Adrenic acid (docosatetraenoic acid), an abundant fatty acid in the vasculature, is produced by a two-carbon chain elongation of arachidonic acid. Despite its abundance and similarity to arachidonic acid, little is known about its role in the regulation of vascular tone. Gas chromatography/mass spectrometric analysis of bovine coronary artery and endothelial cell lysates revealed arachidonic acid concentrations of 2.06 ± 0.01 and 6.18 ± 0.60 μg/mg protein and adrenic acid concentrations of 0.29 ± 0.01 and 1.56 ± 0.16 μg/mg protein, respectively. In bovine coronary arterial rings preconstricted with the thromboxane mimetic U-46619, adrenic acid (10−9–10−5 M) induced concentration-related relaxations (maximal relaxation = 83 ± 4%) that were similar to arachidonic acid relaxations. Adrenic acid relaxations were blocked by endothelium removal and the K+ channel inhibitor, iberiotoxin (100 nM), and inhibited by the cyclooxygenase inhibitor, indomethacin (10 μM, maximal relaxation = 53 ± 4%), and the cytochrome P-450 inhibitor, miconazole (10 μM, maximal relaxation = 52 ± 5%). Reverse-phase HPLC and liquid chromatography/mass spectrometry isolated and identified numerous adrenic acid metabolites from coronary arteries including dihomo (DH)-epoxyeicosatrienoic acids (EETs) and DH-prostaglandins. DH-EET [16,17-, 13,14-, 10,11-, and 7,8- (10−9–10−5 M)] induced similar concentration-related relaxations (maximal relaxations averaged 83 ± 3%). Adrenic acid (10−6 M) and DH-16,17-EET (10−6 M) hyperpolarized coronary arterial smooth muscle. DH-16,17-EET (10−8–10−6 M) activated iberiotoxin-sensitive, whole cell K+ currents of isolated smooth muscle cells. Thus, in bovine coronary arteries, adrenic acid causes endothelium-dependent relaxations that are mediated by cyclooxygenase and cytochrome P-450 metabolites. The adrenic acid metabolite, DH-16,17-EET, activates smooth muscle K+ channels to cause hyperpolarization and relaxation. Our results suggest a role of adrenic acid metabolites, specifically, DH-EETs as endothelium-derived hyperpolarizing factors in the coronary circulation.
- endothelium-dependent relaxation
- cytochrome P-450
- potassium channels
- endothelium-derived hyperpolarizing factor
the polyunsatuarated fatty acid, arachidonic acid (20:4, ω-4), and its metabolites play a major role in the regulation of vascular tone. In the vascular endothelium, arachidonic acid is released from membrane phospholipids by phospholipase and is metabolized by cyclooxygenase (COX), lipoxygenase (LO), or cytochrome P-450 (CYP450) enzymes to numerous metabolites, including the vasodilatory compounds, prostaglandin I2 (PGI2), and epoxyeicosatrienoic acids (EETs) (2, 15, 22). Adrenic acid (7,10,13,16-docosatetraenoic acid, 22:4, ω-6) is identical to arachidonic acid but has two additional carbons at the carboxyl end (12, 18, 19). Adrenic acid is produced by chain elongation of arachidonic acid or elongation and desaturation of linoleic acid (12, 20). It can also be converted to arachidonic acid via β-oxidation (6, 12, 18, 20).
Similar to arachidonic acid, adrenic acid is metabolized by COX and LO. Previously, we demonstrated that adrenic acid is metabolized by COX to dihomo (DH)-PGI2 by human vascular endothelial cells (ECs) (3). DH-PGI2 inhibited thrombin-induced platelet aggregation (3). Additionally, adrenic acid is metabolized in platelets to DH-thromboxane by COX and DH-hydroxyeicosatetraenoic acids (HETE) by 12-LO (24) and in the renal medulla to DH-PGs and DH-thromboxane by COX (23). Metabolism of adrenic acid by CYP450 enzymes has not been demonstrated. However, in isolated porcine coronary arteries, DH-EETs caused dilation (26). All four DH-EET regioisomers showed similar potency with EC50 values averaging 10−11 to 10−12 M and maximal dilations between 77% and 93%. In isolated rat coronary smooth muscle cells (SMCs), DH-13,14-EET activated large-conductance calcium-activated K+ (BKCa) channels in inside-out patches (26).
It is not clear whether the coronary vasculature metabolizes adrenic acid to DH-PGs or DH-EETs, and the physiological role of these metabolites in the regulation of vascular tone remains undefined. Thus the goal of this study was to examine the vascular effect of adrenic acid in bovine coronary arteries (BCAs). Additionally, this study provides the first in-depth examination of adrenic acid metabolism by the coronary vasculature.
