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Am J Physiol Heart Circ Physiol 275: H2113-H2121, 1998;
0363-6135/98 $5.00
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Vol. 275, Issue 6, H2113-H2121, December 1998

14,15-Epoxyeicosatrienoic acid inhibits prostaglandin E2 production in vascular smooth muscle cells

Xiang Fang1, Steven A. Moore2, Lynn L. Stoll3, Gretchen Rich2, Terry L. Kaduce1, Neal L. Weintraub4, and Arthur A. Spector1,4

Departments of 1 Biochemistry, 2 Pathology, 3 Anesthesia, and 4 Internal Medicine, College of Medicine, University of Iowa, Iowa City, Iowa 52242

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

14,15-Epoxyeicosatrienoic acid (EET), a cytochrome P-450 epoxygenase product of arachidonic acid (AA), reduced PGE2 formation by 40-75% in porcine aortic and murine brain microvascular smooth muscle cells. The inhibition was reversed 6-10 h after removal of 14,15-EET from the medium and was regioisomeric specific; 8,9-EET produced a smaller effect, whereas 11,12- and 5,6-EET were ineffective. Although the cells converted 14,15-EET to 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), 14,15-DHET did not inhibit PGE2 formation, and the 14,15-EET-induced inhibition was potentiated by 4-phenylchalcone oxide, an epoxide hydrolase inhibitor. The inhibition occurred when substrate amounts of AA were used and was not accompanied by enhanced production of other PGs, suggesting an effect on PGH synthase; however, in murine cells, 14,15-EET did not reduce PGH synthase mRNA or protein. Moreover, the 14,15-EET-induced decrease in PGE2 production was overcome by increasing the concentration of AA, but not oleic acid (which is not a substrate for PGH synthase). These findings suggest that 14,15-EET competitively inhibits PGH synthase activity in vascular smooth muscle cells. The 14,15-EET-induced inhibition of PGE2 production resulted in potentiation of platelet-derived growth factor-induced smooth muscle cell proliferation, suggesting that the competitive inhibition of PGH synthase by 14,15-EET can affect growth responses in smooth muscle cells.

arachidonic acid; cytochrome P-450 epoxygenase; prostaglandin H synthase; dihydroxyeicosatrienoic acid

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

FOUR EPOXYEICOSATRIENOIC ACID (EET) regioisomers, 5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET, are synthesized from arachidonic acid (AA) in a reaction catalyzed by cytochrome P-450 epoxygenases (5, 21). A major site of action of the EETs is the vascular system, where they act primarily as vasodilators (10, 23, 29, 35). These bioactive lipids are produced by endothelial cells (28), and they are rapidly taken up by arterial smooth muscle cells (SMC) (11-13). The major mechanism of EET-mediated vascular relaxation is activation of smooth muscle Ca2+-activated K+ channels (4, 15, 17). Thus EETs possess some of the properties of endothelium-derived hyperpolarizing factor, an unidentified substance released from the endothelium that also relaxes blood vessels by activating smooth muscle Ca2+-activated K+ channels (6, 20).

EET production increases following stenosis of the canine coronary artery (29). The amount formed by the thoracic aorta also increases when hypercholesterolemia is induced in rabbits (23) and when human endothelial cultures are exposed to atherogenic concentrations of low-density lipoproteins (24). 14,15-EET, the most abundant EET regioisomer formed in each of these cases, reduces basal PGI2 production by the rabbit aorta (23) and inhibits cyclooxygenase activity in human platelets (14). Such alterations in PG production could influence vascular smooth muscle function. However, whether these compounds can also inhibit production of PGs and their functional effects in vascular SMC is not known.

PGE2 is the most abundant eicosanoid produced by vascular SMC in culture (3, 26, 33, 37), and previous studies indicate that it is involved in the regulation of vascular tone and smooth muscle proliferation (1, 3, 27). In the present study, we have examined the effects of EETs on vascular SMC PGE2 production and mitogen-induced SMC proliferation. The results demonstrate that 14,15-EET reduces PGE2 formation in porcine aortic and murine brain microvascular SMC by competitively inhibiting PGH synthase. The reduced PGE2 formation, in turn, results in augmentation of platelet-derived growth factor (PDGF)-induced SMC proliferation.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell culture. A porcine thoracic aortic SMC line was developed from primary cultures isolated from explants and characterized as described previously (34). The cells were grown in DMEM (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT), MEM nonessential amino acids (Sigma, St. Louis, MO), MEM vitamin solution (Sigma), 15 mM HEPES, 2 mM L-glutamine, and 50 µM gentamicin (Schering, Kenilworth, NJ). Cultures were maintained until confluent at 37°C in a humidified atmosphere containing 5% CO2. Stocks were subcultured weekly into six-well plates by trypsinization, and experiments were done with cultures between passages 5 and 12. Under these conditions, the amount of protein harvested from confluent porcine SMC was highly uniform (117.7 ± 6.3 µg/well, n = 9).

