Interleukin-1 Enhances the Ability of Cultured Human Umbilical Vein Endothelial Cells to Oxidize Linoleic Acid
1995; Elsevier BV; Volume: 270; Issue: 29 Linguagem: Inglês
10.1074/jbc.270.29.17279
ISSN1083-351X
AutoresMercedes Camacho, Núria Godessart, Rosa Antón, Montserrat García, L. Vila,
Tópico(s)Cholesterol and Lipid Metabolism
ResumoHuman umbilical vein endothelial cells (HUVEC) were treated with recombinant interleukin (IL)-1β, and the metabolism of exogenous linoleic acid was studied. High performance liquid chromatography, gas chromatography-mass spectrometry, and chiral analysis revealed that HUVEC enzymatically convert linoleic acid mainly into 13-(S)hydroxy-9(Z),11(E)-octadecadienoic (13-HODE) and 9-(R)hydroxy-10(E),12(Z)-octadecadienoic acids, which may isomerize toward all-trans compounds. IL-1β increased the formation of all octadecanoids in a time- and dose-dependent manner with similar EC50 (approximately 1 unit/ml). The apparent Km values of linoleic acid were 15.59 ± 8.39 and 152.9 ± 84 μM (p < 0.05) in IL-1β-treated cells and controls, respectively, indicating a higher substrate affinity in cells stimulated with IL-1β. Ratios of S/R enantiomers for the hydroxyoctadecanoids produced by untreated and IL-1β-treated cells were similar to those from isolated cyclooxygenases (COXs), whereas isolated 15-lipoxygenase yielded 13-HODE with a strict S configuration. The formation of octadecanoids was inhibited in a dose-dependent manner by several COX inhibitors in both controls and IL-1β-treated cells, COX2 selective inhibitors being more effective on IL-1β-treated cells than on controls. COX1 and COX2 protein levels increased less than 2-fold and 8-fold, respectively, after IL-1β treatment. The specificity of COX inhibitors was proven since they did not inhibit 13-HODE formation by human polymorphonuclear leukocytes. Overall, these results indicate that COXs are responsible for the oxidative metabolism of linoleic acid in HUVEC, and IL-1β increases it by inducing the expression of new enzyme, mainly COX2. Human umbilical vein endothelial cells (HUVEC) were treated with recombinant interleukin (IL)-1β, and the metabolism of exogenous linoleic acid was studied. High performance liquid chromatography, gas chromatography-mass spectrometry, and chiral analysis revealed that HUVEC enzymatically convert linoleic acid mainly into 13-(S)hydroxy-9(Z),11(E)-octadecadienoic (13-HODE) and 9-(R)hydroxy-10(E),12(Z)-octadecadienoic acids, which may isomerize toward all-trans compounds. IL-1β increased the formation of all octadecanoids in a time- and dose-dependent manner with similar EC50 (approximately 1 unit/ml). The apparent Km values of linoleic acid were 15.59 ± 8.39 and 152.9 ± 84 μM (p < 0.05) in IL-1β-treated cells and controls, respectively, indicating a higher substrate affinity in cells stimulated with IL-1β. Ratios of S/R enantiomers for the hydroxyoctadecanoids produced by untreated and IL-1β-treated cells were similar to those from isolated cyclooxygenases (COXs), whereas isolated 15-lipoxygenase yielded 13-HODE with a strict S configuration. The formation of octadecanoids was inhibited in a dose-dependent manner by several COX inhibitors in both controls and IL-1β-treated cells, COX2 selective inhibitors being more effective on IL-1β-treated cells than on controls. COX1 and COX2 protein levels increased less than 2-fold and 8-fold, respectively, after IL-1β treatment. The specificity of COX inhibitors was proven since they did not inhibit 13-HODE formation by human polymorphonuclear leukocytes. Overall, these results indicate that COXs are responsible for the oxidative metabolism of linoleic acid in HUVEC, and