INTRODUCTION

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EPOXYEICOSATRIENOIC ACIDS (EETs) are metabolites of arachidonic acid produced by cytochrome P-450 epoxygenases (3). Most commonly, EETs are formed after a stimulus to a cell that results in phospholipase activation and liberation of arachidonic acid. In the cardiovascular system, EETs are produced in the endothelial cells of blood vessels and act as paracrine factors, exerting their primary biological effects on the smooth muscle cells (25). EETs cause a relaxation of vascular tension through regulation of ion fluxes (6, 33). Vascular tension is also influenced by the amount of nitric oxide (NO) produced locally by NO synthase (NOS). The expression of NOS is positively regulated by estrogen, the primary product of aromatase (16). Thus the regulation of aromatase by EETs potentially contributes to the maintenance of vascular tone. EETs are also active in a variety of other tissues, including numerous endocrine glands and the nervous system, where they cause secretion of peptides and neurotransmitters (21, 26).

The cellular site/receptor where EETs initiate their biological action in smooth muscle, or for that matter any other tissue, has not been identified. A plasma membrane site for an EET receptor has been proposed by Wong et. al. (32), who demonstrated that EETs will bind specifically to a protein receptor on guinea pig monocytes. The ability of EETs to increase K⁺ conductance through Ca²⁺-activated K⁺ channels measured using inside-out patch-clamp techniques also suggests that EET has a membrane site of action (10, 13). Additional evidence for a membrane site of action was presented by Node et. al. (22), who showed that EETs mediate the induction of tissue-type plasminogen activator gene transcription through activation of G_s.

Several intracellular molecules are known to bind EETs. These include fatty acid-binding proteins and the peroxisomal proliferator-activated receptor (PPAR). PPARs are activated when they bind a fatty acid such as arachidonic acid or potentially EETs (17, 29). The activated PPAR, when heterodimerized with an RXR, acts as a regulator of gene transcription (17). The PPAR-RXR complex has been implicated as a modulator aromatase gene expression (20). In such a mechanism, the site of EET action would be intracellular rather than at the cell membrane. There have also been proposals that expression of the biological activities of EETs may be dependent on further metabolism of EET or its incorporation into cell lipids (21). Thus there is conflicting evidence concerning the cellular location of a putative receptor for EETs. In an attempt to resolve this question, we examined the role of free and immobilized EETs on regulation of aromatase activity in cultures of aortic smooth muscle.

Aromatase has been reported to be present in vascular smooth muscle, and expression of the aromatase gene is regulated by dibutyryl cAMP in these cells (1, 12). Aromatase activity, in essentially all tissues where it has been studied, is increased after a short lag period of several hours to a maximal induction over a 24- to 48-h period by cAMP. This occurs through control of expression of the aromatase gene (4, 36). When the concentration of cAMP increases in the cytoplasm in response to a receptor-initiated event, cytosolic cAMP-binding protein is activated and moves to the nucleus, where it acts as one of the primary transcription factors that stimulates aromatase gene expression (37). The present results show that the EETs regulate cAMP-stimulated aromatase activity in aortic smooth muscle cells and also provide evidence for an EET receptor that is localized in the plasma membrane.

	METHODS

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Synthesis of 14,15-EET sulfanilamide. 14,15-EET (0.29 mmol) and 0.32 mmol N-hydroxysuccinimide (NHS) were azeotropically dried under benzene and then dissolved in 5 ml anhydrous tetrahydrofuran (THF). After the mixture was cooled to 0°C, 0.32 mmol dicyclohexylcarbodiimide was added, and the mixture was gradually brought to room temperature and stirred for 12 h. After volatile materials were removed under vacuum, the 14,15-EET-NHS derivative was purified by SiO₂ column chromatography. To a solution of sulfanilamide (SA; 0.32 mmol in 5 ml THF) at 78°C, n-butyl lithium (0.32 mmol) was added 1 h before the slow addition of 0.22 mmol 14,15-EET-NHS in THF. This solution was allowed to equilibrate to room temperature over 3 h. The reaction was quenched with a saturated solution of NH₄Cl, and the aqueous layer was extracted with ethyl acetate and combined with the organic phase. The combined fractions were dried under vacuum and the residue, 14,15-EET-SA, was purified by SiO₂ column chromatography.

