Role of Glial Cells for the Basal and Ca2+-Dependent K+-Evoked Release of Transmitter Amino Acids Investigated by Microdialysis

The role of glial cells for the inactivation and synthesis of precursors for amino acid transmitters was studied in the brains of anesthetized rats in vivo using the microdialysis technique. The dialysis probes were inserted stereotactically into each neostriatum. One neostriatum was treated with the gliotoxin fluorocitrate, whereas the contralateral side served as a control. The basal efflux of amino acids, reflecting the extracellular level, was measured as well as the efflux during depolarization with 100 mM K+ in the dialysis stream. The potassium-evoked efflux of transmitter amino acids was calcium dependent and thus considered to reflect release from the transmitter pool. gamma-Aminobutyric acid (GABA) and glutamate release from the treated side was higher than the control value during the first 2-3 h, a result indicating an important role of glial cells in the inactivation of released transmitter. After 6-7 h with fluorocitrate, the release of glutamate was lower than the control value, a result indicating an important role of glial cells in the synthesis of precursors for the releasable pool of glutamate. The role of glutamine for the production of transmitter glutamate and GABA in vivo was further investigated by inhibiting glutamine synthetase with intrastriatally administered methionine sulfoximine. The release of gluatamate into the dialysis probe decreased to 54% of the control value, whereas the release of GABA decreased to 22% of the control value, a result indicating that glutamine may be more important for transmitter GABA than for transmitter glutamate.     

Glutamate as a Putative Transmitter in the Cerebellum: Stimulation by GABA of Glutamic Acid Release from Specific Pools

The aim of the present paper was to determine whether the release of glutamate from putative "glutamergic" terminals in the cerebellum is influenced by gamma-aminobutyric acid (GABA). In a group of preliminary experiments, we present biochemical evidence in favour of a neurotransmitter role of glutamate in the cerebellum: (1) endogenous glutamate was released from depolarized cerebellar synaptosomal preparations in a Ca2+-dependent away; (2) [14C]glutamate was synthesized from [14C]glutamine in cerebellar synaptosomes, and the newly synthesized [14C]glutamate was released released in a Ca2+-dependent way; (3) the elevation of cyclic GMP elicited by depolarization of cerebellar slices in the presence of Ca2+ was partly reversed by the glutamate antagonist glutamic acid diethyl ester, which probably prevented the interaction of endogenously released glutamate with postsynaptic receptors. GABA and muscimol at low concentrations (2--20 micrometers) potentiated the depolarization-induced release of D-[3H]aspartate (a glutamate analogue which labels the glutamate "reuptake pool") from cerebellar synaptosomes. The effect was concentration dependent and was largely prevented by two GABA antagonists, bicuculline and picrotoxin. The stimulation of D-[3H]aspartate release evoked by muscimol was linearly related to the logarithm of K+ concentration in the depolarizing medium. GABA did not affect the overall release of endogenous glutamate, but potentiated, in a picrotoxin-sensitive manner, the depolarization-evoked release of [14C]glutamate previously synthesized from [14C]glutamine. Since nerve endings are the major site of glutamate synthesis from glutamine, GABA and muscimol appear to exert their stimulatory effect at the level of "glutamergic" nerve terminals, probably after interacting with presynaptic GABA receptors. The possible functional significance of these findings is briefly discussed.     

Release of [3H]l-glutamate and [3H]l-glutamine in rat cerebellum slices: A comparison of the effect of veratridine and electrical stimulation

