| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2003: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2002: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
1.The cell-volume regulation by chloride currents was studied in guinea-pig cardiac myocytes, using a microscopic video-image analysis. Application ofhypotonic solution to the cells at normal [K]_0 induced a cell swelling, and the so-swollen cells showed a slight but clear spontaneous cell shrinkage, indicating operation of the mechanism of regulatory volume decrease (RVD)._This RVD could be pronounced at low [Cl]_o, at which E_<Cl> was far more positive than E_m. On the contrary, when the hypotonic solution was applied to the cells at high [K]_o, at which E_<Cl> was negative to E_m, the cells swelled monotonically without showing RVD, the swelling being much greater than that seen at normal [K]_0. Both the RVD at normal [K]_0 and the 'extra' cell inflation at high [K]_0 were suppressed by the inhibitors of I_<Cl, swell>. These findings suggest that the cell inflation activated. I_<Cl, swell>, and that the activation of I_<Cl, swell> induced the RVD or the extra cell inflation, dependi
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ng on the direction and magnitude of the driving force of Cl ions across the membrane.
2.The effects of extracellular ATP on [β-adrenergic activation of CFTR Cl current (I_<Cl, PKA>) were examined with the whole-cell patch clamp method. The cells were initially exposed to isoproterenol (ISO) for 〜3 min to activate I_<Cl, PKA>, and then to 1-100 μM ATP in the presence of ISO. ATP was found to potentiate I_<Cl, PKA>, in most cells examined. With 50 μM ATP, the potentiation, on average, resulted in a 1.3 fold increase of the Cl^-conductance activated by ISO alone (0.02-1μM). The effects of ADP and ATPγS on I_<Cl, PKA> were similar to those of ATP, while AMP and adenosine never potentiated I_<Cl, PKA>. Thus the potentiation was attributed, to a stimulation of P2-purinoceptors. PDBu (0.5μM), an activator of PKC, facilitated I_<Cl, PKA>, and in the presence of PDBu ATP did not further potentiate I_<Cl, PKA>. When BIM (0.2μM), an inhibitor of PKC, was present, ATP did not facilitate I_<Cl, PKA>. These findings suggested involvement of PKC in the observed ATP action. When ATP was removed in the presence of ISO, the potentiated I_<Cl, PKA> decreased (recovered) only slowly, and, if ATP was reapplied during this slowly recovering phase, the subsequent current potentiation was weak. Thus the stimulation of P2 purinoceptors by ATP facilitates the β-adrenergic activation of I_<Cl, PKA> through PKC activation, and this potential appears to persist for several min after removal of ATP. Less
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