Receptors for the 5-Oxo Class of Eicosanoids in Neutrophils
1998; Elsevier BV; Volume: 273; Issue: 49 Linguagem: Inglês
10.1074/jbc.273.49.32535
ISSN1083-351X
AutoresJoseph T. O’Flaherty, Jennifer S. Taylor, Michael J. Thomas,
Tópico(s)Peroxisome Proliferator-Activated Receptors
Resumo5-Hydroxy- and 5-oxo-eicosatetraenoate (5-HETE and 5-oxoETE) activate polymorphonuclear neutrophils (PMNs) through a common, receptor-like recognition system. To define this system, we examined the interaction of these eicosanoids with human PMNs. PMNs esterified 5-[3H]HETE to glycerolipids at 37 and 4 °C. At 37 but not 4 °C, the cells also hydroxylated the label to 5,20-[3H]diHETE. The acyl:CoA synthetase blocker, triacsin C, inhibited esterification but also led to an increase in the hydroxylation of the label. PMNs processed 5-[3H]oxoETE through the same pathways but only or principally after reducing it to 5-[3H]HETE (37 or 4 °C). In the presence of these varying metabolic reactions, PMNs (37 or 4 °C; ± triacsin C) could not be shown to receptor bind either radiolabel.Plasma membranes isolated from PMNs esterified but unlike whole cells did not reduce or hydroxylate 5-[3H]oxoETE. Triacsin C blocked esterification, thereby rendering the membranes unable to metabolize this radiolabel. Indeed, triacsin C-treated membranes bound (K d = 3.8 nm) 5-[3H]oxoETE specifically and reversibly to 86 pmol of sites per 25 μg of membrane protein. 5-OxoETE, 5-HETE, and 5,15-diHETE displaced this binding at concentrations correlating with their potency in eliciting PMN Ca2+ transients. GTP and GTPγS, but not ATP or ATPγS, also reduced 5-[3H]oxoETE binding, whereas 15-HETE, leukotriene B4, platelet-activating factor, IL-8, C5a, and N-formyl-Met-Leu-Phe lacked this effect. We conclude that PMNs and their plasma membranes use an acyl:CoA synthetase-dependent route to esterify 5-HETE and 5-oxoETE into lipids. Blockade of the synthetase uncovers cryptic plasmalemma sites that bind 5-oxoETE with exquisite specificity. These sites apparently mediate responses to the 5-oxo class of eicosanoids and are likely members of the serpentine superfamily of G protein-linked receptors. 5-Hydroxy- and 5-oxo-eicosatetraenoate (5-HETE and 5-oxoETE) activate polymorphonuclear neutrophils (PMNs) through a common, receptor-like recognition system. To define this system, we examined the interaction of these eicosanoids with human PMNs. PMNs esterified 5-[3H]HETE to glycerolipids at 37 and 4 °C. At 37 but not 4 °C, the cells also hydroxylated the label to 5,20-[3H]diHETE. The acyl:CoA synthetase blocker, triacsin C, inhibited esterification but also led to an increase in the hydroxylation of the label. PMNs processed 5-[3H]oxoETE through the same pathways but only or principally after reducing it to 5-[3H]HETE (37 or 4 °C). In the presence of these varying metabolic reactions, PMNs (37 or 4 °C; ± triacsin C) could not be shown to receptor bind either radiolabel. Plasma membranes isolated from PMNs esterified but unlike whole cells did not reduce or hydroxylate 5-[3H]oxoETE. Triacsin C blocked esterification, thereby rendering the membranes unable to metabolize this radiolabel. Indeed, triacsin C-treated membranes bound (K d = 3.8 nm) 5-[3H]oxoETE specifically and reversibly to 86 pmol of sites per 25 μg of membrane protein. 