Functional identification of activity-regulated, high-affinity glutamine transport in hippocampal neurons inhibited by riluzole.
Functional identification of activity-regulated, high-affinity glutamine transport in hippocampal neurons inhibited by riluzole.
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DOI：
10.1111/jnc.14046
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发表时间：
2017-07
影响因子：
4.7
通讯作者：
Erickson JD
中科院分区：
文献类型：
--
作者：
Erickson JD
谷氨酰胺（Gln）被认为是谷氨酸（Glu）神经递质库的首选前体，谷氨酸是哺乳动物中枢神经系统中主要的兴奋性递质。在发育和成熟的富含神经元的海马培养物中，描述了一个活性调节的高亲和力Gln转运系统，该系统被抗谷氨酸药物利鲁唑（IC50为1.3 +/−0.5µM）有效抑制，并被低浓度的2-（甲氨基）异丁酸盐（MeAIB）（一种系统a转运抑制剂）阻断。K+刺激的MeAIB转运对MeAIB的亲和力（Km）为37 +/−1.2µM，在~200µM时达到饱和，依赖于细胞外Ca2+，并通过抑制电压门控Ca2+通道（VGCCs）而受阻。自发MeAIB转运也依赖于细胞外Ca2+和VGCCs，但也被Na+通道阻滞剂河河鱼毒素、Glu受体拮抗剂和GABA阻断，表明其依赖于内源性谷氨酸能活性驱动的完整神经回路。MeAIB本身的转运不依赖于Ca2+，而是依赖于Na+离子，并且对ph值敏感。活性调节的、利鲁唑敏感的自发转运和K+刺激转运在体外7-8天（DIV）最小，在接下来的两周内协调诱导，并在DIV bbbb20中最大程度表达；已知的Glu/Gln周期成熟和调节突触前Glu释放的时期。与各种氨基酸的竞争分析表明，谷氨酰胺是最有可能的生理底物。在星形胶质细胞中未观察到活动调节的Gln/MeAIB运输。海马神经元中活动调节、高亲和力、利鲁唑敏感的Gln/MeAIB转运的功能鉴定可能在活动刺激的突触前Glu释放、星形胶质细胞和神经元之间的Glu/Gln循环以及神经元Glu诱导的兴奋毒性的神经生物学方面具有重要意义。本报告描述了海马神经元富集原代培养中Ca2+调节的“系统a”谷氨酰胺运输系统，该系统依赖于成熟突触中的神经活动，被利鲁唑（突触谷氨酸释放的阻断剂）有效抑制，并且在星形胶质细胞和神经元之间谷氨酸/谷氨酰胺循环和突触谷氨酸释放的功能成熟的关键产后时期上调。本文描述的新型高亲和系统A转运体可能在理解急性和慢性神经退行性疾病中兴奋毒性突触谷氨酸释放的神经生物学方面具有生理和病理意义。
Glutamine (Gln) is considered the preferred precursor for the neurotransmitter pool of glutamate (Glu), the major excitatory transmitter in the mammalian CNS. Here, an activity-regulated, high-affinity Gln transport system is described in developing and mature neuron-enriched hippocampal cultures that is potently inhibited by riluzole (IC50 1.3 +/− 0.5µM), an anti-glutamatergic drug, and is blocked by low concentrations of 2-(methylamino)isobutyrate (MeAIB), a system A transport inhibitor. K+-stimulated MeAIB transport displays an affinity (Km) for MeAIB of 37 +/− 1.2 µM, saturates at ~200 µM, is dependent on extracellular Ca2+, and is blocked by inhibition of voltage gated Ca2+-channels (VGCCs). Spontaneous MeAIB transport is also dependent on extracellullar Ca2+ and VGCCs, but is also blocked by the Na+ channel blocker tetrodotoxin, by Glu receptor antagonists, and by GABA indicating its dependence on intact neural circuits driven by endogenous glutamatergic activity. The transport of MeAIB itself does not rely on Ca2+, but on Na+ ions, and is pH-sensitive. Activity-regulated, riluzole-sensitive spontaneous and K+-stimulated transport is minimal at 7–8 days in vitro (DIV), coordinantly induced during the next two weeks, and is maximally expressed by DIV>20; the known period for maturation of the Glu/Gln cycle and regulated presynaptic Glu release. Competition analyses with various amino acids indicate that Gln is the most likely physiological substrate. Activity-regulated Gln/MeAIB transport is not observed in astrocytes. The functional identification of activity-regulated, high-affinity, riluzole-sensitive Gln/MeAIB transport in hippocampal neurons may have important ramifications in the neurobiology of activity stimulated presynaptic Glu release, the Glu/Gln cycle between astrocytes and neurons, and neuronal Glu-induced excitotoxicity. This report describes a Ca2+-regulated ‘system A’ glutamine transport system in hippocampal neuron-enriched primary cultures that is dependent on neural activity in mature synapses, potently inhibited by riluzole (a blocker of synaptic glutamate release), and that is up-regulated during the critical postnatal period of functional maturation of the glutamate/glutamine cycle between astrocytes and neurons and synaptic glutamate release. The novel high-affinity system A transporter described here may have physiological and pathological implications in understanding the neurobiology of excitotoxic synaptic glutamate release in acute and chronic neurodegenerative diseases.
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