IL-1β increases it by inducing the expression of new enzyme, mainly COX2. Linoleic acid is an essential fatty acid with a cis,cis-pentadiene structure in the molecule which easily reacts with oxygen to yield biologically active compounds. Great amounts of esterified and free hydroperoxyoctadecadienoic acid (HPODE)1 1The abbreviations used are: HPODEhydroperoxyoctadecadienoic acidHODEhydroxyoctadecadienoic acidILinterleukinCOXcyclooxygenase15-HETE15-hydroxyeicosatetraenoic acidNDGAnordihydroguaiaretic acidBSTFAN,O-bis(trimethylsilyl)trifluoroacetamideHPLChigh performance liquid chromatography6-MNA6-methoxy-2-naphthylacetic acidNS-398N-[2-(cyclohexyloxy)-4-nitrophenyl]methanesulfonamideHUVEChuman umbilical vein endothelial cellsPMNpolymorphonuclear leukocyteSP-HPLCstraight phase HPLCGC-MSgas chromatography-mass spectrometryEIelectron impactETYAeicosatetraynoic acid. 1The abbreviations used are: HPODEhydroperoxyoctadecadienoic acidHODEhydroxyoctadecadienoic acidILinterleukinCOXcyclooxygenase15-HETE15-hydroxyeicosatetraenoic acidNDGAnordihydroguaiaretic acidBSTFAN,O-bis(trimethylsilyl)trifluoroacetamideHPLChigh performance liquid chromatography6-MNA6-methoxy-2-naphthylacetic acidNS-398N-[2-(cyclohexyloxy)-4-nitrophenyl]methanesulfonamideHUVEChuman umbilical vein endothelial cellsPMNpolymorphonuclear leukocyteSP-HPLCstraight phase HPLCGC-MSgas chromatography-mass spectrometryEIelectron impactETYAeicosatetraynoic acid. and hydroxyoctadecadienoic acid (HODE) have been detected in psoriatic and atherosclerotic lesions(1Baer A.N. 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Acta. 1987; 921: 135-141Crossref PubMed Scopus (57) Google Scholar) may also form additional metabolites of linoleic acid such as epoxy, trihydroxy, and epoxihydroxy derivatives.Reports concerning the biological activities of HODEs show that they are active mediators in hemostasis, inflammation, and cancer invasion. Both 9-HODE and 13-HODE induce interleukin (IL)-1 release in macrophages, the latter being less active(13Ku G. Thomas C.E. Akeson A.L. Jackson R.L. J. Biol. Chem. 1992; 267: 14183-14188Abstract Full Text PDF PubMed Google Scholar). 13-HODE modulates the mitogenic response to epidermal growth factor (14Glasgow W.C. Afshari C.A. Barrett J.C. Eling T.E. J. Biol. Chem. 1992; 267: 10771-10779Abstract Full Text PDF PubMed Google Scholar, 15Cowlen M.S. Eling T.E. Biochim. Biophys. Acta. 1993; 1174: 234-240Crossref PubMed Scopus (22) Google Scholar) and plays a regulatory role by modulating the activity of several enzymes of the arachidonic acid cascade(16Iversen L. Fogh K. 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IL-1 is a pleiotropic cytokine that plays a major role in the inflammatory response. It orients endothelial cell function in a proinflammatory and prothrombotic sense (25Pober J.S. Cotran R.S. Physiol. Rev. 1990; 70: 427-451Crossref PubMed Scopus (1124) Google Scholar, 26Dinarello C.A. Blood. 1991; 77: 1627-1652Crossref PubMed Google Scholar, 27Mantovani A. Dejana E. Immunol. Today. 1989; 10: 370-374Abstract Full Text PDF PubMed Scopus (419) Google Scholar, 28Montesano R. Orci L. Vasalli P. J. Cell. Physiol. 1985; 122: 422-433Crossref Scopus (86) Google Scholar, 29Bevilaqua M.P. Pober J.S. Majeau G.R. Cotran R.S. Gimbrone Jr., M.A. J. Exp. Med. 1984; 160: 618-623Crossref PubMed Scopus (695) Google Scholar) and induces the expression of adhesion molecules and secondary cytokines that, in turn, induce acute and chronic inflammatory changes(26Dinarello C.A. Blood. 1991; 77: 1627-1652Crossref PubMed Google Scholar, 30Breider M.A. J. Am. Vet. Med. Assoc. 1993; 203: 300-306PubMed Google Scholar, 31Long M.W. Exp. Hematol. (N. Y.). 