Marin P, Nastiuk K L, Daniel N, Girault J A, Czernik A J, Glowinski J, Nairn A C, Prémont J
Chaire de Neuropharmacologie, Institut National de la Santé et de la Recherche Médicale U114, Collège de France, 75231 Paris Cedex 05, France.
J Neurosci. 1997 May 15;17(10):3445-54. doi: 10.1523/JNEUROSCI.17-10-03445.1997.
Postischemic delayed neuronal death is attributed to excitotoxic activation of glutamate receptors. It is preceded by a persistent inhibition of protein synthesis, the molecular basis of which is not known. Here we have examined in cortical neurons in culture the regulation by glutamate of phosphorylation of eukaryotic elongation factor-2 (eEF-2) by eEF-2 kinase, a Ca2+/calmodulin-dependent enzyme. Using a phosphorylation state-specific antibody, we show that glutamate, which triggers a large influx of Ca2+, enhances dramatically the phosphorylation of eEF-2. On the basis of kinetic and pharmacological analysis, we demonstrate a close correlation among the increase in cytosolic Ca2+ concentration, the degree of eEF-2 phosphorylation, and the inhibition of protein synthesis. A 30 min treatment with NMDA induced a transient phosphorylation of eEF-2 and delayed neuronal death. However, pharmacological inhibition of protein translation was not neurotoxic by itself and protected neurons against the toxicity evoked by low concentrations of NMDA. Thus, phosphorylation of eEF-2 and the resulting depression of protein translation may have protective effects against excitotoxicity and open new perspectives for understanding long-term effects of glutamate.
缺血后延迟性神经元死亡归因于谷氨酸受体的兴奋性毒性激活。在此之前会出现蛋白质合成的持续抑制，其分子基础尚不清楚。在这里，我们在培养的皮层神经元中研究了谷氨酸对真核延伸因子2（eEF-2）激酶（一种Ca2+/钙调蛋白依赖性酶）介导的eEF-2磷酸化的调节作用。使用一种磷酸化状态特异性抗体，我们发现谷氨酸会引发大量Ca2+内流，从而显著增强eEF-2的磷酸化。基于动力学和药理学分析，我们证明了胞质Ca2+浓度的增加、eEF-2磷酸化程度以及蛋白质合成抑制之间存在密切相关性。用N-甲基-D-天冬氨酸（NMDA）处理30分钟会诱导eEF-2的短暂磷酸化并导致延迟性神经元死亡。然而，药理学抑制蛋白质翻译本身并无神经毒性，且能保护神经元免受低浓度NMDA诱发的毒性作用。因此，eEF-2的磷酸化以及由此导致的蛋白质翻译抑制可能对兴奋性毒性具有保护作用，并为理解谷氨酸的长期影响开辟了新的视角。
