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• W2021241846 abstract "Arachidonic acid is an essential constituent of cell membranes that is esterified to the sn-2-position of glycerophospholipids and is released from selected lipid pools by phospholipase cleavage. The released arachidonic acid can be metabolized by three enzymatic pathways: the cyclooxygenase pathway forming prostaglandins and thromboxanes, the lipoxygenase pathway generating leukotrienes and lipoxins, and the cytochrome P450 (cP450) pathway producing epoxyeicosatrienoic acids and hydroxyeicosatetraenoic acids. The present study describes a novel group of cP450 epoxygenase-dependent metabolites of arachidonic acid, termed 2-epoxyeicosatrienoylglycerols (2-EG), including two regioisomers, 2-(11,12-epoxyeicosatrienoyl)glycerol (2-11,12-EG) and 2-(14,15-epoxyeicosatrienoyl)glycerol (2-14,15-EG), which are both produced in the kidney and spleen, whereas 2-11,12-EG is also detected in the brain. Both 2-11,12-EG and 2-14,15-EG activated the two cannabinoid (CB) receptor subtypes, CB1 and CB2, with high affinity and elicited biological responses in cultured cells expressing CB receptors and in intact animals. In contrast, the parental arachidonic acid and epoxyeicosatrienoic acids failed to activate CB1 or CB2 receptors. Thus, these cP450 epoxygenase-dependent metabolites are a novel class of endogenously produced, biologically active lipid mediators with the characteristics of endocannabinoids. This is the first evidence of a cytochrome P450-dependent arachidonate metabolite that can activate G-protein-coupled cell membrane receptors and suggests a functional link between the cytochrome P450 enzyme system and the endocannabinoid system. Arachidonic acid is an essential constituent of cell membranes that is esterified to the sn-2-position of glycerophospholipids and is released from selected lipid pools by phospholipase cleavage. The released arachidonic acid can be metabolized by three enzymatic pathways: the cyclooxygenase pathway forming prostaglandins and thromboxanes, the lipoxygenase pathway generating leukotrienes and lipoxins, and the cytochrome P450 (cP450) pathway producing epoxyeicosatrienoic acids and hydroxyeicosatetraenoic acids. The present study describes a novel group of cP450 epoxygenase-dependent metabolites of arachidonic acid, termed 2-epoxyeicosatrienoylglycerols (2-EG), including two regioisomers, 2-(11,12-epoxyeicosatrienoyl)glycerol (2-11,12-EG) and 2-(14,15-epoxyeicosatrienoyl)glycerol (2-14,15-EG), which are both produced in the kidney and spleen, whereas 2-11,12-EG is also detected in the brain. Both 2-11,12-EG and 2-14,15-EG activated the two cannabinoid (CB) receptor subtypes, CB1 and CB2, with high affinity and elicited biological responses in cultured cells expressing CB receptors and in intact animals. In contrast, the parental arachidonic acid and epoxyeicosatrienoic acids failed to activate CB1 or CB2 receptors. Thus, these cP450 epoxygenase-dependent metabolites are a novel class of endogenously produced, biologically active lipid mediators with the characteristics of endocannabinoids. This is the first evidence of a cytochrome P450-dependent arachidonate metabolite that can activate G-protein-coupled cell membrane receptors and suggests a functional link between the cytochrome P450 enzyme system and the endocannabinoid system. When cells are stimulated by relevant growth factors or hormones, the essential constituent of cell membrane, arachidonic acid, is hydrolyzed from the sn-2-position of selected glycerophospholipids by activated phospholipases (1Needleman P. Turk J. Jakschik B.A. Morrison A.R. Lefkowith J.B. Annu. Rev. Biochem. 1986; 55: 69-102Crossref PubMed Google Scholar). We and others (2Capdevila J. Chacos N. Werringloer J. Prough R.A. Estabrook R.W. