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Am J Physiol Heart Circ Physiol 291: H2301-H2307, 2006. First published June 16, 2006; doi:10.1152/ajpheart.00349.2006
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20-Hydroxyeicosatetraenoic acid is a potent dilator of mouse basilar artery: role of cyclooxygenase

Xiang Fang,1 Frank M. Faraci,2,4 Terry L. Kaduce,1 Shawn Harmon,1 Mary L. Modrick,2 Shanming Hu,1 Steven A. Moore,3 J. R. Falck,5 Neal L. Weintraub,2 and Arthur A. Spector1,2

Departments of 1Biochemistry, 2Internal Medicine, 3Pathology, and 4Pharmacology, Carver College of Medicine, University of Iowa, Iowa City, Iowa; and 5Department of Biochemistry, University of Texas Southwestern Medical School, Dallas, Texas

Submitted 31 March 2006 ; accepted in final form 12 June 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
20-Hydroxyeicosatetraenoic acid (20-HETE), an arachidonic acid (AA) metabolite synthesized by cytochrome P-450 {omega}-oxidases, is reported to produce vasoconstriction in the cerebral circulation. However, we find that like 14,15-epoxyeicosatrienoic acid (14,15-EET), 20-HETE produces dilation of mouse basilar artery preconstricted with U-46619 in vitro. Indomethacin inhibited the vasodilation produced by 20-HETE but not by 14,15-EET, suggesting a cyclooxygenase (COX)-dependent mechanism. Metabolic studies indicated several mechanisms that may play a role in this process. Mouse brain endothelial cells (MBEC) converted 20-HETE to 20-OH-PGE2, which was as potent as PGE2 in dilating the basilar artery. 20-HETE also stimulated AA release and PGE2 and 6-keto-PGF1{alpha} production in MBEC. Furthermore, the basilar artery converted 20-HETE to 20-COOH-AA, which also produced COX-dependent dilation of the basilar artery. 20-COOH-AA increased AA release and PGE2 and 6-keto-PGF1{alpha} production by the MBEC, but to a lesser extent than 20-HETE. Whereas the conversion of 20-HETE to 20-OH-PGE2 and production of endogenous prostaglandins probably are primarily responsible for vasodilation, the production of 20-COOH-AA also may contribute to this process.

20-hydroxy-prostaglandin E2; 20-carboxy-arachidonic acid; prostaglandins; cerebral vascular tone

20-HYDROXYEICOSATETRAENOIC acid (20-HETE) is a bioactive eicosanoid synthesized from arachidonic acid (AA) by cytochrome P-450 {omega}-oxidases. 20-HETE modulates vascular tone and cell proliferation, and it appears to play a role in the regulation of blood pressure and myogenic responses (33). Several different intracellular signaling pathways mediate these biological responses to 20-HETE. For example, 20-HETE can activate protein kinase C, mitogen-activated protein kinase, Rho-kinase, and cytosolic phospholipase A2 (20, 25, 33). 20-HETE also elicits the phosphorylation of myosin light chain (24), causes the subcellular translocation of Ras (31), activates L-type Ca2+ channels (15), and inhibits opening of some K+ channels (26). A major metabolite of 20-HETE is 20-carboxy-arachidonic acid (20-COOH-AA), which has been shown to dilate porcine coronary microvessels and inhibit ion transport in kidney tubular cells (7, 8, 19).

The cerebral circulation is one of the important sites of 20-HETE function. 20-HETE is synthesized by cat cerebral microvessels (17) and by cultured rat cerebral vascular muscle (15), and it produces constriction of cerebral blood vessels and may contribute to autoregulation of cerebral blood flow during changes in arterial pressure (16). The concentration of 20-HETE in cerebrospinal fluid is increased after subarachnoid hemorrhage in rats, and this appears to contribute to the resulting acute fall in cerebral blood flow (3). Furthermore, an inhibitor of 20-HETE synthesis was shown to reduce infarct size in an ischemic stroke model (22). Therefore, agents that interfere with the actions of 20-HETE may have therapeutic benefit by preserving cerebral blood flow in the setting of stroke and acute subarachnoid hemorrhage (38).

To explore possibilities for this type of therapeutic approach, we have investigated the metabolism and functional responses to 20-HETE in an experimental model, the mouse basilar artery. As opposed to what has been reported in the rat middle cerebral, rat basilar, and canine basilar arteries (16, 26, 36), we find that 20-HETE produces potent, concentration-dependent dilation of the mouse basilar artery after submaximal constriction with U-46619. Additional studies were done to identify mechanisms responsible for the unexpected 20-HETE-mediated dilation in this preparation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. 20-HETE, 14,15-epoxyeicosatrienoic acid (14,15-EET), 20-hydroxy-PGE2 (20-OH-PGE2), and enzyme immunoassay (EIA) kits for PGE2 and 6-keto-PGF1{alpha} were purchased from Cayman Chemicals (Ann Arbor, MI). Indomethacin, calcium ionophore A-23187, and phorbol 12-myristate 13-acetate (PMA) were obtained from Sigma (St. Louis, MO). 20-COOH-AA and 20-[3H]HETE (16.7 µCi/nmol) were synthesized as described previously (9, 19).

