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Originally published In Press as doi:10.1074/jbc.M306849200 on November 11, 2003

J. Biol. Chem., Vol. 279, Issue 4, 2648-2656, January 23, 2004
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20-Hydroxyeicosatetraenoic Acid (20-HETE) Metabolism in Coronary Endothelial Cells*

Terry L. Kaduce{ddagger}, Xiang Fang{ddagger}, Shawn D. Harmon{ddagger}, Christine L. Oltman§, Kevin C. Dellsperger¶, Lynn M. Teesch||, V. Raj Gopal**, J. R. Falck**, William B. Campbell{ddagger}{ddagger}, Neal L. Weintraub§, and Arthur A. Spector{ddagger}§§§

From the Departments of {ddagger}Biochemistry and §Internal Medicine, Carver College of Medicine, and the ||High Resolution Mass Spectrometry Facility, University of Iowa, Iowa City, Iowa 52242, the Department of Internal Medicine, University of Missouri, Columbia, Missouri 65212, the **Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, and the {ddagger}{ddagger}Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

Received for publication, June 26, 2003 , and in revised form, October 29, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We have investigated the role of endothelial cells in the metabolism of 20-hydroxyeicosatetraenoic acid (20-HETE), a vasoactive mediator synthesized from arachidonic acid by cytochrome P450 {omega}-oxidases. Porcine coronary artery endothelial cells (PCEC) incorporated 20-[3H]HETE primarily into the sn-2 position of phospholipids through a coenzyme A-dependent process. The incorporation was reduced by equimolar amounts of arachidonic, eicosapentaenoic or 8,9-epoxyeicosatrienoic acids, but some uptake persisted even when a 10-fold excess of arachidonic acid was available. The retention of 20-[3H]HETE increased substantially when methyl arachidonoyl fluorophosphonate, but not bromoenol lactone, was added, suggesting that a Ca2+-dependent cytosolic phospholipase A2 released the 20-HETE contained in PCEC phospholipids. Addition of calcium ionophore A23187 [GenBank] produced a rapid release of 20-[3H]HETE from the PCEC, a finding that also is consistent with a Ca2+-dependent mobilization process. PCEC also converted 20-[3H]HETE to 20-carboxy-arachidonic acid (20-COOH-AA) and 18-, 16-, and 14-carbon {beta}-oxidation products. 20-COOH-AA produced vasodilation in porcine coronary arterioles, but 20-HETE was inactive. These results suggest that the incorporation of 20-HETE and its subsequent conversion to 20-COOH-AA in the endothelium may be important in modulating coronary vascular function.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
20-Hydroxyeicosatetraenoic acid (HETE),1 an eicosanoid synthesized from arachidonic acid by cytochrome P450 (CYP) isoforms of the 4A and 4F classes (1–3), produces vasoconstriction of small arteries and regulates salt balance in the kidney (4–6). There is growing interest in 20-HETE because it increases systemic blood pressure, and animal studies indicate that it plays a key role in the pathophysiology of some experimental forms of hypertension (2, 4, 6–8). For example, the CYP4A2 gene is overexpressed during the development of hypertension in the SHR rat (9), and disruption of the CYP4a14 gene causes 20-HETE production and hypertension in male mice (10). Furthermore, treatment with an antisense oligonucleotide reduces CYP4A expression in the vasculature and lowers blood pressure (11), and treatment with a CYP4A inhibitor reduces hypertension induced by angiotensin II (12).

Smooth muscle cells produce 20-HETE when small arteries are subjected to stretch or are exposed to biomediators like angiotensin II, endothelin or norepinephrine (6, 13, 14). The 20-HETE inhibits the smooth muscle Ca2+-activated K+ channels by decreasing their open state probability (15). This depolarizes the membrane, causing Ca2+ influx and vasoconstriction (6). Recent evidence indicates that 20-HETE produces this effect by activating a receptor-dependent process mediated by the protein kinase C signal transduction pathway (16–18). 20-HETE also has major effects on renal function (2, 4, 5, 6). The proximal tubule and thick ascending limb of Henle produce 20-HETE (19), which is incorporated into renal cortex phospholipids, especially the inositol phosphoglycerides (PI) (20). In addition to constricting renal preglomerular arterioles, 20-HETE is mitogenic, it affects ion transport mediated by the Na+/K+-ATPase and Na+/K+/2Cl–-cotransporter, and it mediates tubular-glomerular feedback (4–6,21).

As opposed to its vasoconstrictor effect in the renal and cerebral circulation, 20-HETE acts as a vasodilator in the pulmonary and coronary circulations (22, 23). The pulmonary vasodilator effect is dependent on the presence of endothelium (22), and the vasorelaxation produced by 20-HETE in the coronary artery is mediated by the release of prostacyclin (23). 20-HETE also is converted to a bronchodilator substance by the pulmonary endothelial cyclooxygenase (24). In an attempt to better understand the mechanism of these endothelium-dependent effects, we have investigated the utilization of 20-HETE in porcine coronary artery endothelial cells (PCEC), an experimental model that we have used previously for biochemical studies of eicosanoid metabolism (25–28). To investigate the potential functional significance of these observations, we examined the vasoactivity of 20-HETE and 20-carboxy-arachidonic acid (20-COOH-AA), its main metabolite (19), in an isolated porcine coronary microvessel preparation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Synthesis of 20-[3H]HETE—20-[3H]HETE (16.7 µCi/nmol) was synthesized by incubating 15 nmol of arachidonic acid containing 250 µCi [5,6,8,9,11,12,14,15-3H]arachidonic acid (60–100 Ci/mmol, American Radiolabeled Chemicals, Inc., St. Louis, MO) with commercially available microsomes containing 30 pmol of baculovirus expressed human recombinant CYP4F3B, CYP reductase and cytochrome b5 isolated from BTI-TN-5B1–4 insect cells (Gentest, Woburn, MA). The mixture was incubated in 1 ml of 200 mM phosphate buffered saline, pH 7.4, containing 0.1 µM bovine serum albumin (BSA), 1.3 mM NADP+, 3.3 mM glucose 6-phosphate, 3.3 mM MgCl2, 0.4 units of glucose 6-phosphate dehydrogenase, and 0.05 mM sodium citrate. After 1 h at 37 °C, the reaction was terminated by addition of 1 ml H2O and formic acid to lower the pH to 4, and the lipids were extracted three-times with 8 ml of H2O-saturated ethyl acetate. The ethyl acetate extracts were combined, dried under N2, suspended in 3 ml of acetonitrile, and separated by reverse-phase high-performance liquid chromatography (HPLC) on a 5-µm 4.6 x 250 mm Discovery C18 column (Supelco, Bellefonte, PA) and a dual pump gradient HPLC system (Gilson Medical Electronics, Inc., Middleton, WI) equipped with Model 306 pumps, a Model 117 Dual wavelength UV detector, a Model 231 XL automatic sample injector. The solvent system contained H2O adjusted to pH 4.0 with formic acid and an acetonitrile gradient that began at 30% and was increased to 57% acetonitrile at 30 min, to 65% over the next 20 min, to 100% over 5 min, and then maintained at 100% acetonitrile for 15 min.

