Valine but not leucine or isoleucine supports neurotransmitter glutamate synthesis during synaptic activity in cultured cerebellar neurons
Valine but not leucine or isoleucine supports neurotransmitter glutamate synthesis during synaptic activity in cultured cerebellar neurons
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DOI：
10.1002/jnr.23072
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发表时间：
2012-09-01
影响因子：
4.2
通讯作者：
Waagepetersen, Helle S.
中科院分区：
文献类型：
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作者：
Bak, Lasse K.;Johansen, Maja L.;Waagepetersen, Helle S.
从a-酮戊二酸合成神经元谷氨酸用于神经传递需要氨基氮供体；然而，目前尚不清楚是哪种氨基酸发挥了这一作用。因此，在培养的小鼠小脑（主要是谷氨酸能）神经元中，研究了亮氨酸、异亮氨酸和缬氨酸这三种支链氨基酸（BCAAs）作为氨基氮供体合成泡状神经递质谷氨酸的能力。在15n标记的支链氨基酸存在下进行培养，用300 μ M的n -甲基- d -天冬氨酸脉冲诱导突触活性，导致水疱性谷氨酸释放。在实验结束时，用a- latotoxin （3 nM, 5 min）释放谷氨酸囊泡池。这种实验模式允许对谷氨酸的细胞质和囊泡池进行单独的分析。采用HPLC和LC-MS分析细胞内氨基酸和囊泡谷氨酸的15N标记量和程度。只有当[15N]缬氨酸作为前体时，突触活动后胞质谷氨酸和囊泡谷氨酸的标记才会增加。此外，在突触活动期间，只有[15N]缬氨酸能够维持囊泡谷氨酸的数量。这表明，在支链氨基酸中，只有缬氨酸支持水疱谷氨酸合成的增加需求。(c) 2012 Wiley期刊有限公司
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.