MATERIALS AND METHODS
Adrenic acid, methyl docosatetraenoate, methyl arachidonate, and heptadecanoic acid (C17:0) were purchased from NuChek Prep (Elysian, MN). 1-[14C]adrenic acid (specific activity, 40–60 μCi/mmol) was obtained from American Radiolabeled Chemicals (St. Louis, MO). Arachidonic acid, iberiotoxin, indomethacin, and miconazole were purchased from Sigma Chemical (St. Louis, MO). DH-PGF2α and U-46619 were purchased from Cayman Chemical (Ann Arbor, MI). Indomethacin, U-46619, and miconazole were dissolved in 95% ethanol, and all other drugs were dissolved in distilled water. DH-EETs were synthesized in our laboratory using published procedures (5, 21). Triphenylphosphine and 3-chloroperoxybenzoic acid (PBA) were obtained from Aldrich Chemical (Milwaukee, WI). Boron trifluoride in methanol (14% by weight) was purchased from Pierce (Rockford, IL). All other chemicals and solvents were of analytical or highest purity grades.
Lipid extraction and gas chromatography/mass spectrometry.
Bovine hearts were obtained from a local slaughterhouse. Branches of the left anterior descending coronary artery (diameter <2 mm) were dissected and cleaned of adhering fat and connective tissue. Arterial sections (∼0.1–0.3 g) were homogenized in a glass homogenizer in 1 ml HEPES buffer containing (in mmol/l) 10 HEPES, 150 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, and 6 glucose (pH 7.4), and 100 μl of sample were used for protein analysis (Bio-Rad, Hercules, CA). The extraction procedure for preparation of total lipid extract was adapted from the procedure described by Folch et al. (9). Chloroform and methanol (CHCl3-MeOH = 2:1; 9 ml) were added to the homogenized tissue in a 15-ml glass conical tube. The samples were vortexed and allowed to stand for 30 min. The vortexing and separation were repeated. The organic phase was removed and dried under a stream of N2. The samples were redissolved in 200 μl CHCl3 and stored in −40°C.
BCA ECs were cultured in 75-cm2 flasks as previously described (22). At 75% confluency, the medium was removed and the cells were washed twice with HEPES buffer. The cells were scraped and centrifuged at 200 g for 10 min. The supernatant was discarded. The cell pellet was resuspended in 1 ml of HEPES buffer, and the cells were disrupted with a sonicator-disruptor (Model-185, Branson Ultrasonics, Danbury, CT). A portion of sample (100 μl) was used for protein analysis. The cells were extracted and stored as described above.
Heptadecanoic acid (C17:0) was added as an internal standard. The extracted lipids were hydrolyzed, and fatty acids were esterified to methyl esters as previously reported (13). The solvent was evaporated under a N2 stream, and 0.5 ml benzene and 2 ml of 14% boron trifluoride in methanol were added. The samples were placed in a 100°C water bath for 30 min, cooled to room temperature, and 20 ml of water were then added. The samples were extracted with hexane (3 × 3 ml) and evaporated to dryness under N2.
Gas chromatography/mass spectrometry.
The fatty acid methyl esters were dissolved in 200 μl of acetonitrile and analyzed by gas chromatography/mass spectrometry (GC/MS) as previously described (17). GC/MS was performed using a Hewlett-Packard 5989A mass spectrometer coupled with a 5890 Series II gas chromatograph. The detection was made in the positive ion mode. Fatty acid methyl esters were resolved using a 30-m capillary DB-5 column with a linear increase in temperature from 100° to 300°C. For quantitative measurement, mass-to-charge ratios (m/z) 319, 347, 285, and 327 were monitored for the methyl esters of arachidonic acid, adrenic acid, heptadecanoic acid, and [2H8]arachidonic acid, respectively. Fatty acid methyl ester concentrations were calculated by comparing the ratio of the peak area to the standard curves. Arachidonic and adrenic acid content of BCAs and ECs was expressed (in μg of fatty acid/mg protein).
Coronary arteries were cut into rings (3 mm in length) and suspended on a pair of stainless steel hooks in a 6-ml water-jacketed organ chamber as previously described (4, 213, 1.2 KH2PO4, 1.2 MgSO4, 11 glucose, 0.02 EDTA, and 3.2 CaCl2. The buffer was equilibrated with 95% O2-5% CO2 and maintained at 37°C. Resting tension was adjusted to its length tension maximum of 3.5 g. After equilibration for 1.5 h, KCl (40–60 mM) was added until reproducible contractions were obtained. The thromboxane mimetic U-46619 (10 to 20 nmol/l) was administered to contract the arterial rings to 50% to 75% of KCl-induced contraction. Cumulative additions of arachidonic acid, adrenic acid, or DH-EETs were added. Each concentration was added after stable relaxations to the previous concentration were established, which averaged 4 to 10 min. Arteries were incubated with indomethacin (10−5 M), miconazole (10−5 M), iberiotoxin (10−7 M), or vehicle 10 min before U-46619 contraction. In a subset of experiments, the endothelium was removed by gentle rubbing of the arterial rings with a wooden stick. Results were expressed as percent relaxation with 100% relaxation representing basal tension.
