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Epoxygenase Pathways of Arachidonic Acid Metabolism

2001; Elsevier BV; Volume: 276; Issue: 39 Linguagem: Inglês

10.1074/jbc.r100030200

1083-351X

Darryl C. Zeldin,

Inflammatory mediators and NSAID effects

arachidonic acid cis-epoxyeicosatrienoic acid dihydroxyeicosatrienoic acid hydroxyeicosatetraenoic acid soluble epoxide hydrolase prostaglandin H synthase lipoxygenase tetrahydrofuran phospholipid fatty acid-binding protein endothelial derived hyperpolarizing factor tissue plasminogen activator Arachidonic acid (AA)1 is present in vivo esterified to cell membrane glycerophospholipids. Activation of phospholipases (e.g. cytosolic phospholipase A2) releases free AA from the phospholipid (PL) pools and makes it available for oxidative metabolism by several enzyme systems (Fig. 1). The prostaglandin endoperoxide H synthases (PGHSs) metabolize AA to PGH2, which serves as the precursor of the prostaglandins, thromboxane and prostacyclin (1Smith W.L. Garavito R.M. DeWitt D.L. J. Biol. Chem. 1996; 271: 33157-33160Abstract Full Text Full Text PDF PubMed Scopus (1860) Google Scholar). The lipoxygenases (LOXs) convert AA to labile hydroperoxy intermediates that go on to form the leukotrienes, hydroxyeicosatetraenoic acids (HETEs) and lipoxins (2Brash A.R. J. Biol. Chem. 1999; 274: 23679-23682Abstract Full Text Full Text PDF PubMed Scopus (1155) Google Scholar). The PGHSs and LOXs have been extensively studied, and their eicosanoid products have been shown to play important functional roles in a variety of fundamental biological processes such as inflammation, cellular proliferation, and intracellular signaling (1Smith W.L. Garavito R.M. DeWitt D.L. J. Biol. Chem. 1996; 271: 33157-33160Abstract Full Text Full Text PDF PubMed Scopus (1860) Google Scholar, 2Brash A.R. J. Biol. Chem. 1999; 274: 23679-23682Abstract Full Text Full Text PDF PubMed Scopus (1155) Google Scholar). In contrast, less is known about the “third pathway” of AA metabolism wherein multiple cytochromes P450 metabolize AA to three types of eicosanoid products (3Capdevila J.H. Falck J.R. Harris R.C. J. Lipid Res. 2000; 41: 163-181Abstract Full Text Full Text PDF PubMed Google Scholar, 4Oliw E.H. Prog. Lipid Res. 1994; 33: 329-354Crossref PubMed Scopus (139) Google Scholar, 5Makita K. Falck J.R. Capdevila J.H. FASEB J. 1996; 10: 1456-1463Crossref PubMed Scopus (120) Google Scholar, 6McGiff J.C. Quilley J. Curr. Opin. Nephrol. Hypertens. 2001; 10: 231-237Crossref PubMed Scopus (86) Google Scholar). Allylic oxidation forms several midchain conjugated dienols (5-, 8-, 9-, 11-, 12-, and 15-HETEs). ω-terminal hydroxylation forms C16–C20 alcohols of AA (16-, 17-, 18-, 19-, and 20-HETEs). Olefin epoxidation (also called the epoxygenase reaction) results in the production of fourcis-epoxyeicosatrienoic acids (14,15-, 11,12-, 8,9-, and 5,6-EETs), each of which can be formed as either the R,S or the S,R enantiomer (Fig. 2). Studies have demonstrated that P450 epoxygenase-derived eicosanoids have a multitude of potent biological activities. For example, EETs have been shown to have potent effects on peptide hormone secretion (7Cashman J.R. Hanks D. Weiner R.I. Neuroendocrinology. 1987; 46: 246-251Crossref PubMed Scopus (48) Google Scholar, 8Falck J.R. Manna S. Moltz J. Chacos N. Capdevila J. Biochem. Biophys. Res. Commun. 1983; 114: 743-749Crossref PubMed Scopus (147) Google Scholar, 9Snyder G.D. Yadagiri P. Falck J.R. Am. J. Physiol. 1989; 256: E221-E226PubMed Google Scholar), vascular and bronchial smooth muscle tone (10Campbell W.B. Gebremedhin D. Pratt P.F. Harder D.R. Circ. Res. 1996; 78: 415-423Crossref PubMed Scopus (1115) Google Scholar, 11Proctor K.G. Falck J.R. Capdevila J. Circ. Res. 1987; 60: 50-59Crossref PubMed Scopus (153) Google Scholar, 12Zou A.P. Fleming J.T. Falck J.R. Jacobs E.R. Gebremedhin D. Harder D.R. Roman R.J. Am. J. Physiol. 1996; 270: F822-F832PubMed Google Scholar, 13Zhu D. Bousamra M. Zeldin D.C. Falck J.R. Townsley M. Harder D.R. Roman R.J. Jacobs E.R. Am. J. 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Physiol. 1990; 144: 429-437Crossref PubMed Scopus (92) Google Scholar, 19Chen 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 (123) Google Scholar, 20Sheu H.L. Omata K. Utsumi Y. Tsutsumi E. Sato T. Shimizu T. Abe K. Adv. Prostaglandin Thromboxane Leukotriene Res. 1995; 23: 211-213PubMed Google Scholar), inflammation (21Node K. Huo Y. Ruan X. Yang B. Spiecker M. Ley K. Zeldin D.C. Liao J.K. Science. 1999; 285: 1276-1279Crossref PubMed Scopus (1024) Google Scholar), hemostasis (22Node K. Ruan X.L. Dai J. Yang S.X. Graham L. Zeldin D.C. Liao J.K. J. Biol. Chem. 2001; 276: 15983-15989Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar), and a variety of intracellular signaling pathways (19Chen 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 (123) Google Scholar, 23Burns K.D. Capdevila J. Wei S. Breyer M.D. Homma T. Harris R.C. Am. J. Physiol. 1995; 269: C831-C840Crossref PubMed Google Scholar, 24Chen J.K. Capdevila J. Harris R.C. J. Biol. Chem. 2000; 275: 13789-13792Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 25Dulin N.O. Alexander L.D. Harwalkar S. Falck J.R. Douglas J.