【摘要】 目的 探讨β肾上腺素受体对钠钙交换电流(I_NCX)的调控及可能的信号转导途径。方法 利用免疫磁珠阳性标记表达系统将NCX1.1质粒转染人胚肾293（HEK-293）细胞，选取103个转染阳性的HEK-293细胞，随机分成空白对照组（n=20）、灌流异丙肾上腺素（ISO）组（n=20）、灌流Gi蛋白抑制剂百日咳毒素及ISO组（n=20）、灌流环磷酸腺苷（cAMP）合成激动剂毛喉素（forskolin）及ISO组（n=22）,灌流蛋白激酶A（protein kinase A,PKA）抑制剂H89及ISO组（n=21）。运用全细胞膜片钳技术记录各组I_NCX的变化。采用SPSS 21.0统计软件进行数据分析。根据数据类型，分别采用t检验或ANOVA方差分析进行组间比较。结果 以灌流用药前I_NCX为空白对照组，ISO组可使内向I_NCX密度从（-6.07±1.53）pA/pF增加至（-7.89±1.61）pA/pF（n=20,P<0.05）,平均增加约30%。而预先使用G蛋白-cAMP-PKA通路关键分子激动剂或抑制剂后，ISO对I_NCX的效应均发生明显改变。其中，PTX及forskolin可使ISO对I_NCX密度增加效应更加显著，电流密度分别增至（-10.02±1.99）pA/pF及（-10.78±1.77）pA/pF,与单纯使用ISO相比，差异有统计学意义（n=20,P<0.01）,提示Gi蛋白抑制和环磷酸腺苷激动均可增加ISO的作用。而预先应用H89,ISO增加I_NCX的效应几乎消失，电流密度约为（-6.22±1.70）pA/pF,与对照组相比无显著差异（n=20,P>0.05）,提示抑制PKA可基本阻断ISO对I_NCX的刺激效应。结论 β肾上腺素受体激动可增加NCX1.1内向电流，其可能通过G蛋白-cAMP-PKA信号通路参与调节。更多还原

【Abstract】 Objective To investigate the regulation of β-adrenoceptor on sodium-calcium exchange current(I_NCX) and its possible signal transduction pathway. Methods The NCX1.1 plasmid was transfected into human embryonic kidney-293（HEK-293） cell by immunomagnetic-bead-based positive expression system. Transfected HEK-293 cells totaling 103 with positive transfection were selected and randomly divided into blank control group（n=20）, isoproterenol（ISO） group（n=20）, pertussis toxin（PTX） plus ISO group（n=20）, forskolin plus ISO group（n=22）, and H89 [protein kinase A（PKA） inhibitor] plus ISO group（n=21）. I_NCX changes in each group were recorded with whole-cell patch clamp technique. SPSS statistics 21.0 was used for statistical analysis. According to data type, t-test was used for comparison between two groups, and ANOVA was used for comparison between multiple groups. Results Against control group, ISO increased the inward I_NCX density from（-6.07±1.53） pA/pF to（-7.89±1.61） pA/pF（n=20, P<0.05）, with an average increase of about 30%. However, the effect of ISO on I_NCX current was significantly changed after pre-administration of key molecular agonists or inhibitors of Gi-cAMP-PKA pathway. PTX and forskolin, significantly enhanced the effect of ISO, had a more significant effect on I_NCX density increase, the current density increased to（-10.02±1.99） pA/pF and（-10.78±1.77） pA/pF, respectively. Compared with ISO alone, there was a significant difference（n=20, P<0.01）, suggesting that both the inhibition of Gi protein and activation of cAMP can enhance the effect of ISO. However, the effect of pre-treatment with PKA inhibitor H89 showed no enhancement on I_NCX, and the current density was about（-6.22±1.70） pA/pF, which was not significantly different from that of the control group（n=20,P>0.05）. It suggested that PKA inhibition can basically block the effect of ISO on I_NCX. Conclusion β-adrenoceptor activation may enhance the NCX1.1 inward current, probably through stimulating G protein-cAMP-PKA signaling pathway.更多还原