Involvement of interaction between TRPC1 and Orai1 in calcium sensing receptor-mediated calcium influx and nitric oxide generation in human umbilical vein endothelial cells.
- Author:
La-Mei WANG
1
;
Na TANG
1
;
Hua ZHONG
1
;
Li-Juan PANG
1
;
Chun-Jun ZHANG
1
;
Fang HE
2
Author Information
1. Department of Pathophysiology/Key Laboratory of Xinjiang Endemic and Ethnic Diseases, Ministry of Education, Medical College of Shihezi University, Shihezi 832002, China.
2. Department of Pathophysiology/Key Laboratory of Xinjiang Endemic and Ethnic Diseases, Ministry of Education, Medical College of Shihezi University, Shihezi 832002, China. fangf2002shz@126.com.
- Publication Type:Journal Article
- From:
Acta Physiologica Sinica
2018;70(3):269-280
- CountryChina
- Language:Chinese
-
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 Ca-sensing receptor (CaR)-induced extracellular Ca 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 Ca concentration ([Ca]) was detected using Ca 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+Ca group, Calhex231+ TPA+Spermine+Ca, Ro31-8220+Spermine+Ca and Go6976+Spermine+Ca 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 [Ca] level and NO production in all of the Spermine+Ca, Calhex231+TPA+Spermine+Ca, Ro31-8220+Spermine+Ca and Go6976+Spermine+Ca groups. These results suggest that TRPC1/Orai1 may form a complex that mediates Ca influx and No production via SOC and ROC activation.