Depolarization-elicited release of neurotransmitter glutamate was studied in rat cerebellar slices previously loaded with either [³H]l-glutamate or [³H]l-glutamine. Both depolarization conditions used (e.g. long-lasting tonic depolarization elicited by veratridine, or short repetive electrical pulses) increased 6 to 8 folds the release of labelled glutamate and of another compound, presumably alpha-ketoglutarate, without modifying the release of labeled glutamine. Because of the position of the label in the precursor radioactive molecules, GABA was weakly labeled and aspartate was unlabeled. The properties of the evoked glutamate release from cerebellar slices were those of a neurotransmitter since it was inhibited by tetrodotoxin and was Ca²⁺-dependent. Alpha-ketoglutarate is either coreleased from nerve terminals or is released from astrocytes and could participate in glutamate recycling. The data confirm the generally accepted model implying the presence of two neurotransmitter glutamate pools, a neuronal pool of newly synthesized glutamate and an astrocytic storage pool, but in addition indicate that the former is in rapid isotopic equilibrium with the extracellular compartment. Our present results also indicate that the glutamate/glutamine cycle is not activated in depolarizing conditions.With the technical assistance of O. LEVY¹ and K. WINDISCH²     

Glutamine as a precursor for transmitter glutamate, aspartate and GABA in the cerebellum: a role for phosphate-activated glutaminase

Phosphate-activated glutaminase is present at high levels in the cerebellar mossy fiber terminals. The role of this enzyme for the production of glutamate from glutamine in the parallel-fiber terminals is unclear. In order to address this, we used light miroscopic immunoperoxidase and electron microscopic immunogold methods to study the localization of glutamate in rat cerbellar slices incubated with physiological K+ (3 mmol/L) and depolarizing K+ (40 mmol/L) concentrations, and during depolarizing conditions with the addition of glutamine and the glutaminase inhibitor 6-diazo-5-oxo-l-norleucine. During K+-induced depolarization glutamate labeling was redistributed from parallel-fiber terminals to glial cells. The nerve terminal content of glutamate was sustained when the slices were supplied with glutamine, which also reduced the accumulation of glutamate in glia. In spite of glutamine supplementation, the depolarized slices treated with 6-diazo-5-oxo-l-norleucine showed depletion of glutamate from parallel-fiber terminals and accumulation in glial cells. We conclude that cerebellar parallel-fiber terminals contain a glutaminase activity enabling them to synthesize glutamate from glutamine. Our results confirm that this is also true for the mossy fiber terminals. In addition, we show that, like for glutamate, the levels of aspartate in parallel-fiber terminals and GABA in Golgi fiber terminals can be maintained during depolarization if glutamine is present. This process is dependent on the activity of a glutaminase, as it can be inhibited by 6-diazo-5-oxo-l-norleucine, suggesting that the glutaminase reaction is important for glutamine to act as a precursor also for aspartate and GABA. The low levels of the kidney type of glutaminase that previously has been shown to be present in the parallel and Golgi fiber terminals could be sufficient to produce the transmitter amino acids. Alternatively, the amino acids could be produced from the liver type of glutaminase, which is not yet localized on the cellular level, or from an unknown glutminase.     

Comparison of Transmitter Amino Acid Levels in Rat Globus Pallidus and Neostriatum During Hypoglycemia or After Treatment with Methionine Sulfoximine or γ-Vinyl γ-Aminobutyric Acid

The levels of amino acids in globus pallidus, a structure heavily innervated with gamma-aminobutyric acid (GABA)-ergic terminals but few glutamergic terminals, were compared with the levels in neostriatum, a structure richly innervated with glutamergic terminals but intermediate in GABAergic terminals. The level of glutamate in neostriatum was twice as high as in globus pallidus whereas the level of GABA in globus pallidus was three times higher than in neostriatum. The level of aspartate was similar in both regions whereas the level of glutamine was correlated with the level of glutamate. Methionine sulfoximine, a glutamine synthetase inhibitor, reduced the level of glutamine to 10-20% of control in both structures. This reduction was accompanied by the largest decrease in the level of glutamate in neostriatum, indicating that transmitter glutamate turns over more rapidly than other glutamate pools. Likewise, insulin decreased the levels of glutamate and glutamine more in neostriatum than in globus pallidus. gamma-Vinyl GABA increased the level of GABA in globus pallidus more than in neostriatum although the percent increase was largest in neostriatum. Treatment with gamma-vinyl GABA was accompanied by a large reduction in the level of GABA, indicating that a substantial proportion of the glutamine pool is linked to GABA metabolism.     

