Wang L M, Zhong H, Tang N, Pang L J, Zhang C J, He F
Key Laboratory of Xinjiang Endemic and Ethnic Diseases, Ministry of Education, Department of Pathophysiology, Department of Physiology, Medical College of Shihezi University, Shihezi 832002, China.
Zhonghua Xin Xue Guan Bing Za Zhi. 2017 Nov 24;45(11):978-984. doi: 10.3760/cma.j.issn.0253-3758.2017.11.015.
To investigate the interaction of Ca(2+) protein TRPC1 and STIM1 in extracellular Ca(2+) -sensing receptor (CaR)-induced extracellular Ca(2+) influx and the production of nitric oxide (NO). Human 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) inhibitor 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 Ca(2+) concentration (Ca(2+) ) was detected using the fluorescence Ca(2+) indicator Fura-2/AM, the production of NO was determined by DAF-FM. (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+ Ca(2+) group, Ro31-8220+ Spermine+ Ca(2+) group and Go6976+ Spermine+ Ca(2+) 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+ Ca(2+) group (106.04±2.45)% and (107.78±2.66)% (all <0.05). (3) The influence of joint TRPC1 and STIM1 transfection to four different drugs treated HUVECs on Ca(2+) and NO generation: The changes of two excitation fluorescence intensity ratio and NO net fluorescence intensity values were consistent, Ca(2+) and NO net fluorescence intensity values were significantly lower in the experimental group than the control group and the vehicle group (all <0.05), while which were similar between the vehicle group and control group (all >0.05). Our results indicate that TRPC1 and STIM1 jointly regulate CaR-mediated Ca(2+) influx and nitric oxide generation in HUVECs in the form of binary complex.
研究细胞外钙离子（Ca(2+)）感受受体（CaR）诱导的细胞外Ca(2+)内流及一氧化氮（NO）生成过程中Ca(2+)相关蛋白瞬时受体电位通道蛋白1（TRPC1）和基质相互作用分子1（STIM1）的相互作用。培养人脐静脉内皮细胞（HUVECs），并分别用CaR激动剂精胺（激活储存操纵性阳离子通道（SOC）和受体操纵性通道（ROC））、CaR负性变构调节剂Calhex231（阻断SOC，激活ROC）、ROC类似物佛波酯（TPA，激活ROC，阻断SOC）、蛋白激酶C（PKC）抑制剂Ro31-8220、PKCs和PKCμ抑制剂Go6967（激活SOC，阻断ROC）进行孵育。采用免疫荧光法测定TRPC1与STIM1的相互作用。通过免疫共沉淀检测TRPC1与STIM1之间的相互作用。将HUVECs分为：TRPC1和STIM1短发夹RNA组（shTRPC1 + shSTIM1组）、空载-TRPC1 + 空载-STIM1组和对照组。在上述干预作用下，对细胞进行四种不同处理，使用荧光Ca(2+)指示剂Fura-2/AM检测细胞内Ca(2+)浓度（Ca(2+) ），采用DAF-FM法测定NO生成量。（1）HUVECs中TRPC1和STIM1蛋白水平的表达：在共聚焦显微镜下，TRPC1和STIM1蛋白表达呈阳性，在正常对照组中均位于细胞质中。用Calhex231 + TPA、Ro31-8220和Go6967孵育后，位于细胞质中的TRPC1和STIM1明显减少，TRPC1与STIM1的结合也明显减少。（2）HUVECs中TRPC1与STIM1的相互作用：Calhex231 + TPA + 精胺 + Ca(2+)组、Ro31-8220 + 精胺 + Ca(2+)组和Go6976 + Spermine + Ca(2+)组中STIM1/TRPC1和TRPC1/STIM1的相对比值分别为：(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)%，均显著低于对照组（100.00±4.66)%和(100.00±6.40)%以及精胺 + Ca(2+)组（106.0·4±2.45)%和(107.78±2.66)%（均P<0.05）。（3）联合转染TRPC1和STIM1对四种不同药物处理的HUVECs的Ca(2+) 和NO生成的影响：两个激发荧光强度比值和NO净荧光强度值的变化趋势一致，实验组的Ca(2+) 和NO净荧光强度值显著低于对照组和空载组（均P<0.05），而空载组与对照组之间相似（均P>0.05）。我们的结果表明，TRPC1和STIM1以二元复合物的形式共同调节HUVECs中CaR介导的Ca(2+)内流和一氧化氮生成。
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