【摘要】 目的:探讨小凹蛋白-1(Cav-1)在人脐静脉内皮细胞(HUVECs)钙敏感受体(CaR)介导NO生成中的作用和机制。方法:利用转染技术将构建的Cav-1干扰质粒(Cav-1 shRNA)转染入HUVECs,随机分为:(1)对照组;(2)CaR激动剂(精胺)组;(3)精胺+Ca2+组;(4)CaR负性变构调节剂(Calhex231)+精胺组;(5)Calhex231+精胺+Ca2+组;(6)非律平(filipin)+精胺组;(7)filipin+精胺+Ca2+组;(8)空质粒(vehicle)+精胺+Ca2+组;(9)Cav-1 shRNA+精胺组;(10)Cav-1 shRNA+精胺+Ca2+组。Western blotting检测Cav-1 shRNA转染后HUVECs中CaR和Cav-1蛋白表达,通过NO荧光探针DAF-FM DA负载HUVECs检测细胞内NO的生成;并在提供充足底物的条件下检测细胞内皮型一氧化氮合酶(eNOS)活性。结果:Cav-1干扰后,Cav-1蛋白表达降低,同时CaR的膜蛋白表达降低(P<0.05),CaR的浆蛋白表达无变化(P>0.05)。无论细胞外为零钙液或含钙液时,精胺(2 mmol/L)刺激CaR时均引起eNOS活性和NO含量增加(P<0.05),其中细胞外液为含钙液时,eNOS活性和NO含量增加较细胞外为零钙液时更明显(P<0.05),Calhex231(1μmol/L)可阻断(细胞外为零钙液)或降低(细胞外液为含钙液)精胺介导的上述作用(均P<0.05);此作用亦可被filipin(1.5 mg/L)或Cav-1基因沉默减弱(细胞外液为含钙液)(均P<0.05)。结论:HUVECs中Cav-1对CaR介导的NO生成有促进作用,其机制可能与Cav-1影响CaR膜定位及对激动剂反应性有关。更多还原

【Abstract】 AIM: To study the role of caveolin-1(Cav-1) in extracellular Ca2+-sensing receptor(CaR)-induced production of nitric oxide(NO) in human umbilical vein endothelial cells(HUVECs).METHODS: The expression of Cav-1 gene in HUVECs was silenced by transfection of constructed Cav-1 short hairpin RNA(Cav-1 shRNA) interference plasmids.The HUVECs in second or third passage were divided into 10 groups: control group,spermine group,spermine+Ca2+ group,Calhex231+spermine group,Calhex231+spermine+Ca2+ group,filipin+spermine group,filipin+spermine+Ca2+ group,vehicle+spermine+Ca2+ group,Cav-1 shRNA+spermine group and Cav-1 shRNA+spermine+Ca2+ group.The protein levels of Cav-1 and CaR in HUVECs were detected by Western blotting after transfection.The production of NO and the activity of endothelial nitric oxide synthase(eNOS) were determined using the fluorescent NO indicator,3-amino-4-methylamino-2’,7’-difluorofluorescein diacetate(DAF-FM DA).RESULTS: The protein expression of Cav-1 in HUVECs was decreased after transfected with Cav-1 shRNA.Simultaneously,the CaR membrane protein was decreased,whereas CaR protein level in the cytosol was unaffected.Whether in the culture medium with or without Ca2+,the CaR agonist spermine at concentration of 2 mmol/L resulted in an increase in the activity of eNOS and the production of NO in HUVECs.In the presence of spermine,the production of NO and activity of eNOS in HUVECs were abolished(without Ca2+) or decreased(with Ca2+) after inhibition of CaR by a negative allosteric modulator Calhex231(1 μmol/L,P<0.05).In the presence of Ca2+,the effect of spermine on the increase in the activity of eNOS and the production of NO in HUVECs was also attenuated after acute caveolae disruption with filipin(1.5 mg/L) or transfected with Cav-1 shRNA(P<0.05).CONCLUSION: CaR-mediated production of NO in HUVECs might be promoted by the binding of Cav-1 to CaR.The mechanism is involved in the effect of Cav-1 on the localization of CaR at the plasma membrane,thus changing the responsibility of CaR.更多还原