Ephx2-gene deletion affects acetylcholine-induced relaxation in angiotensin-II infused mice: role of nitric oxide and CYP-epoxygenases

Abstract

Previously, we showed that adenosine A 2A receptor-induces relaxation independent of NO in soluble epoxide hydrolase-null mice (Am J Physiol Regul Integr Comp Physiol 304: R23-R32, 2013). Currently, we hypothesize that Ephx2-gene deletion affects acetylcholine (Ach)-induced relaxation which is independent of A 2A AR but dependent on NO and CYP-epoxygenases. Ephx2 −/− aortas showed a lack of sEH (97.1%, p<0.05) but an increase in microsomal epoxide hydrolase (mEH, 37%, p<0.05) proteins compared to C57Bl/6 mice, and no change in CYP2C29 and CYP2J protein (p>0.05). Ach-induced response was tested with nitro-L-arginine methyl ester (L-NAME) NO-inhibitor; 10 −4 M, N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide (MS-PPOH) (CYP-epoxygenase inhibitor; 10 −5 M), 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, an epoxyeicosatrienoic acid-antagonist; 10 −5 M), SCH-58261 (A 2A AR-antagonist; 10 −6 M) and angiotensin-II (Ang-II, 10 −6 M). In Ephx2 −/− mice, Ach-induced relaxation was not different from C57Bl/6 mice except at 10 −5 M (92.75 ± 2.41 vs. 76.12 ± 3.34, P<0.05). However, Achinduced relaxation was blocked by L-NAME (Ephx2 −/− : 23.74 ± 3.76% and C57Bl/6: 11.61 ± 2.82%), MS-PPOH (Ephx2 −/− : 48.16 ± 6.53% and C57Bl/6: 52.27 ± 7.47%) and 14,15-EEZE (Ephx2 −/− : 44.29 ± 8.33% and C57Bl/6: 39.27 ± 7.47%) vs. non-treated (P<0.05). But, it did not block by SCH-58261 (Ephx2 −/− : 68.75 ± 11.41% and C57Bl/6: 66.26 ± 9.43%, P >0.05) vs. nontreated (P>0.05). Interestingly, Ang-II attenuates less relaxation in Ehx2 −/− vs. C57Bl/6 mice (58.80 ± 7.81% vs. 45.92 ± 7.76, P <0.05). Our data suggests that Ach-induced relaxation in

Adenosine A2A receptor and vascular response: role of soluble epoxide hydrolase, adenosine A1 receptor and angiotensin-II

“…Mice were sacked with pentobarbital sodium (100 mg/kg ip) and extracted their aortas to study protein expression as per our previously published protocol [2,3,12,14,30,[38][39][40][41]. In brief, aortas extracted from both A 2A AR −/− and C57Bl/6 mice were treated with 1 ml of lysis buffer for protein extraction [14].…”

“…In brief, aortas extracted from both A 2A AR −/− and C57Bl/6 mice were treated with 1 ml of lysis buffer for protein extraction [14]. Gel electrophoresis and Western blot analysis were done according to the protocol described by us [2,3,12,14,30,38]. Following the blocking with nonfat dry milk, the nitrocellulose membranes were incubated with monoclonal and polyclonal primary antibodies raised against for A 1 AR, sEH, CYP4A, CYP2C and β-actin [14].…”

“…A 2A AR −/− and C57Bl/6 mice were euthanized with pentobarbital sodium (100 mg/kg intraperitoneal) [14,30,38]. The mouse aorta removed after thoracotomy, the removed and cleaned aorta cut into rings of 3-4 mm in length [14,30,38]. The rings were hung between the two wire hooks [14,30,38].…”

“…The mouse aorta removed after thoracotomy, the removed and cleaned aorta cut into rings of 3-4 mm in length [14,30,38]. The rings were hung between the two wire hooks [14,30,38]. Two rings were suspended in organ baths containing 10 ml of modified Krebs-Henseleit buffer [2,3,14,30,38].…”

“…Epoxyeicosatrienoic acids (EETs) act as EDHFs, which cause hyperpolarization [21][22][23][24][25], and the other endothelial factor, soluble epoxide hydrolase (sEH), an enzyme which metabolize or convert EETs into Dihydroxyeicosatrienoic acids (DHETs, diols), which loses the beneficial effects of EETs, like vasodilation, cardio-protection and anti-inflammatory properties [12,26]. Whereas, sEH inhibition or deletion could increase EETs activity [2][3][4][27][28][29][30]. EETs generated by the action of CYP-epoxygenases on arachidonic acid and bring hyperpolarization of vascular smooth muscle cells by activating Ca 2+ -dependent K + channels as well as Na + -K + -ATPase [21].…”