Brain microvessel SMC were cultured from SJL mice and characterized as described previously (19, 26). Microvessel fractions freshly isolated from 7- to 10-day-old mice and containing fragments of resistance vessels were treated briefly with collagenase and plated onto uncoated plastic dishes. The identity and purity of cultures was characterized by a combination of immunohistochemistry, lectin histochemistry, phase-contrast microscopy, electron microscopy, and fluorescence-activated cell sorting (26). SMC cultures express smooth muscle alpha -actin and contain very few cells expressing endothelial cell characteristics. No astrocytes or microglia were detected. Cultures of 95-98% purity and between passages 8 and 12 were utilized in these studies. The medium utilized to maintain the microvascular cells was essentially the same as described above, except that the FBS was obtained from Sigma. The amount of protein harvested from confluent murine SMC ranged from 110 to 130 µg/well (n = 6). In the case of both porcine and murine SMC, the production of PGE2 did not differ among the passage numbers studied.

Incubation and analysis of radiolabeled products. Before the incubation, radiolabeled (DuPont, Boston, MA) and nonradiolabeled (Cayman Chemical, Ann Arbor, MI) EETs and AA were mixed and added in 10 µl of ethanol to a medium consisting of modified DMEM and 0.1 µM bovine albumin; the final concentration of ethanol in the medium was <0.01%. The cultures were washed and then incubated at 37°C in 1 ml of a medium consisting of serum-free DMEM and 0.1 µM bovine serum albumin. Details about each experiment are given in legends to Tables 1 and 2 and Figs. 1-10. For studies involving radiolabeled PGE2 production, the cultures were incubated for 10-12 h with 1 µM [5,6,8,9,11,12,14,15-3H]arachidonic acid ([3H]AA) in an atmosphere containing 5% CO2. After incubation, the radioactivity contained in the medium was measured by liquid scintillation counting (13). Additional aliquots of the medium were extracted and assayed for radiolabeled products by reverse-phase HPLC (12, 13). Where indicated, some of these media also contained 1 µM 14,15-EET. In some studies, the initial incubation medium was removed, and the cells were washed and then incubated in 1 ml of fresh DMEM medium. Where indicated, this medium contained 1 µM 14,15-EET, 2 µM calcium ionophore A-23187, or both. After 0.5-2 h, the medium was isolated and assayed for radioactivity as described above. In other experiments, cultures were incubated for 2 h in DMEM containing 1 µM [5,6,8,9,11,12,14,15-3H]14,15-EET, and the radiolabeled products were assayed by liquid scintillation counting and HPLC (12).

Aliquots of the medium were extracted twice into 4 ml of ice-cold ethyl acetate saturated with water at pH 5. After the extracts were combined, the solvent was evaporated under N2 and the lipid dissolved in acetonitrile. The lipids were separated by reverse-phase HPLC with a system equipped with a Varian 2010 dual-piston pump, 2050 detector, and 4.6 × 250 mm WQC C-18 spherical silica column (12, 13). 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 that increased from 35 to 95% over 60 min at a flow rate of 1 ml/min (12, 13). The distribution of radioactivity was measured by combining the column effluent with scintillator solution and passing the mixture through a Radiomatic Flo-One/Beta flow detector.

After washing, the cells were harvested by scraping, and the cell lipids were extracted with chloroform-methanol (2:1) (12, 13). The phases were separated, the solvent was removed under a stream of N2, the lipid was dissolved in 0.2 ml chloroform-methanol, and an aliquot was dried under N2 and assayed for radioactivity with a liquid scintillation counter (13). The lipids were separated by thin-layer chromatography on silica gel plates with a solvent system of chloroform-methanol-40% methylamine (65:35:5), and the distribution of radioactivity was determined using a Radiomatic gas flow proportional scanner with automatic peak search and integration (12, 13).

