Objective To illustrate the possible molecular mechanisms of NR2A N - methyl - D - aspartic acid (NM-DA) receptor tyrosine phosphorylation induced by oxygen - glucose deprivation (OGD) in cultured cortical neurons. Methods Cultured cortical neurons were exposed to OGD, and then recovered for certain time (1,6, 12, 18,24h). Samples were immunoprecipitated by anti - phosphotyrosine antibody and blotted by anti - NR2A antibody. Results The tyrosine phosphorylation level of NR2A increased rapidly and peaked (4.9 - fold vs. Sham) at 18 h after OGD and reoxygen. While the protein level of NR2A was not changed during the process. The increase in tyrosine phosphorylation of NR2A was partly inhibited by ethylene glycol tetraacetic acid (EGTA) , dizocilpine (MK - 801) , nimodipine, genistein and KN - 62, which were added to the medium before OGD immediately. Conclusions The results demonstrate that the increase in tyrosine phosphorylation of NR2A may be associated with the extracelluar Ca2 + influx and the opening of NMDA receptor and L -type voltage - gated calcium channel (L - VGCC). L - VGCC may participate in the regulation of NMDA receptor.%目的 研究大鼠皮质神经元缺氧缺糖(OGD)/再灌注诱导N-甲基-D-天冬氨酸(NMDA)受体NR2A酪氨酸磷酸化水平升高的可能分子机制.方法 以培养的大鼠皮质神经元OGD为模型,通过免疫沉淀(IP)及免疫印迹(IB)的方法 研究NR2A酪氨酸磷酸化.结果 OGD/再灌注后NR2A酪氨酸磷酸化水平快速并持续升高,至18 h达高峰(约为sham组的4.9倍);于OGD前加入乙二醇二乙醚二胺四乙酸(EGTA)、地(艹卓)西平(MK-801)、尼莫地平(nimodipine)、染料木黄酮(genistein)和KN-62等,对NR2A酪氨酸磷酸化的升高均有明显的抑制作用.结论 OGD/再灌注诱导NR2A酪氨酸磷酸化水平的升高可能与胞外Ca2+内流有关,并与NMDA受体通道和L-型电压门控钙通道(L-VGCC)的开放有关;而且,NMDA受体通道可以进行自身调控,L-VGCC对NMDA受体也有一定的调控作用.
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