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Am J Physiol Heart Circ Physiol 295: H2068-H2078, 2008. First published September 19, 2008; doi:10.1152/ajpheart.01333.2007
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Role of CYP epoxygenases in A2AAR-mediated relaxation using A2AAR-null and wild-type mice

Mohammed A. Nayeem,1 Samuel M. Poloyac,4 John R. Falck,2 Darryl C. Zeldin,5 Catherine Ledent,3 Dovenia S. Ponnoth,1 Habib R. Ansari,1 and S. Jamal Mustafa1

1Department of Physiology and Pharmacology, Center for Interdisciplinary Research in Cardiovascular Sciences, West Virginia Univeristy, Morgantown, West Virginia; 2Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas; 3Université Libre de Bruxelles, Brussels, Belgium; 4University of Pittsburgh School of Pharmacy, Pittsburgh, Pennslyvania; and 5Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina

Submitted 13 November 2007 ; accepted in final form 12 September 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We hypothesized that A2A adenosine receptor (A2AAR) activation causes vasorelaxation through cytochrome P-450 (CYP) epoxygenases and endothelium-derived hyperpolarizing factors, whereas lack of A2AAR activation promotes vasoconstriction through Cyp4a in the mouse aorta. Adenosine 5'-N-ethylcarboxamide (NECA; 10–6 M), an adenosine analog, caused relaxation in wild-type A2AAR (A2AAR+/+; +33.99 ± 4.70%, P < 0.05) versus contraction in A2AAR knockout (A2AAR–/–; –27.52 ± 4.11%) mouse aortae. An A2AAR-specific antagonist (SCH-58261; 1µM) changed the NECA (10–6 M) relaxation response to contraction (–35.82 ± 4.69%, P < 0.05) in A2AAR+/+ aortae, whereas no effect was noted in A2AAR–/– aortae. Significant contraction was seen in the absence of the endothelium in A2AAR+/+ (–2.58 ± 2.25%) aortae compared with endothelium-intact aortae. An endothelial nitric oxide synthase inhibitor (N-nitro-L-arginine methyl ester; 100 µM) and a cyclooxygenase inhibitor (indomethacin; 10 µM) failed to block NECA-induced relaxation in A2AAR+/+ aortae. A selective inhibitor of CYP epoxygenases (methylsulfonyl-propargyloxyphenylhexanamide; 10 µM) changed NECA-mediated relaxation (–22.74 ± 5.11% at 10–6 M) and CGS-21680-mediated relaxation (–18.54 ± 6.06% at 10–6 M) to contraction in A2AAR+/+ aortae, whereas no response was noted in A2AAR–/– aortae. Furthermore, an epoxyeicosatrienoic acid (EET) antagonist [14,15-epoxyeicosa-5(Z)-enoic acid; 10 µM] was able to block NECA-induced relaxation in A2AAR+/+ aortae, whereas {omega}-hydroxylase inhibitors (10 µM dibromo-dodecenyl-methylsulfimide and 10 µM HET-0016) changed contraction into relaxation in A2AAR–/– aorta. Cyp2c29 protein was upregulated in A2AAR+/+ aortae, whereas Cyp4a was upregulated in A2AAR–/– aortae. Higher levels of dihydroxyeicosatrienoic acids (DHETs; 14,15-DHET, 11,12-DHET, and 8,9-DHET, P < 0.05) were found in A2AAR+/+ versus A2AAR–/– aortae. EET levels were not significantly different between A2AAR+/+ and A2AAR–/– aortae. It is concluded that CYP epoxygenases play an important role in A2AAR-mediated relaxation, and the deletion of the A2AAR leads to contraction through Cyp4a.

epoxyeicosatrienoic acids; dihydroxyeicosatrienoic acids; vasodilation; vasoconstriction; adenosine; cytochrome P-450s

ADENOSINE is an endogenous nucleoside that is involved in the modulation of various physiological activities (45, 58). The physiological effects of adenosine are observed in nearly every tissue and organ (30, 42–48). Adenosine plays an important role in the regulation of vascular tone via the activation of four receptor subtypes: A1, A2A, A2B, and A3 adenosine receptors (ARs). In the coronary artery, vasodilation is primarily caused by the activation of the A2AAR (2, 4, 27, 41, 62). However, it is also well established that adenosine can act as a vasoconstrictor in addition to being a vasodilator. This depends on the interaction with specific subtype and tissue localization. Furthermore, some investigators have suggested that vascular relaxation in response to adenosine may be endothelium independent (55). Others have shown a significant involvement of the endothelium in AR-mediated relaxation (2, 37). However, the precise mechanisms of vasodilation with specific AR subtypes have still not been fully elucidated in relation to the role of the endothelium.

The endothelium produces metabolites of arachidonic acid (AA). This may influence vascular tone through a mediator that elicits hyperpolarization of the underlying smooth muscle, termed as endothelium-derived hyperpolarizing factor (EDHF). Many reports have suggested that EDHFs may be epoxyeicosatrienoic acids (EETs), a class of AA metabolites (23). The AA metabolic process is catalyzed by cytochrome P-450 (CYP) in liver, kidney, lung, and cardiovascular tissues. Many CYP enzyme subfamilies have been identified in the heart, endothelium, and smooth muscle of human blood vessels (24, 31, 65). The link between CYP activity and the generation of EDHF has been investigated extensively. Some EDHFs have demonstrated different pharmacological properties in different vascular beds in different species. However, EDHFs produced by coronary and renal arteries from a number of species, including humans, show characteristics similar to those of CYP-derived metabolites of AA (68). These EETs generated by endothelial CYP epoxygenases bring about the hyperpolarization of vascular smooth muscle cells by activating Ca2+-dependent K+ channels as well as Na+-K+-ATPase (7). 20-Hydroxyeicosatrienoic acid (HETE), a {omega}-hydroxylation product of AA catalyzed by Cyp4a, is one of the essential components of the signal transduction cascade activated by several hormonal systems [e.g., endothelin-1 (53, 54) and angiotensin II (12)] that have central roles in blood pressure regulation (25, 29, 36, 38). Also, 20-HETE (17–19) is believed to be a potent vasoconstrictor in small arteries as well as increasing intracellular Ca2+ and, thus, depolarization of the smooth muscle membrane.

