AIM: To study the role of caveolin-1 (Cav-1) in down-regulating the extracellular Ca2+-sensing receptor ( CaR)-mediated Ca2+ influx in human umbilical vein endothelial cells (HUVECs) and its mechanisms. METHODS : HUVECs were collected and cultured to the second or third passage. Filipin was used to induce acute caveolae disruption. Methyl-p-cyclodextrin (M[$CD) 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+ ] i) 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 membrane (CEM) fractions were isolated and identified by detergent-free (Na2CO3) 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-rnted 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 allosteric modulator calhex231. Conversely, the effect of spermine on the increased [ Ca 2+], in HUVECs was further augmented after acute caveolae disruption by MβCD. No significant difference of the protein levels of CaR and Cav-1 in HUVECs among treating with different concentrations of MβCD was observed. The results of Co-IP showed that the protein levels of CaR and Cav-1 in every group of HUVECs were not significantly different. Compared with control group, the protein expression of CaR and Cav-1 in CEM was decreased in spermine + Ca2+ group, filipin + spermine + Ca2 + group and MfiCD + spermine + Ca2 + group, and that in NCF I was increased. However, the protein expression of Cav-1 increased, and the protein level of CaR was unaffected in NCF II. CONCLUSION: The CaR and Cav-1 co-localize in the same membrane caveolae lipid raft in HUVECs. The function of CaR-induced extracellular Ca2+ influx is down-regulated by binding with Cav-1. This effect might be associated with, at least in part, the inhibitory effect of Cav-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 agonist.%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.
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