High-Affinity Transport of γ-Aminobutyric Acid, Glycine, Taurine, L-Aspartic Acid, and L-Glutamic Acid in Synaptosomal (P2) Tissue: A Kinetic and Substrate Specificity Analysis

In a cortical P2 fraction, [14C]gamma-aminobutyric acid ([14C]GABA), [14C]glycine, [14C]taurine, and [14C]glutamic and [14C]aspartic acids are transported by four separate high-affinity transport systems with L-glutamic acid and L-aspartic acid transported by a common system. GABA transport in cortical synaptosomal tissue occurs by one high-affinity system, with no second, low-affinity, transport system detectable. Only one high-affinity system is observed for the transport of aspartic/glutamic acids; as with GABA transport, no low-affinity transport is detectable. In the uptake of taurine and glycine (cerebral cortex and pons-medulla-spinal cord) both high- and low-affinity transport processes could be detected. The high-affinity GABA and high-affinity taurine transport classes exhibit some overlap, with the GABA transport system being more specific and having a much higher Vmax value. High-affinity GABA transport exhibits no overlap with either the high-affinity glycine or the high-affinity aspartic/glutamic acid transport class, and in fact they demonstrate somewhat negative correlations in inhibition profiles. The inhibition profiles of high-affinity cortical glycine transport and those of high-affinity cortical taurine and aspartic/glutamic acid transport also show no significant positive relationship. The inhibition profiles of high-affinity glycine transport in the cerebral cortex and in the pons-medulla-spinal cord show a significant positive correlation with each other; however, high-affinity glycine uptake in the pons-medulla-spinal cord is more specific than that in the cerebral cortex. The inhibition profile of high-affinity taurine transport exhibits a nonsignificant negative correlation with that of the aspartic/glutamic acid transport class.     

Distinct, Developmentally Regulated Brain mRNAs Direct the Synthesis of Neurotransmitter Transporters

The Xenopus laevis oocyte expression system was utilized to define developmental and structural properties of neurotransmitter transporter mRNAs and the pharmacological characteristics of encoded carriers independent of the complexities of brain tissue preparations. Poly(A)+ RNA from dissected brain regions of neonatal and adult rats was microinjected into Xenopus oocytes and the expression of Na(+)-dependent neurotransmitter transporters determined 48 h later. Transport studies conducted with oocytes injected with RNAs derived from juvenile rat tissues indicate a region- and transporter-specific, postnatal increase in mRNA abundance as a major factor in the developmental changes observed for brain high-affinity amino acid uptake systems. Both L-glutamic acid (Glu) and gamma-aminobutyric acid (GABA) uptake systems were detectable by day 3 in postnatal forebrain mRNA and became progressively enriched during the next 2 weeks of forebrain development. In contrast, brainstem Glu and GABA transporter enrichment was 60-70% of adult values by day 3 and exceeded adult levels by day 10. Parallel determinations of L-glutamic acid decarboxylase mRNA abundance during development argue for distinct regulatory influences on mRNAs directing transmitter synthesis and reuptake. Glycine uptake could not be detected at any point of forebrain development and exhibited a gradual postnatal rise to adult levels over the first 3 postnatal weeks of brainstem development. Uptake studies conducted with well-characterized inhibitors of Glu, GABA, dopamine, and choline transport (D-aspartate, nipecotic acid, nomifensine, and hemicholinium-3, respectively) revealed that oocyte transporters encoded by adult rat brain mRNAs retained antagonist sensitivities exhibited by in vitro brain preparations. In addition, a differential regional sensitivity to the Glu transport antagonist dihydrokainate (1 mM) was observed, lending support to previous reports of region-specific Glu transporter subtypes. To determine the structural diversity present among brain transporter mRNAs, poly(A)+ RNA was size-fractionated on linear (10-31%) sucrose density gradients prior to oocyte injection. These experiments revealed two mRNA size classes (2.4-3.0 kb, 4.0-4.5 kb) independently capable of directing the synthesis of Glu, GABA, and glycine transporters. In regions other than the cerebellum, Glu and GABA transporter activities migrated as single, yet distinct, peaks of 4.0-4.5 kb. In contrast, both Glu and GABA transporters exhibited major peaks of activity at 2.5-3.0 kb with size-fractionated cerebellar mRNA. Brainstem glycine uptake exhibited a broad sedimentation profile, with peaks apparent at 2.4 and 4.0 kb. Taken together, these findings indicate previously unappreciated complexity in mRNA structure and regulation which underlies the expression of amino acid neurotransmitter uptake systems in the rodent CNS.     

