It has been postulated that glutamate levels in the interstitial space are maintained at low levels to prevent excitotoxicity. This is critical since the developing brain has increased sensitivity to excitotoxicity due to up regulation of excitatory amino acid receptors. Elevated extracellular glutamate in perinatal hypoxia/ischemia increases only about 3 fold, yet is a major cause of mortality and morbidity. Survivors are often left with severe, permanent neurological impairment. However, we postulate that a low level of glutamate is necessary in the interstitial space. Baseline levels of glutamate have been shown to interact with metabotropic receptors. This project will examine mechanisms by which extracellular glutamate is generated in both perinatal and adult brain in the normal and in the traumatized perinatal brain following an episode of hypoxia/ischemia. Preliminary results indicate that infusion of glutamine into the rat and mouse brain by microdialysis markedly increases the concentration of extracellular glutamate to excitotoxic levels and that it is synthesized in the extracellular space from glutamine. The concentration of extracellular glutamine in brain is normally 200 -300 mu M providing a ready precursor. We hypothesize that under normal conditions the primary mechanism for extracellular glutamate synthesis is maleate activated glutaminase (MAG), a side reaction of the ectoenzyme gamma-glutamyl transpeptidase (gamma-GTP). We hypothesize a second mechanism predominates in trauma. We propose that intramitochondrial phosphate dependent glutaminase (PDG) is released (or exposed) from damaged cells and converts glutamine to glutamate resulting in excitotoxicity and cell death days after the initial traumatic event. These enzymes may also play a role in conditions such as elevated ammonia. We will determine the enzymatic mechanism for extracellular glutamate formation in normal and 7-day old hypoxic-ischemic rat brain, adult rats under conditions of ammonia toxicity, mice deficient in the enzyme gamma-GTP, and in neuronal cell cultures. Studies will utilize microdialysis and cell culture in the presence and absence of effectors of the enzymes involved. It is anticipated that these studies will lead to new approaches in the care of infants that have experienced hypoxic or ischemic injury during infancy.
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