The acidic amino acids, glutamate and aspartate, are the predominant excitatory neurotransmitters in the mammalian CNS. An extracellular accumulation of these excitatory amino acids (EAAs) causes neuronal cell death. This process known as 'excitotoxicity' contributes to the neurodegeneration that accompanies acute insults to the brain, including stroke, hypoglycemia, and head trauma. Normally, the extracellular concentrations of glutamate and aspartate are maintained in the low micromolar range by a family of Na+-dependent high-affinity glutamate transporters. It is generally thought that both failure and reversed operation of these transporters contribute to the rise in extracellular EAAs during the acute insults. We have recently found that activation of protein kinase C (PKC) rapidly (within min) changes both the activity and cell surface expression of the three forebrain glutamate transporters, EAAC1, GLT-1, and GLAST. PKC increases the activity and cell surface expression of EAAC1, a neuronal transporter that is enriched in brain areas that are exquisitely sensitive to excitotoxic insults. It has the opposite effect on the cell surface expression of the glial transporters. In fact, individual PKC isozymes appear to have different effects on a single transporter. These data imply that activation of PKC can dramatically shift the balance of glial and neuronal EAA clearance. Our data also suggest that EAAC1 couples to the functional antagonist of PKC, protein phosphatase. In our first two aims, we propose using biochemical, cell biological, pharmacological, and molecular biological techniques to study this regulation using both primary cultures derived from neuronal tissue and clonal cell lines. In our final aim, we wish to determine if this PKC-dependent regulation contributes to altered transporter function and activity that is observed during and after an acute insult. Several studies have demonstrated that PKC is activated by hypoxic/ischemic insults. Our preliminary data have prompted the hypothesis that hypoxic/ischemic insults activate PKC-dependent regulation of these transporters. By exploring this hypothesis, we will determine if these signaling pathways contribute to the failure or loss of these transporters. An understanding of these events should lead to the development of alternative strategies that will protect the brain from excitotoxicity.
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