Analysis of mechanisms for intracellular Cl^- homeostasis in cardiac cells and its physiological significances.

心肌细胞内Cl^-稳态机制及其生理意义分析。

• 批准号: 11670095
• 负责人: HOUICHI Yorinaka
• 金额: $ 2.3万
• 依托单位: Kumamoto University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670095/
• 关键词: Intracellular chloride concentration; Cl^--oscillation; Cl^--channels; Cl^--HCO_3-exchanger; Ischemia; reperfusion; Extracellular ATP; Mitochondrial uncouplers; MQAE; Cl^-トランスポート阻害薬; サザンカサポニン; bFGF; 局所心筋層血流量; 不整脈; 細胞内Cl^-ホメオスタシス; ラット心筋梗塞モデル

Our previous studies showed that simulated ischemia increased intracellular chloride concentration ([Cl^-]_i) in guinea pig ventricular muscles (Am. J. Physiol. 1998). In the present study, we investigated possible mechanisms for modulation of Cl^- homeostasis in cardiac cells and its physiological significances.First, using Cl^--fluorescence dye (N-(6-methoyquinolyl) acetoxy-acetyl-ester, MQAE) methods, we observed changes in [Cl^-]_i after application of mitochondrial uncoupleis, m-chlorocarbonylcyanide phenylhydrazone(CCP) and 2,4-dinitrophenol (DNP) in isolated guinea pig ventricular myocytes. CCP and DNP, at a low concentration, induced a transient increase while at a high concentration induced a persistent elevation of [Cl^-]_i. This CCP- or DNP-induced increase in [Cl^-]_i was suppressed by application of stilbene derivatives and lowing Cl^- concentration in perfused solution, indicated that modulation of mitochondrial metabolism plays an important role in regulation of Cl^- hom … More eostasis in cardiac cells. Second, effects of extracellular ATP on [Cl^-]_i were investigated in guinea pig ventricular muscles using double-barreled ion-selective microelectrode techniques. MgATP induced an increase in [Cl^-]_i and triggered a transient acidification of pH_i. Both increases in [Cl^-]_i and intracellular H^+ induced by ATP were prevented by SITS and DDS, or by a Cl^--free solution. Our findings showed that the increased extracellular ATP concentrations might trigger an increase in [Cl^-]_i in ventricular muscles.In light of previous studies showing that cardiac ischemia induced increases in extracellular micleotide concentrations and [Cl^-]_i in ventricular muscles, we propose that ischemia-induced accumulation of ATP concentration in the extracellular space may be an important factor to trigger increment of [Cl^-]_i during ischemia. Third, we investigated changes in [Cl^-]_i during lectin-induced activation and proliferation in human Jurkat T lymphocytes. The [Cl^-]_i was measured using MQAE methods. Lectins, phytohemagglutinin and concanavalin A, dose-dependently increased [Cl^-]_i and triggered intracellular Cl^- oscillation in human Juikat lymphocytes. However, mitochondria metabolism inhibitors, CCP and DNP, increased [Cl^-]_i without triggering Cl^--oscillation. Furthermore, both lectins and metabolism inhibitors-induced elevation in [Cl^-]_i wereblocked by a Cl^--free solution or by application of anthracene-9-carboxylate (9-AC). Since extracellular Cl^--free condition and 9-AC also inhibited PHA-induced proliferation, we suggested that elevation of [Cl^-]_i via activation of Cl^-- channels and increase in incidence of Cl^--oscillation would play an important role in modulation of T cell activation and proliferation.In conclusion, our results suggest that modulation of [Cl^-]_i homeostasis would play an important role in some pathologic conditions (ischemia and reperfusion in cardiac cells) or regulate some important physiological functions (T cell activation in Jurkat T cells). Less

