小凹蛋白1上调人脐静脉内皮细胞钙敏感受体介导一氧化氮合酶激活的机制

Mechanisms of Caveolin-1 Up-regulating the Extracellular Ca2+-Sensing Receptor Mediated Endothelial Nitric Oxide Synthetase Activation in Human Umbilical Vein Endothelial Cells

目的探讨小凹蛋白1(Cav-1)上调人脐静脉内皮细胞钙敏感受体介导内皮型一氧化氮合酶(eNOS)激活的作用机制。方法培养人脐静脉内皮细胞,取同代细胞随机分为:(1)对照组;(2)钙敏感受体激动剂精胺(2.0 mmol/L)+Ca2+组;(3)Caveolae结构破坏剂(Filipin,1.5 mg/L)+精胺+Ca2+组;(4)Cav-1 ShRNA+精胺+Ca2+组;(5)空质粒+精胺+Ca2+组;(6)Filipin不同浓度(1.5、2.0、2.5 mg/L)组。免疫印迹检测人脐静脉内皮细胞中eNOS和磷酸化eNOS(p-eNOS)、Cav-1以及eNOS膜蛋白表达;免疫荧光和免疫共沉淀技术检测Cav-1和eNOS表达、共定位以及相互作用。结果不同浓度Filipin不影响eNOS和p-eNOS蛋白表达(P>0.05);精胺作用下细胞内p-eNOS表达增加(P<0.05),此作用可被Filipin(1.5 mg/L)或Cav-1基因沉默完全阻断(P<0.05);Fili-pin(1.5 mg/L)或Cav-1干扰处理后,eNOS的膜蛋白表达减少(P<0.05),eNOS的蛋白表达无变化(P>0.05);免疫荧光双标显示Filipin(1.5 mg/L)或Cav-1基因沉默后eNOS在小凹定位减少,核周边局部区域聚集增多。与对照组和精胺+Ca2+组比较,Cav-1 ShRNA处理组Cav-1和eNOS相互作用减弱(P<0.05),其他处理组间的相互作用无统计学意义(P>0.05)。结论人脐静脉内皮细胞中Cav-1上调钙敏感受体介导eNOS激活作用机制可能与Cav-1影响eNOS质膜定位及抑制eNOS向细胞器转位有关。

Aim To study the mechanisms of caveolin-1 （Cav-1） up-regulating the extracellular Ca2＋-sensing receptor （CaR）-induced endothelial nitric oxide synthetase （eNOS） activation in human umbilical vein endothelial ceils （HUVECs）. Methods Cultured HUVECs, the same generation of ceils were randomly divided into ： （ 1 ） control group ; （2） CaR agonist （ spermine, 2.0 mmol/L） ＋ Ca2 ＋ group ; （ 3 ） caveolae structural damage （ filipin, 1.5 mg/L） ＋ spermine ＋ Ca2＋ group ; （4） Car-1 short hairpin RNA （ Car-1 ShRNA） ＋ spermine ＋ Ca2 ＋ group ; （ 5 ） vehicle ＋ spermine ＋ Ca2 ＋ group; （6） different concentrations （ 1.5, 2.0, 2. 5 mg/L） fiiipin groups. Western blotting experiments were performed to detect protein expression of Cav-1, eNOS, phosphorated eNOS （p-eNOS） and expressions of Cav-I and eNOS membrane proteins. The interaction and co-localization between eNOS and Cav-1 were determined using eo-immunoprecipitates and immunofiuorescence analysis, respectively. Results Different concentrations filipin did not influence the expression of eNOS and p-eNOS protein in HUVECs. In the presence of Ca2＋ , the CaR agonist spermine at concentration of 2. 0 mmol/L resulted in an increase in the p-eNOS in HUVECs （P 〈 0. 05）, the effect of spermine on the increase of peNOS was also completely blocked after acute caveolae disruption with filipin （ 1.5 mg/L） or transfected with Car-1 ShR- NA （ P 〈 0. 05 ）. The expression of the eNOS membrane protein was decreased in HUVECs after cells were treated by filipin （ 1.5 mg/L） or transfected with Cav-1 ShRNA. Simultaneously, total protein level of eNOS was unaffected. Immunocytochemical results demonstrated that filipin （ 1.5 mg/L） or transfected with Car-1 ShRNA decreased eNOS localization in caveolae, increased in the local area surrounding the nucleus. Compared with control group and spermine ＋ Ca2＋ group, the interaction of eNOS and Cav-1 in Car-1 ShRNA group was attenuated （ P 〈 0. 05 ）. Conclusions Cav-1 might promote CaR-induced eNOS activation. The mechanisms are involved in the effect of Cav-1 on eNOS localization at the plasma membrane and the inhibition of eNOS translocation to the organelles.