AngⅡ/AT_1R通路通过激活人脐静脉内皮细胞PP2A导致eNOS Ser1177磷酸化水平下调

Ang Ⅱ/AT_1R pathway activates PP2A to down-regulate p-eNOS(Ser1177) in human umbilical vein endothelial cells

目的:初步探讨血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)/血管紧张素Ⅱ1型受体(angiotensinⅡtype1 receptor,AT_1R)通路是否通过激活人脐静脉内皮细胞蛋白磷酸酶2A(protein phosphatase 2A,PP2A)导致内皮型一氧化氮合酶(eNOS)Ser1177磷酸化水平下调。方法:将人脐静脉内皮细胞随机分为正常对照(control)组、AngⅡ处理组、单纯坎地沙坦(candesartan,CAN;AT_1R特异性阻断剂)组和CAN预处理+AngⅡ组。用Western blot方法检测各组eNOS总蛋白表达、eNOS Ser1177磷酸化水平、PP2Ac蛋白表达、PP2Ac-Tyr307磷酸化水平和PP2A内源性抑制蛋白I_2^(PP2A)表达水平。采用化学比色法检测各组细胞培养基中的NO含量。结果:与control组相比,AngⅡ处理后eNOS Ser1177磷酸化水平及细胞培养基中的NO含量降低(P<0.05);与同一浓度AngⅡ组相比,CAN预处理可增加eNOS Ser1177磷酸化水平及细胞培养基中的NO含量(P<0.05);各组间eNOS蛋白表达差异无统计学显著性。与control组比较,AngⅡ处理后PP2Ac Tyr307磷酸化水平和I_2^(PP2A)表达降低(P<0.05);与同一浓度AngⅡ组相比,CAN预处理可增加PP2Ac Tyr307磷酸化水平和I_2^(PP2A)表达(P<0.05);各组间PP2Ac蛋白表达差异无统计学显著性。结论:AngⅡ可通过AT_1R通路导致人脐静脉内皮细胞eNOS Ser1177磷酸化水平下调,NO合成减少,这一效应可能与AngⅡ/AT_1R通路降低PP2Ac Tyr307磷酸化水平和I_2^(PP2A)表达水平、导致PP2A活性增强有关。特异性AT_1R阻断剂CAN预处理可通过增加PP2Ac Tyr307磷酸化水平和I_2^(PP2A)表达水平而降低PP2A活性,最终上调eNOS Ser1177磷酸化水平,恢复eNOS活性。

AIM： To investigate whether angiotensinⅡ（ AngⅡ）/angiotensin Ⅱ type 1 receptor（AT_1R）pathway down-regulates endothelial nitric oxide synthase（ eNOS） Ser1177 phosphorylation level in human umbilical vein endothelial cells by activating protein phosphatase 2A（PP2A）. METHODS： Human umbilical vein endothelial cells were randomly divided into normal control（ control） group,Ang II group,candesartan（ CAN; specific AT_1R blocker） group and CAN pretreatment ＋ AngⅡ group. The protein levels of total eNOS,p-eNOS（ Ser1177）,PP2 Ac,I_2^（PP2A） and p-PP2Ac（Tyr307） were determined by Western blot. The content of NO in the cell culture medium was detected by chemical colorimetry. RESULTS： Compared with control group,the level of p-eNOS（ Ser1177） and the content of NO decreased（ P 0. 05）. Compared with the same concentration of Ang Ⅱ group,CAN pretreatment increased the level of p-eNOS（Ser1177） and the content of NO（P 0. 05）,but the protein expression of eNOS showed no significant difference. Compared with control group,the levels of p-PP2Ac（ Tyr307） and I_2^（PP2A） decreased（ P 0. 05）. Compared with the same concentration of AngⅡ group,CAN pretreatment increased the levels of p-PP2Ac（ Tyr307） and I_2^（PP2A）（P 0. 05）,but the protein expression of PP2Ac showed no significant difference. CONCLUSION： AngⅡ down-regulates the level of p-eNOS（Ser1177）,and decreases the production of NO in human umbilical vein endothelial cells via AT_1R pathway. This effect may be related to the reduction of p-PP2Ac（ Tyr307） and protein expression of I_2^（PP2A）,which results in the enhancement of PP2A activity. Pretreatment with AT_1R blocker CAN increases p-PP2Ac（ Tyr307） level and I_2^（PP2A） protein expression,thus reducing the PP2A activity,and ultimately restoring eNOS Ser1177 phosphorylation level and eNOS activity.