title: Valine but not leucine or isoleucine supports neurotransmitter glutamate synthesis during synaptic activity in cultured cerebellar neurons.
abstract: Synthesis of neuronal glutamate from α-ketoglutarate for neurotransmission necessitates an amino group nitrogen donor; however, it is not clear which amino acid(s) serves this role. Thus, the ability of the three branched-chain amino acids (BCAAs), leucine, isoleucine, and valine, to act as amino group nitrogen donors for synthesis of vesicular neurotransmitter glutamate was investigated in cultured mouse cerebellar (primarily glutamatergic) neurons. The cultures were superfused in the presence of (15) N-labeled BCAAs, and synaptic activity was induced by pulses of N-methyl-D-aspartate (300 μM), which results in release of vesicular glutamate. At the end of the superfusion experiment, the vesicular pool of glutamate was released by treatment with α-latrotoxin (3 nM, 5 min). This experimental paradigm allows a separate analysis of the cytoplasmic and vesicular pools of glutamate. Amount and extent of (15) N labeling of intracellular amino acids plus vesicular glutamate were analyzed employing HPLC and LC-MS analysis. Only when [(15) N]valine served as precursor did the labeling of both cytoplasmic and vesicular glutamate increase after synaptic activity. In addition, only [(15) N]valine was able to maintain the amount of vesicular glutamate during synaptic activity. This indicates that, among the BCAAs, only valine supports the increased need for synthesis of vesicular glutamate.
authors.affiliation: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. laba@farma.ku.dk
authors.forename: Lasse K
authors.initials: LK
authors.lastname: Bak
authors.forename: Maja L
authors.initials: ML
authors.lastname: Johansen
authors.forename: Arne
authors.initials: A
authors.lastname: Schousboe
authors.forename: Helle S
authors.initials: HS
authors.lastname: Waagepetersen
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002531:Cerebellum / Q000378:metabolism*; D018698:Glutamic Acid / Q000096:biosynthesis*; D007532:Isoleucine / Q000378:metabolism; D007930:Leucine / Q000378:metabolism; D051379:Mice; D009474:Neurons / Q000378:metabolism*; D009435:Synaptic Transmission / Q000502:physiology*; D014633:Valine / Q000378:metabolism*
chemical_list: D007532:Isoleucine; D018698:Glutamic Acid; D007930:Leucine; D014633:Valine