Glutamate metabolism in the brain of young rats exposed to organic and inorganic lead

The synthesis of glutamate and its conversion to glutamine and GABA were studied using labelled glucose in cerebral cortex, cerebellum and brainstem of rats intoxicated acutely with tetraethyl lead and chronically with lead acetate. To assess the interconversion and the synaptosomal accumulation of these amino acids, the labelling of glutamate, glutamine and GABA were measured in whole tissue and synaptosomes after giving labelled glutamate. The radioactive carbon dioxide production from labelled glutamate by brain slices was measured to evaluate the oxidation of glutamate. The tissue levels of glutamate, glutamine and GABA and the activity of glutamate decarboxylase were also measured in both conditions.In inorganic lead toxicity, even though the glutamate pool size was reduced, the glutamate-glutamine cycling between synaptosomes and astrocytes was increased. The oxidation of glutamate and the glutamate-GABA cycling were reduced. These findings suggest that brain tries to maintain the endogenous glutamate levels by decreasing the oxidation of glutamate and increasing the uptake systems and the cycling through glutamine in inorganic lead toxicity. In organic lead toxicity, the glutamate pool as well as glutamate turnover was reduced markedly resulting in complete distortion of glutamate metabolism.     

Glutamine and Glucose as Precursors of Transmitter Amino Acids: Ex Vivo Studies

Abstract: Radiolabelled glutamine and glucose were infused into lateral ventricles of rats in order to label transmitter amino acid pools in vivo . Brain regions close to the lateral ventricle (hippocampus, corpus striatum, hypothalamus) were labelled more effectively than more distant structures such as cerebral cortex or cerebellum. All regions were labelled to much the same extent over 30-150 min by [U-¹⁴C]glucose, [U-¹⁴C]glutamine, or [³H]glutamine administered alone or together in doublelabel experiments when allowance was made for any differences in precursor specific radioactivities. Slices of cerebral cortex or hippocampus from brains labelled in vivo were incubated and stimulated in vitro with veratrine (75 μ M ); tetrodotoxin (1 μ M ) was present in the control medium. Single-label experiments showed that [U-¹⁴C]- glutamine was more effective than [U-¹⁴C]glucose for labelling releasable glutamate and GABA. Double-label experiments showed that [³H]glutamine and [U-¹⁴C]- glucose given together in vivo labelled glutamate and GABA releasable in vitro to a similar extent. Both types of experiment empbasise the large contribution made by glutamine in vivo to pools of transmitter glutamate and GABA.     

Effect of Glutamine on Glutamate Release from Hippocampal Slices Induced by High K+ or by Electrical Stimulation: Interaction with Different Ca2+ Concentrations

To characterize the effect of glutamine on the release of glutamate, aspartate, and γ-aminobutyric acid (GABA), rat hippocampal slices were superfused with different concentrations of glutamine or Ca²⁺. Amino acids released and retained were analyzed by HPLC. Glutamine (0.5 mmol/L) increased more than threefold the release of glutamate evoked by 50 mmol/L K⁺ in the presence of 2.6 mmol/L Ca²⁺ without a corresponding increase in glutamate content, while the release of aspartate was increased less and that of GABA not at all by glutamine. The evoked release of all three amino acids, including the enhanced release of glutamate in the presence of glutamine, was strongly dependent on Ca²⁺ concentrations between 0.1 and 2.6 mmol/L. The potentiation of glutamate release by glutamine reached a plateau at 0.25 mmol/L glutamine. Intermittent electrical field stimulation increased the release of only glutamate and this release was nearly doubled by glutamine. The increased release was Ca²⁺ dependent and tetrodotoxin (TTX) sensitive. Results suggest that extracellular glutamine promotes primarily the formation of releasable glutamate and this enhancement is dependent on extracellular Ca²⁺.     