Fig. 1 shows the analysis of the radiolabeled material. The main product eluted from the reverse phase HPLC column 24 min before [3H]arachidonic acid (Fig. 1A). As shown in Fig. 1B, this product had the same retention time (RT), 33 min, as a commercially prepared 20-[14,15-3H]HETE standard (PerkinElmer Life Sciences Products, Boston, MA, 45 Ci/mmol, 97% purity by HPLC analysis).



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  		FIG. 1.
Synthesis of 20-[3H]HETE. A, HPLC analysis of the radiolabeled material obtained following incubation of 15 nmol of [3H]arachidonic acid with 30 pmol of recombinant human CYP4F3B, CYP reductase, cytochrome b5, and a NADPH-regenerating system. The incubation was done at 37 °C for 1 h in 200 mM phosphate-buffered saline, pH 7.4. After addition of formic acid to reduce the pH to 4.0, the lipids at were extracted with water-saturated ethyl acetate and separated by reverse phase HPLC. B, HPLC analysis of a commercially prepared 20-[3H]HETE standard. C, mass spectrum of this product obtained by liquid chromatography-mass spectrometry with source collision-induced decomposition.

 
Product containing no radioactivity also was synthesized and isolated by HPLC. The HPLC eluate containing the main product was collected in 0.5 ml fractions and added to 2.5 ml chloroform-methanol (2:1, v/v), and the fractions were pooled. Phosphate-buffered saline was added, and the chloroform phase was separated and collected. After removal of the solvent under N2, the lipid extract was dissolved in acetonitrile and further separated by reverse phase HPLC with a solvent system consisting of H2O adjusted to pH 4.0 with formic acid and an acetonitrile gradient that increased from 30 to 100% over 70 min (29). The product was collected, dried under N2, and analyzed by liquid chromatography combined with mass spectrometry (27). The mass spectrum was obtained by using an Agilent 1100 MSD liquid chromatography-mass spectrometry system with source collision-induced decomposition (27). As shown in Fig. 1C, the mass spectrum contained a molecular ion at m/z 319 and ions at m/z 301 (M-H2O) and m/z 275 (M-CO2), consistent with the structure of 20-HETE.

Synthesis of 20-COOH-AA—20-Hydroxy-5(Z),8(Z),11(Z),14(Z)-eicosatetraenoic acid (10 mg; 0.03 mmol) was dissolved in acetone (3 ml) and added dropwise with stirring to a –10 °C solution of Jones reagent (1.0 ml, 0.14 M solution in water) in acetone (3 ml) under an argon atmosphere (30). The reaction mixture was warmed to 0 °C over 30 min and then quenched with excess isopropyl alcohol. The precipitated chromium salts were removed by filtration, and the filter cake was washed with 5 ml of ethyl acetate. The combined filtrates were diluted with 5 ml of water and extracted three times with 5 ml of ethyl acetate. The combined organic extracts were washed twice with 5 ml of water, a NaCl solution (5 ml), dried over Na2SO4, and concentrated in vacuo. The residue was purified by silica gel preparative thin-layer chromatography (TLC), three elutions, with hexane/ethyl acetate (1:1) to give eicosa-5(Z),8(Z),11(Z),14(Z)-tetraen-1,20-dioic acid (6.9 mg, 74%, Rf = 0.15) as a colorless oil. 1H nuclear magnetic resonance analysis of the purified product (400 MHz, CDCl3): {delta} 1.42-(q, J = 7.3 Hz, 2H), 1.62–1.80 (m, 4H), 2.04–2.20 (m, 4H), 2.32–2.42 (m, 4H), 2.78–2.90 (m, 6H), 5.30–5.50 (m, 8H).

Cell Culture and Incubation—PCEC were isolated and grown in modified Medium 199 supplemented with 10% fetal bovine serum (25–28). The cultures were maintained until confluent at 37 °C in a humidified atmosphere containing 5% CO2. Stocks were subcultured weekly by trypsinization, and cultures were used for experiments between passages 3 and 7. Confluent monolayers were incubated at 37 °C with 1 ml Medium 199 containing 0.1 µM BSA and 20-[3H]HETE in a humidified atmosphere containing 5% CO2. The details concerning concentration of 20-[3H]HETE, time of incubation, and presence of other compounds are described in each experiment.

