A direct correlation between ischemic injury and extracellular glycine concentration in mice with genetically altered activities of the glycine cleavage multi-enzyme system

[背景与目的]脑缺血引起多种氨基酸类神经递质的快速释放。谷氨酸激增激活N-甲基-D-天冬氨酸(NMDA)谷氨酸受体，触发神经元的有害过程。尽管甘氨酸是NMDA受体的共激动剂，但细胞外甘氨酸浓度对缺血损伤的影响仍存在争议。为了探讨这个问题，我们研究了甘氨酸裂解多酶系统(GCS)活性发生遗传变化的小鼠的缺血性损伤，GCS在维持细胞外甘氨酸浓度方面发挥着重要作用。[材料与方法]通过GCS关键成分甘氨酸脱羧酶的转基因表达，建立了GCS活性升高的小鼠系(高GCS小鼠)，获得了GCS活性升高的小鼠(340%的C57BL/6对照鼠)。另一种GCS活性降低的小鼠系(对照的29%)是通过转基因表达甘氨酸脱羧酶的显性负突变(低GCS小鼠)而建立的。我们观察了这些小鼠大脑中动脉短暂性闭塞后的神经元损伤，用微透析法测定细胞外氨基酸浓度。[结果]高GCS组和低GCS组小鼠细胞外甘氨酸基础浓度分别显著低于对照组和高GCS组。在低GCS小鼠中，细胞外甘氨酸在缺血期间达到2-Hold对照水平，脑梗塞体积显著增加69%。相比之下，高GCS组小鼠的脑梗塞体积要小21%。在整个实验过程中，胞外谷氨酸浓度没有显著差异。N-甲基-D-天冬氨酸甘氨酸位点的拮抗剂SM-31900可缩小梗塞面积，提示甘氨酸是通过N-甲基-D-天冬氨酸受体起作用的。[结论]缺血损伤与GCS维持的细胞外甘氨酸浓度有直接关系。

[Background and Purpose] Ischemia elicits rapid release of various amino acid neurotransmitters. Glutamate surge activates N-methyl-D-aspartate (NMDA) glutamate receptors, triggering deleterious process of neurons. Although glycine is a coagonist of the NMDA receptor, the effect of extracellular glycine concentration on ischemic injury remains controversial. To approach this issue we examined ischemic injury in mice with genetically altered activities of the glycine cleavage multi-enzyme system (GCS), which plays a fundamental role in maintaining extracellular glycine concentration. [Materials and Methods] A mouse line with increased GCS activity (340% of C57BL/6 control mice) was generated by transgenic expression of glycine decarboxylase, a key GCS component (high-GCS mice). Another mouse line with reduced GCS activity (29% of controls) was established by transgenic expression of a dominant negative mutant of glycine decarboxylase (low-GCS mice). We examined the neuronal injury after transient occlusion of middle cerebral artery in these mice, measuring extracellular aminoacid concentrations by microdialysis method. [Results] The high-GCS and low-GCS mice had significantly lower and higher basal concentrations of extracellular glycine than controls, respectively. In low-GCS mice, extracellular glycine reached to 2-hold control level during ischemia and the infarction volume was significantly increased by 69% of controls. In contrast, high-GCS mice had significantly smaller infarction volume by 21%. No significant difference was observed in extracellular glutamate concentrations throughout the experiments. An antagonist for the NMDA glycine site, SM-31900, attenuated infarction size, suggesting that glycine operated via NMDA receptor. [Conclusions] There is a direct correlation between ischemic injury and extracellular glycine concentration maintained by the GCS.