Cerebral arteries in vitro. After an overdose of anesthesia (pentobarbital sodium, 200 mg/kg ip), the mouse (C57BL/6) brain was rapidly removed and placed in ice-cold Krebs buffer. As described previously (14, 37), the basilar artery was isolated using a dissecting microscope, cannulated onto glass micropipettes filled with Krebs buffer in an organ chamber, and secured with nylon monofilament suture. Arteries were pressurized to 60 mmHg. With the use of a microscope and a video camera, vessel images were projected on a video monitor. An electronic dimension analyzer was then used to measure lumen diameter. Experimental protocols were approved by the Institutional Animal Care and Use Committee at the University of Iowa, and mice were handled in a manner that meets the guidelines for the National Society of Medical Research and guidelines for the care and use of laboratory animals.

Once prepared for study, basilar arteries were allowed to equilibrate for at least 30 min at a distending pressure of 60 mmHg before protocols were initiated. The basal diameters of the basilar arteries averaged between 134 and 143 µm. We examined changes in diameter of the basilar artery in response to KCl (50 mM) and cumulative doses of agents of interest (20-HETE, etc.). For studies in which vasodilator responses were examined, basilar arteries were constricted by ~30% (~60% of the response to 50 mM KCl) with the thromboxane mimetic U-46619 from 1 x 10–7 to 4 x 10–7 M.

Cell culture. Murine cerebral microvascular endothelial cells (MBEC) were prepared as described previously (23). The cells were grown in modified medium 199 with 20% fetal bovine serum and maintained until confluent at 37°C in a humidified atmosphere containing 5% CO2-95% room air. Stocks were subcultured weekly into 100-mm-diameter dishes or six-well tissue culture plates by trypsinization, and the experiments were performed on culture passages 8–20.

Metabolism of 20-HETE and AA. The metabolic studies were done with either MBEC or an isolated mouse brain vascular preparation consisting of the basilar artery and the circle of Willis with the middle cerebral arteries. The method of incubation and analysis was similar in both cases. The tissues were incubated with 0.1 µM 20-[3H]HETE, or MBEC were incubated with 20-[3H]HETE or [3H]AA for various times. After incubation, the medium was collected, and the tissues were washed twice with cold phosphate-buffered saline solution and harvested. Radioactivity contained in an aliquot of the medium and in the tissue lipid extract was assayed by liquid scintillation counting. The remainder of the medium was extracted with H2O-saturated ethyl acetate and separated by reverse-phase HPLC. A C18 5-µm 4.6 x 150-mm column was used to separate metabolites. For separation of AA metabolites, an elution profile, consisting of water adjusted to pH 4 with formic acid and an acetonitrile gradient that increased from 30% to 100% over 65 min at a flow rate of 0.7 ml/min, was used. An acetonitrile gradient that increased from 10% to 100% over 60 min at a flow rate of 0.7 ml/min was used to separate 20-HETE metabolites (7). The column effluent was combined with scintillator solution and was passed through an in-line flow scintillation detector (IN/US System, Tampa, FL) to determine the distribution of radioactivity (7, 9, 12).

Prostaglandin determination. MBEC were incubated with 0.1 µM [3H]AA for 4 h. After the medium was removed and the cells washed, the incubation was continued for an additional 30 min in a medium containing 0.1 µM BSA and either vehicle, 2 µM 20-HETE, or 20-COOH-AA in the vehicle. The medium was collected and extracted, and the radiolabeled metabolites that accumulated in the medium were analyzed by reverse-phase HPLC. In additional experiments, the cells were incubated with 2 µM 20-HETE or 20-COOH-AA in medium 199 containing 0.1 µM BSA for 4 h. The medium was collected, and PGE2 and 6-keto-PGF1{alpha}, a stable metabolite of prostacyclin, were measured using PGE2 and 6-keto-PGF1{alpha} EIA kits purchased from Cayman. The antibody against PGE2 is specific for PGE2, and it does not cross-react with 20-OH-PGE2 (specificity for PGE2 is 100%; for 20-OH-PGE2, <0.01%).

Identification of 20-HETE metabolites. The structure of lipid metabolites contained in the medium was identified by liquid chromatography combined with mass spectrometry (LC/MS) using a Hewlett-Packard 1100 MSD LC/MS system (19). HPLC separation was done with a C18 5-µm 4.6 x 150-mm column and mobile phase solvents consisting of water: formic acid (100:0.03, vol/vol; solvent A) and acetonitrile (solvent B) at a flow rate of 0.7 ml/min. The gradient was maintained at 30% solvent B for the first 2 min and then linearly increased to 57% solvent B at 20 min, 65% at 40 min, 70% at 45 min, and 95% at 50 min. Negative ion electrospray was used with the fragmentor voltage set at 110 V to produce in-source collision-induced decompositions (CID). N2 nebulizing gas was maintained at 60 bar, whereas the N2 drying gas was set at a flow rate of 10 l/min at 350°C. Data were processed with the Hewlett-Packard Chemstation software program (9, 19).

Western blot analysis. Cells were harvested and sonicated, and the proteins were separated in an SDS-10% polyacrylamide gel with a 5% stacking gel in SDS-Tris-glycine running buffer. The proteins were transferred electrophoretically to a nitrocellulose membrane. After an overnight incubation in 0.02 M Tris-0.15 M NaCl buffer (pH 7.45) with 0.1% Tween 20 buffer containing specific antibody against cyclooxygenase (COX)-2 (1:1,000; Cayman Chemical), the blot was incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:5,000; Roche Diagnostics, Indianapolis, IN) for 1 h at room temperature, and the anti-COX-2 antibodies were detected by using an ECL detection system and exposure to X-ray film. After this was completed, the membrane was stripped and reprobed with antibody against beta-actin (Sigma-Aldrich), and the density of the beta-actin band was used to normalize for protein loading (10).