Synthesis of immobilized 14,15-EET. All solvents were distilled and purged with argon before use. One hundred milligrams of silica microspheres (1 ml of a 10% solid suspension in deionized water, mean diameter 0.50 µm, 0.00627 µeq/mg microspheres; Bangs Laboratories) were transferred to a centrifuge tube and washed with 1 ml deionized water. The contents were centrifuged for 3 min, the supernatant solution was decanted, and the pellet was resuspended by sonication in 1 ml fresh deionized water, centrifuged, and decanted. The entire process was repeated sequentially with deionized water (3 × 3 ml) and anhydrous THF (3 × 3 ml). 1,1'-Carbonyldiimidazole (6.5 mg, 0.039 mmol) was added to 15 mg of microspheres suspended in 1 ml anhydrous THF, and the reaction mixture was shaken for 45 min at room temperature. [1-¹⁴C]14,15-EET-SA (0.032 mmol; 18.26 counts · min¹ · nmol¹) in 1 ml THF was added, and the contents were shaken for 12 h at room temperature, centrifuged, and then washed sequentially with THF (3 × 3 ml), deionized water (3 × 3 ml), and ethyl acetate (3 × 3 ml). Finally, the microspheres were suspended in 1 ml deionized water. Fifty microliters were transferred into a vial containing 5 ml scintillation cocktail, and the radioactivity was determined using a liquid scintillation counter. On the basis of radioactivity, it was calculated that 0.1 mg of 14,15-EET-SA was tethered to 100 mg of microspheres. The EET-bead construct was stored at 4°C in deionized water under nitrogen until use. An aliquot of the bead suspension was pelleted by centrifugation and then resuspended in culture medium. Tethered EET and EET-SA concentrations in culture medium were quantified by scintillation counting. Stability of the bead construct was tested by recovery of beads by centrifugation after incubations. The distribution of the radioactivity in the silica bead pellet (>99%) versus that recovered in culture medium supernatant (<1%) indicates that the covalent linkage of the EET-SA to the bead was stable under these incubation conditions. The structure of the 14,15-EET-SA complex tethered to the silica bead is illustrated in Fig. 1.

View larger version (6K): [in this window] [in a new window]   Fig. 1.   Structure of 14,15-epoxyeicosatrienoic acid (14,15-EET) sulfanilamide (14,15-EET-SA) covalently attached to an ~0.5-µm-diameter silica bead.

Smooth muscle cell culture. Cell cultures were prepared as described by Yuan et al (34). Briefly, the thoracic aorta from Sprague-Dawley rats (150-250 g) was removed under sterile conditions and placed in DMEM, where the vessels were gently stripped of any remaining connective tissue and adventitia, cut longitudinally to expose the lumen, and incubated in DMEM containing 400 U/ml collagenase type 4 (Worthington Biochemical; Freehold, NJ) at 37°C for 10 min. The endothelium was removed with a gentle wash. The tissue was incubated at 37°C under a 5% CO₂ atmosphere for 24 h in DMEM-high glucose medium, which was supplemented with 2 mM L-glutamine (GIBCO-BRL; Grand Island, NY), 10 mM HEPES (Sigma; St. Louis, MO), basal medium Eagle vitamins (GIBCO-BRL), minimum essential medium (MEM) with nonessential amino acids (GIBCO-BRL), and 20% FBS (Intergen; Purchase, NY). The tissue was cut into 1- to 2-mm sections and incubated with 400 U/ml collagenase type 4 (Worthington), 1.5 U/ml elastase (Worthington), and 0.37 mg/ml soybean trypsin inhibitor (Sigma) in DMEM at 37°C for 40 min. After 15 min, the tissue was triturated to speed digestion. The incubation mixture was diluted 20-fold by adding DMEM containing 20% FBS to stop the enzymatic digestion. The cell suspension was centrifuged for 5 min at 800 rpm at room temperature, the supernatant was removed, and the resulting cell pellet was resuspended in DMEM containing 20% FBS. Rat aortic smooth muscle cells were plated into either 60-mm tissue culture dishes or six-well tissue culture plates, both precoated with 0.1% gelatin. The culture medium was changed after 24 h to remove nonadherent cells. Cells were incubated in a humidified atmosphere of 5% CO₂ in air at 37°C. The medium was changed twice weekly, and the cells were used for experiments from passages 3 through 8. Cells were incubated with dibutyryl cAMP (Sigma), 14,15-EET (Cayman Chemical), or EET derivatives for 24 h. All experiments were performed at least three times using triplicate cultures in each experiment. Data are expressed as means ± SE of at least three separate cell culture preparations.