Metabolism of [14C]adrenic acid and [14C]DH-10,11-EET by BCAs.
Arterial rings (0.3 g) were placed in 5 ml HEPES buffer and incubated at 37°C with [1-14C]-adrenic acid (0.8 μCi, 10−7 M) for 30 min (22). The calcium ionophore A-23187 (5 × 10−5 M) was added, and the incubation continued for an additional 15 min. Reactions were stopped by adding ethanol (25% final concentration). The medium was removed and extracted using C18 Bond Elut solid-phase extraction columns (Varian, Harbor City, CA). The metabolites were eluted with ethyl acetate, as previously described (16, 22). The samples were evaporated to dryness under a stream of N2 and stored at −40°C until HPLC analysis. Incubations for liquid chromatography/tandem mass spectrometry (LC/MS/MS) were repeated in larger scale using adrenic acid (10−4 M) and 0.6 g of coronary arteries under similar conditions. For DH-10,11-EET metabolism, [14C]DH-10,11-EET was incubated in 5 ml HEPES buffer with or without bovine coronary arterial rings for 30 min at 37°C. Ethanol was added (25% final concentration), and the metabolites were extracted as above.
Adrenic acid metabolites were resolved by reverse-phase HPLC (Nucleosil-C18 column, 5 μm, 4.6 × 250 mm) using solvent system I as previously described (22). Solvent A was water and solvent B was acetonitrile containing 0.1% glacial acetic acid. The program was a 35-min linear gradient from 50% solvent B to 94% solvent B and flow rate of 1 ml/min. Column eluates were collected in 0.2-ml fractions by a fraction collector, and radioactivity of each fraction was determined by liquid scintillation spectrometry. Metabolites corresponding to the major adrenic acid metabolites were separately collected and extracted with cyclohexane-ethyl acetate (50:50). The solvent was removed under a stream of N2, and the extracts were stored at −40°C until LC/MS/MS analysis.
Liquid chromatography-electrospray ionization mass spectrometry.
The chemical identity of the major metabolites isolated by reverse-phase HPLC was determined by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS) (15). HPLC was performed using a reverse-phase C18 column (Kromasil; 250 × 2 mm) and a Waters 2695 liquid chromatograph (Waters, Milford, MA). Samples were dissolved in acetonitrile, and the volume of injection was 10 μl. The mobile phase consisted of solvent A, water containing 0.01% glacial acetic acid, and solvent B, acetonitrile containing 0.01% glacial acetic acid. The program was a 40-min linear gradient from 50% solvent B to 100% solvent B with a flow rate of 0.2 ml/min. Mass spectrometry was performed using a Micromass Quattro Micro API mass spectrometer (Waters) equipped with an electrospray ionization source. The mass spectrometer was operated in the negative ion mode. Product ion spectra were generated by collision-induced decomposition of the precursor ions [m/z 397 for DH-8-keto-PGF1α, m/z 381 for DH-PGF2α, m/z 363 for DH-DiHETEs, m/z 307 for 14-hydroxy-7,10,12-nonadecatrienoic acid (14-HNT), and m/z 347 for DH-HETEs and DH-EETs, respectively]. Only the precursor ion was allowed to pass through the first quadrupole, and the ion was activated by collision with argon in the second quadrupole. Product ion spectra were recorded for the m/z range of 50 to 420. Data were acquired in the profile mode. Results were processed using Masslynx version 4.0 software (Micromass) (7).
Synthesis and purification of DH-EETs.