The operation of the γ-aminobutyrate bypath of the tricarboxylic acid cycle in brain tissue in vitro

1. Cerebral-cortex slices prelabelled with gamma-amino[1-(14)C]butyrate (GABA) were incubated in a glucose-saline medium. After the initial rapid uptake there was no appreciable re-entry of (14)C into the GABA pool, either from the medium or from labelled metabolites formed in the tissue. The kinetic constants of GABA metabolism were determined by computer simulation of the experimental results by using mathematical procedures. The GABA flux was estimated to be 0.03mumol per min/g, or about 8% of the total flux through the tricarboxylic acid cycle. It was found that the assumption of compartmentation did not greatly affect the estimates of the GABA flux. 2. The time-course of incorporation of (14)C into amino acids associated with the tricarboxylic acid cycle was followed with [1-(14)C]GABA and [U-(14)C]-glucose as labelled substrates. The results were consistent with the utilization of GABA via succinate. This was confirmed by determining the position of (14)C in the carbon skeletons of aspartate and glutamate formed after the oxidation of [1-(14)C]GABA. These results also indicated that under the experimental conditions the reversal of reactions catalysed by alpha-oxoglutarate dehydrogenase and glutamate decarboxylase respectively was negligible. The conversion of [(14)C]GABA into gamma-hydroxybutyrate was probably also of minor importance, but decarboxylation of oxaloacetate did occur at a relatively slow rate. 3. When [1-(14)C]GABA was the labelled substrate there was evidence of a metabolic compartmentation of glutamate since, even before the peak of the incorporation of (14)C into glutamate had been reached, the glutamine/glutamate specific-radioactivity ratio was greater than unity. When [U-(14)C]glucose was oxidized this ratio was less than unity. The heterogeneity of the glutamate pool was indicated also by the relatively high specific radioactivity of GABA, which was comparable with that of aspartate during the whole incubation time (40min). The rates of equilibration of labelled amino acids between slice and medium gave evidence that the permeability properties of the glutamate compartments labelled as a result of oxidation of [1-(14)C]GABA were different from those labelled by the metabolism of [(14)C]glucose. The results showed therefore that in brain tissue incubated under the conditions used, the organization underlying metabolic compartmentation was preserved. The observed concentration ratios of amino acids between tissue and medium were also similar to those obtaining in vivo. These ratios decreased in the order: GABA>acidic acids>neutral amino acids>glutamine. 4. The approximate pool sizes of the amino acids in the different metabolic compartments were calculated. The glutamate content of the pool responsible for most of the labelling of glutamine during oxidation of [1-(14)C]GABA was estimated to be not more than 30% of the total tissue glutamate. The GABA content of the ;transmitter pool' was estimated to be 25-30% of the total GABA in the tissue. The structural correlates of metabolic compartmentation were considered.     

Utilization of alpha-ketoglutarate as a precursor for transmitter glutamate in cultured cerebellar granule cells

Alpha-ketoglutarate together with an amino group donor (alanine) was shown to be able to serve as a precursor for the glutamate pool which is released by potassium-induced depolarization (i.e., transmitter glutamate) in cerebellar granule cells. However, these compounds could not be utilized as precursors for intracellular glutamate or for release of transmitter aspartate. The formation of transmitter glutamate was inhibited by the transamination inhibitor aminooxyacetic acid but not by phenylsuccinate, an inhibitor of the dicarboxylate carrier in the mitochondrial membrane. Both of these inhibitors have previously been found to inhibit synthesis of transmitter glutamate from glutamine. The results support the hypothesis that alpha-ketoglutarate and alanine undergo transamination in the cytosol to form pyruvate and glutamate, and that this glutamate pool is available for transmitter release of glutamate but does not constitute the major intracellular pool of glutamate.     

