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(Circulation Research. 1996;79:784-793.)
© 1996 American Heart Association, Inc.

Articles

Functional Implications of a Newly Characterized Pathway of 11,12-Epoxyeicosatrienoic Acid Metabolism in Arterial Smooth Muscle

Xiang Fang, Terry L. Kaduce, Neal L. Weintraub, Mike VanRollins, Arthur A. Spector

the Departments of Biochemistry (X.F., T.L.K., A.A.S.), Internal Medicine (N.L.W., M.V.R., A.A.S.), and Pharmacology (A.A.S.), University of Iowa, Iowa City.

Correspondence to Dr Arthur A. Spector, Department of Biochemistry, 4-403 BSB, University of Iowa, Iowa City, IA 52242.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Epoxyeicosatrienoic acids (EETs) are potent vasodilators derived from cytochrome P-450 metabolism of arachidonic acid. The rapid conversion of EETs to their corresponding dihydroxyeicosatrienoic acids (DHETs) has been proposed as a process whereby EETs are rendered biologically inactive. However, the vascular metabolism of EETs and the vasoactivities of EET metabolites have not been extensively studied. Accordingly, 11,12-EET metabolism was characterized in porcine aortic smooth muscle cells. The cells converted [³H]11,12-EET to 11,12-DHET and to a newly identified metabolite, 7,8-dihydroxy-hexadecadienoic acid (DHHD). 11,12-DHET accumulation in the medium reached a maximum in 2 to 4 hours and then declined, whereas 7,8-DHHD accumulation increased continuously and exceeded the amount of 11,12-DHET by 8 hours. [³H]11,12-EET conversion to radiolabeled 7,8-DHHD was reduced in the presence of unlabeled 11,12-DHET, indicating that 11,12-DHET is an intermediate in the conversion of 11,12-EET to 7,8-DHHD. This is consistent with a pathway whereby 11,12-EET is converted by an epoxide hydrolase to 11,12-DHET, which then undergoes two ß-oxidations to form 7,8-DHHD. In porcine coronary artery rings contracted with a thromboxane mimetic, 11,12-DHET produced relaxation similar in magnitude to that produced by 11,12-EET (77% versus 64% relaxation at 5 µmol/L, respectively). 7,8-DHHD also produced vasorelaxation. Thus, the vasoactivity of 11,12-EET is not eliminated by conversion to 11,12-DHET and 7,8-DHHD. These results suggest that 11,12-DHET and its metabolite, 7,8-DHHD, may contribute to the regulation of vascular tone in the porcine coronary artery and possibly other vascular tissues.

Key Words: epoxyeicosatrienoic acid • porcine aortic smooth muscle cell • dihydroxyeicosatrienoic acid • cytochrome P-450 • porcine coronary artery

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Arachidonic acid is converted to four EET regioisomers, 5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET, in a monooxygenase reaction catalyzed by cytochrome P-450 epoxygenase.^{1 2 3} EETs dilate systemic blood vessels,^{4 5 6 7 8} producing half-maximal relaxation at a concentration of 1 µmol/L in coronary arteries.⁹ EET-induced vasodilitation has been reported to result, in large part, from activation of Ca²⁺-activated K⁺ channels in vascular smooth muscle cells.^{10 11 12 13} K⁺ channel activation is also the mechanism of vasorelaxation attributed to EDHF,^{14 15} an unidentified substance of endothelial origin that may participate in the regulation of vascular resistance.¹⁶ Indeed, observations that inhibitors of cytochrome P-450 enzyme activity attenuate EDHF-mediated relaxations in a variety of preparations^{13 17 18 19} support the hypothesis that EETs account for the activity of EDHF.^{16 20} Furthermore, recent studies have demonstrated by mass spectrometry that bovine aortic endothelial cultures synthesize and release all four EET regioisomers when incubated with arachidonic acid.²¹

The mechanisms by which EETs are inactivated within the vasculature are unknown. Studies in intact dogs²² and cultured vascular endothelial cells²³ suggest that EETs are rapidly converted to the corresponding DHETs, a process proposed to inactivate EETs.^{24 25} Whether blood vessels convert EETs exclusively to DHETs and whether DHETs lack vasorelaxant properties have not been thoroughly investigated.

Recently, cultured porcine aortic smooth muscle cells were reported to metabolize [³H]11,12-EET* not only to 11,12-DHET but also to an unidentified metabolite (hereafter referred to as compound X).²⁴ Whereas the formation of 11,12-DHET occurred rapidly, the formation of compound X occurred slowly, suggesting that compound X may be produced through the cellular metabolism of 11,12-DHET.²⁴ The existence of such a product suggests that the functional inactivation of 11,12-EET within the vasculature might entail processes in addition to conversion to 11,12-DHET. Alternatively, 11,12-DHET or compound X might themselves possess significant vasoactivity.

The purpose of the present study was to determine (1) the chemical structure of compound X, (2) whether it is derived from 11,12-DHET metabolism, and (3) whether 11,12 DHET and/or compound X possesses vasoactivity. Portions of these results have been published in abstract form.²⁶

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
DMEM, MEM nonessential amino acids, MEM vitamin solution, HEPES, and trypsin were obtained from GIBCO. FBS was purchased from HyClone Laboratories; L-glutamine and U46619, from Sigma Chemical Co; and gentamicin, from Schering Corp. Radiolabeled arachidonic acid was obtained from American Radiolabeled Chemicals Inc and Amersham Corp. EETs and DHETs were obtained from Cayman Chemical Co; fatty acid–free bovine serum albumin, from Miles Laboratories Inc; phospholipid standards, from Avanti Polar Lipids Inc; and Whatman LK5D silica gel TLC plates and silica gel G TLC plates, from Alltech Associates Inc.

Synthesis of [³H]11,12-EET and [1-¹⁴C]11,12-EET
Arachidonic acid was mixed with either [5,6,8,9,11,12,14,15-³H]arachidonic acid to a specific activity of 304 Ci/mol or [1-¹⁴C]arachidonic acid to a specific activity of 13.6 Ci/mol and then methylated with diazomethane.^{23 24 27} 11,12-EET was synthesized, along with the other epoxide regioisomers, as a racemic mixture from the arachidonic acid methyl esters.²⁸ In brief, the arachidonic acid methyl esters were suspended in CH₂Cl₂, and 0.2 equivalents of m-chloroperoxybenzoic acid in CH₂Cl₂ were added dropwise over 1 minute. The solution was mixed for 20 minutes at room temperature, and ice-cold aqueous NaHCO₃ was added. After centrifugation to remove the m-chlorobenzoate, the CH₂Cl₂ phase was washed with water and evaporated under N₂. Recovery of the fatty acid products was 91% to 97% by radioassay.

The epoxide methyl ester products were isolated by normal-phase HPLC using a 4.6x250-mm column packed with 5-µm particles of silicic acid (Ultremex Si, Phenomenex). Products were eluted in hexane/isopropanol (6000:1 [vol/vol]) at a flow rate of 1.5 mL/min. Unreacted arachidonic acid and the EET methyl esters were detected by monitoring absorbance at 192 nm. Under these conditions, methyl arachidonate eluted at 4.5 minutes, whereas the methyl esters of 8,9-EET, 11,12-EET, and 14,15-EET eluted between 14.6 and 16.4 minutes.