High-Affinity Uptake of (RS)-Nipecotic Acid in Astrocytes Cultured from Mouse Brain. Comparison with GABA Transport

Abstract: (RS)-Nipecotic acid is taken up into cultured astrocytes by a saturable high-affinity transport system with a K_m, of 28.8 ± 2.8 μM and a V_max of 0.294 ± 0.022 nmol × min⁻¹× [mg cell protein]⁻¹. The uptake which represents a net inward transport was sodium-dependent, requiring translocation of one sodium ion for each molecule of nipecotic acid taken up. The most potent inhibitors of GABA uptake into astrocytes (GABA, (R)-nipecotic acid, (3RS,4SR)-4-hydroxynipecotic acid, and guvacine) were shown to be potent inhibitors of nipecotic acid uptake (IC₅₀) 20, 25, 25, and 50 μm respectively), GABA being a competitive inhibitor. (S)-2,4-Diaminobutyric acid was a more efficient inhibitor than β-alanine of glial uptake of (RS)-nipecotic acid. It is concluded that astroglial uptake of (RS)-nipecotic acid and GABA is mediated by the same transport system.     

Amino acid neurotransmitter transporters: Structure,function, and molecular diversity

Many biologically active compounds including neurotransmitters, metabolic precursors, and certain drugs are accumulated intracellularly by transporters that are coupled to the transmembrane Na⁺ gradient. Amino acid neurotransmitter transporters play a key role in the regulation of extracellular amino acid concentrations and termination of neurotransmission in the CNS

1 Abbreviations: CNS, central nervous system; GABA, γ-aminobutyric acid; cDNA, complementary deoxyribonucleic acid; mRNA, messenger ribonucleic acid; NMDA, N-methyl-D-aspartate; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; DAG, diacyl glycerol; R59022, DAG kinase inhibitor; AA, arachidonic acid; ACHC, cis-3-aminocyclohexanecarboxylic acid; GAT-A, ACHC-sensitive GABA transporter; GAT-B, β-alanine-sensitive GABA transporter; GLY-1 and GLYT-1, glycine transporters; PROT-1, proline transporter; BGT-1, betaine transporter.

. Transporters for the major amino acid neurotransmitters glutamate, GABA, and glycine are found in both neurons and glial cells. Recent work has resulted in the identification of cDNAs encoding several amino acid neurotransmitter transport proteins, all of which belong to the Na⁺-and Cl^?-dependent transporter gene family. The diversity of this family suggests a degree of transporter heterogeneity that is greater than that indicated by biochemical and pharmacological studies.     

Neurotransmitter Metabolism in Rat Brain Synaptosomes: Effect of Anoxia and pH

Synaptosomes isolated from the rat cerebral cortex by means of a discontinuous Ficoll gradient carry out net, sodium-dependent, veratridine-sensitive accumulation of gamma-aminobutyric acid (GABA), serotonin, norepinephrine, and dopamine. The intrasynaptosomal contents of the four neurotransmitters are: 30.4 nmol/mg protein, 17.4 pmol/mg protein, 13.5 pmol/mg protein, and 21.2 pmol/mg protein, respectively. Anaerobic preincubation of synaptosomes causes an irreversible decrease in the rates of neurotransmitter accumulation but does not affect the rates of their release. The inhibitory effect of anaerobiosis is enhanced by increased concentration of [H+] (decreased pH) in the medium. The most sensitive is the uptake of dopamine, the least that of serotonin. The rates of neurotransmitter efflux are unaffected by anaerobiosis. Synaptosomes leak catecholamines, GABA, and serotonin into the medium when subjected to anaerobiosis, and reintroduction of oxygen is accompanied by a rapid reaccumulation of all four neurotransmitters. It is concluded that: (1) Responses of synaptosomes to anaerobiosis are remarkably similar to the behavior of intact brain in hypoxia and ischemia. (2) Neurotransmitter uptake systems are more sensitive to short periods of anaerobiosis than either the energy metabolism or ion transport. (3) Some neurotransmitter uptake systems are more easily damaged by anaerobiosis than others.     

