title: Role of ω-hydroxylase in adenosine-mediated aortic response through MAP kinase using A2A-receptor knockout mice.
abstract: Previously, we have shown that A(2A) adenosine receptor (A(2A)AR) knockout mice (KO) have increased contraction to adenosine. The signaling mechanism(s) for A(2A)AR is still not fully understood. In this study, we hypothesize that, in the absence of A(2A)AR, ω-hydroxylase (Cyp4a) induces vasoconstriction through mitogen-activated protein kinase (MAPK) via upregulation of adenosine A(1) receptor (A(1)AR) and protein kinase C (PKC). Organ bath and Western blot experiments were done using isolated aorta from A(2A)KO and corresponding wild-type (WT) mice. Isolated aortic rings from WT and A(2A)KO mice were precontracted with submaximal dose of phenylephrine (10(-6) M), and concentration responses for selective A(1)AR, A(2A)AR 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, A(2A)AR 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. A(1) agonist, 2-chloro-N(6)-cyclopentyladenosine (CCPA) produced higher contraction in A(2A)KO aorta than WT (49.2 ± 8.5 vs. 27 ± 5.9% at 10(-6) M, P < 0.05). 20-HETE produced higher contraction in A(2A)KO than WT (50.6 ± 8.8 vs. 21.1 ± 3.3% at 10(-7) M, P < 0.05). Contraction to CCPA in WT and A(2A)KO 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 A(2A)KO aorta was inhibited by PD-98059 and Gö-6976. Western blot analysis indicated the upregulation of A(1)AR, Cyp4a, PKC-α, and phosphorylated-ERK1/2 in A(2A)KO 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 A(2A)AR, Cyp4a induces vasoconstriction through MAPK via upregulation of A(1)AR and PKC-α.
authors.affiliation: Dept. of Physiology and Pharmacology, School of Medicine, West Virginia Univ., Morgantown, WV 26506, USA.
authors.forename: Dovenia S
authors.initials: DS
authors.lastname: Ponnoth
authors.forename: Mohammed A
authors.initials: MA
authors.lastname: Nayeem
authors.forename: Swati S
authors.initials: SS
authors.lastname: Kunduri
authors.forename: Stephen L
authors.initials: SL
authors.lastname: Tilley
authors.forename: Darryl C
authors.initials: DC
authors.lastname: Zeldin
authors.forename: Catherine
authors.initials: C
authors.lastname: Ledent
authors.forename: S Jamal
authors.initials: SJ
authors.lastname: Mustafa
mesh_terms: D000241:Adenosine / Q000494:pharmacology*; D058907:Adenosine A1 Receptor Agonists / Q000494:pharmacology; D058908:Adenosine A2 Receptor Agonists / Q000494:pharmacology; D058646:Adrenergic alpha-1 Receptor Agonists / Q000494:pharmacology; D000818:Animals; D001011:Aorta / Q000187:drug effects* / Q000502:physiology; D000090762:Cytochrome P-450 CYP2J2; D042926:Cytochrome P-450 CYP4A / Q000235:genetics / Q000378:metabolism*; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System; D005260:Female; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology; D011493:Protein Kinase C / Q000378:metabolism / Q000502:physiology; D043682:Receptor, Adenosine A1 / Q000378:metabolism / Q000502:physiology*; D043705:Receptor, Adenosine A2A / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015854:Up-Regulation; D014665:Vasodilator Agents / Q000494:pharmacology*
chemical_list: D058907:Adenosine A1 Receptor Agonists; D058908:Adenosine A2 Receptor Agonists; D058646:Adrenergic alpha-1 Receptor Agonists; D065607:Cytochrome P-450 Enzyme Inhibitors; D043682:Receptor, Adenosine A1; D043705:Receptor, Adenosine A2A; D014665:Vasodilator Agents; D003577:Cytochrome P-450 Enzyme System; D000090762:Cytochrome P-450 CYP2J2; D042926:Cytochrome P-450 CYP4A; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D000241:Adenosine