INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

CYTOCHROME P-450 epoxygenases convert arachidonic acid to four epoxyeicosatrienoic acid (EET) regioisomers, 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET, corresponding to the double-bond position at which the epoxide group is inserted (4, 8, 16, 17). EETs possess a variety of biological actions, including the capacity to potently relax blood vessels by activating Ca²⁺-activated K⁺ (K_Ca) channels (2, 9, 33). Recent studies have established that EETs are produced by the vascular endothelium, and EETs have been identified by Campbell et al. (2, 19-21) as endothelium-derived hyperpolarizing factors in coronary blood vessels.

EETs are avidly incorporated into cell lipids (7, 25), a property that is not shared by other previously characterized endothelium-derived relaxing factors. The biological importance of EET incorporation into cell lipids is suggested by recent reports that these eicosanoids are present esterified to phospholipids in platelets and heart, liver, and kidney tissues (3, 13, 30, 32). The most abundant EET regioisomer present in cell lipids in these tissues was 14,15-EET. Endothelial cells in culture also actively incorporate EETs into phospholipids, from which they are released after agonist-induced phospholipase activation (26). Furthermore, incubation of porcine coronary arteries with 14,15-EET or 11,12-EET resulted in potentiation of endothelium-dependent relaxation to bradykinin, an effect that was prevented when EET incorporation into phospholipids was blocked by inhibition of acyl-CoA synthases (26). These data suggest that EET incorporation into the lipids of cells present in the blood vessel wall can importantly influence vascular function. However, the mechanisms that regulate EET incorporation into vascular cell lipids are unknown.

Besides incorporating EETs into cell lipids, vascular endothelial and smooth muscle cells rapidly convert EETs to the corresponding dihydroxyeicosatrienoic acid (DHET) metabolites by the action of epoxide hydrolases (5, 7, 25, 31). Smaller amounts of DHETs are incorporated into endothelial cell lipids than the corresponding EETs (24, 25). Thus, in contrast to EETs, DHETs are primarily released from endothelial cells. Therefore, one potential mechanism to regulate the amount of EET incorporated into vascular cell lipids is the epoxide hydrolase-mediated conversion to DHETs. In support of this possibility, inhibition of epoxide hydrolases by 4-phenylchalcone oxide (4-PCO) enhanced the incorporation of 14,15-EET into the phospholipids of rat astroglial cells (22).

In the present study, we investigated the effects of inhibition of epoxide hydrolases on endothelial cell incorporation and release of 14,15-EET. We also addressed the functional effects of epoxide hydrolase inhibition on 14,15-EET-induced potentiation of relaxation to bradykinin in porcine coronary arteries.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

14,15-EET and 8,9-EET were purchased from Cayman Chemical (Ann Arbor, MI), U-46619 and bradykinin from Sigma (St. Louis, MO), and triacsin C and 4-PCO from Biomol (Plymouth Meeting, PA). The vehicle used in all experiments in which 4-PCO was added was dimethyl sulfoxide. MEM nonessential amino acids, MEM vitamin solution, HEPES, and trypsin were obtained from GIBCO (Grand Island, NY), fetal bovine serum (FBS) from HyClone Laboratories (Logan, UT), fatty acid-free bovine serum albumin from Miles Laboratories (Elkhardt, IN), and gentamicin from Schering (Kenilworth, NJ). Radiolabeled arachidonic acid was obtained from American Radiolabeled Chemicals (St. Louis, MO) or Amersham (Arlington Heights, IL), and phospholipid standards were obtained from Avanti Polar Lipids (Alabaster, AL). Radiolabeled EETs were synthesized, purified, and quantitated as described previously (7, 25, 26). The specific activity of the ³H-labeled 14,15-EET (14,15-[³H]EET) used for most experiments was 333 Ci/mmol, that of 8,9-[³H]EET was also 333 Ci/mmol, and that of 14,15-[1-¹⁴C]DHET was 14.2 Ci/mmol. Some experiments were performed with 14,15-[³H]EET (232 Ci/mmol) obtained from DuPont-NEN (Boston, MA). Whatman Linear K thin-layer chromatography (TLC) plates were purchased from Alltech (Deerfield, IL). All other reagents were purchased from Sigma.