Increased γ-Aminobutyric Acid Receptor Function in the Cerebral Cortex of Myoclonic Calves with an Hereditary Deficit in Glycine/Strychnine Receptors

Inherited congenital myoclonus (ICM) of Poll Hereford cattle is a neurological disease in which there are severe alterations in spinal cord glycine-mediated neurotransmission. There is a specific and marked decrease, or defect, in glycine receptors and a significant increase in neuronal (synaptosomal) glycine uptake. Here we have examined the characteristics of the cerebral gamma-aminobutyric acid (GABA) receptor complex, and demonstrate that the malfunction of the spinal cord inhibitory system is accompanied by a change in the major inhibitory system in the cerebral cortex. In synaptic membrane preparations from ICM calves, both high-and low-affinity binding sites for the GABA agonist [3H]muscimol were found (KD = 9.3 +/- 1.5 and 227 +/- 41 nM, respectively), whereas only the high-affinity site was detectable in controls (KD = 14.0 +/- 3.1 nM). The density and affinity of benzodiazepine agonist binding sites labelled by [3H]diazepam were unchanged, but there was an increase in GABA-stimulated benzodiazepine binding. The affinity for t-[3H]butylbicyclo-o-benzoate, a ligand that binds to the GABA-activated chloride channel, was significantly increased in ICM brain membranes (KD = 148 +/- 14 nM) compared with controls (KD = 245 +/- 33 nM). Muscimol-stimulated 36Cl- uptake was 12% greater in microsacs prepared from ICM calf cerebral cortex, and the uptake was more sensitive to block by the GABA antagonist picrotoxin. The results show that the characteristics of the GABA receptor complex in ICM calf cortex differ from those in cortex from unaffected calves, a difference that is particularly apparent for the low-affinity, physiologically relevant GABA receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacology of Sodium-Dependent High-Affinity l-[3H]Glutamate Transport in Glial Cultures

Abstract: Pharmacological and molecular biological studies provide evidence for subtypes of sodium-dependent high-affinity glutamate (Glu) transport in the mammalian CNS. At least some of these transporters appear to be selectively expressed in different brain regions or by different cell types. In the present study, the properties of l -[³H]Glu transport were characterized using astrocyte-enriched cultures prepared from cerebellum and cortex. In both brain regions, the kinetic data for sodium-dependent transport were consistent with a single site with K_m values of 91 ± 17 µM in cortical glial cells and 66 ± 23 µM in cerebellar glial cells. The capacities were 6.1 ± 1.6 nmol/mg of protein/min in cortical glial cells and 8.4 ± 0.9 nmol/mg of protein/min in cerebellar glial cells. The potencies of ～40 excitatory amino acid analogues for inhibition of sodium-dependent transport into glial cells prepared from cortex and cerebellum were examined, including compounds that are selective inhibitors of transport in synaptosomes prepared from either cerebellum or cortex. Of the analogues tested, 14 inhibited transport activity by >50% at 1 mM concentrations. Unlike l -[³H]Glu transport in synaptosomes prepared from cerebellum or cortex, there were no large differences between the potencies of compounds for inhibition of transport measured in glial cells prepared from these two brain regions. With the exception of (2S,1′R,2′R)-2-(carboxycyclopropyl)glycine and l -α-aminoadipate, all of the compounds examined were ～10–200-fold less potent as inhibitors of l -[³H]Glu transport measured in glial cells than as inhibitors of transport measured in synaptosomes prepared from their respective brain regions. The pharmacology of transport measured in these glial cells differs from the reported pharmacology of the cloned Glu transporters, suggesting the existence of additional uncloned Glu transporters or Glu transporter subunits.     

