Regulation and effects of intracellular Cl concentrations

细胞内 Cl 浓度的调节和影响

• 批准号: 09470027
• 负责人: INAGAKI Chiyoko
• 金额: $ 6.91万
• 依托单位: Kansai Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09470027/
• 关键词: chloride ion; c-fos expression; brain; ammonia; anion exhanger; protein kinase C; hypoxia; chloride pump; クロライドイオン; c-fos; アミロイドβ蛋白; ナトリウム利尿ペプチド; エタクリン酸; 神経成長因子

(1) Intraneuronal ClィイD1=ィエD1 concentrations ([ClィイD1=ィエD1]i) and immediately early gene (c-fos) expressionIntracerebroventricular administration of a ClィイD1-ィエD1 pump inhibitor, ethacrynic acid, caused convulsions in mice. Expression of c-fos was stimulated biphasically with the peaks at 60min and 10-14 days after the convulsion. Expression of nerve growth factor, damage of GABAergic neurons and development of seizure susceptibility were also observed. A transient increase in neuronal [ClィイD1-ィエD1]i thus appeared to cause biphasic increases in c-fos expression and related modulation of neuronal functions.(2) Mechanisms of ammonia-induced increases in neuronal [ClィイD1=ィエD1]iA neurotoxic factor in hepatic encephalopathy, ammonia (2 mM, 48 hr), increased [ClィイD1-ィエD1]i in cultured rat hippocampal neurons. The increase was found to be due to enhanced expression of anion exchanger (AE3) mediated by protein kinase C activation.(3) ClィイD1=ィエD1 transport in hypoxia/reoxygenaion-induced changes in pHi and {CaィイD12+ィエD1]i in myocytesUnder hypoxia/reoxygenaion conditions, pHi decreased and [CaィイD12+ィエD1]i increased during hypoxia and reoxygenation, respectively. Inhibitors of anion exchanger (SITS and DIDS) and removal of medium ClィイD1-ィエD1 or HCOィイD23ィエD2 attenuated such changes in pHi and [CaィイD12+ィエD1]i. Inhibitors of protein kinase C slightly reduced the changes in pHi. Involvement of anion exchanger in hypoxia-induced pHi changes was first demonstrated with the resulting inhibitory effects on reoxygenation-induced increases in [CaィイD12+ィエD1]i.(4) Subunits and cDNA cloning of neuronal ClィイD1=ィエD1 pumpA new ClィイD1-ィエD1 transporter, ClィイD1-ィエD1 pump, was isolated as 520 kDa protein complex from the rat brain. SDS-PAGE analyses yielded 4 subunits (51, 55, 60 and 62kDa), and 51kDa protein appeared to be a catalytic subunit. cDNA for 55 kDa protein was cloned from the rat brain cDNA library, and its primary structure was assumed to be a new peptide with 475 amino acids.

(1)神经元内Clィイd1=ィエd1浓度([Clィイd1=ィエd1]i)和即刻早期基因(c-fos)表达侧脑室注射氯化ィイd1-ィエd1泵抑制剂乙氰酸可引起小鼠惊厥。C-fos的表达呈双向刺激，高峰出现在惊厥后60min和10~14d。观察神经生长因子的表达、GABA能神经元的损伤及癫痫敏感性的发展。(2)氨可诱导肝性脑病大鼠海马神经元[CLィイD1=ィエD1]IA神经毒性因子的增加，氨水(2 mM，48小时)可使培养的大鼠海马神经元[CLィイD1-ィエD1]i升高。(2)氨诱导肝性脑病大鼠海马神经元[CIィイD1=ィエD1]IA升高的机制(3)缺氧/复氧引起pH i和心肌细胞[CaィイD12+ィエD1]i在缺氧/复氧条件下分别降低，[CaィイD12+ィエD1]i升高。阴离子交换抑制剂(SITS和DIDS)和去掉培养液ClィイD 1-ィエD 1或HcoィイD23ィエD 2可减弱pH i和[CaィイD12+ィエD 1]i的这种变化，蛋白激酶C的抑制剂使pH I的变化略有减少。首次证实阴离子交换器参与低氧诱导的PHI变化，并由此抑制复氧诱导的[CaィイD12+ィエD1]i升高。(4)从大鼠脑中分离出新的CLィイD1=ィエD1 Pumpa新的CLィイD1-ィエD1转运体，CLィイD1-ィエD1泵，作为520 kDa的蛋白复合体。SDS-PAGE分析显示有4个亚基(51、55、60和62 kDa)，其中51 kDa蛋白为催化亚基。从大鼠脑cDNA文库中克隆了55 kDa蛋白的cDNA，推测其一级结构为一种新的多肽，含475个氨基酸。

项目成果

Yagyu, K. 他: "Lithium decreases Cl-ATPase activity and increases intracellular Cl concentration in cultured hippocampal neurons."Brain Res.. 821. 530-534 (1999)

Yagyu, K. 等人：“锂可降低培养的海马神经元中 Cl-ATP 酶活性并增加细胞内 Cl 浓度。”Brain Res.. 821. 530-534 (1999)

稲垣千代子: "(Cl^-チャネルとCl^-輸送体) 「創薬」分担"共立出版(長尾拓他編). 5 (2000)

Chiyoko Inagaki：“（Cl^-通道和 Cl^-转运蛋白）‘药物发现’共享”Kyoritsu Shuppan（由 Taku Nagao 等人编辑）5 (2000)。

Minamino M, et al: "Effects of protein kinase and phosphatase inhibitons ・・" Brain Research. (in press). (1998)

Minamino M 等人：“蛋白激酶和磷酸酶抑制剂的影响··”大脑研究（出版中）。

Kawasaki E. et al.: "Single cell RT-PCR demonstrates expression of"Brain Research. 838. 166-170 (1999)

Kawasaki E. 等人：“单细胞 RT-PCR 证明了”Brain Research 的表达。

Zeng, X.-T., Higashida, T., Hara, M., Hattori, N., Kitagawa, K., Omori, K. and Inagaki, C.: "Antiserum against ClィイD1-ィエD1 pump complex recognized 51 kDa protein, a posible catalytic unit in the rat brain."Neurosci. Lett.. 258. 85-88 (1998)

Zeng, X.-T.、Higashida, T.、Hara, M.、Hattori, N.、Kitakawa, K.、Omori, K. 和 Inagaki, C.：“针对 CliiD1-ieD1 泵复合物识别的 51 kDa 蛋白的抗血清，大鼠大脑中可能的催化单元。“Neurosci. Lett.. 258. 85-88 (1998)