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[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

Received: 2010-08-26　　Accepted: 2010-11-24

Corresponding author: 何芳　　E-mail: fangf2002shz@126.com

Citing This Article：

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).