PGE2 radioimmunoassay. In one set of experiments, the cells were incubated in 1 ml serum-free DMEM containing 0.1 µM bovine serum albumin and 5 µM AA for 10 h. Some of these cultures were treated for 30 min with 1 µM 14,15-EET before the AA was added, and the EET remained in the medium during the subsequent incubation. In other experiments, cells were incubated for 30 min in serum-free DMEM containing 0.1 µM bovine serum albumin and 2 µM A-23187. Some of the incubations contained 1 µM 14,15-EET, and in these cases the cultures were exposed to the EET for 30 min before A-23187 was added. PGE2 production was measured by radioimmunoassay (8) with a PGE2 antibody purchased from PerSeptive Diagnostics (Cambridge, MA). Cross-reactivity of this antibody with other eicosanoids is <0.1%.

PGH synthase mRNA analysis. Total RNA from murine brain microvessel smooth muscle cultures was isolated with RNA STAT-60 reagent (Tel-Test, Friendswood, TX), and the RNA content was measured spectrophotometrically. RNA, 15 µg/lane, was separated by electrophoresis through a 1% agarose-4% formaldehyde gel and transferred to a nylon membrane. cDNA fragments specific for murine PGH synthase (PGHS)-1 and PGHS-2 (Oxford Biomedical Research, Oxford, MI) and the noninducible gene cyclophilin (obtained from J. G. Sutcliffe, The Scripps Research Institute, La Jolla, CA) were labeled with 32P by the random prime method and used for sequential hybridizations. The cDNA probe, 8 × 106 dpm, was added to 5 ml 50% formamide, and hybridization proceeded for 16 h at 42°C. The membrane was washed twice with a sodium chloride-sodium citrate mixture at room temperature, and then at 60°C with a 10-fold dilution of this solution containing 0.1% sodium dodecyl sulfate. The washed membrane was exposed to Fuji RX film at -70°C, and the bands were measured quantitatively by either densitometry with a Bio-Rad GS-670 scanner or with a Packard Instant-Imager electronic autoradiograph. The PGHS values were normalized against those obtained for the cyclophilin mRNA.

PGHS protein analysis. Protein was extracted from murine brain microvascular SMC grown in 100-mm petri dishes (19). The lysis buffer contained 150 mM NaCl, 50 mM Tris, pH 7.5, 100 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM diethyldithiocarbamic acid, 1% Nonidet, and 1% sodium deoxycholate. Protein content was measured colorimetrically (2). The protein was loaded onto a 6% polyacrylamide gel and separated by electrophoresis at 200 V for 45 min. After transfer to nitrocellulose for 1 h at 100 V, the membrane was blocked with 5% nonfat dry milk and incubated for 16 h at 25°C in blocking buffer containing either a rabbit polyclonal anti-PGHS-1 antibody (1:5,000; a gift of Dr. William Smith, Michigan State University) or anti-PGHS-2 antibody (1:1,000; Cayman Chemical). This was followed by incubation for 1 h at 25°C with a horseradish peroxidase-conjugated donkey anti-rabbit antibody (1:5,000). Antibody labeling was detected by enhanced chemiluminescence (26).

DNA synthesis. DNA synthesis in SMC was assessed by measurement of [3H]thymidine incorporation. SMC were grown to subconfluence in 12-well plates and then made quiescent by exposure to serum-free DMEM for 48 h. The cells were incubated with the indicated agents for 30 min and then stimulated with 10 ng/ml of PDGF (recombinant human PDGF-BB; R & D Systems, Minneapolis, MN) for 48 h; [3H]thymidine (1 µCi/well; Amersham, Arlington Heights, IL) was added during the final 24 h. DNA synthesis was assessed by measuring the amount of radioactivity incorporated into the trichloracetic acid-insoluble fraction of the cells.

Statistical analysis. All data are expressed as means ± SE. Differences between mean values of two groups were analyzed by unpaired Student's t-tests, as appropriate. Differences between mean values of multiple groups were analyzed by one-way analysis of variance; when differences among the treatment groups were detected, multiple pairwise comparisons were made using Bonferroni t-tests or the Newman-Keuls test. Probability values of 0.05 or less were considered to be statistically significant.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effects of 14,15-EET on PGE2 production by porcine aortic SMC. The production of PGE2, the main radiolabeled eicosanoid formed under all of the conditions tested, was reduced when the SMC were incubated with 14,15-EET. Figure 1 illustrates the HPLC analysis of the radiolabeled material contained in the extracellular fluid. After 10 h of incubation with 1 µM [3H]AA, 40% less radiolabeled PGE2 was present when the medium contained 1 µM 14,15-EET (Fig. 1, A and B). By contrast, there was no reduction in the amount of three unidentified radiolabeled products with retention times between 42 and 50 min that also were formed under these conditions. Similarly, PGE2 formation was reduced by 40% when cultures previously labeled for 10 h with [3H]AA were subsequently incubated for 1 h with 14,15-EET (Fig. 1, C and D), and by 60% when 2 µM ionophore A-23187 was present during the 1-h incubation (Fig. 1, E and F). To investigate whether the reduced amount of PGE2 was due to enhanced destruction, rather than decreased formation, of PGE2, the cells were incubated with 1 µM [3H]PGE2 in the absence or presence of 1 µM 14,15-EET. Under these conditions, the [3H]PGE2 was metabolized to several metabolites; the formation of these products was not altered by 14,15-EET (data not shown).


