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

我们先前的研究表明，模拟缺血使豚鼠心室肌细胞内氯离子浓度([Cl^-]i)升高。J.Physiol.1998年)。本研究探讨了心肌细胞氯离子稳态的可能调节机制及其生理意义。首先，采用氯离子荧光染料(N-(6-甲氧基喹啉)乙酰氧基乙酸乙酯，MQAE)方法，观察了线粒体解偶联、间氯羰基氰基苯肼(CCP)和2，4-二硝基酚(DNP)作用于豚鼠心肌细胞后[Cl~-]_i的变化。CCP和DNP在低浓度时引起一过性升高，而在高浓度时引起[Cl^-]i持续升高，这种升高可被二苯乙烯类化合物和降低灌流液中的Cl2+浓度所抑制，表明线粒体代谢的调节在调节Cl^-HOM…中起重要作用心肌细胞内有更多的稳定。其次，采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。镁三磷酸腺苷可引起细胞内[Cl-]i升高并引发一过性的pH i酸化，SITS和DDS或无氯溶液均可抑制ATP引起的细胞内[Cl-]i升高和胞内H^+升高。我们的研究结果表明，细胞外ATP浓度的升高可能触发了心肌细胞内[Cl^-]i的升高。鉴于以往的研究表明，心肌缺血引起细胞外微球浓度和心肌细胞内[Cl^-]i的增加，我们认为缺血诱导的细胞外间隙内ATP浓度的增加可能是引发心肌缺血时[Cl^-]i升高的重要因素。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。用MQAE方法测定[Cl~-]_i。凝集素、植物血凝素和刀豆蛋白A呈剂量依赖性地升高人Juikat淋巴细胞[Cl~-]_i并触发细胞内Cl~-振荡。然而，线粒体代谢抑制剂CCP和DNP可增加[Cl^-]_i，但不触发Cl^-振荡。此外，凝集素和代谢抑制剂引起的[Cl^-]_i升高均可被无氯溶液或9-羧酸菲(9-AC)阻断。由于细胞外无氯条件和9-AC也抑制了PHA诱导的增殖，我们认为，通过激活Cl~-通道引起的[Cl~-]_i升高和Cl~+振荡发生率的增加可能在调节T细胞的激活和增殖中起重要作用。较少

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