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CARDIOVASCULAR

Characterization of 14,15-Epoxyeicosatrienoyl-Sulfonamides as 14,15-Epoxyeicosatrienoic Acid Agonists: Use for Studies of Metabolism and Ligand Binding

Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin (W.Y., B.B.H., X.-Y.Y., W.B.C.); and Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas (V.R.G., R.V.K.K., B.S., J.R.F.)

Received January 10, 2007; accepted February 26, 2007.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Epoxyeicosatrienoic acids (EETs) are cytochrome P450 epoxygenase metabolites of arachidonic acid. EETs mediate numerous biological functions. In coronary arteries, they regulate vascular tone by the activation of smooth muscle large-conductance, calcium-activated potassium (BK_Ca) channels to cause hyperpolarization and relaxation. We developed a series of 14,15-EET agonists, 14,15-EET-phenyliodosulfonamide (14,15-EET-PISA), 14,15-EET-biotinsulfonamide (14,15-EET-BSA), and 14,15-EET-benzoyldihydrocinnamide-sulfonamide (14,15-EET-BZDC-SA) as tools to characterize 14,15-EET metabolism and binding. Agonist activities of these analogs were characterized in precontraced bovine coronary arterial rings. All three analogs induced concentration-dependent relaxation and were equipotent with 14,15-EET. Relaxations to these analogs were inhibited by the BK_Ca channel blocker iberiotoxin (100 nM), the 14,15-EET antagonist 14,15-epoxyeicosa-5(Z)-enoylmethylsulfonamide (10 µM), and abolished by 20 mM extracellular K⁺. 14,15-EET-PISA is metabolized to 14,15-dihydroxyeicosatrienoyl-PISA by soluble epoxide hydrolase in bovine coronary arteries and U937 cells but not U937 cell membrane fractions. 14,15-EET-P¹²⁵ISA binding to human U937 cell membranes was time-dependent, concentration-dependent, and saturable. The specific binding reached equilibrium by 15 min at 4°C and remained unchanged up to 30 min. The estimated K_d and B_max were 148.3 ± 36.4 nM and 3.3 ± 0.5 pmol/mg protein, respectively. These data suggest that 14,15-EET-PISA, 14,15-EET-BSA, and 14,15-EET-BZDC-SA are full 14,15-EET agonists. 14,15-EET-P¹²⁵ISA is a new radiolabeled tool to study EET metabolism and binding. Our results also provide preliminary evidence that EETs exert their biological effect through a membrane binding site/receptor.

EETs are rapidly taken up into cells when applied exogenously (Snyder et al., 2002). Whether this uptake is through simple diffusion or a protein transporter is unclear. EETs are actively metabolized by multiple enzyme pathways. The major pathways are hydrolysis of the epoxide groups by soluble epoxide hydrolase (sEH) to the corresponding vicinal-dihydroxyeicosatrienoic acids (DHETs), -oxidation that results in chain-shortened products, and esterification into membrane phosphoglycerolipids (Spector et al., 2004). These studies were done with ¹⁴C- or ³H-labeled EETs synthesized from radiolabeled arachidonic acid. At present, no commercial company markets radiolabeled EETs. The synthesis of radiolabeled EETs from radiolabeled arachidonic acid is inefficient and costly because: 1) yields cannot exceed 50%, or diepoxides are formed; and 2) chemical methods produce all four EET regioisomers in varying amounts (Corey et al., 1979; Rosolowsky and Campbell, 1993). Thus, there is a need to produce radiolabeled EETs efficiently, cheaply and with high specific activity for studies of EET transport, metabolism, and binding.

To serve these purposes, we developed and characterized a series of stable 14,15-EET agonists, 14,15-EET-PISA, 14,15-EET-BSA, and 14,15-EET-BZDC-SA (see Fig. 2). These analogs contain an N-acylsulfonamide group instead of a terminal carboxylic acid. The N-acylsulfonamide group has a similar pK_a as a carboxyl group and obviates -oxidation and phospholipid esterification (Backes and Ellman, 1994; Snyder et al., 2002). For 14,15-EET-PISA, a benzyl ring that may be iodinated with [¹²⁵I] or [¹²⁷I] is attached to the sulfonamide group. For 14,15-EET-BSA and 14,15-EET-BZDC-SA, a biotin or BZDC moiety is covalently attached to the sulfonamide group through a benzyl linker. We characterized 14,15-EET-P¹²⁵ISA as a radioactive tracer to study EET metabolism and as a radioligand for studying the binding kinetics of the putative EET receptor/binding site. It was used as a model for studying the metabolism of other EET-sulfonamides.

View larger version (26K): [in this window] [in a new window]   Fig. 2. Structures of 14,15-EET and 14,15-EET-sulfonamides.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

View larger version (33K): [in this window] [in a new window]   Fig. 1. Synthesis of 14,15-EET-sulfonamides in Schemes 1, 2, and 3.

Synthesis of sulfonamide 4. A solution of 3-(tri-n-butylstannanyl)-benzenesulfonamide (3) (80 mg, 0.179 mmol) and 4-(dimethylamino)pyridine (20 mg, 0.157 mmol) in dry HMPA (1.5 ml) was cannulated into a solution of NHS-ester 2 (60 mg, 0.143 mmol) in HMPA (2.5 ml) under an argon atmosphere. After stirring for 10 min at room temperature, the reaction mixture was warmed to 65°C and allowed to stir for 16 h, then quenched by the addition of water (10 ml). The mixture was extracted with EtOAc (3 x 15 ml), and the combined extracts were washed with water, brine, dried over Na₂SO₄, and concentrated in vacuo. The residue was purified by SiO₂ column chromatography to give stannane 4 (38 mg, 36%). TLC: R_f 0.52 (30% EtOAc/hexanes); ¹H NMR (CDCl₃, 300 MHz) 0.86–0.93 (m, 12H), 1.11 (app t, J = 7.4 Hz, 6H), 1.26 –1.39 (m, 10H), 1.47–1.68 (m, 12H), 2.04 (dd, J = 7.0, 7.0 Hz, 2H), 2.17–2.26 (m, 3H), 2.43–2.51 (m, 1H), 2.69 –2.91 (m, 4H), 3.02–3.12 (m, 2H), 5.20 –5.61 (m, 6H), 7.40 –7.48 (m, 1H), 7.70 (dd, J_H,H = 8.4, J_Sn,H = 34.5 Hz,1H), 7.96 (dd, J = 1.8, 8.4 Hz,1H), 8.08 (dd, J_H,H = 1.8 Hz, J_Sn,H = 34.5 Hz, 1H), 8.99 (br s,1H).