Glutamate-Induced Inhibition of D-Aspartate Uptake in Müller Glia from the Retina

Müller glial cells from the retina "in situ" and in primary culture, mainly express the high-affinity sodium-coupled glutamate/aspartate transporter GLAST-1, which dominates total retinal glutamate (Glu) uptake, suggesting a major role for these cells in the modulation of excitatory transmission. The possible involvement of ionotropic and metabotropic Glu receptors in the regulation of Glu uptake was studied in primary cultures of Müller glia. We demonstrate that exposure to 1 mM L-Glu induces a time-dependent inhibition of D-aspartate (D-Asp) uptake in a Na+-dependent manner, as a result of a reduction in the number of transporters at the plasma membrane. The inhibition of D-Asp uptake by Glu was not mimicked by agonists or modified by antagonists of ionotropic and metabotropic Glu receptors. In contrast, transport was inhibited by GLAST-1 transportable substrates threo-hydroxyaspartate and aspartate-beta-hydroxamate, but not by the nontransportable inhibitors trans-pyrrolidine dicarboxylate or DL-threo-beta-benzyloxyaspartic acid. Under the same experimental conditions, L-Glu did not affect the sodium-dependent transport systems for glycine or GABA. The present results demonstrate that the specific downregulation of glutamate/aspartate transport by L-Glu is unrelated to Glu receptor activation, and results from the internalization of transporter proteins triggered by the transport process itself. Such negative feedback of Glu on Glu transport, could contribute to retinal toxicity under pathological conditions in which high extracellular concentrations of Glu are reached.     

Transporter-associated currents in the gamma-aminobutyric acid transporter GAT-1 are conditionally impaired by mutations of a conserved glycine residue

To determine whether glycine residues play a role in the conformational changes during neurotransmitter transport, we have analyzed site-directed mutants of the gamma-aminobutyric acid (GABA) transporter GAT-1 in a domain containing three consecutive glycines conserved throughout the sodium- and chloride-dependent neurotransmitter transporter family. Only cysteine replacement of glycine 80 resulted in the complete loss of [(3)H]GABA uptake, but oocytes expressing this mutant exhibited the sodium-dependent transient currents thought to reflect a charge-moving conformational change. When sodium was removed and subsequently added back, the transients by G80C did not recover, as opposed to wild type, where recovery was almost complete. Remarkably, the transients by G80C could be restored after exposure of the oocytes to either GABA or a depolarizing pre-pulse. These treatments also resulted in a full recovery of the transients by the wild type. Whereas in wild type lithium leak currents are observed after prior sodium depletion, this was not the case for the glycine 80 mutants unless GABA was added or the oocytes were subjected to a depolarizing pre-pulse. Thus, glycine 80 appears essential for conformational transitions in GAT-1. When this residue is mutated, removal of sodium results in "freezing" the transporter in one conformation from which it can only exit by compensatory changes induced by GABA or depolarization. Our results can be explained by a model invoking two outward-facing states of the empty transporter and a defective transition between these states in the glycine 80 mutants.     

Glycine uptake by cultured human Y79 retinoblastoma cells: effect of changes in phospholipid fatty acid unsaturation

Glycine uptake was investigated in cultured Y79 retinoblastoma cells containing different degrees of phospholipid fatty acid unsaturation. The modifications were produced by growing the retinoblastoma cells in medium supplemented with various unsaturated fatty acids. Glycine was taken up by the retinoblastoma cells through two kinetically distinguishable process. The high-affinity system is totally dependent upon extracellular Na+ and partially dependent upon Ca2+. Of the glycine taken up by retinoblastoma cells, 85-90% remains as free intracellular glycine and less than 30% is incorporated into cellular protein. When the cells are grown in a medium containing 10% fetal bovine serum as the only source of fatty acids, the phospholipids contained 23% polyunsaturated fatty acids. Under these conditions the high-affinity system has a K'm of 34.2 +/- 3.7 micrometers and a V'max of 91.2 +/- 16.2 pmol min-1 mg protein -1. The low-affinity system has a K'm of 2.7 +/- 0.4 mM and a V'max of 4.1 +/- 0.5 nmol min-1 mg protein-1. When the polyunsaturated fatty acid content of the phospholipids was increased by supplementing the medium with linolenic or docosahexaenoic acids (n-3 polyunsaturates) or linoleic or arachidonic acids (n-6 polyunsaturates), the K'm and V'max of the high-affinity glycine uptake system were increased three- to fourfold. By contrast, supplementing the medium with oleic acid, and n-9 monounsaturate, did not significantly alter the K'm or V'max for glycine uptake. The results with this model system suggest that one of the effects of the high polyunsaturated fatty acid content normally present in neural cell membranes may be a modulation of the high-affinity transport system so that it functions more efficiently in regulating glycine uptake.     

