Hsu L, Rockenstein E, Mallory M, Hashimoto M, Masliah E
Department of Neurosciences, University of California, San Diego, La Jolla, 92093-0624, USA.
J Neurosci Res. 2001 Apr 15;64(2):193-202. doi: 10.1002/jnr.1065.
Regulation of extracellular excitotoxins by glial and neuronal glutamate transporters is critical to maintain synaptic terminal integrity. Factors interfering with the normal functioning of these transporters might be involved in neurodegeneration. Among them, recent studies have shown that hypoxia alters glutamate transporter function; however, it is unclear if hypoxia has an effect on the expression of glutamate transporters and which intracellular signaling pathways are involved. The C6 rat glial and GT1--7 mouse neuronal cell lines were exposed to hypoxic conditions (5% CO(2), 95% N(2)) and levels of glutamate transporter mRNA were determined by ribonuclease protection assay. After 21 hr, there was a 100% increase in levels of rat excitatory amino acid transporter 3 (EAAT3) mRNA in C6 cells and a 600% increase in levels of murine EAAT2 mRNA in GT1--7 cells. There was a similar increase in mRNA levels after hypoxia in C6 cells transfected with human EAAT2, whereas reoxygenation normalized the expression levels of glutamate transporters. Although the expression of EAATs was associated with increased immunoreactivity by Western blot, functioning of the transporters was decreased as evidenced by D-aspartate uptake. Finally, although the protein kinase C stimulator phorbol-12-myristate-13-acetate enhanced EAAT2 mRNA levels after hypoxia, protein kinase C inhibitor bisindolylmaleimide I had the opposite effect. Taken together, this study suggests that the hypoxia is capable of upregulating levels of EAATs via a protein kinase C-dependent compensatory mechanism. This increased expression is not sufficient to overcome the decreased functioning of the EAATs associated with decreased ATP production and mitochondrial dysfunction.
神经胶质细胞和神经元谷氨酸转运体对细胞外兴奋性毒素的调节对于维持突触终末的完整性至关重要。干扰这些转运体正常功能的因素可能参与神经退行性变。其中，最近的研究表明缺氧会改变谷氨酸转运体的功能；然而，尚不清楚缺氧是否会影响谷氨酸转运体的表达以及涉及哪些细胞内信号通路。将C6大鼠神经胶质细胞系和GT1-7小鼠神经元细胞系暴露于低氧条件（5%二氧化碳，95%氮气）下，通过核糖核酸酶保护试验测定谷氨酸转运体mRNA水平。21小时后，C6细胞中大鼠兴奋性氨基酸转运体3（EAAT3）mRNA水平增加了100%，GT1-7细胞中鼠源EAAT2 mRNA水平增加了600%。转染人EAAT2的C6细胞在缺氧后mRNA水平也有类似增加，而复氧可使谷氨酸转运体的表达水平恢复正常。虽然通过蛋白质印迹法显示EAATs的表达与免疫反应性增加相关，但D-天冬氨酸摄取表明转运体的功能下降。最后，虽然蛋白激酶C激动剂佛波醇-12-肉豆蔻酸酯-13-乙酸盐在缺氧后可提高EAAT2 mRNA水平，但蛋白激酶C抑制剂双吲哚马来酰亚胺I则有相反作用。综上所述，本研究表明缺氧能够通过蛋白激酶C依赖的代偿机制上调EAATs的水平。这种增加的表达不足以克服与ATP生成减少和线粒体功能障碍相关的EAATs功能下降。