Cell culture and incubations. Porcine coronary artery endothelial cells (PCAEC) were isolated as reported previously (26, 28) and grown in medium 199 (M199) supplemented with 10% FBS, MEM nonessential amino acids, MEM vitamin solution, 2 mmol/l L-glutamate, 50 µmol/l gentamicin, and 15 mmol/l HEPES. The cultures were maintained until confluent in a humidified atmosphere containing 5% CO₂ at 37°C. All experiments were performed on cells confluent at passages 4-9. Incubations were conducted in M199 supplemented as described above but containing 0.1 µmol/l bovine serum albumin instead of 10% FBS. Some experiments were performed with porcine aortic smooth muscle cells (PASMC) isolated as described previously (6). The cells were grown in DMEM supplemented as described above. Incubations were conducted in DMEM containing 0.1 µmol/l bovine serum albumin instead of 10% FBS, and the procedures for incubation with radiolabeled compounds and analysis of the cell lipids were similar to those reported by Bernstrom et al. (1).

Assay of incubation medium. After incubation with 14,15-[³H]EET, 8,9-[³H]EET, or [³H]arachidonic acid, the incubation medium was extracted twice with 4 vols of water-saturated ethyl acetate. After evaporating the solvent under N₂, we dissolved the lipid residue in acetonitrile for separation by reverse-phase HPLC using a Gilson 302 pump plus 2050 ultraviolet (UV) detector and a 4.6 × 250-mm column containing 5-µm spherical particles of EQC C₁₈ (Whatman). The elution profile, developed with a Gilson 715 gradient controller, consisted of water adjusted to pH 3.4 with phosphoric acid and an acetonitrile gradient increasing from 25 to 100% over 60 min at a flow rate of 0.7 ml/min. Radioactivity was measured by combining the column effluent with Budget-Solve scintillation solution and passing the mixture through a Radiomatic Flo-One Beta isotope detector or CR Flo-One, as described previously (6, 26).

Analyses of cell lipids. Cell lipids were extracted and analyzed as described previously (6, 26). Briefly, 6 ml of chloroform-methanol (2:1 vol/vol) followed by 2 ml of acidified saline (4 mmol/l HCl in 0.09% NaCl) solution were added to the scraped cells and the organic phase was removed after centrifugation. The aqueous phase was washed with chloroform-methanol-acidified saline (86:14:1 vol/vol/vol), and the resulting organic layer was combined with the original chloroform extract. The solvent was evaporated under N₂, and the lipids were suspended in chloroform-methanol (2:1 vol/vol). An aliquot of this solution was dried and, after addition of 5 ml of Budget-Solve scintillation solution, assayed for radioactivity.

Effects of 4-PCO on conversion of 14,15-[³H]EET to 14,15-[³H]DHET by porcine coronary arteries. Coronary arteries were dissected from pig hearts immediately after removal at a local slaughterhouse. The arteries were placed in ice-cold Krebs-Ringer bicarbonate solution [KRB (in mmol/l): 118.3 NaCl, 4.7 KCl, 2.5 CaCl₂, 1.2 MgSO₄, 1.2 KH₂PO₄, 26 NaHCO₃, and 11.1 glucose, aerated with 95%O₂-5%CO₂] and transported to the laboratory. Right coronary arteries from three different pigs were cut into rings 3-5 mm in length, and two rings from each animal were placed into separate wells of a 24-well plate containing 1 ml of KRB supplemented with 0.1 µmol/l bovine serum albumin. The plate was slowly rotated at 37°C in 5% CO₂. Two hours later, the KRB was removed and replaced with solution that contained 20 µmol/l 4-PCO or vehicle. After 1 h, 1 µmol/l 14,15-EET and 1 µCi of 14,15-[³H]EET were added and the incubation continued for 30 min. Dimethyl sulfoxide was used as the vehicle at a final concentration of 0.1%, and corresponding amounts of dimethyl sulfoxide were added to the control incubations. The incubation solution was removed, and an aliquot was subjected to scintillation counting. Lipids were extracted from the KRB and analyzed by reverse-phase HPLC as described in Assay of incubation medium. The values were converted to picomoles, calculated from the specific activity of the 14,15-[³H]EET with which the rings were incubated, and normalized to tissue wet weight.