Compartmentation of TCA cycle metabolism in cultured neocortical neurons revealed by 13C MR spectroscopy

Cultured neocortical neurons were incubated in medium containing [U-13C]glucose (0.5 mM) and in some cases unlabeled glutamine (0.5 mM). Subsequently the cells were "superfused" for investigation of the effect of depolarization by 55 mM K+. Cell extracts were analyzed by 13C magnetic resonance spectroscopy and gas chromatography/mass spectrometry to determine incorporation of 13C in glutamate, GABA, aspartate and fumarate. The importance of the tricarboxylic acid (TCA) cycle for conversion of the carbon skeleton of glutamine to GABA was evident from the effect of glutamine on the labeling pattern of GABA and glutamate. Moreover, analysis of the labeling patterns of glutamate in particular indicated a depolarization induced increased oxidative metabolism. This effect was only observed in glutamate and not in neurotransmitter GABA. Based on this a hypothesis of mitochondrial compartmentation may be proposed in which mitochondria associated with neurotransmitter synthesis are distinct from those aimed at energy production and influenced by depolarization. The hypothesis of mitochondrial compartmentation was further supported by the finding that the total percent labeling of fumarate and aspartate differed significantly from each other. This can only be explained by the existence of multiple TCA cycles with different turnover rates.     

Effects of High-Potassium-Induced Depolarization on Amino Acid Chemistry of the Dorsal Cochlear Nucleus in Rat Brain Slices

High K⁺ was used to depolarize glia and neurons in order to study the effects on amino acid release from and concentrations within the dorsal cochlear nucleus (DCN) of brain slices. The release of glutamate, -aminobutyrate (GABA) and glycine increased significantly during exposure to 50 mM K⁺, while glutamine and serine release decreased significantly during and/or after exposure, respectively. After 10 min of exposure to 50 mM K⁺, glutamine concentrations increased in all three layers of DCN slices, to more than 5 times the values in unexposed slices. In the presence of a glutamate uptake blocker, L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC), glutamine concentrations in all layers did not increase as much during 50 mM K⁺. Similar but smaller changes occurred for serine. Mean ATP concentrations were lower in 50 mM K⁺-exposed slices compared to control. The results suggest that depolarization, such as during increased neural activity, can greatly affect amino acid metabolism in the cochlear nucleus.     

Metabolism of [1,6-13C]Glucose and [U-13C]Glutamine and Depolarization Induced GABA Release in Superfused Mouse Cerebral Cortical Mini-slices

Mouse cerebral cortical mini-slices were used in a superfusion system to monitor depolarization-induced (55 mM K⁺) release of preloaded [2,3-³H]GABA and to investigate the biosynthesis of glutamate, GABA and aspartate during physiological and depolarizing (55 mM K⁺) conditions from either [1,6-¹³C]glucose or [U-¹³C]glutamine. Depolarization-induced GABA release could be reduced (50%) by the GABA transport inhibitor tiagabine (25 μM) or by replacing Ca²⁺ with Co²⁺. In the presence of both tiagabine and Co²⁺ (1 mM), release was abolished completely. The release observed in the presence of 25 μM tiagabine thus represents vesicular release. Superfusion in the presence of [1,6-¹³C]glucose led to considerable labeling in the three amino acids, the labeling in glutamate and aspartate being increased after depolarization. This condition had no effect on GABA labeling. For all three amino acids, the distribution of label in the different carbon atoms revealed on increased tricarboxylic acid (TCA) activity during depolarization. When [U-¹³C]glutamine was used as substrate, labeling in glutamate was higher than that in GABA and aspartate and the fraction of glutamate and aspartate being synthesized by participation of the TCA cycle was increased by depolarization, an effect not seen for GABA. However, GABA synthesis reflected TCA cycle involvement to a much higher extent than for glutamate and aspartate. The results show that this preparation of brain tissue with intact cellular networks is well suited to study metabolism and release of neurotransmitter amino acids under conditions mimicking neural activity. Special issue article in honor of Dr. Ricardo Tapia.     

