CYP-epoxygenases contribute to A2A receptor-mediated aortic relaxation via sarcolemmal KATP channels

Previously, we have shown that A2A adenosine receptor (A2AAR) mediates aortic relaxation via cytochrome P-450 (CYP)-epoxygenases. However, the signaling mechanism is not understood properly. We hypothesized that ATP-sensitive K+ (KATP) channels play an important role in A2AAR-mediated relaxation. Organ bath and Western blot experiments were done using isolated aorta from A2AKO and corresponding wild-type (WT) mice. Aortic rings from WT and A2A knockout (KO) mice were precontracted with submaximal dose of phenylephrine (PE, 10-6 M), and concentration-response curves for pinacidil, cromakalim (nonselective KATP openers), and diazoxide (mitochondrial KATP opener) were obtained. Diazoxide did not have any relaxation effect on PE-precontracted tissues, whereas relaxation to pinacidil (48.09 ± 5.23% in WT vs. 25.41 ± 2.73% in A2AKO; P < 0.05) and cromakalim (51.19 ± 2.05% in WT vs. 38.50 ± 2.26% in A2AKO; P < 0.05) was higher in WT than A2AKO aorta. This suggested the involvement of sarcolemmal rather than mitochondrial KATP channels. Endothelium removal, treatment with SCH 58651 (A2AAR antagonist; 10-6 M), NG-nitro-L-arginine methyl ester (L-NAME, nitric oxide synthase inhibitor) and methylsulfonyl-propargyloxyphenylhexanamide (MS-PPOH, CYP-epoxygenases inhibitor; 10-5 M) significantly reduced pinacidil-induced relaxation in WT compared with controls, whereas these treatments did not have any effect in A2AKO aorta. Glibenclamide (KATP channel inhibitor, 10-5 M) blocked 2-p-(2-carboxyethyl)phenethylamino-5′N-ethylcarboxamido adenosine hydrochloride (CGS 21680, A2AAR agonist)-induced relaxation in WT and changed 5′-N-ethylcarboxamide (NECA) (nonselective adenosine analog)-induced response to higher contraction in WT and A2AKO. 5-Hydroxydecanoate (5-HD, mitochondrial KATP channel inhibitor, 10-4 M) had no effect on CGS 21680-mediated response in WT aorta. Our data suggest that A2AAR-mediated vasorelaxation occurs through opening of sarcolemmal KATP channels via CYP-epoxygenases and possibly, nitric oxide, contributing to pinacidil-induced responses. © 2012 the American Physiological Society.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Role of ω-hydroxylase in adenosine-mediated aortic response through MAP kinase using a 2A-receptor knockout mice

Previously, we have shown that A 2A adenosine receptor (A 2AAR) knockout mice (KO) have increased contraction to adenosine. The signaling mechanism(s) for A 2AAR is still not fully understood. In this study, we hypothesize that, in the absence of A 2AAR, ω-hydroxylase (Cyp4a) induces vasoconstriction through mitogen-activated protein kinase (MAPK) via upregulation of adenosine A1 receptor (A 1AR) and protein kinase C (PKC). Organ bath and Western blot experiments were done using isolated aorta from A 2AKO 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 A 1AR, A 2AAR 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 2AAR 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 2AKO 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 2AKO 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 2AKO 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 2AKO aorta was inhibited by PD-98059 and Gö-6976. Western blot analysis indicated the upregulation of A 1AR, Cyp4a, PKC-α, and phosphorylated-ERK1/2 in A 2AKO 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 2AAR, Cyp4a induces vasoconstriction through MAPK via upregulation of A 1AR and PKC-α. © 2012 the American Physiological Society.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Limonene-induced activation of A2A adenosine receptors reduces airway inflammation and reactivity in a mouse model of asthma

Animal models of asthma have shown that limonene, a naturally occurring terpene in citrus fruits, can reduce inflammation and airway reactivity. However, the mechanism of these effects is unknown. We first performed computational and molecular docking analyses that showed limonene could bind to both A2A and A2B receptors. The pharmacological studies were carried out with A2A adenosine receptor knock-out (A2AKO) and wild-type (WT) mice using ovalbumin (OVA) to generate the asthma phenotype. We investigated the effects of limonene on lung inflammation and airway responsiveness to methacholine (MCh) and NECA (nonselective adenosine analog) by administering limonene as an inhalation prior to OVA aerosol challenges in one group of allergic mice for both WT and KO. In whole-body plethysmography studies, we observed that airway responsiveness to MCh in WT SEN group was significantly lowered upon limonene treatment but no effect was observed in A2AKO. Limonene also attenuated NECA-induced airway responsiveness in WT allergic mice with no effect being observed in A2AKO groups. Differential BAL analysis showed that limonene reduced levels of eosinophils in allergic WT mice but not in A2AKO. However, limonene reduced neutrophils in sensitized A2AKO mice, suggesting that it may activate A2B receptors as well. These data indicate that limonene-induced reduction in airway inflammation and airway reactivity occurs mainly via activation of A2AAR but A2B receptors may also play a supporting role.SCOPUS: ar.jinfo:eu-repo/semantics/publishe