5-OxoETE, 5-HETE, and 5,15-diHETE displaced this binding at concentrations correlating with their potency in eliciting PMN Ca2+ transients. GTP and GTPγS, but not ATP or ATPγS, also reduced 5-[3H]oxoETE binding, whereas 15-HETE, leukotriene B4, platelet-activating factor, IL-8, C5a, and N-formyl-Met-Leu-Phe lacked this effect. We conclude that PMNs and their plasma membranes use an acyl:CoA synthetase-dependent route to esterify 5-HETE and 5-oxoETE into lipids. Blockade of the synthetase uncovers cryptic plasmalemma sites that bind 5-oxoETE with exquisite specificity. These sites apparently mediate responses to the 5-oxo class of eicosanoids and are likely members of the serpentine superfamily of G protein-linked receptors. 5(S)-hydroxy-(E,Z,Z,Z)-6,8,11,14-ETE eicosatetraenoic acid 5-oxo-(E,Z,Z,Z)-6,8,11,14-ETE 5(R,S)-hydroxy-(E,Z,Z,Z)-6,8,11,14-ETE 15-diHETE, 5(S),15(S)-dihydroxy-(E,Z,Z,E)-6,8,11,13-ETE 20-diHETE, 5(S),20-dihydroxy-(E,Z,Z,Z)-6,8,11,14-ETE 15(S)-hydroxy-(Z,Z,Z,E)-5,8,11,13-ETE 5-oxo-15(S)-hydro-xy-(E,Z,Z,E)-6,8,11,13-ETE leukotriene B4 platelet-activating factor N-formyl-Met-Leu-Phe polymorphonuclear neutrophil high pressure liquid chromatography bovine serum albumin interleukin. Cells respond to stimulation by converting storage arachidonic acid into products that signal their own or nearby cell responses (1Borgeat P. Nadeau M. Salari H. Poubelle P. Fruteau d.L. Adv. Lipid Res. 1985; 21: 47-77Crossref PubMed Google Scholar,2Chavis C. Vachier I. Chanez P. Bousquet J. Godard P. J. Exp. Med. 1996; 183: 1633-1643Crossref PubMed Scopus (74) Google Scholar). Many such eicosanoids act by binding to receptors (3Halushka P.V. Mais D.E. Mayeux P.R. Morinelli T.A. Annu. Rev. Pharmacol. Toxicol. 1989; 29: 213-239Crossref PubMed Scopus (231) Google Scholar, 4Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (861) Google Scholar, 5Maddox J.F. Hachicha M. Takano T. Petasis N.A. Fokin V.V. Serhan C.N. J. Biol. Chem. 1997; 272: 6972-6978Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Others have a less clear mechanism of action. 5-HETE1 and LTB4, for example, are companion metabolites formed by the attack of 5-lipoxygenase on arachidonic acid in, e.g. human PMNs (1Borgeat P. Nadeau M. Salari H. Poubelle P. Fruteau d.L. Adv. Lipid Res. 1985; 21: 47-77Crossref PubMed Google Scholar,2Chavis C. Vachier I. Chanez P. Bousquet J. Godard P. J. Exp. Med. 1996; 183: 1633-1643Crossref PubMed Scopus (74) Google Scholar, 6Powell W.S. Zhang Y. Gravel S. Biochemistry. 1994; 33: 3927-3933Crossref PubMed Scopus (28) Google Scholar). Although the two eicosanoids have similar stimulatory actions, 5-HETE has little affinity for LTB4 receptors and is resistant to a LTB4 receptor antagonist. In down-regulation assays, furthermore, 5-HETE desensitizes to itself but does not cross-desensitize with other chemotactic factors (LTB4, FMLP, C5a, PAF, or IL-8) or eicosanoids (lipoxins A4 and B4 and prostaglandins D2 and E2) (7O'Flaherty J.T. Jacobson D. Redman J. J. Immunol. 1988; 140: 4323-4328PubMed Google Scholar, 8O'Flaherty J.T. Rossi A.G. J. Biol. Chem. 1993; 268: 14708-14714Abstract Full Text PDF PubMed Google Scholar, 9O'Flaherty J.T. Cordes J.F. Lee S.L. Samuel M. Thomas M.J. Biochim. Biophys. Acta. 1994; 1201: 505-515Crossref PubMed Scopus (37) Google Scholar, 10Powell W.S. Gravel S. MacLeod R.J. Mills E. Hashefi M. J. Biol. Chem. 1993; 268: 9280-9286Abstract Full Text PDF PubMed Google Scholar, 11Wijkander J. O'Flaherty J.T. Nixon A.B. Wykle R.L. J. Biol. Chem. 1995; 270: 26543-26549Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 12Powell W.S. MacLeod R.J. Gravel S. Gravelle F. Bhakar A. J. Immunol. 