Hydrolysis of the epoxide group of EET regioisomers by soluble epoxide hydrolase leads to the generation of dihydroxyeicosatrienoic acids (DHETs). Like EETs, DHETs are also vasoactive. For example, micromolar concentrations of DHETs and EETs dilate coronary conduit vessels (7, 13, 68). Catella et al. (10) developed a method to account for the conversion of EETs to the corresponding DHETs due to chemical hydrolysis during the isolation and purification procedure. They suggested that conversion during chemical or enzymatic hydrolysis to DHETs may partially account for the apparent biological action of EETs in vitro. Some have suggested that the endothelium may limit the vascular actions of EETs through the rapid uptake, hydrolysis, and release of DHETs into the circulation (66). The effects of DHETs on vascular tone vary. 5,6-EET relaxes the rat tail artery; however, 5,6-DHET is without effect (9). In contrast, 11,12-DHET relaxes the porcine coronary artery and is equipotent to 11,12-EET (67). Canine coronary microvessels are much more sensitive to the relaxing effects of 11,12-EET and 11,12-DHET, where 11,12-DHET is more potent than 11,12-EET (52). It is not known whether DHETs act by the same mechanism as EETs and whether these epoxides are involved in adenosine-induced dilation in the mouse aorta as they are in rat renal arteries (8, 9, 34). Therefore, we hypothesized that A2AAR activation causes endothelium-dependent vasorelaxation through CYP epoxygenase-derived metabolites from AA, whereas lack of A2AARs promotes vasoconstriction through CYP {omega}-hydroxylase in the mouse aorta.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals

A2AAR knockout (A2AAR–/–) mice were obtained from C. Ledent, bred, and maintained with their corresponding wild-type (CD-1; A2AAR+/+) mice at our facility at West Virginia University. The generation and initial characterization of A2AAR+/+ and A2AAR–/– mice have been previously described (32). In brief, to produce mice on a homogenous genetic background, first-generation heterozygotes were bred for 14 generations to mice on a CD-1 (Charles River Laboratories) outbred background, with selection for the mutant A2AAR gene at each generation by PCR. Fourteenth-generation heterozygotes were bred together to generate A2AAR–/– and A2AAR+/+ (1:1, their mate controls) mice. All animals used in a given experiment came from the same breeding generation and were matched for age, gender, and weight. All animal care and experimentation protocols were approved and carried out in accordance with the West Virginia University Institutional Animal Care and Use Committee and were in accordance with the principles and guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Preparation of Mouse Aortic Rings

Male A2AAR–/– and A2AAR+/+ mice (12–14 wk old) were used in this study. Mice were euthanized by deep anesthesia with pentobarbital sodium (100 mg/kg ip). After a thoracotomy, the aorta was gently removed, cleaned of fat and connective tissues, and cut transversely into rings of 3–4 mm in length. Extreme care was taken not to damage the endothelium. In some rings, the endothelium was removed by placing a wire in the lumen and rubbing the aortic ring gently over a wet blotting paper. Rings were mounted vertically between two stainless steel wire hooks. Two rings were suspended in 10-ml organ baths containing modified Krebs-Henseleit buffer. The Krebs-Henseleit buffer contained (in mM) 118 NaCl, 4.8 KCl, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 11 glucose, and 2.5 CaCl2. The pH of the buffer was adjusted and maintained at 7.4 with 95% O2-5% CO2 at 37°C. Aortic rings were equilibrated for 90 min with a resting force of 1 g, with changes of the bathing solution at 15-min intervals according to our previously described protocol (62, 63). At the end of the equilibration period, tissues were contracted with KCl (50 mM) to check the viability of the tissue. Aortic rings were then constricted with phenylephrine (PE; 10–7 M), and changes in tension were monitored continuously with a fixed range precision force transducer (TSD, 125 C, BIOPAC Systems) connected to a differential amplifier (DA 100B, BIOPAC Systems). Data were recorded using the MP100 WSW (BIOPAC Systems) digital-acquision system and analyzed using Acknowledge 3.5.7 software (BIOPAC Systems).

The intactness of the endothelium was tested pharmacologically by ACh (10–7 M)-induced vascular responses of PE (10–7 M)-precontracted aortic rings. Also, we used N-nitro-L-arginine methyl ester (L-NAME; 100 µM), indomethacin (10 µM), and methylsulfonyl-propargyloxyphenylhexanamide (MS-PPOH; 10 µM) to check the involvement of nitric oxide (NO), cyclooxygenase (COX), and CYP epoxygenases in ACh-induced responses. Preparations were then washed several times with Krebs-Hanseleit solution and allowed to equilibrate for 30 min before the experimental protocol began. Contraction and relaxation responses are expressed as percent decreases or increases of PE-induced precontraction. The amount of contraction produced by PE (10–7 M) in each ring from its initial resting tension (1 g) was considered as 100%.

Concentration-Response Curves with adenosine 5'-N-Ethylcarboxamide and CGS-21680 in A2AAR–/– and A2AAR+/+ Mouse Aortae

To determine vascular responses to adenosine 5'-N-ethylcarboxamide (NECA; an adenosine analog) and CGS-21680 (a selective A2AAR analog), aortic rings were precontracted with PE (10–7 M), and concentration-response curves were obtained by the cumulative addition of NECA and CGS-21680 to organ baths. Concentrations of NECA and CGS-21680 in the organ bath were increased in 1-log steps. Separate concentration-response curves were constructed for NECA and CGS-21680. All concentration-response curves were performed in pairs of aortic rings from both A2AAR–/– and A2AAR+/+ mice in a parallel fashion in the same organ bath. SCH-58261 (1 µM, a selective A2AAR antagonist) was added 30 min before contraction of the tissue with PE and was present throughout the experiment. These experiments were performed in parallel fashion using four rings from the same aorta; two rings served as the control, and other rings served as treated (+SCH-58261) rings for both A2AAR–/– and A2AAR+/+ mice. In endothelium-denuded experiments, the endothelium was removed from one half of the rings (E–) and was left intact (E+) in the other half of the rings (from both A2AAR–/– and A2AAR+/+ mouse aortae). These experiments were also performed in parallel fashion using four rings from the same aorta; two rings served as the control (E+), and the other rings served as E– rings in both A2AAR–/– and A2AAR+/+ mice. All the drugs were added 30 min before contraction of the tissue with PE. The total time of incubation was ~90 min with the various drugs.