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8098-8102Crossref PubMed Scopus (77) Google Scholar, 26Hoebel B.G. Graier W.F. Eur. J. Pharmacol. 1998; 346: 115-117Crossref PubMed Scopus (45) Google Scholar, 27Rzigalinski 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). This minireview will provide a general overview of the AA epoxygenase pathway with emphasis on the P450 enzymes involved in EET biosynthesis, the metabolic fate of the EETs once they are formed, and the biological relevance of the EETs in the kidney and cardiovascular system.Figure 2EET stereoisomers. Each of the four EET regioisomers can be biosynthesized as either the R,S or theS,R enantiomer.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Studies using purified and/or recombinant P450 enzymes have demonstrated that multiple P450s can metabolize AA to EETs, albeit with different catalytic efficiencies. Thus, members of the mammalian CYP1A, CYP2B, CYP2C, CYP2D, CYP2G, CYP2J, CYP2N, and CYP4A subfamilies have been shown to be capable of EET biosynthesis in vitro(28Rifkind A.B. Lee C. Chang T.K. Waxman D.J. Arch. Biochem. Biophys. 1995; 320: 380-389Crossref PubMed Scopus (219) Google Scholar, 29Keeney D.S. Skinner C. Wei S. Friedberg T. Waterman M.R. J. Biol. Chem. 1998; 273: 9279-9284Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 30Zeldin D.C. DuBois R.N. Falck J.R. Capdevila J.H. Arch. Biochem. Biophys. 1995; 322: 76-86Crossref PubMed Scopus (156) Google Scholar, 31Thompson C.M. Capdevila J.H. Strobel H.W. J. Pharmacol. Exp. Ther. 2000; 294: 1120-1130PubMed Google Scholar, 32Oleksiak M.F. Wu S. Parker C. Karchner S.I. Stegeman J.J. Zeldin D.C. J. Biol. Chem. 2000; 275: 2312-2321Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 33Nguyen X. Wang M.H. Reddy K.M. Falck J.R. Schwartzman M.L. Am. J. Physiol. 1999; 276: R1691-R1700PubMed Google Scholar, 34Wu S. Moomaw C.R. Tomer K.B. Falck J.R. Zeldin D.C. J. Biol. Chem. 1996; 271: 3460-3468Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar, 35Capdevila J.H. Karara A. Waxman D.J. Martin M.V. Falck J.R. Guengerich F.P. J. Biol. Chem. 1990; 265: 10865-10871Abstract Full Text PDF PubMed Google Scholar, 36Laethem R.M. Laethem C.L. Ding X. Koop D.R. J. Pharmacol. Exp. Ther. 1992; 262: 433-438PubMed Google Scholar). In general, the regioselectivity of EET formation is P450 isoform-specific. For example, CYP2C8 forms 14,15-EET and 11,12-EET in a ratio of 1.3:1.0 but does not produce significant amounts of 8,9-EET. In contrast, CYP2C9, which is >80% identical to CYP2C8, produces 14,15-EET, 11,12-EET, and 8,9-EET in a ratio of 2.3:1.0:0.5 (30Zeldin D.C. DuBois R.N. Falck J.R. Capdevila J.H. Arch. Biochem. Biophys. 1995; 322: 76-86Crossref PubMed Scopus (156) Google Scholar, 37Daikh B.E. Lasker J.M. Raucy J.L. Koop D.R. J. Pharmacol. Exp. Ther. 1994; 271: 1427-1433PubMed Google Scholar). The stereoselectivity of EET biosynthesis also depends on the P450 isoform involved in catalysis. Thus, CYP2C8 produces (14R,15S)-EET and (11R,12S)-EET with optical purities of 86 and 81%, respectively. In contrast, CYP2C9 produces (14R,15S)-EET and (11S,12R)-EETs with significantly lower optical purities (63 and 69%, respectively) (30Zeldin D.C. DuBois R.N. Falck J.R. Capdevila J.H. Arch. Biochem. Biophys. 1995; 322: 76-86Crossref PubMed Scopus (156) Google Scholar, 37Daikh B.E. Lasker J.M. Raucy J.L. Koop D.R. J. Pharmacol. Exp. Ther. 1994; 271: 1427-1433PubMed Google Scholar). Multiple P450s may contribute to EET biosynthesis in a given tissue or cell type. Moreover, the contribution of an individual P450 isoform will depend on both its organ/cellular abundance and its catalytic efficiency. For example, in human and rat liver, CYP2C isoforms are highly expressed and likely contribute significantly to EET biosynthesis. Indeed, polyclonal antibodies to CYP2C isoforms inhibit >70% of hepatic microsomal AA epoxygenase activity (37Daikh B.E. Lasker J.M. Raucy J.L. Koop D.R. J. Pharmacol. Exp. Ther. 1994; 271: 1427-1433PubMed Google Scholar, 38Zeldin D.C. Moomaw C.R. Jesse N. Tomer K.B. Beetham J. Hammock B.D. Wu S. Arch. Biochem. Biophys. 1996; 330: 87-96Crossref PubMed Scopus (129) Google Scholar, 39Capdevila J. Zeldin D.C. Karara A. Falck J.F. Adv. Mol. Cell Biol. 1996; 14: 317-339Crossref Scopus (9) Google Scholar). Similarly, in human and rat kidney, CYP2C isoforms are responsible for the majority of EET biosynthesis (30Zeldin D.C. DuBois R.N. Falck J.R. Capdevila J.H. Arch. Biochem. Biophys. 1995; 322: 76-86Crossref PubMed Scopus (156) Google Scholar, 40Holla V.R. Makita K. Zaphiropoulos P.G. Capdevila J.H. J. Clin. Invest. 1999; 104: 751-760Crossref PubMed Scopus (110) Google Scholar). In contrast, in human and rat heart, CYP2J isoforms are particularly abundant and have been proposed to be the predominant enzymes responsible for epoxidation of endogenous AA pools (34Wu S. Moomaw C.R. Tomer K.B. Falck J.R. Zeldin D.C. J. Biol. Chem. 1996; 271: 3460-3468Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar, 41Wu S. Chen W. Murphy E. Gabel S. Tomer K.B. Foley J. Steenbergen C. Falck J.R. Moomaw C.R. Zeldin D.C. J. Biol. Chem. 1997; 272: 12551-12559Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). Both CYP2J and CYP2C isoforms appear to contribute to EET biosynthesis in vascular endothelial cells, although their relative contribution remains unknown (21Node K. Huo Y. Ruan X. Yang B. Spiecker M. Ley K. Zeldin D.C. Liao J.K. Science. 