|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)
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
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