Piezo1通道激活促进大鼠冠脉平滑肌细胞内钙浓度升高的机制研究  

Mechanism of Piezo1 channel activation promoting increase in intracellular calcium concentration of rat coronary smooth muscle cells

作　　者：蔡泳江 郑燕湘 王梓帆 邝素娟 杨慧[2] 饶芳 邓春玉 CAI Yongjiang;ZHENG Yanxiang;WANG Zifan;KUANG Sujuan;YANG Hui;RAO Fang;DENG Chunyu(School of Pharmaceutical Sciences,Southern Medical University,Guangzhou 510515,China;Key laboratory of Clinical Pharmacology,Department of Medical Research,Guangdong Academy of Medical Sciences,Guangdong Provincial Peo-ple's Hospital,Southern Medical University;School of Medicine,South China University of Technology,Guangzhou 510006,China)

机构地区：[1]南方医科大学药学院,广东广州510515 [2]南方医科大学附属广东省人民医院,广东省医学科学院,医学研究部临床药理重点实验室,广东广州510080 [3]华南理工大学医学院,广东广州510006

出　　处：《中国病理生理杂志》2024年第1期9-17,共9页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.82170415,No.82370411);广东省基础与应用基础研究基金资助项目(No.2021A1515011551)。

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Ca^(2+)浓度([Ca^(2+)]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Ca^(2+)成像技术,测量CASMCs[Ca^(2+)]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Ca^(2+)-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[Ca^(2+)]i结果表明:与对照组相比,Piezo1激动剂Yoda1诱导CASMCs的胞外Ca^(2+)内流增加(P<0.01),抑制L型钙通道不影响Yoda1诱导的Ca^(2+)内流;Yoda1诱导CASMCs胞内Ca^(2+)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Ca^(2+)释放;使用毒胡萝卜素(TG)排空内质网中Ca^(2+)后,Yoda1仍能诱导CASMCs中其它细胞器的Ca^(2+)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Ca^(2+)内流减少(P<0.01);敲减Piezo1使TG诱导的CASMCs内质网Ca^(2+)释放增加(P<0.05),但不影响TG诱导的胞外Ca^(2+)内流;抑制L型钙通道和SOCC后,敲减Piezo1导致Yoda1诱导的CASMCs胞内Ca^(2+)释放以及胞外Ca^(2+)内流均减少(P<0.01)。结论:Piezo1激动剂诱导CASMCs胞外Ca^(2+)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Ca^(2+)释放,共同升高[Ca^(2+)]i。AIM:To investigate the mechanism of Piezo1 channel activation promoting the increase in intracellular Ca^(2+)concentration([Ca^(2+)]_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[Ca^(2+)]_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 Ca^(2+)-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 Ca^(2+)influx of CASMCs(P<0.01).However,the Ca^(2+)influx mediated by Yoda1 was not affected by the inhibition of L-type calcium channels.Treatment with Yoda1 increased the intracellular Ca^(2+)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 Ca^(2+)release mediated by Yoda1.After the Ca^(2+)in endoplasmic reticulum was emptied using thapsigargin(TG),Yoda1 also mediated the Ca^(2+)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 Ca^(2+)influx of CASMCs mediated by Yoda1(P<0.01).Treatment with TG increased the release of Ca^(2+)from the endop

关 键 词：Piezo1通道 钙离子 冠状动脉平滑肌细胞 

分 类 号：R541.4[医药卫生—心血管疾病] R363.2[医药卫生—内科学] R329.25[医药卫生—临床医学]