The CYP4A Isoforms Hydroxylate Epoxyeicosatrienoic Acids to Form High Affinity Peroxisome Proliferator-activated Receptor Ligands*

Cytochromes P450 of the CYP2Cand CYP4A gene subfamilies metabolize arachidonic acid to 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs) and to 19- and 20-hydroxyeicosatetraenoic acids (HETEs), respectively. Abundant functional studies indicate that EETs and HETEs display powerful and often opposing biological activities as mediators of ion channel activity and regulators of vascular tone and systemic blood pressures. Incubation of 8,9-, 11,12-, and 14,15-EETs with microsomal and purified forms of rat CYP4A isoforms led to rapid NADPH-dependent metabolism to the corresponding 19- and 20-hydroxylated EETs. Comparisons of reaction rates and catalytic efficiency with those of arachidonic and lauric acids showed that EETs are one of the best endogenous substrates so far described for rat CYP4A isoforms. CYP4A1 exhibited a preference for 8,9-EET, whereas CYP4A2, CYP4A3, and CYP4A8 preferred 11,12-EET. In general, the closer the oxido ring is to the carboxylic acid functionality, the higher the rate of EET metabolism and the lower the regiospecificity for the EET ω-carbon. Analysis of cis-parinaric acid displacement from the ligand-binding domain of the human peroxisome proliferator-activated receptor-α showed that ω-hydroxylated 14,15-EET bound to this receptor with high affinity (K i = 3 ± 1 nm). Moreover, at 1 μm, the ω-alcohol of 14,15-EET or a 1:4 mixture of the ω-alcohols of 8,9- and 11,12-EETs activated human and mouse peroxisome proliferator-activated receptor-α in transient transfection assays, suggesting a role for them as endogenous ligands for these orphan nuclear receptors.

Cytochromes P450 of the CYP4A gene subfamily are structurally and functionally conserved fatty-acid hydroxylases that are expressed in most mammalian tissues, including rat and human kidney and liver (1)(2)(3)(4)(5)(6)(7). These enzymes are selective for the /-1-hydroxylation of saturated and unsaturated fatty acids (1-7) and lack known roles in drug metabolism. The expression of some CYP4A isoforms is under the control of the peroxisome proliferator-activated receptor-␣ (PPAR␣) 1 (8 -13) and regulated by a variety of physiological and pathophysiological stimuli, including dietary fatty acids, hormones, diabetes, and starvation (9 -13). Interest in the molecular and functional properties of these enzymes has been stimulated by the demonstration of their role in the /-1-hydroxylation of arachidonic acid (AA) (4 -7) and the powerful biological activities of 19-and 20-hydroxyeicosatetraenoic acids (HETEs) as modulators of renal ion fluxes and vasoactivity (14 -18). Based on biochemical and functional correlates of CYP4A renal expression, 20-HETE biosynthesis, and the onset of systemic high blood pressure in the SHR/WKY rat model of spontaneous hypertension, a pro-hypertensive role for 20-HETE and CYP4A isoforms was proposed (14).
The cytochrome P450 AA epoxygenase catalyzes the in vivo regio-and enantioselective metabolism of AA to epoxyeicosatrienoic acids (EETs) (16). Studies with microsomal and/or purified cytochrome P450 preparations showed that the AA epoxygenases belong to the CYP2 gene family and that CYP2C isoforms account for most of the epoxygenase activity in rat and human kidney and liver (16). The biological activities attributed to the EETs include mitogenesis; vasodilatation; modulation of cellular Ca 2ϩ , Na ϩ , and K ϩ fluxes; and activation of Ca 2ϩ -dependent K ϩ channels (14 -18). The extensive studies of the functional properties of the cytochrome P450-derived eicosanoids have shown that the metabolites of the epoxygenase and /-1-hydroxylase branches of the cytochrome P450 AA monooxygenase display powerful but often opposing biological activities (14 -18) and that these eicosanoids can be further metabolized by cytochrome P450-dependent and -independent pathways (19 -24). During studies of AA metabolism by microsomal and purified CYP4A isoforms, we observed active /-1hydroxylation of EETs, the products of the AA epoxygenase reaction. We report here that the EETs are excellent substrates for the rat CYP4A isoforms, that they are rapidly oxidized to the corresponding 19-and 20-hydroxylated EETs, and that these products bind with high affinity to the PPAR class of nuclear receptors.