Synthesis of iodide 5. A solution of sodium iodide (40 mg, 0.027 mmol) in water (1.0 ml) was added to a 0°C solution of stannane 4 (20 mg, 0.027 mmol) in 95% EtOH (4 ml) under an argon atmosphere followed by H₂O₂ (30% aq. soln, 24 µl, 0.213 mmol) and peracetic acid (32% in AcOH, 26 µl, 0.213 mmol). The reaction mixture was warmed to room temperature. After 2 h, the reaction mixture was quenched with saturated aqueous sodium thiosulfate (1 ml) and extracted with EtOAc (3 x 15 ml). The combined organic extracts were washed with water, brine, dried over Na₂SO₄, and concentrated in vacuo. The residue was purified by silica gel column chromatography to give 5 (9 mg, 58%). TLC: R_f 0.32 (30% EtOH/hexanes); ¹H NMR (CDCl₃, 400 MHz) 0.92 (t, J = 7 Hz, 3H), 1.32–1.69 (m, 1H), 2.04 (dd, J = 7.0, 7.0 Hz, 2H), 2.18 –2.26 (m, 3H), 2.46 –2.54 (m, 1H), 2.70 –2.94 (m, 4H), 3.06 –3.17 (m, 2H), 5.20 –5.60 (m, 6H), 7.27 (dd, J = 8.4, 8.4 Hz, 1H), 7.95 (dd, J = 1.8, 8.4 Hz, 1H), 7.96 (dd, J = 1.8, 8.4 Hz, 1H), 8.05 (dd, J = 1.8, 8.4 Hz, 1H), 8.33 (dd, J = 1.8, 1.8 Hz, 1H), 9.37 (br s, 1H).

Preparation of 14,15-EET-BZDC-SA (8) (Fig. 1, Scheme 2). Synthesis of aniline 6. n-Butyl lithium (0.21 ml of a 2.5 M solution in hexanes, 0.539 mmol) was added slowly to a –78°C solution of 4-aminobenzenesulfonamide (93 mg, 0.539 mmol) in anhydrous THF (8 ml) and HMPA (2 ml) under an argon atmosphere. After 5 min, a solution of NHS-ester 2 (150 mg, 0.359 mmol) in THF (4 ml) was added at –78°C, and then the mixture was slowly warmed to room temperature. After stirring overnight, the reaction mixture was quenched with saturated aq. NH₄Cl (5 ml) and extracted with EtOAc (3 x 20 ml). The combined organic extracts were washed with water, brine, dried over Na₂SO₄, and concentrated in vacuo. The residue was purified by SiO₂ column chromatography to give 6 (120 mg, 71%). TLC: R_f 0.34 (50% EtOAc/hexanes); ¹H NMR (CDCl₃, 400 MHz) 0.92 (t, J = 7.0 Hz, 3H), 1.32–1.60 (m, 8H), 1.63 (quintet, J = 7.3 Hz, 2H), 2.03 (dd, J = 7.3, 7.3 Hz, 2H), 2.18 –2.26 (m, 2H), 2.42–2.49 (m, 1H), 2.71–2.89 (m, 4H), 3.01–3.07 (m, 2H), 4.31 (br s, 2H), 5.25–5.54 (m, 6H), 6.65 (d, J = 8.6 Hz, 2H), 7.80 (d, J = 8.6 Hz, 2H), 9.01 (br s, 1H).

Synthesis of BZDC 8. A solution of 4-(benzoyl)benzenepropanoic acid NHS ester (Kort et al., 2000) (7) (22.3 mg, 0.063 mmol) and Et₃N (13 µl, 0.126 mmol) in DMF (2 ml) was added dropwise to a solution of 6 (20 mg, 0.042 mmol) in anhydrous DMF (2 ml) followed by 4-(dimethylamino)pyridine (7.7 mg, 0.063 mmol) under an argon atmosphere. After 48 h, the reaction mixture was quenched with water (3 ml), extracted with EtOAc (3 x 15 ml), and the combined organic extracts were washed with water, brine, dried over Na₂SO₄, and concentrated in vacuo. The residue was purified by silica gel column chromatography to yield 8 (13 mg, 30%). TLC: R_f 0.42 (20% EtOAc/CH₂Cl₂); ¹H NMR (CDCl₃, 400 MHz) 0.90 (t, J = 7.0 Hz, 3H), 1.31–1.62 (m, 8H), 1.63 (quintet, J = 7.3 Hz, 2H), 2.41–2.53 (m, 1H), 2.71–2.92 (m, 6H), 3.02–3.20 (m, 4H), 5.20 –5.60 (m, 6H), 7.34 (d, J = 8.2 Hz, 2H), 7.48 (t, J = 7.6 Hz, 2H), 7.56 –7.68 (m, 3H), 7.73 (d, J = 7.9 Hz, 2H), 7.78 (d, J = 7.9 Hz, 2H), 7.88 (d, J = 8.8 Hz, 2H), 8.21 (br s, 1H), 9.35 (br s, 1H).

Preparation of 14,15-EET-BSA (10) (Fig. 1, Scheme 3). Synthesis of acid 9. 4-Carboxybenzenesulfonamide (Aldrich Chemical Co., Milwaukee, WI; 130 mg, 0.358 mmol), dried azeotropically with anhydrous benzene and then dried under high vacuum for 2 h, was dissolved in anhydrous THF (6 ml) and HMPA (1.5 ml) and cooled to –78°C under an argon atmosphere. To this, n-butyl lithium (0.57 ml of a 2.5 M solution in hexanes, 0.862 mmol) was added, and after 10 min, a solution of 2 (180 mg, 0.431 mmol) in THF (4 ml) was slowly added. The reaction mixture was then slowly warmed to room temperature. After stirring overnight, the reaction mixture was quenched with saturated aqueous NH₄Cl solution (15 ml), extracted with EtOAc (3 x 30 ml), and the combined organic extracts were washed with water, brine, dried over Na₂SO₄, and concentrated in vacuo. The residue was purified by silica gel column chromatography to give acid 9 (85 mg, 71%). TLC: R_f 0.23 (50% EtOAc/hexanes); ¹H NMR (CDCl₃, 400 MHz) 0.91 (t, J = 7.0 Hz, 3H), 1.34 –1.60 (m, 8H), 2.02–2.12 (m, 2H), 2.26 (t, J = 8.5 Hz, 2H), 2.42–2.49 (m, 1H), 2.71–2.89 (m, 4H), 3.07–3.16 (m, 2H), 5.23–5.57 (m, 6H), 8.14 (d, J = 8.4 Hz, 2H), 8.23 (d, J = 8.6 Hz, 2H).