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  		Fig. 1.   Effect of 14,15-epoxyeicosatrienoic acid (EET) on conversion of arachidonic acid (AA) to PGE2 by porcine aortic smooth muscle cells (SMC). Cultures were incubated in 1 ml DMEM containing 1 µM [3H]AA and 0.1 µM albumin. After incubation, medium was collected, extracted with ethyl acetate, and assayed by reverse-phase HPLC with an online flow scintillation counter. A: 10-h incubation. B: same as A but with 1 µM 14,15-EET present in medium. C: cells previously labeled for 10 h with [3H]AA were washed and incubated for 1 h in 1 ml DMEM containing 0.1 µM albumin. D: same as C but with 1 µM 14,15-EET present during 1-h incubation. E: same as C but with 2 µM A-23187 added during 1-h incubation period. F: same as E but with 1 µM 14,15-EET present during 1-h incubation. Each chromatogram is from a single culture, but similar results were obtained in every case from 2 or 3 additional cultures.

To determine whether the decreases in PGE2 production might be related in some way to the use of [3H]AA as a tracer to measure eicosanoid production, additional experiments were done in which PGE2 production was measured by radioimmunoassay so that no radiolabeled tracer was necessary. As shown in Table 1, the presence of 14,15-EET reduced PGE2 production by 42% in a 10-h incubation with AA and by 48% during a 30-min incubation with A-23187. These decreases are similar to those obtained with [3H]AA.

                              
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  		Table 1.   Effect of 14,15-EET on PGE2 production by porcine aortic smooth muscle cells

The effectiveness of the four EET regioisomers in reducing PGE2 formation was compared to determine the degree of specificity for 14,15-EET. The cells were incubated for 10 h with 5 µM [3H]AA. Under these conditions, the control cultures incubated without added EET produced 371 ± 2 pmol/ml of PGE2, and those incubated with 1 µM 14,15-EET produced 203 ± 11 pmol/ml, a 46% reduction (n = 3, P < 0.05 vs. control). The corresponding values for the cultures incubated with 1 µM 11,12-EET, 8,9-EET, and 5,6-EET were 322 ± 21 (NS vs. control), 271 ± 25 (P < 0.05 vs. control, a 24% reduction), and 410 ± 60 pmol/ml (NS vs. control), respectively. Thus, although complete selectivity was not observed, 14,15-EET was the most effective inhibitor.

The concentration and time dependence of the inhibition is shown in Fig. 2. In cells previously labeled for 10 h with 1 µM [3H]AA, considerable inhibition of PGE2 production occurred during a subsequent 2-h incubation in the presence of 0.3 µM 14,15-EET, and the degree of inhibition increased only slightly when the 14,15-EET concentration was 1 µM. However, 0.1 µM 14,15-EET was ineffective (Fig. 2A). As seen in Fig. 2B, some reduction in PGE2 formation was apparent within 30 min after addition of 14,15-EET, and congruent 50% decreases were observed at both 1 and 2 h. Further studies indicated that the inhibition was reversible (Table 2). When cells were initially incubated with 14,15-EET, a 36% decrease in radiolabeled PGE2 production still was observed 2 h after the EET was removed from the incubation medium, but no inhibition was detected after 6-10 h.