Rat Treatment and Isolation of Microsomal Fractions-Adult male Sprague-Dawley rats (250 -280 g) were administered Wy 14643 in their drinking water (0.03%, w/v) for 10 days. Liver microsomal fractions were isolated from treated and control rats as described (25). Microsomal pellets were suspended in 10 mM Tris-Cl (pH 7.4) containing 0.25 M sucrose at a protein concentration of ϳ20 mg/ml and stored at 4°C for not more than 48 h.
Expression and Purification of CYP4A Isoforms and Determinations of Enzyme Activity-The CYP4A2 cDNA was expressed using a MAX-BAC baculovirus/Sf9 system (Invitrogen) and purified as described (5). The CYP4A1 cDNA with the described N-terminal modifications (5) in the pCWori vector was expressed in Escherichia coli and purified as described (5). Purified His-tagged CYP4A3 and CYP4A8 were a generous gift from Dr. Paul Ortiz de Montellano (Department of Pharmaceutical Chemistry, University of California at San Francisco). The monooxygenase activities of purified recombinant enzymes were reconstituted in the presence of dilauroylphosphatidylcholine (50 g/ ml) with cytochrome P450, NADPH-cytochrome P450 reductase, and cytochrome b 5 at a 1:10:1 molar ratio. Incubations were performed in a shaking water bath at 35°C in 0.01 M Tris-Cl (pH 7.4) containing 150 mM KCl, 10 mM MgCl 2 , 0.1 unit/ml isocitrate dehydrogenase, and 2 mg/ml isocitric acid (25). 1-14 C-Labeled fatty acids or EETs were added in a small volume of 0.1 M Tris-Cl (pH 8.0). Reactions were initiated by the addition of NADPH (1 mM final concentration). At the indicated times, microsomal reactions were stopped by the addition of ethyl ether containing 0.05% (v/v) acetic acid, and the reaction products were extracted in the presence of aqueous 0.1 M KCl (25). Immuno-inhibition experiments were done by incubating the mixture of microsomes and antibodies for 5 min, prior to the addition of substrate and NADPH. Enzymatic reactions containing purified proteins were stopped by the addition of an equal volume of acetonitrile containing 0.2% HOAc and 0.005% butylated hydroxytoluene and centrifuged at 14,000 ϫ g, and the supernatants were submitted directly to reversed-phase HPLC (RP-HPLC). Reaction products were resolved and quantified using a 5-m Dynamax C 18 column (4.6 ϫ 250 cm; Rainin Instruments Co. Inc., Woburn, MA) with on-line ␤-detection and the following solvent programs: Solvent Program a, AA metabolites, exactly as described (25); and Solvent Program b, lauric acid and EET metabolites, an isocratic mixture composed of CH 3 CN/H 2 O/HOAc (30:70:0.1) for 5 min, followed by a linear solvent gradient to CH 3 CN/H 2 O/HOAc (60:40:0.1) over 25 min, a 5-min isocratic period with CH 3 CN/H 2 O/HOAc (60:40:0.1), and then a linear solvent gradient to CH 3 CN/HOAc (100:0.1) over 20 min at a flow rate of 1 ml/min (R t ϭ 20.4 and 22.2 min for 11-and 12hydroxydodecanoic acids, respectively; R t ϭ 29.4 and 30.3, 29.2 and 30.0, and 29.0 and 29.4 min for the -1-and -alcohols of 8,9-, 11,12-, and 14,15-EETs, respectively). Initial velocities were calculated from the linear portion of product concentration versus time of incubation plots. During these studies, it was observed that the EET /-1-hydroxylase activity of membrane suspensions containing microsomes from Wy 14643-treated animals was particularly sensitive to freezing and thawing and/or to extended storage at temperatures below 0°C. Consequently, the microsomal pellets, obtained after high speed centrifugation (25), were stored at Ϫ80°C as pellets in 50% glycerol. Frozen microsomal pellets were thawed only once and discarded within 48 h of suspension.
Microsomal proteins (20 -40 g) or purified cytochromes P450 (0.5-1.0 pmol each) were resolved by discontinuous SDS-PAGE and transferred to nitrocellulose membranes in Tris/glycine buffer (pH 8.3) (5) under constant current (30 mA) overnight. After blocking, membranes were exposed to affinity-purified rabbit polyclonal antibodies raised against recombinant CYP4A1 or CYP4A2 (5) and then incubated with a commercial horseradish peroxidase-conjugated anti-rabbit IgG (Sigma). Immunoreactive proteins were visualized using a SuperSignal peroxidase kit (Pierce) and exposed to x-ray film.