DH-EETs were synthesized according to the method of Corey et al. (5, 21). Briefly, 10 mg adrenic acid were dried in a 1-ml reacti-vial with a triangular stir bar and under an argon stream, and the following were added in order: CH2Cl2 (50 μl), H2O (20 μl), NaHCO3 (20 mg/50 ml H2O, 25 μl), and meta-chloroperoxybenzoic acid (PBA, 2.2 mg). All solutions were sparged with argon, capped, and stirred rapidly for 15 min. Triphenylphosphine (20 mg/50 ml CH2Cl2, 70 μl) was added to stop the reaction and was stirred for 5 min. The reaction mixture was extracted by CH2Cl2 and dried under an argon stream. The samples were resolved by reverse-phase HPLC (Nucleosil-C18 column, 5 μm, 250 × 4.6 mm) using solvent system I as described in Reverse-phase HPLC. Column eluate (25–30 min) was collected and extracted with cyclohexane-ethyl acetate (50:50). The solvent was removed and dried under a stream of N2. The sample was redissolved in hexane and rechromatographed using normal-phase HPLC (Nucleosil column, 5 μ silica, 250 × 4.6 mm) using solvent system II. The solvent was hexane containing 0.35% isopropanol and 0.1% glacial acetic acid, and the flow rate was 1 ml/min. Three peaks were collected and dried under a stream of N2. The retention time of peak A was ∼17.5 min (DH-16,17-EET and DH-13,14-EET); peak B was ∼19.6 min (DH-10,11-EET); and peak C was ∼30.0 min (DH-7,8-EET). Peak A was chromatographed by using reverse-phase HPLC (Kromasil-C18 column, 5 micron, 250 × 4.6 mm) with solvent system III. Solvent A was water containing 0.01% glacial acetic acid, and solvent B was acetonitrile containing 0.01% glacial acetic acid. The program was a 40-min linear gradient from 70% solvent B to 90% solvent B, and the flow rate was 1.0 ml/min. Two peaks, A1 (DH-16,17-EET) and A2 (DH-13,14-EET), were collected and extracted with cyclohexane-ethyl acetate (50:50) and dried under a nitrogen stream.
BCAs were cut into rings and mounted on a pair of stainless steel hooks in a 6-ml chamber of wire myograph (model 610M, Danish Myo Technology). The arterial rings were equilibrated at 37°C in physiological saline solution containing (in mM) 119 NaCl, 4.7 KCl, 2.5 CaCl2, 1.17 MgSO4, 24 NaHCO3, 1.18 KH2PO4, 0.026 EDTA, and 5.5 glucose, bubbled with 95% O2-5% CO2. SMCs of the equilibrated arteries were impaled with microelectrodes for the measurement of intracellular membrane potential as previously described (4, 11). These studies used glass microelectrodes filled with 3 mol/l KCl with tip resistances of 35 to 80 MΩ and tip potentials of <3 mV. Electrode polarization was eliminated by Ag/AgCl half cells. The main criteria for a successful impalement include a sharp drop in voltage from baseline upon entry of the microelectrode into the cell, a membrane potential of less than −25 mV and a sharp return to 0 mV upon exit from the cell. The arterial rings were preconstricted with U-46619. Adrenic acid (10−6 M) or DH-16,17-EET (10−6 M) was added to the bath solution, and changes in membrane potential were measured.
Whole cell patch clamp.
Coronary SMCs were enzymatically dispersed using published methods (4, 10). Whole cell recordings of K+ currents were obtained in freshly isolated SMCs using an Axopatch 200B amplifier (Axon Instruments), pClamp 8 software (Axon Instruments), and standard methods and solutions as described previously (1, 10). Briefly, macroscopic K+ currents were generated by progressive stepwise 10-mV depolarizing steps (500-ms duration, 5-s intervals) from a constant holding potential of −60 mV. Currents were sampled at 3 kHz and filtered at 1 kHz. After stable control (vehicle) currents were recorded, currents were obtained after application of DH-16,17-EET (10−8–10−6 M) followed by the addition of iberiotoxin (100 nM). Recordings for each condition were performed in triplicate and averaged. Cell membrane capacitance was estimated by integrating the capacitive current generated by a 10-mV hyperpolarizing pulse after electronic cancellation of pipette-patch capacitance. Currents were normalized to cell capacitance and are reported as current density (in pA/pF).
Vascular reactivity and patch-clamp data are expressed as means ± SE. Significant differences between mean values were evaluated by ANOVA followed by the Student-Newman-Keuls multiple-comparison test. Significance was accepted at a value of P < 0.05.
Adrenic and arachidonic acid content of BCAs and ECs.
Lipid extracts from BCAs and ECs were examined for fatty acid content by GC/MS. Arachidonic acid content was 2.06 ± 0.01 and 6.18 ± 0.60 μg/mg protein, and adrenic acid content was 0.29 ± 0.01 and 1.56 ± 0.16 μg/mg protein in lysates from BCAs and ECs, respectively. Thus adrenic acid is a major membrane fatty acid, and its concentrations are 14% and 25% of the corresponding concentrations of arachidonic acid in coronary arteries and the coronary endothelium, respectively.
Coronary vascular response to adrenic acid.