Synthesis of biotin 10. To a –78°C solution of 9 (50 mg, 0.099 mmol) in dry CH₂Cl₂ (3 ml) under an argon atmosphere were added 1-methylpiperidine (Aldrich; 12 µl, 0.099 mmol) followed by isobutyl chloroformate (Aldrich; 13 µl, 0.099 mmol). After stirring for 45 min at the same temperature, the reaction mixture was warmed to –25°C, stirred for another 30 min, and then a solution of biotin hydrazide (Sigma-Aldrich Co.; 100 mg, 0.39 mmol) in DMF (1 ml, prewarmed to 80°C to dissolve) was added. The reaction mixture was slowly warmed to room temperature over 1 h. After stirring at room temperature overnight, the reaction mixture was diluted with CH₂Cl₂ (10 ml), filtered, washed with water, and concentrated in vacuo. The residue was purified by SiO₂ PTLC to give 10 (15 mg, 38%) and recovered starting material (30 mg). TLC: R_f 0.45 (10% MeOH/CH₂Cl₂); ¹H NMR (CDCl₃, 300 MHz) 0.90 (t, J = 6.7 Hz, 3H), 1.28 –1.42 (m, 6H), 1.43–1.84 (m, 10H), 1.99 –2.10 (m, 2H), 2.25 (apparent t, J = 7.0 Hz, 2H), 2.28 –2.33 (m, 2H), 2.36 (apparent t, J = 6.8 Hz, 2H), 2.68 –2.78 (m, 4H), 2.84 (apparent t, J = 5.8 Hz, 2H), 2.91–3.01 (m, 2H), 3.18 –3.28 (m, 1H), 3.32 (br s, 1H, D₂O exchangeable), 3.90 (d, J = 6.4 Hz, 1H, D₂O exchangeable), 4.30–4.65 (m, 1H), 4.48–4.54 (m, 1H), 5.26 –5.40 (m, 4H), 5.42–5.60 (m, 2H), 8.04 (d, J = 8.5 Hz, 2H), 8.10 (d, J = 8.5 Hz, 2H).

Synthesis of 14,15-EET-P¹²⁵ISA and 14,15-DHET-P¹²⁵ISA
To synthesize 14,15-EET-P¹²⁵ISA, carrier-free Na¹²⁵I (2 µl, pH 9 –10, 200 µCi, 17.4 Ci/mg) (GE Healthcare, Arlington Heights, IL) was mixed with NaI (1 µl, 1.08 mg in 10 ml H₂O). 14,15-EET-stannane (4) (10 µgin43 µl of ethanol) was added to the NaI solution followed by H₂O₂ (10.67 nmol in 1.25 µl of H₂O) and peracetic acid (2.69 nmol in 1.68 µl of acetic acid) at 4°C (Fig. 1, scheme 1). The reaction mixture was warmed to room temperature and allowed to react for 45 min. On quenching with saturated aqueous Na₂S₂O₃ (250 µl), the aqueous layer was extracted with ethyl acetate (2 x 300 µl). The combined organic extracts were washed with H₂O (300 µl) and 0.9% NaCl (300 µl) and dried under N₂. The residues were purified by reverse-phase high-performance liquid chromatography (HPLC) as described previously (Campbell et al., 1996). The extracts were redissolved in 100 µl of acetonitrile/glacial acetic acid (999:1) and 100 µl of distilled H₂O and resolved on a Nucleosil C18 reverse-phase column (5 µm, 4.6 x 250 mm; Phenomenex, Torrance, CA). Solvent A was distilled H₂O, and solvent B was acetonitrile/glacial acetic acid (999:1). A linear gradient from 50% solvent B in solvent A to 94% solvent B within 40 min was used at a flow rate of 1 ml/min. Column effluent corresponding to synthetic 14,15-EET-PISA was collected, extracted, and dried under N₂.

To synthesize 14,15-DHET-P¹²⁵ISA, 14,15-EET-P¹²⁵ISA was dissolved in HEPES buffer containing 10 mM HEPES, 150 mM NaCl, 5 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, and 5.5 mM glucose, pH 7.4. Recombinant human sEH (35 µg) was added and incubated for 30 min at 37°C. The residues were extracted with ethyl acetate and purified by HPLC as described above. Specific activities of 14,15-EET-P¹²⁵ISA and 14,15-DHET-P¹²⁵ISA were 257.5 Ci/mmol.

Vascular Reactivity of Bovine Coronary Arteries
Bovine hearts were purchased from a local slaughterhouse. The left anterior descending coronary artery was dissected and cleaned of connective tissue. Arteries of 2-mm diameter were cut into rings (3-mm width) and suspended on a pair of stainless hooks in a 6-ml water-jacketed organ chamber in Krebs' buffer consisting of 119 mM NaCl, 4.8 mM KCl, 24 mM NaHCO₃, 0.2 mM KH₂PO₄, 0.2 mM MgSO₄, 11 mM glucose, 0.02 mM EDTA, and 3.2 mM CaCl₂. The buffer was equilibrated with 95% O₂-5% CO₂ and maintained at 37°C. Tension was continuously recorded as described previously (Campbell et al., 1996). In brief, submaximal concentrations of the thromboxane-mimetic U46619 [GenBank] (10 –20 nM; Cayman Chemical Co., Ann Arbor, MI) were administered to contract the vessels to 50 to 75% of KCl-induced contraction. Cumulative concentrations of 14,15-EET, 14,15-EET-PISA, 14,15-EET-BSA, or 14,15-EET-BZDC-SA were added to the chamber. In some studies, the arteries were incubated with iberiotoxin (IBTX; 100 nM; Sigma-Aldrich Co.), 14,15-epoxyeicosa-5(Z)-enoyl-methylsulfonamide (14,15-EEZE-mSA; 10 µM), or vehicle for 10 min before U46619 [GenBank] contraction (Gauthier et al., 2003). In the high extracellular potassium ([K⁺]_o) studies, K⁺ was increased to 20 mM by substitution of Na⁺. Results are expressed as percent relaxation with 100% relaxation representing basal tension.

Culture of U937 Cells and Membrane Preparation
U937 cells were cultured in suspension in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Hyclone, South Logan, UT), 25 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B. The cultures were maintained at a density of 5 to 10 x 10⁵ cells/ml at 37°C in a humidified atmosphere of 5% CO₂ in air. Membrane extraction was modified from previously reported protocol (Wong et al., 1993). Cells were collected by centrifugation at 1000g for 2 min and washed twice with Hanks' balanced salt solution (HBSS) containing 5 mM CaCl₂, 5 mM MgCl₂, and protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). The pellet was resuspended with HBSS (without CaCl₂ or MgCl₂) containing protease inhibitors and sonicated for 20-s bursts on ice five times at setting 4. The homogenate was centrifuged at 1000g for 10 min at 4°C to remove unbroken cells. The supernatant was then centrifuged at 150,000g for 45 min at 4°C. The pellet represents the membrane fraction and was resuspended in binding buffer consisting of 10 mM HEPES, 5 mM CaCl₂, 5 mM MgCl₂, and 5 mM EDTA, pH 7.4.

Bovine Coronary Artery Homogenate Preparation
Bovine coronary arteries (BCAs) were dissected as described above, cut into small pieces, and homogenized with a glass homogenizer in binding buffer containing miconazole (10 µM; Sigma-Aldrich Co.) and protease inhibitor cocktail (Roche) at 4°C. Unbroken tissue and cell debris were removed by centrifugation at 6000g for 5 min at 4°C. The supernatant represents the BCA homogenate that contains membrane and cytosolic fractions.