Purification of Receptors Expressed in E. coli-For the pET-hPPAR-LBD construct, E. coli BL21(DE3) pLysS cultures were induced according to the pET system manual (Novagen). The cells were harvested by centrifugation at 2000 ϫ g for 10 min at 4°C, washed once with phosphate-buffered saline, and resuspended in cold 50 mM sodium phosphate (pH 8.0) containing 300 mM NaCl. Prior to sonication, the lysates were incubated on ice for 30 min with gentle mixing every 5 min. 0.1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride and 0.2 mM phenylmethylsulfonyl fluoride were added immediately after sonication for 4 ϫ 10 s on ice. The sonicated lysates were centrifuged at 8000 ϫ g for 30 min, and the cleared lysates were loaded onto a column containing a metal ion affinity resin (Talon, CLONTECH). The resin was first washed with 50 mM sodium phosphate (pH 8.0) containing 300 mM NaCl, 0.1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.2 mM phenylmethylsulfonyl fluoride, and 10% glycerol (buffer A) including 0.5% Nonidet P-40 and then with buffer A containing 5 mM imidazole. The resin was further washed with buffer A containing 10 mM imidazole, and then the protein was eluted with buffer A containing 100 mM imidazole. The eluted LBDs were concentrated, and imidazole was removed using Millipore Ultrafree centrifugal filters (BioMax-10). After concentration, protein purity was assessed using SDS-polyacrylamide gels.
Ligand-Receptor Binding Assay-Binding of cis-parinaric acid to purified recombinant hPPAR␣ LBD was measured in a PerkinElmer Life Sciences 650-40 fluorescence spectrophotometer with an excitation wavelength of 330 nm (bandwidth of 4 nm) and an emission wavelength of 413 nm (bandwidth of 10 nm). The binding buffer contained 10 mM Hepes (pH 7.4), 0.5 mM EDTA, and 400 mM NaCl. cis-Parinaric acid was dissolved in ethanol containing 0.05% butylated hydroxytoluene, and its concentration was determined by UV spectroscopy ( max ϭ 304 nm). Titrations of cis-parinaric acid binding were carried out at 24 -26°C using successive additions until a plateau was apparent. The K d for cis-parinaric acid was estimated using the single-site binding, hyperbolic model. Studies of binding affinities for the hPPAR␣ LBD were performed using the displacement method, where PPAR-bound cisparinaric acid was displaced by serial addition of test compounds. Test compounds were dissolved in ethanol containing 0.05% butylated hydroxytoluene and added in doses ranging from 1 nM to 20 M. During all measurements, ethanol concentrations were kept below 0.5%. Each sample and blank were thoroughly mixed with test compound and cis-parinaric acid (0.09 M) and allowed to equilibrate for 10 min at 24 -26°C to allow for stable measurement of the fluorescence signals. Measurements were corrected for background by subtracting values obtained for blank reactions that contained compound or protein only. The IC 50 values and K i constants were calculated according to Cheng and Prusoff (34).
Cell Culture and Transfections-The RK13 cell line was obtained from American Type Culture Collection and maintained in minimal essential medium with Earle's balanced salts (Invitrogen) containing 10% fetal bovine serum (Hyclone Labs, Logan, UT). All reporter and expression constructs were introduced into cultured cells by a modified calcium phosphate coprecipitation procedure (32,33). After an 18-h exposure to the DNA-containing culture medium, the cells were washed twice with medium without serum, and then fresh medium containing the test compounds or an equivalent volume of solvent (0.25% (v/v) ethanol) was added. The medium was supplemented with 10% charcoal/ dextran-treated fetal bovine serum (Hyclone Labs). After a 24-h incubation with the test compounds, the cells were harvested and assayed for luciferase and ␤-galactosidase activities. The luciferase activity was determined as described previously (33). The ␤-galactosidase activity was determined using a Bio-Rad FluorAce ␤-galactosidase reporter assay kit. The luciferase activity obtained for individual wells was expressed relative to the ␤-galactosidase activity obtained from the same preparation of cell lysate.

RESULTS AND DISCUSSION
EET Metabolism by Rat Liver Microsomes-During studies of AA metabolism by rat liver microsomes, we observed a timedependent disappearance of epoxygenase metabolites and the concomitant formation of products with RP-HPLC retention times similar to those of authentic 20,14,15-HEET (data not shown) and to those reported earlier by Oliw et al. (35). The formation of these polar metabolites during the course of AA oxygenation demonstrated that their precursors interacted efficiently with the microsomal oxygenases, even in the presence of excess AA, and suggested a precursor-product relationship between the EETs and these metabolites. Incubation of synthetic 8,9-, 11,12-, or 14,15-[1-14 C]EET with liver microsomes generated products with RP-HPLC retention times similar to those of authentic 20,14,15-HEET and the products generated during long-term incubations with AA ( Fig. 1), indicating that these products were derived from -hydroxylation of the EETs. Under analogous conditions, we also detected NADPH-dependent metabolism of 5,6-EET; however, most of the added 5,6-EET was rapidly hydrated and converted to the ␦-lactone of 5,6-dihydroxyeicosatrienoic acid (data not shown).
