PRECURSORS OF GLUTAMIC-ACID NITROGEN IN PRIMARY NEURONAL CULTURES - STUDIES WITH N-15

We utilized gas chromatography-mass spectrometry to study the transfer of N-15 from [2-N-15]glutamine, [N-15]leucine, [N-15]alanine, or (NH4Cl)-N-15 to [N-15]glutamate and [N-15]aspartate in cultured cerebrocortical GABA-ergic neurons from the mouse. Initial rates of N-15 appearance (atom % excess) were somewhat higher with 2mM [2-N-15]glutamine as a precursor than with 1mM [N-15]leucine or 1mM [N-15]alanine, but initial net formation (nmol [N-15]glutamate/mg protein.min-1) was roughly comparable with all precursors. At steady-state N-15 labeling was about two times greater with 2mM [2-N-15]glutamine as precursor. The subsequent transfer of N-15 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 [N-15]glutamate in the presence of 0.3mM (NH4Cl)-N-15. Reductive amination through glutamate dehydrogenase was much more prominent at a concentration of 3.0mM (NH4Cl)-N-15. 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 alanine 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.