Measurement of porcine coronary arterial ring vasoactivity. Porcine coronary arteries were cut into 35-mm-wide rings and mounted onto stainless steel triangles, which in turn 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 of KRB continuously aerated with 95% O₂-5% CO₂. Basal ring tension was gradually adjusted to 10 g, a value previously reported to yield maximal contractions to KCl (60 mmol/l) in porcine coronary artery rings (27). The rings were contracted several times with 60 mmol/l KCl until maximum levels of tension were achieved. Each ring was then contracted with a thromboxane mimetic, U-46619, to 40-80% of the maximal tension obtained with 60 mmol/l KCl. When tension stabilized, bradykinin (0.3-100 nmol/l) was administered in a cumulative fashion, resulting in >50% relaxation in all experiments. The organ chambers were then rinsed, and ring tension was allowed to return to baseline.

Statistical analysis. All data are expressed as means ± SE. The EC₅₀ of bradykinin was calculated for each concentration-response curve before and after treatment with 14,15-EET (26). The EC₅₀ values (expressed as log[M]) before and after treatment were compared by paired Student's t-tests or repeated-measures ANOVA followed by a Newman-Keuls post hoc analysis (26).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Effects of 4-PCO on conversion of 14,15-[³H]EET to 14,15-[³H]DHET by PCAEC. To investigate whether 4-PCO blocked the conversion of 14,15-[³H]EET to 14,15-[³H]DHET, PCAEC were pretreated with vehicle or 4-PCO (20 µmol/l) for 1 h and incubated with 1 µmol/l 14,15-[³H]EET for an additional 1 h. The incubation medium was removed, and the products were separated by HPLC. At this time, the medium of cultures treated with vehicle contained twice as much 14,15-[³H]DHET as 14,15-[³H]EET (Fig. 1A). Thus, like endothelial cells from other species of animals and from porcine aorta, PCAEC rapidly convert EETs to DHETs (24-27). Treatment with 4-PCO almost completely blocked the formation of 14,15-[³H]DHET (Fig. 1B). Consequently, considerably more of the medium-associated radioactivity remained as 14,15-[³H]EET after treatment with 4-PCO as compared with vehicle. When cells were incubated with 1 µmol/l 8,9-[³H]EET instead of 14,15-[³H]EET under identical conditions, most of the EET was converted to 8,9-[³H]DHET by cells treated with vehicle (Fig. 1C), but appreciable amounts of DHET were not formed when 20 µmol/l 4-PCO was added (Fig. 1D).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Effects of 4-phenylchalcone oxide (4-PCO) on conversion of 3H-labeled 14,15-epoxyeicosatrienoic acid (EET) to 14,15-[3H]dihydroxyeicosatrienoic acid (DHET) (A and B) or 8,9-[3H]EET to 8,9-[3H]DHET (C and D) by porcine coronary artery endothelial cells (PCAEC). Cells were incubated in presence of vehicle (A and C) or 20 µmol/l 4-PCO (B and D) for 1 h. Medium was replaced with fresh medium containing vehicle or 4-PCO + 14,15-[3H]EET or 8,9-[3H]EET (1 µmol/l). Dimethyl sulfoxide was used as vehicle for 4-PCO. One hour later, medium was removed and medium lipids were extracted and analyzed by HPLC (A and B) or cells were washed and treated with 2 µmol/l A-23187 for 30 min, after which medium lipids were extracted and analyzed by HPLC (C and D). In A and B, chromatograms from a single vehicle- and 4-PCO-pretreated culture are shown, but similar results were obtained from 2 additional cultures in each case. In C and D, each chromatogram is from a single culture pretreated with vehicle or 4-PCO.

