Volatile anesthetic modulation of glutamate transporters

描述(申请人提供)谷氨酸转运体(EAAT)在调节细胞外谷氨酸水平方面发挥重要作用，谷氨酸是中枢神经系统中的一种主要兴奋性神经递质。目前的数据表明，细胞外谷氨酸浓度的增加参与了缺血性脑损伤和几种主要的人类神经退行性疾病，如阿尔茨海默病和亨廷顿病，以及肌萎缩侧索硬化症的病理生理过程。因此，EAATs的正常功能对于维持有效的神经传递和预防神经元损伤具有重要意义。我们的长期研究目标是了解EAATs的调节及其在麻醉和神经保护机制中的作用。挥发性麻醉剂可增加EAAT的活性。缺血引起谷氨酸通过EAATs的反向运输。本研究旨在1)确定挥发性麻醉药对EAATs转运和活性的影响及其机制，特别是蛋白激酶C(PKC)在介导这些麻醉效应中的作用；2)研究EAATs对谷氨酸逆向转运的麻醉作用。培养的细胞将暴露在挥发性麻醉剂中，EAAT的细胞分布将通过免疫细胞化学、共聚焦显微镜和亚细胞组分的Western blotting进行研究。潜在的PKC依赖的磷酸化位点在介导3型谷氨酸转运体的麻醉效应中的重要性将通过定点突变来确定。通过使用同工酶特异性抑制剂、下调同工酶和重新引入相关同工酶，也将研究该转运蛋白的麻醉调节对PKC同工酶的依赖性。在存在或不存在挥发性麻醉剂的情况下，将评估缺血/缺氧诱导的谷氨酸在不同脑区(海马区、大脑皮层和小脑)和培养细胞类型(胶质细胞和神经元)中的反向转运。这些研究将为麻醉剂和PKC调节谷氨酸运输提供分子基础，并可能为麻醉或神经保护提供新的靶点。

DESCRIPTION (provided by applicant) Glutamate transporters (EAATs) play an important role in the regulation of extracellular levels of glutamate, a major excitatory neurotransmitter in the central nervous system. Current data suggest that increased extracellular glutamate concentration is involved in the pathophysiology of ischemic brain injury and several major human neurodegenerative disorders, such as Alzheimer's and Huntington's diseases, as well as amyotrophic lateral sclerosis. Thus, normal functioning of EAATs is important for maintaining efficient neurotransmission and preventing neuronal injury. Our long-term research goals are to understand the regulation of EAATs and their role in the mechanisms of anesthesia and neuroprotection. Volatile anesthetics increase the activity of EAATs. Ischemia causes reversed transport of glutamate via EAATs. This proposal aims to 1) determine the effects and mechanisms of volatile anesthetics on the trafficking and activity of EAATs, especially the role of protein kinase C (PKC) in mediating these anesthetic effects; 2) study the anesthetic effects on reversed transport of glutamate via EAATs. Cultured cells will be exposed to volatile anesthetics and cellular distribution of EAATs will be studied by immunocytochemistry with confocal microscopy and Western blotting of the subcellular fractions. The importance of potential PKC-dependent phosphorylation sites in mediating the anesthetic effects on the type 3 glutamate transporter will be determined by site-directed mutagensis. The PKC-isozyme dependence of the anesthetic regulation of this transporter also will be investigated by using isozyme specific inhibitors, down-regulation of isozymes and re-introduction of implicated isozymes. Ischemia-/hypoxia-induced reversed transport of glutamate in different rat brain regions (hippocampus, cerebral cortex and cerebellum) and cultured cell types (glia and neurons) will be evaluated in the presence or absence of volatile anesthetics. These studies will provide a molecular basis for the anesthetic and PKC regulation of glutamate transport and may suggest new targets for anesthesia or neuroprotection.