1996; 156: 336-342PubMed Google Scholar, 13Norgauer J. Barbisch M. Czech W. Pareigis J. Schwenk U. Schroder J.M. Eur. J. Biochem. 1996; 236: 1003-1009Crossref PubMed Scopus (33) Google Scholar, 14Powell W.S. Chung D. Gravel S. J. Immunol. 1995; 154: 4123-4132PubMed Google Scholar, 15Schwenk U. Schroder J.-M. J. Biol. Chem. 1995; 270: 15029-15036Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 16O'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, 17Czech W. Barbisch M. Tenscher K. Schopf E. Schroder J.M. Norgauer J. J. Invest. Dermatol. 1997; 108: 108-112Abstract Full Text PDF PubMed Scopus (36) Google Scholar, 18O'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. Biol. Chem. 1996; 271: 17821-17828Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 19Sozzani S. Zhou D. Locati M. Bernasconi S. Luini W. Mantovani A. O'Flaherty J.T. J. Immunol. 1996; 157: 4664-4671PubMed Google Scholar, 20Rossi A.G. O'Flaherty J.T. Prostaglandins. 1989; 37: 641-653Crossref PubMed Scopus (20) Google Scholar). The data imply that 5-HETE acts through a unique recognition system. However, evidence relating this system to receptor-like binding sites has proven difficult to obtain. Although PMNs process 5-HETE through various metabolic pathways (6Powell W.S. Zhang Y. Gravel S. Biochemistry. 1994; 33: 3927-3933Crossref PubMed Scopus (28) Google Scholar, 7O'Flaherty J.T. Jacobson D. Redman J. J. Immunol. 1988; 140: 4323-4328PubMed Google Scholar, 8O'Flaherty J.T. Rossi A.G. J. Biol. Chem. 1993; 268: 14708-14714Abstract Full Text PDF PubMed Google Scholar,12Powell W.S. MacLeod R.J. Gravel S. Gravelle F. Bhakar A. J. Immunol. 1996; 156: 336-342PubMed Google Scholar, 21O'Flaherty J.T. Wykle R.L. Redman J. Samuel M. Thomas M. J. Immunol. 1986; 137: 3277-3283PubMed Google Scholar, 22Arai M. Imai H. Metori A. Nakagawa Y. Eur. J. Biochem. 1997; 244: 513-519Crossref PubMed Scopus (19) Google Scholar, 23Bonser R.W. Siegel M.I. Chung S.M. McConnell R.T. Cuatrecasas P. Biochemistry. 1981; 20: 5297-5301Crossref PubMed Scopus (64) Google Scholar), one route, esterification, has presented an obstacle for receptor studies. The reaction occurs in PMNs or their isolated plasma membranes at 4 or 37 °C and results in the acylation of 5-[3H]HETE to membrane glycerolipids: PMNs and plasmalemma accumulate 5-HETE almost exclusively in esterified form without evidence of receptor binding (10Powell W.S. Gravel S. MacLeod R.J. Mills E. Hashefi M. J. Biol. Chem. 1993; 268: 9280-9286Abstract Full Text PDF PubMed Google Scholar, 21O'Flaherty J.T. Wykle R.L. Redman J. Samuel M. Thomas M. J. Immunol. 1986; 137: 3277-3283PubMed Google Scholar). Because esterification is a ubiquitous means for processing fatty acids, other cell types are apt also to esterify 5-HETE and thereby obscure the receptor binding of the compound. Despite this difficulty, however, putative receptors for 5-HETE merit study. PMNs, eosinophils, and monocytes dehydrogenate 5-HETE to 5-oxoETE (6Powell W.S. Zhang Y. Gravel S. Biochemistry. 1994; 33: 3927-3933Crossref PubMed Scopus (28) Google Scholar, 13Norgauer J. Barbisch M. Czech W. Pareigis J. Schwenk U. Schroder J.M. Eur. J. Biochem. 1996; 236: 1003-1009Crossref PubMed Scopus (33) Google Scholar, 14Powell W.S. Chung D. Gravel S. J. Immunol. 1995; 154: 4123-4132PubMed Google Scholar, 15Schwenk U. Schroder J.