Multi-point measurement of intracellular chloride concentrations.

细胞内氯离子浓度的多点测量。

• 批准号: 02557012
• 负责人: INAGAKI Chiyoko
• 金额: $ 6.66万
• 依托单位: Kansai Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02557012/
• 关键词: Chloride ion; Cl^-; Cl^--sensitive probe; corneal endothelial cell; retinal pigmemt epithelial cell; hippocampal pyramidal cell; Cl^- pump; Na^+; K^+; Cl^- cotransporter; Cl^-感受性蛍光色素; 細胞内Cl^-濃度; エタクリン酸; フロセミド; 海馬神経細胞

1. Cl^--sensitive probe: A Cl^--sensitive fluorescent dye,N-(6-methoxyquinolyl) acetoethyl ester (MQAE, purity > 99%) was synthesized by the method of Verkman.2. Multi-channel window photometer:The fluorescence intensity of a MQAE-loaded cell was continuously recorded on an inverted microscope equipped with a night vision camera-based multi-channel window photometer, which was devised to measure the photo intensity at desired multiple windows in a microscopic view. Using this equipment, changes in the intracellular Cl^- concentration ([Cl^-]_i) were monitored to elucidate the mechanisms for its regulation.3. Corneal endothelial cell:Stationary [Cl^-]_i of rabbit corneal endothelial cells was 30.1*1.6 mM. Efflux of Cl^- with alpha_2^-adrenoceptor stimulation,and an increase in Cl^- permeability of plasma membranes with beta-adrenoceptor stimulation were suggested.4. Retinal pigment epithelial cell (RPE): Stationary [Cl^-]_i of rabbit RPE was approx. 90 mM. Atrial natriuretic peptide was demonstrated to accumulate through activation of Na^+/K^+/Cl^- co-transporter regulated via cGMP-dependent protein kinase.5. Hippocampal pyramidal cell:Stationary [Cl^-]_i of rat hippocampal pyramidal cells was 6.63*1.56 mM. An ATP-driven Cl^- pump extrudes Cl^- and Na^+/K^+/Cl^- co-transporter accumulates the same anion.

1. Cl^-敏感探针：采用Verkman法合成了氯离子敏感的荧光染料N-（6-甲氧基喹啉基）乙酰乙酯（MQAE，纯度> 99%）.多通道窗口光度计：在配备有基于夜视相机的多通道窗口光度计的倒置显微镜上连续记录MQAE负载的细胞的荧光强度，所述多通道窗口光度计被设计用于测量显微镜视图中所需多个窗口处的光强度。利用该装置，监测细胞内Cl^-浓度（[Cl^-]_i）的变化，以阐明其调节机制.角膜内皮细胞：兔角膜内皮细胞的稳态[Cl^-]_i为30.1*1.6 mM，α_2 ^-肾上腺素能受体刺激可使Cl^-流出，β-肾上腺素能受体刺激可使Cl^-通透性增加.视网膜色素上皮细胞（Retinal pigment epithelial cell，RPE）：视网膜色素上皮细胞的稳态[Cl^-] i约为0.001。心房利钠肽通过激活cGMP依赖性蛋白激酶调节的Na^+/K^+/Cl^-共转运蛋白而蓄积。海马锥体细胞：大鼠海马锥体细胞的静态[Cl^-] i为6.63*1.56 mM，ATP驱动的Cl^-泵排出Cl^-，Na^+/K^+/Cl^-共转运体积累相同的阴离子。

项目成果

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稲垣 千代子(分担): "実験医学:膜電位依存性Cl^-チャネル(イオンチャネルとレセプター)" 羊土社, 6 (1992)

Chiyoko Inagaki（撰稿人）：“实验医学：膜电位门控 Cl^-通道（离子通道和受体）” Yodosha，6 (1992)

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Inagaki,C.,Hara,M. and Inoue,M.: "Cl^- transporting ATPase in the rat brain." Electrogenic Chloride Transporters in Biological Membranes. Springer-Verlag.

稻垣 C.、原 M.