Tissue Incubation of 14,15-EET-P¹²⁵ISA
14,15-EET-P¹²⁵ISA was incubated and extracted using the following protocols: 1) 14,15-EET-P¹²⁵ISA (30 –33 nM) was incubated with 500 µg of BCA homogenate in binding buffer for 1 h at 30°C. The metabolites were extracted by ethyl acetate. In some incubations, 1 µM 1-cyclohexyl-3-dodecylurea (CDU), an sEH inhibitor (Morisseau et al., 2002), was included. 2) 14,15-EET-PISA (10 µM), with tracer amounts of 14,15-EET-P¹²⁵ISA, was incubated with BCA rings (1 g) in Krebs' buffer bubbled with 95% O₂-5% CO₂ for 15 min at 37°C. The metabolites were harvested by solid-phase extraction using the BondElut C₁₈ octadecylsilica column (Varian Inc., Harbor City, CA) as described previously (Nithipatikom et al., 2001). 3) 14,15-EET-P¹²⁵ISA (35 nM) was incubated with U937 cells (800,000 cells) in 200 µl of HBSS for 15 min at 37°C. The metabolites were extracted by liquid-liquid extraction using 500 µl of chloroform/methanol (1:2) followed by the addition of 167 µl of chloroform and 150 µl of H₂O. The organic phase was collected and dried under N₂. 4) 14,15-EET-P¹²⁵ISA (1 nM) was incubated with 50 µg of U937 cell membrane proteins in 200 µl of binding buffer for 15 min at 4°C. In some studies, incubations were performed with buffer alone or boiled membranes. The metabolites were extracted by the same liquid-liquid extraction protocol as for intact U937 cell incubations. The extracts were then analyzed by reverse-phase HPLC as described above. The column effluent was collected in 0.5-ml fractions, and radioactivity was measured by a Packard Cobra-II Auto-Gamma counter (PerkinElmer Life and Analytical Sciences, Boston, MA).

Mass Spectrometry Analysis
To identify the 14,15-EET-PISA metabolite, 14,15-EET-PISA was incubated with BCA homogenate and extracted as described above. The HPLC effluent (21 min) containing the 14,15-EET-PISA metabolite was collected, extracted with cyclohexane/ethyl acetate (50:50), and further analyzed by mass spectrometry (MS). The MS analysis used an Agilent 1100 MSD mass spectrometer (Agilent Technologies, Palo Alto, CA) with electrospray ionization. The residues were introduced into the electrospray chamber by flow injection. Solvent A was water, and solvent B was acetonitrile with both solvents containing 0.005% glacial acetic acid. Fifty percent of each solvent was used at a flow rate of 0.2 ml/min, and data were collected for 3 min. Detection was in the negative ion mode. Results are expressed as relative abundance, with the most abundant ion set as 100%.

Radioligand Binding
U937 membranes (50 µg) were incubated with 14,15-EET-P¹²⁵ISA in the presence of 20 µM 14,15-EEZE-mSA (nonspecific binding) or an equivalent amount of ethanol (total binding) at 4°C with shaking. For the time course study, 1 nM 14,15-EET-P¹²⁵ISA was incubated at various times (1–30 min). For the concentration-dependent study, increasing concentrations of 14,15-EET-P¹²⁵ISA (0.1–150 nM) were incubated for 15 min. Incubations (total volume, 200 µl) were performed in binding buffer and were terminated by filtration through GF/A glass filter paper on a Brandel harvester (Brandel Inc., Gaithersburg, MD). Filters were washed four times with 5 ml of ice-cold binding buffer and counted by a Packard Cobra-II Auto-Gamma counter. Specific binding was defined as total binding minus nonspecific binding. The K_d (equilibrium dissociation constant) and B_max (maximal binding site density) values are determined from saturation studies using nonlinear regression to fit the data to the single-site binding equation using Prism software (GraphPad Inc., San Diego, LA).

Statistical Analysis
Data are expressed as means ± S.E.M. Statistical evaluation was performed by a one-way analysis of variance followed by the Student-Newman-Keuls multiple comparison test when significant differences were present. P < 0.05 was considered statistically significant.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Previously, we characterized 14,15-EET-methylsulfonamide (14,15-EET-mSA; structure shown in Fig. 2) as a full agonist of 14,15-EET (Falck et al., 2003a). The N-acylmethylsulfonamide group has a similar pK_a as the carboxyl group (Backes and Ellman, 1994) and is a commonly used substitute for a carboxyl group (Imig et al., 2001; Snyder et al., 2002). Based on this knowledge, we developed 14,15-EET-PISA, 14,15-EET-BSA, and 14,15-EET-BZDC-SA, which contain an iodine, biotin, and BZDC group, respectively, attaching to the sulfonamide group through a benzyl linker (Fig. 2).

In bovine coronary arteries precontracted with U46619 [GenBank] , 14,15-EET causes concentration-dependent relaxation with an EC₅₀ of 1 µM and a maximum relaxation averaging 80 to 94% at 10 µM. Likewise, 14,15-EET-PISA, 14,15-EET-BSA, and 14,15-EET-BZDC-SA relaxed these arteries with an EC₅₀ of 1 µM and maximal relaxation of 84.5 ± 7.5, 89.6 ± 3.9, and 92.9 ± 5.0% at 10 µM, respectively (Fig. 3A). We tested the effect of K⁺ channel inhibition on these analog-mediated relaxations. Increasing extracellular K⁺ from 4 to 20 mM eliminated relaxations to 14,15-EET-PISA, 14,15-EET-BSA, and 14,15-EET-BZDC-SA (maximum relaxation = 7.8 ± 12.8, 8.6 ± 6.3, and 4.5 ± 3.2%, respectively, Fig. 3, B–D). The BK_Ca channel blocker, IBTX (100 nM), markedly attenuated the 14,15-EET-PISA, 14,15-EET-BSA, and 14,15-EET-BZDC-SA-induced relaxations (maximum relaxation = 39.3 ± 12.8, 20.8 ± 2.6, and 17.3 ± 6.1%, respectively, Fig. 3, B–D). We next examined the effect of 14,15-EEZE-mSA, a 14,15-EET antagonist (Gauthier et al., 2003), on analog-mediated relaxation. Pretreatment with 14,15-EEZE-mSA (10 µM) inhibited 14,15-EET-PISA, 14,15-EET-BSA, and 14,15-EET-BZDC-SA relaxations (maximal relaxation = 23.5 ± 9.1, 21.4 ± 13.1, and 34.7 ± 8.6%, Fig. 3, B–D). These results demonstrate that 14,15-EET-PISA, 14,15-EET-BSA, and 14,15-EET-BZDC-SA caused vascular relaxation by mechanisms similar to 14,15-EET, i.e., they cause relaxation by activating BK_Ca channels, and the relaxations are inhibited by the 14,15-EET antagonist.