Effect of Homo-β-Proline and Other Heterocyclic GAB A Analogues on GABA Uptake in Neurons and Astroglial Cells and on GABA Receptor Binding

Abstract: Two groups of GABA (γ-aminobutyric acid) analogues, one comprising derivatives of β-proline and the other compounds structurally related to nipecotic acid, were investigated as potential inhibitors of high-affinity GABA transport in neurons and glial cells, as well as displacers of GABA receptor binding. In addition to cis -4-hydroxynipecotic acid, which is known as a potent inhibitor of GABA uptake, homo-β-proline was the only compound which proved to be a potent inhibitor of glial as well as neuronal GABA uptake. IC₅₀ values for GABA uptake into glial cells and brain cortex "prisms" were 20 and 75 μM, respectively, and the IC₅₀ value obtained for GABA uptake into cultured neurons was 10 μM. A kinetic analysis of the action of homo-β-proline on GABA uptake into cultured astrocytes and neurons showed that this compound acts as a competitive inhibitor of GABA uptake in both cell types. From the apparent K _m values, K _i values for homo-β-proline of 16 and 6 μM could be calculated for glial and neuronal uptake, respectively. This mechanism of action strongly suggests that homo-β-proline interacts with the GABA carriers. Furthermore, homo-β-proline also displaced GABA from its receptor with an IC₅₀ value of 0.3 μM. The cis -4-hydroxynipecotic acid analogues, cis- and trans-4-mercaptonipecotic acid, had no inhibitory effect on glial or neuronal GABA uptake. Other SH reagents, PCMB, NEM and DTNB, were shown to be relatively weak inhibitors of GABA uptake into cultured astrocytes, suggesting that SH groups are not directly involved in the interaction between GABA and its transport carrier.     

Mutual Inhibition Kinetic Analysis of γ-Aminobutyric Acid, Taurine, and β-Alanine High-Affinity Transport into Neurons and Astrocytes: Evidence for Similarity Between the Taurine and β-Alanine Carriers in Both Cell Types

The transport kinetics of gamma-aminobutyric acid (GABA), taurine, and beta-alanine in addition to the mutual inhibition patterns of these compounds were investigated in cultures of neurons and astrocytes derived from mouse cerebral cortex. A high-affinity uptake system for each amino acid was demonstrated both in neurons (Km GABA = 24.9 +/- 1.7 microM; Km Tau = 20.0 +/- 3.3 microM; Km beta-Ala = 73.0 +/- 3.6 microM) and astrocytes (Km GABA = 31.4 +/- 2.9 microM, Km Tau = 24.7 +/- 1.3 microM; Km beta-Ala = 70.8 +/- 3.6 microM). The maximal uptake rates (Vmax) determined were such that, in neurons, Vmax GABA greater than Vmax beta-Ala = Vmax Tau, whereas in astrocytes, Vmax beta-Ala greater than Vmax Tau = Vmax GABA. Taurine was found to inhibit beta-alanine uptake into neurons and astrocytes in a competitive manner, with Ki values of 217 microM in neurons and 24 microM in astrocytes. beta-Alanine was shown to inhibit taurine uptake in neurons and astrocytes, also in a competitive manner, with Ki values of 72 microM in neurons and 71 microM in astrocytes. However, beta-alanine was found to be a weak noncompetitive inhibitor of neuronal and astrocytic GABA uptake, whereas in reverse experiments, GABA displayed weak noncompetitive inhibition of neuronal and astrocytic uptake of beta-alanine. Likewise, taurine was a weak noncompetitive inhibitor of GABA uptake in neurons and similarly, GABA was a weak noncompetitive inhibitor of taurine uptake into neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in the Uptake of GABA and Taurine During Neuronal and Glial Maturation

Abstract: The influence of the time of culture on GABA and taurine uptake was investigated in spontaneously matured cultures of glial and neuronal origins and in cultures treated with cyclic nucleotides. In the spontaneously matured cultures the capacity of the high-affinity neuronal GABA transport system increased with time in culture. Essentially opposite results were found for the uptake of GABA by glial cultures. In contrast with the neuronal uptake of GABA, the capacity of the taurine transport system was significantly decreased. Uptake of taurine into glia, however, exhibited a progressive increase with the period of culture. The values of Km, for the high-affinity systems were always found to range around 10 μM. It is suggested that, in mature cells, neuronal uptake sites are of prime importance for GABA transport, while taurine uptake may be more specifically directed towards glial cells. When cultures were treated with cyclic nucleotide derivatives, a morphological differentiation was induced, which could not be linked to a stimulation of GABA or taurine uptake systems as compared with the non-treated cultures.     

