Jiang Q, Gu Z, Zhang G, Jing G
Research Center of Biochemistry and Molecular Biology, Xuzhou Medical College, 84 West Huai-hai Road, Xuzhou, China.
Brain Res. 2000 Feb 28;857(1-2):71-7. doi: 10.1016/s0006-8993(99)02364-1.
Glutamate-induced excitotoxicity, with certain characteristics of apoptosis, has been implicated in a variety of neuronal degenerative disorders. In some physiological cases, extracellular signal-regulated kinases (ERK1/2) are activated by stimulation of glutamate receptors. In the present study, the activation (diphosphorylation) and role of ERK1/2 in glutamate-induced apoptotic-like death in cultured cortical neurons were investigated. Protein levels and activation (diphosphorylation) levels of ERK1/2 were examined by Western immunoblot, probed with anti-ERK1/2 and anti-active (diphosphorylated) ERK1/2 antibodies, respectively. Apoptotic-like death was determined by DAPI staining. Before a remarkable increase of apoptotic-like cell death was observed at 9-18 h after 15 min exposure to 50 microM glutamate, diphosphorylation levels of ERK1/2 were rapidly increased, peaked at 5-15 min of the exposure, and reverted to sham control level 3 h after the exposure, while the protein levels of ERK1/2 were unaffected. The glutamate concentration effective for inducing apoptotic-like cell death was correlated with that for inducing ERK1/2 diphosphorylation. Both ERK1/2 diphosphorylation and the apoptotic-like cell death were largely prevented by MK-801, a specific NMDA receptor (a subtype receptor of glutamate) antagonist, or the elimination of extracellular Ca(2+) with EGTA. PD98059, a specific inhibitor of ERK1/2 kinase, completely inhibited ERK1/2 diphosphorylation and partially inhibited the apoptotic-like cell death. These results suggest that largely via NMDA receptor-mediated influx of extracellular Ca(2+), ERK1/2 were rapidly and transiently activated and were involved in glutamate-induced apoptotic-like death in cultured rat cortical neurons.
谷氨酸诱导的具有某些凋亡特征的兴奋性毒性，已被认为与多种神经元退行性疾病有关。在一些生理情况下，细胞外信号调节激酶（ERK1/2）可通过谷氨酸受体的刺激而被激活。在本研究中，研究了ERK1/2在培养的皮质神经元中谷氨酸诱导的凋亡样死亡中的激活（双磷酸化）及其作用。通过蛋白质免疫印迹法检测ERK1/2的蛋白质水平和激活（双磷酸化）水平，分别用抗ERK1/2抗体和抗活性（双磷酸化）ERK1/2抗体进行检测。通过DAPI染色确定凋亡样死亡。在暴露于50μM谷氨酸15分钟后9-18小时观察到凋亡样细胞死亡显著增加之前，ERK1/2的双磷酸化水平迅速升高，在暴露后5-15分钟达到峰值，并在暴露后3小时恢复到假手术对照水平，而ERK1/2的蛋白质水平未受影响。诱导凋亡样细胞死亡的有效谷氨酸浓度与诱导ERK1/2双磷酸化的浓度相关。ERK1/2双磷酸化和凋亡样细胞死亡在很大程度上被MK-801（一种特异性NMDA受体（谷氨酸的一种亚型受体）拮抗剂）或用EGTA消除细胞外Ca(2+)所阻止。PD98059（一种ERK1/2激酶的特异性抑制剂）完全抑制ERK1/2双磷酸化并部分抑制凋亡样细胞死亡。这些结果表明，ERK1/2主要通过NMDA受体介导的细胞外Ca(2+)内流而被快速短暂激活，并参与了培养的大鼠皮质神经元中谷氨酸诱导的凋亡样死亡。