Effects of L-NAME and Indomethacin on NECA Concentration-Response Curves in A2AAR–/– and A2AAR+/+ Mouse Aortae

To rule out the contribution of prostanoids and NO, indomethacin (10 µM, a COX inhibitor) and L-NAME [100 µM, a NO synthase (NOS) inhibitor] were used, respectively, in PE-contracted aortae. These experiments were conducted as described above.

Effects of MS-PPOH, 14,15-Epoxyeicosa-5(Z)-Enoic Acid, Dibromo-Dodecenyl-Methylsulfimide, and HET-0016 on NECA and CGS-21680 Concentration-Response Curves in A2AAR–/– and A2AAR+/+ Mouse Aortae

MS-PPOH (10 µM, a selective CYP epoxygenase inhibitor), 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE; 10 µM, an EET nonselective antagonist), and dibromo-dodecenyl-methylsulfimide (DDMS; 10 µM)-HET-0016 (10 µM) (selective CYP {omega}-hydroxylase inhibitors) were used in PE-contracted tissues. These experiments were conducted as described above.

Western Blot Analysis

In brief, isolated aortae were incubated with and without MS-PPOH for 90 min at 37°C (incubation time similar to the organ bath) from both A2AAR–/– and A2AAR+/+ mice. In another group, isolated aortae were incubated with NECA (10–6 M) + MS-PPOH-1-ABT, similar to the previous protocol. Individual samples were treated with 1 ml lysis buffer [50 mM Tris·HCl (pH 7.4), 1% Triton X-100, 150 mM NaCl, 1 mM EGTA, 1 mM PMSF, 0.25% sodium deoxycholate, 1 µg/ml aprotinin, 1 µg/ml pepstatin, 1 µg/ml leupeptin, 1 mM Na3VO4, and 1 mM NaF] and homogenized in cold. Samples were transferred to dry ice for 5 min and then thawed in cold. After being thawed, samples were vortexed and centrifuged for 5 min at 12,000 rpm at 4°C. Supernatants were sonicated and stored at –80°C. Protein was measured using a Bio-Rad assay based on the Bradford dye binding procedure with BSA as the standard. The protein mixture was divided into aliquots and stored at –80°C. At the time of analysis, samples were thawed, and ~40 µg total protein/lane was loaded onto a slab gel. Proteins were separated by SDS-PAGE using 10% acrylamide gels (1 mm thick). After electrophoresis, proteins on the gel were transferred to nitrocellulose membranes (Hybond-ECL) by electroelution. Protein transfer was confirmed using prestained molecular weight markers (Bio-Rad). After being blocked with nonfat dry milk, nitrocellulose membranes were incubated with monoclonal and polyclonal antibodies that cross-reacted with the primary antibody for Cyp2c29 (no. 202) made against a peptide (GRGSFPMAEKMIKGC) specific to Cyp2c29 (from D. C. Zeldin) and Cyp4a (polyclonal, Affinity Bio-Reagents). The secondary antibody was horseradish peroxidase-conjugated anti-rabbit IgG. Membranes were developed using ECL (Amersham BioSciences) and exposed to X-ray film for the appropriate times. β-Actin was used as an internal standard.

Measurements of EETs and DHETs by Ultra Performance Liquid Chromtography-Tandem Mass Spectrometry in A2AAR–/– and A2AAR+/+ Mouse Aortae

Preparation of mouse aortic microsomes. Aortae were harvested from A2AAR+/+ and A2AAR–/– mice (n = 5–7); each sample consisted of five aortae. For the preparation of microsomes, pooled tissues were homogenized in 50 mmol/l Tris buffer containing 150 mmol/l KCl, 0.1 mmol/l DTT, 1 mmol/l EDTA, and 20% glycerol (pH 7.4). Microsomal fractions were isolated by differential centrifugation as previously described by Poloyac et al. (56). A 25 µM concentration of AA was selected to produce the maximal formation rate of HETE and EET metabolites at saturating concentrations of the enzyme without the observation of substrate inhibition in the microsomal system.

Analysis of mouse aortic microsomes. Microsomal fractions containing 100 µg total protein and 25 µM AA were incubated in a 1-ml volume of buffer (0.12 M potassium phosphate buffer containing 5 mM magnesium chloride). Reactions were initiated by the addition of 1 mM NADPH. Incubations were carried out at 37°C for 60 min, and the reaction was stopped by placing the tubes on ice. To each sample, 0.75 ng of 20-HETE d6 were added as the internal standard. Microsomal incubations were extracted twice with 3 ml diethyl ether, dried under nitrogen gas, and reconstituted in 80:20 methanol-deionized water. HETE, DHET, and EET metabolites were separated via reverse-phase ultra performance light chromotography (UPLC; Waters, Acquity, Milford, MA) with a Acquity UPLC BEH C18 1.7 µM, 2/1 x 100-mm column. The mobile phase flow rate was 0.5 ml/min and consisted of pure acetonitrile (mobile phase A) and 5 mM ammonium acetate with 10% acetonitrile in double-distilled H2O (mobile phase B) to provide a 35% phase A-to-65% phase B initial ratio, which was increased to a 95% phase A-to-5% phase B ratio linearly over 4.1 min. The 95% phase A-to-5% phase B ratio was maintained for 2 min, followed by a return to the baseline 35% phase A-to-65% phase B ratio for 1 min. Mass spectrometric (MS) analysis of DHETs and EETs was performed using a ThermoFinnigan TSQ Quantum Ultra triple quadruple mass spectrometer (Thermo-Finnigan, San Jose, CA) operated in negative electrospray ionization mode. Analysis was carried out on the TSQ operated in negative electrospray ionization-selected reaction monitoring mode with unit resolutions at both Q1 and Q3 set at 0.70 full width at half maximum. The selected reaction monitoring transitions that were monitored were as follows: 20-HETE mass-to-charge ratio (m/z) 319.3 -> 245.0; 12-HETE m/z 319.3 -> 179.0; 15-HETE and 14,15-EET m/z 319.3 -> 219.0; 11,12-EET m/z 319.3 -> 167.0; 8,9-EET m/z 319.3 -> 127.0; 5,6-EET m/z 319.3 -> 145.0; 14,15-DHET m/z 337.0 -> 207.0; 11,12-DHET m/z 337.0 -> 167; 8.9-DHET m/z 337.0 -> 127.0; 5,6-DHET m/z 337.0 -> 144.9; and 20-HETE-d6 (internal standard) m/z 325.3 -> 251.0. Collision energy was optimized for each transition and ranged from 11 to 25 eV with a total scan time of 0.01 s. Parameters were optimized to obtain the highest [M-H]+ ion abundance and were as follows: capillary temperature 270°C, spray voltage 3,800 kV, and source collision-induced dissociation set at 1 V. Sheath gas, auxiliary gas, and ion sweep gas pressures were set to 60, 50, and 0 psi, respectively. Collision gas pressure was set to 1.2 mTorr. DHET and EET concentrations were quantified from the standard curve as their area divided by the internal standard peak areas.