For structural analysis, the products of the microsomal metabolism of 8,9-, 11,12-, and 14,15-EETs were purified by RP-HPLC as described for Fig. 1; converted to the corresponding pentafluorobenzyl ester, trimethylsilyl ether derivatives; and analyzed by NICI/GC/MS. The NICI/MS properties of theand -1-hydroxylated 8,9-, 11,12-, and 14,15-EETs were similar, with base peaks at m/z 407 (loss of pentafluorobenzyl) and carbon and hydrogen isotopic fragment ions at m/z 408 and 409 (Fig. 2). These values showed that the metabolites contained a hydroxyl moiety and that the EET oxido and triene functionalities remained intact. Two low intensity fragment ions, derived from the loss of oxygen and water, were also observed at m/z 391 and 389, respectively (Fig. 2) (30). Under the conditions of analysis, all three -hydroxylated EETs eluted with a GC R t of ϳ6 min, whereas the corresponding -1-hydroxylated isomers eluted at ϳ5.7 min. The regiochemistry of the hydroxyl group in 20,14,15-HEET was tentatively assigned by comparisons of its electron impact/GC/MS fragmentation patterns with that of authentic 20,14,15-EET (data not shown).
A comparison of the rates of microsomal EET hydroxylation with those obtained using, under identical conditions, AA or lauric acid as substrate (0.37 Ϯ 0.01 and 0.60 Ϯ 0.04 nmol of product/min/mg of protein, respectively) showed that the /-1-hydroxylation of 8,9-and 11,12-EETs by control rat liver microsomes proceeded at rates considerably faster than that of AA and, notably, lauric acid. As shown in Table I, the selectivity of the microsomal enzymes for the EET 19-and 20-carbon atoms was regioisomer-dependent, with 8,9-and 14,15-EETs hydroxylated preferentially at the -position. Animal treatment with Wy 14643, a PPAR␣ ligand and an inducer of rat CYP4A isoforms (11)(12)(13), led to significant increases in the rates of microsomal 8,9-, 11,12-, and 14,15-EET hydroxylation (ϳ3.7-, 4.0-, and 2.3-fold, respectively) and in the selectivity of the microsomal enzymes for the -carbon, with 14,15-EET hydroxylated now almost exclusively at this carbon (Table I).
The rat CYP4A gene subfamily is composed of four members, CYP4A1, CYP4A2, CYP4A3, and CYP4A8 (3-7), 2 of which CYP4A1 and CYP4A2 are the major CYP4A isoforms expressed in the male liver (5). Under the exposure times used in Fig. 3A, the mRNAs coding for CYP4A1 and CYP4A2 were nearly undetectable in the livers of untreated rats; however, longer exposure confirmed that they are the predominant CYP4A isoforms expressed in male rat liver (data not shown). Treatment of the animals with Wy 14643 caused marked increases in the levels of hepatic mRNAs coding for CYP4A1 and CYP4A2/3 (Fig. 3A). In contrast, Wy 14643 had only a minor effect on the concentrations of CYP4A8 mRNA transcripts, suggesting its regulation by a PPAR␣-independent mechanism (Fig. 3A). CYP4A8 is regulated by androgens in rat kidney (3), and the increased expression of the androgen-sensitive murine homolog of CYP4A8, CYP4A12, has been linked to the development of hypertension (36). Finally, immunoelectrophoresis of microsomal fractions isolated from control and Wy 14643-treated rats using polyclonal antibodies raised against recombinant CYP4A1 (unreactive toward CYP4A2 and CYP4A3) and CYP4A2 (cross-reactive toward CYP4A3 and CYP4A8, but not CYP4A1) demonstrated that the Wy 14643 effects shown in Fig. 3A led to increases in the microsomal levels of anti-CYP4A1 and anti-CYP4A2/4A3 immunoreactive proteins (Fig.  3B). These results suggest that the CYP4A1 and CYP4A2/4A3 isoforms play an important role in the catalysis of microsomal EET /-1-hydroxylation.