Aromatase assay. Aromatase enzymatic activity was measured using a modification (19) of the method described by Weisz (28). The assay uses a radiolabeled substrate ([³H]androstenedione) that releases a tritium-labeled water molecule for each molecule of substrate converted to estradiol. Vascular smooth muscle cell cultures (sets of three dishes/point) were incubated in covered petri dishes with 150 pmol androstenedione/ml medium for intervals of 2-4 h in a humidified 37°C incubator to minimize evaporation. Afterward, the assay was terminated by chilling the cell cultures and medium to 4°C for 10 min. The culture medium was recovered, protein was precipitated by the addition of cold 100% TCA (final concentration, 3%) and sedimented by centrifugation, and the steroids (substrate and estrogens) were removed from the supernatant by extraction into CHCl₃ (2× vol). The aqueous phase was extracted again to remove any remaining radiolabeled steroids. This was done by the addition of an equal volume of dextran-coated charcoal (0.5% dextran, 5% activated charcoal), vigorous mixing, and then centrifugation to remove the charcoal. More than 99.8% of the substrate ([³H]androstenedione) was removed from the final product (³H₂O) by this procedure. An aliquot of the supernatant solution was assayed for radioactivity to quantify the amount of ³H₂O produced during the incubation. Media from corresponding incubations that did not contain cells ("blanks") were assayed for radioactivity, and these values were subtracted from the experimental values. Cell protein content was determined by the Bradford procedure (2). Aromatase activity was expressed as picomoles of androstenedione converted to H₂O per milligram of cell protein.

Chromatographic separation of EETs and phospholipids. EETs, their metabolites, and their derivatives in the culture medium were analyzed by reverse-phase HPLC using a Gilson dual-pump gradient system equipped with model 306 pumps, a model 117 dual-wavelength UV detector, a model 231XL automatic sample injector (Gilson Medical Electronics; Middleton, WI), and a 3-µm, 4.6 × 150-mm Spherisorb C₁₈ column obtained from Alltech (Deerfield, IL). The mobile phase consisted of water adjusted to pH 3.4 with formic acid and an acetonitrile gradient that increased from 30-100% over 60 min at a flow rate of 0.7 ml/min. The distribution of radioactivity was measured by combining the column effluent with scintillator solution and passing the mixture through an on-line flow detector (IN/US Systems; Tampa, FL).

	RESULTS

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Stability and metabolism of free 14,15-EET-SA and 14,15-EET-SA bound to beads. To determine whether EETs have a biological effect on smooth muscle cells that is due to the EET interacting with a cell surface receptor molecule rather than acting at a site inside the cell, a 14,15-EET derivative, 14,15-EET-SA, was covalently attached to silica beads (average diameter = 0.5 µm). Cultures of rat aorta smooth muscle cells were incubated with medium containing free 1 µM [1-¹⁴C]14,15-EET-SA (specific activity, 915 counts · min¹ · nmol¹) or 1 µM [1-¹⁴C]14,15-EET-SA that was bound to silica beads for either 1 or 24 h. After 1 h, ~2.4% free 14,15-EET-SA was recovered with the cells, and this amount increased only slightly after 24 h. In contrast, after 1 h, <1% 14,15-EET-SA radioactivity attached to silica beads was recovered with the cells and only ~2% was cell associated after 24 h. Approximately 98% of the radioactivity remained in the culture medium and was recovered as 14,15-EET-SA after either 1 or 24 h of incubation (Table 1).