The EET methyl esters were saponified with methanolic 0.04N KOH for 16 hours at 25°C. After the pH was adjusted to 8.0 with 1 mmol/L Na₂HPO₄ buffer, pH 6.0, the compounds were extracted with 19 vol of ice-cold ethyl acetate saturated with water. The resulting epoxy fatty acids were identified by isocratic normal-phase HPLC with a mixture of hexane/isopropanol/glacial acetic acid (180:0.58:0.01). The elution time of 11,12-EET was 14.4 minutes. These products were assayed by coelution with authentic standards, GC, and GC/MS. [¹⁴C]11,12-EET and [³H]11,12-EET were dissolved in ethanol and stored under N₂ at -80°C.

Tissue Culture and Incubation
Smooth muscle cells from porcine thoracic aorta were grown in DMEM supplemented with MEM nonessential amino acids, MEM vitamin solution, 15 mmol/L HEPES, 2 mmol/L L-glutamine, and 50 µmol/L gentamicin.²⁴ Primary cultures were isolated and suspended in this medium containing 10% FBS. The suspensions were counted with a hemocytometer and plated into 25-cm² flasks, and the cultures were maintained until confluent at 37°C in a humidified atmosphere containing 5% CO₂. Stocks were subcultured weekly after trypsinization.

These cells were characterized as smooth muscle by elongated bipolar morphology, production of prostaglandin E₂, and positive staining with an antibody specific for smooth muscle -actin.²⁹ The cultures were used between passage numbers 3 and 12.

Before incubation with washed porcine aortic smooth muscle cell cultures, radiolabeled 11,12-EET was mixed with modified DMEM containing 0.1 µmol/L bovine albumin. The final ethanol concentration in the medium was <0.01%. The smooth muscle cells were then incubated in 1 mL of this medium at 37°C in an atmosphere containing 5% CO₂. The incubation was terminated by removing the medium and washing the cells twice with 1 mL ice-cold buffer solution containing (mmol/L) NaCl 137, KCl 3, CaCl₂ 1, MgCl₂ 0.5, Na₂HPO₄ 8, and KH₂PO₄ 1.5, pH 7.4. Cells were harvested by scraping into methanol. Studies with radioactive fatty acids demonstrated that this scraping procedure did not cause appreciable hydrolysis of tissue lipids.³⁰

Assay of Incubation Medium
To quantitatively measure the metabolites released into the extracellular fluid, the incubation medium was extracted twice with 2.5 mL of ethyl acetate saturated with water. After evaporating the solvent under N₂, the lipid residue was dissolved in acetonitrile for separation by reverse-phase HPLC. A Varian 2010 dual-piston pump plus 2050 UV detector and a 4.6x250-mm column containing 5-µm spherical particles of EQC C₁₈ (Whatman) were used. The elution profile, developed with an ISCO 2360 low-pressure gradient controller, consisted of water adjusted to pH 3.4 with phosphoric acid and an acetonitrile gradient increasing from 35% to 95% over 60 minutes at a flow rate of 0.9 mL/min. Radioactivity was measured by combining the column effluent with scintillator solution and passing the mixture through a Radiomatic model CR Flo-One beta isotope detector.²³ Some HPLC effluents also were monitored with a Perkin-Elmer 480 diode-array detector.²³

Analyses of Cell Lipids
Cell lipids were extracted with 20 vol of chloroform-methanol (2:1) using a procedure described by Folch et al,³¹ except that the phases were separated with a solution containing (mmol/L) NaCl 137 and Na₂HPO₄ 8, pH 7.4. After removal of the chloroform phase, the aqueous phase was washed with 5 vol of chloroform/methanol/Na₂HPO₄ (pH 7.4) solution (86:14:1), and the resulting organic layer was combined with the original chloroform extract. The solvent was evaporated under N₂, and the lipids were suspended in 200 µL chloroform/methanol (2:1). An aliquot of this solution was dried and assayed for radioactivity after addition of liquid scintillation solution. Radioactivity was measured with a Packard 4640 liquid scintillation spectrometer (Canberra Corp), and quenching was monitored with a ²²⁶Ra external standard.

The cell lipid extracts were separated by TLC. Neutral lipids were separated on silica gel G plates with hexane ethyl ether/acetic acid/methanol (90:20:2:3),³² and phospholipids were separated on Whatman LK50 plates with chloroform/methanol/40% methylamine (65:36:5).³³ The distribution of radioactivity on the TLC plate was determined with a gas-flow proportional scanner (Radiomatic model R) as described and validated previously.³⁴ Radioactive lipid standards were added to each plate.

Some of the cell lipid extracts were saponified for 1 hour at 50°C with 0.5 mL methanolic 0.2N NaOH containing 10% H₂O. After the pH was brought to 8.0 with 0.1 mol/L Na₂HPO₄ (pH 7.4), the lipids were extracted twice with 5 mL ice-cold ethyl acetate saturated with water. This solvent was removed under N₂, and the lipids were dissolved in acetonitrile and assayed by reverse-phase HPLC.^{23 24}

Identification of Metabolites by GC/MS
Saponified cell lipids amd media extracts were resuspended in anhydrous methanol and incubated for 10 minutes at 22°C with 2 vol of freshly prepared ethereal diazomethane.³⁵ After evaporation of the organic solvent under N₂, lipids were suspended in methanol, and the derivatization reaction was repeated. Trimethylsilylether derivatives were prepared by incubation of the fatty acid methyl esters with 50 µL of bis(trimethylsilyl)-trifluoroacetamide containing 1% trimethylchlorosilane (Pierce Chemical Co) for 1 hour at 60°C. These derivatives were prepared because they exhibit good chromatographic properties. Regioisomers of the eicosatrienoate diols are resolved by capillary GC,³⁶ and as opposed to alkyl cyclic boronates, the trimethylsilylethers direct cleavages that facilitate identification of diol positions.³⁷

Aliquots of the methylated derivatives were hydogenated using platinum oxide as a catalyst.³⁸ Briefly, the methyl esters were suspended in methanol, and after addition of platinum (IV) oxide, H₂ was bubbled through the mixture for 1 hour at 22°C. Platinum was removed by filtration. After evaporating the solvent under N_2, trimethylsilylethers were prepared as described above.

Metabolites dissolved in n-heptane were separated on a Hewlett-Packard 5890 gas chromatograph using a 0.32 mmx15 m 1-µm DB-1 column (J&W Scientific Inc). Both the on-column injector and the transfer line were heated to 250°C. The initial oven temperature of 180°C was maintained for 5 minutes and then ramped to 250°C at 5°/min. Electron impact spectra were obtained using a Trio I quadrupole spectrometer set at 50 eV and with a 1000-amu range (VG Analytical).

