H_(2)O_(2)对人胚肾293细胞钠钙交换体内向电流的作用及机制  

Effect of H_(2)O_(2) on Na^(+)/Ca^(2+)-exchanger inward current in human embryonic kidney cells and its mechanism

作　　者：项国剑 柯永鑫 陈茜 王庆峰[5] 曾琳雯 李泱 张建成[1,2] XIANG Guojian;KE Yongxin;CHEN Qian;WANG Qingfeng;ZENG Linwen;LI Yang;ZHANG Jiancheng(Cardiology Department,Fujian Provincial Hospital,Fuzhou,Fujian 35000l,China;Provincial Clinical Medicine College of Fujian Medical University;Department of Cardiovascular Medicine,Fuzhou Changle District Hospital;Department of Intensive Care Medicine,Fujian Provincial Hospital;Department of Electrocardiogram Diagnosis,Fujian Provincial Hospital;Cardiology Department,General Hospital of People's Liberation Army)

机构地区：[1]福建省立医院心血管内科,福州福建350001 [2]福建医科大学省立临床医学院心血管内科 [3]福州市长乐区医院心血管内科 [4]福建省立医院重症医学科 [5]福建省立医院心电诊断科 [6]解放军总医院老年心血管病研究所

出　　处：《中华高血压杂志》2023年第11期1079-1085,共7页Chinese Journal of Hypertension

基　　金：国家自然科学基金(82070341)。

摘　　要：目的 探讨H_(2)O_(2)对钠钙交换体(NCX)内向电流的调控及可能的机制。方法 利用绿色荧光蛋白标记表达系统将NCX1.1质粒转染人胚肾293细胞(HEK293),全细胞膜片钳技术记录NCX内向电流,观察H_(2)O_(2)对NCX1.1内向电流和I-V曲线的作用及浓度依赖性作用。选取120个转染阳性的HEK293细胞,随机均分成对照组、H_(2)O_(2)处理组、Gi蛋白抑制剂百日咳毒素(PTX)及H_(2)O_(2)处理组,蛋白激酶A(PKA)抑制剂H89及H_(2)O_(2)处理组,记录对比处理前后NCX内向电流的变化。Western-blot法检测HEK293组、H_(2)O_(2)组、PTX+H_(2)O_(2)组、H89+H_(2)O_(2)组处理24 h后细胞NCX1.1蛋白水平的变化。结果 H_(2)O_(2)可使NCX1.1内向电流增加,使其电流密度从(-6.22±0.53)pA/pF增加至(-12.92±1.12)pA/pF(n=60,t=2.54,P<0.01),在细胞外灌流不同浓度的H_(2)O_(2),NCX1.1内向电流增加呈浓度依赖性特征。在倒斜坡刺激模式中,H_(2)O_(2)对内向电流的作用存在电压依赖性。Gi蛋白选择性抑制剂PTX可以逆转H_(2)O_(2)的增加效应[H_(2)O_(2):(-12.92±1.12)比H_(2)O_(2)+PTX:(-6.76±0.60)pA/pF,t=3.61,n=60,P<0.001]。PKA抑制剂H89同样可以逆转H_(2)O_(2)的增加效应[H_(2)O_(2):(-12.92±1.12)比H_(2)O_(2)+H89:(-7.22±1.60)pA/pF,n=60,t=4.17,P<0.005]。应用H_(2)O_(2)与HEK293细胞培养24 h后,观察到细胞NCX1.1蛋白表达增加,而PTX及H89可以逆转H_(2)O_(2)对NCX1.1蛋白的增加效应。结论 H_(2)O_(2)刺激可增加NCX1.1内向电流,Gi-环磷酸腺苷(cAMP)-PKA信号通路参与该过程的调节。Objective To investigate the regulation and possible mechanism of H_(2)O_(2) on the inward current of Na^(+)/Ca^(2+)-exchanger(NCX). Methods The NCX1.1 plasmid was transfected into human embryonic kidney cells(HEK293 cell) by green fluorescent protein labeling expression system. The whole cell patch clamp technique was used to record NCX current, and the concentration-dependent effect of H_(2)O_(2) on the current and Ⅰ-Ⅴ curve of NCX1.1 were recorded. A total of 120 positive HEK293 cells were selected and divided into control group, H_(2)O_(2) treatment group, pertussis toxin(Gi protein inhibitor) and H_(2)O_(2) treatment group. inhibitor H89(protein kinase A) and H_(2)O_(2) treatment. Analysis of I_(NCX) before and after treatment. Western-blot assay was used to detect the changes of NCX1.1 protein level in H_(2)O_(2) and HEK293 cell group, PTX+H_(2)O_(2) and HEK293 cell group, H89+ H_(2)O_(2) and HEK293 cell group after 24 h culture. Results H_(2)O_(2)increased the inward current of I_(NCX)[ from(-6.22±0.53) to(-12.92±1.12) pA/pF(n=60, t=2.54, P<0.01)], and the inward current of NCX1.1 was increased in a concentration-dependent manner by extracellular perfusion with different concentrations of H_(2)O_(2). The results of inverted ramp stimulation mode showed that the effect of H_(2)O_(2 )on the inward current of NCX1.1 was voltage dependent. The selective inhibitor of Gi protein PTX was added, and PTX was found to reverse the increase effect of H_(2)O_(2)[H_(2)O_(2):(-12.92±1.12) vs H_(2)O_(2)+PTX:(-6.76±0.60)pA/pF, n=60, t=3.61, P<0.001]. The PKA inhibitor H89 also reversed the increase effect of H_(2)O_(2)[H_(2)O_(2):(-12.92±1.12) vs H_(2)O_(2)+H89:(-7.22±1.60)pA/pF, n=60, t=4.17, P<0.005]. In addition, the expression of NCX1.1 protein in HEK293 cells was increased after 24h culture with H_(2)O_(2). However, PTX and H89 can reverse the increasing effect of H_(2)O_(2) on the expression of NCX1.1 protein. Conclusion H_(2)O_(2) stimulation can increase the inward current of NCX1.1, and this process is m

关 键 词：钠钙交换体 钠钙交换电流 H_(2)O_(2) 全细胞膜片钳技术 

分 类 号：R54[医药卫生—心血管疾病]