The efflux of 20-HETE previously incorporated by the PCEC was investigated using a pulse-chase experimental design. PCEC cultures were incubated initially for 1 h with 20-[3H]HETE and after removal of this medium, the cells were washed and incubated for 20 min to 16 h in a culture medium containing BSA. Following incubation, the medium and cells were separated, and the lipid-soluble material was extracted and assayed for total radioactivity content. In some experiments, ionophore A23187 [GenBank] was added to determine whether activation of a calcium-mediated signaling pathway increased the efflux of 20-[3H]HETE from the PCEC (25–28). In other experiments, either methyl arachidonoyl fluorophosphonate (MAFP) or bromoenol lactone (BEL), both obtained from Biomol (Plymouth Meeting, PA), was added to investigate the role of phospholipase A2 (PLA2) in mediating the efflux of 20-[3H]HETE from the cells.

Assay of Incubation Medium—The amount of radioactivity remaining in the medium after incubation was measured by liquid scintillation counting. Lipids present in the medium were extracted twice with 4 ml of ice-cold ethyl acetate saturated with water. Extracts were combined, the ethyl acetate was evaporated under N2, and the lipids were dissolved in acetonitrile and separated by reverse-phase HPLC with the dual pump gradient system and Discovery C18 column. The solvent system contained H2O adjusted to pH 4.0 with formic acid and an acetonitrile gradient that increased from 30 to 100% over 70 min at a flow rate of 0.7 ml/min (27). Radioactivity was measured by combining the column effluent with scintillator solution and then passing the mixture through a flow scintillation detector (IN/US Systems, Inc., Tampa, FL).

Analyses of Cell Lipids—After the medium was removed and the PCEC monolayer washed with ice-cold buffer, the lipids were extracted from the cells with 6 ml of chloroform-methanol (2:1, v/v). Phosphate-buffered saline, 1.5 ml containing 0.05 N HCl was added, the phases were separated, the chloroform was removed and dried under N2. The lipid residue was dissolved in 200 µl of chloroform-methanol, and an aliquot was dried under N2 and assayed for radioactivity in a liquid scintillation spectrometer (31).

To determine the distribution of the radioactivity in the cell lipids, aliquots of the extract were separated by thin layer chromatography (TLC) on Whatman LK5D silica gel plates (Whatman Inc., Clifton, NJ) with a solvent system of chloroform-methanol-40% methylamine-H2O (60:36:1.5:1, v/v/v) (27). The radioactivity contained in the separated lipids was determined with an AR-200 Imaging Scanner (Bioscan, Inc., Washington, D. C.). Phospholipid standards (Avanti Polar Lipids, (Naperville, IL) were added to each chromatogram, and the separated lipids were visualized by staining with 1-anilinonaphthalene 8-sulfonic acid.

Studies also were done to determine the positional distribution of the radioactivity incorporated into the PCEC phospholipids (28, 32). After incubation with 20-[3H]HETE, the phospholipids contained in the cell lipid extract were separated by TLC as indicated above and each phospholipids fraction was extracted and suspended in 1 ml of phosphate buffer (pH 7.5) containing 2 mM CaCl2. Snake venom PLA2 (obtained from Sigma, 125 units/ml) dissolved in this buffer solution was added, and the samples were sealed under N2 and incubated at 37 °C for 45 min. The hydrolysates were extracted with chloroform-methanol (2:1 v/v), dried under N2, and fractionated by TLC using a solvent mixture containing chloroform, methanol, 40% methylamine, water (60:36:1.5: 1.0, v/v/v/v), and the distribution of radioactivity in the chromatogram was determined with the AR-200 Imaging Scanner.

To determine whether the radioactivity incorporated in the PCEC lipids remained as 20-HETE or was converted to metabolites, additional aliquots of the cell extract were hydrolyzed for 1 h at 50 °C in 0.5 ml methanol containing 50 µl of 2 N NaOH (32). After the pH was adjusted to 7.0 with formic acid/H2O (1:10, v/v) and the volume adjusted to 1 ml with H2O, the hydrolyzed lipid products were extracted twice with 4 ml of ice-cold ethyl acetate saturated with H2O. The ethyl acetate was removed under N2, and the lipids were dissolved in acetonitrile and separated by reverse-phase HPLC on the Discovery C18 column with a gradient containing H2O adjusted with formic acid to pH 4.0 and acetonitrile (27).

Mass Spectrometry—Liquid chromatography tandem mass spectrometry (LC/MS-MS) experiments to separate and identify the metabolites formed by the PCEC from 20-HETE were performed using a ThermoFinnigan LCQ Deca quadrupole ion trap mass spectrometer interfaced with a ThermoFinnigan Surveyor liquid chromatograph. The same HPLC parameters were used as described above, except a Supelco C18 5 µm 2.1 x 150 mm Discovery column was used and the flow rate was 200 µl/min. Negative electrospray ionization was also used. The N2 sheath gas and auxiliary gas were maintained at 40 and 30 arbitrary units, respectively, the capillary was heated to 300 °C, and the normalized collision energy was 45% for the LC/MS-MS experiments. ThermoFinnigan Xcalibur 1.3 software was used for data processing.

Porcine Coronary Microvessel Studies—Porcine hearts were obtained from a local abattoir. The hearts were quickly harvested and immediately placed in cold (4 °C), oxygenated Krebs bicarbonate buffer solution for dissection. A standard in vitro pressurized arteriole preparation was used to study coronary microvessels (33, 34). Ventricular microvessels of 100–175 µm intraluminal diameter and ~1 mm in length were carefully removed from the myocardium, cleaned with the aid of a dissecting microscope and placed in an organ chamber. Each end of the microvessel was cannulated with a glass micropipette and secured with 10-0 ophthalmic suture. The organ chamber was placed on the stage of an inverted microscope. Attached to the microscope were a video camera, a video monitor, and a calibrated video caliper. The organ chamber was connected to a rotary pump that continuously circulated oxygenated Krebs Henseleit solution containing in mM: NaCl 120.0; KCl 4.7; CaCl2 2.5; MgSO4 1.2; NaHCO3 23.0; KH2PO4 1.2; glucose 11.0 and EDTA 0.025. Solutions were aerated with 20% O2, 5% CO2, and 75% N2 and maintained at 37 °C, pH 7.4. An image of the microvessel was displayed on the video monitor and intraluminal diameters were measured by manually adjusting the video micrometer. The resolution of the system allowed measurement of very small (1–2 µm) changes in vessel diameter.