-M. J. Biol. Chem. 1995; 270: 15029-15036Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 25Zhang Y. Powell W.S. FASEB J. 1994; 8: A356Crossref PubMed Scopus (0) Google Scholar). 5-OxoETE is ∼10-fold stronger than 5-HETE in stimulating PMNs and monocytes (9O'Flaherty J.T. Cordes J.F. Lee S.L. Samuel M. Thomas M.J. Biochim. Biophys. Acta. 1994; 1201: 505-515Crossref PubMed Scopus (37) Google Scholar, 10Powell W.S. Gravel S. MacLeod R.J. Mills E. Hashefi M. J. Biol. Chem. 1993; 268: 9280-9286Abstract Full Text PDF PubMed Google Scholar, 11Wijkander J. O'Flaherty J.T. Nixon A.B. Wykle R.L. J. Biol. Chem. 1995; 270: 26543-26549Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 12Powell W.S. MacLeod R.J. Gravel S. Gravelle F. Bhakar A. J. Immunol. 1996; 156: 336-342PubMed Google Scholar, 13Norgauer J. Barbisch M. Czech W. Pareigis J. Schwenk U. Schroder J.M. Eur. J. Biochem. 1996; 236: 1003-1009Crossref PubMed Scopus (33) Google Scholar, 19Sozzani S. Zhou D. Locati M. Bernasconi S. Luini W. Mantovani A. O'Flaherty J.T. J. Immunol. 1996; 157: 4664-4671PubMed Google Scholar). It is even more active on eosinophils (14Powell W.S. Chung D. Gravel S. J. Immunol. 1995; 154: 4123-4132PubMed Google Scholar, 15Schwenk U. Schroder J.-M. J. Biol. Chem. 1995; 270: 15029-15036Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 16O'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, 17Czech W. Barbisch M. Tenscher K. Schopf E. Schroder J.M. Norgauer J. J. Invest. Dermatol. 1997; 108: 108-112Abstract Full Text PDF PubMed Scopus (36) Google Scholar), eliciting the chemotaxis response of this cell at concentrations 10,000-, 1000-, and >10-fold lower than LTB4, 5-HETE, or other chemotactic factors, respectively (18O'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. Biol. Chem. 1996; 271: 17821-17828Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). 5-OxoETE down-regulates these cells to itself and 5-HETE but not to LTB4 or other stimuli (7O'Flaherty J.T. Jacobson D. Redman J. J. Immunol. 1988; 140: 4323-4328PubMed Google Scholar, 8O'Flaherty J.T. Rossi A.G. J. Biol. Chem. 1993; 268: 14708-14714Abstract Full Text PDF PubMed Google Scholar, 9O'Flaherty J.T. Cordes J.F. Lee S.L. Samuel M. Thomas M.J. Biochim. Biophys. Acta. 1994; 1201: 505-515Crossref PubMed Scopus (37) Google Scholar, 10Powell W.S. Gravel S. MacLeod R.J. Mills E. Hashefi M. J. Biol. Chem. 1993; 268: 9280-9286Abstract Full Text PDF PubMed Google Scholar, 11Wijkander J. O'Flaherty J.T. Nixon A.B. Wykle R.L. J. Biol. Chem. 1995; 270: 26543-26549Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 12Powell W.S. MacLeod R.J. Gravel S. Gravelle F. Bhakar A. J. Immunol. 1996; 156: 336-342PubMed Google Scholar, 13Norgauer J. Barbisch M. Czech W. Pareigis J. Schwenk U. Schroder J.M. Eur. J. Biochem. 1996; 236: 1003-1009Crossref PubMed Scopus (33) Google Scholar, 14Powell W.S. Chung D. Gravel S. J. Immunol. 1995; 154: 4123-4132PubMed Google Scholar, 15Schwenk U. Schroder J.-M. J. Biol. Chem. 1995; 270: 15029-15036Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 16O'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, 17Czech W. Barbisch M. Tenscher K. Schopf E. Schroder J.M. Norgauer J. J. Invest. Dermatol. 1997; 108: 108-112Abstract Full Text PDF PubMed Scopus (36) Google Scholar, 18O'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. Biol. Chem. 1996; 271: 17821-17828Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 19Sozzani S. Zhou D. Locati M. Bernasconi S. Luini W. Mantovani A. O'Flaherty J.T. J. Immunol. 