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Analysis of radioactivity contained in the incubation medium. A and B: HPLC of the medium extract after incubation of radiolabeled 14,15-EET-SA in the cell culture medium for 24 h without cells (A) or with a confluent cell layer (B). C and D: HPLC of the medium extract after 24 h of incubation of radiolabeled 14,15-EET in the cell culture medium without cells (C) or with a confluent cell layer (D). Data are representative of 3 experiments. 14,15-DHET, 14,15-dihydroxyeicosatrienoic acid.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Analysis of radioactivity contained in the cells. A and B: distribution of 14,15-EET (A) or 14,15-EET-SA (B) radioactivity in lipids extracted from cultured smooth muscle cells that had been incubated with 1 µM [1-14C]14,15-EET or 1 µM [1-14C]14,15-EET-SA for 4 h. The lipid extract was separated by thin-layer chromotography and scanned for radioactivity. Data are representative of 3 experiments. CPM, counts per minute; PI, phosphatidylinositol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; NL, neutral lipids.

Effect of EETs on vascular smooth muscle aromatase activity. Incubation of low passage (<8) cultures of rat aorta smooth muscle cells for 24 h with 1 mM dibutyryl cAMP caused a doubling of aromatase activity (Fig. 4). Incubation of the cells for 24 h with 1 µM 14,15-EET, a concentration that inhibits aromatase activity in granulosa luteal cells (27), did not significantly affect the basal activity of aromatase. However, when 1 µM 14,15-EET was included in the culture medium containing dibutyryl cAMP, the increase in aromatase activity produced by dibutyryl cAMP was almost entirely inhibited (Fig. 4). Higher concentrations of EETs (i.e., up to 10 µM) showed no greater inhibition. The inhibition of aromatase by EETs was maximal at 24 h and could no longer be detected after 72 h.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Effect of 1 µM 14,15-EET on aromatase activity in rat smooth muscle cells in the absence or presence of 1 mM dibutyryl cAMP (DBcAMP). Data are means ± SE of 4 experiments. +P < 0.05 vs. control; *P < 0.05 vs. DBcAMP stimulation.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Effect of 1 µM 11,12-EET and 1 µM 11,12-DHET on DBcAMP-induced aromatase activity in rat aorta smooth muscle cell cultures. Data are means ± SE of 4 cell cultures. *P < 0.05 vs. DBcAMP-induced cells.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Comparison of the inhibitory action of 1.0 and 0.2 µM free 14,15-EET-SA and immobilized 14,15-EET-SA-beads on DBcAMP-stimulated aromatase activity in rat aortic smooth muscle cells. Data are means ± SE of 3 experiments. *P < 0.05 by ANOVA and Newmann-Keuls range test vs. DBcAMP-stimulated cultures.

	DISCUSSION

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Several previous reports have indicated that EETs are able to affect membrane-associated signal transduction mechanisms. Such membrane-associated activities stimulated by EETs include tyrosine phosphorylation (5), ADP ribosylation (18), G_s-mediated responses (10, 18), and protein kinase A activation (15). Although these reports support the hypothesis that EETs can act on processes that are initiated at the cell membrane, they do not provide direct evidence that EET biological responses are initiated at the cell membrane. Our experiments were designed to determine whether EETs retained biological activity if they were restricted to the cell surface, and thus the results of this study provide direct evidence for a cell membrane locus for EET action. To restrict EET to the outside of the cell, EETs were covalently linked via a SA derivative to silica beads. To ensure that EETs attached to the bead were restricted from entering the cell cytoplasm, EET-SA radioactivity was measured after incubation of the bead complex with the cells for up to 24 h. In both 1- and 24-h incubations, no more than 2% of the EET-associated radioactivity was recovered with the cell fraction. By observation of the cells with phase microscopy after removal of the culture medium and gentle washes, we were able to observe small numbers of beads that appeared to be adherent to the cells or trapped between adjacent cells. Therefore, we attribute residual cell-associated EET radioactivity to beads that could not be removed by gentle washing. We conclude from these experiments that an appreciable amount of EET tethered to the beads did not transverse the cell membrane and thus did not gain access to any potential EET targets in the cell interior.

