中文摘要：

目的 ：探讨蛋白激酶A（protein kinase A,PKA）对小鼠皮层神经元酸敏感离子通道（acid sensing ion channels,ASICs）功能的影响及机制。方法：取原代培养小鼠皮层神经元,通过应用钙影像、全细胞膜片钳、免疫荧光和Western Blot等技术,分别观察PKA激动剂和抑制剂对ASICs功能的影响及机制。结果：（1）在给予PKA激动剂或抑制剂前神经元ASICs电流幅值约为（224.2±17.59）pA（n=15）,预孵育PKA激动剂forskolin（10μmol/L）使电流幅值减小为（108±14.67）pA（n=9）,与对照组相比,差异有统计学意义（P〈0.01）,而预孵育PKA抑制剂PKAI（5μmol/L）则使电流幅值增加为（306.3±22.9）pA（n=12）,与对照组相比,差异有统计学意义（P〈0.01）;（2）在给予PKA激动剂或抑制剂前,酸诱导的胞内钙△F/F值为（0.708±0.07）（n=20）,PKA激动剂forskolin（10μmol/L）使其减少为（0.140±0.013）（n=17）,与对照组相比,差异有统计学意义（P〈0.01）,PKA抑制剂PKAI（5μmol/L）使其增加为（0.978±0.108）（n=19）,与对照组相比,差异有统计学意义（P〈0.01）;（3）在加入forskolin或PKAI 24 h后,神经元ASIC1总荧光密度无明显改变（P〉0.05）,但神经元ASIC1膜荧光密度从对照组的（78.58±7.42）（n=32）减少到forskolin孵育后的（31.41±3.38）（n=57）,与对照组相比,差异有统计学意义（P〈0.01）,而PKAI孵育后神经元ASIC1膜荧光密度增加到（118.29±11.50）（n=34）,与对照组相比,差异有统计学意义（P〈0.01）。结论 ：PKA通过影响ASICs膜分布负性调节小鼠皮层神经元ASICs电流及由酸诱导的胞内钙增加。

英文摘要：

Objective：To explore the effects and mechanisms of protein kinase A（PKA） on the function of acid sensing ion channels（ASICs） in mouse cortical neurons.Methods： Explore the effects of the agonist and inhibitor of PKA on the function of ASICs by using the techniques of whole cell patch clamp,calcium imaging,Western Blot,and immunofluorescence in cultured cortical neurons.Results：（1）ASICs currents were induced with the amplitude of（224.2±17.59） pA（n=15） in cortical neurons before the treatment of the agonist and antagonist of PKA.Pre-incubation of the agonist of PKA forskolin（10 μmol/L） reduced the amplitude of ASICs currents to（108±14.67） pA（n=9,P〈0.01 vs control）,whereas pre-incubation of the inhibitor of PKA fragment 6-22 amide（PKAI,5 μmol/L） raised it to（306.3±22.9） pA（n=12,P〈0.01 vs control）.（2）The normalized variation of i（△F/F） was（0.708±0.07）（n=20） in cortical neurons before the treatment of PKA agonist or inhibitor.Pre-incubation of forskolin（10 μmol/L） reduced the value of △F/F to（0.140±0.013）（n=17,P〈0.01 vs control）,whereas pre-incubation of PKAI（5 μmol/L） raised it to（0.978±0.108）（n=19,P0.05 vs control）.（3）After the treatment of forskolin or PKAI for 24 h,the fluorescence density of total ASICs were not changed significantly（P0.05 vs control）.However,the fluorescence density of membrane ASICs was reduced from（78.58±7.42）（n=32） to（31.41±3.38）（n=57,P〈0.01,vs control） after forskolin incubation,and the fluorescence density of membrane ASICs was increased to（118.29±11.50）（n=34,P〈0.01,vs control） after PKAI incubation.Conclusion： PKA negatively regulates the currents of ASICs and the elevation of intracellular Ca2＋ induced by acid via influencing the membrane distribution of ASICs in mouse cortical neurons.