Microvessels were allowed to equilibrate for 30 min at a hydrostatic distending pressure of 60 mm Hg under conditions of no flow. KCl (75 mM) was added to the bath to test constrictor capacity. After washing with fresh Krebs buffer, the vessel diameter returned to baseline. Endothelin-1 (0.40–1.2 nM, Phoenix Peninsula Laboratories, Inc., San Carlos, CA) was used to constrict the microvessels to 30–60% of their resting diameter. Cumulative concentration-response relationships were evaluated for 20-HETE (10–10 to 10–6 M), 20-COOH-AA, (10–10 to 10–6 M), or sodium nitroprusside (10–10 to 10–4 M) by adding the compound directly to the organ bath. A single dose of sodium nitroprusside (10–4 M, Sigma Chemical) was given at the end of each experiment to determine the maximal diameter. All solutions and vasoactive agents were prepared fresh on the day of the experiment.

Statistical Analysis—Data are expressed as mean ± S.E. Sigmastat software (Jandel Scientific) was used for statistical analyses. All concentration response curves were evaluated for differences using 2-way repeated measures analysis of variance followed by the Fisher LSD correction for multiple comparisons. Differences with p < 0.05 are considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
20-HETE Uptake—The PCEC readily incorporated 20-[3H]HETE from the extracellular fluid. Fig. 2 shows that the amount taken up was dependent on the time of incubation and the concentration of 20-HETE. During a 24-h incubation in a medium containing 1 µM 20-[3H]HETE and 0.1 µM BSA, the amount incorporated by the PCEC increased rapidly, reached a maximum at 4 h, and then declined by 30% over the ensuing 20 h (Fig. 2A). After 30 min, 21% of the 20-[3H]HETE initially present in the medium was incorporated by the cells. This increased to 41% at 4 h and then declined to 28% at 24 h. The amount present in the cells after a 2-h incubation increased over the entire range of 20-[3H]HETE concentrations tested, 0.25–7.5 µM (Fig. 2B). However, the percentage uptake of available 20-[3H]HETE decreased as the concentration in the medium increased, from 50% at 0.25 µM to 17% at 7.5 µM.



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  		FIG. 2.
Uptake of 20-[3H]HETE by PCEC. A, time dependence of uptake during a 24 h incubation at 37 °C of confluent PCEC cultures with 1 ml of 1 µM 20-[3H]HETE in modified M199 medium containing 0.1 µM BSA. B, dependence of uptake on the concentration of 20-[3H]HETE in a 2-h incubation. Each point is the average of values obtained from two separate cultures.

 
Although the capacity of the PCEC to incorporate 20-HETE is substantial, it is less than arachidonic acid under the same conditions. For example, at a concentration of 1 µM, the PCEC cultures took up 43% of the available 20-HETE, whereas comparable cultures took up 73% of the available arachidonic acid.

Formation of 20-HETE Metabolites—HPLC analysis of the medium at various times during the 24-h incubation with 1 µM 20-[3H]HETE demonstrated that the PCEC converted 20-HETE to a number of metabolites (Fig. 3). The major radiolabeled metabolites detected during the incubation are designated in the chromatograms as Products I-IV. More than 95% of the lipid-soluble radioactivity recovered from the medium after 1 h of incubation remained as 20-HETE, which had an RT of 32.2 min in this HPLC analysis (Fig 3A). However, 20-[3H]HETE accounted for only 40% of the radioactivity remaining in the medium after 4 h, and Product III (RT, 22.5 min) was the most abundant radiolabeled metabolite (Fig. 3B). At this time, the four main metabolites accounted for 12% of the 20-[3H]HETE added to the medium at the start of the incubation. After 8 h, little radiolabeled 20-HETE or Product II remained, Product I (RT, 31 min) was the major metabolite, and Product IV (RT, 17 min) increased (Fig. 3C). The only prominent metabolite remaining in the medium at the end of the 24-h incubation was Product I (Fig. 3D). Increasing amounts of radiolabeled material that eluted with the solvent front also accumulated as the incubation progressed. Preliminary analysis indicated that this material consisted of a mixture of short-chain products, and no further analysis of this material was attempted.



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  		FIG. 3.
Radiolabeled 20-HETE metabolites detected in the incubation medium. Confluent PCEC cultures were incubated with 1 µM 20-[3H]HETE as described in Fig. 2A, and the radioactivity contained in the medium at each of the incubation times was separated by reverse phase HPLC and assayed by passing the effluent through an in-line scintillation spectrometer. The chromatograms shown were obtained after the following incubation times: A, 1 h; B, 4 h; C, 8 h; D, 24 h.

 
The main 20-HETE metabolites were identified by LC/MS-MS. Fig. 4 shows the MS-MS spectra of the precursor (M-H)– ions of 20-HETE (Fig. 4A) and the metabolites that were detected (Fig. 4, B–E). The fragmentation seen in these spectra are consistent with the following structures for the products: Fig. 4B, 18-OH-18:3 (m/z 293); Fig. 4C, 16-OH-16:3 (m/z 265); Fig. 4D, 14-OH-14:2 (m/z 239); and Fig. 4E, 20-COOH-AA (m/z 333). The MS-MS fragmentation of both the metabolites with the terminal OH-group and the dicarboxylic acid metabolite show losses of water, loss of two water molecules, loss of CO2, and loss of water and CO2 (–18, –36, –44, and –62, respectively). The unique series of fragment ions from the metabolites with the terminal OH group is due a rearrangement reaction that involves cleavage along the carbon chain and includes the OH group: –CH2O (–30), C2H6O (–46), and C4H10O (–74). Another unique fragment ion to the OH metabolites is m/z 113. One possibility is that it originates from the deprotonation of the OH-group to form the (M-H)– ion and is formed by a rearrangement reaction that cleaves the carbon backbone. These MS-MS results indicate that 20-HETE undergoes two oxidative processes in the PCEC, conversion to the dicarboxylic acid and {beta}-oxidation.