Statistical analyses. The data are expressed as means ± SD. A one-way ANOVA was used to analyze differences between mean values of multiple groups, followed by the Newman-Keuls method. Probability values ≤0.05 were considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of 20-HETE and 14,15-EET on vascular tone. Figure 1 illustrates the comparative effects of 20-HETE and 14,15-EET on the diameter of the mouse basilar artery. 20-HETE produced potent, concentration-dependent relaxation of basilar arteries preconstricted with U-46619; 25% dilation occurred with 50 nM 20-HETE, and 1 µM 20-HETE produced 85% dilation (Fig. 1A). 14,15-EET, a lipid mediator synthesized from AA by cytochrome P-450 epoxygenases (35), also produced dilation of the basilar artery (Fig. 1B). Responses of the basilar artery to 20-HETE were completely inhibited by 10 µM indomethacin, a nonselective COX inhibitor. By contrast, indomethacin did not affect the vasodilation produced by 14,15-EET, indicating that the inhibitory effect was selective for 20-HETE.


Figure 1
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  		Fig. 1. Vasoactive effects of 20-HETE and 14,15-epoxyeicosatrienoic acid (14,15-EET) in mouse basilar arteries. Arteries were constricted by 30% of resting diameter with U-46619. Cumulative concentration-response relationships were evaluated for 20-HETE (A) and 14,15-EET (B) in the absence or presence of 10 µM indomethacin (Indo). Data are means ± SE obtained from 3 to 4 separate experiments. **P < 0.01 compared with 20-HETE in the presence of Indo at same concentration.

 
Production and function of 20-COOH-AA. Metabolic studies were done with 20-HETE to investigate the mechanism of the vasodilator response. When 20-[3H]HETE was incubated in the medium in the absence of tissues for 24 h, a single radiolabeled peak with a reverse-phase HPLC retention time (RT) of 43 min, which comigrated with a standard 20-[3H]HETE, was detected (Fig. 2A). When a preparation consisting of mouse cerebral arteries was incubated with 20-[3H]HETE, a radiolabeled metabolite with a RT of 41 min was detected in the medium (Fig. 2B). This product was previously identified as 20-COOH-AA, a {omega}-oxidation product of 20-HETE (7, 19). 20-[3H]COOH-AA accounted for 5% of the total radioactivity recovered from the medium after incubation for 3 h. Formation of 20-[3H]COOH-AA continued to increase as the incubation progressed, accounting for 35% of total radioactivity in the medium at the end of the 24-h incubation. The increase in 20-COOH-AA was associated with a concomitant decrease in 20-[3H]HETE (Fig. 2C).


Figure 2
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  		Fig. 2. Formation of 20-COOH-arachidonic acid (AA) and its effect on vascular tone. A and B: representative chromatograms showing radiolabeled compounds present in medium after 24 h of incubation of 0.1 µM 20-[3H]HETE in the absence (A) or presence (B) of cerebral artery preparation. C: time-dependent changes in radiolabeled 20-HETE and 20-COOH-AA contained in the medium. These data were converted to values (in pmol) using the specific activity of radiolabeled 20-[3H]HETE added to cultures. D: changes in diameter of basilar artery produced by 20-COOH-AA in the absence or presence of 10 µM indomethacin (Indo) in U-46619-preconstricted arteries. Procedure was the same as described in Fig. 1. Data in in D are means ± SE obtained from 3 to 4 separate experiments. **P < 0.01 compared with 20-COOH-AA in the presence of Indo at same concentration.

 
Previous studies (19) indicated that 20-COOH-AA produced dilation of porcine coronary microvessels. Therefore, studies were done to determine whether it might have a similar effect in the mouse cerebral vasculature. As shown in Fig. 2D, 20-COOH-AA produced dilation of the basilar artery, but the response was somewhat smaller than that produced by 20-HETE. However, as was observed with 20-HETE, the vasodilation produced by 20-COOH-AA was completely inhibited by 10 µM indomethacin (Fig. 2D).

Prostaglandin production. The inhibition of dilation produced by indomethacin suggested that 20-HETE and 20-COOH-AA produce vasodilation through a COX-dependent mechanism. This would be consistent with previous results demonstrating that COX-1 and COX-2 are expressed in the MBEC (5, 11). To assess this possibility, we investigated whether these compounds stimulate production of vasodilator prostaglandins in mouse cerebrovascular tissue. These studies were done with cultures of MBEC because the amount of endothelium present in intact blood vessels that can be obtained from the mouse is too small to produce detectable amounts of prostaglandins.

The cultures of MBEC were incubated with 0.1 µM [3H]AA for 4 h, washed, and subsequently incubated in a fresh medium containing either vehicle (control), 2 µM 20-HETE, or 2 µM 20-COOH-AA. The results are shown in Fig. 3. A radiolabeled compound with a RT of 15 min, which is identical to the RT of a PGE2 standard in this HPLC gradient, was detected in the control cultures (Fig. 3A). This radiolabeled product was also observed when the MBEC were incubated with 20-HETE, together with a second product with a RT corresponding to a 6-keto-PGF1{alpha} standard (9 min) (Fig. 3B). Although radiolabeled material with the RT of PGE2 was also detected in the cultures incubated with 20-COOH-AA, a distinct component corresponding to 6-keto-PGF1{alpha} was not observed (Fig. 3C).


