Pharmacological assessment of the role of the glycine transporter GlyT-1 in mediating high-affinity glycine uptake by rat cerebral cortex and cerebellum synaptosomes

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 muM, respectively. NFPS potently inhibited uptake in GlyT-1c cells (IC50 value 0.22 +/-0.03 muM), being around 500-fold more potent than glycine or sarcosine, but had no effect on uptake in GlyT-2 cells (IC50 >10 muM). Efflux of pre-loaded [H-3]-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. (C) 2001 Elsevier Science Ltd. All rights reserved.