Ana María López-Colomé

Glial transporters for glutamate, glycine, and GABA III. Glycine transporters

The termination of chemical neurotransmission in the CNS involves the rapid removal of neurotransmitter from synapses by specific transport systems. Such mechanism operates for the three major amino acid neurotransmitters glutamate, γ‐aminobutyric acid (GABA) and glycine. To date, five different high‐affinity Na+‐dependent glutamate (Glu) transporters have been cloned: GLT1, GLAST, EAAC1, EAAT4 and EAAT5. The first two are expressed mainly by glial cells, and seem to be the predominant Glu transporters in the brain. A major function of Glu uptake in the nervous system is to prevent extracellular Glu concentrations from raising to neurotoxic levels in which glial transporters seem to play a critical role in protecting neurons from glutamate‐induced excitotoxicity. Under particular conditions, glial GluTs have been shown to release Glu by reversal of activity, in a Ca2+‐ and energy‐independent fashion. Furthermore, an activity of these transporters as ion channels or transducing units coupled to G‐proteins has recently been reported. The localization, stoichiometry, and regulation of glial GluTs are outlined, as well as their possible contributions to nervous system diseases as ALS, AD and ischemic damage. J. Neurosci. Res. 63:453–460, 2001.

NMDA receptors in cultured radial glia

© 1997 Federation of European Biochemical Societies.

Sodium-dependent glutamate transport in Müller glial cells : regulation by phorbol esters

The regulation of the Na(+)-dependent high affinity glutamate/aspartate transporter system expressed in cultured Müller glia cells from chick retina was studied. Treatment of the cells with the Ca(2+)/diacylglycerol dependent protein kinase C (PKC) activator, phorbol 12-tetradecanoil-13-acetate (TPA) produced a decrease in [(3)H]D-aspartate uptake which was reversed by staurosporine and partially by H7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochoride], two PKC inhibitors. Long-term treatment with TPA resulted in a drastic decrease in the uptake activity, correlated with a substantial fall in the expression of the transporter protein. These findings suggest that PKC is involved in transport modulation at two different levels: phosphorylation and transporter expression in retinal Müller glial cells.

K+-stimulated release of labeled γ-aminobutyrate, glycine and taurine in slices of several regions of rat central nervous system

The effect of depolarization by high K+ concentration (68.5 mm) on the release of [3H] γ-aminobutyrate (GABA), [14C]glycine and [35S]taurine was studied in superfused slices of rat cerebellum, striatum, hypothalamus, colliculi, cerebral cortex and ventral and dorsal halves of spinal cord. The release of [3H]GABA was notably stimulated by K+-depolarization in all regions studied, particularly in the cerebral cortex and the hypothalamus. The Ca2+-dependence of this phenomenon was studied in the cortex and ventral spinal cord; in both regions the K+-stimulated release was abolished when Ca2+ was omitted from the superfusing medium. The release of [14C]glycine was also stimulated in all regions, except the cerebellum, but to a lesser extent than that of GABA. This stimulation was Ca2+-dependent in the ventral spinal cord but not in the cerebral cortex. The release of [35S]taurine was not affected by K+-depolarization in any of the regions studied. These results are consistent with a widely distributed neurotransmitter role for GABA. The Ca2+-dependence of glycine release in the spinal cord is in agreement with a role of this amino acid as a transmitter in this region. The finding that [35S]taurine release was not stimulated by K+-depolarization in any of the regions studied, under experimental conditions identical to those resulting in an enhancement of [3H]GABA and [14C]glycine release, argues against a neurotransmitter role of this amino acid in brain and spinal cord.

Potassium-stimulated release of GABA, glycine, and taurine from the chick retina.

The effect of depolarizing potassium concentration on the release of [14C]glycine, [3H]GABA, and [35S]taurine was investigated in the whole chick retina and in a synaptosomal fraction prepared from the chick retina. In the whole retina, increasing potassium concentration above 40 mM resulted in an increased release of the three amino acids. The release of glycine was the most stimulated and that of taurine, the least. The potassium-evoked release of glycine and GABA was calcium dependent. In the synaptosomal fraction, 68.5 mM potassium significantly stimulated the efflux of GABA and glycine by a calcium-dependent mechanism. The release of taurine from this fraction was unaffected by high potassium.

Taurine effects on45Ca2+ transport in retinal subcellular fractions

The effect of taurine on 45Ca2+ transport by subcellular fractions from the chick retina was examined. An inhibitory action of taurine on 45Ca2+ uptake was observed in retinal fractions incubated for 1--5 min in a Krebs--bicarbonate medium, pH 7.4. In the crude nuclear fraction, 25 mM taurine produced a decrease of 50% in 45Ca2+ uptake; in the crude synaptosomal fraction, taurine reduced 45Ca2+ accumulation by 70%; the maximum inhibitory effect of taurine on 45Ca2+ uptake (80%) was observed in a fraction containing outer segments and pigment epithelium cells. Taurine effect was specific, dose-dependent and related to osmotically sensitive particles. The results suggest a role for taurine in the regulation of calcium fluxes in the retina.

