脑缺血再灌注后NMDA受体亚基2B酪氨酸磷酸化的调节机制(英文)

目的:研究沙土鼠脑缺血再灌注后海马突触体N-甲基-D-天冬氨酸(NMDA)受体亚基2B(NR2B)酪氨酸磷酸化调节的机制。方法:沙土鼠双侧颈总动脉结扎形成前脑缺血模型;NR2B酪氨酸磷酸化通过免疫沉淀和免疫印渍分析。结果:脑缺血15分钟导致蛋白酪氨酸磷酸化水平明显下降;再灌注引起包括180kDa蛋白在内的多种蛋白酪氨酸磷酸化水平快速(再灌注15分钟)、持续(至少48小时)升高。免疫沉淀和免疫印渍证实,180 kDa条带为NR2B。缺血15分钟,再灌注6小时,NR2B酪氨酸磷酸化明显高于对照组,为对照组的1.8倍,而NR2B蛋白表达量则无变化。缺血前腹腔注射非竞争性NR拮抗剂氯胺酮或L-型电压门控钙通道(L-type VGCC)阻滞药硝苯地平,对NR2B酪氨酸磷酸化水平升高有明显的拮抗作用,而对NR2B蛋白表达量均无影响。在此条件下,非NMDA受体拮抗剂6,7-二硝基喹恶啉土卫四(DNQX)对NR2B酪氨酸磷酸化水平无影响。酪氨酸蛋白磷酸酶(PTP)抑制剂钒酸钠使脑缺血再灌注诱导的NR2B酪氨酸磷酸化进一步增加,而酪氨酸蛋白激酶(PTK)抑制剂金雀异黄素则使其减少。Src能与NR2B免疫共沉淀。结论:沙土鼠脑缺血再灌注NR2B的酪氨酸磷酸化的升高是通过NR和L-type VGCC介导的;PTK和PTP参与脑缺血再灌注NR2B酪氨酸磷酸化的调节,与NR2B以物理方式结合的Src可能在这种

Mechanisms of regulation of tyrosine phosphorylation of NMDA receptor subunit 2B after cerebral ischemia/reperfusion

To study the mechanisms of the regulation of the tyrosine phosphorylation of TV-methyl-D-aspartate (NM-DA) receptor subunit 2B(NR2B) in the gerbil hippocam-pal synaptosomes following ischemia/reperfusion (I/R). METHODS: Transient (15 min) cerebral ischemia was produced by bilateral carotid artery occlusion procedure. The tyrosine phosphorylation of NR2B was analyzed by immunoprecipitation and immunoblot assay. RESULTS: Transient forebrain ischemia for 15 min caused a marked decrease in the levels of tyrosine phosphorylation of many protein bands including 180 kDa protein. Transient ischemia followed by reperfusion induced rapid (within 15 min of reperfusion), and sustained (for at least 48 h) increase in the tyrosine phosphorylation of many protein bands including 180 kDa protein. Immunoprecipitation and immunoblot confirmed that NR2B is among the phosphorylated 180 kDa protein. Maximal phosphorylation of 180 kDa band corresponding to NR2B (1.8 fold relative to sham-operated controls) was reached at 6 h of reperfusion following 15 min of cerebral ischemia. But the level of protein expression of NR2B did not change. Administration of ketamine (KT), a non-competitive NMDA receptor antagonist, or nifedipine (ND), an L-type voltage gated calcium channel (L-type VGCC) blocker, 20 min before ischemia attenuated stimulation of the tyrosine phosphorylation of NR2B withoutaffecting the level of protein expression of NR2B. Under these conditions, non-NMDA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) had no effect on the level of tyrosine phosphorylation. Protein tyrosine phosphatase (FTP) inhibitor vanadate and protein tyrosine kinase (PTK) inhibitor genestein resulted in the increase and the decrease of the tyrosine phosphorylation of NR2B, respectively. Src coprecipitated with NR2B protein. CONCLUSION: The increase of the tyrosine phosphorylation of NR2B induced by I/R has relation to NR and L-type VGCC; PTK and FTP participate in the regulation of the tyrosine phosphorylation of NR2B during I/R. Src that associates with NR2B may play an important role in the regulation of the tyrosine phosphorylation of NR2B during I/R.