期刊
JOURNAL OF NEUROSCIENCE RESEARCH
卷 90, 期 9, 页码 1768-1775出版社
WILEY-BLACKWELL
DOI: 10.1002/jnr.23072
关键词
branched-chain amino acid; neuron; glutamate; vesicular; neurotransmitter
资金
- Alfred Benzon Foundation
- Lundbeck Foundation
- Carlsberg Foundation
- Horslev Foundation
- Danish Medical Research Council [09-063399, 09-063393]
Synthesis of neuronal glutamate from a-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 mu M), which results in release of vesicular glutamate. At the end of the superfusion experiment, the vesicular pool of glutamate was released by treatment with a-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. (c) 2012 Wiley Periodicals, Inc.
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