Chemicals, Drugs, and Antibodies

PE and ACh were dissolved in distilled water. NECA, CGS-21680, SCH-58261, indomethacin, and L-NAME (Sigma Chemicals, St. Louis, MO) were dissolved in 100% DMSO as 10 mM stock solutions, followed by serial dilutions in distilled water. MS-PPOH and 14,15-EEZE were kindly provided by J. R. Falck. DDMS, HET-0016, HETEs, EET, and DHET metabolites were purchased from Cayman Chemical (Ann Arbor, MI) and were dissolved in 100% ethanol. Cyp2c29 (polyclonal, from D. C. Zeldin) and Cyp4a (polyclonal, Affinity Bio-Reagents) antibodies were used for Western blot experiments.

Statistical Analysis

Data are expressed as means ± SE. Comparisons among different groups were analyzed by one-way ANOVA followed by Tukey's multiple-comparison test. Comparisons between two groups were assessed by an unpaired t-test. A P value of <0.05 was considered as significant. Furthermore, densitometry values for Western blots are expressed as means ± SE. Statistical analyses were performed using Graph Pad Prism.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
ACh-Dependent Vascular Responses in A2AAR–/– and A2AAR+/+ Mouse Aortae

ACh (10–7 M)-dependent vasorelaxation was significantly higher in PE-precontracted A2AAR+/+ (+74.09 ± 7.22%, P < 0.05) compared with A2AAR–/– (+50.26 ± 3.93%) aortae. The vascular response showed a significant (P < 0.05) difference between E+ (+74.09 ± 7.22%) and E– (–2.66 ± 1.58%) A2AAR+/+ tissues with ACh. Similarly, ACh-dependent vasodilation was significantly higher in E+ (+52.65 ± 3.18%, P < 0.05) compared with E– (+1.04 ± 1.36%) A2AAR–/– tissues. L-NAME (100 µM) was able to completely block ACh-dependent relaxation in A2AAR+/+ (–7.03 ± 4.15%, P < 0.05) as well as A2AAR–/– (–1.26 ± 2.36%, P < 0.05) mouse aortae compared with their respective controls. Indomethacin (10 µM) did not block ACh-dependent relaxation in A2AAR+/+ (+68.69 ± 6.68%, P > 0.05) or A2AAR–/– (+47.53 ± 4.00%, P > 0.05) aortae compared with their respective controls. MS-PPOH (10 µM) was able to significantly block ACh-dependent relaxation in A2AAR+/+ (+49.66 ± 9.97%, P < 0.05) compared with control aortae, whereas in A2AAR–/– aortae, there was no effect (+54.01 ± 5.30%, P > 0.05) compared with their controls.

Effects of SCH-58261, L-NAME, and Indomethacin on Concentration-Response Curves for NECA in A2AAR–/– and A2AAR+/+ Mouse Aortae

NECA produced a concentration-dependent relaxation in aortae from A2AAR+/+ mice as opposed to contraction in A2AAR–/– mice (Figs. 1 and 2). Concentration-response curves showed a significant difference in relaxation responses to NECA (P < 0.05) in A2AAR+/+ compared with A2AAR–/– aortae. At 10–6 M NECA, the relaxation response was +33.99 ± 4.70% in A2AAR+/+ aortae compared with contraction (–27.52 ± 4.11%, P < 0.05) in A2AAR–/– aortae (Figs. 1 and 2). Furthermore, there was a significant blockade (P < 0.05) of NECA-induced relaxation with SCH-58261 (1 µM), a selective A2AAR antagonist, in A2AAR+/+ aortae compared with nontreated controls (P < 0.05; Fig. 2). At 10–6 M NECA, the relaxation response with SCH-58261 was –35.82 ± 4.69% in A2AAR+/+ aortae compared with their controls (+33.99 ± 4.70%, P < 0.05; Fig. 2). No significant changes in response to NECA were observed with SCH-58261 in A2AAR–/– aortae compared with their controls (Fig. 2).


Figure 1
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  		Fig. 1. Actual tracings of relaxation/contraction responses to ACh, adenosine 5'-N-ethylcarboxamide (NECA), and CGS-21680 in phenylephrine (PE)-preconstricted A2A adenosine receptor (A2AAR) knockout (A2AAR–/–) and wild-type (A2AAR+/+) mouse aortic rings.

 

Figure 2
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  		Fig. 2. Relaxation/contraction responses to NECA and its antagonism by SCH-58261 (1 µM) using A2AAR–/– and A2AAR+/+ mouse aortic rings. Values are means ± SE; n = 6. *P < 0.05 between A2AR+/+ and A2AR–/– mouse aortic rings.

 
A significant difference (P < 0.05) in the vascular response to NECA was observed in the presence (E+) and absence of the endothelium (E–) in A2AAR+/+ mouse aortae. NECA (10–6 M) caused 33.99 ± 4.70% relaxation in the E+ group (the same control as in the other experiments) in contrast to the contraction seen in A2AAR+/+ aortae in the E– group (–2.58 ± 2.25%, P < 0.05). No significant differences were observed in concentration-response curves between the A2AAR+/+ E–, A2AAR–/– E+, and A2AAR–/– E– groups (Fig. 3).