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  		Fig. 2.   Concentration and time dependence of 14,15-EET inhibition of PGE2 formation by porcine aortic SMC. In A, cultures were incubated initially for 12 h in 1 ml DMEM containing 0.1 µM albumin and 5 µM [3H]AA. After removal of medium and washing twice with 1 ml DMEM, cultures were incubated in 1 ml fresh DMEM containing 0.1 µM albumin, 2 µM A-23187, and indicated amounts of 14,15-EET for 1 h, and these media were assayed by HPLC for radiolabeled PGE2 production. Each point is mean ± SE of values obtained from 3 separate cultures. Values are given in picomole amounts, calculated from specific radioactivity of [3H]AA added in initial incubation. In B, cells were labeled with 1 µM [3H]AA and, after washing, were incubated for up to 2 h in fresh DMEM containing 0.1 µM albumin with or without 1 µM 14,15-EET. Formation of radiolabeled PGE2 during 2-h incubation was measured by HPLC, and each point is average of values obtained from 2 separate cultures that were within 10% agreement.

                              
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  		Table 2.   Recovery of PGE2 production following exposure of aortic smooth muscle cells to 14,15-EET

Effects of 14,15-dihydroxyeicosatrienoic acid on PGE2 production. Previous studies demonstrated that SMC rapidly convert 14,15-EET to 14,15-dihydroxyeicosatrienoic acid (DHET) (11). To determine whether this product might be involved in inhibiting PGE2 formation, we first examined the effect of 14,15-DHET on porcine aortic SMC PGE2 production. Under conditions in which the control cultures produced 100 ± 14 pmol PGE2 and those treated with 1 µM 14,15-EET produced 34 ± 2.6 pmol/ml (n = 3, P < 0.05 vs. control), cultures treated with 1 µM 14,15-DHET produced 78 ± 22 pmol/ml (NS vs. control). This finding suggests that 14,15-EET, not 14,15-DHET, mediates the inhibition of PGE2 formation.

To examine whether blocking the conversion of 14,15-EET to 14,15-DHET might modulate the 14,15-EET-induced inhibition of PGE2 production, we investigated the effects of 4-phenylchalcone oxide (4-PCO), an epoxide hydrolase inhibitor (28). Figure 3 illustrates the efficacy of 4-PCO in blocking the conversion of [3H]14,15-EET to 14,15-DHET by the porcine SMC. The HPLC tracing in Fig. 3A illustrates that in the absence of 4-PCO, much of the radiolabeled EET added to the medium was converted to DHET. However, considerably less 14,15-DHET was formed when 4-PCO was present (Fig. 3B). More radiolabeled 14,15-EET was recovered in the cells that were incubated with 4-PCO (635 ± 38 pmol) than in the control cells (517 ± 26 pmol) (n = 3, P < 0.05). However, there was no difference in the distribution of the radioactivity in the cell lipids; ~70% was present in choline phosphoglycerides and 10% in inositol phosphoglycerides in both cases.


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  		Fig. 3.   Metabolites produced from 14,15-EET by porcine aortic SMC. Cultures were incubated for 10 h in 1 ml DMEM containing 0.1 µM albumin and 1 µM [3H]14,15-EET. Medium was extracted and assayed for radiolabeled products by HPLC as described in Fig. 1. Elution times of [3H]14,15-EET and [3H]14,15-dihydroxyeicosatrienoic acid (DHET) are shown. A: chromatogram from control culture. B: comparison of radiolabeled 14,15-DHET formed by control cultures and those incubated with 2 µM 4-phenylchalcone oxide (4-PCO) (n = 3; * P < 0.01).

As shown in Fig. 4, the inhibitory effect of 14,15-EET on PGE2 production was potentiated when 4-PCO was present. A 47% reduction in PGE2 formation occurred with 14,15-EET alone, and this increased to 82% when 4-PCO was added together with the 14,15-EET. In contrast, 4-PCO alone (in the absence of 14,15-EET) did not significantly reduce PGE2 production.


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  		Fig. 4.   Epoxide hydrolase inhibitor 4-PCO potentiates inhibitory effect of 14,15-EET on PGE2 formation in porcine aortic SMC. Cultures were preincubated for 1 h in 1 ml DMEM containing 0.1 µM albumin without (control) or with 2 µM 4-PCO. 14,15-EET (1 µM) was then added to some of the control (EET) and 4-PCO-treated (4-PCO + EET) cultures, and 30 min later, 1 µM [3H]AA was added and incubation continued for 10 h. Radiolabeled PGE2 production was measured by HPLC. Each bar is mean ± SE of values obtained from 3 separate cultures. * P < 0.05 vs. control; + P < 0.05 vs. EET; # P < 0.05 vs. 4-PCO.