Glial transporters for glutamate, glycine, and GABA III. Glycine transporters: Glial Transporters

Glial cells possess transport systems for the three major amino acid neurotransmitters glutamate, γ‐aminobutyric acid (GABA) and glycine, involved in the arrest of neurotransmission mediated by these compounds. Two glycine transporters have been cloned: GLYT1, mainly expressed by glial cells and shown to colocalize with NMDA receptors, and GLYT2, exclusively expressed by neurons and colocalized with the inhibitory glycine receptors. The way in which the regulation of extracellular glycine concentration by glial glycine transporters affects physiological and pathological conditions is discussed. The presence, differential pharmacology and specific regulation of glycine transporters in glial cells strongly support an important role for glia in the modulation of both, excitatory and inhibitory neurotransmission. J. Neurosci. Res. 64:218–222, 2001.

Thrombin promotes actin stress fiber formation in RPE through Rho/ROCK-mediated MLC phosphorylation

The retinal pigment epithelium (RPE) forms the outer blood–retina barrier (BRB). Most retinal diseases involve BRB breakdown, whereupon thrombin contained in serum directly contacts the RPE. Thrombin is known to promote actin stress fiber formation, an important determinant in eye diseases involving the epithelial–mesenchymal transition (EMT) and migration of RPE cells, such as proliferative vitreoretinopathy. We analyzed thrombin effect on signaling pathways leading to myosin light chain (MLC) phosphorylation and actin stress fiber formation in primary cultures of rat RPE cells, in order to support a role for thrombin in RPE transdifferentiation. MLC phosphorylation was measured by Western blot; actin cytoskeleton was visualized using immunofluorescent phalloidin, and Rho GTPase activation was assessed by ELISA. We showed that thrombin/PAR‐1 induces the time‐ and dose‐dependent phosphorylation of MLC through the activation of Rho/ROCK and myosin light chain kinase (MLCK). ROCK increased phospho‐MLC by phosphorylating MLC and by inhibiting MLC phosphatase. Thrombin effect was abolished by the ROCK inhibitor Y‐27632, whereas MLCK inhibitor ML‐7 and PLC‐β inhibitor U73122 attenuated MLC phosphorylation by ≈50%, suggesting the activation of MLCK by PLC‐β‐mediated calcium increase. Additionally, thrombin‐induced MLC phosphorylation was blocked by the inhibitory PKCζ pseudosubstrate, wortmannin, and LY294002, indicating IP3/PKCζ involvement in the control of MLC phosphorylation. Moreover, we demonstrated that thrombin effect on MLC induces actin stress fiber formation, since this effect was prevented by inhibiting the pathways leading to MLC phosphorylation. We conclude that thrombin stimulation of MLC phosphorylation and actin stress fiber formation may be involved in thrombin‐induced RPE cell transformation subsequent to BRB dysfunction. J. Cell. Physiol. 226: 414–423, 2011.

AMPA/KA receptor expression in radial glia.

The expression of four genes (GluR 1; 2; 3; 4) encoding functional subunits of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/low affinity kainate (KA) subtype of glutamate receptors was investigated in chick radial glia, namely Bergmann and Müller glial cells, using Northern blot analysis with oligonucleotide probes. Both cell types expressed the transcripts GluR 1; 3; 4, whereas the GluR 2 mRNA could not be detected. The synaptic localization of these receptors, their ion-channel properties and their regulation further strengthen the putative role of glial cells in the modulation of synaptic efficacy and plasticity.

High-affinity binding ofl-glutamate to chick retinal membranes

Binding ofl-[3H]glutamate to membranes from whole chick retina and from subcellular fractions enriched with photoreceptor terminals (P1), or terminals from the inner plexiform layer (P2) was studied. Na+-dependent and Na+-independent binding to these membranes was demonstrated. Na+-independent binding was stereospecific. Kinetic analysis of the binding process indicated a single high-affinity system (KB=0.55 μM) with a capacity of approximately 20 pmoles/mg protein in all the membrane fractions. [3H]Glutamate binding to P1 and P2 fractions was effectively displaced by several structural analogues of glutamate. Glutamate diethyl-ester appreciably displaced binding, whereas kainic acid did not displace bound glutamate. Data indicate the binding of [3H]glutamate to physiologically relevant receptors in the chick retina.