipore) as described previously (32Powell W.S. Prostaglandins. 1980; 20: 947-957Crossref PubMed Scopus (539) Google Scholar). The methyl formate fraction was evaporated to dryness under a stream of nitrogen, and the residue (containing 5-oxo-12-epi-6-trans-LTB4) was incubated with the 200,000 × g supernatant fraction from human neutrophils for 90 min at 37 °C in the presence of calcium (1 mm) and NADPH (1 mm). The products were extracted using octadecylsilyl silica as described above. RP-HPLC analysis of an aliquot of the methyl formate fraction after the first extraction (material from the incubation with the 1500 ×g supernatant) confirmed that the major product was 5-oxo-12-epi-6-trans-LTB4 (18Powell W.S. Gravelle F. Gravel S. J. Biol. Chem. 1992; 267: 19233-19241Abstract Full Text PDF PubMed Google Scholar). Dihydro-5-oxo-12-epi-6-trans-LTB4, the major product of the incubation with the 200,000 ×g supernatant fraction, was purified by RP-HPLC as described above and incubated at a concentration of 2 μm with the 200,000 × g pellet obtained from porcine neutrophils, prepared as described previously (6Wainwright S.L. Powell W.S. J. Biol. Chem. 1991; 266: 20899-20906Abstract Full Text PDF PubMed Google Scholar), in the presence of NAD+ (1 mm). The products of the reaction were analyzed by precolumn extraction/RP-HPLC as described above. Protein concentrations were determined as described by Bradford (33Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216377) Google Scholar). Calcium levels were measured in indo-1-loaded neutrophils as described previously (34Powell W.S. MacLeod R.J. Gravel S. Gravelle F. Bhakar A. J. Immunol. 1996; 156: 336-342PubMed Google Scholar), using a Photon Technology International (PTI) Deltascan 4000 spectrofluorometer with a temperature-controlled cuvette holder equipped with a magnetic stirrer. We had previously shown that 12-epi-6-trans-LTB4 is converted to dihydro and dihydro-5-oxo metabolites by a 1500 × gsupernatant fraction from human neutrophils. To investigate the subcellular localization of the olefin reductase required for the formation of these products, 5-oxo-12-epi-6-trans-LTB4 was incubated with subcellular fractions from neutrophils in the presence of different cofactors. When 5-oxo-12-epi-6-trans-LTB4 was incubated with a microsomal fraction from neutrophils in the presence of NADPH, the major metabolite was the ketoreductase product, 12-epi-6-trans-LTB4(12e-6t-B4) (Fig.1 A). Only a small amount of a dihydro product (dh-12e-B4) was formed under these conditions. In contrast, the major product formed when 5-oxo-12-epi-6-trans-LTB4 was incubated with the cytosolic fraction from neutrophils in the presence of NADPH was its dihydro metabolite (dh-5o-12e-B4) (Fig. 1 B). The amounts of the above metabolites of 5-oxo-12-epi-6-trans-LTB4 formed by different subcellular fractions from neutrophils are shown in TableI. The major product formed by both the 10,000 and 200,000 × g pellets was the ketoreductase product 12-epi-6-trans-LTB4. Formation of this product by particulate fractions was dependent upon the presence of NADPH, NADH being much less effective. It was formed to a lesser extent by the 200,000 × g supernatant, but in this case its formation was not affected by the addition of cofactors, perhaps because the level of endogenous cofactor was sufficient. In contrast, the two particulate fractions tested displayed relatively little or no olefin reductase activity, whereas the 200,000 ×g supernatant was quite active in the formation of dihydro metabolites of 5-oxo-12-epi-6-trans-LTB4. The cytosolic olefin reductase activity was highly dependent on the presence of NADPH, with much smaller amounts of dihydro metabolites being formed in the absence of exogenous cofactors or in the presence of NADH.Table IMetabolism of 5-oxo-12-epi-6-trans-LTB4 by subcellular fractions from human neutrophilsFractionCofactorProduct12e-6t-B4dh-5o-12e-B4dh-12e-B4pmol/ml/30 min10,000 pelletsNone12NDaND, not detectable.NDNADH24NDNDNADPH322NDND200,000 pelletsNone10NDNDNADH154NDNDNADPH862ND106200,000 supernatantNone23846NDNADH222166NDNADPH2509101745-Oxo-12-epi-6-trans-LTB4 (2 μm) was incubated for 30 min at 37 °C with different subcellular fractions from human neutrophils (equivalent to 2 × 107 cells/ml) in the presence or absence of NADH (1 mm) or