| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2001: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2000: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1999: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Research Abstract |
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
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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
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