To further document the participation of CYP4A isoforms in EET hydroxylation, we incubated EETs with microsomes from untreated and Wy 14643-treated rats in the presence of nonimmune rabbit IgG or anti-CYP4A1 or anti-CYP4A2 antibody. As shown in Fig. 4, the extent of inhibition of the EET /-1hydroxylases by these antibodies differed significantly between the microsomes from control and Wy 14643-treated animals. In general, EET metabolism by control microsomes was less susceptible to immuno-inhibition than metabolism by Wy 14643induced microsomes (Fig. 4). Furthermore, the hydroxylation of 14,15-and 8,9-EETs was significantly more sensitive to inhibition by anti-CYP4A1 antibody (30 and 29% of control rates, respectively) than to inhibition by anti-CYP4A2 antibody (69 and 73% of control rates, respectively) (Fig. 4). On the other hand, anti-CYP4A1 antibody caused only a small reduction in microsomal 11,12-EET /-1-hydroxylation (81% of the control rate), and anti-CYP4A2 antibody was without effect (Fig. 4). Using reconstituted systems containing purified NADPH-cytochrome P450 reductase, cytochrome b 5 , and purified recombinant CYP4A1, CYP4A2, or CYP4A8, it was shown that the metabolism of 8,9-, 11,12-, and 14,15-EETs by these isoforms was inhibited by anti-CYP4A1 and anti-CYP4A2 antibodies (data not shown). These results indicate that CYP4A1 accounts for the majority of microsomal 14,15-and 8,9-EET hydroxylation and that other cytochrome P450 isoforms are probably responsible for most of the microsomal 11,12-EET hydroxylation. The /-1-hydroxylation of arachidonic acid and of several eicosanoids by CYP1A, CYP2C, CYP2J, and CYP4F isoforms has been documented (37)(38)(39)(40)(41).
The role of CYP4A1 and CYP4A2 as the predominant EET hydroxylases in microsomes from Wy 14643-treated animals was indicated by the nearly complete inhibition of EET /-1hydroxylation by anti-CYP4A1 and anti-CYP4A2 antibodies (Fig. 4). Titration of the inhibitory potency of these antibodies by varying the antibody/microsomal protein ratio demonstrated the following. (a) At antibody/microsomal protein ratios of 1, anti-CYP4A1 antibody blocked ϳ60 and 50% of the 14,15and 11,12-EET /-1-hydroxylase activities, respectively. Un-

FIG. 3. Expression of hepatic CYP4A isoforms in control and
Wy 14643-treated rats. A, Northern blot analysis of total RNA samples isolated from the livers of control (C) and Wy 14643 (W)-treated male rats. Nucleic acids were fractionated by gel electrophoresis, transferred to nitrocellulose membranes, and hybridized to 32 P-labeled genespecific probes as described under "Materials and Methods." After several high stringency washes, the membranes were exposed to x-ray films for 3 h. B, immunoelectrophoresis of liver microsomes from control and Wy 14643-treated rats. Microsomes (30 g each) or purified CYP4A2, CYP4A3, or CYP4A8 (5 pmol each) was submitted to discontinuous SDS-PAGE as described under "Materials and Methods." After electrophoretic transfer, the polyvinylidene difluoride membranes were incubated with a solution of affinity-purified anti-CYP4A1 or anti-CYP4A2 antibody (1-4 g/ml). Immunoreactive proteins were visualized using horseradish peroxidase-conjugated anti-rabbit IgG and a SuperSignal substrate Western blotting kit (Pierce).

FIG. 4. Effects of anti-CYP4A1 and anti-CYP4A2 antibodies on
the EET /-1-hydroxylase activity of microsomes from untreated and Wy 14643-treated rats. Microsomal fractions (0.25-0.5 mg/ml protein) were incubated with either nonimmune rabbit IgG or purified rabbit anti-CYP4A1 or CYP4A2 antibody (2.5-5.0 mg/ml protein each) for 5 min, prior to the addition of the EET substrate (70 -90 M each) and NADPH (1 mM). After 10 min at 35°C, the reaction products were extracted and quantified as described under "Materials and Methods." Shown are the results of one of two experiments that were performed using different microsomal preparations and that yielded values that differed by Ͻ15%. der similar conditions, anti-CYP4A2 antibody inhibited only 17 and 14% of these activities, respectively (data not shown). (b) At protein ratios from 1 to 10 (mg of antibody protein/mg of microsomal protein), anti-CYP4A1 and anti-CYP4A2 antibodies were nearly equally as effective in blocking 8,9-EET metabolism by Wy 14643-induced microsomes (data not shown). Based on the above results, we conclude that (a) CYP4A1 and CYP4A2 are the predominant EET /-1-hydroxylases present in microsomes from Wy 14643-treated rats; (b) CYP4A1 is responsible for most of the 11,12-and 14,15-EET hydroxylase activities induced by the PPAR␣ ligand; and (c) CYP4A1 and CYP4A2 mediate most of the induced metabolism of 8,9-EET.
