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

Nayeem MA, Poloyac SM, Falck JR, Zeldin DC, Ledent C, Ponnoth DS, Ansari HR, Mustafa SJ. Role of CYP epoxygenases in A(2A)AR-mediated relaxation using A(2A)AR-null and wild-type mice. Am J Physiol Heart Circ Physiol 295: H2068-H2078, 2008. First published September 19, 2008; doi: 10.1152/ajpheart.01333.2007.-We hypothesized that A(2A) adenosine receptor (A(2A)AR) activation causes vasorelaxation through cytochrome P-450 (CYP) epoxygenases and endothelium-derived hyperpolarizing factors, whereas lack of A(2A)AR 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 A(2A)AR (A(2A)AR(+/+); + 33.99 +/- 4.70%, P < 0.05) versus contraction in A(2A)AR knockout (A(2A)AR(+/+); + 27.52 +/- 4.11%) mouse aortae. An A(2A)AR-specific antagonist (SCH-58261; 1 mu M) changed the NECA (10(-6) M) relaxation response to contraction (-35.82 +/- 4.69%, P < 0.05) in A(2A)AR(+/+) aortae, whereas no effect was noted in A(2A)AR(-/-) aortae. Significant contraction was seen in the absence of the endothelium in A(2A)AR(+/+) (-2.58 +/- 2.25%) aortae compared with endothelium intact aortae. An endothelial nitric oxide synthase inhibitor (N-nitroL-arginine methyl ester; 100 mu M) and a cyclooxygenase inhibitor (indomethacin; 10 mu M) failed to block NECA-induced relaxation in A(2A)AR(-/-) aortae. A selective inhibitor of CYP epoxygenases (methylsulfonyl-propargyloxyphenylhexanamide; 10(-6) M) 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 A(2A)AR(+/+) aortae, whereas no response was noted in A(2A)AR(-/-) aortae. Furthermore, an epoxyeicosatrienoic acid (EET) antagonist [14,15-epoxyeicosa-5(Z)-enoic acid; 10 mu M] was able to block NECAinduced relaxation in A(2A)AR(+/+) aortae, whereas omega-hydroxylase inhibitors (10 mu M dibromo-dodecenyl-methylsulfimide and 10 mu M HET-0016) changed contraction into relaxation in A(2A)AR (-/-) aorta. Cyp2c29 protein was upregulated in A(2A)AR(+/+) aortae, whereas Cyp4a was upregulated in A(2A)AR(-/-) aortae. Higher levels of dihydroxyeicosatrienoic acids (DHETs; 14,15-DHET, 11,12-DHET, and 8,9-DHET, P < 0.05) were found in A(2A)AR(+/+) versus A(2A)AR(-/-) aortae. EET levels were not significantly different between A(2A)AR(+/+) and A(2A)AR(-/-) aortae. It is concluded that CYP epoxygenases play an important role in A(2A)AR-mediated relaxation, and the deletion of the A(2A)AR leads to contraction through Cyp4a.