Investigation of the Vasoactivity of 11,12-EET and Its Metabolites
Coronary arteries were dissected from pig or cow hearts immediately after removal at a local slaughterhouse. The arteries were placed into ice-cold Krebs-Ringer bicarbonate solution composed of (mmol/L) NaCl 118.3, KCl 4.7, CaCl₂ 2.5, MgSO₄ 1.2, KH₂PO₄ 1.2, NaHCO₃ 26, and glucose 11.1, aerated with 95% O₂/5% CO₂, transported to the laboratory, and maintained at 4°C. The arteries were cut into rings (3 to 5 mm in width) and mounted onto stainless steel triangles, which were attached by thread to isometric force transducers coupled to a polygraph, for continuous recording of ring tension. The rings were suspended in water-jacketed (37°C) organ baths containing 5 mL Krebs-Ringer bicarbonate solution, which was continuously aerated with 95% O₂/5% CO₂. Basal ring tension was gradually adjusted to 10 g for porcine coronary artery rings³⁹ and 5 g for bovine coronary artery rings.¹⁷ The rings were contracted with KCl (60 mmol/L) until tension stabilized, the organ baths were rinsed thoroughly, and tension was allowed to return to baseline. Each ring was then contracted with a thromboxane mimetic, U46619, within 40% to 80% of the tension obtained with KCl (60 mmol/L), and 11,12-EET or 11,12-DHET was randomly administered in cumulative fashion. The baths were rinsed, and after tension returned to baseline, the rings were contracted again with U46619 to a level of tension similar to that for the previous contraction. When a stable level of tension was achieved, either 11,12-EET or 11,12-DHET (whichever was not tested during the previous dose-response determination) was administered. In separate experiments, the effects of compound X and ethanol, the vehicle for 11,12-EET and its metabolites, on U46619-contracted porcine coronary artery rings were also examined. Relaxation responses were expressed as the percent decrease from the U46619-induced tension.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Detection of 11,12-EET Metabolites
Fig 1 illustrates the products detected in the medium when cultured porcine aortic smooth muscle cells were incubated with [³H]11,12-EET or [1-¹⁴C]11,12-EET. After a 2-hour incubation with 1 µmol/L [³H]11,12-EET (Fig 1A), only 3% of the radioactivity in the medium remained as 11,12-EET. The predominant product in the medium had an RT of 26.5 minutes and was identified by GC/MS as 11,12-DHET, confirming our previous findings.²⁴ Compound X (RT, 18.5 minutes) was also present by 2 hours and contained at least 15% of the radioactivity present in the medium. Several additional radiolabeled components were detected, but together they constituted <3% of the radioactivity. When the incubation with [³H]11,12-EET was extended to 10 hours, more compound X (55%) than 11,12-DHET (30%) was present (Fig 1B). In addition, two minor radiolabeled products (RTs, 16 and 21.5 minutes) increased to a small extent. As opposed to these findings, all of the radioactivity remained as EET when [³H]11,12-EET was incubated for 24 hours in the absence of cells (Fig 1C).

View larger version (27K): [in this window] [in a new window]   Figure 1. Comparison of the radiolabeled products formed from [3H]11,12-EET and [1-14C]11,12-EET by porcine aortic smooth muscle cells. Cells were incubated for 2 hours (A and D) or 10 hours (B and E) with [3H]11,12-EET (A and B) or [1-14C]11,12-EET (D and E) at a concentration of 1 µmol/L. After incubation, lipids were extracted from the medium and separated by HPLC, and radioactivity was assayed with an on-line flow scintillation detector. The radiolabeled components comigrating with 11,12-EET and 11,12-DHET are indicated, and the main additional product formed from [3H]11,12-EET is designated as X. Each panel contains a chromatogram from a single culture, but similar results were obtained from a duplicate culture in every case. The radiolabeled EETs also were incubated in these media, but without cells, for 2 to 24 hours to determine whether conversion to these products was tissue dependent. Chromatograms are shown for the 24-hour cell-free incubations with [3H]11,12-EET (C) and [1-14C]11,12-EET (F).

In similar incubations with 1 µmol/L [1-¹⁴C]11,12-EET, radiolabeled 11,12-EET and 11,12-DHET were observed after 2 hours, but compound X was not detected (Fig 1D). After 10 hours of incubation, the only prominent radiolabeled product was 11,12-DHET; neither compound X nor the metabolites with RTs of 16 and 21.5 minutes were detected (Fig 1E). This indicates that the carboxyl-carbon was removed when 11,12-EET was converted to compound X and the additional products. All of the radioactivity remained as EET when [1-¹⁴C]11,12-EET was incubated in a cell-free medium (Fig 1F), again demonstrating that the conversion of 11,12-EET to these products was mediated by the smooth muscle cells.

Kinetics of 11,12-DHET and Compound X Formation
The time course of conversion of 11,12-EET to 11,12-DHET and compound X was investigated by incubating porcine aortic smooth muscle cells with [³H]11,12-EET for up to 24 hours. Little radioactivity remained in the medium as 11,12-EET by the end of 1 hour of incubation (Fig 2A). The predominant radiolabeled metabolite early in the incubation was 11,12-DHET; its concentration reached a maximum between 2 and 4 hours and declined thereafter. In contrast, compound X accumulated slowly and continuously, accounting for 80% of the radioactivity in the medium after 24 hours.

View larger version (22K): [in this window] [in a new window]   Figure 2. Time- and concentration-dependent metabolism of [3H]11,12-EET by smooth muscle cells. Product formation was determined as described in Fig 1. The concentration of [3H]11,12-EET was 1 µmol/L in the time-dependence study (A). In the concentration-dependence study (B), the time of incubation was 10 hours. The results are expressed in picomole values that were calculated from the specific radioactivity of the added [3H]11,12-EET.

To further explore the possibility that 11,12-DHET might be a precursor of compound X, as suggested by the above data, nonradiolabeled 11,12-DHET was added to the incubation medium to determine whether it would block the conversion of [³H]11,12-EET to compound X. As seen in the Table, 11,12-DHET (20 µmol/L) did not inhibit the conversion of the [³H]11,12-EET to [³H]11,12-DHET by the smooth muscle cells; rather, the amount of [³H]11,12-DHET increased by 38%. However, 11,12-DHET (20 µmol/L) inhibited the formation of radiolabeled compound X by 80%. These results are consistent with a precursor-product relationship between 11,12-DHET and compound X.

View this table: [in this window] [in a new window]   Table 1. Effects of Nonradioactive 11,12-DHET on Conversion of [3H]11,12-EET to Radiolabeled Compounds

Fig 2B illustrates the dependence of radiolabeled 11,12-DHET and compound X formation on the concentration of [³H]11,12-EET added to the smooth muscle cell cultures during a 10-hour incubation. The production of compound X was maximal when the initial 11,12-EET concentration was between 5 and 10 µmol/L. Therefore, the cells were incubated with 10 µmol/L 11,12-EET to generate maximum amounts of compound X for structural identification.

