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.
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