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 5362-5366Crossref PubMed Scopus (259) Google Scholar, 3Morrison A.R. Pascoe N. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7375-7378Crossref PubMed Scopus (114) Google Scholar, 4Oliw E.H. Lawson J.A. Brash A.R. Oates J.A. J. Biol. Chem. 1981; 256: 9924-9931Abstract Full Text PDF PubMed Google Scholar) have previously documented that metabolism of the released arachidonic acid by the cytochrome P450 enzyme system (cP450) 2The abbreviations used are:cP450cytochrome P450 enzyme systemEETepoxyeicosatrienoic acid2-EG2-epoxyeicosatrienoylglycerol2-11,12-EG2-(11,12-epoxyeicosatrienoyl)glycerol2-14,15-EG2-(14,15-epoxyeicosatrienoyl)glycerol11,12-EG11,12-epoxyeicosatrienoylglycerol14,15-EG14,15-epoxyeicosatrienoylglycerol2-AG2-arachidonylglycerolCBcannabinoidCB1 and -2cannabinoid receptor 1 and 2; respectivelyhCB1 and -2human CB1 and CB2, respectivelyERKextracellular signal-regulated kinaseESIelectrospray ionizationLCliquid chromatographyMSmass spectrometryHPLChigh pressure liquid chromatographyCHOChinese hamster ovarySRMselective reaction monitoringTICtotal ion current. 2The abbreviations used are:cP450cytochrome P450 enzyme systemEETepoxyeicosatrienoic acid2-EG2-epoxyeicosatrienoylglycerol2-11,12-EG2-(11,12-epoxyeicosatrienoyl)glycerol2-14,15-EG2-(14,15-epoxyeicosatrienoyl)glycerol11,12-EG11,12-epoxyeicosatrienoylglycerol14,15-EG14,15-epoxyeicosatrienoylglycerol2-AG2-arachidonylglycerolCBcannabinoidCB1 and -2cannabinoid receptor 1 and 2; respectivelyhCB1 and -2human CB1 and CB2, respectivelyERKextracellular signal-regulated kinaseESIelectrospray ionizationLCliquid chromatographyMSmass spectrometryHPLChigh pressure liquid chromatographyCHOChinese hamster ovarySRMselective reaction monitoringTICtotal ion current. produces biologically active lipid mediators, epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids. Subsequent studies revealed that EETs can serve as intracellular second messengers (5Graier W.F. Simecek S. Sturek M. J. Physiol. (Lond.). 1995; 482: 259-274Crossref Scopus (209) Google Scholar, 6Chen J.K. Wang D.W. Falck J.R. Capdevila J. Harris R.C. J. Biol. Chem. 1999; 274: 4764-4769Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), activate Ca2+ signaling (7Rzigalinski B.A. Willoughby K.A. Hoffman S.W. Falck J.R. Ellis E.F. J. Biol. Chem. 1999; 274: 175-182Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), and promote cell proliferation (8Chen J.K. Falck J.R. Reddy K.M. Capdevila J. Harris R.C. J. Biol. Chem. 1998; 273: 29254-29261Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 9Chen J.K. Capdevila J. Harris R.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6029-6034Crossref PubMed Scopus (82) Google Scholar). EETs also act as endothelium-derived hyperpolarizing factors by opening Ca2+-activated K+ channels (10Campbell W.B. Gebremedhin D. Pratt P.F. Harder D.R. Circ. Res. 1996; 78: 415-423Crossref PubMed Scopus (1100) Google Scholar, 11Fisslthaler B. Popp R. Kiss L. Potente M. Harder D.R. Fleming I. Busse R. Nature. 1999; 401: 493-497Crossref PubMed Scopus (799) Google Scholar) and play an anti-inflammatory role by inhibiting NF-κB-mediated vascular cell adhesion molecule 1 expression (12Node K. Huo Y. Ruan X. Yang B. Spiecker M. Ley K. Zeldin D.C. Liao J.K. Science. 1999; 285: 1276-1279Crossref PubMed Scopus (1000) Google Scholar). Recent studies indicate that EETs are downstream effectors of anandamide that activate TRPV4 (13Watanabe H. Vriens J. Prenen J. Droogmans G. Voets T. Nilius B. Nature. 