Activation Of BK_Ca Channels Mediates Hippocampal Neuronal Apoptosis In Culture Induced By Hypoxia/reoxygenation

We have previously shown that hyperactivity of BKca channels is involved in ischemia-induced hippocampal CA1 degeneration. As an in vitro model of ischemia/reperfusion,cultured hippocampal neurons subjected to hypoxia (95% N2/5% CO2) for 6 h and than reoxygenation for up 72 h were applied to test the hypothesis that an upmodulation of potassium channels may play a key role in neuronal apoptosis. Following reoxygenation,neurons showed a progressive death with typical apoptotic features such as increased TUNEL positive cells and activation of caspase-3. Activities of BKca channels were studied at the single-channel level with inside-out patch-clamp configuration.. BKca channels in neurons after reoxygenation for 6 h showed a significant increase in the unitary conductance and open probability compared with control. After reoxygenation for 24 h,both the conductance and the open probability recovered to the control level. Application of TEA,an extensive K+ channel blocker,protected neuronal cell death in a concentration-dependent manner. TEA at the concentration of 500 uM showed a maximal protection while TEA at the concentration of 10 mM produced a neurotoxicity. The neuroprotection by TEA was not eliminated by blocking voltage-gated Ca2-1" channels. TEA treatment also reduced the number of TUNEL positive cells and caspase-3 activation. Then several kinds of selective K+ channel blockers were applied to further clarify the specific type of K+ channels involved in apoptosis. Both BKca channel blockers,IBTX and CHTX (at the concentration of 100 nM),significantly reduced neuronal death and number of positive TUNEL-staining cells and inhibited caspase-3 activation. However,A-type channel blocker 4-AP and channel blocker apamin displayed no effect on cell death andpercentage of TUNEL positive cells and caspase-3 activation. Furthermore,K+ ionophore valinomycin and K+ channel opener diazoxide alone induced neuronal apoptosis and caspase-3 activation. Besides,KATP channel blockers tolbutamide and glybenclamide also showed neuroprotective effects and decreased caspase-3 activation after hypoxia/reoxygenation. While the role of KATP channels in neuronal apoptosis is not clear. These data suggested that following hypoxia/reoxygenation,over-activity of BKca channels may mediate cultured hippocampal neuronal apoptosis induced by hypoxia/reoxygenation via activation of caspase-3.