1999; 285: 1276-1279Crossref PubMed Scopus (1024) Google Scholar, 42Fisslthaler B. Popp R. Kiss L. Potente M. Harder D.R. Fleming I. Busse R. Nature. 1999; 401: 493-497Crossref PubMed Scopus (809) Google Scholar, 43Campbell W.B. Trends Pharmacol. Sci. 2000; 21: 125-127Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 44Zeldin D.C. Liao J.K. Trends Pharmacol. Sci. 2000; 21: 127-128Abstract Full Text Full Text PDF PubMed Google Scholar). In most tissues, however, the relative contribution of different P450s to EET biosynthesis remains unknown due to the absence of P450 isoform-specific chemical inhibitors and inhibitory antibodies. EET biosynthesis can be altered by factors that affect P450 expression and/or activity. Treatment of rats with phenobarbital induces CYP2B subfamily P450s leading to increased hepatic AA epoxygenase activity and altered regio- and stereochemical selectivity of rat liver EETs (35Capdevila J.H. Karara A. Waxman D.J. Martin M.V. Falck J.R. Guengerich F.P. J. Biol. Chem. 1990; 265: 10865-10871Abstract Full Text PDF PubMed Google Scholar, 45Karara A. Dishman E. Blair I. Falck J.R. Capdevila J.H. J. Biol. Chem. 1989; 264: 19822-19827Abstract Full Text PDF PubMed Google Scholar). In contrast, treatment of rats with the aromatic hydrocarbon β-naphthoflavone results in reduced hepatic AA epoxygenase activity and a concomitant increase in the formation of ω-terminal HETEs (35Capdevila J.H. Karara A. Waxman D.J. Martin M.V. Falck J.R. Guengerich F.P. J. Biol. Chem. 1990; 265: 10865-10871Abstract Full Text PDF PubMed Google Scholar). The effects of aromatic hydrocarbons on P450 epoxygenase activity are species-dependent. Treatment of fish and avian species with benzo(a)pyrene or 2,3,7,8-tetrachlorodibenzo-p-dioxin increases hepatic EET biosynthesis, likely by inducing CYP1A subfamily P450s (46Schlezinger J.J. Parker C. Zeldin D.C. Stegeman J.J. Arch. Biochem. Biophys. 1998; 353: 265-275Crossref PubMed Scopus (34) Google Scholar, 47Gilday D. Bellward G.D. Sanderson J.T. Janz D.M. Rifkind A.B. Toxicol. Appl. Pharmacol. 1998; 150: 106-116Crossref PubMed Scopus (31) Google Scholar). Nutritional factors can modulate P450-dependent AA metabolism. Fasting leads to reduced hepatic CYP2C11 expression and decreased epoxygenase activity in rats (48Qu W. Rippe R.A. Ma J. Scarborough P. Biagini C. Fiedorek F.T. Travlos G.S. Parker C. Zeldin D.C. Mol. Pharmacol. 1998; 54: 504-513Crossref PubMed Scopus (35) Google Scholar). EET biosynthesis is increased in vascular tissue isolated from rabbits fed a cholesterol-rich diet compared with control rabbits suggesting that dietary cholesterol alters P450 AA metabolism (49Pfister S.L. Falck J.R. Campbell W.B. Am. J. Physiol. 1991; 261: H843-H852PubMed Google Scholar). Dietary salt has been shown to induce rat kidney CYP2C23 resulting in increased renal EET biosynthesis and enhanced urinary secretion of epoxygenase metabolites (40Holla V.R. Makita K. Zaphiropoulos P.G. Capdevila J.H. J. Clin. Invest. 1999; 104: 751-760Crossref PubMed Scopus (110) Google Scholar, 50Capdevila J.H. Wei S. Yan J. Karara A. Jacobson H.R. Falck J.R. Guengerich F.P. DuBois R.N. J. Biol. Chem. 1992; 267: 21720-21726Abstract Full Text PDF PubMed Google Scholar, 51Oyekan A.O. Youseff T. Fulton D. Quilley J. McGiff J.C. J. Clin. Invest. 1999; 104: 1131-1137Crossref PubMed Scopus (102) Google Scholar). Genetic variation in human P450 genes is well described; however, little is known about the effect of P450 genetic polymorphism on AA metabolic pathways. A coding polymorphism in the humanCYP2C8 gene (CYP2C8*3), which includes both R139K and K399R amino acid substitutions, has been shown to result in significantly reduced AA epoxygenase activity (52Dai D. Zeldin D.C. Blaisdell J.A. Chanas B. Coulter S.J. Ghanayem B.I. Goldstein J.A. Pharmacogenetics. 2001; 11: 1-11Crossref PubMed Scopus (441) Google Scholar). We have recently identified several polymorphisms within the CYP2J2gene that result in nonconservative amino acid substitutions that affect P450 enzyme function. 2L. King and D. C. Zeldin, unpublished data. It is not yet known whether the frequency of these polymorphisms is altered in diseased patients. Once formed, the EETs can be further metabolized along a number of pathways as illustrated in Fig. 3. Capdevila and co-workers (53Capdevila J.H. Kishore V. Dishman E. Blair I.A. Falck J.R. Biochem. Biophys. Res. Commun. 