High-salt diet enhances mouse aortic relaxation through adenosine A _2A receptor via CYP epoxygenases

We hypothesize that A_2A adenosine receptors (A_2A AR) promote aortic relaxation in mice through cytochrome P450 (CYP)-epoxygenases and help to avoid salt sensitivity. Aortas from male mice maintained on a high-salt (HS; 7% NaCl) or normal-salt (NS; 0.45% NaCl) diet for 4-5 wks were used. Concentration-response curves (10^-11-10^-5 M) for 5′-N-ethylcarboxamidoadenosine (NECA; a nonselective adenosine analog) and CGS 21680 (A_2A AR agonist) were obtained with different antagonists including ZM 241385 (A_2A AR antagonist; 10^-6 M), SCH 58261 (A_2A AR antagonist; 10^-6 M), N^ω-nitro-L- arginine methyl ester (L-NAME; endothelial nitric oxide synthase inhibitor; 10^-4 M) and inhibitors including methylsulfonyl- propargyloxyphenylhexanamide (MS-PPOH; CYP epoxygenases inhibitor; 10 ^-5M), 14,15-epoxyeicosa-5(z)-enoic acid (14,15-EEZE; EET antagonist; 10^-5M), dibromo-dodecenyl-methylsulfimide (DDMS; CYP4A inhibitor; 10^-5M), and HET0016 (20-HETE inhibitor; 10^-5M). At 10 ^-7 M of NECA, significant relaxation in HS (+22.58 ± 3.12%) was observed compared with contraction in NS (-10.62 ± 6.27%, P < 0.05). ZM 241385 changed the NECA response to contraction (P < 0.05) in HS. At 10^-7 M of CGS 21680, significant relaxation in HS (+32.04 ± 3.08%) was observed compared with NS (+10.45 ± 1.34%, P < 0.05). SCH 58261, L-NAME, MS-PPOH, and 14,15-EEZE changed the CGS 21680-induced relaxation to contraction (P < 0.05) in HS. Interestingly, DDMS and HET0016 changed CGS 21680 response to relaxation (P < 0.05) in NS; however, there was no significant difference found between DDMS, HET0016-treated HS and NS vs. nontreated HS group (P > 0.05). CYP2C29 protein was 55% and 74% upregulated in HS vs. NS (P < 0.05) mice aorta and kidney, respectively. CYP4A protein was 30.30% and 35.70% upregulated in NS vs. HS (P < 0.05) mice aorta and kidneys, respectively. A₁ AR was downregulated, whereas A _2A AR was upregulated in HS compared with NS. These data suggest that HS may activate CYP2C29 via A_2A AR, causing relaxation, whereas NS may contribute to the upregulation of CYP4A causing contraction.