Murine brain microvascular SMC. Because the 14,15-EET-induced decrease in PGE2 formation occurred when substrate amounts of AA were present and the decrease was not associated with the accumulation of other eicosanoid products, the most likely mechanism was a direct effect on PGHS. We thus proceeded to investigate the effects of 14,15-EET on vascular SMC PGHS mRNA and protein. Because cDNA probes and antibodies for porcine PGHS are not available, these studies were performed in murine brain microvascular SMC. First, we determined the effects of incubation with 14,15-EET on PGE2 formation by the murine cells (Fig. 5). A different column and solvent gradient were utilized for these HPLC separations, accounting for the differences in retention times relative to those shown in Fig. 1. As was observed with the porcine cells, murine cultures incubated without added EET produced 75% more PGE2 (Fig. 5A) than those exposed to 1 µM 14,15-EET (Fig. 5B). Additional radiolabeled products with retention times similar to those of hydroxyeicosatetraenoic acids (30-34 min) also were detected, but, as in the case of the porcine cells (Fig. 1, A and B), the formation of these unidentified products was not appreciably decreased by the presence of 14,15-EET.


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  		Fig. 5.   Effect of 14,15-EET on conversion of AA to radiolabeled eicosanoids by murine brain microvessel SMC. Cultures were incubated initially with 1 µM [3H]AA for 10 h followed by 1-h incubation with 2 µM A-23187, and medium was assayed for radiolabeled eicosanoid formation as described in Fig. 1. No EET was present in A, and 1 µM 14,15-EET was added to medium in B. Chromatograms from single cultures are shown, but similar results were obtained from 2 additional cultures in each case.

The effect of 14,15-EET on PGHS-1 and PGHS-2 expression was determined in the murine smooth muscle cultures. Figure 6 demonstrates that the amounts of PGHS-1 mRNA and protein were not reduced when 14,15-EET was added to the incubation medium. As seen in Fig. 7, PGHS-2 mRNA and protein increased during a 2-h incubation with A-23187. However, a similar increase occurred when 14,15-EET was present in the incubation medium, and the amounts present at the end of the incubation were similar to those in corresponding cultures incubated without 14,15-EET.


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  		Fig. 6.   Effects of 14,15-EET on PGH synthase (PGHS)-1 mRNA and protein content in murine microvascular SMC. Cultures were initially incubated in 1 ml DMEM containing 0.1 µM albumin and 1 µM AA for 12 h, washed twice with DMEM, and incubated for 2 h in fresh medium containing 2 µM A-23187. Total RNA or protein was isolated from cultures and assayed by Northern or Western blotting, respectively. Cyclophilin mRNA was utilized to control for amount of PGHS mRNA loaded onto membrane.


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  		Fig. 7.   Effects of 14,15-EET on PGHS-2 mRNA and protein content in murine microvascular SMC. Cultures were incubated and assayed by Northern and Western blotting as described in Fig. 6, except that time of incubation with A-23187 varied as indicated. Cyclophilin mRNA was utilized to control amount of PGHS mRNA loaded onto membrane. Protein from lipopolysaccharide-activated RAW macrophages (RAW) was utilized as a source of control murine PGHS-2 protein.

Because these results are consistent with a posttranslational inhibitory mechanism, we determined whether the inhibitory effect of 14,15-EET could be overcome by increasing the concentration of AA. As seen in Fig. 8, the inhibition of PGE2 formation by 1 µM 14,15-EET was decreased by AA in a dose-dependent manner. 14,15-EET produced a 59% inhibition of PGE2 formation in the presence of 1 µM AA; however, when the AA concentration was raised to 20 µM, only a 5% inhibition of PGE2 formation was observed. In contrast, the inhibitory effect of 14,15-EET on PGE2 formation was not overcome by incubating the cells with the combination of 1 µM AA and 19 µM oleic acid (which is not a substrate for PGHS) [45.5 ± 2.8 (control) vs. 21.0 ± 0.8 (14,15-EET) pmol PGE2/ml, P < 0.05, a 54% reduction].


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  		Fig. 8.   Dose-dependent effects of AA on 14,15-EET-induced inhibition of PGE2 formation in murine microvascular SMC. Cultures were initially incubated with or without 1 µM 14,15-EET for 30 min and then incubated with 1 to 20 µM [3H]AA for 4 h. Media were removed and extracted, and radiolabeled PGE2 production was measured by HPLC. Percentage inhibition of PGE2 formation was calculated from picomole values at each AA concentration in absence or presence of 14,15-EET. Each bar is mean ± SE of values obtained from 3 to 6 separate cultures.

