|Budget Amount *help
¥3,200,000 (Direct Cost : ¥3,200,000)
Fiscal Year 1999 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1998 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1997 : ¥1,800,000 (Direct Cost : ¥1,800,000)
Purpose : Analysis of Na,K-pump-induced currents in astrocytes and the role in brain ischemia
Method : Using nystatin-perforated patch clamp techniques, Na,K-pump currents (Ip) were recorded from cultured astrocytes isolated from rat cerebral cortex.
Results : 1. In the presence of intracellular Na([Na]I), Ip were activated by addition of extracellular K([K]o) in a dose-dependent manner with a Kd of O.8mM and a Hill coefficient of 1.7. 2.Ip were blocked by ouabain in a dose-dependent manner with a relatively high IC50 of about 0.1mM, suggesting the existence of a ouabain-resistant isoform in astrocytes. 3.Ip were increased in depolarized potentials and were decreased in hyperpolarized potential. Consequently, I-V relation of Ip showed a sigmoid shape. 4.Ip could be activated by [K]o even in the absence of [Na]I, suggesting the existence of non-inactivating Na currents that are not sensitive to tetrodotoxin. 5. In the presence of [Na]I, [K]o-induced Ip were augmented by removal of extracellular Na ([Na]o). 6. Metabolic inhibitor, sodium cyanide did not affect Ip function, suggesting that Ip activities are kept during brain ischemia. 7. Exogenous glutamate augmented Ip by increasing [Na]I. In some cases, the augmentation of Ip was evident after removal of glutamate, which is probably due to the phosphorylation of Na,K-ATPase via metabotropic-type glutamate receptors.
Conclusion : From the all results, it is suggested that the Na,K-pump function in astrocytes is maintained during brain ischemia, and the activity is maximized by both elevation of [K]o and glutamate and reduction of [Na]o. These facts indicates that the Na,K-pump activity in astrocytes has a neuroprotective role in the removal of extracellular K accumulation during brain ischemia.