As a free molecule, EET-SA is readily taken up into the cell, as can be seen from the data in Fig. 3. However, the total amount of 14,15-EET compared with 14,15-EET-SA recovered from the cells was much greater due to the ability of the EET to be incorporated into cell lipids. The ability of both compounds to pass through the plasma membrane into the cell by diffusion is likely to be similar because both EET and EET-SA possess similar polarities, as can be inferred from the relatively small difference in the elution profiles of 14,15-EET-SA (retention time, 42 min) and 14,15-EET (retention time, 44 min) on reverse-phase HPLC (Fig. 2, A vs. C). Therefore, it can be argued that 14,15-EET-SA and 14,15-EET molecules would diffuse across a membrane in a similar fashion. The data presented in Table 1 clearly show that EET-SA was able to pass through the plasma membrane into the cells and quickly establish an equilibrium with EET-SA in the culture medium that remained constant for 24 h. On the other hand, EET-SA, once it gains access to the cytoplasm either by a specific transporter or by diffusion, did not become incorporated in the cell lipids. Thus EET-SA, as opposed to free EET, does not accumulate in the cells. Although the addition of sulfanilamide to EET does not appear to influence the passage of EET into the cell, the presence of sulfanilamide on the carboxyl carbon prevents the cell from metabolizing EET.

There was no observable conversion of 14,15-EET-SA to 14,15-DHET-SA or other more polar metabolites under the conditions tested. Therefore, EET-SA is not a substrate for the normal metabolic pathways for EETs or hydration to DHET by epoxide hydrolase (35) or -oxidation (7). SA added to the carboxyl group also prevents the acylation of 14,15-EET-SA into cell lipids, which is where EETs are normally stored in the cell (8). This obviously is not due to an inability of EET-SA to cross the plasma membrane and reach the cytoplasm because the TLC analysis indicates that there is a substantial amount of free 14,15-EET-SA present in cells that have been incubated with 14,15-EET-SA for as little as 4 h, even though no 14,15-EET-SA was detected in either neutral lipids or phospholipids.

The data presented in this study show that 14,15-EET and 14-15-EET-SA are equipotent in their ability to inhibit the increase in aromatase activity induced by cAMP. However, without physically restricting EET to either the external or cytoplasmic domain, no distinction can be made as to whether EET or EET-SA are acting via an effector molecule on the plasma membrane or binding to and affecting a molecule within the cell. Because 14,15-EET-SA is not incorporated into the cell lipids, it appears that there is no necessity for EET incorporation into phospholipids to produce its inhibitory action on dibutyryl cAMP-stimulated aromatase activity. Thus acylation of EETs into membrane phospholipids is most likely a storage mechanism rather than an essential part of the process through which EET exerts its effect on the expression of aromatase activity.

When 14,15-EET is covalently bound to silica beads, the EET cannot pass through the plasma membrane and reach potential intracellular sites of action. Our findings demonstrate that EET, when available only at the cell surface in the form of the 14,15-EET-SA-bead complex, completely retains the ability to inhibit cAMP-stimulated aromatase activity. The observation that the beads alone had no effect on the dibutyryl cAMP-stimulated aromatase activity supports our contention that the EET-SA construct attached to the bead is producing the effect. Taken together, these results provide strong evidence that EETs produce their biological effect on aromatase activity by acting on a receptor that is present on or near the outer surface of the cell membrane. What remains to be resolved is the discrepancy that exists between the nanomolar K_d reported for EET binding to a monocyte plasma membrane protein by Wong et al. (32) and the need for micromolar concentrations to obtain an aromatase biological response. This discrepancy, however, may be artifical and dependent on the sensitivity of the biological assay being employed, because there are reports of low nanomolar biological responses to EETs where the assay used is quite sensitive (36).

This study also provides some structure-function data regarding the binding site of EET involved in its biological activity. Because the carboxyl carbon is unavailable in the tethered form or in the free SA derivative, this region of the EET molecule is apparently not involved in its binding to the putative EET target. These data, along with those of Wong et al. (30, 31) that show specific EET binding to a putative monocyte membrane protein, are consistent with a mechanism in which EET biological activity is mediated through a receptor located in the plasma membrane of cells. We hypothesize that this receptor, upon binding EET, initiates a signal cascade (15). This results in interference with the cAMP-initiated mechanism (37) that stimulates transcription of the aromatase gene.