View larger version (24K): [in this window] [in a new window]   Fig. 3. Effects of 14,15-EET and 14,15-EET-sulfonamides on vascular tone of U46619 preconstricted bovine coronary arteries. A, relaxations to 14,15-EET, 14,15-EET-PISA, 14,15-EET-BSA, and 14,15-EET-BZDC-SA. B to D, effects of the BKCa channel inhibitor, IBTX (100 nM); the EET antagonist, 14,15-EEZE-mSA (10 µM); or increased extracellular K+ (20 mM) on 14,15-EET-PISA (B), 14,15-EET-BSA (C), and 14,15-EET-BZDC-SA (D) relaxations. *, significantly different from control, P < 0.05.

14,15-EET is metabolized through three major pathways including -oxidation, epoxide hydration, and phospholipid esterification. 14,15-EET-mSA, the parent compound of 14,15-EET-PISA, does not undergo -oxidation or incorporation into membrane lipids (Snyder et al., 2002). It is reasonable to predict that 14,15-EET-mSA derived analogs are likewise not metabolized by these pathways. However, the 14,15-epoxide group is susceptible to hydration. We used 14,15-EET-P¹²⁵ISA as a radioactive tracer to study its metabolism in BCA homogenates. 14,15-EET-P¹²⁵ISA was incubated with buffer or BCA homogenate in the presence and absence of 1 µMCDU for 1 h at 30°C. The metabolites were extracted and resolved on reverse-phase HPLC. With buffer, 94.8% of radioactivity was extracted into the organic phase. The HPLC chromatogram showed one single peak at 29 min (peak 29), which comigrates with the synthetic 14,15-EET-PISA standard (Fig. 4A). After 30-min incubation with BCA homogenate at 30°C, an additional more polar metabolite migrating at 21 min (peak 21) was produced. The radioactivity ratio of peak 21 to peak 29 was 1.27 (Fig. 4B). In the presence of 1 µM CDU, an sEH inhibitor, the production of peak 21 was inhibited. The peak 21/peak 29 ratio was reduced to 0.22 (Fig. 4C). Similar metabolism was observed with 90 min incubation and similar inhibition was obtained using another sEH inhibitor, adamantyl dodecanoic acid urea (1 µM) (data not shown). This suggests that peak 21 is likely the sEH metabolite of 14,15-EET-PISA, 14,15-DHET-PISA. To identify this metabolite, peak 21 was collected, re-extracted, and analyzed by negative electrospray ionization MS. The most abundant ion in the mass spectrum of this peak was at m/z 602.1 (M-H) (Fig. 4E). This indicated a molecular weight of 603, which is consistent with the molecular weight of 14,15-DHET-PISA. Thus, peak 21 was identified as 14,15-DHET-PISA. We then tested 14,15-EET-P¹²⁵ISA metabolism by bovine coronary arterial rings using conditions under which the vascular activities were tested. After a 15-min incubation at 37°C, the majority of radioactivity eluted at 29 min, and a small portion of radioactivity was converted into peak 21. The peak 21/peak 29 ratio was 0.18 (Fig. 4D). These data indicate that 14,15-EET-PISA is hydrolyzed to 14,15-DHET-PISA by sEH in both BCA homogenate and arterial rings. The arterial rings showed less conversion. There was no evidence of the metabolism of 14,15-EET-PISA by -oxidation.

View larger version (35K): [in this window] [in a new window]   Fig. 4. Metabolism of 14,15-EET-P125ISA by BCA homogenate and arterial rings. A to C, HPLC chromatographs of extracted 14,15-EET-P125ISA metabolites after incubation with buffer, BCA homogenate (500 µg) in the presence or absence of CDU (1 µM) for 30 min at 30°C, respectively. D, HPLC chromatograph of extracted 14,15-EET-P125ISA metabolites after incubation with arterial rings for 15 min at 37°C. E. Negative ion electrospray ionization MS spectrum of 14,15-EET-PISA metabolite (peak 21).

Similar results were obtained when 14,15-EET-P¹²⁵ISA was incubated with U937 cells for 15 min at 37°C (Fig. 5A). The major metabolite eluted at 29 min, and a small radioactive product eluted at 21 min. Only the 29-min radioactive metabolite was observed when the cells were incubated at 4°C (data not shown). Thus, U937 cells metabolized 14,15-EET-PISA to 14,15-DHET-PISA, as did coronary arteries. Next, we investigated the stability of 14,15-EET-P¹²⁵ISA under the experimental conditions of radioligand binding. After incubation with U937 membranes at 4°C for 15 min, the majority of the radioactivity was extracted with an organic solvent, and the extraction efficiency was 93.6 ± 1.0% (n = 3). When 14,15-EET-P¹²⁵ISA was incubated with boiled and control U937 membranes, the percentage of radioactivity in the organic phase was 95.8 ± 0.1 and 95.6 ± 0.2%, respectively (n = 3). The HPLC chromatograms showed a single peak eluting at 29 min (Fig. 5, B–D). These results indicate that 14,15-EET-P¹²⁵ISA is metabolically stable under the conditions for radioligand binding.

View larger version (20K): [in this window] [in a new window]   Fig. 5. Metabolism of 14,15-EET-P125ISA by U937 cells and U937 membrane protein. A, 14,15-EET-P125ISA (35 nM) was incubated with intact U937 cells (800,000 cells) for 15 min at 37°C. B to D, 14,15-EET-P125ISA (1 nM) was incubated with buffer (B), boiled U937 membrane protein (C), or U937 membrane protein (50 µg, D) for 15 min at 4°C. HPLC chromatographs of extracted 14,15-EET-P125ISA metabolites from the incubations were shown.

To determine whether 14,15-EET-P¹²⁵ISA is a radioligand useful for the characterization of 14,15-EET binding, we used membrane fractions from U937 cells, a cell line that was reported to contain an EET binding site (Wong et al., 1997). To test whether 14,15-EET-P¹²⁵ISA binds to U937 membrane in a time-dependent manner, 1 nM 14,15-EET-P¹²⁵ISA was incubated with U937 membranes (50 µg of protein) at 4°C for 1 to 30 min. The binding of 14,15-EET-P¹²⁵ISA to U937 membranes was rapid and specific. Equilibrium was reached in 15 min and remained stable until 30 min (Fig. 6A). Then, we tested the saturability of the specific 14,15-EET-P¹²⁵ISA binding. Various concentrations of 14,15-EET-P¹²⁵ISA (0.1–150 nM) were incubated with U937 membrane (50 µg of protein) at 4°C for 15 min. 14,15-EET-P¹²⁵ISA-specific binding was concentration-dependent and reached an apparent plateau at 100 nM (Fig. 6B). The data were fit to a one-site saturation model, and the predicted K_d and B_max were 148.3 ± 36.4 nM and 3.3 ± 0.5 pmol/mg protein, respectively. To confirm the specificity of this binding site to 14,15-EET-PISA, the binding of 14,15-DHET-P¹²⁵ISA to U937 membranes was determined. Specific binding of 14,15-DHET-P¹²⁵ISA to U937 membranes was very low with a maximum binding of 0.3 pmol/mg protein (Fig. 6C). The data poorly fit with saturation models. This suggests that 14,15-DHET-PISA does not specifically bind to U937 membranes. Therefore, the 14,15-epoxide group is required for 14,15-EET-P¹²⁵ISA binding to U937 membranes. Taken together, binding of 14,15-EET-P¹²⁵ISA to U937 membranes is specific, time-dependent, concentration-dependent, and saturable.

