Roles Of TRPC1and TRPC3in The CaR Mediated Ca²⁺ Entry And NO Generation And Its Mechanisms In Human Umbilical Vein Endothelial Cells

Objective: To study the role of Transient Receptor Potential Canonical1、3（TRPC1、3）in extracellular Ca²⁺‐sensing receptor （CaR）‐induced extracellular Ca²⁺ influx and the production of nitric oxide （NO）, and its mechanisms in human umbilical vein endothelial cells （HUVEC）.Methods:（1） HUVEC were cultured using trypsinzation method and identified by growth pattern and immunofluorescent staining.（2） Co‐incubating the cells using store‐operated cation channels blockers （SOC） and/or receptor‐operated cation channels （ROC） blockers, Western blotting experiments were performed to detect proteins expression of TRPC1and TRPC3.（3） The interaction and co‐localization of TRPC1and TRPC3were determined using the co‐immunoprecipitate.（4） We silenced the expression of TRPC1、TRPC3gene in HUVEC by transfection constructed TRPC1RNA、TRPC3RNA interference plasmids. The efficiency of interference and TRPC1、3protein and mRNA level were determined by Western blotting and real time RT‐PCR, respectively.（5） The second to fifth passage of HUVEC were divided into: TRPC1short hairpin RNA （TRPC1shRNA）+spermine+Ca²⁺group and TRPC3‐004short hairpin RNA （TRPC3‐004ShRNA）+spermine+Ca²⁺group; control group(spermine+Ca²⁺ group) and vehicle+spermine+Ca²⁺ group. The four groups of cells were incubated with CaR agonist spermine（activating ROC and SOC）, ROC analog TPA and negative allosteric modulators of CaR Calhex231（activating ROC, blocking SOC）, PKC inhibitors Ro31‐8220and classic‐type PKCs and PKCμ inhibitor Go6976（activating SOC, blocking ROC）, respectively. Intracellular Ca²⁺ concentration ([Ca²⁺]i) was detected using the fluorescence Ca²⁺ indicator Fura‐2/AM, the production of NO was determined by DAF‐FM（NO fluorescent probe） of every group in HUVEC.（6） The SPSS17.0was used. Data are reported as means±SE. Statistical comparisons were made using t‐test or chi‐square test for the paired and the unpaired groups. An analysis of variance was used when multiple comparisons were performed. A difference was considered significant at p<0.05.Results:（1）HUVEC were confirmed by cell morphology and immunocytochemistry measuring â…§ factor antigen.（2）Compared with control group, the TRPC1and TRPC3protein levels in different groups were not considered significant, respectively （P>0.05）.（3）Immunocytochemical results showed that TRPC1and TRPC3protein were expressing in the cytoplasm, and co‐localized in the cytoplasm.（4） The results of transfection constructed TRPC1RNA and TRPC3RNA interference plasmids were as follows: that shRNA targeted to the TRPC1and TRPC3gene decreased TRPC1and TRPC3protein levels by83.4%and71.8%, respectively （p<0.05）, simultaneously, the TRPC1and TRPC3mRNA levels was decreased by39.0%and74.0%, respectively （p<0.05）.（5） The results of dynamic changes of intracellular Ca²⁺ fluorescence intensity ratio by Fura‐2/AM and net NO fluorescence intensity by DAF‐FM DA in HUVEC showed: Compared with spermine+Ca²⁺ group （△ratio=7.54±0.18,846.39±68.13）, the [Ca²⁺]i and the net NO fluorescence intensity of spermine+Ca²⁺ ShTRPC1group （△ratio=5.73±0.15,137.82±14.22） were decreased,respectively （p<0.05）; but spermine+Ca²⁺+Vehicle group （△ratio=7.66±0.13,724.96±20.72） and spermine+Ca²⁺+ShTRPC3‐004group （△ratio=7.37±0.12,794.685±20.60） were not changed （p＞0.05）. Compared with Calhex231+TPA+spermine+Ca²⁺ group （△ratio=4.64±0.07,176.20±14.13）, the [Ca²⁺]i and the net NO fluorescence intensity of Calhex231+TPA+spermine+Ca²⁺ ShTRPC1group （△ratio=2.67±0.07,58.65±5.05） were decreased, respectively （p<0.05）; but Calhex231+TPA+spermine+Ca²⁺+Vehicle group（△ratio=4.43±0.14,175.31±16.89） and Calhex231+TPA+spermine+Ca²⁺+ShTRPC3‐004group（△ratio=4.51±0.12,150.71±6.08） were not changed （p＞0.05）. Compared with Ro31‐8220+spermine+Ca²⁺ group （△ratio=6.03±0.05,214.96±7.83）, the [Ca²⁺]i and the net NO fluorescence intensity of Ro31‐8220+spermine+Ca²⁺ ShTRPC1group （△ratio=3.41±0.09,57.97±7.14） were decreased, respectively （p<0.05）; but Ro31‐8220+spermine+Ca²⁺+Vehicle group （△ratio=5.90±0.03,207.34±10.2） and Ro31‐8220+spermine+Ca²⁺+ShTRPC3‐004group （△ratio=5.90±0.07,192.52±19.21） were not changed （p＞0.05）. Compared with Go6976+spermine+Ca²⁺ group （△ratio=5.98±0.02,199.16±6.93）, the [Ca²⁺]i and the net NO fluorescence intensity of Go6976+spermine+Ca²⁺ ShTRPC1group （△ratio=4.22±0.11,57.44±6.65） were decreased respectively （p<0.05）; but Go6976+spermine+Ca²⁺+Vehicle group（△ratio=6.03±0.13,199.92±12.19） and Go6976+spermine+Ca²⁺+ShTRPC3‐004group （△ratio=6.15±0.15,187.5±1.5） were not changed （p＞0.05）.Conclusion:（1） TRPC1plays an important role in CaR‐mediating Ca²⁺ influx and NO production in HUVEC.（2） TRPC1is a key component in CaR‐mediated Ca²⁺ influx and NO production though SOC and ROC. TRPC3does not participate in this processes.