NADPH (1 mm). The products were quantitated by RP-HPLC as described in the legend to Fig. 1. The abbreviations are as described in the legend to Fig. 1.a ND, not detectable. Open table in a new tab 5-Oxo-12-epi-6-trans-LTB4 (2 μm) was incubated for 30 min at 37 °C with different subcellular fractions from human neutrophils (equivalent to 2 × 107 cells/ml) in the presence or absence of NADH (1 mm) or NADPH (1 mm). The products were quantitated by RP-HPLC as described in the legend to Fig. 1. The abbreviations are as described in the legend to Fig. 1. We had previously suggested that 12-epi-6-trans-LTB4 was converted to a 6,11-dihydro metabolite by intact neutrophils (16Powell W.S. Gravelle F. J. Biol. Chem. 1988; 263: 2170-2177Abstract Full Text PDF PubMed Google Scholar). However, identification of the positions of the double bonds in this product was not very conclusive, because the diagnostic fragment ions in its mass spectrum were not very intense and could possibly have arisen as a result of rearrangements. A recent study employing mass spectral analysis of fragments formed by oxidative ozonolysis provided evidence that it is the 6,7-double bond of 6-trans isomers of LTB4that is reduced by these cells (17Wheelan P. Murphy R.C. J. Biol. Chem. 1995; 270: 19845-19852Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). We used a different approach to investigate the position of the reduced double bond of 12-epi-6-trans-LTB4. As shown in Fig.2, 5-oxo-12-epi-6-trans-LTB4 could potentially be reduced to three products by an olefin reductase, resulting in 6,7-dihydro, 6,11-dihydro, or 10,11-dihydro metabolites. Reduction of the 9,10-double bond in the middle of the triene chromophore is unlikely, whereas reduction of the 14,15-double bond is theoretically possible but would not result in a change in the UV spectrum of the product. Reduction of the 10,11-double bond of 12-epi-6-trans-LTB4 (Fig. 2) can also be excluded, since the resulting product would have a λmax around 280 nm, which is not observed. To determine whether 5-oxo-12-epi-6-trans-LTB4 is converted to a 6,7-dihydro or a 6,11-dihydro metabolite, this substance was incubated with microsomal 12-hydroxyeicosanoid dehydrogenase from porcine neutrophils in the presence of NAD+. Oxidation of the 12-hydroxyl group of 6,7-dihydro-5-oxo-12-epi-LTB4 by 12-hydroxyeicosanoid dehydrogenase would give a 5,12-dioxo product absorbing at 280 nm, whereas oxidation of the 12-hydroxyl group of 6,11-dihydro-5-oxo-12-epi-LTB4 would not result in any change in the λmax of the substrate (Fig. 2). RP-HPLC analysis of the metabolites of dihydro-5-oxo-12-epi-LTB4 formed by porcine microsomes indicated that two fewer polar products were formed, presumably due to oxidation of the 12-hydroxyl group (Fig.3 A). The major product had a λmax at 280 nm and was therefore a 6,7-dihydro product (structure I in Fig. 2), whereas a minor product had a λmax at 231 nm and was probably identical to the 6,11-dihydro compound (structure II in Fig. 2) (Fig. 3 B). This demonstrates that the cytosolic fraction from human neutrophils reduces 5-oxo-12-epi-6-trans-LTB4 principally by 1,2-addition to the triene chromophore and suggests that 1,6-addition may also occur to some extent. For the purpose of clarity, this activity will be referred to below as Δ6-reductase activity. Other 5-oxoeicosanoids were also metabolized by cytosolic fractions from neutrophils in a manner analogous to that shown for 12-epi-6-trans-LTB4 in Fig.1. 5-Oxo-15-HETE was converted to 5,15-diHETE and two dihydro products, presumably 6,7-dihydro-5-oxo-15-HETE and 6,7-dihydro-5,15-diHETE. The time course for the formation of these three metabolites is shown in Fig. 4. The initial Δ6-reductase and ketoreductase products (dihydro-5-oxo-15-HETE and 5,15-diHETE) were formed fairly rapidly and reached maximal levels by about 90 min, after which time the amounts declined. The product formed by a combination of the two pathways (dihydro-5,15-diHETE) was formed much more slowly and did not appear to have reached maximal levels by 120 min. To investigate the substrate specificity of the neutrophil cytosolic olefin reductase, a variety of oxoeicosanoids were prepared either chemically or biochemically and incubated with neutrophil cytosol in the presence of NADPH. The products were analyzed by