急性缺氧对大鼠海马神经元钙激活钾通道的作用

目的:本实验采用膜片钳单通道技术研究缺氧条件下对培养新生大鼠海马神经元钙激活钾通道的作用;方法:取1 ～3天的SD大鼠海马神经元组织,制成细胞悬液,加入含80 %DMEM(dulbecco' s modified eagle' s medium)、20 %小牛血清培养基进行培养,将培养3 ～5天的形态典型的神经元置于对称性高钾溶液中,用膜钳技术记录单通道电流,其通道电流通过膜片钳放大器(CEZ -2200)放大,滤波(3KHz)后,经A/D、D/A转换器输入计算机,采用pClamp6 .0 .6软件采样分析;结果:在细胞贴附式下,应用氰化钠(20μmol/L)造成细胞急性缺氧,在缺氧早期(5 ～20 min)通道开放概率增加(P<0 .01或P<0 .05 ,n=5) ,而缺氧后期(20 ～30 min) ,通道开放概率明显降低,表明缺氧时间对通道的开放概率有明显影响。结论:缺氧早期神经元钙激活钾通道有自身激活作用,当钙激活钾通道激活时,钾离子外流增加,产生膜的复极化,使去极化不易产生,从而稳定细胞膜及降低细胞的兴奋性,这可能是缺氧早期神经元自身的一种代偿机制。

Effect of acute hypoxia on calcium-activated potassium channels in Rat Hippocampal neurons

Objective:The object of this study was to investigate the effect of acute hypoxia on calcium-activated potassium chantels in cultured newbern rat hJnpocampal neurons in hypoxic conditions with patch-Clamp technique;Method:The rat hippocampal neurons were isolated from 1-3 days old Sprague-Dawly rat and cultured in media(80%DMEM+20%bovine serum)for 3～5 days.Single channel current signals were recorded with patch-Clamp amplifier(CEZ-2200),then input to computer via A/D,D/A converter for analysis using pClamp Software(version 6.0.6);Result:In cell-attached patch,when sodium cyanide(NaCN 20μmol/L)which can cause cell hypoxia was added to the bath solution,the increase of open probability occurred in early hypoxic stage(5～20min)(P<0.01 or P<0.05,n=5),the decrease of open probability in late hypoxic stage(20～30min),this indicates that open probability changes with hypoxic stage;Conclusion:The result indicates that in early hyoxic stage there is self-activation of the Ca 2+-activated potassium channels in rat hippocampal neurons.When the K_ ca channels were activated,K+ effluxes increase,which induces cell membrane hyperpolarization against excessive neurons depolarization,and so stabilizes cell membrane and decreases cell excitability.It may be an autocompensating mechanism of neurons in early hypoxic stage.