Herdon H J, Godfrey F M, Brown A M, Coulton S, Evans J R, Cairns W J
Neuroscience Research, GlaxoSmithKline, Harlow, Essex CM19 5AW, UK.
Neuropharmacology. 2001 Jul;41(1):88-96. doi: 10.1016/s0028-3908(01)00043-0.
Two distinct types of glycine transporter, GlyT-1 and GlyT-2, have been characterised. GlyT-1 and GlyT-2 are known to be differentially expressed amongst CNS areas, but direct functional evidence for their relative contributions to high-affinity glycine uptake by brain tissues is lacking. In the present study, we have used the selective GlyT-1 inhibitor N[3-(4"-fluorophenyl)-3-(4"-phenylphenoxy)propyl]sarcosine (NFPS) to investigate the role of GlyT-1 in mediating glycine uptake. HEK293 cells expressing human GlyT-1c or GlyT-2 showed high levels of Na(+)-dependent glycine uptake, with K(m) values of 117+/-13 and 200+/-22 microM, respectively. NFPS potently inhibited uptake in GlyT-1c cells (IC(50) value 0.22+/-0.03 microM), being around 500-fold more potent than glycine or sarcosine, but had no effect on uptake in GlyT-2 cells (IC(50) >10 microM). Efflux of pre-loaded [3H]-glycine from GlyT-1c cells was increased by glycine or sarcosine, whereas NFPS had no effect on its own but blocked the effects of glycine or sarcosine. These results confirm that NFPS is a potent, selective and non-transportable GlyT-1 inhibitor. Rat cortex and cerebellum synaptosomes also showed a high-affinity Na(+)-dependent component of glycine uptake, with affinities similar to those observed for uptake in GlyT-1c or GlyT-2 cells. In cortex synaptosomes, NFPS and sarcosine produced the same maximal inhibition of uptake as glycine itself. However, in cerebellum synaptosomes, the maximal inhibition produced by NFPS and sarcosine was only half that produced by glycine. In both tissues NFPS was around 1000-fold more potent than glycine or sarcosine. Overall, our findings indicate that high-affinity glycine uptake in cerebral cortex occurs predominantly via GlyT-1. However, in cerebellum, only a part of the high-affinity uptake is mediated by GlyT-1, with the remaining NFPS-insensitive component most likely mediated by GlyT-2.
已鉴定出两种不同类型的甘氨酸转运体，即甘氨酸转运体-1（GlyT-1）和甘氨酸转运体-2（GlyT-2）。已知GlyT-1和GlyT-2在中枢神经系统区域中的表达存在差异，但缺乏关于它们对脑组织高亲和力甘氨酸摄取相对贡献的直接功能证据。在本研究中，我们使用了选择性GlyT-1抑制剂N-[3-(4''-氟苯基)-3-(4''-苯基苯氧基)丙基]肌氨酸（NFPS）来研究GlyT-1在介导甘氨酸摄取中的作用。表达人GlyT-1c或GlyT-2的HEK293细胞表现出高水平的钠依赖性甘氨酸摄取，其米氏常数（K(m)）值分别为117±13和200±22微摩尔。NFPS能有效抑制GlyT-1c细胞中的摄取（半数抑制浓度（IC(50)）值为0.22±0.03微摩尔），其效力比甘氨酸或肌氨酸高约500倍，但对GlyT-2细胞中的摄取没有影响（IC(50)>10微摩尔）。预加载的[3H]-甘氨酸从GlyT-1c细胞中的外流因甘氨酸或肌氨酸而增加，而NFPS自身没有影响，但能阻断甘氨酸或肌氨酸的作用。这些结果证实NFPS是一种强效、选择性且不可转运的GlyT-1抑制剂。大鼠皮质和小脑突触体也表现出高亲和力的钠依赖性甘氨酸摄取成分，其亲和力与在GlyT-1c或GlyT-2细胞中观察到的摄取亲和力相似。在皮质突触体中，NFPS和肌氨酸对摄取产生的最大抑制作用与甘氨酸本身相同。然而，在小脑突触体中，NFPS和肌氨酸产生的最大抑制作用仅为甘氨酸的一半。在这两种组织中NFPS的效力比甘氨酸或肌氨酸高约1000倍。总体而言，我们的研究结果表明，大脑皮质中的高亲和力甘氨酸摄取主要通过GlyT-1发生。然而，在小脑中，只有一部分高亲和力摄取是由GlyT-1介导的，其余对NFPS不敏感的成分很可能由GlyT-2介导。
