Precursors of glutamic acid nitrogen in primary neuronal cultures: Studies with15N

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Abstract
We utilized gas chromatography-mass spectrometry to study the transfer of15N from [2-15N]glutamine, [15N]leucine, [15N]alanine, or15NH4Cl to [15N]glutamate and [15N]aspartate in cultured cerebrocortical GABA-ergic neurons from the mouse. Initial rates of15N appearance (atom % excess) were somewhat higher with 2mM [2-15N]glutamine as a precursor than with 1mM [15N]leucine or 1mM [15N]alanine, but initial net formation (nmol [15N]glutamate/mg protein.min−1) was roughly comparable with all precursors. At steady-state15N labeling was about two times greater with 2mM [2-15N]glutamine as precursor. The subsequent transfer of15N from glutamate to aspartate was extremely rapid, the labelling pattern of these two amino acid pools being virtually indistinguishable. We observed little reductive amination of 2-oxo-glutarate to yield [15N]glutamate in the presence of 0.3mM15NH4Cl. Reductive amination through glutamate dehydrogenase was much more prominent at a concentration of 3.0mM15NH4Cl. Glutamate formation via reductive amination was unaffected by inclusion of 1 mM 2-oxo-glutarate in the incubation medium. These results indicate that glutamate synthesis in cultured GABA-ergic neurons is derived not only from the glutaminase reaction, but also from transamination reactions in which both leucine and alamine are efficient N donors. Reductive amination of 2-oxo-glutarate in the glutamate dehydrogenase pathway plays a relatively minor role at lower concentrations of extracellular ammonia but becomes quite active at 3mM ammonia.