【摘要】 目的：探讨机械敏感性离子通道Piezo1激活促进冠状动脉平滑肌细胞（CASMCs）内Ca²⁺浓度([Ca²⁺]_i)升高的机制。方法：采用原代成年大鼠CASMCs为研究对象，用细胞免疫荧光技术观察Piezo1在CASMCs中的表达与亚细胞定位情况；利用Western blot检测用si RNA敲减CASMCs中Piezo1和基质相互作用分子1（STIM1）后CASMCs中的蛋白表达情况；利用激光共聚焦显微镜，通过基于Flou-4 AM负载的细胞内Ca²⁺成像技术，测量CASMCs[Ca²⁺]_i的变化。结果：细胞免疫荧光染色结果显示，Piezo1在原代大鼠CASMCs中表达；Piezo1与肌浆/内质网Ca²⁺-ATP酶2（SERCA2）、线粒体外膜蛋白TOM20和核膜蛋白lamin B1共定位程度较高。Western blot结果表明，si RNA转染后STIM1和Piezo1蛋白表达下调（P<0.05）。激光共聚焦显微镜检测[Ca²⁺]_i结果表明：与对照组相比，Piezo1激动剂Yoda1诱导CASMCs的胞外Ca²⁺内流增加（P<0.01），抑制L型钙通道不影响Yoda1诱导的Ca²⁺内流；Yoda1诱导CASMCs胞内Ca²⁺释放增加（P<0.01），抑制内质网上的钙通道（雷诺丁受体和1,4,5-三磷酸肌醇受体）不影响Yoda1诱导的胞内Ca²⁺释放；使用毒胡萝卜素（TG）排空内质网中Ca²⁺后，Yoda1仍能诱导CASMCs中其它细胞器的Ca²⁺释放（P<0.01）；抑制L型钙通道后，使用钙库操纵性钙通道（SOCC）抑制剂BTP2或敲减STIM1使Yo‐da1诱导CASMCs胞外Ca²⁺内流减少（P<0.01）；敲减Piezo1使TG诱导的CASMCs内质网Ca²⁺释放增加（P<0.05），但不影响TG诱导的胞外Ca²⁺内流；抑制L型钙通道和SOCC后，敲减Piezo1导致Yoda1诱导的CASMCs胞内Ca²⁺释放以及胞外Ca²⁺内流均减少（P<0.01）。结论：Piezo1激动剂诱导CASMCs胞外Ca²⁺内流主要通过细胞膜上的Piezo1通道和间接激活的SOCC，也可触发胞内细胞器Ca²⁺释放，共同升高[Ca²⁺]_i。更多还原

【Abstract】 AIM:To investigate the mechanism of Piezo1 channel activation promoting the increase in intracellular Ca²⁺concentration ([Ca²⁺]_i) of rat coronary artery smooth muscle cells （CASMCs）.METHODS:The primary CASMCs of SD rats were cultured,and the expression and subcellular localization of Piezo1 in the cells were observed by immunofluorescence staining.The Piezo1 and stromal interaction molecule 1 （STIM1） in CASMCs were knocked down by si RNA transfection,and the expression levels of the proteins were detected by Western blot.Utilizing laser confocal microscopy,the change of[Ca²⁺]_i in CASMCs was detected by Fluo-4 AM fluorescent probes.RESULTS:It was confirmed by immunofluorescence staining that the expression of Piezo1 existed in primary rat CASMCs.Immunofluorescence staining also showed that Piezo1 was co-located with sarco-/endoplasmic reticulum Ca²⁺-ATPase 2 （SERCA2）,mitochondrial outer membrane protein TOM20 and nuclear membrane protein lamin B1.Western blot results showed that the protein expression levels of STIM1 and Piezo1 were significantly down-regulated by si RNA transfection （P<0.05）.Compared with control group,Yoda1,the agonist of Piezo1,could increase the extracellular Ca²⁺influx of CASMCs （P<0.01）.However,the Ca²⁺influx mediated by Yoda1 was not affected by the inhibition of L-type calcium channels.Treatment with Yoda1 increased the intracellular Ca²⁺release of CASMCs （P<0.01）.However,inhibition of calcium channels on endoplasmic reticulum,ryanodine receptor and inositol 1,4,5-triphosphate receptor,did not affect intracellular Ca²⁺release mediated by Yoda1.After the Ca²⁺in endoplasmic reticulum was emptied using thapsigargin （TG）,Yoda1 also mediated the Ca²⁺release of other organelles in CASMCs （P<0.01）.After inhibition of L-type calcium channels,treatment with store-operated calcium channel （SOCC） inhibitor BTP2 or knockdown of STIM1 led to the decrease in extracellular Ca²⁺influx of CASMCs mediated by Yoda1 （P<0.01）.Treatment with TG increased the release of Ca²⁺from the endoplasmic reticulum of CASMCs after knockdown of Piezo1 （P<0.05）,but the extracellular Ca²⁺influx mediated by TG was not affected.After inhibition of L-type calcium channels and SOCC,knockdown of Piezo1 led to the decreases in intracellular Ca²⁺release and extracellular Ca²⁺influx induced by Yoda1 （P<0.01）.CONCLUSION:The Piezo1 agonist orchestrates the influx of extracellular Ca²⁺by activating Piezo1 channels on the cell membrane and inducing the indirect activation of SOCC.Moreover,it facilitates the release of Ca²⁺from organelles.Consequently,these pathways synergistically elevate the[Ca²⁺]_i of rat CASMCs.更多还原