Effects of 4-PCO on EET incorporation into cell lipids. Depending on the conditions of incubation, from 10 to 50 times more EETs than DHETs are incorporated into endothelial cell lipids (24, 25). We therefore investigated whether inhibition of epoxide hydrolases would enhance the amount of radioactivity incorporated into PCAEC lipids during incubation with 14,15-[³H]EET or 8,9-[³H]EET. The cells were pretreated with 4-PCO (20 µmol/l) or vehicle, and 1 h later, 14,15-[³H]EET or 8,9-[³H]EET (1 µmol/l) was added. After 1 h the cultures were washed to remove EET that had not been taken up into the cells, and fresh medium containing 4-PCO or vehicle was added. After 30 min the cells were washed, and cell lipids were extracted and assayed for radioactivity. Treatment with 4-PCO enhanced the amount of radioactivity retained in cells during incubations with 14,15-[³H]EET or 8,9-[³H]EET (Fig. 2A). TLC analysis of vehicle- and 4-PCO-treated cells indicated that >80% of the radioactivity was present in phospholipids. In cells incubated with 14,15-[³H]EET, the order of distribution of radioactivity was phosphatidylinositol (PI) > phosphatidylcholine (PC) > phosphatidylethanolamine (PE) (Fig. 2B), with smaller amounts present in neutral lipids and diglycerides. Each fraction contained more incorporated radioactivity when the cultures were treated with 4-PCO, indicating that the increase in retention was not caused by selective incorporation into specific lipids.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Effects of 4-PCO on incorporation of [3H]EET into PCAEC. Cells were pretreated with vehicle or 4-PCO (20 µmol/l) for 1 h and then incubated in medium containing 14,15-[3H]EET or 8,9-[3H]EET (1 µmol/l). Dimethyl sulfoxide was used as vehicle for 4-PCO. After 1 h, medium was removed, cells were washed, and cell lipids were extracted and assayed for radioactivity to determine total uptake (A). After incubation with 14,15-[3H]EET, lipids were separated by thin-layer chromatography (TLC), and distribution of radioactivity on TLC plate was determined with a gas flow proportional scanner (B). Main radiolabeled lipid fractions are phosphatidylinositol (PI), phosphatidylcholine (PC), phosphatidylethanolamine (PE), neutral lipids (NL), and diglycerides (DG). Results are expressed in picomoles/well (means ± SE), calculated from specific activity of [3H]EET with which cells were incubated; n = 3-6 (A) and 3 (B). In both panels, differences between 4-PCO- and vehicle-treated cells were detected by unpaired t-testing and individual P values are listed.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Effect of 4-PCO concentration on uptake of 14,15-EET and its conversion to 14,15-DHET by PCAEC. Cultures were pretreated with vehicle or indicated concentrations of 4-PCO for 1 h and then incubated with 1 µmol/l 14,15-[3H]EET for 1 h. Dimethyl sulfoxide was used as vehicle for 4-PCO. Radioactivity taken up by PCAEC (A) was assayed as described in Fig. 2, and distribution of radioactivity remaining in medium (B) was determined by HPLC as described in Fig. 1. Each point is average of 2 separate cultures, and the 2 values were within 10% agreement.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Separation of radiolabeled products incorporated into PCAEC lipids during incubation with 14,15-[3H]EET in presence of vehicle (A) or 4-PCO (B). After incubation of cells as described in Fig. 2, cell lipid extracts were hydrolyzed with 0.5 ml of methanolic 0.2 N NaOH containing 10% H2O. After extraction twice with 5 ml of ice-cold ethyl acetate saturated with water, lipids were dissolved in acetonitrile and assayed by reverse-phase HPLC.

