Deitmer Joachim W, Bröer Angelika, Bröer Stefan
School of Biochemistry & Molecular Biology, Australian National University, Canberra, Australia.
J Neurochem. 2003 Oct;87(1):127-35. doi: 10.1046/j.1471-4159.2003.01981.x.
The neurotransmitter glutamate, once released into the synaptic cleft, is largely recycled by the glutamate-glutamine cycle, which involves uptake into astrocytes, conversion into glutamine and subsequent release of glutamine from astrocytes as a precursor for neuroneal glutamate synthesis. We analysed glutamine efflux from cultured astrocytes by pre-loading cells with labelled glutamine for 30 min and subsequently measured glutamine efflux for 30 min. Efflux of pre-loaded glutamine was rapid and almost complete after 30 min with a first order rate of 0.11 +/- 0.01/min. Efflux was 50% reduced when cells were depleted of intracellular Na+. Increasing intracellular Na+ concentration had a small stimulatory effect on glutamine efflux, indicating the participation of a Na+-dependent transport mechanism. About 50% of the basal efflux could not be inhibited by depletion of the intracellular Na+, suggesting the presence of an additional Na+-independent transport mechanism. Glutamine efflux was stimulated two- to threefold by addition of extracellular neutral amino acids, such as alanine or leucine. The stimulatory effects of alanine and leucine had a Na+-dependent and a Na+-independent component, suggesting the presence of two antiport mechanisms one involving Na+. When compared to the expression of glutamine transporter mRNAs in cultured astrocytes it appeared likely that glutamine efflux was mediated by SN1, LAT2, ASCT2 and an additional, yet unidentified, transporter that mediates about 40% of the basal efflux.
神经递质谷氨酸一旦释放到突触间隙，很大程度上会通过谷氨酸-谷氨酰胺循环进行再循环，该循环包括被星形胶质细胞摄取、转化为谷氨酰胺以及随后谷氨酰胺从星形胶质细胞释放，作为神经元谷氨酸合成的前体。我们通过用标记的谷氨酰胺预加载细胞30分钟，然后测量30分钟内的谷氨酰胺流出，来分析培养的星形胶质细胞中的谷氨酰胺流出情况。预加载的谷氨酰胺流出迅速，30分钟后几乎完全流出，一级速率为0.11±0.01/分钟。当细胞内的钠离子耗尽时，流出减少50%。增加细胞内钠离子浓度对谷氨酰胺流出有轻微的刺激作用，表明存在一种钠离子依赖性转运机制。约50%的基础流出不能被细胞内钠离子的耗尽所抑制，这表明存在一种额外的非钠离子依赖性转运机制。通过添加细胞外中性氨基酸，如丙氨酸或亮氨酸，谷氨酰胺流出受到两到三倍的刺激。丙氨酸和亮氨酸的刺激作用有一个钠离子依赖性成分和一个非钠离子依赖性成分，表明存在两种反向转运机制，其中一种涉及钠离子。与培养的星形胶质细胞中谷氨酰胺转运体mRNA的表达相比，谷氨酰胺流出似乎是由SN1、LAT2、ASCT2以及另一种尚未确定的转运体介导的，后者介导约40%的基础流出。