2003; 424: 434-438Crossref PubMed Scopus (783) Google Scholar). Since cP450 arachidonate metabolites have been demonstrated to play important roles in regulating salt and fluid balance, vascular tone, systemic blood pressure, and local blood supply in vital organs such as the brain, heart, and kidney, the cP450-dependent metabolic pathway has emerged as a potential target for treatment of hypertension and ischemic organ damage (14McGiff J.C. Quilley J. Curr. Opin. Nephrol. Hypertens. 2001; 10: 231-237Crossref PubMed Scopus (86) Google Scholar, 15Pratt P.F. Medhora M. Harder D.R. Curr. Opin. Investig. Drugs. 2004; 5: 952-956PubMed Google Scholar, 16Sarkis A. Lopez B. Roman R.J. Curr. Opin. Nephrol. Hypertens. 2004; 13: 205-214Crossref PubMed Scopus (70) Google Scholar, 17Seubert J.M. Zeldin D.C. Nithipatikom K. Gross G.J. Prostaglandins Other Lipid Mediat. 2007; 82: 50-59Crossref PubMed Scopus (121) Google Scholar).Cell surface receptors that mediate the biological effects of the arachidonic acid metabolites derived from cyclooxygenase and lipoxygenase pathways have been well characterized (18Breyer R.M. Bagdassarian C.K. Myers S.A. Breyer M.D. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 661-690Crossref PubMed Scopus (849) Google Scholar, 19Brink C. Dahlen S.E. Drazen J. Evans J.F. Hay D.W. Nicosia S. Serhan C.N. Shimizu T. Yokomizo T. Pharmacol. Rev. 2003; 55: 195-227Crossref PubMed Scopus (261) Google Scholar). However, no study to date has identified any cP450-dependent arachidonate metabolites that exert their biological effects through cell membrane receptor-mediated mechanisms. In the present study, using ESI/LC/MS/MS analysis, we identified two novel cP450-dependent metabolites of arachidonic acid, termed 2-(11,12-epoxyeicosatrienoyl)glycerol (2-11,12-EG) and 2-(14,15-epoxyeicosatrienoyl)glycerol (2-14,15-EG), and determined that these compounds are potent cannabinoid receptor agonists. This report provides the first direct evidence that cP450-dependent arachidonate metabolites can exert their biological effects by directly binding to and activating cell surface receptors coupled to Gi/o proteins.EXPERIMENTAL PROCEDURESChemicals and Antibodies—Arachidonic acid was purchased from Nu Chek Prep (Elysian, MN). [3H]CP55940 (specific activity 168 Ci/mmol) was from PerkinElmer Life Sciences. 2-Arachidonylglycerol (2-AG), WIN55212-2, AM251, and AM630 were from Tocris Cookson Inc. (Ellisville, MO). A deuterated standard of 2-arachidonoylglycerol was from Cayman Chemical (Ann Arbor, MI). Wortmannin, LY294002, H89, MDL12,330A, and U73122 were purchased from EMD Biosciences/Calbiochem. Polyclonal antibodies to total and phospho-ERKs were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Other chemicals were from Sigma.Synthesis of EET, 2-EG, and ACU—11,12-EET and 14,15-EET were synthesized as we described previously (8Chen J.K. Falck J.R. Reddy K.M. Capdevila J. Harris R.C. J. Biol. Chem. 1998; 273: 29254-29261Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). 2-11,12-EG and 2-14,15-EG were synthesized as we have described recently (20Chen J. Chen J.K. Falck J.R. Guthi J.S. Anjaiah S. Capdevila J.H. Harris R.C. Mol. Cell. Biol. 2007; 27: 3023-3034Crossref PubMed Scopus (27) Google Scholar). 1-Adamantyl-3-cyclohexylurea was synthesized as described (21Hwang S.H. Tsai H.J. Liu J.Y. Morisseau C. Hammock B.D. J. Med. Chem. 2007; 50: 3825-3840Crossref PubMed Scopus (205) Google Scholar, 22Morisseau C. Goodrow M.H. Newman J.W. Wheelock C.E. Dowdy D.L. Hammock B.D. Biochem. Pharmacol. 