1992; 20: 288-301PubMed Google Scholar). IL-1 also stimulates the release of prostaglandins and promotes the expression of cyclooxygenase (COX)(32Maier J.A.M. Hla T. Maciag T. J. Biol. Chem. 1990; 265: 10805-10808Abstract Full Text PDF PubMed Google Scholar, 33Raz A. Wyche A. Siegel N. Needleman P. J. Biol. Chem. 1988; 263: 3022-3028Abstract Full Text PDF PubMed Google Scholar, 34Korn J.H. Downie E. Roth G.J. Ho S.Y. Clin. Immunol. Immunopathol. 1989; 50: 196-204Crossref PubMed Scopus (22) Google Scholar, 35Jones D.A. Carlton D. McIntyre M.T. Zimmerman G.A. Prescott S.M. J. Biol. Chem. 1993; 268: 9049-9054Abstract Full Text PDF PubMed Google Scholar). Two COX isoenzymes COX1 and COX2 encoded by different genes have been characterized. COX1 is expressed in a constitutive manner, and COX2 is the inducible isoenzyme by mitogens, which is overexpressed in many inflammatory processes(36Smith W.L. Am. J. Physiol. 1992; 263: F181-F191PubMed Google Scholar, 37Hla T. Ristimäki A. Appleby S. Barriocanal J.G. Ann N. Y. Acad. Sci. 1993; 696: 197-204Crossref PubMed Scopus (159) Google Scholar). In general, enzymatic oxidation of linoleic acid may involve both COX and 15-lipoxygenase activities(9Reinaud O. Delaforge M. Boucher J.L. Rocchiccioli F. Mansuy D. Biochem. Biophys. Res. Commun. 1989; 161: 883-891Crossref PubMed Scopus (56) Google Scholar, 38Hamberg M. Samuelsson B. J. Biol. Chem. 1967; 242: 5344-5354Abstract Full Text PDF PubMed Google Scholar). In fact, there are conflicting reports concerning the COX and/or 15-lipoxygenase origin of 13-HODE from linoleic acid, and 15-HETE from arachidonic acid, in endothelial cells(4Buchanan M.R. Haas T.A. Lagarde M. Guichardant M. J. Biol. Chem. 1985; 260: 16056-16059Abstract Full Text PDF PubMed Google Scholar, 5Buchanan M.R. Butt R.W. Magas Z. Van Ryn J. Hirsh J. Nazir D.J. Thromb. Haemostasis. 1985; 53: 306-311Crossref PubMed Scopus (59) Google Scholar, 6Kaduce T.L. Figard P.H. Leifur R. Spector A.A. J. Biol. Chem. 1989; 264: 6823-6830Abstract Full Text PDF PubMed Google Scholar, 21Haas T.A. Bertomeu M.C. Bastida E. Buchanan M.R. Biochim. Biophys. Acta. 1990; 1051: 174-178Crossref PubMed Scopus (25) Google Scholar, 22Bertomeu M.C. Gallo S. Lauri D. Haas T.A. Orr F.W. Bastida E. Buchanan M. Clin. Exp. Metastasis. 1993; 11: 243-250Crossref PubMed Scopus (39) Google Scholar, 39Hopkins N.K. Oglesby T.D. Bundy G.L. Gorman R.R. J. Biol. Chem. 1984; 259: 14048-14053Abstract Full Text PDF PubMed Google Scholar, 40López S. Vila L. Breviario F. de Castellarnau C. Biochim. Biophys. Acta. 1993; 1170: 17-24Crossref PubMed Scopus (27) Google Scholar, 41Takayama H. Gimbrone Jr., M.A. Schafer A.I. Thrombos. Res. 1987; 45: 803-816Abstract Full Text PDF PubMed Scopus (43) Google Scholar, 42Kühn H. Pönicke K. Halle W. Wiesner R. Schewe T. Försten W. Prostaglandins Leukotrienes Med. 1985; 17: 291-303Abstract Full Text PDF PubMed Scopus (39) Google Scholar, 43Setty B.N.Y. Stuart M.J. J. Clin. Invest. 1986; 77: 202-211Crossref PubMed Scopus (78) Google Scholar).The fact that IL-1 is a crucial mediator of the interactions among endothelial cells, immunocompetent cells, and tumor cells and that part of these interactions may be mediated by octadecanoids prompted us to investigate the effect of this cytokine on the metabolism of linoleic acid in endothelial cells.DISCUSSIONHUVEC formed 13(S)-HODE(Z,E) and 9(R)-HODE(E,Z) as the mayor enzymatic products from linoleic acid. Minor nonenzymatic isomerizations toward 13- and 9-hydroxy all-trans-isomers from both 13-HPODE(Z,E) and 9-HPODE(E,Z) and/or the corresponding hydroxides may also occur. Isomerization of very small amounts of 9-HODE(E,Z) toward 13-HODE(Z,E) and 