Aroeira Rita I, Sebastião Ana M, Valente Cláudia A
Institute of Pharmacology and Neurosciences, Faculty of Medicine, and Unit of Neurosciences, Institute of Molecular Medicine, University of Lisbon, Av. Prof. Egas Moniz, 1649-028, Lisbon, Portugal.
Brain Struct Funct. 2014 May;219(3):817-30. doi: 10.1007/s00429-013-0537-3. Epub 2013 Mar 26.
GlyT1 and GlyT2 are the transporters responsible for glycine uptake from the synaptic cleft. The expression and function of these two glycine transporters in rat cortical cultured astrocytes over several maturation stages (10, 18 and 24 days in vitro) were herein investigated. Quantitative PCR and western blot showed that both GlyT1 and GlyT2 transcripts and protein were expressed in astrocytes in the examined maturation stages. Double detection of Glial fibrillary acidic protein (GFAP) and GlyT1/GlyT2 revealed that both transporters were detected in the cell body and in the processes of astrocytes. Furthermore, the double immunofluorescence analysis carried out in P21 rat brain slices corroborated the presence of both transporters in cortical and hippocampal astrocytes. The functional characterization of GlyT1 and GlyT2 in cultured astrocytes performed by [(3)H]glycine uptake experiments revealed that both transporters take up glycine in a concentration-dependent way, but with a very distinct affinity. Kinetic analysis revealed a K m of 51.15 ± 4.96 μM and a V max of 379.30 ± 10.31 pmol/min/mg for GlyT1 and a K m of 1,801 ± 148.9 μM and a V max of 5,730 ± 200.2 pmol/min/mg for GlyT2. It is concluded that astrocytes express functional GlyT2, which challenge previous findings that those cells would express only GlyT1, whereas GlyT2 was supposed to be restricted to the glycinergic nerve terminals. Therefore, the work herein reported provides new insights about glycinergic neurotransmission in the brain.
甘氨酸转运体1（GlyT1）和甘氨酸转运体2（GlyT2）是负责从突触间隙摄取甘氨酸的转运蛋白。本文研究了这两种甘氨酸转运蛋白在大鼠皮质培养星形胶质细胞的几个成熟阶段（体外培养10、18和24天）的表达和功能。定量PCR和蛋白质印迹显示，在检测的成熟阶段，星形胶质细胞中均表达了GlyT1和GlyT2的转录本及蛋白质。胶质纤维酸性蛋白（GFAP）和GlyT1/GlyT2的双重检测显示，在星形胶质细胞的胞体和突起中均检测到了这两种转运蛋白。此外，在出生后21天的大鼠脑切片上进行的双重免疫荧光分析证实，皮质和海马星形胶质细胞中均存在这两种转运蛋白。通过[³H]甘氨酸摄取实验对培养的星形胶质细胞中GlyT1和GlyT2进行功能特性分析，结果显示这两种转运蛋白均以浓度依赖性方式摄取甘氨酸，但亲和力非常不同。动力学分析显示，GlyT1的米氏常数（Km）为51.15±4.96μM，最大反应速度（Vmax）为379.30±10.31pmol/min/mg；GlyT2的Km为1801±148.9μM，Vmax为5730±200.2pmol/min/mg。得出的结论是，星形胶质细胞表达功能性GlyT2，这对之前认为这些细胞仅表达GlyT1而GlyT2仅限于甘氨酸能神经末梢的研究结果提出了挑战。因此，本文报道的工作为大脑中甘氨酸能神经传递提供了新的见解。
