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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

王振焕, 胡清华, 钟华, 邓峰美, 何芳^*

新疆石河子大学医学院病理生理教研室/新疆地方与民族高发病教育部重点实验室，石河子 832002；华中科技大学同济医学院病理生理学系/卫生部呼吸疾病重点实验室，武汉 430030

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 Ca2+-sensing receptor, CaR)介导Ca2+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Ca2+内流的影响。Fura-2/AM负载检测细胞内Ca2+浓度(intracellular Ca2+ concentration, [Ca2+]i)。结果显示，HUVECs中CaR对不同浓度细胞外Ca2+刺激无反应。无论细胞外为零钙液或含钙液时，精胺(Spermine, 2 mmol/L)刺激CaR时均引起[Ca2+]i升高(P<0.05)，其中细胞外液为含钙液时，[Ca2+]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 (1 μmol/L)均可完全阻断Spermine刺激引起的[Ca2+]i升高(P<0.05)；相反，Spermine 升高[Ca2+]i作用可被Filipin (1.5 μg/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Ca2+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。

关键词： 小凹蛋白-1; 内皮细胞; 钙敏感受体; 钙信号

分类号：R331

[Extracellular Ca(2+)-sensing receptor-induced extracellular Ca(2+) influx is down-regulated by caveolin-1 in human umbilical vein endothelial cells.] [Article in Chinese]

WANG Zhen-Huan, HU Qing-Hua, ZHONG Hua, DENG Feng-Mei, HE Fang

Department of Pathophysiology/Key Laboratory of Education Ministry of Xinjiang Endemic and Ethnic Diseases, Medical College of Shihezi University, Shihezi 832002, China； Department of Pathophysiology, Tongji Medical College, Huazhong University of Science and Technology/Key Laboratory of Respiratory Diseases, Health Ministry of China, Wuhan 430030, China

Abstract

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 and attenuating the CaR response to the agonist.

Key words: caveolin-1; endothelial cells; calcium-sensing receptors; calcium signaling

收稿日期：2010-08-26　　录用日期：2010-11-24

通讯作者：何芳　　E-mail: fangf2002shz@126.com

引用本文：

王振焕, 胡清华, 钟华, 邓峰美, 何芳. 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[J]. 生理学报 2011; 63 (1): 39-47.

WANG Zhen-Huan, HU Qing-Hua, ZHONG Hua, DENG Feng-Mei, HE Fang. [Extracellular Ca(2+)-sensing receptor-induced extracellular Ca(2+) influx is down-regulated by caveolin-1 in human umbilical vein endothelial cells.] [Article in Chinese]. Acta Physiol Sin 2011; 63 (1): 39-47 (in Chinese with English abstract).