2002; 63: 1599-1608Crossref PubMed Scopus (158) Google Scholar).Analysis of Endogenous 2-EG by ESI/LC/MS/MS Analysis—Synthetic mixtures of glycerol 1- and 2-positional isomers of 11,12- and 14,15-EG were injected onto a reversed phase HPLC column (Kromasil 100 C18, 3 μm, 150 × 2.1 mm) and eluted using a solvent gradient from initially 30% H2O, 70% CH3OH containing 25 μm AgBF4 to 100% CH3OH containing 25 μm AgBF4 in 10 min and at a flow of 0.2 ml/min. Column eluents were monitored by ESI/MS/MS using a TSQ Quantum 700 triple quadrupole mass spectrometer. Freshly isolated samples of rat spleen, kidney, or brain (1 g each) were homogenized in CHCl3/CH3OH (2:1) containing half a volume of aqueous 0.15 m KCl and a mixture of the 1- and 2-positional isomers of synthetic 13C3-labeled 11,12- and 14,15-EG (30 ng each) as internal standards. After low speed centrifugation, the organic phases were collected and evaporated under argon. The dry extracts were suspended in a minimal volume of 30% ethyl ether, 49.5% hexane, 0.5% HOAc and loaded onto a SiO2 column (0.5 × 5 cm), and the column was washed with the same solvent to remove free fatty acids and nonesterified eicosanoids. The sample EGs were then eluted using a mixture of 92.5% hexane, 7% EtOH, 0.5% HOAc and, after solvent evaporation, purified by reversed phase HPLC on a Dynamax C18 column (150 × 4.6 mm; 5 μm), using a liner solvent gradient from 49.9% H2O, 50% CHCN3, 0.1% HOAc to 99.9% CHCN3, 0.1% HOAc in 40 min at 1 ml/min. Fractions with the retention times of authentic 11,12- and 14,15-EG (between 19 and 21 min) were collected and analyzed by ESI/LC/MS/MS-selected ion monitoring of fragmentation product ions from the 13C3-labeled 11,12- and 14,15-EG at m/z 504.2 or M + 3, from the 11,12- and 14,15-EG of biological origin at m/z 501.2 as well as m/z 426.5–427.5, 302.5–303.5, and 342.5–343.5.To compare the levels of endogenous 2-11,12- and 2-14,15-EG with that of 2-AG in the fresh tissues, including kidney, spleen, and brain, both 2-EGs and 2-AG were measured in the same batch of lipid extract samples by ESI/LC/MS/MS analysis and calculated based on their internal standards, respectively.Cell Culture—Chinese hamster ovary (CHO) cells were cultured in F-12K medium. Human promyelocytic leukemia HL-60 cells were grown in RPMI 1640 medium and N18TG2 neuroblastoma cells in Dulbecco's modified Eagle's medium supplemented with 6-thioguanine, as described previously (23Merkler D.J. Chew G.H. Gee A.J. Merkler K.A. Sorondo J.P. Johnson M.E. Biochemistry. 2004; 43: 12667-12674Crossref PubMed Scopus (37) Google Scholar).cDNA Constructs and Transfection—Expression plasmids carrying the entire coding region of human cannabinoid receptor 1 (hCB1) were kindly provided by Dr. Tom Bonner (NIMH, National Institutes of Health, Bethesda, MD), whereas human CB2 expression cDNA construct (hCB2) was kindly provided by Dr. Ken Mackie (University of Washington, Seattle, WA). The hCB1 expression plasmids, hCB2 expression plasmids, or empty vector alone were transfected into CHO cells, respectively, using Lipofectamine (Invitrogen), as we described previously (6Chen J.K. Wang D.W. Falck J.R. Capdevila J. Harris R.C. J. Biol. Chem. 1999; 274: 4764-4769Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 24Chen J.K. Capdevila J. Harris R.C. J. Biol. Chem. 2000; 275: 13789-13792Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The cells were deprived of serum overnight before being used for signaling studies or isolation of plasma membranes for radioligand binding assays.Radioligand Binding Assay—Sprague-Dawley rat cerebellum, spleen, or CHO cells transfected with human CB1 or CB2 receptor were Dounce-homogenized in a freshly prepared, ice-cold buffer containing 50 mm Tris-HCl, pH 7.4, 3 mm MgCl2, 1 mm EDTA, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 2 μg/ml pepstatin A, 1 mm benzamidine, 1 mm phenylmethylsulfonyl fluoride, and the membranes were pelleted from the post-nuclear supernatant at 90,000 × g for 20 min, washed, and then resuspended in a freshly prepared, ice-cold binding buffer containing 50 mm Tris-HCl, pH 7.4, 3 mm MgCl2, 1 mm EDTA, 3 mm CaCl2, 1 mm phenylmethylsulfonyl fluoride, and 1 mm diisopropyl fluorophosphate. Binding of [3H]CP55940 (specific activity 168 Ci/mmol; PerkinElmer Life Sciences) to plasma membranes (50 μg of membrane protein/150-μl reaction) was performed essentially as described previously (25Munro S. Thomas K.L. Abu-Shaar M. Nature. 