【摘要】 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。更多还原

【Abstract】 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 cells(HUVECs).Methods Cultured HUVECs,the same generation of cells 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)Cav-1 short hairpin RNA(Cav-1 ShRNA)+spermine+Ca2+ group;(5)vehicle+spermine+Ca2+ group;(6)different concentrations(1.5,2.0,2.5 mg/L) filipin groups.Western blotting experiments were performed to detect protein expression of Cav-1,eNOS,phosphorated eNOS(p-eNOS) and expressions of Cav-1 and eNOS membrane proteins.The interaction and co-localization between eNOS and Cav-1 were determined using co-immunoprecipitates and immunofluorescence analysis,respectively.ResultsDifferent 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 p-eNOS was also completely blocked after acute caveolae disruption with filipin(1.5 mg/L) or transfected with Cav-1 ShRNA(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 Cav-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 Cav-1 ShRNA group was attenuated(P<0.05).ConclusionsCav-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.更多还原