Role of CYP epoxygenases in A2AAR-mediated relaxation using A2AAR-null and wild-type mice

Abstract

We hypothesized that A2Aadenosine 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; 10−6M), 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 (10−6M) 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 10−6M) and CGS-21680-mediated relaxation (−18.54 ± 6.06% at 10−6M) 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 ω-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.