1996; 157: 4664-4671PubMed Google Scholar). Possibly, therefore, putative 5-HETE receptors and their preferred ligand, 5-oxoETE, participate in recruiting eosinophils to sites of allergic reactivity (18O'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. Biol. Chem. 1996; 271: 17821-17828Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). These receptors might also mediate the remodeling of bone, growth of prostate cancer cells, contraction of uterus, and transmission of nerve impulses (26Gallwitz W.E. Mundy G.R. Lee C.H. Qiao M. Roodman G.D. Raftery M. Gaskell S.J. Bonewald L.F. J. Biol. Chem. 1993; 268: 10087-10094Abstract Full Text PDF PubMed Google Scholar, 27Ghosh J. Myers C.E. Biochem. Biophys. Res. Commun. 1997; 235: 418-423Crossref PubMed Scopus (236) Google Scholar, 28Edwin S.S. Romero R.J. Munoz H. Branch D.W. Mitchell M.D. Prostaglandins. 1996; 51: 403-412Crossref PubMed Scopus (26) Google Scholar, 29Foley T.D. Biochem. Biophys. Res. Commun. 1997; 235: 374-376Crossref PubMed Scopus (30) Google Scholar). We report here on studies identifying PMN membrane sites that bind 5-oxoETE with the specificity and other properties anticipated for receptors of the 5-oxo class of eicosanoids. Cells and membranes were suspended in a modified Hanks' balanced salt solution (9O'Flaherty J.T. Cordes J.F. Lee S.L. Samuel M. Thomas M.J. Biochim. Biophys. Acta. 1994; 1201: 505-515Crossref PubMed Scopus (37) Google Scholar) containing 1.4 mm CaCl2 and 0.7 mmMgCl2 unless indicated otherwise. Stimuli, glycerolipids, and other reagents were obtained commercially (8O'Flaherty J.T. Rossi A.G. J. Biol. Chem. 1993; 268: 14708-14714Abstract Full Text PDF PubMed Google Scholar, 9O'Flaherty J.T. Cordes J.F. Lee S.L. Samuel M. Thomas M.J. Biochim. Biophys. Acta. 1994; 1201: 505-515Crossref PubMed Scopus (37) Google Scholar). Triacsin C was purchased from Biomol (Plymouth Meeting, PA). We synthesized LTB4, 5-HETE, 5,20-diHETE, rac-5-HETE, 5,15-diHETE, and 5-oxoETE (9O'Flaherty J.T. Cordes J.F. Lee S.L. Samuel M. Thomas M.J. Biochim. Biophys. Acta. 1994; 1201: 505-515Crossref PubMed Scopus (37) Google Scholar, 21O'Flaherty J.T. Wykle R.L. Redman J. Samuel M. Thomas M. J. Immunol. 1986; 137: 3277-3283PubMed Google Scholar,30Rossi A.G. Thomas M.J. O'Flaherty J.T. FEBS Lett. 1988; 240: 163-166Crossref PubMed Scopus (8) Google Scholar, 31O'Flaherty J.T. Thomas M.J. Prostaglandins Leukotrienes Med. 1985; 17: 199-212Abstract Full Text PDF PubMed Scopus (21) Google Scholar). Before use, 5-[3H]HETE and [3H]LTB4 (∼250 Ci/mmol; NEN Life Science Products) were purified on reversed-phase HPLC (0.8 × 30 cm μ-Bondapak column; methanol:water:acetate, 750:250:0.1, v/v; 1 ml/min (24Richards C.F. Campbell W.B. Prostaglandins. 1989; 38: 565-580Crossref PubMed Scopus (12) Google Scholar, 31O'Flaherty J.T. Thomas M.J. Prostaglandins Leukotrienes Med. 1985; 17: 199-212Abstract Full Text PDF PubMed Scopus (21) Google Scholar)). To make 5-[3H]oxoETE, 5-[3H]HETE (600 pmol) was treated with 5 μmol of dichlorodicyanobenzoquinone in dry ethyl ether (25 μl) for 5 min at 20 °C. Reactions were stopped with 20 μl of isopropanol, reduced to 10 μl under a stream of N2, diluted with 0.5 ml of water, made pH 3 with HCl, and exhaustively extracted into hexane. Pooled extracts were evaporated to 5 μl, dissolved in 30 μl of isopropanol, and purified by normal-phase HPLC (0.8 × 30 cm μ-Porasil column, hexane:isopropanol:acetic acid, 984:16:1, v/v; 1 ml/min). 