Valine but not leucine or isoleucine supports neurotransmitter glutamate synthesis during synaptic activity in cultured cerebellar neurons

TY - JOUR

T1 - Valine but not leucine or isoleucine supports neurotransmitter glutamate synthesis during synaptic activity in cultured cerebellar neurons

AU - Bak, Lasse Kristoffer

AU - Johansen, Maja L.

AU - Schousboe, Arne

AU - Waagepetersen, Helle S

N1 - Copyright © 2012 Wiley Periodicals, Inc.

PY - 2012/9

Y1 - 2012/9

N2 - 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 15N-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 15N labeling of intracellular amino acids plus vesicular glutamate were analyzed employing HPLC and LC-MS analysis. Only when [15N]valine served as precursor did the labeling of both cytoplasmic and vesicular glutamate increase after synaptic activity. In addition, only [15N]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.

AB - 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 15N-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 15N labeling of intracellular amino acids plus vesicular glutamate were analyzed employing HPLC and LC-MS analysis. Only when [15N]valine served as precursor did the labeling of both cytoplasmic and vesicular glutamate increase after synaptic activity. In addition, only [15N]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.

U2 - 10.1002/jnr.23072

DO - 10.1002/jnr.23072

M3 - Journal article

C2 - 22589238

SN - 0360-4012

VL - 90

SP - 1768

EP - 1775

JO - Journal of Neuroscience Research

JF - Journal of Neuroscience Research

IS - 9

ER -