View larger version (12K): [in this window] [in a new window]   Fig. 6. 14,15-EET-P125ISA binding to U937 membrane protein. A, time course of 14,15-EET-P125ISA binding. 14,15-EET-P125ISA (1 nM) was incubated with 50 µg of protein at 4°C for various time (1–30 min). n = 2. B, concentration-dependent 14,15-EET-P125ISA binding. Increasing concentrations of 14,15-EET-P125ISA (0.1–150 nM) were incubated with 50 µg of protein at 4°C for 15 min. n = 2–7. C, 14,15-DHET-P125ISA binding to U937 membranes. Increasing concentrations of 14,15-DHET-P125ISA (2.5–100 nM) were incubated with 50 µg of protein at 4°C for 15 min. n = 2–3.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Our present study developed and characterized a series of stable 14,15-EET-mSA derivatives, 14,15-EET-PISA, 14,15-EET-BSA, and 14,15-EET-BZDC-SA, as tools to study EET transport, metabolism, and binding. The analogs caused concentration-dependent relaxation of bovine coronary arteries and retained full agonist potency and activity compared with 14,15-EET. Attachment of a bulky group at the C1 of 14,15-EET did not alter its vascular activity. The relaxations to the analogs were blocked by inhibition of K⁺ efflux by 20 mM extracellular K⁺ and BK_Ca channel blockade by iberiotoxin. These data suggest that these agonists, like 14,15-EET, mediate vascular relaxation by activation of BK_Ca channels and membrane hyperpolarization. 14,15-EEZE-mSA, a known 14,15-EET antagonist, inhibited the relaxations (Gauthier et al., 2003). It is likely that these agonists bind to the same binding site or receptor as 14,15-EET and act through the similar cellular mechanisms as 14,15-EET.

EET-mediated activation of vascular smooth muscle BK_Ca channels requires active Gs protein and may involve ADP-ribosylation of Gs (Li and Campbell, 1997; Li et al., 1999). Likewise, Gs is required for 11,12-EET-mediated activation of BK_Ca channels expressed in human embryonic kidney 293 cells (Fukao et al., 2001). Because G proteins characteristically couple to membrane receptors, it is likely that EETs initiate their signaling events by binding to a G protein-coupled receptor. Node et al. (2001) showed that 11,12-EET stimulated GTP-binding activity of Gs in human saphenous vein endothelial cells and activated endothelial t-PA gene expression through a cAMP-dependent mechanism. Taken together, this suggests that EET may mediate their cellular events through a G protein-coupled receptor signaling pathway. However, characterization and purification of such a membrane receptor/binding site have been limited due to lack of proper tools. In addition, the mechanisms of EETs uptake and secretion by cells are unclear. Many eicosanoids such as prostaglandins (Kanai et al., 1995), leukotrienes (Leier et al., 1994), and arachidonic acid (Krischer et al., 1998) have been described to enter and leave cells through transporter-facilitated diffusion. It is unknown whether a similar transporter is present for EETs.

We designed 14,15-EET-mSA derivatives as tools to study EET transport, metabolism, and binding. Each agonist contains a functional group, i.e., a biotin, a photolinking reagent, or a radioiodinated group. With aid of convenient avidin conjugates such as agarose beads and fluorophore conjugates, 14,15-EET-BSA can be used to pull down an EET binding protein or image 14,15-EET binding sites. Upon ultraviolet radiation, 14,15-EET-BZDC-SA cross-links with molecules in close proximity. This reaction may add a tag to the target molecule, which may be detected by mass spectrometric analysis. 14,15-EET-PISA can be radiolabeled with [¹²⁵I] to provide a new radioactive 14,15-EET agonist. Radioactive EET analogs are valuable tools to study their metabolism and binding.

Currently, radiolabeled 14,15-EET is made by epoxidation of ¹⁴C- or ³H-labeled arachidonic acid, which results in all four EET regioisomers and less than 25% production of 14,15-EET (Corey et al., 1979; Rosolowsky and Campbell, 1993). In contrast, radioiodination of 14,15-EET-P¹²⁵ISA from 14,15-EET-stannane is specific and efficient. In addition, [¹²⁵I] has higher specific activity than [³H], which results in increased sensitivity for radioligand binding assays. Therefore, 14,15-EET-P¹²⁵ISA is a radioactive 14,15-EET agonist with high specific activity, low production cost, and efficient yields.

-Oxidation and phospholipid esterification of 14,15-EET require the C-1 carboxyl group. Other routes of 14,15-EET metabolism include sEH hydration, cytochrome P450 -hydroxylation (Cowart et al., 2002), and glutathione conjugation (Spearman et al., 1985) and are independent of the C-1 carboxyl group. Therefore, 14,15-EET-P¹²⁵ISA is a useful tool to study C1-carboxyl-independent metabolism of 14,15-EET. Like 14,15-EET, 14,15-EET-PISA is hydrolyzed into its corresponding 14,15-DHET-PISA. This metabolism is effectively inhibited by sEH inhibitors. Among the tested tissues, the hydration was most active in BCA homogenate where sEH is exposed, markedly less active in BCA rings and U937 cells where sEH is intracellular and less accessible, and absent in U937 membrane fractions where sEH is not localized. These data indicate that 14,15-EET-PISA is actively metabolized into 14,15-DHET-PISA by sEH.

Previously in the human leukemic monocyte lymphoma cell line U937 cells, a 14,15-EET binding site was characterized by whole-cell radioligand binding using [³H]14,15-EET. The K_d and B_max were 13.84 ± 2.58 nM and 3.54 ± 0.28 pmol/10⁶ cells, respectively (Wong et al., 1997). We used U937 cell membrane fraction to test 14,15-EET-P¹²⁵ISA binding. 14,15-EET-P¹²⁵ISA remained stable and unchanged when it was incubated with U937 membrane proteins at 4°C for 15 min. In agreement with the [³H]14,15-EET whole-cell binding data, 14,15-EET-P¹²⁵ISA bound to U937 membranes in a time-dependent, concentration-dependent, and saturable manner. Interestingly, the binding of 14,15-EET-P¹²⁵ISA to U937 membranes was with a lower affinity and lower receptor density. 14,15-EET-P¹²⁵ISA binding to U937 membranes reached equilibrium by 15 min and remained unchanged to 30 min at 4°C. The K_d was 148.3 ± 36.4 nM, which was 10-fold higher than that observed with [³H]14,15-EET in whole cells. The B_max of 14,15-EET-P¹²⁵ISA binding to U937 membranes was 3.3 ± 0.5 pmol/mg protein, which was lower than that observed in whole cells. The reason for these discrepant data could be due to esterification of [³H]14,15-EET into phospholipids or conjugation to cellular components. This could contribute to the observed binding in whole cells, whereas these processes are absent or less active in membrane fractions. It is also possible that intracellular components maintain the binding sites in a conformation that favors binding to the ligand. This intracellular factor is missing in extracted membranes, which may change the receptor conformation to a ligand-unfavorable form. In other words, a smaller portion of the receptors in the membrane fractions than whole cell is available for the ligand, which results in a decreased receptor density. In addition, ligands differ in affinity. The addition of the sulfonamide group to C-1 may reduce the affinity for the binding site. The variability between U937 cell lines could also account for the discrepancies. The 14,15-EET-P¹²⁵ISA binding requires the 14,15-epoxide because its hydrolyzed product, 14,15-DHET-P¹²⁵ISA, did not bind to U937 membranes. Taken together, our binding data validate the use of 14,15-EET-P¹²⁵ISA as a radiolabeled tool to study 14,15-EET binding.