5-[3H]OxoETE and 5-[3H]HETE eluted at 8.5 and 30 min, respectively. Label eluting at ∼8.5 min was further purified on the reversed-phase HPLC system described above. We stress that our synthesis used reagent amounts and conditions that favor formation of the natural, as opposed to the 8–9-trans,isomer of 5-oxoETE. Almost exclusive production of natural 5-[3H]oxoETE was confirmed based on its elution at 30 min on reversed-phase HPLC (8–9-trans-5-[3H]oxoETE eluted at 32 min (9O'Flaherty J.T. Cordes J.F. Lee S.L. Samuel M. Thomas M.J. Biochim. Biophys. Acta. 1994; 1201: 505-515Crossref PubMed Scopus (37) Google Scholar)). The final product (30% recovery; 99% radiological purity) migrated with authentic 5-oxoETE on reversed- and normal-phase HPLC as well as TLC systems I and II (see below). The label was stored in elution solvent at 5 × 106 dpm/ml under argon at 4 °C. Stored in this way, it retained radiological purity for at least 12 weeks. Just before use, 5-[3H]HETE and 5-[3H]oxoETE were reduced to 10–30 μl volumes under a N2 stream, diluted in Hanks' buffer containing 2.5 mg/ml BSA, and added to reactions so that the final solvent and BSA levels were <0.1% and 250 μg/ml, respectively. Control studies treated PMNs with an equivalent amount of solvent and BSA. PMNs were isolated from normal donor blood (7O'Flaherty J.T. Jacobson D. Redman J. J. Immunol. 1988; 140: 4323-4328PubMed Google Scholar). Ca2+ transients were done on fura2-AM-loaded PMNs suspended in Hanks' buffer (+Ca2+, no MgCl2) (7O'Flaherty J.T. Jacobson D. Redman J. J. Immunol. 1988; 140: 4323-4328PubMed Google Scholar). Binding was done on PMNs (107) suspended in 1 ml of Hanks' buffer (37 °C) and treated with triacsin C or Me2SO for 30 min. Suspensions were placed on ice for 30 min, incubated (4 °C) with label for 0–120 min, layered on 0.4 ml of silicone oil, and centrifuged (12,000 × g for 1 min at 4 °C). Isolated supernatant fluids and pellets were counted for radioactivity (32O'Flaherty J.T. Redman J.F. Jacobson D.P. J. Cell. Physiol. 1990; 142: 299-308Crossref PubMed Scopus (12) Google Scholar). Results are given as the percentage of total recovered radioactivity in pellets. For membrane binding assays, plasma membranes (see below) were incubated (37 °C) with radiolabel in 250 μl of Hanks' buffer and passed through GF/C filters. Filters were washed with 5 ml of Hanks' buffer (no CaCl2 or MgCl2; 4 °C), air-dried, and counted for radioactivity (32O'Flaherty J.T. Redman J.F. Jacobson D.P. J. Cell. Physiol. 1990; 142: 299-308Crossref PubMed Scopus (12) Google Scholar). PMNs (107 in 1 ml of Hanks' buffer) were incubated at 37 °C with triacsin C or Me2SO and placed on ice for 30 min (for 4 °C experiments) or used directly (for 37 °C experiments). Suspensions were incubated (4 or 37 °C, respectively) with a radiolabel for 2.5–120 min and centrifuged (12,000 × g for 5 s at 4 °C). Cell pellets were twice washed with 1 ml of Hanks' buffer (4 °C; no CaCl2 or MgCl2) and then suspended in 0.5 ml of the same buffer. For plasmalemmal studies, plasma membranes were isolated from PMNs (see below), suspended in Hanks' buffer (37 °C), treated with triacsin C or Me2SO for 30 min, and incubated (37 °C) with label. Final washed PMN suspensions, pools of the supernatant, and washes of the original cell suspensions and membrane suspensions were twice extracted with an equal volume of chloroform:methanol, 2:1, v/v, containing enough