小凹蛋白-1下调人脐静脉内皮细胞外钙敏感受体介导的钙内流的作用机制

Role of caveolin-1 in down-regulating extracellular Ca^(2+)-sensing receptor-mediated Ca^(2+) influx in human umbilical vein endothelial cells

目的:研究小凹蛋白-1(Cav-1)对人脐静脉内皮细胞(HUVECs)细胞外钙敏感受体(CaR)介导钙内流的作用机制。方法:取2～3代HUVECs,采用细胞膜穴样凹陷(caveolae)结构破坏剂非律平(filipin)和甲基-β-环糊精(MβCD)及Cav-1基因沉默,配合CaR激动剂精胺(spermine)和负性变构调节剂Calhex 231。Fura-2/AM负载检测细胞内Ca2+浓度([Ca2+]i)。Western blotting检测HUVECs中Cav-1以及CaR蛋白表达,免疫共沉淀技术检测Cav-1和CaR相互作用。用蔗糖密度梯度离心的方法提取并鉴定caveolae,Western blotting检测caveolae的标志蛋白Cav-1和浮舰蛋白1(flotillin-1)及CaR表达。同时检测胞膜、胞浆和高尔基体标志蛋白:转铁蛋白受体(TfR)、β-肌动蛋白(β-actin)和β-外被体蛋白(β-COP)。检测富含Cav-1膜(CEM)区、非caveolae I区(NCF I)和II区(NCF II)的Cav-1和CaR表达。结果:(1)细胞外液为含钙液时,Calhex 231完全阻断精胺刺激引起的[Ca2+]i升高(P<0.05),MβCD可加强精胺升高[Ca2+]i的作用(P<0.05)。不同浓度MβCD处理后各组Cav-1和CaR相互作用的差异无统计学意义(P>0.05);(2)免疫共沉淀结果显示,各组Cav-1和CaR蛋白表达的差异无统计学意义(P>0.05);(3)与control组比较,spermine+Ca2+组、filipin+spermine+Ca2+组和MβCD+spermine+Ca2+组CEM区的Cav-1和CaR蛋白表达均降低(P<0.05),NCF I区的Cav-1和CaR蛋白表达增加(P<0.05),NCF II区Cav-1蛋白表达增加(P<0.05),而CaR蛋白表达的差异无统计学意义(P>0.05)。结论:在HUVECs中,Cav-1和CaR共定位于同一caveolae区,同时Cav-1对CaR介导的Ca2+内流有下调作用,其机制可能与Cav-1抑制CaR膜定位、促使其向非caveolae区转位及减弱其对激动剂的反应性有关。

AIM： To study the role of caveolin-1 （Car-1） in down-regulating the extracellular Ca2 ＋ -sensing receptor （CaR）-mediated Ca2＋ influx in human umbilical vein endothelial cells （HUVECs） and its mechanisms. METH- ODS： HUVECs were collected and cultured to the second or third passage. Filipin was used to induce acute caveolae dis- ruption. Methyl-β-cyclodextrin （MβCD） or shRNA targeting Cav-1 combined with CaR agonist spermine and negative al- losteric modulator Calhex 231 was also used in HUVECs. Intracellular concentration of Ca2＋ （ [ Ca2＋ ]1） Was measured by Fura-2/AM loading. The protein expression of Cav-1 and CaR was examined by Western blotting. The interaction and co- localization of Cav-1 and CaR were determined by the method of co-immunoprecipitation （Co-IP）. Caveolae-enriched mem- brane （CEM） fractions were isolated and identified by detergent-free （Na2C03） sucrose density gradient centrifugation. The protein levels of Cav-1, CaR, flotillin-1, β-coat protein （ β-COP）, β-actin and transferrin receptor （TfR） were detec-ted by Western blotting. Noncaveolar fraction I （ NCF I） and noncaveolar fraction II （ NCF II） in the CEM fractions were separated. RESULTS： Using extracellular buffer with Ca2~ , the increase in [ Ca2 ~ ]i induced by spermine in HUVECs was abolished after inhibition of CaR by its negative allosterie modulator calhex231. Conversely, the effect of spermine on the increased [ Ca2 ~ ] i in HUVECs was further augmented after acute caveolae disruption by MI^CD. No significant differ- ence of the protein levels of CaR and Cav-1 in HUVECs among treating with different concentrations of MI3CD was ob- served. The results of Co-IP showed that the protein levels of CaR and Cav-1 in every group of HUVECs were not signifi- cantly different. Compared with control group, the protein expression of CaR and Cav-I in CEM was decreased in spermine ＋ Ca2 ＋ group, filipin ＋ spermine ＋ Ca2 ＋ group and MIBCD ＋ spermine ＋ Ca2 ~ group, and that in NCF I was increased. How- ever, the protein expression of Cav-1 increased, and the protein level of CaR was unaffected in NCF II. CONCLUSION： The CaR and Cav-I co-localize in the same membrane caveolae lipid raft in HUVECs. The function of CaR-induced extra- cellular Ca2＋ influx is down-regulated by binding with Cav-1. This effect might be associated with, at least in part, the in- hibitory effect of Gay-1 on CaR localization at the plasma membrane by a translocation of CaR from the caveolar fractions to noncaveolar fractions, thus attenuating the CaR response to the agunist.