Compound X Identification
Fig 3 shows the electron ionization mass spectra of compound X and its hydrogenated product. The top panel shows the spectrum of the methyl ester trimethylsilylether derivative of the metabolite. A molecular ion was not observed, but the ions m/z 427 [M-CH₃] and m/z 411 [M-CH₃O] were detected. Ions m/z 331 [M-CH₃(CH₂)₄(CH=CH)CH₂] and m/z 315 [M-CH₂(CH=CH)(CH₂)₂CO₂CH₃] localize the trimethylsilylether groups to C₇ and C₈, counting from the carbomethoxy group. Other prominent ions are m/z 241 [M-{CH₃(CH₂)₄(CH=CH)CH₂+(CH₃)₃SiOH}], m/z 229 [M-CH₃(CH₂)₄(CH=CH)CH₂CHOSi(CH₃)₃], and m/z 213 [M-(CH₃)₃SiOCHCH₂(CH=CH)(CH₂)₂CO₂CH₃].

View larger version (28K): [in this window] [in a new window]   Figure 3. Electron impact mass spectra of compound X, which was formed from 11,12-EET. Compound X was isolated by HPLC after a 24-hour incubation of the smooth muscle cultures with 10 µmol/L 11,12-EET. For GC/MS analysis, the methyl ester was converted to a trimethylsilylether, without (top) or with prior hydrogenation (bottom).

A molecular ion also was not observed with the methyl ester trimethylsilylether derivative of the hydrogenated derivative (Fig 3, bottom panel). Ion m/z 439 was determined by further analysis to be an artifact due to a contaminant in the system. Ion m/z 415 [M-CH₃], when compared with m/z 411 in the top panel, indicates that the original compound X contained two double bonds. Ions m/z 317 [M-(CH₂)₅CO₂CH₃] and m/z 215 [M-(CH₃)₃SiOCH(CH₂)₅CO₂CH₃], when compared with m/z 315 and m/z 213 in the top panel, confirm that one double bond is between the methyl end and the trimethylsilylether groups. Likewise, when compared with m/z 241 and m/z 229 in compound X, ions m/z 243 [M-{CH₃(CH₂)₇+(CH₃)₃SiOH}] and m/z 231 [M-CH₃(CH₂)₇CHOSi(CH₃)₃] indicate that the other double bond is located between the carbomethoxy group and the trimethylsilylether groups. Ion m/z 304 reflects the migration of a trimethylsilyl group and the loss of HCO. These findings, when considered together with the known double-bond positions in the 11,12-EET and 11,12-DHET precursors, suggest that the structure of compound X is 7,8-DHHD.

Production and Disposition of Metabolites Formed From Smooth Muscle Cell–Incorporated 11,12-EET
Porcine aortic smooth muscle cells can take up [³H]11,12-EET when it is present in the medium and incorporate this compound into cell lipids.²⁴ Therefore, we investigated whether [³H]11,12-EET contained in the cells can be converted to radiolabeled 7,8-DHHD and whether the 7,8-DHHD formed in this way can be retained in cell lipids. Cultures incubated for 1 hour with 1 µmol/L [³H]11,12-EET incorporated substantial amounts of radioactivity, almost all of which was present in the cell lipids. Fig 4A illustrates the distribution of radioactivity as determined by TLC. More than 85% was present in phospholipids. The PC fraction contained the largest amount of radioactivity, but sizable amounts also were present in the PE and PI fractions. Further studies were performed to determine whether the distribution of radioactivity changes during subsequent incubation. Fig 4B shows the distribution of cell-incorporated radioactivity present at the end of a 16-hour incubation in media that contained no supplemental 11,12-EET. Whereas the PC fraction still contained the largest amount of radioactivity, the percentage distribution into PI, PE, and the neutral lipids was increased relative to that observed after the initial 1-hour incubation.

View larger version (23K): [in this window] [in a new window]   Figure 4. Distribution of 11,12-EET radioactivity in smooth muscle cell lipids. After incubation, the cell lipids were extracted and separated by TLC, and radioactivity was monitored using a plate scanner. A, 1-Hour incubation with 1 µmol/L [3H]11,12-EET. B, 1-Hour incubation with 1 µmol/L [3H]11,12-EET, followed by a 16-hour incubation in fresh medium containing no added 11,12-EET. The chromatograms were obtained from single cultures, but similar distributions were observed with two additional cultures in each case. The main radiolabeled fractions of the lipid extract are PI, PC, PE, and neutral lipids (NL).

To determine whether the incorporated radioactivity consisted primarily of 11,12-EET, the smooth muscle lipids were hydrolyzed by saponification, and the resulting lipid-soluble material was separated by reverse-phase HPLC. Fig 5A shows that >70% of the radioactivity contained in the smooth muscle lipids at the end of the 1-hour preincubation was 11,12-EET (RT, 42.5 minutes). Two prominent radiolabeled components with RTs of 37 minutes (peak P) and 49 minutes (peak Q) were observed but not identified. These metabolites contained 15% and 7% of the radioactivity, respectively. A radiolabeled component with an RT of 27.5 minutes, which contained <5% of the radioactivity, also was observed. This component, which comigrated with 11,12-DHET on HPLC, was not further identified. The RT of 7,8-DHHD is shown in this figure, but no radiolabeled 7,8-DHHD was detected in the cells after 1 hour of incubation with [³H]11,12-EET. When these labeled cells were incubated subsequently for 4 to 16 hours in fresh medium that did not contain any added 11,12-EET, the percentage of radioactivity present in the cell lipids as 11,12-EET declined, whereas that in the peak that comigrated with 11,12-DHET increased (Fig 5B through 5D). However, measurable amounts of 7,8-DHHD were not detected in the cells at any of the longer incubation times. Therefore, 7,8-DHHD does not accumulate in the cells even when it builds up to high levels in the extracellular fluid.

View larger version (26K): [in this window] [in a new window]   Figure 5. The 11,12-EET products esterified in smooth muscle cell lipids. Porcine aortic smooth muscle cultures were incubated for 1 hour with 1 µmol/L [3H]11,12-EET (A) and for an additional 4 hours (B), 8 hours (C), and 16 hours (D) in fresh medium containing no 11,12-EET. The cell lipids were extracted, subjected to alkaline hydrolysis, and separated by HPLC as indicated in Fig 1. Chromatograms from single cultures are shown, but similar results were obtained from two additional cultures in each case.

Additional studies were undertaken to obtain quantitative estimates of the amounts of cell-associated 11,12-EET that were released into the medium. Smooth muscle cultures were incubated for 1 hour with [³H]11,12-EET and washed. One set of cells was assayed to determine the amount of radioactivity taken up during this initial 1-hour incubation. The remaining labeled cultures were incubated for 2 to 16 hours in fresh medium containing no added 11,12-EET, and the cells were separated from the medium. Fig 6A shows the total lipid-soluble radioactivity remaining in the cells and the amount released into the medium during the 16-hour incubation, and Fig 6B shows the distribution of the radioactivity released into the medium as determined by HPLC analysis. As seen in Fig 6A, 60% of the radioactivity incorporated into the cells during the 1-hour preincubation with [³H]11,12-EET was present in the medium by the end of the subsequent 16-hour incubation. Almost all of this released radioactivity was recovered in lipid-soluble form. Calculations indicate that 65% of the radioactivity released into the medium over the 16-hour period was derived from smooth muscle cell PC.

