Synthesis of vesicular GABA from glutamine involves TCA cycle metabolism in neocortical neurons

In contrast to the classic concept of direct conversion of glutamine to gam ma-aminobutyric acid (GABA; via glutamate), this process may involve a-keto glutarate as an intermediary metabolite and tricarboxylic acid (TCA) cycle activity. To obtain information about a possible differential role of these pathways for the synthesis of cytosolic and vesicular GABA, cultured neoco rtical neurons were incubated in medium containing [U-C-13]glucose (0.5 mM) and in some cases unlabeled glutamine (0.5 mM). Subsequently, the cells we re "superfused" for investigation of the effect of depolarization by 55 mM K+. To make sure that depolarization by 55 mM K+ released only vesicular GA BA, tiagabin, a nontransportable inhibitor of the plasma membrane GABA carr iers, was included in the medium to prevent GABA release from the cytoplasm ic pool by reversal of the carriers. The importance of the TCA cycle for co nversion of the carbon skeleton of glutamine to GABA was evident from the e ffect of glutamine on the labeling pattern of GABA. Percentage of labeling by GABA released into the depolarizing medium was the same as that in the c orresponding cell extracts and was unaffected by the presence of glutamine during incubation. Despite the existence of multiple forms of glutamate dec arboxylase, compartmentation of glutamate pools, and functionally different compartments within neurons, there appears to be full equilibration betwee n the vesicular and cytosolic pools of GABA. However, during depolarization , the newly synthesized pool of GABA from glutamine does not rapidly equili brate with the vesicular pool. (C) 1999 Wiley-Liss, Inc.