论文部分内容阅读
内皮源性一氧化氮合酶(eNOS)是一氧化氮(NO)参与的血管稳态调节过程中的关键酶.多种体液因子和机械刺激都可以通过磷酸化修饰调节eNOS的活性,但具体的信号转导通路因刺激物不同而异.最近发现花生四烯酸细胞色素P450(CYP)表氧化酶代谢产物表氧化二十碳三烯(EETs)可以显著上调eNOS的蛋白表达并增强其活性,但其分子机制尚不清楚.通过在4代以内培养的牛主动脉内皮细胞中直接加入外源性EETs和转染CYP表氧化酶基因CYP2C11和CYPF87V,并同时给予实验组不同信号转导抑制剂进行干预,观察其对总的eNOS表达及其在Ser1179和Thr497位点磷酸化水平的影响.结果显示,内外源性EETs均可以显著上调eNOS的蛋白表达并增强及其在Ser1179和Thr497位点的磷酸化水平;PI3K抑制剂LY294002可以阻断EETs对eNOS-Ser1179的磷酸化上调作用,但它对eNOS-Thr(P)497并无影响,而Akt抑制剂却可以抑制eNOS在这两个位点的磷酸化,且这两种抑制剂都可以阻断EETs对eNOS的蛋白表达上调作用.结果提示:(i)EETs对eNOS的活性调节可能与PI3K/Akt所介导的eNOS-Ser1179和Akt所介导的eNOS-Thr497磷酸化水平改变相关;(ii)PI3K/Akt信号通路可能参与了EETs对eNOS的蛋白表达上调过程.
Endothelial nitric oxide synthase (eNOS) is a key enzyme in the steady-state regulation of blood vessels involved in nitric oxide (NO) .A variety of humoral and mechanical stimuli can regulate eNOS activity through phosphorylation modification, but specific Of the signal transduction pathways vary with the stimulus.Recently, the discovery that arachidonic acid cytochrome P450 (CYP) epoxide oxidase metabolite epoxidized eicosapentaenoic (EETs) can significantly upregulate eNOS protein expression and enhance its activity , But its molecular mechanism is not yet clear.Endogenous EETs were directly transfected into bovine aortic endothelial cells cultured within 4 passages and CYP2C11 and CYPF87V were transfected CYP2C11 and CYPF87V and different signal transduction inhibition Were used to observe the effect of eNOS on the expression of eNOS and the phosphorylation of Ser1179 and Thr497 sites.The results showed that both exogenous and exogenous EETs could upregulate the expression of eNOS and enhance its expression at Ser1179 and Thr497 sites PI3K inhibitor LY294002 can block the phosphorylation of eNOS-Ser1179 by EETs, but it has no effect on eNOS-Thr (P) 497, while Akt inhibitor can inhibit eNOS at these two positions point Phosphorylation of both EETs and EETs.The results suggest that: (i) the regulation of eNOS activity by EETs may be related to the activation of eNOS-Ser1179 and Akt mediated by PI3K / Akt Mediated changes in eNOS-Thr497 phosphorylation; (ii) PI3K / Akt signaling pathway may be involved in the up-regulation of eNOS protein expression by EETs.