Fatty Acid Metabolism by CYP4A Isoforms-The CYP4A isoforms are largely responsible for microsomal fatty acid /-1hydroxylation in liver and kidney (1)(2)(3)(4)(5)(6)(7)13); and as shown in Table II, all four rat CYP4A isoforms were at least 10-fold more active toward lauric acid than toward AA (6). However, despite the differences between lauric acid and AA in carbon chain length, degree of saturation, and rates of metabolism, all four isoforms showed a marked similarity in their regioselectivity of fatty acid /-1-hydroxylation (Table II). The chemistry of the reaction products as well as the effects of Wy 14643 suggest that the CYP4A isoforms play a dominant role in the /-1hydroxylation of EETs by liver microsomes. We therefore incubated samples of 8,9-, 11,12-, and 14,15-[1-14 C]EETs with reconstituted systems containing purified recombinant CYP4A1, CYP4A2, CYP4A3, or CYP4A8. As shown in Table III, EETs were hydroxylated by the four CYP4A enzymes at rates significantly higher than AA and approaching the hydroxylation rates of lauric acid, a fatty acid that is present in mammalian tissues at nearly undetectable levels and that is, however, the best reported substrate for these isoforms (Table II) (6). Importantly, under similar conditions, we failed to detect significant 8,9-, 11,12-, or 14,15-EET /-1-hydroxylation by recombinant CYP4F5, CYP2C11, or CYP2C23 (data not shown).
Although previous activity studies have not found significant differences in fatty acid substrate selectivity among the rat CYP4A isoforms (4 -7), the results in Table III show that 8,9-EET was the preferred substrate for CYP4A1, whereas CYP4A2, CYP4A3, and CYP4A8 favored 11,12-EET. Therefore, the addition of an oxido group to the AA carbon chain conferred a degree of CYP4A isoform substrate selectivity for the /-1hydroxylases and increased the rates of hydroxylation such that AA, one of the poorest substrates, was converted to an excellent one, with rates paralleling those of lauric acid (Tables  II and III). Additionally, the presence of a polar oxygen atom along the AA hydrocarbon chain led to significant increases in the regioselectivity of the CYP4A isoforms for the EET -carbon (Table IV). This is specially evident with CYP4A1, where the selectivity for the EET -carbon was almost complete (Table IV). In general, the closer the oxido ring is to the methyl end of the EETs, the lower the overall rates of metabolism (Table  III) and catalytic efficiency (Tables III and V) and the higher the regioselectivity of the CYP4A isoforms for the substrate -carbon (Table IV). Thus, for example, 14,15-EET was hydroxylated by all four isoforms almost exclusively at its -carbon (Table IV), but also the 14,15-EET hydroxylases showed the lowest degree of catalytic efficiency (Tables III and V). Displacement of the EET oxido ring toward the carboxylic acid functionality significantly increased the catalytic efficiency of the CYP4A /-1-hydroxylases, at the expense of their regioselectivity (Tables III-V).
Potent biological activities for products of the epoxygenase and /-1-hydroxylase pathways of cytochrome P450-mediated AA metabolism have been described (14 -18). That they often have opposing functional effects (14 -18) suggests an interaction between these pathways. These interactions may be functional, i.e. the products of one pathway have opposing activities to the products of the other pathway, and/or, as we have demonstrated herein, biochemical, i.e. the products of one pathway are substrates for the other, such that modulation of -hydroxylase activity may significantly alter epoxygenase product profiles and affect steady-state EET levels. The liver is a major site of cytochrome P450-mediated in vivo EET formation (30,42) and, as indicated by their regio-and stereochemical properties (37,42), the likely source of the EETs present in circulating plasma lipoproteins (43). Analysis of EET levels in the liver and plasma of Wy 14643-treated rats suggests that, in addition to phospholipid esterification (21), enzymatic hydration (20), and ␤-oxidation (22), the hepatic /-1-hydroxylation of these bioactive lipids could play important functional roles by altering either their bioactivity profiles and/or potency or by participating in their catabolism and disposition. As shown in Table VI  Rates of fatty acid /-1-hydroxylation by purified recombinant CYP4A isoforms The CYP4A hydroxylases were reconstituted in the presence of cytochrome b 5 , purified rat liver cytochrome P450 reductase, and 50 g/ml of dilauroylphosphatidylcholine. After 15 min at room temperature, the enzyme mixtures were incubated with the 1-14 C-labeled fatty acids in the presence of NADPH. Reaction products were resolved and quantified as described under "Materials and Methods."

