Inhibition of glutamine transport depletes glutamate and GABA neurotransmitter pools: further evidence for metabolic compartmentation Caroline Rae,* Nathan Hare,* William A. Bubb,* Sally R. McEwan,* Angelika Bro ¨ er,  James A. McQuillan,* Vladimir J. Balcar,à Arthur D. Conigrave* and Stefan Bro ¨er  *Discipline of Biochemistry, School of Molecular & Microbial Biosciences, The University of Sydney, New South Wales, Australia  School of Biochemistry and Molecular Biology, Australian National University, Acton, Australian Capital Territory, Australia àDepartment of Anatomy & Histology, The University of Sydney, New South Wales, Australia Abstract The role of glutamine and alanine transport in the recycling of neurotransmitter glutamate was investigated in Guinea pig brain cortical tissue slices and prisms, and in cultured neuroblastoma and astrocyte cell lines. The ability of exogenous (2 mM) glutamine to displace 13 C label sup- plied as [3- 13 C]pyruvate, [2- 13 C]acetate, L-[3- 13 C]lactate, or D-[1- 13 C]glucose was investigated using NMR spectroscopy. Glutamine transport was inhibited in slices under quiescent or depolarising conditions using histidine, which shares most transport routes with glutamine, or 2-(methylamino)isobutyric acid (MeAIB), a specific inhibitor of the neuronal system A. Glutamine mainly entered a large, slow turnover pool, probably located in neurons, which did not interact with the glutamate/glutamine neurotransmitter cycle. This uptake was inhibited by MeAIB. When [1- 13 C]glucose was used as substrate, glutamate/glutamine cycle turnover was inhibited by histidine but not MeAIB, suggesting that neuronal system A may not play a prominent role in neurotransmitter cycling. When transport was blocked by histidine under depolarising conditions, neurotransmitter pools were depleted, showing that glutamine transport is essential for maintenance of glutamate, GABA and alanine pools. Alanine labelling and release were decreased by histidine, showing that alanine was released from neurons and returned to astrocytes. The resultant implications for metabolic compartmentation and regulation of metabolism by transport processes are dis- cussed. Keywords: alanine transport, glutamate/glutamine cycle, glutamine transport, metabolic compartmentation, System A. J. Neurochem. (2003) 85, 503–514. The glutamate/glutamine cycle represents a fast, energy- efficient way for the brain to recycle neurotransmitter glutamate. It allows for rapid removal of potentially neurotoxic amounts of glutamate from the synapse with resultant maintenance of synaptic signal-to-noise ratio, and for the return of glutamate to the neuron in the ‘defused’ form of glutamine. The recycling process is necessary because astrocytes would otherwise deprive neurons of TCA cycle intermediates. In addition, recycling of glutam- ate may be favoured under conditions of functional activation and high glutamate demand, where transient inadequacies in the supply of oxygen and glucose may occur (Madsen et al. 1999; Dienel and Hertz 2001). Estimates of neuronal glutamate derived from the glutamate/ glutamine cycle range from 60 to 70% (Lieth et al. 2001; Sibson et al. 2001). Glutamate is removed from the extracellular space by high affinity, high capacity, Na + -dependent glutamate transporters of the excitatory amino acid transporter (EAAT) family, which have been extensively characterised (Vandenberg 1998; Danbolt 2001). Experimental data indicate that the transport of glutamate by EAAT(s) is an integral part of the Received August 9, 2002; revised manuscript received December 15, 2002; accepted January 23, 2003. Address correspondence and reprint requests to Dr Caroline Rae, School of Molecular & Microbial Biosciences, The University of Sydney, NSW, 2006, Australia. E-mail: c.rae@mmb.usyd.edu.au Abbreviations used: ASCT-2, system ASC amino acid transporter; ATA1 and ATA2, system A amino acid transporters; EAAT, excitatory amino acid transporter; HBSS, HEPES-buffered salt solution; MeAIB, 2-(methylamino)isobutyric acid; RPMI, Roswell Park Memorial Institute Medium 1640. Journal of Neurochemistry , 2003, 85, 503–514 doi:10.1046/j.1471-4159.2003.01713.x Ó 2003 International Society for Neurochemistry, J. Neurochem. (2003) 85, 503–514 503