Effects of 4-PCO on release of incorporated 14,15-[³H]EET from PCAEC lipids. To determine whether the enhanced incorporation of 14,15-[³H]EET into PCAEC lipids consequent to treatment with 4-PCO would result in an increase in the amount of 14,15-[³H]EET subsequently released from the cells, PCAEC were pretreated with 4-PCO or vehicle and then incubated with 1 µmol/l 14,15-[³H]EET, as described in Effects of 4-PCO on EET incorporation into cell lipids. After 1 h the incubation medium was removed, and the cells were washed and incubated in the absence (control) or presence of 2 µmol/l A-23187 for 30 min. The medium was then removed and analyzed as described previously. During the 30-min control incubation (in the absence of A-23187), little radioactivity was released from cells treated with either 4-PCO or vehicle and no differences were detected between the two groups (Fig. 5A). Exposure to A-23187 resulted in marked increases in the amount of radioactivity released from both 4-PCO- and vehicle-treated cells; however, only slightly more radioactivity was released from cells treated with 4-PCO than from vehicle-treated cells.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Effects of 4-PCO on release of incorporated 14,15-[3H]EET from PCAEC. Cells were pretreated with vehicle or 4-PCO and incubated with 14,15-[3H]EET as described in Fig. 2. Cells were then washed, and incubations were continued in absence (control) or presence of A-23187 (2 µmol/l) for 30 min. Incubation medium was removed and assayed for radioactivity, and cell lipids were extracted and analyzed as described in Fig. 2. A: total release of radiolabeled compounds into medium. Results are expressed in pmol/well (means ± SE), calculated from specific activity of 14,15-[3H]EET with which cells were incubated; n = 3. * P < 0.05 vs. corresponding control; + P < 0.05 vs. vehicle. B: distribution of radiolabeled products in cell lipids after treatment with A-23187. Main radiolabeled peaks are abbreviated as described in Fig. 2. Similar chromatograms were obtained from 2 other identically treated sets of cultured cells.

[³H]arachidonic acid incorporation into and release from PCAEC lipids. We next examined [³H]arachidonic acid uptake and release in the absence and presence of treatment with 4-PCO. Experiments were performed exactly as described for EETs, except that after pretreatment with vehicle or 4-PCO the cells were incubated with 1 µmol/l [³H]arachidonic acid instead of 14,15-[³H]EET or 8,9-[³H]EET. Treatment with 4-PCO did not alter the amount of [³H]arachidonic acid incorporated into cell lipids (Fig. 6A) or the amount released into the medium during control incubations or in response to A-23187 (Fig. 6B). In vehicle-pretreated cells, 263 ± 27 pmol/well of arachidonic acid was incorporated into cell lipids, more than twice as much the amount of 14,15-[³H]EET incorporated under the same conditions (Fig. 2A). However, in response to treatment with A-23187, the cells released 24 ± 1 pmol of [³H]arachidonic acid into the medium, only one-third as much as the amount of 14,15-[³H]EET that was released from the cells (Fig. 5A). As was observed after incubation with 14,15-[³H]EET, most of the incorporated [³H]arachidonic acid was contained in phospholipids in both vehicle- and 4-PCO treated cells (not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 6.   [3H]arachidonic acid incorporation into and release from PCAEC. Cells were treated with vehicle or 20 µmol/l 4-PCO for 1 h as described in Fig. 2, followed by incubation with 1 µmol/l [3H]arachidonic acid. After 1 h, medium was removed and cells were washed. In some experiments (A), cell lipids were then extracted and assayed for radioactivity, whereas in other experiments (B), cells were incubated in fresh medium without (control) or with A-23187 for 30 min, after which incubation medium was removed and analyzed as described in Fig. 4. Results are expressed in picomoles/well (means ± SE), calculated from specific activity of [3H]arachidonic acid with which cells were incubated; n = 3 (A and B). * P < 0.01, + P < 0.02 vs. corresponding control value (unpaired t-test).

Effects of inhibition of acyl-CoA synthase activity on release of incorporated 14,15-[³H]EET and on formation of 14,15-[³H]DHET by PCAEC. Previous work from our laboratories (25, 26) indicates that EETs are rapidly incorporated into endothelial lipids via an acyl-CoA synthase-dependent pathway, with the maximal rate of uptake occurring at 30 min. We therefore investigated whether, in the presence of 4-PCO, some of the incorporated 14,15-[³H]EET might have been released from the cells in response to A-23187 but then reincorporated during the 30-min incubation period. To test this possibility, cells were pretreated with 4-PCO and then incubated with 1 µmol/l 14,15-[³H]EET for 1 h as described previously. The medium was removed, and the cells were washed and incubated for 20 min in medium containing 4-PCO and 2 µmol/l triacsin C or vehicle. This concentration of triascin C, an inhibitor of acyl-CoA synthase (20), selectively blocks 14,15-EET incorporation into PCAEC lipids (26). Incubations were then continued for 30 min in the absence (control) or presence of A-23187 (2 µmol/l). Under these conditions, treatment with triacsin C did not alter the amount of 14,15-[³H]EET recovered in the medium after control incubations [30.0 ± 1.6 (triacsin C) vs. 31.6 ± 0.7 (vehicle) pmol; n = 3, P > 0.05] or after incubation with A-23187 [68.2 ± 2.8 (triacsin C) vs. 71.1 ± 0.2 (vehicle) pmol; n = 3, P > 0.05]. Furthermore, the amount of incorporated radioactivity present in cell lipids after the 30-min incubation with A-23187 did not differ between the two groups [112.6 ± 6.7 (triacsin C) vs. 119.5 ± 6.0 (vehicle) pmol/well; n = 3, P > 0.05]. These results suggest that in cells treated with 4-PCO, there was little reincorporation of released, unesterified 14,15-[³H]EET during the 30-min incubation with A-23187.