Figure 3
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  		Fig. 3. Effects of 20-HETE and 20-COOH-AA on prostaglandin formation. Mouse brain endothelial cells (MBEC) were incubated with 0.1 µM [3H]AA for 4 h. After incubation, cells were washed and then incubated with medium containing 0.1 µM BSA and either vehicle (A), 2 µM 20-HETE (B), or 2 µM 20-COOH-AA (C) for 30 min. Medium was collected and radiolabeled products analyzed by reverse-phase HPLC. Representative chromatograms are shown.

 
To obtain quantitative results, additional cultures of MBEC were incubated with 2 µM 20-HETE or 20-COOH-AA, and the medium was collected and assayed for PGE2 and 6-keto-PGF1{alpha} by EIA. When compared with the control cultures, PGE2 production was sevenfold higher when the MBEC were incubated with 20-HETE and 4.5-fold higher when incubated with 20-COOH-AA (Fig. 4A). Likewise, when compared with the control cultures, 6-keto-PGF1{alpha} production increased 3.9-fold and 1.8-fold during incubation with 20-HETE and 20-COOH-AA, respectively (Fig. 4B).


Figure 4
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  		Fig. 4. Effects of 20-HETE and 20-COOH-AA on endogenous PGE2 (A) and 6-keto-PGF1{alpha} production (B). MBEC were treated with either vehicle, 2 µM 20-HETE, or 20-COOH-AA. After 4 h of incubation, medium was collected, and the amounts of PGE2 and 6-keto-PGF1{alpha} in medium were assayed by enzyme immunoassay. Bars in graph are means ± SE obtained from 3 separate cultures. *P < 0.05 compared with control.

 
To investigate the mechanism of the increases in prostaglandin production, we determined whether 20-HETE and 20-COOH-AA increased the expression of COX-2 in MBEC. As shown in Fig. 5, neither 20-HETE nor 20-COOH-AA increased the expression of COX-2 protein, whereas incubation with PMA (100 nM, positive control) produced a large increase. However, 20-HETE and 20-COOH-AA increased AA mobilization. The MBEC initially were incubated for 4 h with 0.1 µM [3H]AA. After this, the medium was removed and the cells washed. The cultures were then incubated for 30 min in a fresh medium containing either 0.1 µM BSA as a control or BSA with either 2 µM 20-HETE or 2 µM 20-COOH-AA. The results, which also are illustrated in Fig. 5, show that both compounds increased [3H]AA release compared with that of the control cultures. The MBEC incubated with 20-HETE released 2.3-times more radioactivity than the corresponding controls, whereas those incubated with 20-COOH-AA released 35% more than the controls. These results suggest that the increase in prostaglandin formation produced by 20-HETE and 20-COOH-AA is secondary to increased AA mobilization from endothelial lipids.


Figure 5
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  		Fig. 5. Effect of 20-HETE and 20-COOH-AA on expression of cyclooxygenase (COX)-2 protein and AA release. MBEC were incubated with 2 µM 20-HETE or 20-COOH-AA or 100 nM phorbol 12-myristate 13-acetate (PMA) for 4 h. Medium was removed, and cells were harvested and sonicated. COX-2 protein in cell lysates was detected by Western blot analysis using a specific antibody against COX-2. For AA release experiments, MBEC were incubated with 0.1 µM [3H]AA for 4 h. After incubation, cells were washed and then treated with medium containing 0.1 µM BSA and either vehicle (C) or 2 µM 20-HETE (20-H) or 2 µM 20-COOH-AA (20-C) for 30 min. Medium was collected and radioactivity measured by liquid scintillation counting. Values are results obtained from 5 separate cultures, expressed as means ± SE. Specific activity of added [3H]AA was used to calculate amounts (in pmol). *P < 0.05 compared with control. Std, standard.

 
20-OH-PGE2 production and function. Studies in other experimental systems indicated that 20-HETE is a COX substrate (18, 27, 34). To further examine potential mechanisms whereby COX might mediate the vasodilation produced by 20-HETE in mouse cerebral arteries, we investigated whether 20-HETE is a substrate for COX metabolism in this preparation. In the initial experiment, MBEC were incubated with 2 µM 20-[3H]HETE with or without indomethacin, and the medium was collected and analyzed by HPLC. The main radiolabeled metabolite had a RT of 34 min, consistent with that of 16-OH-hexadecatrienoic acid, a partial beta-oxidation product of 20-HETE that was previously identified in porcine coronary artery endothelial cultures (19). Several other radiolabeled polar metabolites were also detected, including peak X (RT = 13 min) that accounted for 4% of radioactivity present in the medium (Fig. 6A). Formation of peak X was completely inhibited by indomethacin (Fig. 6B), suggesting that compound X is a COX metabolite of 20-HETE. Production of the radiolabeled COX metabolite was substantially increased by treatment of the MBEC with 100 nM PMA. About 3% of the radiolabeled products that accumulated in the medium were present as COX metabolites in the control cultures, whereas in the presence of PMA, COX metabolites accounted for 8% of the radiolabeled products in the medium.


Figure 6
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  		Fig. 6. Conversion of 20-HETE to 20-OH-PGE2: effect of Indo. MBEC were treated with or without Indo (10 µM) for 30 min. Medium was removed, and incubation was continued in fresh medium containing 2 µM 20-[3H]HETE in the absence (A) or presence (B) of Indo. Medium was collected and radiolabeled products analyzed by reverse-phase HPLC. Representative chromatograms are shown, but similar results were obtained from 2 additional cultures in each case. 16-OH-16:3, 16-OH-hexadecatrienoic acid.

