Issue Archive

Involvement of interaction between TRPC1 and Orai1 in calcium sensing receptor-mediated calcium influx and nitric oxide generation in human umbilical vein endothelial cells

WANG La-Mei¹, TANG Na¹, ZHONG Hua¹, PANG Li-Juan^1,2, ZHANG Chun-Jun¹, HE Fang^1,*

¹Department of Pathophysiology/Key Laboratory of Xinjiang Endemic and Ethnic Diseases, Ministry of Education, Medical College of Shihezi University, Shihezi 832002, China；²Department of Pathology, Medical College of Shihezi University, Shihezi 832002, China

Abstract

The present study was to investigate the role of the interaction between canonical transient receptor potential channel 1 (TRPC1) and calcium release-activated calcium modulator 1 (Orai1) in extracellular Ca2+-sensing receptor (CaR)-induced extracellular Ca2+ influx and nitric oxide (NO) production. Human umbilical vein endothelial cells (HUVECs) were incubated with CaR agonist Spermine [activating store-operated calcium channels (SOC) and receptor-operated calcium channels (ROC)] alone or in combination with the following reagents: CaR negative allosteric modulator Calhex231 plus ROC analogue TPA (activating ROC and blocking SOC), Ro31-8220 (PKC inhibitor that activates SOC and blocks ROC) or Go6967 (PKCs and PKCµ inhibitor that activates SOC and blocks ROC). The protein expressions and co-localization of TRPC1 and Orai1 were determined using immunofluorescent staining. The interaction between TRPC1 and Orai1 was examined by co-immunoprecipitation. We silenced the expressions of their genes in the HUVECs by transfection of constructed TRPC1 and Orai1 shRNA plasmids. Intracellular Ca2+ concentration ([Ca2+]i) was detected using Ca2+ indicator Fura-2/AM, and NO production was determined by DAF-FM staining. The results showed that TRPC1 and Orai1 protein expressions were co-located on the cell membrane of the HUVECs. Compared with Spermine+Ca2+ group, Calhex231+ TPA+Spermine+Ca2+, Ro31-8220+Spermine+Ca2+ and Go6976+Spermine+Ca2+ groups exhibited down-regulated protein expressions of TRPC1 and Orai1 in cytoplasm and decreased co-localization on the cell membrane. Co-immunoprecipitation results showed that the interaction between TRPC1 and Orai1 was reduced by Calhex231 plus TPA, Ro31-8220 or Go6976 addition in the Spermine-stimulated HUVECs. Double knockdown of Trpc1 and Orai1 genes significantly decreased [Ca2+]i level and NO production in all of the Spermine+Ca2+, Calhex231+TPA+Spermine+Ca2+, Ro31-8220+Spermine+Ca2+ and Go6976+Spermine+Ca2+ groups. These results suggest that TRPC1/Orai1 may form a complex that mediates Ca2+ influx and No production via SOC and ROC activation.

Key words: canonical transient receptor potential channel 1; calcium release-activated calcium modulator 1; Ca2+-sensing receptor; Ca2+; nitric oxide

Received: 2017-09-22　　Accepted: 2017-12-12

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

DOI: 10.13294/j.aps.2018.0022

Citing This Article：

WANG La-Mei, TANG Na, ZHONG Hua, PANG Li-Juan, ZHANG Chun-Jun, HE Fang. Involvement of interaction between TRPC1 and Orai1 in calcium sensing receptor-mediated calcium influx and nitric oxide generation in human umbilical vein endothelial cells. Acta Physiol Sin 2018; 70 (3): 269-280 (in Chinese with English abstract).