Figure 3
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  		Fig. 3. Effect of NECA-induced vascular responses in the presence (E+) and absence (E–) of the endothelium in A2AAR–/– and A2AAR+/+ mouse aortic rings. The control curve is the same as in Fig. 2. Values are means ± SE; n = 6. *P < 0.05 between E+ and E– of A2AAR+/+ mouse aortic rings.

 
L-NAME (100 µM) did not alter vascular responses in both treated (+25.93 ± 4.04% with 10–6 NECA) and control A2AAR+/+ (+29.99 ± 4.70%, P > 0.05) tissues (Fig. 4). There were no significant differences in concentration-response curves between treated (L-NAME) and control A2AAR–/– aortae (–21.56 ± 5.39 vs. –27.52 ± 4.11 with 10–6 NECA; Fig. 4). Similarly, indomethacin (10 µM) also did not alter concentration-response curves in A2AAR+/+ (30.25 ± 0.61%, P > 0.05, with 10–6 NECA) compared with nontreated (33.99 ± 4.70%) aortae (Fig. 5). Finally, there were no significant differences in concentration-response curves between treated (indomethacin) and control A2AAR–/– tissues (–14.41 ± 6.16 vs. –13.81 ± 9.75 with 10–6 NECA; Fig. 5).


Figure 4
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  		Fig. 4. Effect of N-nitro-L-arginine methyl ester (L-NAME; 100 µM) on NECA-induced vascular responses in A2AAR–/– and A2AAR+/+ mouse aortic rings. The control curve is the same as in Fig. 2. Values are means ± SE; n = 6. *P < 0.05 between treated and control groups.

 

Figure 5
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  		Fig. 5. Effect of indomethacin (10 µM) on NECA-induced vascular responses in A2AAR–/– and A2AAR+/+ mouse aortic rings. The control curve is the same as in Fig. 2. Values are means ± SE; n = 6. *P < 0.05 between treated and control groups.

 
Effects of MS-PPOH on Concentration-Response Curves for NECA in A2AAR–/– and A2AAR+/+ Mouse Aortae

Concentration-response curves showed a significant blockade (P < 0.05) of NECA-induced relaxation with MS-PPOH (10 µM, a highly selective CYP epoxygenase inhibitor) compared with control in A2AAR+/+ aortae. At 10–6 M NECA, MS-PPOH changed the relaxation response into contraction in A2AAR+/+ (–22.74 ± 5.11%) aortae compared with their controls (P < 0.05; Fig. 6). Conversely, NECA concentration responses were unchanged among MS-PPOH-treated A2AAR+/+, MS-PPOH-treated A2AAR–/–, and control A2AAR–/– aortae (Fig. 6).


Figure 6
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  		Fig. 6. Effect of methylsulfonyl-propargyloxyphenylhexanamide (MS-PPOH; 10 µM) on NECA-induced vascular responses in A2AAR–/– and A2AAR+/+ mouse aortic rings. The control curve is the same as in Fig. 2. Values are means ± SE; n = 6. *P < 0.05 between treated and control groups.

 
Effects of MS-PPOH on Concentration-Response Curves for CGS-21680 in A2AAR–/– and A2AAR+/+ Mouse Aortae

CGS-21680 produced a concentration-dependent relaxation in aortae similar to NECA responses from A2AAR+/+ aortae as opposed to insignificant responses in A2AAR–/– aortae (Fig. 7). The CGS-21680-induced concentration response was significantly different (P < 0.05) in A2AAR+/+ compared with A2AAR–/– aortae. At 10–6 M CGS-21680, the relaxation response in A2AAR+/+ (+28.44 ± 4.36%) aortae was changed to contraction in A2AAR–/– (0.19 ± 4.98%, P < 0.05; Fig. 7) aortae. Also, MS-PPOH (10 µM) significantly blocked the response to CGS-21680, which changed from relaxation to contraction in A2AAR+/+ (–18.54 ± 6.06%) aortae compared with their controls (P < 0.05; Fig. 7). Conversely, the CGS-21680-induced relaxation response was unchanged between MS-PPOH-treated A2AAR–/– and control aortae (Fig. 7).


Figure 7
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  		Fig. 7. Effect of MS-PPOH on CGS-21680-induced vascular responses in A2AAR–/– and A2AAR+/+ mouse aortic rings. The control curve is the same as in Fig. 6. Values are means ± SE; n = 6. *P < 0.05 between treated and control groups.

 
Effects of 14,15-EEZE on Concentration-Response Curves for NECA in A2AAR–/– and A2AAR+/+ Mouse Aortae

A significant blockade was found in the concentration-dependent response (P < 0.05) in NECA-induced relaxation with 14,15-EEZE (10 µM, an EET-nonselective antagonist) compared with control in A2AAR+/+ aortae. At 10–6 M NECA, 14,15-EEZE changed the relaxation response into contraction in A2AAR+/+ (–24.38 ± 8.79%) aortae compared with controls (+33.99 ± 4.70%, P < 0.05; Fig. 8). The NECA concentration response did not change among 14,15-EEZE-treated A2AAR+/+, 14,15-EEZE-treated A2AAR–/–, and control A2AAR–/– aortae (P > 0.05; Fig. 8).


Figure 8
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  		Fig. 8. Effect of 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE; 10 µM) on NECA-induced vascular responses in A2AAR–/– and A2AAR+/+ mouse aortic rings. The control curve is the same as in Fig. 2. Values are means ± SE; n = 6. *P < 0.05 between treated and control groups.

 
Effects of DDMS and HET-0016 on Concentration-Response Curves for NECA in A2AAR–/– and A2AAR+/+ Mouse Aortae

DDMS (10 µM), a {omega}-hydroxylase inhibitor, significantly blocked NECA (10–6 M)-induced contraction (–27.52 ± 4.11%) in controls, which was changed to relaxation in A2AAR–/– aortae (+15.86 ± 4.51%, P < 0.05; Fig. 9). No significant changes were observed with NECA between control and DDMS-treated A2AAR+/+ groups (Fig. 9). Similarly, another {omega}-hydroxylase inhibitor (HET-0016, 10 µM) also significantly changed the contraction (–27.52 ± 4.11%) into relaxation in A2AAR–/– aortae (+30.01 ± 5.01%, P < 0.05). No significant changes were observed between control and HET-0016-treated A2AAR+/+ groups with 10–6 M NECA.


