Adenosine A2A receptor modulates vascular response in soluble epoxide hydrolase-null mice through CYP-epoxygenases and PPARγ

Abstract

The interaction between adenosine and soluble epoxide hydrolase (sEH) in vascular response is not known. Therefore, we hypothesized that lack of sEH in mice enhances adenosine-induced relaxation through A(2A) adenosine receptors (AR) via CYP-epoxygenases and peroxisome proliferator-activated receptor γ (PPARγ). sEH(-/-) showed an increase in A(2A) AR, CYP2J, and PPARγ by 31%, 65%, and 36%, respectively, and a decrease in A(1)AR and PPARα (30% and 27%, respectively) vs. sEH(+/+). 5'-N-ethylcarboxamidoadenosine (NECA, an adenosine receptor agonist), CGS 21680 (A(2A) AR-agonist), and GW 7647 (PPARα-agonist)-induced responses were tested with nitro-l-arginine methyl ester (l-NAME) (NO-inhibitor; 10(-4) M), ZM-241385, SCH-58261 (A(2A) AR-antagonists; 10(-6) M), 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, an epoxyeicosatrienoic acid-antagonist; 10(-5) M), 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA; 10 μM) or trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB, sEH-inhibitors; 10(-5) M), and T0070907 (PPARγ-antagonist; 10(-7) M). In sEH(-/-) mice, ACh response was not different from sEH(+/+) (P > 0.05), and l-NAME blocked ACh-responses in both sEH(-/-) and sEH(+/+) mice (P < 0.05). NECA (10(-6) M)-induced relaxation was higher in sEH(-/-) (+12.94 ± 3.2%) vs. sEH(+/+) mice (-5.35 ± 5.2%); however, it was blocked by ZM-241385 (-22.42 ± 1.9%) and SCH-58261(-30.04 ± 4.2%). CGS-21680 (10(-6) M)-induced relaxation was higher in sEH(-/-) (+37.4 ± 5.4%) vs. sEH(+/+) (+2.14 ± 2.8%). l-NAME (sEH(-/-), +30.28 ± 4.8%, P > 0.05) did not block CGS-21680-induced response, whereas 14,15-EEZE (-7.1 ± 3.7%, P < 0.05) did. Also, AUDA and t-AUCB did not change CGS-21680-induced response in sEH(-/-) (P > 0.05), but reversed in sEH(+/+) (from +2.14 ± 2.8% to +45.33 ± 4.1%, and +63.37 ± 7.2, respectively). PPARα-agonist did not relax as CGS 21680 (-2.48 ± 1.1 vs. +37.4 ± 5.4%) in sEH(-/-), and PPARγ-antagonist blocked (from +37.4 ± 5.4% to +9.40 ± 3.1) CGS 21680-induced relaxation in sEH(-/-). Our data suggest that adenosine-induced relaxation in sEH(-/-) may depend on the upregulation of A(2A) AR, CYP2J, and PPARγ, and the downregulation of A(1) AR and PPARα.

Fig. 1.

Representative Western blots and densitometric analysis for A_2A AR (∼45 kDa; A) and A₁AR (∼37 kDa; B) proteins in aortas of sEH^−/− and sEH^+/+ mice. Values are expressed as means ± SE. *P < 0.05, sEH^+/+ compared with sEH^−/− aortas; n = 6.

Fig. 2.

Representative Western blots and densitometric analysis for CYP2J5 (∼58 kDa; A) and CYP4A (∼50 kDa; B) proteins in aortas of sEH^−/− and sEH^+/+ mice. Values are expressed as means ± SE. *P < 0.05, sEH^+/+ compared with sEH^−/− aortas; n = 6.

Fig. 3.

Representative Western blots and densitometric analysis for PPARγ (∼58 kDa; A) and PPARα (∼52 kDa; B) proteins in aortas of sEH^−/− and sEH^+/+ mice. Values are expressed as means ± SE. *P < 0.05, sEH^+/+ compared with sEH^−/− aortas; n = 6.

Fig. 4.