13-HODE(Z,E) toward 9-HODE(E,Z) were also observed. These nonenzymatic isomerizations (mainly all-trans, which are thermodynamically favorable) probably occurred due to rearrangements of radical species formed during the incubation and/or further manipulation.Exposure of HUVEC to IL-1β increases their ability to transform linoleic acid into HODEs enzymatically in a time- and dose-dependent fashion. Blocking the stimulating action of IL-1β on the biosynthesis of HODEs by cycloheximide and actinomycin D indicates that de novo synthesis of protein is required for this effect to occur. Biosynthesis of 13-HODE may be catalyzed by both COX and 15-lipoxygenase(9Reinaud O. Delaforge M. Boucher J.L. Rocchiccioli F. Mansuy D. Biochem. Biophys. Res. Commun. 1989; 161: 883-891Crossref PubMed Scopus (56) Google Scholar, 38Hamberg M. Samuelsson B. J. Biol. Chem. 1967; 242: 5344-5354Abstract Full Text PDF PubMed Google Scholar, 49Hamberg M. Samuelsson B. Biochim. Biophys. Acta. 1980; 617: 545-547Crossref PubMed Scopus (36) Google Scholar), whereas significant amounts of 9-HODE are produced by COX(38Hamberg M. Samuelsson B. J. Biol. Chem. 1967; 242: 5344-5354Abstract Full Text PDF PubMed Google Scholar, 49Hamberg M. Samuelsson B. Biochim. Biophys. Acta. 1980; 617: 545-547Crossref PubMed Scopus (36) Google Scholar). The absence of significant differences in the stimulating effect of IL-1β in the production of 13- and 9-HODEs in terms of EC50 suggests the involvement of a common enzymatic pathway in the effect of IL-1β on the production of the two position isomers.The exact enzymatic pathway for the biosynthesis of HODEs in HUVEC, specially 13-HODE, remains controversial. The location of the hydroxyl group in the molecule of 15-HETE or 13-HODE led other authors to infer the presence of 15-lipoxygenase in HUVEC, but as mentioned before 15-HETE and 13-HODE can also be formed by COX(38Hamberg M. Samuelsson B. J. Biol. Chem. 1967; 242: 5344-5354Abstract Full Text PDF PubMed Google Scholar, 49Hamberg M. Samuelsson B. Biochim. Biophys. Acta. 1980; 617: 545-547Crossref PubMed Scopus (36) Google Scholar, 50Hecker M. Ullrich V. Fischer C. Meese C.O. Eur. J. Biochem. 1987; 169: 113-123Crossref PubMed Scopus (54) Google Scholar). The 15-lipoxygenase origin of 13-HODE and 15-HETE in nonstimulated HUVEC has been claimed by several authors, although no direct evidence of this has been reported(4Buchanan M.R. Haas T.A. Lagarde M. Guichardant M. J. Biol. Chem. 1985; 260: 16056-16059Abstract Full Text PDF PubMed Google Scholar, 5Buchanan M.R. Butt R.W. Magas Z. Van Ryn J. Hirsh J. Nazir D.J. Thromb. Haemostasis. 1985; 53: 306-311Crossref PubMed Scopus (59) Google Scholar, 21Haas T.A. Bertomeu M.C. Bastida E. Buchanan M.R. Biochim. Biophys. Acta. 1990; 1051: 174-178Crossref PubMed Scopus (25) Google Scholar, 22Bertomeu M.C. Gallo S. Lauri D. Haas T.A. Orr F.W. Bastida E. Buchanan M. Clin. Exp. Metastasis. 1993; 11: 243-250Crossref PubMed Scopus (39) Google Scholar, 39Hopkins N.K. Oglesby T.D. Bundy G.L. Gorman R.R. J. Biol. Chem. 1984; 259: 14048-14053Abstract Full Text PDF PubMed Google Scholar, 41Takayama H. Gimbrone Jr., M.A. Schafer A.I. Thrombos. Res. 1987; 45: 803-816Abstract Full Text PDF PubMed Scopus (43) Google Scholar, 43Setty B.N.Y. Stuart M.J. J. Clin. Invest. 1986; 77: 202-211Crossref PubMed Scopus (78) Google Scholar). Buchanan et al.(5Buchanan M.R. Butt R.W. Magas Z. Van Ryn J. Hirsh J. Nazir D.J. Thromb. Haemostasis. 