Effects of 4-PCO on 14,15-EET-induced vasorelaxation and potentiation of bradykinin-induced relaxation in porcine coronary arteries. We next investigated the functional consequences of inhibition of epoxide hydrolases on 14,15-EET-induced relaxation and potentiation of relaxation to bradykinin in porcine coronary arteries. To demonstrate the efficacy of 4-PCO to block conversion of 14,15-[³H]EET to 14,15-[³H]DHET by porcine coronary arteries, arterial rings were treated with vehicle or 4-PCO (20 µmol/l) for 1 h and then incubated with 14,15-[³H]EET for an additional 30 min, after which the incubation buffer was removed and analyzed by HPLC. Rings treated with vehicle formed 1.4 ± 0.4 pmol/g of 14,15-[³H]DHET (Fig. 7A) during the 30-min incubation, which represented a conversion of ~10% of the added 14,15-EET. No detectable 14,15-[³H]DHET was produced by rings treated with 4-PCO (Fig. 7B).

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Effects of 4-PCO on conversion of 14,15-[3H]EET to 14,15-[3H]DHET by porcine coronary arteries. Arterial rings were pretreated with vehicle (A) or 20 µmol/l 4-PCO (B) and incubated in Krebs-Ringer bicarbonate solution (KRB) supplemented with 0.1 µmol/l bovine serum albumin and 1 µmol/l 14,15-[3H]EET. Dimethyl sulfoxide was used as vehicle for 4-PCO. After 30 min, KRB was removed, and lipids were extracted and separated by HPLC. Chromatograms from a single experiment are shown, but similar results were obtained from 2 additional preparations in each case.

View larger version (15K): [in this window] [in a new window]   Fig. 8.   Effects of 4-PCO on relaxation to 14,15-EET (A) and on 14,15-EET-induced potentiation of relaxation to bradykinin (B-D). Porcine coronary artery rings were submaximally contracted with U-46619, and when tension stabilized, bradykinin (0.3-100 nmol/l) was administered in cumulative fashion. B: effect of 14,15-EET. C: effect of 14,15-EET + 4-PCO. After washing, rings were treated with vehicle (dimethyl sulfoxide) or 4-PCO (20 µmol/l) in dimethyl sulfoxide for 1 h and contracted with U-46619. When tension stabilized, 14,15-EET (0.1-5 µmol/l) was administered in ethanol, while tension was monitored (A). Final 14,15-EET dose (5 µmol/l) remained in organ chambers for 30 min before rinsing with KRB to remove EET that had not been taken up by tissue. 4-PCO or vehicle (dimethyl sulfoxide) was reintroduced, and after 30 min rings were contracted with U-46619 and bradykinin was administered (depicted in B as 14,15-EET and in C as 14,15-EET + 4-PCO). Effect of 4-PCO concentration on bradykinin EC50 is plotted in D. In each panel, n = 4-8.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In the present study, we found that treatment of coronary endothelial cells with an inhibitor of epoxide hydrolases blocked the conversion of 14,15-EET and 8,9-EET to their corresponding DHETs and enhanced the amount of EET incorporated into cell lipids. Moreover, in coronary arteries, the inhibitor 4-PCO enhanced the 14,15-EET-induced potentiation of relaxation to bradykinin, a phenomenon that we recently showed is related to EET incorporation into cell lipids (26). Taken together, these observations suggest that endothelial epoxide hydrolases may play an important role in regulating EET incorporation into complex lipids, which in turn may modulate endothelial function.