1993; 365: 61-65Crossref PubMed Scopus (4074) Google Scholar). Different concentrations of unlabeled CP55940, 2-EGs, EETs, arachidonic acid, 2-AG, AM251, or AM630 were used to conduct competition binding in the presence of 0.5 nm [3H]CP55940. Specific binding was determined by subtracting the nonspecific binding component measured in the presence of excess unlabeled CP55940 (1 μm). Competition binding data were analyzed using Prism 4.01 software (GraphPad) by entering Kd values of 2.3 and 0.608 nm as determined for [3H]CP55940 in cell membranes expressing either CB1 or CB2 receptors, respectively (26Pertwee R.G. Pharmacol. Ther. 1997; 74: 129-180Crossref PubMed Scopus (1282) Google Scholar).Immunoblotting—CHO cells transfected with human CB1 or CB2 receptor were treated with the indicated agents, and cell lysates were prepared and subjected to immunoblotting analysis with the indicated antibodies as described previously (6Chen J.K. Wang D.W. Falck J.R. Capdevila J. Harris R.C. J. Biol. Chem. 1999; 274: 4764-4769Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 8Chen J.K. Falck J.R. Reddy K.M. Capdevila J. Harris R.C. J. Biol. Chem. 1998; 273: 29254-29261Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 9Chen J.K. Capdevila J. Harris R.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6029-6034Crossref PubMed Scopus (82) Google Scholar, 24Chen J.K. Capdevila J. Harris R.C. J. Biol. Chem. 2000; 275: 13789-13792Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar).In Vivo Studies to Measure Cannabimimetic Effects of 2-EG—Male, 10-week-old C57BL/6J mice (Jackson) were used to examine the biological effects of 2-EG. Each animal was used only once in the sequential experiments. 2-EG, 2-AG, or vehicle alone was administered by tail-vein injection. Core temperature was determined by a thermistor probe (inserted 25 mm) and a telethermometer (Yellow Springs Instrument Co.); the difference in temperature before and 30 min after injection was calculated for each animal. The open field mobility experiments were performed at the Vanderbilt Murine Neurobehavioral Core Laboratory of the Center for Molecular Neuroscience, essentially as described previously (27Schramm N.L. McDonald M.P. Limbird L.E. J. Neurosci. 2001; 21: 4875-4882Crossref PubMed Google Scholar). Mice were placed individually in the center of each open field activity chamber equipped with three vectors of infrared photo beam emitters and detectors spaced 1.5 cm apart (Med Associates). Spontaneous locomotor activity was recorded as the total distance traveled in cm for each 5-min block of the continuous 30-min session.Vasodilatory Response of the Renal Glomerular Afferent Arteriole to 2-EG—Experiments were conducted using the in vitro perfused juxtamedullary nephron preparation as described (28Imig J.D. Navar L.G. Roman R.J. Reddy K.K. Falck J.R. J. Am. Soc. Nephrol. 1996; 7: 2364-2370PubMed Google Scholar). Briefly, the isolated rat kidney was perfused with a reconstituted red blood cell-containing solution, and renal artery perfusion pressure was set to 100 mm Hg. Effects of 2-11,12-EG or 2-14,15-EG on glomerular afferent arterioles preconstricted with 500 nm norepinephrine were determined with or without pretreatment by the CB receptor antagonists, AM251 or AM630 (each at 1 μm for 20 min), respectively. Data are presented as percentage relaxation, with 100% relaxation equal to the control diameter before the addition of norepinephrine (28Imig J.D. Navar L.G. Roman R.J. Reddy K.K. Falck J.R. J. Am. Soc. Nephrol. 