目的:初步探讨血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)/血管紧张素Ⅱ1型受体(angiotensinⅡtype 1 receptor,AT1R)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—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;AT1R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IPP2A2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IPPP2A2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IPP2A2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AT1R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/AT1R通路降低PP2Ac Tyr307磷酸化水平和IPP2A2表达水平、导致PP2A活性增强有关.特异性AT1R阻断剂CAN预处理可通过增加PP2Ac Tyr307磷酸化水平和IPP2A2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 angiotensin Ⅱ (Ang Ⅱ)/angiotensin Ⅱ type 1 receptor (AT1 R)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 AT1R blocker) group and CAN pretreatment + Ang Ⅱ group.The protein levels of total eNOS,p-eNOS (Ser1177),PP2Ac,IPP2A2 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 IPP2A2 decreased (P ＜ 0.05).Compared with the same concentration of Ang Ⅱ group,CAN pretreatment increased the levels of p-PP2Ac (Tyr307) and IPP2A2 (P ＜ 0.05),but the protein expression of PP2Ac showed no significant difference.CONCLUSION:Ang Ⅱ down-regulates the level of p-eNOS (Ser1177),and decreases the production of NO in human umbilical vein endothelial cells via AT1 R pathway.This effect may be related to the reduction of p-PP2Ac (Tyr307) and protein expression of IPP2A2,which results in the enhancement of PP2A2 activity.Pretreatment with AT1 R blocker CAN increases p-PP2Ac (Tyr307) level and IPP2A2 protein expression,thus reducing the PP2A activity,and ultimately restoring eNOS Ser1177 phosphorylation level and eNOS activity.
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