Although 4-PCO increased the amount of 14,15-[³H]EET and 8,9-[³H]EET incorporated into PCAEC lipids, it did not alter the of distribution of incorporated radioactivity in phospholipids and neutral lipids. Furthermore, the compound did not alter the amount of structurally similar arachidonic acid incorporated into, or released from, PCAEC. These observations suggest that 4-PCO did not directly affect EET uptake or release. Accordingly, our findings suggest that the 4-PCO-induced enhancement in EET incorporation most likely resulted from a reduced conversion of EET to DHET, resulting in an increased availability EET for incorporation into cell lipids.

The largest incorporation of 14,15-EET radioactivity in the PCAEC occurred in PI, followed by PC and PE. This differs from what was observed in murine mast cells, in which PE contained the largest amount of the incorporated radioactivity (1). Most likely, this is caused by either tissue or species differences, because substantial incorporation of 14,15-EET into PI also has been observed in porcine aortic endothelial cells (25).

Substantial effects on 14,15-EET uptake by PCAEC and conversion to DHET were observed at 4-PCO concentrations as low as 0.1-1 µmol/l. Also, the effects of 4-PCO on 14,15-EET-induced potentiation of relaxation to bradykinin appeared maximal at a concentration of 1 µmol/l. No toxicity was observed in either the PCAEC or coronary rings when we added up to 30 µmol/l 4-PCO. Many of the coronary ring studies required prolonged incubations with changes of the medium and repeated washing of the tissue. To ensure that adequate amounts of the inhibitor were present throughout these experiments, a concentration of 20 µmol/l 4-PCO was used routinely in the coronary ring studies and, to permit comparisons, in the PCAEC studies.

Compared with the other EET regioisomers, 14,15-EET was reported to have the highest rate of hydration by cytosol preparations from rat and rabbit liver (5, 22, 31), suggesting that it may be a preferred substrate for cytosolic epoxide hydrolases. Furthermore, Shivachar et al. (22) recently reported that substantially more DHET was formed when rat brain astrocytes were incubated with 14,15-[³H]EET than with 8,9-[³H]EET, whereas far more 8,9-[³H]EET than 14,15-[³H]EET was incorporated into cell lipids. However, we previously reported (25) that after incubation of porcine aortic endothelial cells with 14,15-[³H]EET, [³H]11,12-EET or 8,9-[³H]EET for 2 h, most of the radioactivity present in the medium was in the form of DHETs, suggesting that the epoxide hydrolases of endothelial cells may not be regioisomeric specific. In the present study, similar results were obtained when PCAEC were incubated with 8,9-[³H]EET. Perhaps more importantly, inhibition of epoxide hydrolases clearly enhanced the amount of 8,9-[³H]EET incorporated into PCAEC lipids. Thus epoxide hydrolase-mediated conversion of EETs to DHETs may broadly limit the incorporation of EET regioisomers within the endothelium. Because 4-PCO can inhibit membrane-bound and cytosolic epoxide hydrolases (11, 16), the present results do not necessarily prove where this conversion occurs.

14,15-EET and 14,15-DHET were found previously to produce comparable amounts of relaxation of porcine coronary arteries (26). This observation raised the possibility that 14,15-DHET could mediate 14,15-EET-induced vasorelaxation. However, treatment with 4-PCO, which blocked 14,15-DHET production by PCAEC and porcine coronary arteries, did not reduce the magnitude or duration of 14,15-EET-induced relaxation, suggesting that the vasorelaxation does not require the formation of 14,15-DHET.