Adrenic acid caused concentration-dependent relaxations of precontracted BCAs (Fig. 1). Maximal relaxations were 83 ± 4% at 10−5 M. Relaxations to adrenic acid were nearly identical to the relaxations induced by arachidonic acid (Fig. 1A). The adrenic acid relaxations were abolished by endothelium removal (Fig. 1B) and attenuated by pretreatment with indomethacin (10 μM), a COX inhibitor (maximal relaxations = 53 ± 4% at 10−5 M, P < 0.05), or miconazole (10 μM), an inhibitor of CYP450 (maximal relaxation = 52 ± 5% at 10−5 M, P < 0.05) (Fig. 1C). The combination of indomethacin and miconazole resulted in a greater inhibition than of either inhibitor alone (maximal relaxation = 24 ± 4% at 10−5 M, P < 0.05) and is comparable with the response observed with the removal of the endothelium. Pretreatment with the K+ channel inhibitor iberiotoxin (100 nM) eliminated the adrenic acid-induced relaxations (Fig. 1D). These findings suggest that EC metabolites derived from COX and CYP450 pathways mediate the relaxations to adrenic acid, and the relaxations are dependent on K+ channel activation.
Metabolism of [14C]adrenic acid by BCAs.
Because both COX and CYP450 metabolites contribute to adrenic acid-induced relaxation, we investigated the metabolism of adrenic acid by BCAs. Arteries were incubated with [14C]adrenic acid, and the medium was extracted and purified by HPLC (Fig. 2). [14C]adrenic acid was metabolized to four major radioactive peaks. The four peaks were collected and analyzed by LC-ESI/MS (Table 1). Metabolites associated with peak 1 (fractions 15–34) comigrated with DH-PGs. From this fraction, a metabolite eluting at 6.26 min (peak 1B) comigrated with the DH-PGF2α standard and gave a similar mass spectrum. Major ions were detected at m/z 381 [M-H]−, 363 [M-H-H2O]−, 345 [M-H-2H2O]−, and 337 [M-H-C2H4O]−, which indicates a molecular weight of 382 (Table 1). From collisional dissociation of the m/z 381, the product ions of m/z 291, 275, and 193 were also detected. These ions followed the fragmentation pattern of PGF2α (14). Peak 1A eluted on LC-ESI/MS at 4.08 min and mass spectral analysis indicated a molecular weight of 398 (Table 1 and Fig. 3A). Major MS/MS ions were m/z 397 [M-H]−, 379 [M-H-H2O]−, 361 [M-H-2H2O]−, 343 [M-H-3H2O]−, and 317 [M-H-2H2O-CO2]−. Product ions of m/z 273, 235, and 191 follow the fragmentation pattern of 6-keto-PGF1α (14). Based on these results, we conclude that peak 1 contains DH-8-keto-PGF1α and DH-PGF2α.
Metabolites in peak 2 (fractions 78–103) eluted on LC-ESI/MS between 18.6 and 24.3 min (Table 1). There were three groups of metabolites in peak 2. The molecular weight of the first group (peaks 2A–2F) was 364, which exceeds the molecular weight of dihydroxyeicosatetraenoic acid (diHETE, molecular weight = 336) by 28 (2CH2). The metabolites eluted at 20.53, 21.03, 21.60, 22.04, 22.97, and 23.73 min. For the peak with a retention time of 20.53 min (peak 2A), major m/z were 363 [M-H]−, 345 [M-H-H2O]−, 327 [M-H-2H2O]−, 283 [M-H-2H2O-CO2]−, 263 (cleavage between C16 and C17), 183 (cleavage between C10 and C11), and 155 (cleavage between C9 and C10), indicating DH-10,17-diHETE. For the other peaks (peaks 2B–2F), their spectra showed similar ions at 363 [M-H]−, 345 [M-H-H2O]−, and 327 [M-H-2H2O]− (with the exception of peak 2B). For the 21.03-min peak (peak 2B), characteristic ions were 263 (cleavage between C16 and C17) and 195 (cleavage between C11 and C12), indicating DH-11,17-diHETE (Table 1 and Fig. 3B). For the 21.60-min peak (peak 2C), characteristic ions were 263 (cleavage between C16 and C17) and 221 (cleavage between C13 and C14), indicating DH-13,17-diHETE. For the 22.04-min peak (peak 2D), characteristic ions were 283 [M-H-2H2O-CO2]−, 263 (cleavage between C16 and C17), 219, and 143 (cleavage between C7 and C8), indicating DH-7,17-diHETE. For the 22.97-min peak (peak 2E), characteristic ions were 223 (cleavage between C13 and C14), 181 (cleavage between C10 and C11), and 153 (cleavage between C9 and C10), indicating DH-10,14-diHETE. For the 23.73-min peak (peak 2F), characteristic ions were 185 (cleavage between C10 and C11), 143, and 219 (cleavage between C7 and C8), indicating DH-7,11-diHETE. Thus six metabolites from the first group of peak 2 were identified as DH-10,17-, DH-11,17-, DH-13,17-, DH-7,17-, DH-10,14-, and DH-7,11-diHETE.