 
The chemical structure of compound X was identified by LC/MS using in-source CID (Fig. 7A). The mass spectrum contained a molecular ion (M-H)–1 mass/charge (m/z) 367 and fragmented with loss of H2O (m/z 349), loss of 2H2O (m/z 331), loss of CO2 and 2H2O (m/z 287), loss of C6H13O2 (m/z 233), and loss of C7H13O4 (m/z 189). This fragmentation pattern is identical to that obtained with authentic 20-OH-PGE2 under the same LC/MS conditions (Fig. 7B).


Figure 7
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  		Fig. 7. Mass spectra of 20-HETE metabolite. Mass spectra were obtained by liquid chromatography combined with mass spectrometry (LC/MS) with in-source collision-induced decompositions (CID). To obtain sufficient quantities of products for structural identification, it was necessary to utilize 75-cm2 cultures of MBEC. HPLC analysis indicated that pattern of metabolite formation by endothelial cell cultures was similar to that shown in Fig. 5. Major polar metabolite (A) exhibited a mass spectrum that was identical to that of a 20-OH-PGE2 standard (B).

 
To determine whether 20-OH-PGE2 formation might play a role in the observed COX-dependent vasodilatation produced by 20-HETE, we compared effects of the PGE2 and 20-OH-PGE2 in the basilar artery. In arteries preconstricted with U-46619, PGE2 produced dose-dependent vasodilation when the concentration was raised from 50 to 500 nM, but dilation partially abated when the concentration was raised to 1 µM PGE2 (Fig. 8A). In contrast, 50 nM to 1 µM 20-OH-PGE2 produced only concentration-dependent vasodilation (Fig. 8B). To determine whether this dilation might be due to a PGE2- or 20-OH-PGE2-induced increase in AA mobilization, these compounds were incubated with MBEC previously loaded with [3H]AA. Unlike 20-HETE and 20-COOH-AA, neither 20-OH-PGE2 nor PGE2 increased radiolabeled AA mobilization when compared with control cultures. However, calcium ionophore A-23187 produced a 2.5-fold greater increase in radiolabeled AA release compared with that of the controls cultures (Fig. 8C).


Figure 8
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  		Fig. 8. Effects of 20-OH-PGE2 and PGE2 on mouse basilar artery vasoactivity and on AA release from MBEC. Cumulative concentration-response relationships were determined for PGE2 (A) and 20-OH-PGE2 (B). Data are presented as means ± SE obtained from 4 separate experiments. For AA release experiments (C), MBEC were incubated with 0.1 µM [3H]AA for 4 h. After incubation, cells were treated with medium containing 0.1 µM BSA and either vehicle (control), 2 µM 20-OH-PGE2, 2 µM PGE2, or 1 µM A-23187 for 30 min. Medium was collected and radioactivity measured by liquid scintillation counting. Values are expressed as means ± SE of 3 separate cultures of MBEC. Specific activity of added [3H]AA was used to calculate amounts (in pmol). **P < 0.01 compared with control.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we found that 1) 20-HETE produced potent dilation of the mouse basilar artery; 2) 20-HETE was converted to 20-COOH-AA by the artery, and the 20-COOH-AA also produced dilation of the basilar artery; 3) indomethacin inhibited this response in both cases; 4) 20-HETE and 20-COOH-AA increased AA release and the production of PGE2 and 6-keto-PGF1{alpha} in MBEC; 5) 20-HETE was converted to 20-OH-PGE2 by MBEC; and 6) similar to 20-HETE, 20-OH-PGE2 also produced potent dilation of the basilar artery. These findings suggest that the vasodilation produced by 20-HETE in the mouse basilar artery occurs through a COX-dependent mechanism.

20-HETE is a potent vasoconstrictor in several vascular beds, including kidney, cerebral, and coronary arteries (33). In contrast, EETs, which are cytochrome P-450 epoxygenase metabolites of AA, are vasodilators in most vascular beds (33, 35). These findings have led to the generally accepted view that the balance between these two classes of cytochrome P-450 eicosanoid products contributes to the regulation of vascular tone. For example, the formation of 20-HETE antagonizes endothelium-derived hyperpolarization factor-mediated relaxation in porcine coronary arterioles (32). Consistent with previous results, we found that 14,15-EET caused potent dilation of the mouse basilar artery. Surprisingly, 20-HETE also caused dilation of this artery. However, the mechanism differs in these two cases because unlike 20-HETE, responses to 14,15-EET were not inhibited by indomethacin.

20-HETE is generally viewed as a vasoconstrictor, and cerebral vasoconstrictor effects of 20-HETE were observed in the cat, rat, and dog (33). However, there are systems where 20-HETE functions as a vasodilator. For example, 20-HETE produces vasodilation in the rabbit kidney, bovine coronary, and bovine pulmonary arteries (4, 30, 39). These vasodilatory responses have been attributed to nitric oxide release (39) and prostacyclin formation (4, 30). In the present study, we found that the addition of 20-HETE to mouse brain endothelial cultures increased the production of PGE2 and prostacyclin (measured as the stable product 6-keto-PGF1{alpha}). A mechanism involving an increase in COX expression seems unlikely because COX-1 and COX-2 are constitutive in MBEC (5, 11), and the expression of COX-2 was not increased when the cells were incubated with 2 µM 20-HETE. A more likely possibility is that prostaglandin formation resulted from the increased release of AA, perhaps triggered by a 20-HETE-mediated increase in intracellular free Ca2+ (24). It should be noted that some previous studies (1, 38) demonstrating constrictor effects of 20-HETE in the cerebral artery were performed in the presence of indomethacin, which would mask a vasodilatory effect of 20-HETE that was dependent on COX activity.