In confirmation of our previous observations (26), preincubation of porcine coronary arteries with 14,15-EET potentiated bradykinin-induced relaxation. In the former studies, the potentiation was obviated by triacsin C, suggesting a dependence on incorporation of 14,15-EET into cell lipids (26). In the present study, 4-PCO markedly increased the amount of 14,15-EET incorporated into endothelial cell lipids and modestly enhanced the amount subsequently released from the cells in response to A-23187. Moreover, treatment with 4-PCO augmented the 14,15-EET-induced potentiation of relaxation to bradykinin. Increased incorporation and/or release of EET could potentiate vasorelaxation by acting on either endothelial or smooth muscle cells. For example, EETs have been shown to enhance Ca²⁺ influx into endothelial cells, thereby stimulating production of autocoids that could produce vasorelaxation (10). Once released from endothelial cells, EETs could also interact with smooth muscle cell receptors or G proteins to elicit vasorelaxation (15, 29). Taken together, these findings provide additional support for the contention that incorporated EETs potentiate endothelium-dependent relaxation. Whether potentiation is directly related to the amount of EET esterified to a specific membrane phospholipid or to the amount of EET released from the cells in response to agonist stimulation remains to be determined.

Consistent with our previous reports (25), we found that after a 1-h incubation of PCAEC with 14,15-[³H]EET in the absence of treatment with 4-PCO, most of the medium-associated radioactivity was in the form of 14,15-[³H]DHET, whereas ~90% of the cell-associated radioactivity remained as 14,15-[³H]EET. A very small amount of 14,15-[³H]DHET was detected in cell lipids, along with a larger amount of an unidentified metabolite that eluted from the reverse-phase HPLC column after 14,15-[³H]EET. When the cells were treated with triacsin C to inhibit 14,15-EET incorporation into PCAEC lipids, the conversion of 14,15-[³H]EET to 14,15-[³H]DHET was enhanced. These observations demonstrate that unesterified 14,15-EET probably is the main substrate for the epoxide hydrolases present in endothelial cells. Because the amount of 14,15-[³H]EET incorporated into cell lipids was enhanced when the epoxide hydrolases were inhibited by 4-PCO, it appears that epoxide hydrolases and acyl-CoA synthases compete for the same 14,15-EET substrate so that inhibition of one enzymatic pathway enhances the amount of 14,15-EET available for the other pathway.

Although PCAEC incorporated more than twice as much [³H]arachidonic acid as 14,15-[³H]EET into cell lipids, the amount of [³H]arachidonic acid recovered in the medium after exposure to A-23187 was far less. The reason for this difference is not clear. We considered the possibility that, unlike 14,15-EET, arachidonic acid might be rapidly reincorporated after its release from cell lipids. However, pretreatment with triacsin C did not affect the amount of arachidonic acid present in medium or cell lipids after a 30-min incubation with A-23187 (n = 3, data not shown), suggesting that rapid reincorporation of arachidonic acid did not occur. We also considered whether the arachidonic acid might have been distributed to different classes of intracellular lipids than 14,15-EET. However, analyses of cell lipids after incubations with either 14,15-[³H]EET or [³H]arachidonic acid demonstrated that most of the radioactivity was contained in phospholipids, particularly PI and PC. Although 14,15-EET and arachidonic acid were similarly distributed, the phospholipid pools in which the two fatty acids were incorporated could be located within different intracellular sites, with different accessibility to activated phospholipases. Alternatively, phospholipids to which oxidized fatty acids are esterified may be preferred substrates for phospholipase A₂, phospholipase C, or platelet-activating factor acetylhydrolase (18). Further studies are required to investigate these possibilities.

The biological importance of EET incorporation into cell lipids is suggested by recent reports that these eicosanoids are present esterified to phospholipids in platelets and heart, liver, and kidney tissues (3, 13, 30, 32). It is possible that such endogenous EETs may have contributed to the effects produced by 4-PCO in the coronary rings. Furthermore, Karara et al. (14) reported that most endogenous EETs in rat plasma were esterified to phospholipids in circulating lipoproteins; in fact, only 3% was present as the free fatty acid. These observations suggest that, normally, the amount of EET incorporated into cell lipids greatly exceeds the concentration of unesterified EET, as is the case with arachidonic acid (12). In contrast, much less DHETs are incorporated into phospholipids. Our results therefore suggest that by converting EETs to DHETs, endothelial epoxide hydrolases may play an important role in regulating EET incorporation into complex lipids, a function that could modulate the biological effects of EETs within the vasculature.