HCl to make the final mixture pH 3–4. Pooled extracts were evaporated to dryness, taken up in 40 μl of chloroform:methanol (2:1, v/v), and applied to heat-activated (180 °C, 3 h) Silica Gel G TLC plates (Analtech, Newark, DE). Plates were developed to 15 cm in TLC system I (ethyl ether:hexane:acetic acid, 60:40:1, v/v) or II (ethyl ether:hexane:NH3(OH), 60:40:1, v/v) and scraped in 0.5-cm zones. Scrapings were suspended in 200 μl of methanol for 10 min and counted for radioactivity (18O'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. Biol. Chem. 1996; 271: 17821-17828Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). In selected experiments, extracts were evaporated to dryness and suspended in 500 μl of Tris (1m) buffer (pH 8.0) containing 500 ng of 5-oxoETE, 500 ng of rac 5-HETE, and 5 μg of triglyceride. Reactions were incubated (20 °C) with BSA or triglyceride lipase (Calbiochem, 50,000 units/mg, or Sigma, 60,000 units/mg) for 20 min and then extracted and examined by TLC. In other analyses, extracts of supernatant fluids were dried, taken up in 30 μl of methanol or isopropanol, and analyzed by reversed-phase or normal-phase HPLC, respectively. 2–4 × 108 PMNs in 5 ml of normal saline were treated with 2 mm diisopropyl fluorophosphate for 5 min at 4 °C. Cells were washed three times, taken up in 8 ml of relaxation buffer, subjected to N2-cavitation, and treated with 2 mm EDTA, 50 mm 2-mercaptoethanol, and 1 mmphenylmethylsulfonyl fluoride. Cavitates were freed of whole cells and nuclei by centrifugation and resolved on Percoll gradients (32O'Flaherty J.T. Redman J.F. Jacobson D.P. J. Cell. Physiol. 1990; 142: 299-308Crossref PubMed Scopus (12) Google Scholar). Fractions enriched with markers for plasma membrane ([3H]conconavalin A-labeled cell surface glycoproteins and cell surface alkaline phosphatase (18O'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. Biol. Chem. 1996; 271: 17821-17828Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar)) were pooled. Pools had 90% of the label. On TLC system I, cellular radioactivity migrated with triglyceride and to a small extent phospholipid (Fig. 1, top left panel), whereas medium radioactivity moved with 5,20-diHETE (Fig. 1, bottom left panel). In studies at 4 °C, in contrast, PMNs took up only ∼25% of label, and most of this migrated with triglyceride. Media label migrated with 5-HETE (Fig. 2,left panels). The pattern of these results was not limited to 80-min incubations. Analyses of suspensions incubated (37 or 4 °C) with label for 5, 10, 20, 40, or 60 min, as well as 80 min, by TLC, HPLC, and other systems revealed that PMNs contained glycerolipid-associated but virtually no intact 5-[3H]HETE (21O'Flaherty J.T. Wykle R.L. Redman J. Samuel M. Thomas M. J. Immunol. 1986; 137: 3277-3283PubMed Google Scholar). We next examined the effect of triacsin C with the expectation that this drug would block the charging of 5-HETE with CoA and thereby reduce its esterification while increasing its accumulation as intact, receptor-bound ligand. Triacsin C (≥ 1 μm) did in fact reduce esterification. PMNs treated with a 20 μm concentration of the drug had only a small amount of triglyceride-associated radioactivity during an 80-min incubation with 5-[3H]HETE at 37 or 4 °C (Figs. 1 and 2,top right panels). In 37 °C experiments, cells contained no detectable intact 5-[3H]HETE (Fig. 1, top right panel) while media contained radioactivity migrating