Effects of 14,15-EET on PDGF-induced SMC proliferation. Because the mitogen PDGF stimulates the production of PGE2, which, in turn, inhibits SMC growth, the effects of 14,15-EET on PDGF-induced DNA synthesis (an index of cell proliferation) were examined (1, 27). Pretreatment of quiescent porcine aortic SMC with 2 µM 14,15-EET, indomethacin, or 4-PCO did not change basal DNA synthesis relative to control cultures (data not shown). Addition of PDGF (10 ng/ml) produced two- to threefold increases in [3H]thymidine uptake over control (Fig. 9). Indomethacin (2 µM), a PGHS inhibitor, blocked the production of PGE2 (data not shown) and augmented PDGF-induced SMC proliferation [15.2 ± 0.24 × 103 (PDGF alone) vs. 33.9 ± 2.0 × 103 (PDGF + indomethacin) cpm/well, P < 0.01]. Pretreatment with 14,15-EET also enhanced the PDGF-induced mitogenic response, and this effect was further potentiated by 43% in the presence of 2 µM 4-PCO (Fig. 9). In contrast, 14,15-DHET did not affect PDGF-induced [3H]thymidine uptake (data not shown). In murine SMC, 14,15-EET also enhanced PDGF-induced [3H]thymidine uptake [33.7 ± 2.4 × 103 (PDGF alone) vs. 40.7 ± 2.1 × 103 cpm/well, P < 0.05]. Furthermore, treatment of the murine cells with PDGF (10 ng/ml) for 4 h significantly increased PGHS-2 protein expression (Fig. 10). Treatment of the cells with either indomethacin or 14,15-EET did not attenuate the induction of PGHS-2 protein expression by PDGF.


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  		Fig. 9.   Effects of 14,15-EET and 4-PCO on platelet-derived growth factor (PDGF)-induced [3H]thymidine uptake in porcine aortic SMC. Quiescent subconfluent cells were incubated initially with vehicle, 14,15-EET (EET, 2 µM), or 4-PCO (PCO, 2 µM) for 30 min and then stimulated with PDGF (10 ng/ml) for 48 h. [3H]thymidine uptake was determined during final 24 h. Each bar is mean ± SE of values obtained from 5 or 6 separate cultures. * P < 0.05 vs. control; + P < 0.05 vs. PDGF; z P < 0.05 vs. PDGF + 14,15-EET.


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  		Fig. 10.   Induction of PGHS-2 protein by PDGF in murine microvascular SMC. Cultures were initially incubated with or without 2 µM 14,15-EET (EET) or 2 µM indomethacin (Indo) for 30 min, followed by treatment with or without (control) PDGF (10 ng/ml) in serum-free DMEM containing 0.1 µM BSA for 4 h. PGHS-2 protein in cell lysates was detected by Western blot analysis.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The present results demonstrate that 14,15-EET, at concentrations between 0.3 and 1 µM, decreases PGE2 production in vascular smooth muscle. When released from activated platelets, the local EET concentration can approach 1 µM (39). This suggests that the inhibitory effect is possible under physiological or pathological conditions. Two observations suggest that the inhibition takes place at the PGHS reaction. First, the reduction occurs when substrate amounts of AA are available in the extracellular fluid. This excludes an effect on the phospholipase-mediated mobilization of AA. Second, alternative products are not formed when PGE2 synthesis is reduced. If the inhibition occurred subsequent to the PGHS reaction, some accumulation of either PGH2 or one of its other metabolites would be expected. However, no other radiolabeled products were detected by HPLC even though 14,15-EET reduced PGE2 production from [3H]AA by 45% or more.

These findings suggest that besides having a direct effect on Ca2+-activated K+ channels (4, 15, 17), 14,15-EET may influence vascular smooth muscle function by reducing either basal or stimulated PGE2 production. This decrease is consistent with previous data demonstrating that 14,15-EET reduces PGI2 production by the aorta and thromboxane production by platelets (14, 23). As observed in the present study, 8,9-EET produced less inhibition of platelet thromboxane production than 14,15-EET, and 11,12-EET was ineffective (14). 14,15-EET also inhibited ram seminal vesicle PGHS, and as observed in the intact platelet, 8,9-EET was less effective and 11,12-EET was noninhibitory (14). These correlations support the conclusion that inhibition in the intact cell probably results from an effect on PGHS. Because the inhibitory effect can be partially overcome by adding high concentrations of AA, but not oleic acid (which is not a substrate for PGHS), it is likely that 14,15-EET acts as a competitive inhibitor of the enzyme. The structural basis for the regioisomer specificity presumably is due to differences in binding of the various EETs to PGHS, but this remains to be demonstrated.