Figure 9
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  		Fig. 9. Effect of dibromo-dodecenyl-methylsulfimide (DDMS) on NECA-induced vascular responses in A2AAR–/– and A2AAR+/+ mouse aortic rings. The control curve is the same as in Fig. 2. Values are means ± SE; n = 6. *P < 0.05 between treated and control groups.

 
Effects of MS-PPOH on the Expression of Cyp2c29 in A2AAR–/– and A2AAR+/+ Mouse Aortae

In A2AAR+/+ mouse aortae, the expression of Cyp2c29 (~50 kDa) protein showed a significant decrease (43.24%, P < 0.05) with MS-PPOH compared with control (Fig. 10, lanes 1 and 2). A significant downregulation of Cyp2c29 was observed in A2AAR–/– compared with A2AAR+/+ aortae (Fig. 10, lanes 1 and 3; ~71%). Furthermore, there were no significant differences between MS-PPOH-treated and control A2AAR–/– aortae (Fig. 10, lanes 3 and 4).


Figure 10
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  		Fig. 10. Representative Western blots (top) with densitometric analysis (bottom) in A2AAR+/+ and A2AAR–/– mouse aortae. CYP, cytochrome P-450. Values are means ± SE; n = 3. *P < 0.05 between A2AAR+/+ (control, lane 1 and bar 1) and A2AAR+/+ [MS-PPOH (10 µM), lane 2 and bar 2] groups and between A2AAR–/– (control, lane 3 and bar 3) and A2AAR–/– [MS-PPOH (10 µM), lane 4 and bar 4] groups.

 
Effects of MS-PPOH on the Expression of Cyp4a in A2AAR–/– and A2AAR+/+ Mouse Aortae

Western blot analysis showed a significant increase in the expression of Cyp4a protein in treated (MS-PPOH, ~50%, P < 0.05) compared with control A2AAR+/+ mouse aortae (Fig. 11, lanes 1 and 2). Also, there was a significant increase in the expression of Cyp4a protein in A2AAR–/– compared with A2AAR+/+ mouse aortae, where an upregulation of Cyp4a protein (~45%) was found (Fig. 11, lanes 1 and 3). Furthermore, there were no significant differences between MS-PPOH-treated and control A2AAR–/– aortae (Fig. 11, lanes 3 and 4).


Figure 11
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  		Fig. 11. Representative Western blots (top) with densitometric analysis (bottom) in A2AAR+/+ and A2AAR–/– mouse aortae. Values are means ± SE; n = 3. *P < 0.05 between A2AAR+/+ (control, lane 1 and bar 1) and A2AAR+/+ [MS-PPOH (10 µM), lane 2 and bar 2] groups and between A2AAR–/– (control, lane 3 and bar 3) and A2AAR–/– [MS-PPOH (10 µM), lane 4 and bar 4] groups.

 
Detection of EETs and DHETs by UPLC-MS/MS in A2AAR–/– and A2AAR+/+ Mouse Aortae

Figure 12 shows the levels of AA-derived metabolites, in the form of EETs and DHETs, by the action of CYP epoxygenases (Cyp2c and Cyp2c29). EETs and DHETs were detected in isolated mouse aortic microsomes of both A2AAR+/+ and A2AAR–/– mice. UPLC-MS/MS analysis showed a 1.4-fold (P > 0.05) increase of 14,15-EET (unstable form) in A2AAR+/+ (Fig. 12A) compared with A2AAR–/– mouse aortae, whereas a 2-fold (P < 0.05) increase in 14,15-DHET (stable form) was observed in A2AAR+/+ (1.66 ± 0.25 pmol·mg–1·min–1; Fig. 12B) compared with A2AAR–/– (0.83 ± 0.17 pmol·mg–1·min–1) mouse aortae. Also, a 1.4-fold (P > 0.05) increase in 11,12-EET was detected in A2AAR+/+ (Fig. 12C) compared with A2AAR–/– mouse aortae, whereas an ~2-fold (P < 0.05) increase in 11,12-DHET was observed in A2AAR+/+ (1.39 ± 0.20 pmol·mg–1·min–1; Fig. 12D) compared with A2AAR–/– (0.84 ± 0.12 pmol·mg–1·min–1) mouse aortae. Furthermore, a 1.36-fold increase of 8,9-EET was noted in A2AAR+/+ (7.44 ± 2.55 pmol·mg–1·min–1, P > 0.05; Fig. 12E) compared with A2AAR–/– (5.44 ± 1.20 pmol·mg–1·min–1) mouse aortae, whereas a 2.54-fold increase of 8,9-DHET was observed in A2AAR+/+ (0.89 ± 0.15 pmol·mg–1·min–1, P < 0.05; Fig. 12F) compared with A2AAR–/– (0.35 ± 0.08 pmol·mg–1·min–1) mouse aortae.


Figure 12
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  		Fig. 12. Levels of CYP epoxygenase-derived metabolites in A2AAR+/+ and A2AAR–/– mouse aortae. A: 14,15-epoxyeicosatrienoic acid (EET). B: 14,15-dihydroxyeicosatrienoic acid (DHET). C: 11,12-EET. D: 11,12-DHET. E: 8,9-EET. F: 8,9-DHET. *P < 0.05.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
In this study, with the use of A2AAR knockout mouse aortae, the observed vascular responses to NECA and CGS-21680 were found to be significantly different from wild type. Furthermore, the data showed that NECA and CGS-21680 cause concentration-dependent vasorelaxation in A2AAR+/+ mouse aortae compared with vasoconstriction in A2AAR–/– mouse aortae. Similar vasorelaxation responses with A2AAR activation have been reported in preglomerular microvessels, retinal arterioles, pial arterioles, and mesenteric arteries in rats, in porcine coronary arteries, and in coronary arterioles in patients with heart disease (8, 11, 20, 28, 51, 57, 60). Furthermore, NECA and CGS-21680-induced responses were blocked by SCH-58261 (an A2AAR-selective antagonist) in A2AAR+/+ but not A2AAR–/– mouse aortae. These findings further confirmed that there is a potential role for the A2AAR in vascular relaxation, similar to reports in other vascular beds (11, 60). Recent work from our laboratory (64) has shown a partial NO-dependent increase in coronary flow to both NECA and CGS-21680 in A2AAR+/+ compared with A2AAR–/– mice. Also, they have shown coronary A2BAR upregulation in A2AAR-deficient mice. These data are different from the present findings, where we did not find a NO-dependent response to NECA in aortic relaxation from A2AAR+/+ and A2AAR–/– mice. These data support the variations in vascular responses to adenosine analogs in various tissues in relation to their role in NO-dependent relaxation and AR expression.