PGE2 and prostacyclin are potent dilators in the cerebral circulation (21, 29). In agreement with these results, we observed that PGE2 caused marked dilation of the mouse basilar artery at concentrations between 50 to 500 nM. Thus increased production of PGE2 and prostacyclin might contribute to 20-HETE-induced vasorelaxation in the basilar artery. In addition, COX-dependent production of reactive oxygen species has been reported to produce dilation of cerebral blood vessels in several species (13). Whether such an oxidative mechanism also might contribute to 20-HETE-induced dilation of the mouse basilar arteries remains to be determined.

COX regulates renal arterial 20-HETE levels, and the metabolism of 20-HETE by COX is proposed to be a regulatory mechanism in vascular tone (2, 6, 18, 34). 20-HETE is converted to 20-OH-PGG2 and 20-OH-PGH2 by COX in ram seminal vesicle microsomes. However, these COX metabolites produce contraction of the rat aorta (34). Rabbit airway epithelial cells also convert 20-HETE to a COX metabolite that produces bronchial relaxation (18). In addition, lung microsomes convert 20-HETE to a prostanoid product (2). Consistent with these results, we found that 20-HETE is converted to 20-OH-PGE2 by the COX-dependent mechanism in MBEC. The production of 20-OH-PGE2 was enhanced by treatment of the cells with PMA, suggesting the involvement of COX-2 in this process. Like PGE2, 20-OH-PGE2 causes vasodilation in the kidney and increases renal cortical and medullary blood flow (28). Likewise, we observed that 20-OH-PGE2 produced potent dilation of the basilar artery. However, unlike 20-HETE, 20-OH-PGE2 did not increase AA mobilization in MBEC. Because PGE2 also did not increase AA mobilization, it is likely that both compounds produced vasodilation by a direct effect rather than indirectly through AA mobilization.

We found that 20-COOH-AA, like 20-HETE, produced dilation of the basilar artery and increased formation of PGE2 and 6-keto-PGF1{alpha}. Although these results raise the possibility that conversion to 20-COOH-AA might also contribute to 20-HETE-induced vasodilation, this seems less likely for several reasons. 20-COOH-AA was less potent than 20-HETE in producing these effects, and we were unable to detect the formation of a radiolabeled prostaglandin derivative when the brain endothelial cultures were incubated with 20-[3H]COOH-AA (data not shown). In addition, it is unlikely that 20-COOH-AA mediates the rapid vasodilatory response to 20-HETE, because 3 h was the earliest time that radiolabeled 20-COOH-AA was detected when the isolated cerebral artery preparation was incubated with 20-[3H]HETE. However, it is possible that 20-COOH-AA may prolong the vasodilatory response produced by 20-HETE in the basilar artery.

In summary, these findings indicate that 20-HETE produces dilation of the mouse basilar artery through a COX-dependent mechanism. The most likely possibilities are conversion of 20-HETE to 20-OH-PGE2 or increased production of PGE2 and prostacyclin due to mobilization of AA. While 20-HETE is generally considered to be a cerebral vasoconstrictor, modulation of its actions by COX could play an important counterregulatory role in the cerebral circulation in some species or under some conditions.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
These studies were supported in part by American Heart Association Scientist Development Grant 0230096N (to X. Fang); National Institutes of Health Grants HL-072845 (to A. A. Spector); HL-38901, NS-24621, and HL62984 (to F. M. Faraci); HL-070860, HL-076684, and HL-062984 (to N. L. Weintraub); DK-038226 and GM-31278 (to J. R. Falck); and support from the Robert A. Welch Foundation (to J. R. Falck).


   FOOTNOTES
 

Address for reprint requests and other correspondence: X. Fang, Dept. of Medicine, Harbor Hospital Center, 3001 S. Hanover St., Baltimore MD 21225 (e-mail: xiang.fang01{at}gmail.com)

