中文摘要：

为了探讨小凹蛋白-1（caveolin-1,Cav-1）在人脐静脉内皮细胞（human umbilical vein endothelial cells,HUVECs）细胞外钙敏感受体（extracellular Ca^2＋-sensing receptor,CaR）介导Ca^2＋内流中的作用,本实验研究了细胞膜穴样凹陷（caveolae）结构破坏剂Filipin或Cav-1基因沉默后对CaR介导Ca^2＋内流的影响。Fura-2/AM负载检测细胞内Ca^2＋浓度（intracellular Ca^2＋ concentration,[Ca^2＋]i）。结果显示,HUVECs中CaR对不同浓度细胞外Ca^2＋刺激无反应。无论细胞外为零钙液或含钙液时,精胺（Spermine,2mmol/L）刺激CaR时均引起[Ca^2＋]i升高（P〈0.05）,其中细胞外液为含钙液时,[Ca^2＋]i升高较细胞外为零钙液时更明显（P〈0.05）,CaR的负性变构调节剂Calhex231（1μmol/L）均可完全阻断Spermine刺激引起的[Ca^2＋]i升高（P〈0.05）;相反,Spermine升高[Ca^2＋]i作用可被Filipin（1.5μg/mL）或Cav-1基因沉默进一步加强（均P〈0.05）。免疫荧光技术检测显示HUVECs中有CaR和Cav-1表达,两者共定位于膜上。Western blot检测结果显示Cav-1干扰后,Cav-1蛋白表达降低,同时CaR的膜蛋白表达也降低（P〈0.05）,CaR的总蛋白表达无变化（P〉0.05）。上述结果表明：Cav-1和CaR共定位于HUVECs膜,Cav-1对CaR介导的Ca^2＋内流有下调作用,其机制可能与Cav-1影响CaR膜定位及减弱其对激动剂反应性有关。

英文摘要：

Although the function of extracellular Ca^2＋-sensing receptor（CaR） is known,the regulatory mechanism of the CaR function remains to be clarified.The purpose of the present study was to investigate the effect of caveolin-1（Cav-1） on CaR-induced extracellular Ca^2＋ influx by using acute caveolae disruption with Filipin or siRNA targeted to the Cav-1 in human umbilical vein endothelial cells（HUVECs）.Intracellular Ca^2＋ concentration（[Ca^2＋]i） was detected by Fura-2/AM loading.The results showed that different concentrations of extracellular Ca^2＋ failed to increase [Ca^2＋]i,while the CaR agonist Spermine（2 mmol/L） resulted in an increase in [Ca^2＋]i that was diminished in buffer without Ca^2＋（P〈0.05）.No matter in buffer with or without 2 mmol/L Ca^2＋,the [Ca^2＋]i increase induced by Spermine in HUVECs was abolished after inhibition of CaR by a negative allosteric modulator Calhex231（1 μmol/L）（P〈0.05）,conversely,the effect of Spermine on the increase in [Ca^2＋]i in HUVECs was further augmented after acute caveolae disruption with Filipin（1.5 μg/mL） or transfection with siRNA targeted to the Cav-1（P〈0.05）.This indicated that Cav-1 produced an inhibition of CaR-induced extracellular Ca^2＋ influx.As to the biological mechanism of Cav-1-induced inhibition,immunofluorescence technique showed that both CaR and Cav-1 were present in HUVECs,and confocal microscopy supported the co-localization of CaR and Cav-1 on the plasma membrane.Functionally,the Cav-1 protein expression was decreased in HUVECs transfected with siRNA targeted to the Cav-1（P〈0.05）;simultaneously,the CaR membrane protein expression was decreased（P〈0.05）,whereas CaR total protein level was unaffected（P〈0.05）.In conclusion,the present study suggests that CaR and Cav-1 co-localize on the plasma membrane in HUVECs and CaR-induced Ca^2＋ influx is down-regulated by binding with Cav-1,and the mechanism involves the effect of Cav-1 on CaR localization on the plasma membrane