EETs are key regulators of vascular tone and active mediators in pathophysiologic conditions such as anti-inflammation, fibrinolysis, angiogenesis, and ischemic reperfusion protection (Seubert et al., 2004; Nithipatikom et al., 2006). However, whether they initiate their signaling through binding to specific protein(s)/receptor(s) is unclear. 14,15-EET-PISA, 14,15-EET-BSA, and 14,15-EET-BZDC-SA are 14,15-EET agonists that serve as tools to identify and purify EET binding protein(s). 14,15-EET-P¹²⁵ISA, in particular, is a radioactive tracer to study EET metabolism and a radioligand to characterize EET binding kinetics.

	Acknowledgements

 
We thank Dr. Bruce Hammock for the kind gift of recombinant sEH and sEH inhibitors, Dr. Kathy Gauthier for review of the manuscript, and Gretchen Barg for secretarial assistance.

	Footnotes

 
This work was supported by the National Institutes of Health (Grants HL-51055 and GM-31278) and by the Robert A. Welch Foundation.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.119651.

ABBREVIATIONS: EET, epoxyeicosatrienoic acid; BK_Ca, large-conductance, calcium-activated potassium channel; sEH, soluble epoxide hydrolase; DHET, dihydroxyeicosatrienoic acid; 14,15-EET-PISA, 14,15-EET-phenyliodosulfonamide; 14,15-EET-BSA, 14,15-EET-biotinsulfonamide; 14,15-EET-BZDC-SA, 14,15-EET-benzoyldihydrocinnamide-sulfonamide; THF, tetrahydrofuran; NHS, N-hydroxysuccinimide; TLC, thin layer chromatography; HPLC, high-performance liquid chromatography; IBTX, iberiotoxin; 14,15-EEZE-mSA, 14,15-epoxyeicosa-5(Z)-enoyl-methylsulfonamide; HBSS, Hanks' balanced salt solution; BCA, bovine coronary artery; CDU, 1-cyclohexyl-3-dodecylurea; MS, mass spectrometry; 14,15-EET-mSA, 14,15-EET-methylsulfonamide; U46619 [GenBank] , 9,11-dideoxy-9, 11-methanoepoxy-prostaglandin F₂; HMPA, hexamethyl phosphoramide; DMF, N,N-dimethylformamide.

Address correspondence to: Dr. William B. Campbell, Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226. E-mail: wbcamp{at}mcw.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

Backes BJ and Ellman JA (1994) Carbon-carbon bond-forming methods on solid support: utilization of Kenner's "safety-catch" linker. J Am Chem Soc 116: 11171–11172.[CrossRef]

Campbell WB, Gebremedhin D, Pratt PF, and Harder DR (1996) Identification of epoxyeicosatrienoic acids as endothelium-derived hyperpolarizing factors. Circ Res 78: 415–423.[Abstract/Free Full Text]

Capdevila J, Parkhill L, Chacos N, Okita R, Masters BS, and Estabrook RW (1981) The oxidative metabolism of arachidonic acid by purified cytochromes P-450. Biochem Biophys Res Commun 101: 1357–1363.[CrossRef][Medline]

Corey EJ, Niwa H, and Falck JR (1979) Selective epoxidation of eicosa-cis-5,8,11,14-tetraenoic (arachidonic) acid and eicosa-cis-8,11,14-trienoic acid. J Am Chem Soc 101: 586–587.

Cowart LA, Wei S, Hsu MH, Johnson EF, Krishna MU, Falck JR, and Capdevila JH (2002) The CYP4A isoforms hydroxylate epoxyeicosatrienoic acids to form high affinity peroxisome proliferator-activated receptor ligands. J Biol Chem 277: 35105–35112.[Abstract/Free Full Text]

Falck JR, Krishna UM, Reddy YK, Kumar PS, Reddy KM, Hittner SB, Deeter C, Sharma KK, Gauthier KM, and Campbell WB (2003a) Comparison of vasodilatory properties of 14,15-EET analogs: structural requirements for dilation. Am J Physiol 284: H337–H349.

Falck JR, Reddy LM, Reddy YK, Bondlela M, Krishna UM, Ji Y, Sun J, and Liao JK (2003b) 11,12-epoxyeicosatrienoic acid (11,12-EET): structural determinants for inhibition of TNF-alpha-induced VCAM-1 expression. Bioorg Med Chem Lett 13: 4011–4014.[CrossRef][Medline]

Fisslthaler B, Popp R, Kiss L, Potente M, Harder DR, Fleming I, and Busse R (1999) Cytochrome P450 2C is an EDHF synthase in coronary arteries. Nature (Lond) 401: 493–497.[CrossRef][Medline]

Fukao M, Mason HS, Kenyon JL, Horowitz B, and Keef KD (2001) Regulation of BK(Ca) channels expressed in human embryonic kidney 293 cells by epoxyeicosatrienoic acid. Mol Pharmacol 59: 16–23.[Abstract/Free Full Text]

Fulton D, Mahboubi K, McGiff JC, and Quilley J (1995) Cytochrome P450-dependent effects of bradykinin in the rat heart. Br J Pharmacol 114: 99–102.[Medline]

Gauthier KM, Edwards EM, Falck JR, Reddy DS, and Campbell WB (2005) 14,15-epoxyeicosatrienoic acid represents a transferable endothelium-dependent relaxing factor in bovine coronary arteries. Hypertension 45: 666–671.[Abstract/Free Full Text]

Gauthier KM, Falck JR, Reddy LM, and Campbell WB (2004) 14,15-EET analogs: characterization of structural requirements for agonist and antagonist activity in bovine coronary arteries. Pharmacol Res 49: 515–524.[CrossRef][Medline]

Gauthier KM, Jagadeesh SG, Falck JR, and Campbell WB (2003) 14,15-epoxyeicosa-5(Z)-enoic-mSI: a 14,15- and 5,6-EET antagonist in bovine coronary arteries. Hypertension 42: 555–561.[Abstract/Free Full Text]