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  		FIG. 4.
Tandem mass spectra of compounds detected in the medium following incubation of the endothelial cells with 20-HETE. To obtain sufficient amounts of each product for analysis, 75-cm2 PCEC cultures were incubated for 4 h with 5 µM 20-HETE. The mass spectra shown are: A, 20-HETE; B, Product II, 18-OH-18:3; C, Product III, 16-OH-16:3; D, Product IV, 14-OH-14:2; E, Product I, 20-COOH-AA.

 
The time and concentration dependence of the production of 20-COOH-AA, the main 20-HETE metabolite that accumulated in the medium, are shown in Fig. 5. After a lag period of 1 h, the amount of 20-[3H]COOH-AA continued to increase throughout a 24-h incubation with 1 µM 20-[3H]HETE (Fig. 5A). Likewise, the amount of 20-[3H]COOH-AA that accumulated during a 2 h incubation increased progressively over the range of 20-[3H]HETE concentrations tested, 0.25–7.5 µM (Fig. 5B). Thus, PCEC have the capacity to convert substantial amounts of 20-HETE to 20-COOH-AA.



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  		FIG. 5.
Effect of incubation time and 20-HETE concentration on 20-COOH-AA formation by the endothelial cells. Confluent PCEC cultures were incubated at 37 °C with 1 µM 20-[3H]HETE and 0.1 µM BSA in 1 ml of modified M199 medium, and the medium was assayed for 20-[3H]COOH-AA by HPLC, as described in Fig. 3. A, time-dependent incubation with 1 µM 20-[3H]HETE. B, concentration dependence in a 2-h incubation. The quantity of 20-COOH-AA contained in the 1 ml of medium was calculated from the specific activity of the 20-[3H]HETE with which the cells were incubated. Each point is the average of values obtained from two separate cultures.

 
Cell Lipid Analysis—A study was done to determine whether these metabolites accounted for a substantial amount of the radioactivity that accumulated in the cells. Following incubations of the PCEC for various times between 2 and 24 h with 1 µM 20-[3H]HETE, the cell lipids were hydrolyzed by saponification, and the radioactivity contained in the resulting lipid extract was separated and assayed by HPLC. Fig. 6 demonstrates that essentially all of the radioactivity contained in the cell lipids remained as 20-HETE at the end of the 24 h incubation. Similar chromatograms were obtained from the cells at each of the earlier time points (data not shown). Additional studies indicated that when the PCEC were incubated for 2 h with 20-[3H]HETE concentrations between 0.25 and 7.5 µM, between 94 and 100% of the radioactivity incorporated into the cell lipids remained as 20-HETE (data not shown). Thus, under all of the conditions tested, the 20-HETE that was retained in the PCEC was not structurally modified, and none of the radiolabeled metabolites that were observed in the culture medium were detected in the cells.



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  		FIG. 6.
HPLC analysis of the 20-HETE radioactivity retained in the endothelial cells. Confluent PCEC cultures were incubated for 24 h with 1 µM 20-[3H]HETE as described in Fig. 2A. After removal of the medium and washing, the cells were extracted with a chloroform-methanol mixture and the lipids contained in the isolated chloroform phase were hydrolyzed by saponification. The radioactivity contained in the resulting lipid extract was separated and assayed by reverse phase HPLC. A single chromatogram is shown, but similar results were obtained from a second culture. Likewise, similar results were observed in chromatograms obtained from duplicate cultures incubated for 2, 4, 8, and 16 h.

 
The distribution of the incorporated 20-[3H]HETE in the PCEC lipids, as determined by TLC analysis, is illustrated in Fig. 7. Following a 1-h incubation with 1 µM 20-[3H]HETE, phospholipids contained 78% of the incorporated radioactivity, 16% was contained in neutral lipids (NL), and 6% was present as unesterified 20-HETE. The distribution in phospholipids was 31% in PI, 39% in choline phosphoglycerides (PC) and 30% in ethanolamine phosphoglycerides (PE). When 5 µM of the acyl CoA synthase inhibitor triacsin C obtained from Biomol was added to the incubations, the total uptake of 20-[3H]HETE decreased by 58%, and the incorporation in phospholipids and NL decreased by 63%. The magnitude of the decreases in the PI, PC, PE, and NL fractions were similar, indicating that the incorporation of 20-HETE into PCEC lipids is CoA-dependent. However, no decrease was observed in the amount of unesterified 20-HETE present in the cells.



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  		FIG. 7.
Distribution of radiolabeled 20-HETE in endothelial cell lipids. Confluent PCEC cultures were incubated for 1 h at 37 °C with 1 ml of 1 µM 20-[3H]HETE in modified M199 medium containing 0.1 µM BSA and, where indicated, 5 µM triacsin C. After the cells were washed, the cell lipids were extracted with a chloroform-methanol mixture and assayed for radioactivity by liquid scintillation counting. Aliquots of the extracts were separated by TLC, and the distribution of the radioactivity that co-migrated with indicated lipid standards was determined by scanning the chromatogram. Each bar is the mean ± S.E. of values obtained from three separate cultures, *, p < 0.01. The amounts are calculated based on the specific activity of the 20-[3H]HETE incubated with the cultures.

 
A study was done to determine the positional distribution of the 20-[3H]HETE incorporated into the PCEC phospholipids. After a 1 h incubation with 1 µM 20-[3H]HETE, the PI, PC, and PE fractions were separated by TLC, and each fraction was isolated and incubated with PLA2. After extraction and separation of the products by TLC, all of the radioactivity originally contained in PI and PC, and 92% of the radioactivity in PE, co-migrated with the 20-HETE standard (data not shown). Therefore, almost all of the 20-[3H]HETE was incorporated into the sn-2 position of the PCEC phospholipids.

