Waagepetersen Helle S, Døring Søren, Schousboe Arne
Department of Pharmacology and Pharmacotherapy, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark.
Neurochem Res. 2008 Aug;33(8):1610-7. doi: 10.1007/s11064-008-9695-8. Epub 2008 Apr 25.
Mouse cerebral cortical mini-slices were used in a superfusion system to monitor depolarization-induced (55 mM K(+)) release of preloaded [2,3-(3)H]GABA and to investigate the biosynthesis of glutamate, GABA and aspartate during physiological and depolarizing (55 mM K(+)) conditions from either [1,6-(13)C]glucose or [U-(13)C]glutamine. Depolarization-induced GABA release could be reduced (50%) by the GABA transport inhibitor tiagabine (25 microM) or by replacing Ca(2+) with Co(2+). In the presence of both tiagabine and Co(2+) (1 mM), release was abolished completely. The release observed in the presence of 25 microM tiagabine thus represents vesicular release. Superfusion in the presence of [1,6-(13)C]glucose led to considerable labeling in the three amino acids, the labeling in glutamate and aspartate being increased after depolarization. This condition had no effect on GABA labeling. For all three amino acids, the distribution of label in the different carbon atoms revealed on increased tricarboxylic acid (TCA) activity during depolarization. When [U-(13)C]glutamine was used as substrate, labeling in glutamate was higher than that in GABA and aspartate and the fraction of glutamate and aspartate being synthesized by participation of the TCA cycle was increased by depolarization, an effect not seen for GABA. However, GABA synthesis reflected TCA cycle involvement to a much higher extent than for glutamate and aspartate. The results show that this preparation of brain tissue with intact cellular networks is well suited to study metabolism and release of neurotransmitter amino acids under conditions mimicking neural activity.
小鼠大脑皮质微小切片被用于灌注系统，以监测去极化诱导（55 mM K⁺）下预加载的[2,3-(³H)]GABA的释放，并研究在生理和去极化（55 mM K⁺）条件下，从[1,6-(¹³C)]葡萄糖或[U-(¹³C)]谷氨酰胺合成谷氨酸、GABA和天冬氨酸的情况。GABA转运抑制剂噻加宾（25 μM）或用Co²⁺替代Ca²⁺可使去极化诱导的GABA释放减少（50%）。在同时存在噻加宾和Co²⁺（1 mM）时，释放完全被消除。因此，在25 μM噻加宾存在下观察到的释放代表囊泡释放。在[1,6-(¹³C)]葡萄糖存在下进行灌注导致这三种氨基酸有相当程度的标记，去极化后谷氨酸和天冬氨酸的标记增加。这种情况对GABA标记没有影响。对于所有三种氨基酸，不同碳原子上标记的分布显示去极化期间三羧酸（TCA）活性增加。当使用[U-(¹³C)]谷氨酰胺作为底物时，谷氨酸的标记高于GABA和天冬氨酸，并且通过TCA循环参与合成的谷氨酸和天冬氨酸的比例因去极化而增加，GABA未观察到这种效应。然而，GABA合成反映TCA循环参与的程度比谷氨酸和天冬氨酸高得多。结果表明，这种具有完整细胞网络的脑组织制剂非常适合在模拟神经活动的条件下研究神经递质氨基酸的代谢和释放。
