Study On The Permeability And Regulatory Mechanisms Of Hippocampal Neuronal Hemichannels During Brain Ischemia

Cerebrovascular disease is a brain damage caused by variety of cerebral vascular diseases and is one of the major causes of the human death and disability.This disease is mainly related to the decrease of cerebral blood flow and ischemic brain injury. Many secondary factors play an important role in ischemic brain injury. Hemichannels in apposed plasma membranes of neighbouring cells can dock to each other and form gap junctions.At least 21 connexin genes and 3 pannexin genes probably code for gap junction proteins in humans.It was reported that ischemia could open hippocampal neuronal pannexin1 hemichannels and trigger necrotic cellular death within a few minutes as a result of ions dysregulation and glucose,ATP leakage.However,it is not yet clear that the regulatory mechanisms of pannexin1 hemichannels.To clarify the regulatory mechanisms of pannexin1 hemichannels,the oxygen glucose deprivation(OGD)and metabolic inhibition(MI),two models of in vitro cerebral ischemia,and middle cerebral artery occlusion(MCAO),the model of in vivo cerebral ischemia,were established at first.Then the regulatory mechanisms of hemichannels were studied by cellular and molecular biology technologies.At last, some related signal pathways were also examined.The dye coupling and detection by confocal were adopted to examine the permeability of hemichannels during OGD and MI.The results showed that OGD and MI could induce calcein leakage from neuron gap junction hemichannels;nitric oxide (NO)kept a sharp rise in the hippocampal neurons in the first 15 min of OGD and 20 min of MI,followed by a gradual increase at later time.To clarify the role of NO in the regulatory mechanisms of hemichannel,the donor of NO and the NO synthase (NOS)inhibitors were used in this study.The data revealed that the donor of NO could induce calcein leakage obviously and the calcein leakage during OGD and MI was inhibited by NOS inhibitors.These results implied that the enhanced NO might take part in the regulation of hemichannel permeability. To understand whether the NO related signal pathway had an effect on the regulation of hemichannel permeability,the NS2028,NEM,DTT,GSH and GSH-EE were applied in this study.The results of our study implied that NO could increase the permeability of hemichannel and the NO/cGMP-independent S-nitrosylation might play an important role in the regulation of hemichannel permeability.Besides the increase of hemichannel permebiltiy induce by NO,the insertion of additional hemichannels into the surface membrane and the expression of pannexin1 might also take part in these events.Therefore,related studies were performed on the distribution and expression of panenxin1 after 1 hr OGD.The distribution of pannexin1 was analyzed by immunocytochemistry and Western blot.Finally, immunocytochemical and immunoblotting analysis revealed that,after 1 hr OGD,the distribution and expression of panenxin1 showed no significant difference compared with control.The mRNA expression was also studied.The results showed that the pannexin1 mRNA was elevated after 1 hr OGD and a sustained increase was maintained during 1 hr OGD/reperfusion 3,6,9 hr and 1 hr MCAO/reperfusion 24 hr.In conclusion,our results suggested that in the early stage of ischemia,the enhanced pannexin1 hemichannels opening made a major contribution to the increased permeability of hippocampal pyramidal neurons and the NO might take part in this regulatory mechanism.Furthermore,S-nitrosylation might play a more important role than cGMP-dependent pathway in these processes.In physiological environment,the redox state keeps in a homeostasis.But under many pathological conditions such as ischemia and reperfusion,excessive NO may induce many downstream responses such as the enhanced pannexin1 hemichannels opening.These results are consistent with the fact that the restricted opening of hemichannels in normal cells is adequate to allow the release of signaling molecules and with greater opening,cell death may be accelerated under pathological conditions such as ischemia and reperfusion,in which there is enhanced NO generation.