Role of ω-hydroxylase in adenosine-mediated aortic response through MAP kinase using A2A-receptor knockout mice

Abstract

Previously, we have shown that A2A adenosine receptor (A2AAR) knockout mice (KO) have increased contraction to adenosine. The signaling mechanism(s) for A2AAR is still not fully understood. In this study, we hypothesize that, in the absence of A2AAR, ω-hydroxylase (Cyp4a) induces vasoconstriction through mitogen-activated protein kinase (MAPK) via upregulation of adenosine A1 receptor (A1AR) and protein kinase C (PKC). Organ bath and Western blot experiments were done using isolated aorta from A2AKO and corresponding wild-type (WT) mice. Isolated aortic rings from WT and A2AKO mice were precontracted with submaximal dose of phenylephrine (10−6 M), and concentration responses for selective A1AR, A2AAR agonists, angiotensin II and cytochrome P-450-epoxygenase, 20-hydroxyeicosatrienoic acid (20-HETE) PKC, PKC-α, and ERK1/2 inhibitors were obtained. 2- p-(2-Carboxyethyl)-phenethylamino-5′- N-ethylcarboxamidoadenosine hydrochloride (CGS-21680, A2AAR agonist) induced concentration-dependent relaxation in WT, which was blocked by methylsulfonyl-propargyloxyphenylhexanamide (cytochrome P-450-epoxygenase inhibitor; 10−5 M) and also with removal of endothelium. A1 agonist, 2-chloro- N6-cyclopentyladenosine (CCPA) produced higher contraction in A2AKO aorta than WT (49.2 ± 8.5 vs. 27 ± 5.9% at 10−6 M, P < 0.05). 20-HETE produced higher contraction in A2AKO than WT (50.6 ± 8.8 vs. 21.1 ± 3.3% at 10−7 M, P < 0.05). Contraction to CCPA in WT and A2AKO aorta was inhibited by PD-98059 (p42/p44 MAPK inhibitor; 10−6 M), chelerythrine chloride (nonselective PKC blocker; 10−6 M), Gö-6976 (selective PKC-α inhibitor; 10−7 M), and HET0016 (20-HETE inhibitor; 10−5 M). Also, contraction to 20-HETE in WT and A2AKO aorta was inhibited by PD-98059 and Gö-6976. Western blot analysis indicated the upregulation of A1AR, Cyp4a, PKC-α, and phosphorylated-ERK1/2 in A2AKO compared with WT ( P < 0.05), while expression of Cyp2c29 was significantly higher in WT. CCPA (10−6 M) increased the protein expression of PKC-α and phosphorylated-ERK1/2, while HET0016 significantly reduced the CCPA-induced increase in expression of these proteins. These data suggest that, in the absence of A2AAR, Cyp4a induces vasoconstriction through MAPK via upregulation of A1AR and PKC-α.