Perinatal changes of transport systems for amino acids in slices of mouse brain

Perinatal changes in the uptake of amino acids were measured in slices of fetal (15- and 19-day) and newborn (4-, 24-, and 48-hr-old) mouse brain. Uptake increased with age; smaller changes occurred with basic and neutral amino acid transport systems, and the largest changes occurred in fetal brain with amino acids of putative neurotransmitter function (taurine, glycine, GABA, and the acidic amino acids). The pattern of increase in uptake was similar at high and at low external amino acid concentrations. Developmental changes in tissue content of Na⁺, K⁺, or ATP were small during this period, and so are unlikely to be responsible for the observed changes in uptake. It appears that by the 15th day of fetal life, the transport systems for essential amino acids are fairly well developed in the brain, and the transport systems for neurotransmitter amino acids are not so well developed, but undergo a rapid increase in the 15–19-day period. From birth to adulthood, the concentrative capacity of slices of mouse brain for nonessential (putative neurotransmitter) amino acids is much greater than for essential amino acids.This research was supported in part by NIH Grant No. RR05707.     

Transport of G AB A, β-Alanine and Glutamate into Perikarya of Postnatal Rat Cerebellum

Abstract: Cells dissociated from the postnatally developing rat cerebellum retain their high-affinity carrier-mediated transport systems for [³H]GABA ( K _t=1.9 μM, V = 1.8 pmol/10⁶ cells/min) and [³H]glutamate ( K _t= 10 μM, V = 7.9 pmol/10⁶ cells/min). Using a unit gravity sedimentation technique it was demonstrated that [3H]GABA was taken principally into fractions that were enriched in inhibitory neurons (Purkinje, stellate and basket cells). [³H]β-alanine (which is taken up specifically by the glial GABA transport system) and [³H]glutamate were concentrated by glial-enriched fractions. However [³H]glutamate uptake was minimal in fractions enriched in precursors of granule cells, which may utilise this amino acid as their neurotransmitter. These results are discussed in relation to reports of high-affinity [³H]glutamate uptake by glia. The role of glutamate transport in glutamatergic cells is also considered. The data suggest that high-affinity glutamate transport is a property of glial cells but not granule neurons.     

Effects of GABAergic Drugs In Vivo on High-Affinity Choline Uptake In Vitro in Mouse Hippocampal Synaptosomes

The effects of several gamma-aminobutyric acid (GABA)-ergic drugs on sodium-dependent high-affinity choline uptake (HACU) were investigated in the hippocampus. HACU was measured in vitro after in vivo administration of the drug to mice. HACU was inhibited by those drugs that enhance GABA transmission. The convulsant 3-mercaptopropionic acid, which decreases GABA levels, stimulated HACU. From these results and previous findings, it appears that GABA mediates a tonic inhibitory effect on the septal-hippocampal cholinergic system.     

Developmental changes in high-affinity uptake of GABA by cultured neurons

High-affinity uptake of [³H]-aminobutyric acid (GABA) was studied in cultures of neonatal rat cortical neurons grown on pre-formed monolayers of non-neuronal (glial) cells. Both the maximum rate (V _max) and, to a smaller extent, theK _m of [³H]GABA uptake increased with time. In addition, in parallel with these changes, 2,4-diaminobutyric acid and cis-3-aminocyclohexane-1-carboxylic acid (ACHC), compounds which are considered typical substrate/inhibitors of GABA uptake in neurons, became progressively stronger inhibitors of [³H]GABA uptake. Consequently, the present results may mean that the studies using uptake, of [³H]GABA, [³H]ACHC, or [³H]DABA as a specific marker for GABAergic neurons differentiating during the ontogenetic development of the central nervous system may have to be interpreted with caution.     