1987; 146: 638-644Crossref PubMed Scopus (36) Google Scholar, 54Karara A. Dishman E. Falck J.R. Capdevila J.H. J. Biol. Chem. 1991; 266: 7561-7569Abstract Full Text PDF PubMed Google Scholar) have documented the presence of glycerophospholipids that contain an epoxyeicosatrienoate moiety esterified to the glycerol-sn-2 position. These novel EET-PLs are formed in vivo by a multienzyme process initiated by P450 epoxidation of AA followed by ATP-dependent activation to epoxyeicosatrienoyl-CoA derivatives and finally regio- and stereoselective lysolipid acylation (54Karara A. Dishman E. Falck J.R. Capdevila J.H. J. Biol. Chem. 1991; 266: 7561-7569Abstract Full Text PDF PubMed Google Scholar). In fact, the majority (>85%) of endogenous EETs present in mammalian tissues are esterified to cellular glycerophospholipids. Importantly, it has recently been shown that 1-palmitoyl-2-epoxyeicosatrienoyl phosphatidylcholine significantly inhibits the activity of porcine l-type Ca2+channels reconstituted into planar lipid bilayers, suggesting that these EET-PLs may be involved in the regulation of membrane ion permeabilities (55Chen J. Capdevila J.H. Zeldin D.C. Rosenberg R.L. Mol. Pharmacol. 1999; 55: 288-295Crossref PubMed Scopus (90) Google Scholar). EETs can be hydrated to their correspondingvic-dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH) (56Chacos N. Capdevila J. Falck J.R. Manna S. Martin-Wixtrom C. Gill S.S. Hammock B.D. Estabrook R.W. Arch. Biochem. Biophys. 1983; 223: 639-648Crossref PubMed Scopus (171) Google Scholar, 57Zeldin D.C. Kobayashi J. Falck J.R. Winder B.S. Hammock B.D. Snapper J.R. Capdevila J.H. J. Biol. Chem. 1993; 268: 6402-6407Abstract Full Text PDF PubMed Google Scholar, 58Zeldin D.C. Wei S. Falck J.R. Hammock B.D. Snapper J.R. Capdevila J.H. Arch. Biochem. Biophys. 1995; 316: 443-451Crossref PubMed Scopus (119) Google Scholar, 59Moghaddam M. Motoba K. Borhan B. Pinot F. Hammock B.D. Biochim. Biophys. Acta. 1996; 1290: 327-339Crossref PubMed Scopus (72) Google Scholar). The microsomal epoxide hydrolase can also metabolize EETs, albeit at significantly lower rates. The documentation of DHETs as endogenous constituents of liver, lung, and urine confirms that EET hydration occurs in vivo (15Zeldin D.C. Plitman J.D. Kobayashi J. Miller R.F. Snapper J.R. Falck J.R. Szarek J.L. Philpot R.M. Capdevila J.H. J. Clin. Invest. 1995; 95: 2150-2160Crossref PubMed Scopus (105) Google Scholar, 38Zeldin D.C. Moomaw C.R. Jesse N. Tomer K.B. Beetham J. Hammock B.D. Wu S. Arch. Biochem. Biophys. 1996; 330: 87-96Crossref PubMed Scopus (129) Google Scholar, 60Catella F. Lawson J. Braden G. Fitzgerald D.J. Shipp E. FitzGerald G.A. Adv. Prostaglandin Thromboxane Leukotriene Res. 1991; 21A: 193-196PubMed Google Scholar). The metabolism of EETs by sEH is highly regioselective with 14,15-EET being the preferred substrate. The hydration of 11,12-EET and 8,9-EET by sEH proceeds at significantly lower rates, and 5,6-EET is a very poor substrate for this enzyme (57Zeldin D.C. Kobayashi J. Falck J.R. Winder B.S. Hammock B.D. Snapper J.R. Capdevila J.H. J. Biol. Chem. 1993; 268: 6402-6407Abstract Full Text PDF PubMed Google Scholar). The metabolism of EETs by sEH is also highly stereoselective for the (14R,15S)-EET, (11S,12R)-EET, and (8S,9R)-EET enantiomers (57Zeldin D.C. Kobayashi J. Falck J.R. Winder B.S. Hammock B.D. Snapper J.R. Capdevila J.H. J. Biol. Chem. 1993; 268: 6402-6407Abstract Full Text PDF PubMed Google Scholar). Because DHETs are incorporated into PLs to a much lesser extent than EETs, Weintraubet al. (61Weintraub N.L. Fang X. Kaduce T.L. VanRollins M. Chatterjee P. Spector A.A. Am. J. Physiol. 1999; 277: H2098-H2108PubMed Google Scholar) have postulated that sEH functionally regulates this process. Whereas the rapid conversion of EETs to their corresponding diols has generally been viewed as a process whereby EETs are rendered biologically inactive, DHETs have been shown to be vasoactive in the coronary circulation and are inhibitors of the hydroosmotic effect of arginine vasopressin in the kidney (62Fang X. Kaduce T.L. Weintraub N.L. VanRollins M. Spector A.A. Circ. Res. 1996; 79: 784-793Crossref PubMed Scopus (90) Google Scholar, 63Hirt D.L. Capdevila J. Falck J.R. Breyer M.D. Jacobson H.R. J. Clin. Invest. 1989; 84: 1805-1812Crossref PubMed Scopus (61) Google Scholar, 64Oltman C.L. Weintraub N.L. VanRollins M. Dellsperger K.C. Circ. Res. 1998; 83: 932-939Crossref PubMed Scopus (209) Google Scholar). Incubations of EETs with rat or mouse liver microsomal P450 results in the production of a series of diepoxyeicosadienoic acids (diepoxides) and monohydroxyepoxyeicosatrienoic acids (epoxyalcohols) (59Moghaddam M. Motoba K. Borhan B. Pinot F. Hammock B.D. Biochim. Biophys. Acta. 1996; 1290: 327-339Crossref PubMed Scopus (72) Google Scholar, 65Capdevila J.H. Mosset P. Yadagiri P. Lumin S. Falck J.R. Arch. Biochem. Biophys. 1988; 261: 122-133Crossref PubMed Scopus (28) Google Scholar). The diepoxides can be further metabolized by sEH to diol epoxides, which cyclize to the corresponding tetrahydrofuran-diols (THF-diols) (59Moghaddam M. Motoba K. Borhan B. Pinot F. Hammock B.D. Biochim. Biophys. Acta. 1996; 1290: 327-339Crossref PubMed Scopus (72) Google Scholar). Little is known regarding the biological relevance of the diepoxides, epoxyalcohols, and THF-diols. Both 8,9-EET and 5,6-EET are substrates for PGHS (66Zhang J.Y. Prakash C. Yamashita K. Blair I.A. Biochem. Biophys. Res. Commun. 