Excitatory Amino Acid Synthesis in Hypoxic Brain Slices: Does Alanine Act as a Substrate for Glutamate Production in Hypoxia?

Abstract: Excitatory amino acids are an important cause of cell death in the hypoxic and ischaemic brain. Neuronal glutamate stores are depleted rapidly in hypoxia, but alanine production rises under such conditions and has been suggested to be a potential precursor of glutamate. To test this hypothesis, we have investigated amino acid metabolism using ¹³C NMR with superfused guinea pig cortical slices subjected to varying degrees of hypoxia. During severe hypoxia, brain slices metabolising 5 mM [2-¹³C]pyruvate exported [2-¹³C]alanine into the superfusion fluid. The metabolic fate of alanine during normoxia and hypoxia was tested by superfusion of brain slices with 10 mM glucose and 2 mM [2-¹³C, ¹⁵N]alanine. Metabolism of exogenous alanine leads to the release of aspartate into the superfusion fluid. The pattern of labelling of aspartate indicated that it was synthesised via the glial-specific enzyme pyruvate carboxylase. ¹³C-labelled glutamate was produced with both normoxia and hypoxia, but concentrations were 30-fold lower than for labelled aspartate. Thus, although substantial amounts of glutamate are not synthesised from alanine in hypoxia, there is significant production of aspartate, which also may have deleterious effects as an excitatory amino acid.