RP-HPLC, and the amounts of dihydro products (dihydro plus dihydro-oxo) were determined. The cytosolic olefin reductase converted a variety of 5-oxoeicosanoids to dihydro metabolites (TableII). Of these, the best substrates were 5-oxo-6-trans-LTB4 and 8-trans-5-oxo-15-HETE. 5-Oxo-ETE and its 8-trans isomer appeared to be metabolized more slowly, but this may have been due at least in part to the conversion of these substances by a competitive pathway to 15-hydroxy products due to the presence of 15-lipoxygenase in the cytosol. The Δ6-reductase appears to prefer substrates with an 8-trans double bond, since both 8-trans-5-oxo-ETE and 8-trans-5-oxo-15-HETE were metabolized more rapidly than the corresponding 8-cis compounds. However, of all of the products tested, 15-oxo-5-HETE was by far the best substrate, being metabolized at a rate at least 3 times that of the 5-oxoeicosanoids tested. This raised the possibility that the cytosolic reductase was actually a Δ13-reductase that was also capable of reducing the 6,7-double bond of 5-oxoeicosanoids.Table IISubstrate specificity of cytosolic olefin reductases in human neutrophilsSubstrateOlefin reductase activityCytosolaValues are sums of dihydro-oxo (olefin reductase) and dihydro (olefin reductase plus ketoreductase) products.DEAE boundDEAE flow-throughpmol/min/mg protein5-Oxo-6-trans-LTB411.5 ± 0.8NDbND, not detectable.5-Oxo-12-epi-6-trans-LTB44.93 ± 0.27ND5-Oxo-15-HETE (8-cis)7.0 ± 0.94.13 ± 0.23ND8-trans-5-Oxo-15-HETE12.6 ± 0.95.30 ± 0.10ND5-Oxo-ETE (8-cis)2.0 ± 0.1cThese values are underestimates because both 5-oxo-ETE and 8-trans-5-oxo-ETE, but not the other substrates, were converted to 15-hydroxy products by cytosolic 15-lipoxygenase.1.73 ± 0.18ND8-trans-5-Oxo-ETE5.3 ± 0.5cThese values are underestimates because both 5-oxo-ETE and 8-trans-5-oxo-ETE, but not the other substrates, were converted to 15-hydroxy products by cytosolic 15-lipoxygenase.6.13 ± 0.28ND15-Oxo-5-HETE38.1 ± 0.8d15-oxo-5-HETE was converted only to a dihydro-oxo product.ND32.67 ± 1.2615-Oxo-PGF2αND48.53 ± 4.29Various oxoeicosanoids (1 μm) were incubated at 37 °C in the presence of NADPH (1 mm) with neutrophil cytosol (30 min; 1 mm Ca2+), the 250 mm NaCl fraction following chromatography on DEAE-Sepharose (60 min; 1 mm Ca2+), or the DEAE-Sepharose flow-through fraction (30 min; 1 mm EGTA). The products were analyzed by precolumn extraction/RP-HPLC as described under “Experimental Procedures.” The values are means ± S.E. (n = 3).a Values are sums of dihydro-oxo (olefin reductase) and dihydro (olefin reductase plus ketoreductase) products.b ND, not detectable.c These values are underestimates because both 5-oxo-ETE and 8-trans-5-oxo-ETE, but not the other substrates, were converted to 15-hydroxy products by cytosolic 15-lipoxygenase.d 15-oxo-5-HETE was converted only to a dihydro-oxo product. Open table in a new tab Various oxoeicosanoids (1 μm) were incubated at 37 °C in the presence of NADPH (1 mm) with neutrophil cytosol (30 min; 1 mm Ca2+), the 250 mm NaCl fraction following chromatography on DEAE-Sepharose (60 min; 1 mm Ca2+), or the DEAE-Sepharose flow-through fraction (30 min; 1 mm EGTA). The products were analyzed by precolumn extraction/RP-HPLC as described under “Experimental Procedures.” The values are means ± S.E. (n = 3). All of the experiments described above were performed in the presence of calcium (1 mm). To determine whether the conversion of oxoeicosanoids to dihydro products was affected by calcium, neutrophil cytosol was incubated with various substrates in calcium-free medium in the presence of EGTA (1 mm). Removal of calcium inhibited the conversion to dihydro metabolites of the three 5-oxoeicosanoids tested (5-oxo-6-trans-LTB4, 5-oxo-15-HETE, and 8-trans-5-oxo-15-HETE) by between 70 and 80% (p < 0.01) (Fig. 5). In contrast, conversion of 15-oxo-5-HETE to its dihydro metabolite was stimulated by about 25% (p < 0.05) in the presence of EGTA. This experiment thus provides strong evidence that neutrophil cytosol contains at least two distinct olefin reductases and that the activity of one of these is enhanced by calcium. To investigate the possibility that the calcium dependence of the Δ6-reductase is mediated by calmodulin, the effect of the calmodulin inhibitor" @default.
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