A study on mechanisms of anesthetic actions on ion channels using expression systems of AMPA type glutamate receptors.

1.表达系统我们构建了含Lacz基因报告基因的单纯疱疹病毒载体，用该载体转染PC12细胞和培养的大鼠皮层神经元，发现转染效率高，细胞损伤小。电生理研究我们研究了挥发性麻醉药对脂双层的影响，发现挥发性麻醉药增加脂双层对离子的渗透性，这些作用的大小取决于麻醉药和脂质成分，但通常很小。我们报道了静脉麻醉药，硫喷妥钠，异丙酚和氯胺酮均强烈抑制PC12细胞神经元烟碱受体介导的电流，但不影响P2x嘌呤能反应。这些结果表明，P2x嘌呤受体缺乏高亲和力的网站，麻醉剂不像神经元烟碱受体，反映缺乏结构homology.We研究了巴比妥类药物对本地AMPA受体使用大鼠培养的皮层神经元的影响。我们发现，巴比妥酸盐的抗惊厥异构体和惊厥异构体抑制红藻氨酸诱导的电流，尽管惊厥异构体缺乏麻醉作用，这表明抑制AMPA受体对巴比妥酸盐的麻醉作用贡献不大。

1.Expression systemWe constructed a Herpes Simplex viral vector including Lacz gene as a reporter and found that efficiency of trasfection is very high and cell damages are minimal when PC12 cells and rat cultured cortical neurons were transfected with this vector. We also produced an expression system of NMDA type glutamate receptor subunits in rat cortical neurons using Herpes Simplex viral vector2.Electrophysiological studyWe studied effects of volatile anesthetics on lipid bilayrs, and found that volatile anesthetics increase permeability of lipid bilayrs to ions and that magnitudes of these effects depend on anesthetics and lipid composition but are generally small.We reported that intravenous anesthetics, thiopental, propofol and ketamine, all inhibited the neuronal nicotinic receptor-mediated current strongly but did not affect P2x purinergic responses in PC12 cells. These results indicate that P2x purine receptors lack high affinity sites for anesthetics unlike neuronal nicotinic receptors, reflecting lack of structural homology.We have studied effects of barbiturates on native AMPA receptors using rat cultured cortical neurons. We found that both depressant and convulsant isomers of a barbiturate suppress the kainate-induced current despite convulsant isomer lacks the anesthetic effect, suggesting that the inhibition of AMPA receptors contributes little to the anesthetic action of barbiturates.