我们之前的研究表明，模拟缺血增加了豚鼠心室肌细胞内氯离子浓度（[Cl^-]_i）。生理学杂志。1998)。在本研究中，我们探讨了心肌细胞Cl^-稳态调节的可能机制及其生理意义。首先，采用Cl^-荧光染料(N-（6-甲氧基喹啉）乙酰氧基酯（MQAE）方法，观察了线粒体解偶联剂、间氯羰基氰基苯腙（CCP）和2,4-二硝基苯酚（DNP）对豚鼠离体心室肌细胞[Cl^-]_i的影响。CCP和DNP在低浓度时诱导短暂性升高，而在高浓度时诱导[Cl^-]_i持续升高。CCP-或dnp诱导的[Cl^-]_i升高被苯乙烯衍生物的应用和灌注液中Cl^-浓度的降低所抑制，表明线粒体代谢的调节在心肌细胞Cl^- -的调节中起重要作用。其次，利用双管离子选择微电极技术研究了细胞外ATP对豚鼠心室肌[Cl^-] i的影响。MgATP诱导了[Cl^-]_i的增加，并引发了pH_i的短暂酸化。ATP诱导的[Cl^-]_i和细胞内H^+的增加均被sit和DDS或无Cl^-溶液所阻止。我们的研究结果表明，细胞外ATP浓度的增加可能引发心室肌[Cl^-]_i的增加。鉴于先前的研究表明心脏缺血引起心室肌细胞外微肽浓度和[Cl^-]_i的增加，我们提出缺血引起的细胞外ATP浓度的积累可能是缺血期间触发[Cl^-]_i增加的重要因素。第三，我们研究了凝集素诱导的人Jurkat T淋巴细胞活化和增殖过程中[Cl^-]_i的变化。采用MQAE法测定[Cl^-]_i。凝集素，植物血凝素和豆豆蛋白A，剂量依赖性地增加[Cl^-]_i并触发人Juikat淋巴细胞内的Cl^-振荡。然而，线粒体代谢抑制剂，CCP和DNP，增加了[Cl^-]_i而不触发Cl^-振荡。此外，凝集素和代谢抑制剂诱导的[Cl^-]_i升高被无Cl^-溶液或应用蒽-9-羧酸盐（9-AC）阻断。由于细胞外无Cl^-条件和9-AC也抑制pha诱导的增殖，我们认为通过激活Cl^-通道来升高[Cl^-]_i和增加Cl^-振荡的发生率在T细胞活化和增殖的调节中起重要作用。综上所述，我们的研究结果表明，[Cl^-]_i稳态的调节可能在某些病理条件（心肌细胞缺血和再灌注）或调节一些重要的生理功能（Jurkat T细胞中的T细胞活化）中发挥重要作用。少

项目成果

頼仲 方一: "心筋虚血期間に細胞内クロライドイオンの動態とその意義"Acta Acad Med Jiangxi. 41(2). 25-32 (2001)

Hoichi Yorinaka：“心肌缺血时细胞内氯离子的动态及其意义”江西医学学报41（2）25-32。

頼 仲方: "心筋虚血期間に細胞内クロライドイオンの動態と其の意義"Acta Academiae Medicinae Jiangxi. 41(2). 25-32 (2001)

中方佑里：“心肌缺血时细胞内氯离子的动态及其意义”江西医学科学院学报41（2）（2001）。

頼 仲方, 邵 占強, 西 勝英: "サザンカサポニンのマウス心筋虚血再灌流モデルでの保護作用と可能なメカニズム"日本薬理学雑誌. 121(3). 72 (2003)

Nakagata Lai、Zhanqiang Shao、Katsuhide Nishi：“Southern casaponin 对小鼠心肌缺血再灌注模型的保护作用及其可能的机制”日本药理学杂志 121(3) (2003)。

Lai Z.-F.and Nishi K.: "Extracellular ATP induced increase in intracellular Cl-concentrations and its role on spontaneous electrical activities in guinea pig ventricular muscle."Jap.J.Pharmacol.. 82(Supp I). 308 (2000)

Lai Z.-F. 和 Nishi K.：“细胞外 ATP 诱导细胞内 Cl 浓度增​​加及其对豚鼠心室肌​​自发电活动的作用。”Jap.J.Pharmacol.. 82（补充 I）。

Lai Z-F, Chen Y-Z, Nishimura Y, Nishi K.: "An amiloride sensitive and voltage-dependent Na^+-channel in an HLA-DR-restricted human T cell clone"The Journal of Immunology. 165. 83-90 (2000)

Lai Z-F、Chen Y-Z、Nishimura Y、Nishi K.：“HLA-DR 限制性人 T 细胞克隆中的阿米洛利敏感且电压依赖性 Na + 通道”免疫学杂志。