Metabolites from the second group of peak 2 (peaks 2G–2I) were present in lower amounts than were the DH-diHETEs. Their molecular weight was 366, which exceeds the molecular weight of dihydroxyeicosatrienoic acid (DHET, molecular weight = 338) by 28 (2CH2). They eluted on LC-ESI/MS at 18.6, 20.53, and 21.1 min. For the peak with a retention time of 18.6 min (peak 2G), major ions were 365 [M-H]−, 347 [M-H-H2O]−, and 235 (cleavage between C15 and C16), indicating DH-16,17-DHET (Table 1). The spectra of the 20.53- and 21.1-min peaks (peaks 2H and 2I) showed similar ions at 365 [M-H]− and 347 [M-H-H2O]−. For the 20.53-min peak (peak 2H), characteristic ions were 213 (cleavage between C11 and C12), 183 (cleavage between C10 and C11), and 155 (cleavage between C9 and C100), indicating DH-10,11-DHET (Table 1 and Fig. 3C). For the 21.1-min peak, characteristic ions were 195 (cleavage between C12 and C13), indicating DH-13,14-DHET. Thus the three metabolites from the second group of peak 2 were identified as DH-16,17-, DH-10,11-, and DH-13,14-DHET.
A third metabolite in peak 2 (peak 2J) eluted on LC-ESI/MS at 21.7 min (Table 1 and Fig. 3D). It had a molecular weight of 308, which exceeds the molecular weight of 12-hydroxy-5,8,10-heptadecatrienoic acid (12-HHT, molecular weight = 280) by 28 (2CH2). Its major ions were 307 [M-H]−, 289 [M-H-H2O]−, and 207 [cleavage between C13 and C14], indicating 14-HNT.
Metabolites eluting in peak 3 (fractions 105–123) were resolved by LC-ESI/MS and had elution times of 27.3, 28.2, 28.8, and 29.3 min (Table 1). These four compounds had identical molecular weights of 348, which exceed the molecular weight of the HETE (molecular weight = 320) by 28 (2CH2), suggesting DH-HETE. For the peak with a retention time of 27.3 min (peak 3A), major ions were 347 [M-H]−, 329 [M-H-H2O]−, and 247 (cleavage between C16 and C17), indicating DH-17-HETE (Table 1 and Fig. 3E). The other three compounds in peak 3 (peaks 3B–3D) showed similar ions at 347 [M-H]− and 329 [M-H-H2O]−. For the 28.2-min peak (peak 3B), a characteristic ion was 195 (cleavage bond between C12 and C13), indicating DH-13-HETE. For the 28.8-min peak (peak 3C), a characteristic ion was 207 (cleavage between C13 and C14), indicating DH-14-HETE. For the 29.3-min peak (peak 3D), characteristic ions were 203 and 143 m/z (cleavage between C7 and C8), indicating DH-7-HETE. Thus the four metabolites from peak 3 were identified as DH-17-, DH-13-, DH-14-, and DH-7-HETE.
Metabolites eluting in peak 4 (fractions 123–150) comigrated on LC-ESI/MS with authentic DH-EET standards. Elution times were 33.0, 34.3, and 35.3 min. Their molecular weight of 348 was identical to DH-EET. The MS/MS spectra of the three metabolites (peaks 4A–4C) were identical to the DH-16,17-, DH-13,14-, and DH-7,8-EET standards, respectively. For the peak with the retention time of 33.0 min (peak 4A), major ions were 347 [M-H]−, 329 [M-H-H2O]−, and 247 (cleavage between C16 and C17, the epoxide bond), indicating DH-16,17-EET (Table 1 and Fig. 3F). For the 34.3-min peak (peak 4B), major ions were 347 [M-H]−, 329 [M-H-H2O]−, 236 (cleavage between C14 and C15), 207 (cleavage between C13 and C14, the epoxide bond), and 195 (cleavage between C12 and C13), indicating DH-13,14-EET. For the 35.3-min peak (peak 4C), major ions were 347 [M-H]−, 329 [M-H-H2O]−, 285 [M-H-H2O-CO2]−, and 143 (cleavage between C7 and C8, the epoxide bond), indicating DH-7,8-EET.
Coronary vascular response to DH-EETs.