Effect of l-NAME (100 μM) on ACh-induced vascular response in aortic rings of sEH^+/+ and sEH^−/− mice. Values are expressed as means ± SE. *P < 0.05, sEH^+/+ controls vs. sEH^+/+ mice treated with l-NAME. ^#P < 0.05, sEH^−/− controls vs. ^#sEH^−/− mice treated with l-NAME; n = 8.

Fig. 5.

Effect of ZM 241385 (1 μM; A) and SCH 58261 (1 μM; B) on NECA-induced vascular response in aortic rings of sEH^+/+ and sEH^−/− mice. @P < 0.05 between sEH^+/+ vs. sEH^−/− mice. #P < 0.05, sEH^−/− vs. sEH^−/− mice treated with ZM 241385. *P < 0.05, sEH^+/+ vs. sEH^+/+ mice treated with ZM 241385, n = 8 (A). @P < 0.05, between sEH^+/+ vs. sEH^−/− mice. *P < 0.05, sEH^+/+ vs. sEH^+/+ mice with SCH 58261. #P < 0.05, sEH^−/− vs. sEH^−/− mice with SCH 58261; n = 8 (B). Values are expressed as means ± SE.

Fig. 6.

Effect of l-NAME (100 μM; A) and 14,15-EEZE (10 μM; B) on CGS 21680-induced vascular response in aortic rings of sEH^+/+ and sEH^−/− mice. *P < 0.05, sEH^+/+ vs. sEH^−/− mice. #P < 0.05, sEH^+/+ vs. sEH^−/− treated with l-NAME, and sEH^+/+ treated with l-NAME vs. sEH^−/− treated with l-NAME; n = 8 (A). ^@P < 0.05, between sEH^+/+vs. sEH^−/− mice. ^@P < 0.05, sEH^+/+ mice treated with 14,15-EEZE vs. sEH^−/−; n = 8 (B). #P < 0.05, sEH^−/− vs. sEH^−/− mice treated with 14,15-EEZE. Values are expressed as means ± SE.

Fig. 7.

Effect of AUDA (10 μM) on CGS 21680 induced vascular response in aortic rings of sEH^+/+ and sEH^−/− mice (A). The control curve is the same as in Fig. 6. #P < 0.05, sEH^+/+vs. sEH^−/− mice. *P < 0.05, sEH^+/+ vs. sEH^+/+ mice treated with AUDA. ^@P < 0.05, sEH^+/+ vs. sEH^−/− treated with AUDA; n = 8 (A). Effect of t-AUCB (10 μM) on CGS 21680-induced vascular response in aortic rings of sEH^+/+ and sEH^−/− mice (B). The control curve is the same as in Fig. 6. *P < 0.05, sEH^+/+ vs. sEH^−/− mice. @P < 0.05, sEH^+/+ vs. sEH^+/+ mice treated with t-AUCB. $P < 0.05, sEH^+/+ vs. sEH^−/− mice treated with t-AUCB; n = 8 (B). Values are expressed as means ± SE.

Fig. 8.

GW 7647 (PPARα-agonist)-induced vascular response in aortic rings of sEH^+/+ and sEH^−/− mice. P > 0.05 for GW 7647-sEH^+/+ vs. GW 7647-sEH^−/−; n = 8 (A). CGS 21680-induced vascular response in aortic rings of sEH^+/+ and sEH^−/− mice. ^@P < 0.05, CGS 21680-sEH^+/+ vs. CGS 21680-sEH^−/− mice; n = 8 (B). Effect of PPARγ-antagonist (0.1 μM; B) on CGS 21680-induced vascular response in aortic rings of sEH^+/+ and sEH^−/− mice. ^@P < 0.05, sEH^+/+vs. sEH^−/− mice. ^@P < 0.05, sEH^+/+ treated with PPARγ-antagonist vs. sEH^−/− mice alone. ^@P < 0.05, sEH^−/− treated with PPARγ-antagonist vs. sEH^−/− mice alone; n = 8 (C). Values are expressed as means ± SE.