Based on the concentration-response curves, CGS-21680 is more potent in relaxing the aorta through A2AARs than NECA. NECA-induced relaxation in A2AAR+/+ mouse aortae may have a biphasic response around 10–7 M, suggesting the involvement of another AR. On the other hand, the CGS-21680-induced vascular response did not have this biphasic characteristic. Since both A1ARs and A3ARs have a higher affinity for NECA than A2AARs, this slight biphasic response in A2AAR+/+ mouse aortae may be due to the presence of A1ARs and A3ARs, whereas CGS-21680, being a highly selective A2AAR agonist, did not show this kind of a response. However, the major difference between NECA and CGS-21680 was observed in A2AAR–/– mouse aortae. NECA induced a larger contraction in A2AAR–/– mouse aortae, whereas CGS-21680 did not elicit any response.

Our findings in this report support the involvement of CYP in NECA- and CGS-21680-induced vascular relaxation in A2AAR+/+ mouse aortae, as investigated by others in preglomerular microvessels of rats (8, 34). The data show that the endothelium-dependent relaxation produced with NECA and CGS-21680 was totally blocked with MS-PPOH, a highly selective CYP epoxygenase inhibitor. The expression Cyp2c29 was downregulated in MS-PPOH-treated mouse aortae compared with controls. It has been reported that at the concentration used, MS-PPOH has no effect on pathways other than the CYP patheway (5, 39, 40). EDHF stimulates hyperpolarization of smooth muscle cells and thus induces vasodilation (16). The relationship between CYP activity and the generation of EDHF has been investigated for many years by others (15, 22, 49, 69). Coronary and renal arteries from humans and rats have shown properties similar to those of CYP-derived metabolites of AA (22). Interestingly, in the present study, the Western blot data showed a significant expression of Cyp2c, including Cyp2c29 protein, in A2AAR+/+ compared with A2AAR–/– mouse aortae. The present findings are in agreement with those reported by Fleming et al. (16) in the porcine coronary artery. The vascular responses seen in our study were similar to those in experiments where CYP epoxygenases played a role in the generation of EET's, leading to vasorelaxation in rat preglomerular microvessels (8, 11).

We also observed that the removal of the endothelium significantly attenuated ACh-dependent vasorelaxation compared with the intact endothelium in A2AAR+/+ mouse aortae. Similarly, NECA-induced relaxation was totally abolished in the E– aorta compared with the E+ aorta of A2AAR+/+ mice. There were no significant differences among E+ A2AAR–/–, E– A2AAR–/–, and E– A2AAR+/+ mouse aortae. Our present data show that ACh- and NECA-induced relaxations are dependent on the endothelium in A2AAR+/+ mouse aortae. Similar findings have been reported previously from this laboratory, where adenosine-induced relaxation in the porcine coronary artery was found to be endothelium dependent (1). Our data also show that L-NAME was able to block ACh-dependent relaxation in A2AAR+/+ mouse aortae. However, NECA-induced relaxation was not significantly affected by L-NAME (Fig. 4), indicating that NO does not contribute to adenosine-induced relaxation in A2AAR+/+ mouse aortae. These responses in our study were similar to those of studies where NO-independent adenosine-induced vascular relaxation in rat preglomerular microvessels was observed (8, 11). Furthermore, ACh- and NECA-dependent responses were not affected by indomethacin in A2AAR+/+ mouse aortae (Fig. 5), suggesting the noninvolvement of COX in this response. In addition to NOS and COX, the vascular endothelium is a site for CYP epoxygenases as well as the site for the generation of EDHF (EETs). We used MS-PPOH, a highly selective CYP epoxygenase blocker, to check whether ACh-, NECA-, and CGS-21680-dependent vascular responses would be altered in A2AAR+/+ mouse aortae. Indeed, MS-PPOH was able to significantly block ACh-dependent relaxation in A2AAR+/+ compared with A2AAR–/– mouse aortae. Similar findings have been observed by Archer et al. (3) with ACh-dependent relaxation, which was blocked by MS-PPOH in human left internal mammary arteries. Another study (61) has also shown that sulphaphenazole, a selective Cy2c9 inhibitor, blunted vasodilation response to ACh in hypertensive patients. Our data and these findings (3, 61) suggest that CYP epoxygenases may also be involved in ACh-induced responses. Furthermore, the adenosine analog (NECA and CGS-216800)-induced relaxation in A2AAR+/+ mouse aortae (Figs. 6 and 7) also suggests that this relaxation is through A2AARs via CYP epoxygenases in the downstream pathway. These results were further confirmed when NECA-induced relaxation was blocked by SCH-58261, a selective A2AAR antagonist, in A2AAR+/+ mouse aortae (Fig. 2). CYP epoxygenases lead to the generation of 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET from AA. We were able to block NECA-induced vasodilation through 14,15-EEZE, a blocker of EET formation, in A2AAR+/+ mouse aortae (Fig. 8). Our data suggest that the endothelium-dependent NECA- and CGS-21680-induced vascular relaxation is mediated by CYP epoxygenases and EETs in A2AAR+/+ mouse aortae.

