. The overall purpose of the proposal is to understand how the brain produces and consumes glutamate and glutamine. Neither amino acid efficiently crosses the blood-brain barrier, so the brain must make them from external precursors. The carbon of glutamate and glutamine is derived from glucose, the primary metabolic substrate. The central hypothesis is that the alpha-N of glutamate and glutamine is formed in large part from the branched-chain amino acids (BCAA), which donate amino groups via BCAA transaminase, e.g. leucine + a- ketoglutarate--> a-ketoisocaproate + glutamate. Both the BCAA and the cognate branched-chain ketoacids quickly cross into brain. Indeed recent studies of arteriovenous differences of amino acids across the human brain indicate that leucine is utilized faster than any other amino acid. Their studies, both in vitro and in vivo (preliminary results), show that transamination of the BCAA furnished about 30 percent of the amino nitrogen of glutamate and glutamine. The purpose of this proposal is to characterize the BCAA-glutamate interchange and to explore its regulation. They will also study the """"""""reversal"""""""" of the BCAA transaminase reaction, which involves reamination of BCKA (glutamate + BCKA--> BCAA + a-ketoglutarate). They have found that the reversal of the transaminase can lower intracellular glutamate, thereby affording a mechanism for temporary """"""""buffering"""""""" of intracellular [glutamate]. Reducing intra-neuronal glutamate might even attenuate the excitotoxic insult that is thought to be an important component of hypoxic brain injury. They have obtained preliminary evidence to suggest that in astrocytes the sequence leu--> glu--> gln predominates while in neurons the sequence glu-->leu is more prominent. This arrangement suggests the possibility of a BCAA-Glutamate Cycle functioning between astrocytes and neurons. Studies will be performed both in vivo and in cultures of astrocytes and neurons and using the """"""""neighbor"""""""" coculture system. The in vivo model involves perfusion of the common carotid artery with 15N and 13C- labelled isotopes and the subsequent tracing of these species in brain. The carotid perfusion will be combined with in vivo microdialysis in order to determine the metabolic origin(s) of the glutamate in the extracellular fluid. The goal is to define the BCAA-glutamate relationship in the CNS, to explore aspects of its regulation, its anatomic compartmentalization, and its therapeutic implications. The flux of N between the BCAA and glutamate will be measured with 15N and 13C as tracers with gas chromatography-mass spectrometry as a analytical tool. The PI has extensively applied this approach in prior investigations of brain metabolism. Reliable measurements of isotopic abundance can be made even in a medium or microdialysate fluid that contains just a few picomoles of analyte. The technology is ideal for studies in a biologic fluid (culture medium, brain extract, microdialysis fluid) that contains both the tracer, e.g. [15N]leucine, and the remaining (unlabelled) amino acids that are found in the ECF. The application of this technique should afford fresh insight into the critical but neglected metabolic relationship between glutamate and the branched-chain amino acids.
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