钙敏感受体介导的人脐静脉内皮细胞钙内流及一氧化氮生成过程中TRPCI与STIM1的相互作用

Interaction between TRPC1 and STIM1 in calcium sensing receptor mediated calcium influx and nitric oxide production in human umbilical vein endothelial cells

目的探讨经典瞬时受体电位通道1（TRPC1）与基质相互作用分子1（STIM1）在钙敏感受体（CaR）介导的人脐静脉内皮细胞（HUVEC）钙离子（Ca2＋）内流和一氧化氮（NO）生成中的相互作用。 方法取华中科技大学同济医学院附属同济医院妇产科健康孕产妇剖宫产新生儿的新鲜脐带，原代培养HUVEC并传代。将所得HUVEC分别与CaR激动剂精胺[激活钙池操纵性钙通道（SOC）和受体操纵性钙通道（ROC）]、ROC模拟剂12－O－十四烷酰佛波醇－13－乙酸酯（TPA）＋CaR负性变构调节剂Calhex231（阻断SOC阻、激活ROC）、蛋白激酶C（PKC）抑制剂Ro31－8220及经典型PKCs和PKCμ抑制剂Go6967（激活SOC、阻断ROC）共孵育。采用免疫荧光技术检测HUVEC中TRPC1与STIM1的蛋白表达，免疫共沉淀法检测TRPC1与STIM1之间的相互作用。取2～3代HUVEC进行分组，将构建的TRPC1、STIM1干扰质粒（shTRPC1、shSTIM1）联合转染HUVEC即shTRPC1＋shSTIM1组 （实验组）、vehicle－shTRPC1＋vehicle－shSTIM1组 （空质粒组）和对照组，将3组细胞分别加入上述4种不同的药物进行干预。采用荧光探针Fura－2/AM检测HUVEC细胞内钙离子浓度（[Ca2＋]i）的变化，NO荧光探针DAF－FM负载方法同步检测HUVEC中NO生成的变化。 结果（1） HUVEC中TRPC1与STIM1的蛋白表达：激光共聚焦显微镜下观察，正常对照组HUVEC中TRPC1和STIM1蛋白的表达呈阳性，二者共定位于胞浆。Calhex231＋TPA＋精胺＋Ca2＋组、Ro31－8220＋精胺＋Ca2＋组和Go6976＋精胺＋Ca2＋组HUVEC中定位于胞浆中的TRPC1、STIM1均减少，二者结合也减少。（2） HUVEC中TRPC1与STIM1的相互作用：Calhex231＋TPA＋精胺＋Ca2＋组、Ro31－8220＋精胺＋Ca2＋组和Go6976＋精胺＋Ca2＋组STIM1/TRPC1和TRPC1/STIM1的相对比值的百分数分别为（25.98±2.17）%和（44.10±4.01）%、（20.85±1.01）%和（46.31±3.47）%、（23.88±2.05）%和（39.65±2.91）%，均明显低于对照组的（100.00±4.66）%和（100.00±6.40）%以及精胺＋Ca2＋组的（106.04±2.45）%和（107.78±2.66）%（P均〈0.05）。（3） 联合转染TRPC1与STIM1对4种不同药物诱导的HUVEC中[Ca2＋]i和NO生成的影响：联合转染TRPC1与STIM1对4种不同药物诱导的HUVEC中[Ca2＋]i两激发光荧光强度比的变化值（Δratio）和NO净荧光强度值的影响趋势是一致的，均为实验组[Ca2＋]iΔratio值和NO净荧光强度值均明显低于对照组和空质粒组（P均〈0.05），而空质粒组与对照组比较二者差异均无统计学意义（P均〉0.05）。 结论TRPC1与STIM1以二元复合物的形式共同调节CaR介导HUVEC Ca2＋内流及NO生成。

ObjectiveTo investigate the interaction of Ca2＋ protein TRPC1 and STIM1 in extracellular Ca2＋ -sensing receptor （CaR）-induced extracellular Ca2＋ influx and the production of nitric oxide （NO）. MethodsHuman umbilical vein endothelial cells （HUVECs） were cultured and incubated with CaR agonist spermine （activating store-operates cation channels （SOC） and receptor-operated channels （ROC））, CaR negative allosteric modulator Calhex231 （blocking SOC, activating ROC） and ROC analogue TPA （activating ROC, blocking SOC）, protein kinase C （PKC） inhibitr Ro31-8220, PKCs and PKCμ inhibitor Go6967（activate SOC, blocking ROC）, respectively. The interaction of TRPC1 and STIM1 was determined using the immunofluorescence methods. The interaction between TRPC1 and STIM1 were examined by Co-immuno precipitation. The HUVECs were divided into： TRPC1 and STIM1 short hairpin RNA group （shTRPC1＋ shSTIM1 group）, vehicle-TRPC1＋ vehicle-STIM1 group and control group. The cells were incubated with four different treatments under the action of above mentioned interventions, intracellular Ca2＋ concentration （[Ca2＋ ]i） was detected using the fluorescence Ca2＋ indicator Fura-2/AM, the production of NO was determined by DAF-FM. Results（1） The expression of TRPC1 and STIM1 proteins levels in HUVECs： Under the confocal microscope, TRPC1 and STIM1 protein expression showed masculine gender, both located in cytoplasm in the normal control group. Post incubation with Calhex231＋ TPA, Ro31-8220 and Go6967, TRPC1 and STIM1 positioned in cytoplasm was significantly reduced, and the combined TRPC1 and STIM1 was also significantly reduced. （2） The interaction of TRPC1 and STIM1 in HUVECs： The relative ratios of Calhex231＋ TPA＋ Spermine＋ Ca2＋ group, Ro31-8220＋ Spermine＋ Ca2＋ group and Go6976＋ Spermine＋ Ca2＋ group STIM1/TRPC1 and TRPC1/STIM1 were as follows： （25.98±2.17）% and （44.10±4.01）%, （20.85±1.01）% and （46.31±3.47）%, （23.88±2.05）% and （39.65±2.91）%, which were significantly lower than those in the control group （100.00±4.66）% and （100.00±6.40）% and in the Spermine＋ Ca2＋ group （106.04±2.45）% and （107.78±2.66）% （all P〈0.05）. （3） The influence of joint TRPC1 and STIM1 transfection to four different drugs treated HUVECs on [Ca2＋ ]i and NO generation： The changes of two excitation fluorescence intensity ratio and NO net fluorescence intensity values were consistent, [Ca2＋ ]i and NO net fluorescence intensity values were significantly lower in the experimental group than the control group and the vehicle group （all P〈0.05）, while which were similar between the vehicle group and control group （all P〉0.05）.ConclusionsOur results indicate that TRPC1 and STIM1 jointly regulate CaR-mediated Ca2＋ influx and nitric oxide generation in HUVECs in the form of binary complex.