Competition for Incorporation—Experiments were carried out to determine whether structurally related compounds that are taken up by endothelial cells would reduce the incorporation of 20-HETE (Fig. 8). Each of these compounds was tested at a concentration of 1 µM, and the concentration of 20-[3H]HETE also was 1 µM. As seen in Fig. 8A, substantial reductions in 20-[3H]HETE incorporation into the PCEC occurred when the medium contained either arachidonic acid (20:4, p < 0.02) or eicosapentaenoic acid (20:5, p < 0.01), while 8,9-epoxyeicostrienoic acid (EET) produced a modest decrease (p < 0.05). Endothelial cells incorporate each of these compounds (35–37). However, 11,12-EET, 14,15-EET, 12-HETE, 15-HETE, 11,12-dihydroxyeicosatrienoic acid, and oleic acid, which also are taken up by endothelial cells (37–41), did not reduce 20-[3H]HETE incorporation into the PCEC (p > 0.1). The concentration dependence of the effect produced by arachidonic acid is shown in Fig. 8B. A 60% decrease in 20-[3H]HETE uptake occurred when 1 µM arachidonic acid was added to the medium, and a further reduction occurred at higher arachidonic acid concentrations. However, some 20-[3H]HETE incorporation persisted even when a 10-fold excess of arachidonic acid was available.



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  		FIG. 8.
Effects of structurally related compounds on incorporation of 20-HETE into the endothelial cells. A, comparative effects of equimolar concentrations of structurally related compounds. Confluent PCEC cultures were incubated in modified M199 medium containing 1 µM BSA, 1 µM 20-[3H]HETE, and 1 µM of the competing compound. The amount of 20-[3H]HETE present in the cells was measured following a 1-h incubation at 37 °C. B, concentration-dependent effects of arachidonic acid. The conditions were similar to those described in A, except that the incubation time was 2 h, and the concentration of arachidonic acid varied from 1–10 µM. Each point and bar represents the mean ± S.E. of values obtained from three separate cultures; *, p < 0.02 versus control; **, p < 0.01 versus control; ***, p < 0.05 versus control. Abbreviations: 18:1, oleic acid; 20:4, arachidonic acid; 20:5, eicosapentaenoic acid; HETE, hydroxyeicosatetraenoic acid; EET, epoxyeicosatrienoic acid; DHET, dihydroxyeicosatrienoic acid.

 
Retention in Cells—The retention of 20-HETE and arachidonic acid in the PCEC was compared under basal conditions (Fig. 9). In both cases cultures were labeled for 1 h with either 20-[3H]HETE or [3H]arachidonic acid, washed, and then incubated in fresh medium for up to 16 h. About 60% of the 20-[3H]HETE initially present in the PCEC was eliminated during the subsequent 16 h incubation (Fig. 9A). Most of the loss of 20-[3H]HETE from the cells occurred in the first 4 h. In corresponding cultures containing [3H]arachidonic acid, much less radioactivity was released during the 16 h incubation, and no decrease was observed after the first hour. Consistent with these findings, the amount of lipid-soluble radioactivity that was released into the medium during the 16-h incubation was 4 times greater from the PCEC containing 20-[3H]HETE as compared with those containing [3H]arachidonic acid (Fig. 9B). The amount released from the cells labeled with 20-[3H]HETE continued to increase during the first 8 h, whereas it reached a maximum within 2 h in the case of the cells labeled with [3H]arachidonic acid.



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  		FIG. 9.
Comparison of the amount of radiolabeled 20-HETE and arachidonic acid retained in the endothelial cells. Confluent PCEC were labeled for 1 h at 37 °C with either 0.5 µM 20-[3H]HETE or [3H]arachidonic acid. After the medium was removed and the cells washed, three cultures from each group were assayed to determine the uptake of radioactivity. The remaining cultures were washed and then incubated at 37 °C for up to 16 h in 1.5 ml of modified M199 medium containing 5 µM BSA. After incubation, the medium and cells were extracted and assayed for lipid-soluble radioactivity by liquid scintillation counting. A, time-dependent comparison of the retention of 20-[3H]HETE and [3H]arachidonic acid in the cells. The results are expressed as a percentage of the radiolabeled material present in the cells at the start of the 16-h incubation. B, time-dependent accumulation of lipid-soluble radiolabeled material in the culture medium. These results are expressed in pmol amounts calculated from the specific activity of the 20-[3H]HETE and [3H]arachidonic acid, respectively, with which the cells were incubated.

 
Because more than 75% of the 20-[3H]HETE present in the labeled PCEC was contained in phospholipids and almost all of this was present in the sn-2 position, we tested whether PLA2 inhibition of would prevent the release of 20-HETE from the cells. Two inhibitors were tested: MAFP, a dual cytosolic PLA2 (cPLA2) and calcium-independent PLA2 (iPLA2) inhibitor (42, 43); and BEL, a selective iPLA2 inhibitor (43, 44). The experimental conditions were similar to those described above, except that the time of incubation of the labeled cells was reduced to 3 h because of the possibility that the inhibitors might produce cytotoxicity if the exposure was prolonged. The release of 20-[3H]HETE from the PCEC was reduced by 53% when 10 µM MAFP was added (55 ± 2 pmol in the control cultures as compared with 26 ± 1 pmol in this treated with MAFP, n = 3). By contrast, the amount of 20-[3H]HETE that was released into the medium was not reduced appreciably by either 10 µM or 25 µM BEL (48 ± 1 and 50 ± 4 pmol, respectively, n = 3). These concentrations of BEL are reported to be effective against iPLA2-mediated processes in other systems (43, 44). The time dependence of the reduction in 20-[3H]HETE release from the PCEC produced by 15 µM MAFP is shown in Fig. 10. Less 20-[3H]HETE accumulated in the medium throughout this period when MAFP was added, and a 62% reduction occurred at the end of the 3 h incubation. The differential effects of MAFP and BEL suggest that a substantial amount of the efflux of 20-HETE is mediated by cPLA2.