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


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Alonso-Galicia M, Hudetz AG, Shen H, Harder DR, and Roman RJ. Contribution of 20-HETE to vasodilator actions of nitric oxide in the cerebral microcirculation. Stroke 30: 2727–2734, 1999.[Abstract/Free Full Text]
  2. Birks EK, Bousamra KM, Presberg K, Marsh JA, Effros RM, and Jacobs ER. Human pulmonary arteries dilate to 20-HETE, an endogenous eicosanoid of lung tissue. Am J Physiol Lung Cell Mol Physiol 272: L823–L829, 1997.[Abstract/Free Full Text]
  3. Cambj-Sapunar L, Yu M, Harder DR, and Roman RJ. Contribution of 5-hydroxytryptamine1B receptors and 20-hydroxyeiscosatetraenoic acid to fall in cerebral blood flow after subarachnoid hemorrhage. Stroke 34: 1269–1275, 2003.[Abstract/Free Full Text]
  4. Carroll MA, Garcia MP, Falck JR, and McGiff JC. Cyclooxygenase dependency of the renovascular actions of cytochrome P450-derived arachidonate metabolites. J Pharmacol Exp Ther 260: 104–109, 1992.[Abstract/Free Full Text]
  5. Chen P, Hu S, Yao J, Moore SA, Spector AA, and Fang X. Induction of cyclooxygenase-2 by anandamide in cerebral microvascular endothelium. Microvasc Res 69: 28–35, 2005.[CrossRef][Web of Science][Medline]
  6. Cheng MK, McGiff JC, and Carroll MA. Renal arterial 20-hydroxyeicosatetraenoic acid levels: regulation by cyclooxygenase. Am J Physiol Renal Physiol 284: F474–F479, 2003.[Abstract/Free Full Text]
  7. Collins XH, Harmon SD, Kaduce TL, Berst KB, Fang X, Moore SA, Raju TV, Falck JR, Weintraub NL, Duester G, Plapp BV, and Spector AA. {omega}-Oxidation of 20-hydroxyeicosatetraenoic acid (20-HETE) in cerebral microvascular smooth muscle and endothelium by alcohol dehydrogenase 4. J Biol Chem 280: 33157–33164, 2005.[Abstract/Free Full Text]
  8. Escalante B, Erlij D, Falck JR, and McGiff JC. Effect of cytochrome P450 arachidonate metabolites on ion transport in rabbit kidney loop of Henle. Science 251: 799–802, 1991.[Abstract/Free Full Text]
  9. Fang X, Kaduce TL, Weintraub NL, Harmon S, Teesch LM, Morisseau C, Thompson DA, Hammock BD, and Spector AA. Pathways of epoxyeicosatrienoic acid metabolism in endothelial cells. Implications for the vascular effects of soluble epoxide hydrolase inhibition. J Biol Chem 276: 14867–14874, 2001.[Abstract/Free Full Text]
  10. Fang X, Moore SA, Stoll LL, Kaduce TL, Rich G, Weintraub NL, and Spector AA. 14,15-Epoxyeicosatrienoic acid inhibits prostaglandin E2 production in vascular smooth muscle cells. Am J Physiol Heart Circ Physiol 275: H2113–H2121, 1998.[Abstract/Free Full Text]
  11. Fang X, Moore SA, Nwankwo JO, Weintraub NL, Oberley LW, Snyder GD, and Spector AA. Induction of cycloxygenase-2 by overexpression of human catalase gene in cerebral microvascular endothelial cells. J Neurochem 75: 614–623, 2000.[CrossRef][Web of Science][Medline]
  12. Fang X, Weintraub NL, McCaw RB, Hu SM, Harmon SD, Rice JB, Hammock BD, and Spector AA. Effect of soluble epoxide hydrolase inhibition on epoxyeicosatrienoic acid metabolism in human blood vessels. Am J Physiol Heart Circ Physiol 287: H2412–H2420, 2004.[Abstract/Free Full Text]
  13. Faraci FM. Reactive oxygen species: influence on cerebral vascular tone. J Appl Physiol 100: 739–743, 2006.[Abstract/Free Full Text]
  14. Faraci FM, Lamping KG, Modrick ML, Ryan MJ, Sigmund CD, and Didion SP. Cerebral vascular effects of angiotensin II: new insights from genetic models. J Cereb Blood Flow Metab 26: 449–455, 2006.[CrossRef][Web of Science][Medline]
  15. Gebremedhin D, Lange AR, Narayanan J, Aebly MR, Jacobs ER, and Harder D. Cat cerebral arterial smooth muscle cells express cytochrome P450 4A2 enzyme and produce the vasoconstrictor 20-HETE which enhances L-type Ca2+ current. J Physiol 507: 771–781, 1998.[Abstract/Free Full Text]
  16. Gebremedhin D, Lange AR, Lowry TF, Taheri MR, Birks EK, Hudetz AG, Narayanan J, Falck JR, Okamoto H, Roman RJ, Nithipatikom K, Campbell WB, and Harder D. Production of 20-HETE and its role in autoregulation of cerebral blood flow. Circ Res 87: 60–65, 2000.[Abstract/Free Full Text]
  17. Harder DR, Gebremedhin D, Narayanan J, Jefcoat C, Falck JR, Campbell WB, and Roman R. Formation and action of a P-450 4A metabolite of arachidonic acid in cat cerebral microvessels. Am J Physiol Heart Circ Physiol 266: H2098–H2107, 1994.[Abstract/Free Full Text]
  18. Jacobs ER, Effros RM, Falck JR, Reddy KM, Campbell WB, and Zhu D. Airway synthesis of 20-hydroxyeicosatetraenoic acid: metabolism by cyclooxygenase to a bronchodilator. Am J Physiol Lung Cell Mol Physiol 276: L280–L288, 1999.[Abstract/Free Full Text]
  19. Kaduce TL, Fang X, Harmon SD, Oltman CL, Dellsperger KC, Teesch LM, Gopal VR, Falck JR, Campbell WB, Weintraub NL, and Spector AA. 20-Hydroxyeicosatetraenoic acid (20-HETE) metabolism in coronary endothelial cells. J Biol Chem 279: 2648–2656, 2004.[Abstract/Free Full Text]