with 5,20-diHETE and to a lesser extent 5-HETE in TLC system I (Fig. 1,bottom right panel), TLC system II (not shown), and HPLC (Fig. 3, top panel). In 4 °C experiments, cells had small amounts ( 70%), of radioactivity that moved with 5-HETE in TLC system I (Fig. 2, bottom right panel), TLC system II (data not shown), and reversed-phase HPLC (data not shown). Analysis of triacsin C-treated PMNs exposed to 5-[3H]HETE (4 or 37 °C) for 20 min gave results paralleling those shown in Figs. 1and 2. In particular, these PMNs never amassed more than 3% of total radioactivity as intact 5-[3H]HETE. We conclude that triacsin C blocks the esterification of 5-HETE into glycerolipids. It also leads to an increase in the oxidation of the fatty acid at 37 °C and promotes accumulation of intact 5-[3H]HETE with cells at 4 °C.Figure 2Effect of triacsin C on the metabolism of 5-HETE at 4 °C. PMN suspensions were treated with Me2SO (left panels) or 20 μmtriacsin C (right panels) for 30 min, placed on ice for 30 min, incubated with 100 pmol of 5-[3H]HETE at 4 °C for 80 min, and separated into pellet (top panels) and supernatant (bottom panels) fractions. Fractions were analyzed and compared with standards (numbered areas) as in Fig. 1. Results are typical of studies on cells from four different donorsView Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3HPLC analysis of the radioactivity in the supernatant fractions of PMNs incubated with 100 pm5-[3H]HETE (top panel) or 5-[3H]oxoETE (bottom panel). PMNs were treated with 100 μm triacsin C, incubated with the indicated label at 37 °C for 80 min, and processed as in Fig. 1. Extracts of supernatants were analyzed on reversed-phase HPLC and collected in 250-μl fractions. Results are percentages of recovered radioactivity per 250 μl of eluent and are typical of experiments on two donor cells. The numbered areas give the elution of 5,20-diHETE (1), 5-HETE (2), and 5-oxoETE (3). The first peak of radioactivity in each panel eluted with the exclusion volume of the column and represented at least in part material broken down during preparation.View Large Image Figure ViewerDownload Hi-res image Download (PPT) PMNs incubated with 100 pmol of 5-[3H]oxoETE, ± 20 μm triacsin C, for 80 min at 4 or 37 °C incorporated and processed the label in a fashion similar to their processing of 5-[3H]HETE. More particularly, cell label migrated with glycerolipids, media label migrated with 5,20-diHETE, and triacsin C reduced glycerolipid-co-migrating and increased 5,20-diHETE-co-migrating radioactivity (Figs. 4 and 5). However, the media from these incubations also had radioactivity that migrated with 5-HETE in TLC system I (Figs. 4 and 5, bottom panels), TLC system II (data not shown), reversed-phase HPLC (Fig. 3, bottom panel), and normal-phase HPLC (data not shown). Hence, PMNs readily reduce 5-[3H]oxoETE and might process the latter metabolite further. We accordingly examined the radioactivity migrating with lipids and 5,20-diHETE in greater detail. Cell extracts were digested with triglyceride lipase. The label recovered from these digests migrated with 5-HETE rather than 5-oxoETE on TLC (Fig. 6) and reversed-phase HPLC (not shown). Similarly, label in the media from these suspensions eluted on reversed-phase HPLC not only with 5-HETE but also with 5,20-diHETE (Fig. 3, bottom panel). We did not characterize the latter species because stud
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