TABLE III
Rates of EET /-1-hydroxylation by purified recombinant CYP4A isoforms The CYP4A EET /-1-hydroxylase activities were reconstituted in the presence of cytochrome b 5 , purified rat liver cytochrome P450 reductase, and 50 g/ml dilauroylphosphatidylcholine. After 15 min at room temperature, the enzyme mixtures were incubated with 1-14 Clabeled EETs in the presence of NADPH. Reaction products were resolved and quantified as described under "Materials and Methods." Initial rates were calculated from the linear portion of rate versus time of incubation plots, and are the means Ϯ S.E. of at least three different experiments. Isoform 8,9-EET 11,12-EET 14,15-EET and liver, and they were the most affected by Wy 14643 treatment, with their levels reduced to between 40 and 50% of control values. On the other hand, Wy 14643 caused only moderate reductions in plasma and liver 11,12-EET levels (88 and 73% of control values, respectively). The levels of 11,12-EET in plasma and liver were less than half the levels of 8,9-and 14,15-EETs (Table VI), and microsomal 11,12-EET metabolism was the least affected by anti-CYP4A1 and anti-CYP4A2 antibodies (Fig. 4). However, the interpretation of these in vivo studies is complicated by the facts that (a) as indicated above, /-1-hydroxylation is but one of the known routes for EET metabolism; and (b) Wy 14643 also causes down-regulation of liver CYP2C11, a known AA epoxygenase (45). Nevertheless, the data in Table VI show that Wy 14643 has profound effects on the levels of bioactive EETs in liver and plasma and indicate that /-1-hydroxylation may be an important component of the in vivo reactions that regulate the steady state of these metabolites and, presumably, their biological properties. These reactions may be of special relevance under pathophysiological conditions such as hypertension (14), diabetes (9 -13), and starvation (9 -13), all known to regulate CYP4A expression and/or PPAR␣ function (9 -13). It was recently demonstrated that chemical inhibition of cytosolic epoxide hydrolase reduces the blood pressure of hypertensive spontaneously hyperactive rats (46) and that targeted disruption of the gene coding for this enzyme has hypotensive effects in mice (47). These effects, attributed to increases in the levels of antihypertensive EETs caused by reduced enzymatic EET hydration and disposition, point to the potential functional importance of pathways that control the organ levels of bioactive EETs. However, it may also be possible that the HEETs have functions distinct from and/or more potent than their parent compounds. Current efforts to develop mass spectral methods for HEET quantification in biological samples will help to clarify the role of the /-1hydroxylases in EET disposition and/or bioactivity.
Binding of HEETs to PPAR␣-Although synthetic PPAR ligands such as thiazolidinediones and fibric acid derivatives have been used in the treatment of disease for some time, the discovery that their targets were the PPAR family of nuclear receptor transcription factors prompted a search for endogenous ligands (48 -51). Several eicosanoids, including 15-deoxy-⌬ 12,14 -prostaglandin J 2 , (8S)-HETE, and leukotriene B 4 , have been reported to bind to purified PPAR isoforms with affinities in the M to nM range (52)(53)(54)(55)(56)(57)(58). However, questions remain concerning the identities of endogenous ligands, as it is unknown whether some of these compounds are formed in vivo or achieve sufficiently high nuclear concentrations for PPAR activation. Data indicating that fatty acids bind to the different PPAR isoforms in the low M range (56 -58) suggest that the endogenous PPAR ligands may be fatty acid-derived. Samples of 20,14,15-HEET and of a mixture of 20,8,[9][10][11][12][13][14][15][16][17][18][19][20]11, were tested as ligands for hPPAR␣. As described under "Materials and Methods," binding to purified PPAR isoforms was estimated from the changes in cis-parinaric acid fluorescence caused by its ligand-induced displacement from the nuclear receptor ligand-binding domain (Fig. 5) (57,58). As shown in Table VII, the K i for cis-parinaric acid displacement from hPPAR␣ by 20,14,15-HEET is ϳ26-fold lower than that for Wy 14643. At saturating concentrations, 20,14,15-HEET displaced ϳ70% of the cis-parinaric acid bound to hPPAR␣ (Fig. 5). Although we could not obtain accurate K i values for the mixture of 20,8,9-and 20 -11,12-HEETs, its affinity for the hPPAR␣ LBD appears to be lower compared with 20,14,15-HEET (Table VII). A comparison of the binding properties of these metabolites with those of lauric acid and AA (two known CYP4A substrates), 20-HETE (the major product of AA metabolism by most CYP4A isoforms), and their metabolic precur- sors, the EETs, showed that HEETs bound to hPPAR␣ with at least an order of magnitude higher affinity (Table VII).