1992; 183: 138-143Crossref PubMed Scopus (42) Google Scholar, 67Carroll M.A. Balazy M. Margiotta P. Falck J.R. McGiff J.C. J. Biol. Chem. 1993; 268: 12260-12266Abstract Full Text PDF PubMed Google Scholar, 68Homma T. Zhang J.Y. Shimizu T. Prakash C. Blair I.A. Harris R.C. Biochem. Biophys. Res. Commun. 1993; 191: 282-288Crossref PubMed Scopus (28) Google Scholar). 5,6-EET is converted to both 5-hydroxy-PGI1 and 5,6-epoxy-PGE1 in rabbit kidney. Interestingly, the 5,6-epoxy-PGE1 is equipotent to PGE2 as a renal vasodilator whereas 5-hydroxy-PGI1 is without activity (67Carroll M.A. Balazy M. Margiotta P. Falck J.R. McGiff J.C. J. Biol. Chem. 1993; 268: 12260-12266Abstract Full Text PDF PubMed Google Scholar). 8,9-EET is converted to 11-hydroxy-8,9-epoxyeicosatrienoic acid and 15-hydroxy-8,9-epoxyeicosatrienoic acid by PGHS. Importantly, the 11-hydroxy-8,9-epoxyeicosatrienoic acid has been shown to be a potent mitogen for rat glomerular mesangial cells (68Homma T. Zhang J.Y. Shimizu T. Prakash C. Blair I.A. Harris R.C. Biochem. Biophys. Res. Commun. 1993; 191: 282-288Crossref PubMed Scopus (28) Google Scholar). All four EETs can also serve as substrates for cytosolic glutathione S-transferase to form a series of glutathione conjugates, the biological relevance of which is unknown (69Spearman M.E. Prough R.A. Estabrook R.W. Falck J.R. Manna S. Leibman K.C. Murphy R.C. Capdevila J. Arch. Biochem. Biophys. 1985; 242: 225-230Crossref PubMed Scopus (67) Google Scholar). Fang et al. (62Fang X. Kaduce T.L. Weintraub N.L. VanRollins M. Spector A.A. Circ. Res. 1996; 79: 784-793Crossref PubMed Scopus (90) Google Scholar) have described several novel pathways of EET metabolism in endothelial cells. 11,12-EET is converted to 7,8-dihydroxy-18:2 via epoxide hydration and β-oxidation. This compound possesses vasoactive properties in the coronary circulation. In the presence of epoxide hydrolase inhibition, these investigators observed the biosynthesis of the several chain-shortened β-oxidation products including 10,11-epoxy-16:2, 7,8-epoxy-16:2, and 8,9-epoxy-14:1, as well as a chain elongation product 16,17-epoxy-22:3 (70Fang X. Kaduce T.L. Weintraub N.L. Harmon S. Teesch L.M. Morisseau C. Thompson D.A. Hammock B.D. Spector A.A. J. Biol. Chem. 2001; 276: 14867-14874Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Intracellular fatty acid-binding proteins (FABPs) may differentially bind EETs and DHETs, thus modulating their metabolism, activities, and/or targeting. Widstrom et al. (71Widstrom R.L. Norris A.W. Spector A.A. Biochemistry. 2001; 40: 1070-1076Crossref PubMed Scopus (64) Google Scholar) have recently evaluated the relative affinities of FABPs for several P450 epoxygenase-derived eicosanoids. The affinity of heart FABP for 5,6-EET and 11,12-EET (Kd ∼0.4 μm) was ∼20-fold greater than for DHETs (Kd ∼8 μm). The homologous proteins, liver FABP and intestinal FABP, also displayed selective affinity for EETs versusDHETs. These investigators postulated that FABP binding of EETs may facilitate their intracellular retention whereas the lack of FABP affinity for DHETs may partially explain their release from cells. The biological relevance of EETs and/or DHETs in various tissues has been the subject of a number of excellent reviews (3Capdevila J.H. Falck J.R. Harris R.C. J. Lipid Res. 2000; 41: 163-181Abstract Full Text Full Text PDF PubMed Google Scholar, 4Oliw E.H. Prog. Lipid Res. 1994; 33: 329-354Crossref PubMed Scopus (139) Google Scholar, 5Makita K. Falck J.R. Capdevila J.H. FASEB J. 1996; 10: 1456-1463Crossref PubMed Scopus (120) Google Scholar, 6McGiff J.C. Quilley J. Curr. Opin. Nephrol. Hypertens. 2001; 10: 231-237Crossref PubMed Scopus (86) Google Scholar, 39Capdevila J. Zeldin D.C. Karara A. Falck J.F. Adv. Mol. Cell Biol. 1996; 14: 317-339Crossref Scopus (9) Google Scholar, 72Jacobs E.R. Zeldin D.C. Am. J. Physiol. 2001; 280: H1-H10Crossref PubMed Google Scholar,73Roman R.J. Maier K.G. Sun C.W. Harder D.R. Alonso-Galicia M. Clin. Exp. Pharmacol. Physiol. 2000; 27: 855-865Crossref PubMed Scopus (113) Google Scholar). Herein, I will focus only on the role of these eicosanoids in kidney and cardiovascular function (summarized in TableI).Table IRenal and cardiovascular effects of P450 epoxygenase metabolitesBiological effectsRef.Renal Effects on Na+ and K+transport(16Harris R.C. Homma T. Jacobson H.R. Capdevila J. J. Cell. Physiol. 1990; 144: 429-437Crossref PubMed Scopus (92) Google Scholar, 17Romero M.F. Madhun Z.T. Hopfer U. Douglas J.G. Adv. Prostaglandin Thromboxane Leukotriene Res. 1991; 21A: 205-208PubMed Google Scholar, 78Madhun Z.T. Goldthwait D.A. McKay D. Hopfer U. Douglas J.G. J. Clin. Invest. 1991; 88: 456-461Crossref PubMed Scopus (117) Google Scholar, 80Satoh T. Cohen H.T. Katz A.I. J. Clin. Invest. 1993; 91: 409-415Crossref PubMed Scopus (128) Google Scholar) Increases in intracellular Ca2+(17Romero M.F. Madhun Z.T. Hopfer U. Douglas J.G. Adv. Prostaglandin Thromboxane Leukotriene Res. 1991; 21A: 205-208PubMed Google Scholar, 23Burns K.D. Capdevila J. Wei S. Breyer M.D. Homma T. Harris R.C. Am. J. Physiol. 1995; 269: C831-C840Crossref PubMed Google Scholar, 78Madhun Z.T. Goldthwait D.A. McKay D. Hopfer U. Douglas J.G. J. Clin. Invest. 1991; 88: 456-461Crossref PubMed Scopus (117) Google Scholar, 79Sakairi Y. Jacobson H.R. Noland T.D. Capdevila J.H. Falck J.R. Breyer M.D. Am. J. Physiol. 