View larger version (22K): [in this window] [in a new window]   Figure 6. Time-dependent losses of 11,12-EET products from smooth muscle cells. Porcine aortic smooth muscle cultures were preincubated for 1 hour with 1 µmol/L [3H]11,12-EET, washed, and then incubated for up to 16 hours in fresh medium containing no added 11,12-EET. A, The release of radioactivity from the labeled cells and its accumulation in the fresh medium during the 16-hour incubation is shown. B, The distribution of the released radioactivity between 11,12-DHET and 7,8-DHHD during the 16-hour incubation, as determined by HPLC analysis of these media, is shown. The values are expressed in picomoles, calculated from the specific radioactivity of the 11,12-EET with which the cells were initially labeled. Each point is the average of values obtained from two separate cultures; the values in each case agreed within 10%.

HPLC analysis of the medium demonstrated that the major radiolabeled products released from the labeled cells during incubations of 2 to 16 hours were 11,12-DHET and 7,8-DHHD. Fig 6B shows the picomole amounts of radiolabeled 11,12-DHET and 7,8-DHHD that accumulated in the medium during the course of the 16-hour incubation. The amounts were calculated using the specific radioactivity of the [³H]11,12-EET with which the cells were incubated during the initial 1-hour loading period. The amount of 11,12-DHET increased early during the course of the incubation, reached a peak at 4 hours, and then subsequently declined. By contrast, 7,8-DHHD gradually accumulated throughout the 16-hour incubation and accounted for most of the radioactivity at the longer incubation periods.

Taken together, these results indicate that 11,12-EET present in cell lipids can be converted to 7,8-DHHD. However, the 7,8-DHHD that is formed is not retained or incorporated into lipids but, rather, is released from the cells.

Vasoactivity of 11,12-EET Metabolites
The effects of 11,12-EET, 11,12-DHET, and 7,8-DHHD on isolated coronary artery rings submaximally contracted with a thromboxane mimetic, U46619 (0.8 to 30 nmol/L), were examined. 11,12-EET (1.0 and 5.0 µmol/L) produced concentration-dependent relaxation of porcine coronary artery rings (Fig 7, top). A similar result has been reported previously.¹⁷ The administration of 11,12-DHET (1.0 and 5.0 µmol/L) to U46619-contracted porcine coronary artery rings also resulted in concentration-dependent relaxation (Fig 7, middle). Likewise, 11,12-DHET (0.1 to 5.0 µmol/L) produced a concentration-dependent relaxation of bovine coronary artery rings contracted with U46619 (data not shown).

View larger version (11K): [in this window] [in a new window]   Figure 7. Representative tracings showing relaxation induced by 11,12-EET, 11,12-DHET, and 7,8-DHHD. Porcine coronary artery rings were contracted with U46619, and 11,12-EET (top), 11,12-DHET (middle), or 7,8-DHHD (bottom) was administered at concentrations of 1 µmol/L (solid arrow) and 5 µmol/L (dashed arrow).

In separate experiments, we examined the effects of 7,8-DHHD on isolated coronary artery rings submaximally contracted with U46619. 7,8-DHHD was obtained from the medium of cultured porcine aortic smooth muscle cells incubated with 10 µmol/L 11,12-EET for 24 hours. The lipids were extracted and subjected to HPLC, and the fraction eluting at 18.5 minutes, which contained 7,8-DHHD (Fig 1B), was collected. The structure of the 7,8-DHHD contained in this fraction was confirmed by GC/MS analysis. 7,8-DHHD (1.0 and 5.0 µmol/L) produced concentration-dependent relaxation of porcine coronary rings (Fig 7, bottom). Care was taken in these experiments to ensure that ring tension was stable before administering each dose of 11,12-EET or its metabolites. To depict the sustained relaxation responses produced by these compounds, the entire contraction phase had to be removed from Fig 7, and this gives the appearance of unstable contractions.

The magnitude of the relaxation produced by 11,12-EET and 11,12-DHET was compared over a concentration range of 0.05 to 5.0 µmol/L. As seen in Fig 8, the magnitude of the effects produced by both compounds was similar. These are paired experiments conducted in a randomized fashion to limit, as much as possible, interanimal variability and effects of incubation conditions. Similar results were obtained in other unpaired experiments (data not shown). Because of the small quantity of material available, the concentration-dependent effects of 7,8-DHHD could not be compared with those of 11,12-EET and 11,12-DHET.

View larger version (20K): [in this window] [in a new window]   Figure 8. Dose-dependent relaxation of porcine coronary artery rings produced by 11,12-EET and 11,12-DHET. The coronary artery rings were contracted with a thromboxane mimetic, U46619, and the compounds were administered in cumulative fashion. Each point represents the mean±SE of 4 determinations. The responses to ethanol, the vehicle for 11,12-EET and 11,12-DHET, also are shown. This was done in separate experiments (n=4, mean±SE), but the values for the SE are too small to be visible.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Recently, a great deal of attention has been directed toward elucidating the role of cytochrome P-450–derived arachidonic acid metabolites in regulating vascular tone. The production of EET regioisomers has been demonstrated in a variety of blood vessels, including cerebral¹¹ and coronary arteries.^{4 9 13} The enzymatic activity responsible for the production of EETs in blood vessels has been localized to the intima, suggesting that EETs are produced predominantly by vascular endothelial cells,⁴⁰ and the conversion of arachidonic acid to EETs has recently been demonstrated in cultured bovine aortic endothelium.²¹ By virtue of their production in the vascular endothelium and their ability to relax blood vessels through Ca²⁺-activated K⁺ channel activation,^{10 11 12} EETs can be considered as EDHFs. Indeed, a number of recent studies suggest that EETs may account for the activity attributed to EDHF in some blood vessels.^{13 17 18 19 41}

Although there are a large number of reports describing the production and activity of EETs within the vasculature, little has been reported about the vascular inactivation of EETs. Previous studies indicate that both cultured porcine aortic endothelial²³ and smooth muscle²⁴ cells are capable of efficiently converting EETs to corresponding DHETs. Thus, EETs could be inactivated within the vasculature through conversion to DHETs, provided that DHETs are devoid of vasoactivity. Although no published studies have compared the vascular effects of DHETs with those of EETs, DHETs have been reported to lack certain biological activities attributed to EETs, such as inhibition of isoproterenol-induced renin release from the renal cortex,⁴² stimulation of ADP ribosylation of a cytosolic protein,⁴³ and inhibition of platelet aggregation.⁴⁴ However, reports that DHETs inhibit Na⁺,K⁺-ATPase⁴⁵ and block the action of vasopressin on rabbit cortical collecting ducts⁴⁶ indicate that DHETs possess some biological activities. The latter findings support the contention that the conversion of EETs to DHETs might not be the sole process whereby EETs become inactivated within the vasculature.

