内外源性EETs对血管内皮一氧化氮合酶的表达及其在Thr-495位磷酸化的作用

The effects of exogenous and endogenous epoxyeicosatrienoic acids on the expression of endothelial nitric oxide synthase and its phosphorylation at Thr-495

目的 研究细胞色素P4 5 0表氧化酶基因转染和直接加入EETs对内皮细胞eNOS表达及其在Thr 4 95位磷酸化的影响。方法 在原代培养的牛主动脉内皮细胞中 ,分别加入生理浓度的 8,9 EET(1 0 0nmol·L-1 )、1 1 ,1 2 EET(1 0 0nmol·L-1 )、1 4 ,1 5 EET(1 0 0nmol·L-1 )孵育 4h ,或直接用重组腺相关病毒介导的花生四烯酸表氧化酶转染牛主动脉内皮细胞2wk ,以产生内源性EETs ,用Westernblot法检测总eNOS蛋白的表达及eNOSThr 4 95磷酸化的水平 ;此外 ,从大鼠尾静脉注射真核表达质粒pCB6、pCB6 2C1 1OR、pCB6 2J2和pCB6 F87V ,2wk后检测大鼠主动脉总eNOS表达及eNOSThr 4 95磷酸化的水平。结果 与空白和溶媒组比较 ,外源性EETs明显促进内皮细胞总eNOS表达 ,增加eNOSThr 4 95的磷酸化 ,而CYP4 5 0抑制剂 (1 7 ODYA)则可明显降低eNOS表达和eNOSThr 4 95的磷酸化水平 ;与相应的对照组比较 ,体内和体外转染不同的表氧化酶基因均能明显上调内皮细胞eNOS的表达 ,增加eNOSThr 4 95的磷酸化水平。结论 EETs和CYP表氧化酶不仅能明显促进血管内皮细胞总eNOS蛋白的表达 ,而且还通过其翻译后修饰来增加其Thr 4

AIM The present study investigated the effect of exogenous and endogenous epoxyeicosatrienoic acids (EETs) on the expression of endothelial nitric oxide synthase(eNOS) and its phosphorylation at Thr 495 in primarily cultured bovine aortic endothelium cells(BAEC). METHODS BAECs were incubated with exogenous 8,9 EET, 11,12 EET and 14,15 EET(100 nmol·L -1 )for 4 h. Furthermore, cytochrome P450 epooxygenase (CYP450) genes, CYP2C11, CYP2J2 and CYPBM 3 F87V were transfected into cultured BAECs and Sprague Dawley (SD) rats using recombinant adeno associated viral vetor (rAAV) and plasmid vector pCB6, respectively, to produce endogenous EETs. The levels of total eNOS protein synthesis and Thr 495 phospho eNOS were determined by western blots. RESULTS Compared with control and vehicle group, three exogenous EETs significantly up regulated the total eNOS protein expression levels and increased phosphorylation of Thr 495, while CYP450 inhibitor, 17 ODYA, can inhibit eNOS protein expression and decreased phosphorylation of Thr 495 eNOS. Compared with corresponding controls, both in vivo and in vitro transfections of three epoxygenase genes all significantly increased the eNOS protein expression and increased phosphorylation of Thr 495 eNOS. CONCLUSION These results indicate that EETs and epoxygenases not only up regulate eNOS protein expression but also increase phosphorylation of eNOS at Thr 495 site, which may affect eNOS function.