1995; 268: F931-F939PubMed Google Scholar) Mitogenic effects(16Harris R.C. Homma T. Jacobson H.R. Capdevila J. J. Cell. Physiol. 1990; 144: 429-437Crossref PubMed Scopus (92) Google Scholar, 23Burns K.D. Capdevila J. Wei S. Breyer M.D. Homma T. Harris R.C. Am. J. Physiol. 1995; 269: C831-C840Crossref PubMed Google Scholar) Effects on water transport(63Hirt D.L. Capdevila J. Falck J.R. Breyer M.D. Jacobson H.R. J. Clin. Invest. 1989; 84: 1805-1812Crossref PubMed Scopus (61) Google Scholar) Effects on renal vascular tone(12Zou A.P. Fleming J.T. Falck J.R. Jacobs E.R. Gebremedhin D. Harder D.R. Roman R.J. Am. J. Physiol. 1996; 270: F822-F832PubMed Google Scholar, 75Katoh T. Takahashi K. Capdevila J. Karara A. Falck J.R. Jacobson H.R. Badr K.F. Am. J. Physiol. 1991; 261: F578-F586PubMed Google Scholar) Stimulation of prostaglandin biosynthesis(79Sakairi Y. Jacobson H.R. Noland T.D. Capdevila J.H. Falck J.R. Breyer M.D. Am. J. Physiol. 1995; 268: F931-F939PubMed Google Scholar)Cardiovascular Vasodilation(10Campbell W.B. Gebremedhin D. Pratt P.F. Harder D.R. Circ. Res. 1996; 78: 415-423Crossref PubMed Scopus (1115) Google Scholar, 42Fisslthaler B. Popp R. Kiss L. Potente M. Harder D.R. Fleming I. Busse R. Nature. 1999; 401: 493-497Crossref PubMed Scopus (809) Google Scholar, 87Quilley J. McGiff J.C. Trends Pharmacol. Sci. 2000; 21: 121-124Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) Antiinflammatory effects(21Node K. Huo Y. Ruan X. Yang B. Spiecker M. Ley K. Zeldin D.C. Liao J.K. Science. 1999; 285: 1276-1279Crossref PubMed Scopus (1024) Google Scholar) Fibrinolytic effects(22Node K. Ruan X.L. Dai J. Yang S.X. Graham L. Zeldin D.C. Liao J.K. J. Biol. Chem. 2001; 276: 15983-15989Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar) Effects on cardiomyocyte Na+ transport(89Lee H.C. Lu T. Weintraub N.L. VanRollins M. Spector A.A. Shibata E.F. J. Physiol. 1999; 519: 153-168Crossref PubMed Scopus (88) Google Scholar) Effects on cardiomyocyte Ca2+ transport(55Chen J. Capdevila J.H. Zeldin D.C. Rosenberg R.L. Mol. Pharmacol. 1999; 55: 288-295Crossref PubMed Scopus (90) Google Scholar, 90Moffat M.P. Ward C.A. Bend J.R. Mock T. Farhangkhoee P. Karmazyn M. Am. J. Physiol. 1993; 264: H1154-H1160PubMed Google Scholar, 91Xiao Y.F. Huang L. Morgan J.P. J. Physiol. 1998; 508: 777-792Crossref PubMed Scopus (95) Google Scholar) Effects on cardiomyocyte contractile function(90Moffat M.P. Ward C.A. Bend J.R. Mock T. Farhangkhoee P. Karmazyn M. Am. J. Physiol. 1993; 264: H1154-H1160PubMed Google Scholar) Protective effects following ischemia(41Wu S. Chen W. Murphy E. Gabel S. Tomer K.B. Foley J. Steenbergen C. Falck J.R. Moomaw C.R. Zeldin D.C. J. Biol. Chem. 1997; 272: 12551-12559Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 92Yang B. Graham L. Dikalov S. Mason R.P. Falck J.R. Liao J.K. Zeldin D.C. Mol. Pharmacol. 2001; 60: 1-11Crossref PubMed Scopus (150) Google Scholar) Open table in a new tab EETs are endogenous constituents of human and rodent kidney, and EET biosynthesis occurs throughout the nephron (74Karara A. Dishman E. Jacobson H. Falck J.R. Capdevila J.H. FEBS Lett. 1990; 268: 227-230Crossref PubMed Scopus (66) Google Scholar, 75Katoh T. Takahashi K. Capdevila J. Karara A. Falck J.R. Jacobson H.R. Badr K.F. Am. J. Physiol. 1991; 261: F578-F586PubMed Google Scholar, 76Ma J. Qu W. Scarborough P.E. Tomer K.B. Moomaw C.R. Maronpot R. Davis L.S. Breyer M.D. Zeldin D.C. J. Biol. Chem. 1999; 274: 17777-17788Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 77Omata K. Abraham N.G. Schwartzman M.L. Am. J. Physiol. 1992; 262: F591-F599PubMed Google Scholar). Moreover, EETs have been shown to contribute to integrated renal function, either by directly affecting tubular transport processes, vascular tone, and cellular proliferation or by mediating the actions of renal hormones including renin, angiotensin II, and arginine vasopressin (3Capdevila J.H. Falck J.R. Harris R.C. J. Lipid Res. 2000; 41: 163-181Abstract Full Text Full Text PDF PubMed Google Scholar, 5Makita K. Falck J.R. Capdevila J.H. FASEB J. 1996; 10: 1456-1463Crossref PubMed Scopus (120) Google Scholar, 6McGiff J.C. Quilley J. Curr. Opin. Nephrol. Hypertens. 2001; 10: 231-237Crossref PubMed Scopus (86) Google Scholar,73Roman R.J. Maier K.G. Sun C.W. Harder D.R. Alonso-Galicia M. Clin. Exp. Pharmacol. Physiol. 2000; 27: 855-865Crossref PubMed Scopus (113) Google Scholar). For example, in the proximal tubule, EETs inhibit Na+transport and mediate the angiotensin II-induced rise in cytosolic Ca2+ (17Romero M.F. Madhun Z.T. Hopfer U. Douglas J.G. Adv. Prostaglandin Thromboxane Leukotriene Res. 1991; 21A: 205-208PubMed Google Scholar, 78Madhun Z.T. Goldthwait D.A. McKay D. Hopfer U. Douglas J.G. J. Clin. Invest. 1991; 88: 456-461Crossref PubMed Scopus (117) Google Scholar). In the collecting duct, P450 epoxygenase metabolites inhibit the hydroosmotic effect of arginine vasopressin, decrease net Na+ reabsorption and K+ secretion, and increase cytosolic Ca2+ concentrations (63Hirt D.L. Capdevila J. Falck J.R. Breyer M.D. Jacobson H.R. J. Clin. Invest. 