Why glycine transporters have different stoichiometries

In the brain, neurons and glial cells compete for the uptake of the fast neurotransmitters, glutamate, GABA and glycine, through specific transporters. The relative contributions of glia and neurons to the neurotransmitter uptake depend on the kinetic properties, thermodynamic coupling and density of transporters but also on the intracellular metabolization or sequestration of the neurotransmitter. In the case of glycine, which is both an inhibitory transmitter and a neuromodulator of the excitatory glutamatergic transmission as a co-agonist of N-methyl D-aspartate receptors, the glial (GlyT1b) and neuronal (GlyT2a) transporters differ at least in three aspects: (i) stoichiometries, (ii) reverse uptake capabilities and (iii) pre-steady-state kinetics. A 3 Na(+)/1 Cl(-)/gly stoichiometry was established for GlyT2a on the basis of a 2 charges/glycine flux ratio and changes in the reversal potential of the transporter current as a function of the extracellular glycine, Na(+) and Cl(-) concentrations. Therefore, the driving force available for glycine uphill transport in neurons is about two orders of magnitude larger than for glial cells. In addition, GlyT2a shows a severe limitation for reverse uptake, which suggests an essential role of GlyT2a in maintaining a high intracellular glycine pool, thus facilitating the refilling of synaptic vesicles by the low affinity, low specificity vesicular transporter VGAT/VIAAT. In contrast, the 2 Na(+)/1 Cl(-)/gly stoichiometry and bi-directional transport properties of GlyT1b are appropriate for the control of the extracellular glycine concentration in a submicromolar range that can modulate N-methyl D-aspartate receptors effectively. Finally, analysis of the pre-steady-state kinetics of GlyT1b and GlyT2a revealed that at the resting potential neuronal transporters are preferentially oriented outward, ready to bind glycine, which suggests a kinetic advantage in the uptake contest.     

Amino acid and putative neurotransmitter transport in human Y79 retinoblastoma cells. Effect of insulin and insulin-like growth factor

The binding of insulin and insulin-like growth factor I (IGF-I) and their effect on amino acid and neurotransmitter transport was studied in cultured human Y79 retinoblastoma cells. Y79 cells possess specific receptors for both insulin and IGF-I. Insulin binding to Y79 cells is characterized by a curvilinear Scatchard plot suggesting a two-site or two-affinity binding system. In contrast, IGF-I binding has a linear plot indicative of a one-site, one-affinity binding system. The uptake of glycine, a putative neurotransmitter in the retina occurs by a specific transport system in Y79 cells, independent of the uptake of other neutral amino acids. The uptake of glycine was increased 25-50% by either insulin or IGF-I. The response to insulin or IGF-I on glycine uptake is gradual and concentration dependent. The accumulation of other amino acids and putative retinal neurotransmitters by Y79 cells was not significantly affected by insulin of IGF-I. In addition, the activity of Na+/K+-ATPase was not influenced. The analysis of high affinity glycine uptake indicates that insulin and IGF-I are stimulating glycine transport by increasing the V'max without significantly affecting the K'm. Further analysis suggests that insulin and IGF-I are causing a recruitment of additional glycine transporters at the cell surface or activating otherwise nonfunctional transporters by an unexplained mechanism. Because of the implication that glycine responds as a neuroactive amino acid in Y79 cells these studies suggest that insulin and IGF-I may influence neuroactivity in the human retina by regulating the transport of glycine.     

Morphological and Biochemical Changes in the Cerebellum Induced by Kainic Acid In Vivo

Abstract: Injection Of nanomole quantities Of kainic acid into The Rat cerebellum resulted in a rapid and selective destruction Of neurons. At all times Studied, the purkinje cells were depleted following injection, and this loss was paralleled by a decrease in GABA levels, high-affinity neuronal GABA uptake, and in glutamate decarboxylase activity. For the first 3 days following injection The granule cells were not reduced in numbers and the high-affinity uptake of glutamate was similarly not decreased. After 4-6 days, however, the granule cells were substantially depleted, although No clear changes in glutamate up-take were observed. The normal laminar structure of the cerebellum was extensively disrupted and glial proliferation was evident. Measurement of endogenous amino acid levels showed a decrease in glutamate and aspartate levels after kainic acid injection. glycine levels were consistently increased while glutamine and alanine were unchanged. GABA receptor binding was decreased after injection, but glutamate binding was enhanced.