As noted previously (13), the cells converted much of the 14,15-EET to 14,15-DHET, probably through the action of epoxide hydrolases (38). The observation that 14,15-DHET failed to inhibit PGE2 production suggests that 14,15-EET, rather than its DHET metabolite, was the actual inhibitor. This was substantiated by demonstrating that the epoxide hydrolase inhibitor 4-PCO (28), which blocked the conversion of 14,15-EET to 14,15-DHET, potentiated the 14,15-EET-induced inhibition of PGE2 production. Furthermore, the inhibitory effects of 14,15-EET were completely reversible 6-10 h after its removal from the incubation medium. We previously reported that over a similar time period, most of the radiolabeled 11,12-EET that had been taken up into porcine SMC was converted to 11,12-DHET and its metabolites and released from the cells (12). We also found that 14,15-EET can be rapidly taken up and hydrated by SMC (11). Therefore, a likely explanation for the reversibility is that the 14,15-EET remaining in the cells was gradually inactivated through conversion to 14,15-DHET, thereby removing the inhibitory substance. The inhibition of conversion of 14,15-EET to 14,15-DHET by 4-PCO was accompanied by an increase in the amount of 14,15-EET incorporated into cell lipids. Similar results were previously reported with 4-PCO in rat brain astrocytes (32). These observations are also consistent with prior data from our laboratory indicating that EETs are more avidly incorporated into cell lipids than their DHET metabolites (36). Because blood vessels synthesize EETs (4, 23, 29) and also are continuously exposed to EETs circulating in the blood (18), the conversion of EETs to DHETs by epoxide hydrolases may act to limit the amount of AA epoxygenase products incorporated in the blood vessel wall.

PDGF is an important mitogen that has been suggested to contribute to proliferation of vascular SMC in pathological conditions such as atherosclerosis (30). Others have reported that PDGF-induced mitogenic responses are modulated by PGE2 formation through induction of PGHS-2 (1, 27). Consistent with these prior reports, we found that PGHS-2 protein was induced by PDGF in murine SMC and that PDGF-induced [3H]thymidine uptake was enhanced by indomethacin or 14,15-EET, but not 14,15-DHET. Furthermore, the inhibition of PGE2 formation and augmentation of PDGF-induced [3H]thymidine uptake resulting from treatment with 14,15-EET were further potentiated by 4-PCO. In contrast, neither indomethacin nor 14,15-EET affected basal [3H]thymidine uptake, an observation that suggests that the two compounds possess similar mechanisms of action (i.e., inhibition of PGE2 synthesis). The stimulation of SMC proliferation consequent to inhibition of PGHS by 14,15-EET may help to explain prior observations that EETs stimulate vascular SMC growth (31) and enhance mitogenesis in glomerular mesangial cells (16).

It is difficult to predict how the 14,15-EET-induced decrease in PGE2 formation might affect vasomotor tone. PGE2 has been shown to either contract or relax vascular smooth muscle, depending on conditions or the preparation (7, 8). In rat tail artery SMC, PGE2 inhibits K+ currents and produces contraction (25). By contrast, it dilates the pial arterioles of the cerebral circulation in newborn pigs (22). Therefore, a decrease in PGE2 formation might be expected to augment the vasodilator response to EETs in those vessels where PGE2 produces vasoconstriction, whereas it might attenuate the EET vasodilator response in those vessels where PGE2 produces relaxation.

In summary, these findings suggest that 14,15-EET can affect vascular SMC function by reducing PGE2 formation. The most likely mechanism is competitive inhibition of PGHS. The inhibition of PGE2 formation, in turn, can modulate PDGF-induced mitogenic responses. Because 14,15-EET produces similar inhibition of PGHS in endothelium and platelets (14, 23), it is possible that a major function of this cytochrome P-450 product in the vascular system is to modulate PG synthesis.

    ACKNOWLEDGEMENTS

These studies were supported by Research Grants HL-49264, NS-24621, and NS-09858 from the National Institutes of Health and by an American Heart Association Clinician-Scientist Award (96004540) and Grant-in-Aid (9650661N).

    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: A. A. Spector, Dept. of Biochemistry, 4-403 BSB, Univ. of Iowa, Iowa City, IA 52242.

Received 6 July 1998; accepted in final form 24 August 1998.

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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Heart Circ Physiol 275(6):H2113-H2121
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