In the present study, we elucidated a novel pathway for 11,12-EET metabolism in porcine aortic smooth muscle cells. As reported previously,²⁴ these cells metabolized [³H]11,12-EET predominantly to [³H]11,12-DHET in short-term incubations. However, during longer incubations, another major radiolabeled metabolite (compound X) was detected and identified as 7,8-DHHD. The concentration of 7,8-DHHD increased concomitantly with a decline in the concentration of 11,12-DHET, suggesting that 7,8-DHHD is produced from 11,12-DHET. The inhibition of radiolabeled 7,8-DHHD production by unlabeled 11,12-DHET is consistent with the conclusion that 11,12-DHET is a precursor of 7,8-DHHD. Most likely, 7,8-DHHD is formed through two cycles of ß-oxidation at the carboxyl end of 11,12-DHET.³⁸ In a previous study, the conversion of 15-hydroxyeicosatetraenoic acid to 11-hydroxyhexadecatrienoic acid in vascular endothelial cells was also observed to occur through a ß-oxidation pathway.⁴⁷ Although we did not investigate whether ß-oxidation products are formed from other EET regioisomers, such a process could explain the unidentified metabolites formed during prolonged incubations of porcine aortic smooth muscle cells with [³H]8,9-EET and [³H]14,15-EET.²⁴

We did not determine the stereochemistry of the 11,12-DHET and 7,8-DHHD that are formed. Cytosolic epoxide hydrolases isolated from rat liver and lung convert 11,12-EET nonstereoselectively to DHET, resulting in approximately equal proportions of R,R and S,S vicinal diols.^{48 49} This differs from the stereoselective conversion of 8,9-EET and 14,15-EET.^{48 49} The data in Fig 1 demonstrate that the formation of 11,12-DHET and 7,8-DHHD is tissue dependent. If the epoxide hydrolase of the vascular smooth muscle cells is similar to that present in liver and lung, roughly equal proportions of the R,R and S,S 11,12-DHET stereoisomers would be expected. It is likely that the same stereoisomerism, equal R,R and S,S forms, is maintained in 7,8-DHHD, because the stereochemistry of 11,12-DHET should not change when it undergoes partial ß-oxidation.

To address the functional consequences of 11,12-EET conversion to 11,12-DHET, we examined the effects of the compounds on U46619-induced tension in isolated coronary artery rings. As reported previously,¹⁷ 11,12-EET produced concentration-dependent relaxation of porcine coronary artery rings. Surprisingly, 11,12-DHET also produced relaxation that was virtually identical to that produced by 11,12-EET. 11,12-DHET also relaxed bovine coronary artery rings contracted with U46619. To our knowledge, this is the first report demonstrating that DHETs are vasoactive compounds. Because unesterified DHETs are formed when smooth muscle cells are exposed to EETs, the present findings suggest that DHETs may also contribute to the regulation of vascular tone.

Since the vasorelaxant activity of 11,12-EET was not diminished by its conversion to 11,12-DHET, we investigated the possibility that the vasoactivity might be lost through the subsequent ß-oxidation of the 11,12-DHET to 7,8-DHHD. However, 7,8-DHHD also produced relaxation of U46619-contracted porcine coronary artery rings. Thus, the vasorelaxant activity of 11,12-DHET is not eliminated by conversion to 7,8-DHHD. Since we were unable to compare the magnitude of vasorelaxation produced by 7,8-DHHD with that produced by 11,12-EET and 11,12-DHET, whether the compound possesses less vasoactivity remains to be determined.

The fact that DHET and DHHD produce relaxation raises the question as to whether other vicinal diols might produce a similar response. In this regard, lipoxin A₄ and B₄, which contain the vicinal diol structure, cause vasodilation of cerebral arterioles in newborn pigs.⁵⁰ Whether the mechanism is the same as in DHET- and DHHD-induced relaxation remains to be determined.

The slow conversion of 11,12-EET to 11,12-DHET and 7,8-DHHD (Figs 2A and 6) compared with the relatively rapid onset of vasorelaxation (Fig 7) suggests that the relaxation initially induced by exposure to 11,12-EET is not mediated by these metabolites. This interpretation is in agreement with other results indicating that the vascular actions of EETs are independent of their conversion to secondary products.¹³ However, 11,12-DHET accumulates to a small extent in the smooth muscle lipids (Fig 5), and it is possible that (like EETs) it may produce vasodilatation if it is released in response to physiological stimuli. Furthermore, the finding that DHET produces vasorelaxation suggests that a mechanism other than hydration is responsible for terminating the response produced by 11,12-EET. Since 7,8-DHHD is not present to any measurable extent in the cell lipids, it may act to transiently prolong the relaxation response produced by 11,12-EET or 11,12-DHET.

The biological function of EET incorporation into cell lipids is unknown. Such incorporation could conceivably alter membrane properties and/or ion transport pathways. Moreover, Zhu et al⁵¹ reported that EETs incorporated into platelet phospholipids were released by stimulation with phospholipase A₂, thrombin, and platelet activating factor. This observation suggests that the presence of EETs in phospholipids might influence membrane signal transduction processes. In addition, a recent study demonstrated that modulation of protein kinase C activity affected the amount of 14,15-EET incorporated into cell phospholipids, suggesting that the incorporation of EETs might be a cell-regulated process.²⁵ Taken together, these reports suggest that EETs incorporated into cell lipids are not devoid of biological activity. Thus, the ß-oxidation pathway described in this report might serve to reduce some of the activities of 11,12-EET by facilitating its removal from cell lipids through conversion to 7,8-DHHD.

The present findings suggest that factors affecting the amount of 11,12-EET incorporated into cell lipids versus the amount that undergoes hydration followed by ß-oxidation could influence the vascular effects of 11,12-EET. Studies with rat astrocytes indicate that phorbol myristate acetate stimulates the uptake of 14,15-EET and reduces its conversion to 14,15-DHET, probably by inhibiting the cytosolic epoxide hydrolase.²⁵ Likewise, interleukin-1ß, interleukin-6, and tumor necrosis factor- downregulate epoxide hydrolase in cultured hepatocytes.⁵² Although we have not investigated the effects of these substances, the astrocyte and hepatocyte data suggest that similar effects of growth factors on EET metabolism may occur in the arterial smooth muscle cells.

In summary, we describe a novel pathway of 11,12-EET metabolism whereby porcine aortic smooth muscle cells convert 11,12-EET to 11,12-DHET, which is subsequently converted to a newly identified metabolite, 7,8-DHHD. Both 11,12-DHET and 7,8-DHHD possess vasorelaxant activity. 11,12-EET and 11,12-DHET, but not 7,8-DHHD, are incorporated to an appreciable extent into cell lipids. These results suggest that the vascular inactivation of EETs entails additional processes besides conversion to their corresponding DHETs. By reducing the amount of 11,12-EET and 11,12-DHET incorporated into smooth muscle cell lipids, however, the pathway of conversion to 7,8-DHHD could serve to reduce some of the biological activities of 11,12-EET and 11,12-DHET.