The relative affinity of 20,14,15-HETE for the hPPAR␣ LBD is comparable to that for (8S)-HETE, a potent ligand for human and mouse PPAR␣ (52,58). Murakami et al. (59) reported a 50-fold lower binding affinity for (8S)-HETE compared with Wy 14643 using a radiolabeled competitive binding assay and the hPPAR␣ LBD protein. Similar results for mPPAR␣ were reported by Forman et al. (56) using ligand-induced DNA binding assays. In the latter study, the K d value for (8S)-HETE was estimated to be 5-fold lower than that for Wy 14643 and 4-fold lower than that for the synthetic compound carbaprostacyclin (58). Another eicosanoid, leukotriene B 4 , was shown to be a weak ligand for mPPAR␣ and hPPAR␣ (55,59). In summary, the data in Table VII document 20,14,8,HETE and/or 20,11,12-HEET as high affinity ligands for hPPAR␣. A single human CYP4A isoform, CYP4A11, has been cloned, expressed, and shown to catalyze AA and lauric acid /-1-hydroxylation (1, 6). 2 Although CYP4A11 is the human homolog of rat CYP4A8, it is presently unknown whether this enzyme catalyzes the /-1-hydroxylation of EETs.
The ability of 20,14,15-HEET and the mixture of 20,8,9-and 20,11,12-HEETs (ϳ1:4) to activate PPAR␣ in transient transfection assays using RK13 cells was also studied. At 1 M, these compounds could transactivate a peroxisomal proliferator responsive element containing luciferase reporter via full-length hPPAR␣ and mPPAR␣ to levels similar to those seen with 100 M Wy 14643 (Fig. 6A). The HEETs activated not only the full-length receptor, but also a Gal4 chimeric transcription factor (Fig. 6B). The Gal4-mPPAR␣-LBD chimera activates its cognate reporter in the absence of the PPAR DNA-binding domain, and it does not require the retinoid X receptor, the PPAR dimerization partner, thus limiting interference by endogenous PPARs present in the cell line. When the Gal4-mPPAR␣-LBD chimera was used, 20,11,12-HEET (20 M) and 20,14,15-HEET (10 M) caused a 2-and 3-fold transactivation, respectively. Due to cellular toxicity, it was not possible to establish a maximum activation value for the HEETs. Nonetheless, the activation of the Gal4-mPPAR␣-LBD chimera clearly demonstrates that these compounds activate PPAR via binding to its LBD. Finally, under conditions similar to those used for Fig. 6, 50 M Wy 14643 caused a 12-fold (50 M) activation of the Gal4-mPPAR␣-LBD chimera (data not shown).
The high affinity and efficacy of the binding interactions between 20,14,15-HEET and hPPAR␣ suggest a role for this compound as an endogenous ligand for this receptor. Studies with PPAR␣ knockout mice have shown the key roles played by this receptor in peroxisomal fatty acid ␤-oxidation; mitochondrial fatty acid ␤-oxidation; microsomal fatty acid /-1-hydroxylation; lipoprotein metabolism; energy metabolism; and ultimately, hepatic lipolysis (31,60). Because CYP4A isoforms are positively regulated by PPAR␣, CYP4A-mediated HEET formation may mediate feedback regulation of PPAR␣-dependent gene transcription and thus provide a functional link between fatty acid /-1-hydroxylation and the regulation of lipid homeostasis. Indeed, studies with a mouse strain carrying disrupted copies of the genes coding for peroxisomal fatty-acid acyl-CoA oxygenase and PPAR␣ suggest a role for CYP4A isoforms in PPAR␣ signaling and liver lipid homeostasis (44).
In summary, these studies document a novel route for efficient EET oxidative metabolism and identify a new endogenous substrate for members of the rat CYP4A gene subfamily and a novel and potent agonist for PPAR␣. The results with AA and EETs indicate that mid-chain epoxidation increases catalytic turnover at the fatty acid /-1-position and that, because the  chemistry of the /-1-carbons remains unaltered, it also facilitates the proper alignment and/or proximity between the heme iron-bound reactive oxygen and the fatty acid acceptor carbon(s). Based on the high binding affinities displayed by the products of these reactions for hPPAR␣, we propose a role for these reactions in the regulation of transcriptional activities by this receptor.