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GlyT1 and GlyT2 in brain astrocytes: expression, distribution and function

Abstract

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 [3H]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.

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Acknowledgments

The authors thank Dr. Manuel Miranda-Arango (Texas) for kindly supplying the antibodies against GlyT1 and GlyT2. This work was supported by Fundação para a Ciência e a Tecnologia (FCT), Portugal. Rita I Aroeira is in receipt of a fellowship (SFRH/BD/62831/2009) from FCT. The authors declare that this research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Correspondence to Cláudia A. Valente.

Electronic supplementary material

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Supplementary material 1 Online Resource 1 melting curves of GlyT1, GlyT2 and β-actin transcripts analyzed by qPCR. Y axis represents the first derivate of raw fluorescence and X axis corresponds to an increase in temperature. Each curve has a single melting peak, which indicates that a single PCR product is being amplified. (PPT 126 kb)

Supplementary material 2 Online Resource 2 transport progression curves using two concentrations of glycine, 50 µM to study GlyT1 (a) and 1,500 µM to study GlyT2 (b). Incubation time ranging from 30 to 240 s was tested. Arrows point to the incubation time used in the subsequent experiments. (PPT 176 kb)

Supplementary material 3 Online Resource 3 concentration-dependent uptake of glycine in rat cultured cortical astrocytes by GlyT inhibitors. GlyT1-specific inhibitor Org 24598 (a), GlyT2-blockers ALX 1393 (b) and amoxapine (c) were used in several concentrations. Note the differences in X axis scale between graphs. Y axis represents [3H]glycine uptake as percentage of the control value (absence of inhibitors) in the same experiment. Arrows point to the concentration of inhibitor used in the subsequent experiments to isolate GlyT1 and GlyT2 specific transport. (PPT 134 kb)

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Aroeira, R.I., Sebastião, A.M. & Valente, C.A. GlyT1 and GlyT2 in brain astrocytes: expression, distribution and function. Brain Struct Funct 219, 817–830 (2014). https://doi.org/10.1007/s00429-013-0537-3

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Keywords

  • Glycine transporter 1
  • Glycine transporter 2
  • Astrocytes
  • Brain
  • Glycine uptake
  • Inhibitory neurotransmission