1985; 53: 306-311Crossref PubMed Scopus (59) Google Scholar) first reported the production by HUVEC of a lipoxygenase-derived product, lipoxygenase X, which inhibited adhesion of platelets to endothelial surface; lipoxygenase X was later identified as 13-HODE(4Buchanan M.R. Haas T.A. Lagarde M. Guichardant M. J. Biol. Chem. 1985; 260: 16056-16059Abstract Full Text PDF PubMed Google Scholar). These authors studied the content of free 13-HODE in HUVEC, showing that it is dependent on the turnover of linoleic acid in the triacylglycerol pool(21Haas T.A. Bertomeu M.C. Bastida E. Buchanan M.R. Biochim. Biophys. Acta. 1990; 1051: 174-178Crossref PubMed Scopus (25) Google Scholar). The amount of free 13-HODE present in cells after stimulation with thrombin or calcium ionophore was lower than in controls(4Buchanan M.R. Haas T.A. Lagarde M. Guichardant M. J. Biol. Chem. 1985; 260: 16056-16059Abstract Full Text PDF PubMed Google Scholar). Thrombin and calcium ionophore cause the release of free arachidonic acid; interestingly, it was found that when HUVEC were incubated with 200 μM labeled arachidonic acid lipoxygenase X (13-HODE) was formed(5Buchanan M.R. Butt R.W. Magas Z. Van Ryn J. Hirsh J. Nazir D.J. Thromb. Haemostasis. 1985; 53: 306-311Crossref PubMed Scopus (59) Google Scholar). The fact that the cytosolic fraction was able to produce 13-HODE, together with the inhibition by ETYA, was supporting evidence for the conclusion that 15-lipoxygenase is the origin of 13-HODE in HUVEC(4Buchanan M.R. Haas T.A. Lagarde M. Guichardant M. J. Biol. Chem. 1985; 260: 16056-16059Abstract Full Text PDF PubMed Google Scholar, 5Buchanan M.R. Butt R.W. Magas Z. Van Ryn J. Hirsh J. Nazir D.J. Thromb. Haemostasis. 1985; 53: 306-311Crossref PubMed Scopus (59) Google Scholar). COX2 protein and COX activity have also been observed in the cytosol(51Rimachin J.A. Jacobson J.A. Szabo P. Maclouf J. Creminon C. Weksler B.B. Arterioscler. Thromb. 1994; 14: 1021-1031Crossref PubMed Google Scholar, 52Godessart N. Vila L. Puig L. de Moragas J.M. J. Invest. Dermatol. 1994; 102: 98-104Abstract Full Text PDF PubMed Google Scholar).The fact that lipoxygenase inhibitors such as ETYA and/or NDGA inhibit the production of 15-HETE and 13-HODE(4Buchanan M.R. Haas T.A. Lagarde M. Guichardant M. J. Biol. Chem. 1985; 260: 16056-16059Abstract Full Text PDF PubMed Google Scholar, 5Buchanan M.R. Butt R.W. Magas Z. Van Ryn J. Hirsh J. Nazir D.J. Thromb. Haemostasis. 1985; 53: 306-311Crossref PubMed Scopus (59) Google Scholar, 39Hopkins N.K. Oglesby T.D. Bundy G.L. Gorman R.R. J. Biol. Chem. 1984; 259: 14048-14053Abstract Full Text PDF PubMed Google Scholar, 41Takayama H. Gimbrone Jr., M.A. Schafer A.I. Thrombos. Res. 1987; 45: 803-816Abstract Full Text PDF PubMed Scopus (43) Google Scholar, 42Kühn H. Pönicke K. Halle W. Wiesner R. Schewe T. Försten W. Prostaglandins Leukotrienes Med. 1985; 17: 291-303Abstract Full Text PDF PubMed Scopus (39) Google Scholar, 43Setty B.N.Y. Stuart M.J. J. Clin. Invest. 1986; 77: 202-211Crossref PubMed Scopus (78) Google Scholar) and the oxidative action of HUVEC on lipoproteins (53Parthasarathy S. Wieland E. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1046-1050Crossref PubMed Scopus (375) Google Scholar) has been used as the supporting evidence for the 15-lipoxygenase involvement in such events in HUVEC. ETYA actually is a competitive arachidonic acid analogue that inhibits many dioxygenases including COXs(54Tobias L.D. Hamilton J.C. Lipids. 1979; 14: 181-193Crossref PubMed Scopus (118) Google Scholar, 55Anderson K.M. Ondrey F. Harrys J.E. Clin. Biochem. 1992; 25: 1-9Crossref PubMed Scopus (17) Google Scholar). In the present work, we found that 50 μM ETYA completely inhibited HODEs formation by HUVEC (not shown). Furthermore, we found that NDGA inhibited 13-HODE(Z,E) formation by HUVEC in a concentration-dependent manner. However, NDGA showed higher efficiency in inhibiting 9-HODE(E,Z) than 13-HODE(Z,E), especially in resting cells. NDGA is a potent inhibitor of lipoxygenases but also inhibits COX (40López S. Vila L. Breviario F. de Castellarnau C. Biochim. Biophys. 