1996; 7: 2364-2370PubMed Google Scholar).Bradykinin-increased 2-EG Production in Renal Microvessels—Renal microvessels were isolated as we described previously (29Imig J.D. Falck J.R. Wei S. Capdevila J.H. J. Vasc. Res. 2001; 38: 247-255Crossref PubMed Scopus (104) Google Scholar) and incubated at 37 °C in a vial containing Dulbecco's modified Eagle's medium solution gassed with 95% O2 and 5% CO2 for 30 min to determine 2-EG levels. In a subset of renal microvessel incubations, 1 μm bradykinin was included. Incubations were terminated by placing the samples on ice and adding 5 mg of triphenylphosphine. Samples were then snap frozen in liquid N2, and lipids were extracted and analyzed for 2-EG levels using LC/MS/MS.Cell Migration Assay—HL-60 cells were treated with 100 nm 1,25-(OH)2 vitamin D3 for 5 days to induce differentiation into macrophage-like cells. 1 × 106 differentiated cells suspended in 0.1 ml of RPMI 1640 medium containing 0.1% bovine serum albumin were plated into each Transwell™ insert (pore size, 5 μm) with 0.6 ml of the same cell-free medium in the lower compartment of 24-well culture plates (Corning Costar), followed by the addition of increasing concentrations of 2-11,12-EG, 2-14,15-EG, 11,12-EET, 14,15-EET, 2-AG, or vehicle (0.1% ethanol) alone to the lower compartment. After a 4-h treatment at 37 °C in the cell culture incubator (95% air and 5% CO2), cells that migrated from the upper compartment to the lower compartment were counted using a hemocytometer. For the experiments with inhibitors, the cells were pretreated with or without either AM251 or AM630 at 1 μm for 15 min or pertussis toxin (100 ng/ml) for 18 h before exposure to 1 μm 2-11,12-EG or 11,12-EET.Statistical Analysis—Data are presented as means ± S.E. for at least four separate experiments (each in triplicate). An unpaired Student's t test was used for statistical analysis, and analysis of variance and Bonferroni t tests were used for multiple group comparisons. A value of p < 0.05 in comparison was considered statistically significant.RESULTSCharacterization of the Mass Spectral Properties of Synthetic 2-EG—Our initial experiments characterized the mass spectral properties of synthetic 2-14,15-EG and 2-11,12-EG using Ag+ cationization and positive ion ESI/LC/MS/MS analysis. Since 2-acyl-glycerols isomerize spontaneously to mixtures of the corresponding 1- and 2-acyl-derivatives, the synthetic standards contain variable mixtures of the 1- and 2-positional isomers. As shown in Fig. 1 (A and B), collision-induced dissociation of the 107Ag adduct of synthetic 11,12- and 14,15-EG produced molecular ions at m/z 501.3 ([M + 107Ag]+) and yielded common ion fragments at m/z 427.2 (base peak, loss of glycerol) and 273. Regioisomer-specific EG fragments originating from transannular cleavage of the oxido functions are found at m/z 302.9 and 342.9, which are diagnostic for 11,12-EG (Fig. 1A) and 14,15-EG (Fig. 1B), respectively. A similar analysis using [13C3]glycerol-labeled 11,12- and 14,15-EG yielded the corresponding molecular ions at m/z 504.3 ([M + 107Ag] + 3) and the same common and diagnostic ion fragments observed with both the unlabeled standards (Fig. 1, A and B) and fragments generated after the loss of [13C3]glycerol from the m/z 504.3 molecular ion.Identification of Endogenous 2-EG in Fresh Tissues by ESI/LC/MS/MS Analysis—We then utilized collision-induced fragmentation of the m/z 501.3 ion ([M + 107Ag]+) and selective reaction monitoring (SRM) at m/z 342.5–343.5 (for 14,15-EG), 302.5–303.5 (for 11,12-EG), and 426.5–427.5 (common for both EG isoforms) to analyze an equimolar mixture of 11,12-EG and 14,15-EG (2 ng each). As