title: [Extracellular Ca(2+)-sensing receptor-induced extracellular Ca2+ influx is down-regulated by caveolin-1 in human umbilical vein endothelial cells].
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.
authors.affiliation: Department of Pathophysiology/Key Laboratory of Education Ministry of Xinjiang Endemic and Ethnic Diseases, Medical College of Shihezi University, Shihezi 832002, China.
authors.forename: Zhen-Huan
authors.initials: ZH
authors.lastname: Wang
authors.forename: Qing-Hua
authors.initials: QH
authors.lastname: Hu
authors.forename: Hua
authors.initials: H
authors.lastname: Zhong
authors.forename: Feng-Mei
authors.initials: FM
authors.lastname: Deng
authors.forename: Fang
authors.initials: F
authors.lastname: He
mesh_terms: D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D051242:Caveolin 1 / Q000819:agonists / Q000502:physiology*; D002478:Cells, Cultured; D015536:Down-Regulation; D005372:Filipin / Q000494:pharmacology; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology* / Q000378:metabolism*; D006801:Humans; D044169:Receptors, Calcium-Sensing / Q000502:physiology*; D013096:Spermine / Q000494:pharmacology
chemical_list: D015220:Calcium Channels; D051242:Caveolin 1; D044169:Receptors, Calcium-Sensing; D013096:Spermine; D005372:Filipin; D002118:Calcium