Because miconazole attenuated the adrenic acid-induced relaxations, CYP450 metabolites may contribute to the vascular responses. To verify this possibility, we tested the effect of DH-16,17-, DH-13,14-, DH-10,11-, and DH-7,8-EETs on vascular tone of precontracted coronary arteries (Fig. 4A). Relaxations to all four DH-EET regioisomers were equipotent with a threshold relaxation occurring at 10−8 M. Maximal relaxations averaged 83 ± 3% at 10−5 M. Relaxations to DH-16,17-EET or a combination of 50% DH-16,17-EET plus 50% 14,15-EET were nearly identical to relaxations induced by 14,15-EET (Fig. 4B). DH-16,17-EET-induced relaxations were not affected by the addition of indomethacin (10 μmol/l) and miconazole (10 μmol/l) in combination or endothelium removal (Fig. 4C). Additionally, relaxations to DH-16,17-EET were nearly abolished by pretreatment with iberiotoxin (100 nM, Fig. 4D). Thus DH-EETs cause similar relaxations of BCAs, and the relaxation induced by DH-16,17-EET is mediated by the activation of K+ channels. The combination of DH-16,17-EET plus 14,15-EET causes identical relaxations to either one alone, suggesting that no synergistic or antagonistic interaction occurs in the relaxation response induced by these compounds (Fig. 4B).
Effects of adrenic acid and DH-16,17-EET on membrane potential.
In the presence of U-46619, the membrane potential of coronary smooth muscle was 31.6 ± 1.3 mV (Fig. 5). Both adrenic acid (10−6 M) and DH-16,17-EET (10−6 M) increased the membrane potential to 42.3 ± 3.3 and 46.0 ± 3.2 mV, respectively.
DH-16,17-EET activation of SMC K+ currents.
Macroscopic, whole cell, outward K+ currents were generated by 10-mV depolarizing steps from −60 to +60 mV in isolated bovine coronary SMCs (Fig. 6). DH-16,17-EET (10−8–10−6 M) activated outward K+ currents in a concentration-dependent manner. At +60 mV, DH-16,17-EET (10−6 M) increased current density by 212%. A subsequent addition of iberiotoxin reduced current density 51% below control values. Membrane capacitance, an indicator of cell membrane area, averaged 20.0 ± 3.2 pF. These results demonstrate that DH-16,17-EET activates iberiotoxin-sensitive K+ channels of isolated bovine coronary SMCs.
Metabolism of 14[C]DH-10,11-EET by BCAs.
We evaluated the metabolism of DH-10,11-EET to determine whether DH-EETs are converted via β-oxidation to the vasoactive EETs. 14[C]DH-10,11-EET was incubated in HEPES buffer with or without bovine coronary arterial rings, and the medium was extracted and analyzed by reverse-phase HPLC. 14[C]DH-10,11-EET incubated with buffer produced a single peak (Fig. 7A). [14C]DH-10,11-EET incubated with the arterial rings showed metabolism to DH-10,11-DHET but not 8,9-EET or 8,9-DHET (Fig. 7B). This demonstrates that relaxations to DH-EETs are not mediated by metabolism to EETs.
Adrenic acid is produced by chain elongation of arachidonic acid or elongation and desaturation of linoleic acid (6, 12, 18, 19). Through retroconversion, a portion of the adrenic acid present in EC lipids can be converted to arachidonic acid via chain shortening by β-oxidation (12). In this regard, Mann and colleagues (12) proposed that adrenic acid might serve as a supplementary source of arachidonic acid in the vascular endothelium. Alternatively, adrenic acid may serve as a substrate for the production of vasoactive metabolites.
Previously, we demonstrated that adrenic acid is a component of the cellular lipids of cultured human umbilical vein ECs (3). In these cells, cellular lipid ratio of adrenic to arachidonic acid was 34%. The present study similarly demonstrates that adrenic acid is a component of cellular lipids in BCAs and ECs with adrenic acid-to-arachidonic acid ratios of 14% and 25%, respectively. Interestingly, in bovine aortic ECs, preloaded with either radiolabeled arachidonic or adrenic acid, cellular release of adrenic or arachidonic acid during calcium ionophore stimulation was 12.9% and 15.6% of the initial content of 120,000 and 409,500 dpm, respectively (12). This demonstrates that, similar to arachidonic acid, the vascular endothelium maintains concentrations of adrenic acid that may be assessable for the production of vasoactive metabolites.
Exogenous adrenic acid caused concentration-related relaxations of bovine coronary arterial rings that were nearly identical to relaxations induced by arachidonic acid. In addition, adrenic acid-induced relaxations were reduced by the COX inhibitor, indomethacin, and the CYP450 inhibitor, miconazole, and were eliminated by EC removal or the K+ channel inhibitor, iberiotoxin. In addition, adrenic acid hyperpolarized the coronary smooth muscle. Therefore, relaxations induced by adrenic acid are mediated by EC COX and CYP450 metabolites and activation of K+ channels that cause SMC hyperpolarization.