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  		FIG. 10.
Time-dependent effects of PLA2 inhibition on the amount of 20-HETE released from the endothelial cells. Confluent PCEC cultures were incubated for 3 h with 1.0 µM 20-[3H]HETE, washed, and then incubated for up to 3 h in 1.5 ml of modified M199 medium containing 1.0 µM BSA, with or without 15 µM MAFP, an inhibitor of PLA2. The radioactivity contained in the medium was assayed at various times during the second incubation period. The pmol values were calculated from the specific activity of the 20-[3H]HETE with which the cells were incubated. Each point is the average of values obtained from two separate cultures.

 
Ca2+-stimulated 20-HETE Release—To further explore the Ca2+-dependence of 20-HETE efflux, the effect of a Ca2+ ionophore was determined. The results are shown in Fig. 11. PCEC cultures were incubated for 1 h with 20-[3H]HETE. After the medium was removed and the cells washed, the cultures were incubated for 20 min in a fresh medium containing 5 µM BSA, with or without 4 µM ionophore A23187 [GenBank] . The cultures incubated with A23187 [GenBank] released 4.5 times more radioactivity than the corresponding controls (Fig 11A). HPLC analysis of the incubation medium indicated that radioactivity contained in the medium of the cultures exposed to A23187 [GenBank] was almost entirely 20-HETE (Fig. 11B). These data demonstrate that a rapid release of some 20-HETE contained in the PCEC can occur and that this process is Ca2+-activated.



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  		FIG. 11.
Ca2+ ionophore-stimulated release of radiolabeled 20-HETE from the endothelial cells. Confluent PCEC cultures were incubated at 37 °C for 1 h with 0.5 µM 20-[3H]HETE in modified M199 medium containing 0.1 µM BSA. After removal of the medium and washing, the labeled cultures were incubated for 20 min in modified M199 medium containing 5 µM BSA, with or without 4 µM ionophore A23187 [GenBank] . The media were collected, the lipids extracted, and aliquots were assayed for radioactivity by liquid scintillation counting. A, quantity of released material in pmol, calculated from the specific activity of the 20-[3H]HETE with which the cells were incubated. Each bar is the mean ± S.E. of values obtained from three separate cultures, *, p < 0.01. B, HPLC analysis of the lipids released into the medium following treatment with A23187 [GenBank] . A single chromatogram is shown, but a similar result was obtained from a second culture.

 
Vascular Reactivity—The effects of 20-HETE and 20-COOH-AA on vascular function in porcine coronary arterioles is illustrated in Fig. 12. In vessels preconstricted with endothelin, 20-COOH-AA produced concentration-dependent vasodilation at concentrations as low as 100 nM. The magnitude of vasodilation was significantly less than that produced by the potent vasodilator sodium nitroprusside. In contrast, 20-HETE produced no demonstrable vasodilation at the concentrations studied. Thus, our findings suggest that under these defined experimental conditions, 20-COOH-AA produced moderately potent vasodilation, while the same concentrations of 20-HETE did not alter microvascular diameter.



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  		FIG. 12.
Reactivity of isolated coronary microvessels. Porcine coronary microvessels were preconstricted with endothelin and then treated with sodium nitroprusside (SNP, solid circles), 20-COOH-AA (open triangles), and 20-HETE (closed squares). Values represent mean ± S.E.; n = 3–4 pigs tested in each group; # indicates p < 0.05, versus sodium nitroprusside; * indicates p < 0.05, 20-COOH-AA versus 20-HETE.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
These results indicate that the endothelium has the potential to modulate the function of 20-HETE in the vascular wall. We find that cultured endothelial cells have a large capacity to take up and metabolize 20-HETE in the concentration range that produces functional responses in vascular and renal tissues (6, 14, 16, 45). When 1 µM 20-[3H]HETE was initially available, the cells took up a 45% in 1 h. After 4 h, 9.5% of the initially available 20-[3H]HETE was converted to the radiolabeled metabolites detected in the medium. As observed in the rabbit kidney (20), the PCEC incorporated 20-HETE primarily in phospholipids and to a small extent in neutral lipids. The 20-HETE was distributed in PC, PI, and PE and was present almost entirely in the sn-2 position of these phospholipids, a generally similar pattern to what has been observed in the cortex and medulla of the rabbit kidney (20). A small amount of unesterified 20-HETE also was detected in the PCEC, possibly due in part to binding to a cytosolic fatty acid-binding protein (46).

Triacsin C, an acylcoenzyme A synthase inhibitor (47), reduced the amount of 20-HETE present into phospholipids and neutral lipids, indicating that the incorporation into these endothelial lipids is coenzyme A-dependent. Importantly, we show that the 20-HETE incorporated into endothelial cell lipids can be released in response to agonist-induced phospholipase activation, a mechanism that has the potential to modulate endothelium-dependent vasomotor responses.

Previous studies have shown that angiotensin II stimulates 20-HETE release from rat renal microvessels and that 20-HETE is the second messenger for endothelin-1 in the rat proximal tubule (48, 49). Based on these findings, it has been proposed that 20-HETE, like EETs, is stored in the kidney and is released by peptide hormone activation of an acylhydrolase (20). These physiological agonists were not tested in the PCEC cultures. However, the fact that 20-HETE is present in the sn-2 position of the PCEC phospholipids and that its release was stimulated by the addition of a Ca2+ ionophore suggests that a similar signal transduction mechanism may be operative in the endothelium. This hypothesis also is consistent with the observation that 20-HETE release from the PCEC was reduced substantially by MAFP, a cPLA2 inhibitor (42, 43).