  20. Lange A, Gebremedhin D, Narayanan J, and Harder D. 20-Hydroxyeicosatetraenoic acid-induced vasoconstriction and inhibition of potassium current in cerebral vascular smooth muscle is dependent on activation of protein kinase C. J Biol Chem 272: 27345–27352, 1997.[Abstract/Free Full Text]
  21. Leffler CW, Mirro R, Pharris LJ, and Shibata M. Permissive role of prostacyclin in cerebral vasodilation to hypercapnia in newborn pigs. Am J Physiol Heart Circ Physiol 267: H285–H291, 1994.[Abstract/Free Full Text]
  22. Miyata N, Seki T, Tanaka Y, Omura T, Taniguchi K, Doi M, Bandou K, Kametani S, Sato M, Okuyama S, Cambj-Sapunar L, and Harder DR. Beneficial effects of a new 20-hydroxyeicosatetraenoic acid synthesis inhibitor, TS-011 [N-(3-chloro-4-morpholin-4-yl) phenyl-N'-hydroxyimido formamide], on hemorrhagic and ischemic stroke. J Pharmacol Exp Ther 314: 77–85, 2005.[Abstract/Free Full Text]
  23. Moore SA, Figard PH, Spector AA, and Hart MN. Brain microvessels produce 12-hydroxyeicosatetraenoic acid. J Neurochem 53: 376–382, 1989.[Web of Science][Medline]
  24. Muthalif MM, Benter IF, Karzoun N, Fatima S, Harper J, Uddin MR, and Malik KU. 20-Hydroxyeicosatetraenoic acid mediates calcium/calmodulin-dependent protein kinase II-induced mitogen-activated protein kinase activation in vascular smooth muscle cells. Proc Natl Acad Sci USA 95: 12701–12706, 1998.[Abstract/Free Full Text]
  25. Nakayama K, Obara K, Tanabe Y, and Ishikawa T. 20-Hydroxyeicosatetraenoic acid potentiates contractile activation of canine basilar artery in response to stretch via protein kinase C{alpha}-mediated inhibition of calcium-activated potassium channel. Adv Exp Med Biol 538: 411–416, 2003.[Web of Science][Medline]
  26. Obara K, Koide M, and Nakayama K. 20-Hydroxyeicosatetraenoic acid potentiates stretch-induced contraction of canine basilar artery via PKC{alpha}-mediated inhibition of KCa channel. Br J Pharmacol 137: 1362–1370, 2002.[CrossRef][Web of Science][Medline]
  27. Oliw EH, Fahlstadius P, and Hamberg M. Isolation and biosynthesis of 20-hydroxyprostaglandins E1 and E2 in ram seminal fluid. J Biol Chem 261: 9216–9221, 1986.[Abstract/Free Full Text]
  28. Oyekan AO. Differential effects of 20-hydroxyeicosatetraenoic acid on intrarenal blood flow in the rat. J Pharmacol Exp Ther 313: 1289–1295, 2005.[Abstract/Free Full Text]
  29. Parfenova H and Leffler CW. Functional study on vasodilator effects of prostaglandin E2 in the newborn pig cerebral circulation. Eur J Pharmacol 278: 133–142, 1995.[CrossRef][Web of Science][Medline]
  30. Pratt PF, Falck JR, Reddy KM, Kurian JB, and Campbell WB. 20-HETE relaxes bovine coronary arteries through the release of prostacyclin. Hypertension 31: 237–241, 1998.[Abstract/Free Full Text]
  31. Randriamboavonjy V, Busse R, and Fleming I. 20-HETE-induced contraction of small coronary arteries depends on the activation of Rho-kinase. Hypertension 41: 801–806, 2003.[Abstract/Free Full Text]
  32. Randriamboavonjy V, Kiss L, Falck JR, Busse R, and Fleming I. The synthesis of 20-HETE in small porcine coronary arteries antagonizes EDHF-mediated relaxation. Cardiovasc Res 65: 487–494, 2005.[Abstract/Free Full Text]
  33. Roman RJ. P-450 metabolites of arachidonic acid in the control of cardiovascular function. Physiol Rev 82: 131–185, 2002.[Abstract/Free Full Text]
  34. Schwartzman ML, Falck JR, Yadagiri P, and Escalante B. Metabolism of 20-hydroxyeicosatetraenoic acid by cyclooxygenase. Formation and identification of novel endothelium-dependent vasoconstrictor metabolites. J Biol Chem 264: 11658–11662, 1989.[Abstract/Free Full Text]
  35. Spector AA, Fang X, Snyder GD, and Weintraub NL. Epoxyeicosatrienoic acids (EETs): metabolism and biochemical function. Prog Lipid Res 43: 55–90, 2004.[CrossRef][Web of Science][Medline]
  36. Takeuchi K, Miyata N, Renic M, Harder DR, and Roman RJ. Hemoglobin, NO, and 20-HETE interactions in mediating cerebral vasoconstriction following SAH. Am J Physiol Regul Integr Comp Physiol 290: R84–R89, 2006.[Abstract/Free Full Text]
  37. Yamada M, Lamping KG, Duttaroy A, Zhang W, Cui Y, Bymaster FP, McKinzie DL, Felder CC, Deng CX, Faraci FM, and Wess J. Cholinergic dilation of cerebral blood vessels is abolished in M5 muscarinic acetylcholine receptor knockout mice. Proc Natl Acad Sci USA 98: 14096–14101, 2001.[Abstract/Free Full Text]
  38. Yu M, Cambj-Sapunar L, Kehl F, Maier KG, Takeuchi K, Miyata N, Ishimoto T, Reddy LM, Falck JR, Gebremedhin D, Harder DR, and Roman RJ. Effects of a 20-HETE antagonist and agonists on cerebral vascular tone. Eur J Pharmacol 486: 297–306, 2004.[CrossRef][Web of Science][Medline]
  39. Yu M, McAndrew RP, Al-Saghir P, Maier KG, Medhora M, Roman RJ, and Jacobs ER. Nitric oxide contributes to 20-HETE-induced relaxation of pulmonary arteries. J Appl Physiol 93: 1391–1399, 2002.[Abstract/Free Full Text]



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