	Selected Abbreviations and Acronyms

 

DHET = dihydroxyeicosatrienoic acid DHHD = 7,8-dihydroxyhexadeca-4,10-dienoic acid EDHF = endothelium-derived hyperpolarizing factor EET = epoxyeicosatrienoic acid GC = gas-liquid chromatography m/z = mass-to-charge ratio MS = mass spectrometry PC = phosphatidylcholine PE = phosphatidylethanolamine PI = phosphatidylinositol RT = retention time TLC = thin-layer chromatography

	Acknowledgments

 
This work was supported by grant HL-49264 from the National Heart Lung and Blood Institute, National Institutes of Health. The authors thank Ruzicka's Meat Processing in Solon, Iowa, for supplying arterial tissue.

	Footnotes

 
Presented in part at the 68th Scientific Sessions of the American Heart Association, Anaheim, Calif, November 13-16, 1995, and published in abstract form (Circulation. 1995;92[suppl]:I-37).

*[³H]11,12-EET: ³H is present at carbons 5, 6, 8, 9, 11, 12, 14, and 15. The 11,12- refers to the location of the epoxide group. Likewise, 11,12- in 11,12-DHET and the 7,8- in 7,8-DHHD refer to the location of hydroxyl groups, not the ³H.

Received February 20, 1996; accepted June 14, 1996.

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				T. L. Kaduce, X. Fang, S. D. Harmon, C. L. Oltman, K. C. Dellsperger, L. M. Teesch, V. R. Gopal, J. R. Falck, W. B. Campbell, N. L. Weintraub, et al. 20-Hydroxyeicosatetraenoic Acid (20-HETE) Metabolism in Coronary Endothelial Cells J. Biol. Chem., January 23, 2004; 279(4): 2648 - 2656. [Abstract] [Full Text] [PDF]

				T. Thum and J. Borlak Mechanistic Role of Cytochrome P450 Monooxygenases in Oxidized Low-Density Lipoprotein-Induced Vascular Injury: Therapy Through LOX-1 Receptor Antagonism? Circ. Res., January 9, 2004; 94 (1): e1 - e13. [Abstract] [Full Text] [PDF]

				X. Zhao, D. M. Pollock, D. C. Zeldin, and J. D. Imig Salt-Sensitive Hypertension After Exposure to Angiotensin Is Associated With Inability to Upregulate Renal Epoxygenases Hypertension, October 1, 2003; 42(4): 775 - 780. [Abstract] [Full Text] [PDF]

				K. M. Gauthier, S. G. Jagadeesh, J. R. Falck, and W. B. Campbell 14,15-Epoxyeicosa-5(Z)-Enoic-mSI: A 14,15- and 5,6-EET Antagonist in Bovine Coronary Arteries Hypertension, October 1, 2003; 42(4): 555 - 561. [Abstract] [Full Text] [PDF]

				T. He, N. L. Weintraub, P. C. Goswami, P. Chatterjee, D. M. Flaherty, F. E. Domann, and L. W. Oberley Redox factor-1 contributes to the regulation of progression from G0/G1 to S by PDGF in vascular smooth muscle cells Am J Physiol Heart Circ Physiol, July 11, 2003; 285(2): H804 - H812. [Abstract] [Full Text] [PDF]

				M. Miyazono, D. Zhu, R. Nemenoff, E. R. Jacobs, and E. P. Carter Increased Epoxyeicosatrienoic Acid Formation in the Rat Kidney during Liver Cirrhosis J. Am. Soc. Nephrol., July 1, 2003; 14(7): 1766 - 1775. [Abstract] [Full Text] [PDF]

				X. Zhao, D. M. Pollock, E. W. Inscho, D. C. Zeldin, and J. D. Imig Decreased Renal Cytochrome P450 2C Enzymes and Impaired Vasodilation Are Associated With Angiotensin Salt-Sensitive Hypertension Hypertension, March 1, 2003; 41(3): 709 - 714. [Abstract] [Full Text] [PDF]

				J. R. Falck, U. M. Krishna, Y. K. Reddy, P. S. Kumar, K. M. Reddy, S. B. Hittner, C. Deeter, K. K. Sharma, K. M. Gauthier, and W. B. Campbell Comparison of vasodilatory properties of 14,15-EET analogs: structural requirements for dilation Am J Physiol Heart Circ Physiol, January 1, 2003; 284(1): H337 - H349. [Abstract] [Full Text] [PDF]

				X. Fang, N. L. Weintraub, C. L. Oltman, L. L. Stoll, T. L. Kaduce, S. Harmon, K. C. Dellsperger, C. Morisseau, B. D. Hammock, and A. A. Spector Human coronary endothelial cells convert 14,15-EET to a biologically active chain-shortened epoxide Am J Physiol Heart Circ Physiol, December 1, 2002; 283(6): H2306 - H2314. [Abstract] [Full Text] [PDF]

				B. B. Davis, D. A. Thompson, L. L. Howard, C. Morisseau, B. D. Hammock, and R. H. Weiss Inhibitors of soluble epoxide hydrolase attenuate vascular smooth muscle cell proliferation PNAS, February 6, 2002; (2002) 261710799. [Abstract] [Full Text] [PDF]

				R. J. Roman P-450 Metabolites of Arachidonic Acid in the Control of Cardiovascular Function Physiol Rev, January 1, 2002; 82(1): 131 - 185. [Abstract] [Full Text] [PDF]

				T. Lu, T. Hoshi, N. L Weintraub, A. A Spector, and H.-C. Lee Activation of ATP-sensitive K+ channels by epoxyeicosatrienoic acids in rat cardiac ventricular myocytes J. Physiol., December 15, 2001; 537(3): 811 - 827. [Abstract] [Full Text] [PDF]

				I. Fleming Cytochrome P450 and Vascular Homeostasis Circ. Res., October 26, 2001; 89(9): 753 - 762. [Abstract] [Full Text] [PDF]

				T. Lu, P. V G Katakam, M. VanRollins, N. L Weintraub, A. A Spector, and H.-C. Lee Dihydroxyeicosatrienoic acids are potent activators of Ca2+-activated K+ channels in isolated rat coronary arterial myocytes J. Physiol., August 1, 2001; 534(3): 651 - 667. [Abstract] [Full Text] [PDF]

				F. M. Faraci, C. G. Sobey, S. Chrissobolis, D. D. Lund, D. D. Heistad, and N. L. Weintraub Arachidonate dilates basilar artery by lipoxygenase-dependent mechanism and activation of K+ channels Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2001; 281(1): R246 - R253. [Abstract] [Full Text] [PDF]

				R. Rastaldo, N. Paolocci, A. Chiribiri, C. Penna, D. Gattullo, and P. Pagliaro Cytochrome P-450 metabolite of arachidonic acid mediates bradykinin-induced negative inotropic effect Am J Physiol Heart Circ Physiol, June 1, 2001; 280(6): H2823 - H2832. [Abstract] [Full Text] [PDF]

				C. Benoit, B. Renaudon, D. Salvail, and E. Rousseau EETs relax airway smooth muscle via an EpDHF effect: BKCa channel activation and hyperpolarization Am J Physiol Lung Cell Mol Physiol, May 1, 2001; 280(5): L965 - L973. [Abstract] [Full Text] [PDF]

