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.
ABSTRACT
This paper aims to investigate the effect of store-operated calcium channels (SOC) and receptor-operated calcium channels (ROC) on Ca(2+)-sensing receptor (CaR)-induced extracellular Ca(2+) influx and nitric oxide (NO) generation in human umbilical vein endothelial cells (HUVEC). SOC blocker, non-selective cation channel blocker, ROC agonist and ROC blocker were used separately and combined. Intracellular Ca(2+) concentration ([Ca(2+)]i) was measured by Fura-2/AM loading. The activity of endothelial nitric oxide synthase (eNOS) and the production of NO were determined by the DAF-FM diacetate (DAF-FM DA). The results showed that increases of [Ca(2+)]i, eNOS activity and NO generation induced by CaR agonist Spermine were all reduced after single blocking the SOC or ROC, respectively (P < 0.05). ROC agonist can partially abolish the ROC blocker's effect (P < 0.05). The above mentioned effects evoked by CaR agonist Spermine were further reduced when blocking both SOC and ROC than single blocking SOC or ROC in HUVEC (P < 0.05). In conclusion, these results suggest that the SOC and ROC participate in the processes of CaR-evoked extracellular Ca(2+) influx and NO generation by a synergistic manner in HUVEC.