1989; 84: 1805-1812Crossref PubMed Scopus (61) Google Scholar, 79Sakairi Y. Jacobson H.R. Noland T.D. Capdevila J.H. Falck J.R. Breyer M.D. Am. J. Physiol. 1995; 268: F931-F939PubMed Google Scholar, 80Satoh T. Cohen H.T. Katz A.I. J. Clin. Invest. 1993; 91: 409-415Crossref PubMed Scopus (128) Google Scholar). The EETs also have potent effects on renal vascular tone and are mitogenic in glomerular mesangial cells (12Zou A.P. Fleming J.T. Falck J.R. Jacobs E.R. Gebremedhin D. Harder D.R. Roman R.J. Am. J. Physiol. 1996; 270: F822-F832PubMed Google Scholar, 16Harris R.C. Homma T. Jacobson H.R. Capdevila J. J. Cell. Physiol. 1990; 144: 429-437Crossref PubMed Scopus (92) Google Scholar, 75Katoh T. Takahashi K. Capdevila J. Karara A. Falck J.R. Jacobson H.R. Badr K.F. Am. J. Physiol. 1991; 261: F578-F586PubMed Google Scholar). In addition to the well documented effects of EETs on renal vascular tone and fluid/electrolyte transport, several lines of evidence suggest that the P450 epoxygenase pathway may be involved in the pathogenesis of hypertension. First, the rat renal epoxygenases are under regulatory control by dietary salt, and their inhibition leads to the development of salt-dependent hypertension (40Holla V.R. Makita K. Zaphiropoulos P.G. Capdevila J.H. J. Clin. Invest. 1999; 104: 751-760Crossref PubMed Scopus (110) Google Scholar, 50Capdevila J.H. Wei S. Yan J. Karara A. Jacobson H.R. Falck J.R. Guengerich F.P. DuBois R.N. J. Biol. Chem. 1992; 267: 21720-21726Abstract Full Text PDF PubMed Google Scholar, 51Oyekan A.O. Youseff T. Fulton D. Quilley J. McGiff J.C. J. Clin. Invest. 1999; 104: 1131-1137Crossref PubMed Scopus (102) Google Scholar, 81Makita K. Takahashi K. Karara A. Jacobson H.R. Falck J.R. Capdevila J.H. J. Clin. Invest. 1994; 94: 2414-2420Crossref PubMed Scopus (177) Google Scholar). Second, spontaneously hypertensive rats have altered renal epoxygenase and epoxide hydrolase activities, and treatment of these animals with agents that either deplete renal P450 or inhibit sEH normalizes blood pressure (82Sacerdoti D. Abraham N.G. McGiff J.C. Schwartzman M.L. Biochem. Pharmacol. 1988; 37: 521-527Crossref PubMed Scopus (72) Google Scholar, 83Yu Z. Xu F. Huse L.M. Morisseau C. Draper A.J. Newman J.W. Parker C. Graham L. Engler M.M. Hammock B.D. Zeldin D.C. Kroetz D.L. Circ. Res. 2000; 87: 992-998Crossref PubMed Scopus (418) Google Scholar, 84Yu Z. Huse L.M. Adler P. Graham L. Ma J. Zeldin D.C. Kroetz D.L. Mol. Pharmacol. 2000; 57: 1011-1020PubMed Google Scholar, 85Gebremedhin D. Ma Y.H. Imig J.D. Harder D.R. Roman R.J. J. Vasc. Res. 1993; 30: 53-60Crossref PubMed Scopus (53) Google Scholar). Third, the salt-sensitive phenotype in the Dahl rat model of genetic hypertension is associated with an inability to increase renal epoxygenase activity in response to dietary salt intake (81Makita K. Takahashi K. Karara A. Jacobson H.R. Falck J.R. Capdevila J.H. J. Clin. Invest. 1994; 94: 2414-2420Crossref PubMed Scopus (177) Google Scholar). Fourth, targeted disruption of the sEH gene lowers blood pressure in male mice in the absence and presence of dietary salt loading (86Sinal C.J. Miyata M. Tohkin M. Nagata K. Bend J.R. Gonzalez F.J. J. Biol. Chem. 2000; 275: 40504-40510Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar). Fifth, the urinary excretion of epoxygenase metabolites is profoundly increased in women with pregnancy-induced hypertension (60Catella F. Lawson J. Braden G. Fitzgerald D.J. Shipp E. FitzGerald G.A. Adv. Prostaglandin Thromboxane Leukotriene Res. 1991; 21A: 193-196PubMed Google Scholar). Together these data suggest a role for P450 epoxygenases and sEH in blood pressure regulation and identify this pathway as an attractive target for therapeutic intervention. EETs have been proposed to be endothelial derived hyperpolarizing factors (EDHFs) in that they relax vascular smooth muscle by opening large conductance, Ca2+-activated K+ channels (BKCa) in the smooth muscle cell membrane (10Campbell W.B. Gebremedhin D. Pratt P.F. Harder D.R. Circ. Res. 1996; 78: 415-423Crossref PubMed Scopus (1115) Google Scholar, 42Fisslthaler B. Popp R. Kiss L. Potente M. Harder D.R. Fleming I. Busse R. Nature. 1999; 401: 493-497Crossref PubMed Scopus (809) Google Scholar, 87Quilley J. McGiff J.C. Trends Pharmacol. Sci. 2000; 21: 121-124Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Recent studies suggest that the mechanism by which EETs activate the BKCa channels involves ADP-ribosylation of the guanine nucleotide-binding protein GSα with a subsequent membrane-delimited action on the channel (88Li P.L. Chen C.L. Bortell R. Campbell W.B. Circ. Res. 1999; 85: 349-356Crossref PubMed Scopus (95) Google Scholar). Treatment of porcine coronary arteries with β-naphthoflavone induces a CYP2C isoform, enhances EET biosynthesis, and increases EDHF-mediated hyperpolarization resulting in vasorelaxation (42Fisslthaler B. Popp R. Kiss L. Potente M. Harder D.R. Fleming I. Busse R. Nature. 1999; 401: 493-497Crossref PubMed Scopus (809) Google Scholar). Moreover, transfection with an antisense oligonucleotide to CYP2C results in decreased CYP2C expression and attenuation of EDHF-mediated vascular responses, providing further evidence that the EDHF synthase in the porcine coronary vascular bed is a CYP2C isoform (42Fisslthaler B. Popp R. Kiss L. Potente M. Harder D.R. Fleming I. Busse R. Nature. 