Adrenic acid is metabolized to DH-thromboxane by COX and DH-14-HETE by 12-LO in human platelets (24) and to DH-PGs and DH-thromboxane by the renal medulla (23). Additionally, in human ECs, adrenic acid is metabolized to DH-PGI2 by COX (3). Results from this present study indicate that adrenic acid is metabolized by the bovine coronary endothelium by COX to the DH-PGs, LO to DH-HETEs, and DH-diHETEs and CYP450 to DH-EETs and DH-DHETs (Table 1 and Fig. 8). The DH-PGs include DH-8-keto-PGF1α (the DH-PGI2 metabolite), DH-PGF2α, and 14-HNT (analog of 12-HHT). The DH-HETEs include DH-17-, DH-13-, DH-14-, and DH-7-HETEs. The DH-diHETEs include DH-10,17-, DH-11,17-, DH-13,17-, DH-7,17-, DH-10,14-, and DH-7,11-diHETEs. The DH-EETs include DH-16,17-, DH-13,14-, and DH-7,8-EETs. The DH-DHETs include DH-16,17-, DH-13,14-, and DH-10,11-DHETs. Identification of these metabolites was based on reverse-phase HPLC comigration with known DH-PG and DH-EET standards and MS/MS spectra. Thus adrenic acid is metabolized by BCAs to a myriad of DH-eicosanoids by COX, LO, and CYP450 enzymatic pathways.
Zhang and colleagues (26) reported that DH-7,8-, 10,11-, 13,14-, and 16,17-EET caused concentration-related dilations of small porcine coronary arteries. Correspondingly, we found that the DH-EET regioisomers caused concentration-related relaxations of BCAs. Relaxations to DH-16,17-EET were nearly identical to 14,15-EET. A coaddition of DH-16,17-EET plus 14,15-EET did not alter the relaxation response, demonstrating no synergism or antagonism. Relaxations to DH-EET were not secondary to EET conversion. Under conditions of the incubation with BCAs, 14[C]DH-10,11-EET was not converted to 8,9-EET. However, DH-10,11-EET was metabolized to DH-10,11-DHET. In the coronary endothelium, EETs are metabolized to DHETs by epoxide hydrolase (1, 25). Likewise, in BCAs, DH-10,11-EET is converted to DH-10,11-DHET.
Relaxations to DH-16,17-EET were blocked by the BKCa channel inhibitor iberiotoxin. In isolated bovine coronary SMCs, DH-16,17-EET activates iberiotoxin-sensitive outward K+ currents and hyperpolarizes vascular smooth muscle. Similarly, in rat coronary SMCs, DH-13,14-EET activates BKCa channels (26). In the coronary circulation, EETs act as endothelium-derived hyperpolarizing factors (EDHFs) (4). They are produced by the endothelium, diffuse to the smooth muscle, and activate membrane K+ channels to cause hyperpolarization and vascular relaxation (2, 4, 8). This suggests that similar to the EETs, DH-EETs may function as EDHFs.
Vasoactive properties of the specific adrenic acid COX and LO metabolites were not evaluated in this current study. LO metabolites appear to play a minor role because adrenic acid relaxations were nearly eliminated by the combination of COX and CYP450 inhibition. Previous studies have demonstrated biological activity of DH-PGs. Like PGI2, DH-PGI2 inhibited thrombin-induced human platelet aggregation (3) and, in renal medullary cells, DH-PGE2 and DH-PGI2 increased cellular cyclic AMP (23). Because relaxations to adrenic acid were inhibited by indomethacin, it is probable that DH-PGs also cause vascular relaxation; however, DH-PGI2 is no longer commercially available for testing.
Results from this study represent an initial report addressing the role of adrenic acid and its metabolites in the regulation of coronary vascular tone. We demonstrated that adrenic acid relaxations are mediated by endothelial COX and CYP450 metabolites. BCAs metabolize adrenic acid by COX, LO, and CYP450 to DH-8-keto-PGF1α, DH-PGF2α, DH-HETEs, DH-diHETEs, and DH-EETs. The DH-EETs caused vascular relaxation, and DH-16,17-EET activated vascular smooth muscle K+ current and caused membrane hyperpolarization. These results suggest a possible role of adrenic acid metabolites, specifically, DH-EETs as EDHFs in the coronary circulation. At the current time, the physiological function of these metabolites in agonist and/or flow-mediated relaxations and dilations is unknown. Isolating and characterizing their vascular function will be complex since inhibitors that block COX, LO, and CYP450 metabolism will alter arachidonic as well as adrenic acid metabolism. Regardless, the contribution of DH-EETs and other metabolites of adrenic acid should be considered in future studies that evaluate fatty acid regulation of vascular tone.
These studies were supported by National Institutes of Health Grants HL-51055, HL-83297 and GM-31278 and by the Robert A. Welch Foundation. The LC-MS/MS was provided by a National Institute of Research Resources Grant (RR-17824).
We thank Gretchen Barg for secretarial assistance.
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- Copyright © 2007 by the American Physiological Society