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 [(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.

BDNF modulates glycine uptake in hippocampal synaptosomes by decreasing membrane insertion of glycine transporter 2

“…1B, squares in merge panel), which most probably correspond to gliosomes (Raiteri et al, 2008). This observation is in accordance with our findings that astrocytes do express GlyT2 (Aroeira et al, 2014). It was also possible to observe several SV2 positive…”

“…Immunocytochemistry assays were performed in the absence of the primary antibodies in order to exclude the possibility of cross reactions, as described previously (Aroeira et al, 2014). with a LP 615 nm filter.…”

“…Indeed, GlyT2 is regarded as a marker for glycine-containing nerve endings (Poyatos et al, 1997). Recently, it was demonstrated that functional GlyT2 was also expressed in cortical astrocytes, although with a much lower affinity for glycine than glial GlyT1, most probably contributing, together with astrocytic GlyT1, to shape glycinergic transmission (Aroeira et al, 2014).…”

A Glycine-Insulin Autocrine Feedback Loop Enhances Insulin Secretion From Human β-Cells and Is Impaired in Type 2 Diabetes

“…Indeed, we believe the local extracellular glycine concentration in islets is significantly lower due to the GlyT-dependent clearance of glycine. These transporters are very effective at clearing glycine (V max = 379 pmol/min/mg for GlyT1 and 5730 pmol/min/mg for GlyT2 [42]), and GlyT activity is stimulated as the cell becomes more hyperpolarized (i.e., during resting periods or between the characteristic slow wave depolarizations of b-cells [34]). Our experiments show that inhibition of GlyT via Li + replacement results in nearly a doubling of the endogenous glycine signal, suggesting that GlyT not only clear plasma glycine but also regulate glycine signaling in cells.…”

Glycine transporters type 1 inhibitor promotes brain preconditioning against NMDA-induced excitotoxicity

“…For instance, the stimulation of purinergic receptors (PY2R) led to a functional inhibition of GlyT2 and stimulation of GlyT1 through the same signal transduction pathways, whereas lithium exposition inhibits GlyT1 and stimulate GlyT2 (Jim enez et al, 2011;P erez-Siles et al, 2011). In the hippocampus, GlyT-1 is widely expressed and associated with glutamatergic neurotransmission where functionally interact with NMDAR, whereas GlyT2 is sparsely expressed in hippocampal interneurons, being associated with glycinergic terminals (Zafra et al, 1995a;Zafra et al, 1995b;Cubelos et al, 2005;Aroeira et al, 2013). GlyT2 has a greater carrying capacity when compared to GIyT1, probably due to the characteristic that it did not operate on the reverse mode as GlyT1, which gives it an advantage in maintaining high cytosolic concentrations of glycine (Aubrey et al, 2007;Raiteri et al, 2008).…”