shown by the HPLC elution profiles of the total ion current (TIC) and of the common ion at m/z 427.2, the mixture of synthetic 11,12- and 14,15-EG was partially resolved into three fractions eluting from the column between 8.6 and 9.8 min and containing the 1- and 2-glycerol positional isomers of 14,15-EG, followed by those derived from 11,12-EG (Fig. 2A). By monitoring the elution profiles of the respective diagnostic ion fragments, 11,12-EG was able to be selectively detected in the presence of 14,15-EG, and vice versa (Fig. 2A). These results confirmed the validity of the diagnostic ESI/LC/MS/MS SRM method for the analysis of mixtures of these EGs in biological samples. SRM monitoring of EG diagnostic ions derived from the collision-induced fragmentation of the 13C3-labeled (internal standard; m/z 504.3) and unlabeled (biological; m/z 501.3) 11,12- and 14,15-EG and comparisons of HPLC retention times and diagnostic ion intensities at m/z 342.5–343.5 and 302.5–303.5 provided evidence for the presence of mixtures of the glycerol 1- and 2-positional isomers of these EGs in samples extracted from rat spleen (Fig. 2B) and kidney (Fig. 2C). By comparison of diagnostic ion intensities for the labeled and nonlabeled 2-EGs, we detected 750 and 1500 pg of 2-EGs (320 and 700 pg of 11,12-EG and 430 and 800 pg of 14,15-EG)/g of rat kidney and spleen tissues, respectively (Fig. 2D). In contrast, only 11,12-EG was detected in rat brain (210 pg/g of tissue) (Fig. 2D). The presence of endogenous 2-EG in rat kidney, spleen, and brain was confirmed in additional experiments in which the EG pools were extracted and purified by reversed HPLC and then submitted to hydrolysis in methanolic KOH (30Capdevila J.H. Dishman E. Karara A. Falck J.R. Methods Enzymol. 1991; 206: 441-453Crossref PubMed Scopus (72) Google Scholar). The resulting products were then converted to the corresponding pentafluorobenzyl esters by reaction with pentafluorobenzyl bromide and characterized as EETs using NICI/GC/MS (data not shown) (30Capdevila J.H. Dishman E. Karara A. Falck J.R. Methods Enzymol. 1991; 206: 441-453Crossref PubMed Scopus (72) Google Scholar).FIGURE 2Identification of endogenous 2-EG in fresh spleen and kidney tissues by ESI/LC/MS/MS analysis. A, HPLC elution profile of product ions derived from the collision induced fragmentation of mixtures of synthetic 11,12- and 14,15-EG. Shown are the ion products derived from the fragmentation of the EG molecular ions at m/z 501.3 ([M + 107Ag]+) and detected by SRM at m/z 426.5–427.5 (for 11,12- and 14,15-EG), 302.5–303.5 (for 11,12-EG), and 342.5–343.5 (for 14,15-EG). TIC, total ion current. B and C, chromatograms of endogenous 11,12- and 14,15-EG detection in fresh tissues of spleen (B) and kidney (C). SRM-HPLC chromatograms of the EG-derived ions fragmented from the 107Ag adduct of the biological, unlabeled EGs at m/z 501.3 (B2, C2 and B3, C3) and the 13C3-labeled EG internal standard at m/z 504.3 (B4, C4 and B5, C5). The HPLC elution profiles of the TIC and of the common ion at m/z 427.2 are also shown (B1, C1 and B6, C6), respectively; shown are representative data from four separate experiments with similar results. D, levels of endogenous 11,12- and 14,15-EG present in rat kidney, spleen, and brain quantified from the ratios of intensities of the diagnostic ion fragments originated for the internal standard and the biological EG isomers (n = 3–6).View Large Image Figure ViewerDownload Hi-res image Download (PPT)To compare the endogenous levels of 2-EGs with that of 2-AG, we measured the levels of 2-AG in the same tissues using the same methodology side by side. By comparison with the internal 2-AG standard, we detected 7.41, 11.42, and 5.21 ng of 2-AG in each gram of fresh kidney, spleen, and