1999; 401: 493-497Crossref PubMed Scopus (809) Google Scholar). EETs have recently been shown to play important nonvasodilatory roles within the vasculature. Physiological concentrations (100 nm) of EETs or overexpression of human CYP2J2 decreased cytokine-induced endothelial cell adhesion molecule expression, and EETs prevented leukocyte adhesion to the vascular wall (21Node K. Huo Y. Ruan X. Yang B. Spiecker M. Ley K. Zeldin D.C. Liao J.K. Science. 1999; 285: 1276-1279Crossref PubMed Scopus (1024) Google Scholar). The mechanism for the anti-inflammatory effect of the EETs was shown to involve inhibition of the transcription factor NF-κB and IκB kinase (21Node K. Huo Y. Ruan X. Yang B. Spiecker M. Ley K. Zeldin D.C. Liao J.K. Science. 1999; 285: 1276-1279Crossref PubMed Scopus (1024) Google Scholar). In vascular endothelial cells, addition of EETs or overexpression of CYP2J2 increased tissue plasminogen activator (tPA) expression and fibrinolytic activity (22Node K. Ruan X.L. Dai J. Yang S.X. Graham L. Zeldin D.C. Liao J.K. J. Biol. Chem. 2001; 276: 15983-15989Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Induction of tPA gene transcription by EETs was associated with increased GSα GTP-binding activity, increased intracellular cAMP levels, and cAMP-driven tPA promoter activation (22Node K. Ruan X.L. Dai J. Yang S.X. Graham L. Zeldin D.C. Liao J.K. J. Biol. Chem. 2001; 276: 15983-15989Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Neither the anti-inflammatory effects nor the hemostatic actions of the EETs was blocked by BKCa channel inhibitors suggesting that these EET effects were independent of their membrane-hyperpolarizing actions (21Node K. Huo Y. Ruan X. Yang B. Spiecker M. Ley K. Zeldin D.C. Liao J.K. Science. 1999; 285: 1276-1279Crossref PubMed Scopus (1024) Google Scholar, 22Node K. Ruan X.L. Dai J. Yang S.X. Graham L. Zeldin D.C. Liao J.K. J. Biol. Chem. 2001; 276: 15983-15989Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Together these data suggest that EETs possess homeostatic properties in the vasculature in addition to their vasodilatory actions. EETs have been shown to have effects on cardiomyocyte function. For example, 8,9-EET inhibits cardiac Na+ channels and produces a hyperpolarized shift in the steady-state membrane potential (89Lee H.C. Lu T. Weintraub N.L. VanRollins M. Spector A.A. Shibata E.F. J. Physiol. 1999; 519: 153-168Crossref PubMed Scopus (88) Google Scholar). Moffat et al. (90Moffat M.P. Ward C.A. Bend J.R. Mock T. Farhangkhoee P. Karmazyn M. Am. J. Physiol. 1993; 264: H1154-H1160PubMed Google Scholar) demonstrated that both 5,6-EET and 11,12-EET significantly increase guinea pig cardiac myocyte cell shortening and intracellular calcium concentrations. Xiao et al. (91Xiao Y.F. Huang L. Morgan J.P. J. Physiol. 1998; 508: 777-792Crossref PubMed Scopus (95) Google Scholar) showed that 11,12-EET enhanced l-type Ca2+ current and intracellular cAMP content in intact ventricular myocytes and that the P450 epoxygenase inhibitor clotrimazole suppressed cardiac myocyte cell shortening. In contrast, we have shown that 11,12-EET has direct inhibitory effects on cardiacl-type Ca2+ channels reconstituted into planar lipid bilayers suggesting that EETs can either increase or decrease Ca2+ channel activity depending on the metabolic and regulatory state of the cells (55Chen J. Capdevila J.H. Zeldin D.C. Rosenberg R.L. Mol. Pharmacol. 1999; 55: 288-295Crossref PubMed Scopus (90) Google Scholar). In an isolated-perfused rat heart model, 11,12-EET significantly improved recovery of heart contractile function following prolonged, global ischemia (41Wu S. Chen W. Murphy E. Gabel S. Tomer K.B. Foley J. Steenbergen C. Falck J.R. Moomaw C.R. Zeldin D.C. J. Biol. Chem. 1997; 272: 12551-12559Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). During the past two decades, the contributions of many laboratories have led to an improved understanding of the biochemical, pharmacological, and physiological relevance of the AA epoxygenase pathway. However, a number of important questions remain. Which specific P450 isoforms are primarily responsible for EET biosynthesis in various tissues? What are the signaling pathways and molecular mechanisms that underlie the biological actions of the EETs? What is the functional relevance of epoxygenase gene polymorphisms and are they associated with increased susceptibility to human disease? The application of modern molecular biological techniques such as gene overexpression and/or gene disruption, the development of specific P450 epoxygenase inhibitors and inhibitory antibodies, and the generation of stable EET receptor agonists/antagonists will facilitate future studies that attempt to address these and other critical questions. I am grateful to Drs. John R. Falck, William B. Campbell, and Jorge H. Capdevila for their helpful comments during the preparation of this manuscript. I apologize for omitting many relevant studies because of space constraints.