In the present study, interestingly, we found significant upregulation of Cyp4a in A2AAR–/– compared with A2AAR+/+ mouse aorta (Fig. 11), whereas Cyp2c, including Cyp2c29, were upregulated significantly in A2AAR+/+ compared with A2AAR–/– mouse aortae (Fig. 10). Cyp2c isoforms, including Cyp2c29, are epoxygenase enzymes that produce EETs. In particular, Cyp2c29 generates 14,15-EET from AA (35), leading to vasodilation (15). These findings suggest that the A2AAR is involved in NECA- and CGS-21680-induced endothelium-dependent relaxation through Cyp2c, including Cyp2c29, in A2AAR+/+ mouse aortae. The upregulation of Cyp4a, an {omega}-hydroxylase enzyme, elicits vasoconstriction (33, 50). In our study, the increased expression of Cyp4a in A2AAR–/– compared with A2AAR+/+ mouse aortae may be responsible for the vasoconstriction seen in A2AAR–/– aortae. This conclusion is further supported by the fact that NECA- and CGS-21680-dependent relaxation were changed into contraction. Also, an increase in the expression of Cyp4a was observed when A2AAR+/+ mouse aortae were treated with MS-PPOH. Furthermore, DDMS (Fig. 9) and HET-0016, which are {omega}-hydroxylase inhibitors, were able to block vascular contraction in A2AAR–/– mouse aortae. This suggests that the vascular contraction was induced due to the absence of A2AARs through the Cyp4a enzyme, whereas no significant changes at 10–6 M NECA were observed between treated (DDMS and HET-0016) and nontreated A2AAR+/+ mouse aortae. Hydroxylation of AA would yield 20-HETE formation in the presence of {omega}-hydroxylase enzymes, and the measurement of these metabolites in the present study would have provided additional important data related to its role in vascular constriction in A2AAR–/– mouse aortae.

Our data suggest a link between the activation of A2AARs through the endothelium and upregulation of Cyp2c29, leading to NECA- and CGS-21680-mediated relaxation in A2AAR+/+ mouse aortae. Conversely, in the absence of A2AARs, there is an upregulation of Cyp4a, leading to NECA- and CGS-21680-mediated vasoconstriction in mouse aortae. The present study also showed that treatment with MS-PPOH increases Cyp4a expression in A2AAR+/+ mouse aortae, leading to NECA- and CGS-21680-induced vasoconstriction. Furthermore, we observed an upregulation of A1ARs in A2AAR–/– mouse aortae compared with A2AAR+/+ control and treated (MS-PPOH) tissues (data not shown).

We also measured the levels of 14,15-EET, 11,12-EET, and 8,9-EET and their corresponding diols, such as 14,15-DHET, 11,12-DHET, and 8,9-DHET, in A2AAR+/+ and A2AAR–/– mouse aortae. EETs are endothelium-derived eicosanoids that mediate the vasodilator effects of agonists such as bradykinin and ACh (7, 15, 21, 26, 59). 11,12-DHET is also vasoactive in the porcine coronary artery, producing relaxation similar to that with 11,12-EET (13). In the present study, we found significantly (P < 0.05) higher levels of 14,15-DHET, 11,12-DHET, and 8,9-DHET along with upregulation of CYP2C, leading to NECA- and CGS-21680-dependent relaxation in A2AAR+/+ compared with A2AAR–/– mouse aortae. Usually, cells convert EETs to DHETs, and CYP epoxygenases help the formation of EET, particularly Cyp2c29 and Cyp2c39, to generate 14,15-EET from AA (35), leading to relaxation (15). No significant differences were noted in the levels of 14,15-EET, 11,12-EET, and 8,9-EET in A2AAR+/+ compared with A2AAR–/– mouse aortae (although there was a trend toward a higher level in A2AAR+/+ compared with A2AAR–/– mouse aortae). These findings may indicate that EETs could have been rapidly converted into DHETs, similar to the findings of Catella et al. (10), where intravenous administration of 14,15-[3H]EET in dogs markedly increased DHET levels in plasma. They also suggested that the conversion from EETs to DHETs is important, and DHETs may partially account for the apparent biological actions of EETs in vitro. Van Rollins et al. (66) suggested that the endothelium may limit the vascular actions of EETs through the rapid uptake, hydration, and release of DHETs into the circulation. Another report by Fang et al. (14) suggested that EETs also convert to the corresponding DHETs during incubation and that additional metabolites that do not retain the EET carboxyl groups are formed. They also suggested that most of these products are released into the medium and that some DHETs have less polarity than EETs. In the present study, there are two functional possibilities: first, these EETs could have played a role in the vascular relaxation in the muscle bath by NECA and CGS-21680 in A2AAR+/+ mouse aortae, whereas during the microsomal preparation and incubation with AA, 14,15-EET, 11,12-EET, and 8,9-EET might have been converted to the corresponding 14,15-DHET, 11,12-DHET, and 8,9-DHET. Similar findings of conversion from EETs to DHETs have been reported in pregnancy-induced hypertension in humans (10) as well as EET metabolism in cell cultures (14). The second possibility is that DHETs (14,15-DHET, 11,12-DHET, and 8,9-DHET) may be responsible for the NECA- and CGS-21680-induced vascular relaxation in A2AAR+/+ compared with A2AAR–/– mouse aortae. This is similar to the findings of Weintraub et al. (68), Oltman et al. (52), and Campbell et al. (6), where they have shown the involvement of DHETs in vascular relaxation from coronary microvessels. 11,12-DHET relaxed coronary microvessels in concentrations as low as 10–18 M and was 1,000 times more potent than 11,12-EET (52).

In summary, the present data provide evidence that NECA- and CGS-21680-induced endothelium-dependent relaxation was significantly higher in A2AAR+/+ compared with A2AAR–/– mouse aortae. The data support the relationship between the presence of A2AARs and the upregulation of Cyp2c, including Cyp2c29, leading to endothelium-dependent relaxation with NECA and CGS-21680 in A2AAR+/+ mouse aortae. In contrast, the A2AAR–/– data provide evidence that there may be a link between the absence of A2AARs and the upregulation of Cyp4a leading to vasoconstriction with NECA and CGS-21680 in mouse aortae. Further studies will be necessary to unravel the mechanism(s) by which the A2AAR plays a role in the regulation of vascular tone in the mouse aorta through epoxidation, hyperpolarization, {omega}-hydroxylation, and depolarization in isolated endothelial and smooth muscle cells.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants HL-027339, HL-094447, GM-31278, RO1-NS-052315, S10-RR-023461, and RO1-NS-052315 and by the Intramural Research Program of the National Institute of Environmental Health Sciences.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. J. Mustafa, Dept. of Physiology and Pharmacology, Center for Interdisciplinary Research in Cardiovascular Sciences, Robert C. Byrd Health Science Center, West Virginia Univ., Morgantown, WV 26506 (e-mail: smustafa{at}hsc.wvu.edu)

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
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
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