Huang A, Sun D, Jacobson A, Carroll MA, Falck JR, and Kaley G (2005) Epoxyeicosatrienoic acids are released to mediate shear stress-dependent hyperpolarization of arteriolar smooth muscle. Circ Res 96: 376–383.[Abstract/Free Full Text]

Imig JD, Falck JR, Wei S, and Capdevila JH (2001) Epoxygenase metabolites contribute to nitric oxide-independent afferent arteriolar vasodilation in response to bradykinin. J Vasc Res 38: 247–255.[CrossRef][Medline]

Kanai N, Lu R, Satriano JA, Bao Y, Wolkoff AW, and Schuster VL (1995) Identification and characterization of a prostaglandin transporter. Science (Wash DC) 268: 866–869.[Abstract/Free Full Text]

Kort Md, Luijendijk J, Marel GAvd, and Boom JHv (2000) Synthesis of photoaffinity derivatives of adenophostin A. Eur J Org Chem 2000: 3085–3092.[CrossRef]

Kozak W, Kluger MJ, Kozak A, Wachulec M, and Dokladny K (2000) Role of cytochrome P-450 in endogenous antipyresis. Am J Physiol 279: R455–R460.

Krischer SM, Eisenmann M, and Mueller MJ (1998) Transport of arachidonic acid across the neutrophil plasma membrane via a protein-facilitated mechanism. Biochemistry 37: 12884–12891.[CrossRef][Medline]

Leier I, Jedlitschky G, Buchholz U, Cole SP, Deeley RG, and Keppler D (1994) The MRP gene encodes an ATP-dependent export pump for leukotriene C4 and structurally related conjugates. J Biol Chem 269: 27807–27810.[Abstract/Free Full Text]

Li PL and Campbell WB (1997) Epoxyeicosatrienoic acids activate K⁺ channels in coronary smooth muscle through a guanine nucleotide binding protein. Circ Res 80: 877–884.[Abstract/Free Full Text]

Li PL, Chen CL, Bortell R, and Campbell WB (1999) 11,12-Epoxyeicosatrienoic acid stimulates endogenous mono-ADP-ribosylation in bovine coronary arterial smooth muscle. Circ Res 85: 349–356.[Abstract/Free Full Text]

Michaelis UR and Fleming I (2006) From endothelium-derived hyperpolarizing factor (EDHF) to angiogenesis: epoxyeicosatrienoic acids (EETs) and cell signaling. Pharmacol Ther 111: 584–595.[CrossRef][Medline]

Morisseau C, Goodrow MH, Newman JW, Wheelock CE, Dowdy DL, and Hammock BD (2002) Structural refinement of inhibitors of urea-based soluble epoxide hydrolases. Biochem Pharmacol 63: 1599–1608.[CrossRef][Medline]

Nithipatikom K, Grall AJ, Holmes BB, Harder DR, Falck JR, and Campbell WB (2001) Liquid chromatographic-electrospray ionization-mass spectrometric analysis of cytochrome P450 metabolites of arachidonic acid. Anal Biochem 298: 327–336.[CrossRef][Medline]

Nithipatikom K, Moore JM, Isbell MA, Falck JR, and Gross GJ (2006) Epoxyeicosatrienoic acids in cardioprotection: ischemic versus reperfusion injury. Am J Physiol 291: H537–H542.

Node K, Huo Y, Ruan X, Yang B, Spiecker M, Ley K, Zeldin DC, and Liao JK (1999) Anti-inflammatory properties of cytochrome P450 epoxygenase-derived eicosanoids. Science (Wash DC) 285: 1276–1279.[Abstract/Free Full Text]

Node K, Ruan XL, Dai J, Yang SX, Graham L, Zeldin DC, and Liao JK (2001) Activation of Galpha s mediates induction of tissue-type plasminogen activator gene transcription by epoxyeicosatrienoic acids. J Biol Chem 276: 15983–15989.[Abstract/Free Full Text]

Pinto A, Abraham NG, and Mullane KM (1987) Arachidonic acid-induced endothelial-dependent relaxations of canine coronary arteries: contribution of a cytochrome P-450-dependent pathway. J Pharmacol Exp Ther 240: 856–863.[Abstract/Free Full Text]

Rosolowsky M and Campbell WB (1993) Role of PGI2 and epoxyeicosatrienoic acids in relaxation of bovine coronary arteries to arachidonic acid. Am J Physiol 264: H327–H335.[Medline]

Rosolowsky M and Campbell WB (1996) Synthesis of hydroxyeicosatetraenoic (HETEs) and epoxyeicosatrienoic acids (EETs) by cultured bovine coronary artery endothelial cells. Biochim Biophys Acta 1299: 267–277.[Medline]

Seubert J, Yang B, Bradbury JA, Graves J, Degraff LM, Gabel S, Gooch R, Foley J, Newman J, Mao L, et al. (2004) Enhanced postischemic functional recovery in CYP2J2 transgenic hearts involves mitochondrial ATP-sensitive K⁺ channels and p42/p44 MAPK pathway. Circ Res 95: 506–514.[Abstract/Free Full Text]

Snyder GD, Krishna UM, Falck JR, and Spector AA (2002) Evidence for a membrane site of action for 14,15-EET on expression of aromatase in vascular smooth muscle. Am J Physiol 283: H1936–H1942.

Spearman ME, Prough RA, Estabrook RW, Falck JR, Manna S, Leibman KC, Murphy RC, and Capdevila J (1985) Novel glutathione conjugates formed from epoxyeicosatrienoic acids (EETs). Arch Biochem Biophys 242: 225–230.[CrossRef][Medline]

Spector AA, Fang X, Snyder GD, and Weintraub NL (2004) Epoxyeicosatrienoic acids (EETs): metabolism and biochemical function. Prog Lipid Res 43: 55–90.[CrossRef][Medline]

Wong PY, Lai PS, and Falck JR (2000) Mechanism and signal transduction of 14 (R), 15 (S)-epoxyeicosatrienoic acid (14,15-EET) binding in guinea pig monocytes. Prostaglandins Other Lipid Mediat 62: 321–333.[CrossRef][Medline]

Wong PY, Lai PS, Shen SY, Belosludtsev YY, and Falck JR (1997) Post-receptor signal transduction and regulation of 14(R), 15(S)-epoxyeicosatrienoic acid (14,15-EET) binding in U-937 cells. J Lipid Mediat Cell Signal 16: 155–169.[CrossRef][Medline]

Wong PY, Lin KT, Yan YT, Ahern D, Iles J, Shen SY, Bhatt RK, and Falck JR (1993) 14(R),15(S)-epoxyeicosatrienoic acid (14(R),15(S)-EET) receptor in guinea pig mononuclear cell membranes. J Lipid Mediat 6: 199–208.[Medline]

Yang W, Gauthier KM, Reddy LM, Sangras B, Sharma KK, Nithipatikom K, Falck JR, and Campbell WB (2005) Stable 5,6-epoxyeicosatrienoic acid analog relaxes coronary arteries through potassium channel activation. Hypertension 45: 681–686.[Abstract/Free Full Text]

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