The Role Of BK Channel In Hippocampal Neuronal Death Induced By Oxygen And Glucose Deprivation

Stroke has a high incidence and is harmful to human health,it is a leading cause of death and the third cause of disability.The loss of neurological functions following stroke is caused by massive loss of neurons resulting from hypoxic-ischemie insults. The mechanism underlying the brain ischemic injury is unclear and there is no effective clinical treatment yet.Up to now,several hypotheses have been put forward, for example,Ca²⁺ overload,free radical and so on.The acute neuronal damage is followed by a second round of neuronal injury that occurs hours to days after brain ischemia in the neighboring areas,which called delayed neuronal death(DND).So it's very important to study the mehanism underlying DND for prolonging the timescale of clinical treatment.Most recently,it is suggested that over-activation of K+ channel may be involved in neuronal death induced by different insults.We have previously shown that the enhancement of BK channel activation is an important mechanism mediating hippocampal neuronal damage both in vivo and in vitro.The present study focuses on the memchanism underlying enhancement of BK channel activation after hypoxic-ischemic and the neuroprotective timescale of BK channel antagonist.Cultured neurons were prepared from neonatal Sprague-Dawley rats.Neurons cultured for 12 days in vitro were subjected to OGD for 4 hours and then reoxygenated for untill being treated,and cell death was assessed by the fuorescent dye DAPI or MTT cell viability assay.Statistical comparisons of pooled data were performed independent samples t-tests and one-way analysis of variance(ANOVA) (SPSS 13.0),statistical differences were evaluated by one-way analysis of variance followed by LSD-t test or S-N-K for multiple comparison.The results showed that OGD induced about 20%of neuronal death at 48 h after reoxygenation(P＜0.05). Treatment with EBSS containing 1g/L glucose didn't affect the survial rate of normal cultured neurons.To investigate the memchanism underlying enhancement of BK channel activation after hypoxic-ischemic,membrane protein was extracted by biotinylation assay and performed western blot with whole cell lysis together.The total BK channel expression decreased just after reoxygenation and returned to normal level after 1 hour(P=0.028),while the surface level increased by about 2 folds 6 hours after reoxygenation((P＜0.05).Application of a specific BK channel blocker,paxilline(5μM,10μM and 20μM) after reoxygenation showed completly protective effect in a concentration-dependent manner(P＜0.05) and achieved maximal protective effect at 10μM.Paxilline didn't affect the survial rate of normal cultured neurons.To investigate the neuroprotective timescale of paxilline, paxilline(10μM) was added at different time points(0h,6h,12h ) after reoxygenation. Data showed that paxilline could completly protect nerons from death induced by OGD.Further prolonging the timescale,paxilline showed neuroprotective effect at 24h after reoxygenation.Treatment with a specific opener of BK channel,NS 1619(20μmol/L) for 4 hours or 48 hours,both caused about 30%normal cultured cells died (P＜0.05).In order to make sure that paxilline tageted at BK channel,paxilline was added to medium with NS1619.Our data showed that NS1619 could counteract the neuroprotective effect of paxilline but didn't aggravate OGD-induced cell damage.In conclusion,The total BK channel expression decreased just after reoxygenation and return to normal 1h later,while the surface level remained increased throughout the 24h time period after reoxygenation.Application of a specific BK channel blocker,paxilline protected neuronal death completely and toke effect even 24h after reoxygenation.A specific opener of BK channel,NS1619, caused about 30%normal cultured cells died and it could also counteract the protective effect of paxilline..Accordingly,the BK channel is a potential molecular target for neuroprotective therapy in stroke.