				M. H. Zink, C. L. Oltman, T. Lu, P. V. G. Katakam, T. L. Kaduce, H.-C. Lee, K. C. Dellsperger, A. A. Spector, P. R. Myers, and N. L. Weintraub 12-Lipoxygenase in porcine coronary microcirculation: implications for coronary vasoregulation Am J Physiol Heart Circ Physiol, February 1, 2001; 280(2): H693 - H704. [Abstract] [Full Text] [PDF]

				Z. Yu, F. Xu, L. M. Huse, C. Morisseau, A. J. Draper, J. W. Newman, C. Parker, L. Graham, M. M. Engler, B. D. Hammock, et al. Soluble Epoxide Hydrolase Regulates Hydrolysis of Vasoactive Epoxyeicosatrienoic Acids Circ. Res., November 24, 2000; 87(11): 992 - 998. [Abstract] [Full Text] [PDF]

				M. B. Engler and M. M. Engler Docosahexaenoic Acid-Induced Vasorelaxation in Hypertensive Rats: Mechanisms of Action Biol Res Nurs, October 1, 2000; 2(2): 85 - 95. [Abstract] [PDF]

				W.-G. Li, F. J. Miller Jr, M. R. Brown, P. Chatterjee, G. R. Aylsworth, J. Shao, A. A. Spector, L. W. Oberley, and N. L. Weintraub Enhanced H2O2-Induced Cytotoxicity in "Epithelioid" Smooth Muscle Cells : Implications for Neointimal Regression Arterioscler. Thromb. Vasc. Biol., June 1, 2000; 20(6): 1473 - 1479. [Abstract] [Full Text] [PDF]

				X. Fang, T. L. Kaduce, M. VanRollins, N. L. Weintraub, and A. A. Spector Conversion of epoxyeicosatrienoic acids (EETs) to chain-shortened epoxy fatty acids by human skin fibroblasts J. Lipid Res., January 1, 2000; 41(1): 66 - 74. [Abstract] [Full Text]

				X. Fang, N. L. Weintraub, L. L. Stoll, and A. A. Spector Epoxyeicosatrienoic Acids Increase Intracellular Calcium Concentration in Vascular Smooth Muscle Cells Hypertension, December 1, 1999; 34(6): 1242 - 1246. [Abstract] [Full Text] [PDF]

				N. L. Weintraub, X. Fang, T. L. Kaduce, M. VanRollins, P. Chatterjee, and A. A. Spector Epoxide hydrolases regulate epoxyeicosatrienoic acid incorporation into coronary endothelial phospholipids Am J Physiol Heart Circ Physiol, November 1, 1999; 277(5): H2098 - H2108. [Abstract] [Full Text] [PDF]

				M. R. Brown, F. J. Miller Jr, W.-G. Li, A. N. Ellingson, J. D. Mozena, P. Chatterjee, J. F. Engelhardt, R. M. Zwacka, L. W. Oberley, X. Fang, et al. Overexpression of Human Catalase Inhibits Proliferation and Promotes Apoptosis in Vascular Smooth Muscle Cells Circ. Res., September 17, 1999; 85(6): 524 - 533. [Abstract] [Full Text] [PDF]

				H.-C. Lee, T. Lu, N. L Weintraub, M. VanRollins, A. A Spector, and E. F Shibata Effects of epoxyeicosatrienoic acids on the cardiac sodium channels in isolated rat ventricular myocytes J. Physiol., August 15, 1999; 519(1): 153 - 168. [Abstract] [Full Text] [PDF]

				X. Fang, T. L. Kaduce, and A. A. Spector 13-(S)-Hydroxyoctadecadienoic acid (13-HODE) incorporation and conversion to novel products by endothelial cells J. Lipid Res., April 1, 1999; 40(4): 699 - 707. [Abstract] [Full Text]

				C. Thollon, J. P. Bidouard, C. Cambarrat, I. Delescluse, N. Villeneuve, P. M. Vanhoutte, and J. P. Vilaine Alteration of Endothelium-Dependent Hyperpolarizations in Porcine Coronary Arteries With Regenerated Endothelium Circ. Res., March 5, 1999; 84(4): 371 - 377. [Abstract] [Full Text] [PDF]

				X. Fang, S. A. Moore, L. L. Stoll, G. Rich, T. L. Kaduce, N. L. Weintraub, and A. A. Spector 14,15-Epoxyeicosatrienoic acid inhibits prostaglandin E2 production in vascular smooth muscle cells Am J Physiol Heart Circ Physiol, December 1, 1998; 275(6): H2113 - H2121. [Abstract] [Full Text] [PDF]

				C. L. Oltman, N. L. Weintraub, M. VanRollins, and K. C. Dellsperger Epoxyeicosatrienoic Acids and Dihydroxyeicosatrienoic Acids Are Potent Vasodilators in the Canine Coronary Microcirculation Circ. Res., November 2, 1998; 83(9): 932 - 939. [Abstract] [Full Text] [PDF]

				M. Dumoulin, D. Salvail, S. B. Gaudreault, A. Cadieux, and E. Rousseau Epoxyeicosatrienoic acids relax airway smooth muscles and directly activate reconstituted KCa channels Am J Physiol Lung Cell Mol Physiol, September 1, 1998; 275(3): L423 - L431. [Abstract] [Full Text] [PDF]

				N. L. Weintraub, X. Fang, T. L. Kaduce, M. VanRollins, P. Chatterjee, and A. A. Spector Potentiation of Endothelium-Dependent Relaxation by Epoxyeicosatrienoic Acids Circ. Res., August 19, 1997; 81(2): 258 - 267. [Abstract] [Full Text]

				X. Fang, T. L. Kaduce, N. L. Weintraub, S. Harmon, L. M. Teesch, C. Morisseau, D. A. Thompson, B. D. Hammock, and A. A. Spector Pathways of Epoxyeicosatrienoic Acid Metabolism in Endothelial Cells. IMPLICATIONS FOR THE VASCULAR EFFECTS OF SOLUBLE EPOXIDE HYDROLASE INHIBITION J. Biol. Chem., April 27, 2001; 276(18): 14867 - 14874. [Abstract] [Full Text] [PDF]

				D. C. Zeldin Epoxygenase Pathways of Arachidonic Acid Metabolism J. Biol. Chem., September 21, 2001; 276(39): 36059 - 36062. [Full Text] [PDF]

				B. B. Davis, D. A. Thompson, L. L. Howard, C. Morisseau, B. D. Hammock, and R. H. Weiss Inhibitors of soluble epoxide hydrolase attenuate vascular smooth muscle cell proliferation PNAS, February 19, 2002; 99(4): 2222 - 2227. [Abstract] [Full Text] [PDF]

				W. B. Campbell, C. Deeter, K. M. Gauthier, R. H. Ingraham, J. R. Falck, and P.-L. Li 14,15-Dihydroxyeicosatrienoic acid relaxes bovine coronary arteries by activation of KCa channels Am J Physiol Heart Circ Physiol, May 1, 2002; 282(5): H1656 - H1664. [Abstract] [Full Text] [PDF]

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