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Based on these results we think that caution should be exercised in defining the role of 15-lipoxygenase in the metabolic pathways based only on inhibitions by NDGA or ETYA.The limited effect of aspirin in the production of 15-HETE has also been considered as supportive evidence of the 15-lipoxygenase origin of this eicosanoid in endothelial cells(41Takayama H. Gimbrone Jr., M.A. Schafer A.I. Thrombos. Res. 1987; 45: 803-816Abstract Full Text PDF PubMed Scopus (43) Google Scholar). We reported that aspirin, even at 1 mM concentration, was unable to suppress totally 15-HETE formation in HUVEC and human dermal fibroblasts treated with IL-1β(40López S. Vila L. Breviario F. de Castellarnau C. Biochim. Biophys. Acta. 1993; 1170: 17-24Crossref PubMed Scopus (27) Google Scholar, 52Godessart N. Vila L. Puig L. de Moragas J.M. J. Invest. Dermatol. 1994; 102: 98-104Abstract Full Text PDF PubMed Google Scholar). This phenomenon can be explained not only by the 15-lipoxygenase origin of 15-HETE but also by the COX2 origin, since 15-HETE is the main eicosanoid produced by COX2 treated with aspirin(60Meade E.A. Smith W.L. DeWitt D.L. J. Biol. Chem. 1993; 268: 6610-6614Abstract Full Text PDF PubMed Google Scholar). However, 100 μM aspirin totally inhibited all octadecanoids, even 13-HODE(Z,E), in both controls and IL-1β-treated cells (not shown).All COX inhibitors tested in the present study inhibited the synthesis of both products 13-HODE(Z,E) and 9-HODE(E,Z) in untreated as well as in IL-1β-treated cells in a dose-dependent fashion. The greater ability to inhibit 9-HODE(E,Z) than 13-HODE(Z,E) formation, especially in untreated cells, was a common feature of all inhibitors tested (Table 1). This is consistent with the lower inhibitory strength of indomethacin on 15-HETE than 11-HETE or prostaglandin formation from arachidonic acid by HUVEC and dermal fibroblasts(40López S. Vila L. Breviario F. de Castellarnau C. Biochim. Biophys. Acta. 1993; 1170: 17-24Crossref PubMed Scopus (27) Google Scholar, 52Godessart N. Vila L. Puig L. de Moragas J.M. J. Invest. Dermatol. 1994; 102: 98-104Abstract Full Text PDF PubMed Google Scholar). Nevertheless, only indomethacin was able to suppress HODEs formation totally at the concentrations used. Consistent with reports that 100 μM indomethacin did not inhibit 15-lipoxygenase activity in cells transfected with reticulocyte 15-lipoxygenase(61Sigal E. Grunberger D. Highland E. Gross C. Dixon R.A.F. Craik C.S. J. Biol. Chem. 1990; 265: 5113-5120Abstract Full Text PDF PubMed Google Scholar), we found that 50 μM indomethacin did not inhibit 13-HODE(Z,E) formation by PMN.Furthermore, lipoxygenases render compounds with a strict S stereospecificity(62Kühn H. Schewe T. Rapoport S.M. Adv. Enzymol. 1986; 58: 273-311PubMed Google Scholar), whereas COX forms R- and S-isomer mixtures(49Hamberg M. Samuelsson B. Biochim. Biophys. Acta. 1980; 617: 545-547Crossref PubMed Scopus (36) Google Scholar). Results from chiral analysis indicate that the main products formed by HUVEC, irrespective of their treatment or not with IL-1β, were 13(S)-HODE and 9(R)-HODE. The ratio of enantiomers in controls or IL-1β-treated HUVEC is quite similar to that obtained when HODEs were synthesized by isolated COX1 or COX2 independently and is different from that obtained in the incubations of linoleic acid with reticulocyte 15-lipoxygenase, which yielded only 13(S)-HODE(Z,E). These results agree with those obtained by Hamberg and Samuelsson(49Hamberg M. Samuelsson B. Biochim. Biophys. Acta. 1980; 617: 545-547Crossref PubMed Scopus (36) Google Scholar), Baer et al.