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

利用 AMPA 型谷氨酸受体表达系统研究离子通道麻醉作用机制。

• 批准号: 08671761
• 负责人: ANDOH Tomio
• 金额: $ 1.34万
• 依托单位: Yokohama City University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08671761/
• 关键词: glutamete receptors; anesthetics; PC12 cells; viral vectors; ion channels; gene transfer; 大脳皮質ニューロン; ウィルスベクター

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.

1.表达系统我们构建了包含Lacz基因作为报告基因的单纯疱疹病毒载体，发现用该载体转染PC12细胞和大鼠培养的皮层神经元时，转染效率非常高，细胞损伤最小。我们还使用单纯疱疹病毒载体在大鼠皮层神经元中构建了 NMDA 型谷氨酸受体亚基的表达系统。 我们报道，静脉麻醉药硫喷妥钠、异丙酚和氯胺酮均强烈抑制神经元烟碱受体介导的电流，但不影响 PC12 细胞中的 P2x 嘌呤能反应。这些结果表明，与神经元烟碱受体不同，P2x 嘌呤受体缺乏对麻醉剂的高亲和力位点，反映出缺乏结构同源性。我们使用大鼠培养的皮层神经元研究了巴比妥类药物对天然 AMPA 受体的影响。我们发现，尽管惊厥异构体缺乏麻醉作用，但巴比妥的镇静剂和惊厥异构体都能抑制红藻氨酸诱导的电流，这表明对 AMPA 受体的抑制对巴比妥类的麻醉作用影响不大。

项目成果

T.Andoh: "Differential effects of thiopental on neuronal nicotinic acetylcholine receptors and P_<2X> purinergic receptors in PC12 cells." Anesthesiology. 89. 1199-1209 (1997)

T.Andoh：“硫喷妥钠对 PC12 细胞中神经元烟碱乙酰胆碱受体和 P_<2X> 嘌呤能受体的不同影响。”

T.Andoh.: "Differential effects of thiopental on neuronal nicotinic acetylcholine receptors and Pzx purinergic receptors in PC12 cells." Anesthesiology. 87. 1199-1209 (1997)

T.Andoh.：“硫喷妥钠对 PC12 细胞中神经元烟碱乙酰胆碱受体和 Pzx 嘌呤能受体的不同影响。”

T.Andoh.: "Volatile anaesthetic effects on calcium condactance of plana lipid bilayers formed with synthetic lipids or extracted lipids from sarcoplasmic reticulum." British Journal of Anaesthesia.78. 66-74 (1997)

T.Andoh.：“挥发性麻醉剂对合成脂质或从肌浆网提取的脂质形成的平面脂质双层的钙电导产生影响。”

S.Kawamoto: "Expression of NMDA receptor channel subunit proteins using Baculovirus and Herpesvirus Vectors" Methods in Molecular Biology. (in press).

S.Kawamoto：“使用杆状病毒和疱疹病毒载体表达 NMDA 受体通道亚基蛋白”分子生物学方法。

服部 聡: "神経生物学のための遺伝子導入発現研究法." シュプリンガー・フェアラーク東京, (1997)

Satoshi Hattori：“神经生物学的基因转移表达研究方法”，Springer-Verlag Tokyo，（1997）