In addition to being incorporated into endothelial lipids, 20-HETE was metabolized by the endothelial cells through two oxidative processes, {omega}-oxidation that formed 20-COOH-AA, and {beta}-oxidation. The conversion of 20-HETE to 20-COOH-AA was first detected in rabbit kidney cells isolated from the thick ascending loop of Henle (19). We find, as reported previously in the kidney studies (19), that the difference in the reverse-phase HPLC RTs of 20-COOH-AA and 20-HETE is less than 2 min. Therefore, it is likely that the formation of 20-COOH-AA may be overlooked in work with 20-HETE unless the RTs of compounds detected by HPLC are carefully compared with standards and the structure is confirmed by tandem mass spectrometry. The formation of 20-COOH-AA by the PCEC occurred slowly as compared with 20-HETE incorporation into endothelial lipids. However, 20-COOH-AA continued to accumulate in the medium during relatively long incubations, indicating that endothelial cells do not rapidly degrade this metabolite.

Previous studies by Escalante et al. (19) demonstrated that 20-COOH-AA was as effective as 20-HETE in inhibiting 86Rb uptake by the kidney tubular cells, demonstrating that it is a biologically active metabolite and not simply a degradation product of 20-HETE. We also find that 20-COOH-AA is a moderately potent vasodilator in the porcine coronary microvessels, providing additional evidence that this metabolite has biological activity. As opposed to 20-COOH AA, 20-HETE failed to produce vasodilation in this preparation. Therefore, 20-COOH-AA may have functional effects that are distinct from those of 20-HETE. Taken together, these findings suggest that the endothelium may be a site of 20-COOH-AA production, and that the vascular effects of 20-HETE may be modulated by endothelial conversion to 20-COOH-AA.

Partial {beta}-oxidation metabolites also were produced from 20-HETE by the PCEC. As opposed to 20-COOH-AA, these products appeared sequentially in the medium and then were converted to chain-shortened derivatives. Endothelial cells synthesize partial {beta}-oxidation products from other eicosanoids, including intra-chain HETEs and EETs (27, 39, 50, 51), and studies with mutant human skin fibroblasts indicate that this occurs through peroxisomal {beta}-oxidation (52, 53). While it seems likely that the {beta}-oxidation pathway functions to degrade 20-HETE, the possibility that one or more of the chain-shortened metabolites may have functional activity cannot be excluded.

20-HETE is converted to 20-hydroxy-prostaglandin G2 and H2 by cyclooxygenase in rat aortic endothelium and ram seminal vesicle microsomes (54). These prostaglandin endoperoxide derivatives produce vasoconstriction of the rat aorta. Lung microsomes also convert 20-HETE to a prostanoid product (22), and rabbit airway epithelial cells convert 20-HETE to a cyclooxygenase product that causes bronchial relaxation (24). We did not detect appreciable amounts of prostaglandin-like products, as determined by either HPLC or mass spectrometry, following incubation of the PCEC with 20-HETE. In this regard it should be noted that more recent results with bovine coronary arteries suggest an alternative mechanism for the cyclooxygenase-dependent vasorelaxant effect of 20-HETE (23). This work indicates that 20-HETE increases the production of prostacyclin in the coronary artery endothelium. The rapid and substantial incorporation of 20-HETE into PCEC phospholipids that we observed is consistent with such a mechanism. Incorporation of 20-HETE would be expected to displace some endogenous arachidonic acid from the sn-2 position of phospholipids and thereby make increased amounts available to the endothelial cyclooxygenase.

In conclusion, we suggest that through uptake and {beta}-oxidation, the endothelium can reduce the availability of 20-HETE to the vascular smooth muscle. We also suggest that through incorporation into phospholipids, 20-HETE can affect the functional properties of the endothelium. In particular, the release of incorporated 20-HETE during agonist-induced activation of endothelial cells could modulate endothelium-dependent vasomotor responses, and the formation of 20-COOH-AA may further affect this process. Therefore, we propose the hypothesis that the endothelium can have an important influence on the vasoactive effects of 20-HETE.


   FOOTNOTES
 
* This study was supported by National Institutes of Health Grants HL72845 (to A. A. S.), HL62984 and HL070860 (to N. L. W.), HL51055 (to W. B. C.), GM31278 (to J. R. F.), and Shared Instrumentation Grant RR 13799. Support also was provided by American Heart Association Research Grant 0230096N (to X. F.), Department of Veterans Affairs Merit Review Entry Program Grant (to C. L. O.) and Merit Review Award (to K. C. D.), and the Robert A. Welch Foundation (to J. R. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§§ To whom correspondence should be addressed: Dept. of Biochemistry, 4-403 BSB, University of Iowa, 51 Newton Rd, Iowa City, IA 52242. Tel.: 319-335-7913; Fax: 319-335-9570; E-mail: arthurspector{at}uiowa.edu.

1 The abbreviations used are: HETE, hydroxyeicosatetraenoic acid; CYP, cytochrome P450; PI, inositol phosphoglycerides; PCEC, porcine coronary artery endothelial cells; 20-COOH-AA, 20-carboxy-arachidonic acid; BSA, bovine serum albumin; HPLC, high-performance liquid chromatography; RT, retention time; TLC, thin-layer chromatography; MAFP, methyl arachidonoyl fluorophosphonate; BEL, bromoenol lactone; PLA2, phospholipaseA2; LC/MS-MS, liquid chromatography and tandem mass spectrometry; NL, neutral lipid; PC, choline phosphoglycerides; PE, ethanolamine phosphoglycerides; cPLA2; cytosolic phospholipase A2; iPLA2, calcium-independent phospholipase A2; EET, epoxyeicosatrienoic acid. Back


   ACKNOWLEDGMENTS
 
We thank Bud's Custom Meats, Inc., Riverside, IA, for supplying pig hearts.



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