(63Baer A.N. Costello P.B. Green F.A. Biochim. Biophys. Acta. 1991; 1085: 45-52Crossref PubMed Scopus (26) Google Scholar), and Reinaud et al.(9Reinaud O. Delaforge M. Boucher J.L. Rocchiccioli F. Mansuy D. Biochem. Biophys. Res. Commun. 1989; 161: 883-891Crossref PubMed Scopus (56) Google Scholar) with pure COX, bovine aortic endothelial cells, and human leukocytes, respectively.Recent studies have shown that 15-lipoxygenase expressed in macrophages is regulated by inflammatory cytokines, and only IL-4 induces specifically 15-lipoxygenase mRNA and enzyme activity in cultured human monocytes(64Conrad D.J. Kühn H. Mulkins M. Highland E. Sigal E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 217-221Crossref PubMed Scopus (342) Google Scholar). We were unable to find 15-lipoxygenase mRNA in untreated and in IL-4- or IL-1β-treated HUVEC, whereas IL-4, but not IL-1β, effectively induced 15-lipoxygenase mRNA on cultured monocytes (40López S. Vila L. Breviario F. de Castellarnau C. Biochim. Biophys. Acta. 1993; 1170: 17-24Crossref PubMed Scopus (27) Google Scholar). Overall, these results indicate that formation of both 13-HODE(Z,E) and 9-HODE(E,Z) was mediated by COX rather than by 15-lipoxygenase in resting and IL-1β-treated HUVEC.According to data reported by Jones et al.(35Jones D.A. Carlton D. McIntyre M.T. Zimmerman G.A. Prescott S.M. J. Biol. Chem. 1993; 268: 9049-9054Abstract Full Text PDF PubMed Google Scholar), both COX1 and COX2 were detected in nonstimulated HUVEC. COX1 was induced slightly by IL-1β (less than 2-fold), whereas COX2 increased 8-fold as a result of IL-1β exposure. The ratio of HODE biosynthetic activity between IL-1β-treated cells and controls at substrate concentrations ≤ 50 μM (from data in Fig. 5: IL-1β/controls 5-8) is consistent, but not absolutely coincident, with the increment of COX proteins. Kinetics of linoleic acid concentration show a 1.5-fold increase of apparent Vmax and a 9.8-fold decrease in the apparent Km in IL-1β-treated cells with respect to controls. That the increased amount of COX2 caused by IL-1β was observed mainly as a reduction of the apparent Kmvalue may be due to a combination of two factors: (i) the higher affinity of COX2 than COX1 for linoleic acid, and (ii) a substrate inhibition at high substrate concentrations which led to an undervalued Vmax, even though the experimental values fit the Michaelis-Menten equation.The higher strength of COX2-specific inhibitors NS-398 and 6-MNA (60Meade E.A. Smith W.L. DeWitt D.L. J. Biol. Chem. 1993; 268: 6610-6614Abstract Full Text PDF PubMed Google Scholar, 65Futaki N. Takahashi S. Yokoyama M. Arai I. Higuchi S. Otomo S. Prostaglandins. 1994; 47: 55-59Crossref PubMed Scopus (798) Google Scholar) for inhibiting HODEs in IL-1β-treated cells is consistent with the fact that IL-1β induced mainly the expression of new COX2. The strength of COX2-selective inhibitors NS-398 and 6-MNA on HODE formation was similar to those reported with arachidonic acid as substrate(60Meade E.A. Smith W.L. DeWitt D.L. J. Biol. Chem. 1993; 268: 6610-6614Abstract Full Text PDF PubMed Google Scholar, 65Futaki N. Takahashi S. Yokoyama M. Arai I. Higuchi S. Otomo S. Prostaglandins. 1994; 47: 55-59Crossref PubMed Scopus (798) Google Scholar, 66DeWitt D.L. Meade E.A. Smith W.L. Am. J. Med. 1993; (Suppl. 2A 95): 40S-44SAbstract Full Text PDF PubMed Scopus (133) Google Scholar). As mentioned before, except indomethacin none of the COX inh
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