brain tissues, respectively (the values are mean of n = 3 animals).2-EG but Not EET Bound both CB1 and CB2 Cannabinoid Receptors with High Affinity—After confirmation of the existence of endogenous 2-EG in multiple tissues, we attempted to identify potential receptors for these cP450-dependent arachidonic acid metabolites. Radioligand binding experiments revealed that both 2-11,12-EG and 2-14,15-EG dose-dependently competed with [3H]CP55940 for binding to cerebellar plasma membranes (Fig. 3A), which express high levels of CB1 receptors (31Matsuda L.A. Lolait S.J. Brownstein M.J. Young A.C. Bonner T.I. Nature. 1990; 346: 561-564Crossref PubMed Scopus (4160) Google Scholar), with a Ki value of 23 ± 3 and 40 ± 5 nm, respectively. [3H]CP55940 is a well characterized synthetic ligand specific for both CB1 and CB2 cannabinoid receptor subtypes with equally high affinity (26Pertwee R.G. Pharmacol. Ther. 1997; 74: 129-180Crossref PubMed Scopus (1282) Google Scholar, 32Thomas B.F. Gilliam A.F. Burch D.F. Roche M.J. Seltzman H.H. J Pharmacol. Exp. Ther. 1998; 285: 285-292PubMed Google Scholar). Since the spleen is the organ that expresses the highest levels of CB2 receptor (25Munro S. Thomas K.L. Abu-Shaar M. Nature. 1993; 365: 61-65Crossref PubMed Scopus (4074) Google Scholar), we also isolated plasma membranes from rat spleen and performed competition binding assays. As shown in Fig. 3B, both 2-EG isoforms also competed with [3H]CP55940 for binding to spleen plasma membranes, with a Ki of 75 ± 6 nm for 2-11,12-EG and 138 ± 9 nm for 2-14,15-EG. As indicated in Table 1, both 2-11,12-EG and 2-14,15-EG bind CB1 and CB2 receptors with an affinity comparable with 2-AG, a well established endogenous ligand for both CB1 and CB2 receptors (33Mechoulam R. Ben-Shabat S. Hanus L. Ligumsky M. Kaminski N.E. Schatz A.R. Gopher A. Almog S. Martin B.R. Compton D.R. Pertweee R.G. Griffine G. Bayewitchf M. Bargf J. Vogelf Z. Biochem. Pharmacol. 1995; 50: 83-90Crossref PubMed Scopus (2315) Google Scholar, 34Sugiura T. Kondo S. Sukagawa A. Nakane S. Shinoda A. Itoh K. Yamashita A. Waku K. Biochem. Biophys. Res. Commun. 1995; 215: 89-97Crossref PubMed Scopus (1795) Google Scholar, 35Sugiura T. Kondo S. Kishimoto S. Miyashita T. Nakane S. Kodaka T. Suhara Y. Takayama H. Waku K. J. Biol. Chem. 2000; 275: 605-612Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 36Sugiura T. Kishimoto S. Oka S. Gokoh M. Prog. Lipid Res. 2006; 45: 405-446Crossref PubMed Scopus (279) Google Scholar, 37Di Marzo V. Petrosino S. Curr. Opin. Lipidol. 2007; 18: 129-140Crossref PubMed Scopus (255) Google Scholar). In contrast, neither arachidonic acid nor EETs competed with [3H]CP55940 for binding to CB1 receptors (Fig. 3C) or CB2 receptors (Fig. 3D). Included as positive controls, the CB1-selective antagonist AM251 and the CB2-selective antagonist AM630, each at 1 μm, almost completely blocked 0.5 nm [3H]CP55940 binding to CB1 receptors (Fig. 3C) and CB2 receptors (Fig. 3D), respectively. Thus, these new cP450-dependent arachidonate metabolites are endogenous ligands for both CB1 and CB2 receptors.FIGURE 32-EG but not EET bound both CB1 and CB2 receptors with high affinity. A and B, both 2-11,12-EG and 2-14,15-EG competed with [3H]CP55940 (0.5 nm) for binding to rat cerebellar membranes, which express high levels of CB1 (A), and rat spleen membranes, which express high levels of CB2 (B). C an" @default.
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• W2021241846 date "2008-09-01" @default.
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• W2021241846 title "Identification of Novel Endogenous Cytochrome P450 Arachidonate Metabolites with High Affinity for Cannabinoid Receptors" @default.
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