ÈVIDENCE FOR THE COMPARTMENTATION OF GLUTAMATE METABOLISM IN ISOLATED RAT RETINA

—Glucose is a major precursor of glutamate and related amino acids in the retina of adult rats. ¹⁴C from labelled glucose appears to gain access to a large glutamate pool, and the resulting specific activity of glutamate labelled from glucose is always higher than that of glutamine or the other amino acids. Radioactive acetate appeared to label a small glutamate pool. The specific activity of glutamine labelled from acetate relative to that of glutamate was always greater than 1.0. Other precursors of the small glutamate pool were found to include glutamate, aspartate, GABA, serine, leucine and sodium bicarbonate. The level of radioactivity present in retinae incubated with [U-¹⁴C]glucose or [1-¹⁴C]sodium acetate was reduced in the presence of 10^?5m -ouabain. Under these conditions, the relative specific activity of glutamine labelled from [1-¹⁴C]sodium acetate was lowered, but it was raised when [U-¹⁴C]glucose was used as substrate. Ouabain also considerably reduced the synthesis of GABA from [1-¹⁴C]sodium acetate. In all cases ouabain caused a fall in the tissue levels of the amino acids. Aminooxyacetic acid (10^?4m ) almost completely abolished the labelling of GABA from both [U-¹⁴C]glucose and [1-¹⁴C]sodium acetate, while the RSA of glutamine labelled from the latter substrate was significantly increased. Aminooxyacetic acid raised the tissue concentration of glutamate, but caused a fall in the tissue concentrations of glutamine, aspartate and GABA. The results suggest that there are separate compartments for the metabolism of glutamate in retina and that these can be modified in different ways by different drugs.     

Distinct changes in neuronal and astrocytic amino acid neurotransmitter metabolism in mice with reduced numbers of synaptic vesicles

The relations between glutamate and GABA concentrations and synaptic vesicle density in nerve terminals were examined in an animal model with 40–50% reduction in synaptic vesicle numbers caused by inactivation of the genes encoding synapsin I and II. Concentrations and synthesis of amino acids were measured in extracts from cerebrum and a crude synaptosomal fraction by HPLC and ¹³C nuclear magnetic resonance spectroscopy (NMRS), respectively. Analysis of cerebrum extracts, comprising both neurotransmitter and metabolic pools, showed decreased concentration of GABA, increased concentration of glutamine and unchanged concentration of glutamate in synapsin I and II double knockout (DKO) mice. In contrast, both glutamate and GABA concentrations were decreased in crude synaptosomes isolated from synapsin DKO mice, suggesting that the large metabolic pool of glutamate in the cerebral extracts may overshadow minor changes in the transmitter pool. ¹³C NMRS studies showed that the changes in amino acid concentrations in the synapsin DKO mice were caused by decreased synthesis of GABA (20–24%) in cerebral neurons and increased synthesis of glutamine (36%) in astrocytes. In a crude synaptosomal fraction, the glutamate synthesis was reduced (24%), but this reduction could not be detected in cerebrum extracts. We suggest that lack of synaptic vesicles causes down-regulation of neuronal GABA and glutamate synthesis, with a concomitant increase in astrocytic synthesis of glutamine, in order to maintain normal neurotransmitter concentrations in the nerve terminal cytosol.     

Evoked release from guinea pig cerebral cortex slices of endogenous 14C-labelled amino acids, labelled via D-[U-14C]glucose.

Release of endogenous amino acids labelled via D-[U-14C]glucose was compared with that of several exogenous labelled amino acids using slices of guinea pig cerebral cortex. Electrical field stimulation evoked a selective release of endogenous [14C]glutamate, [14C]aspartate, and gamma-amino[14C]butyrate (14C-labelled GABA). The selectivity of release correlated well with 14C incorporation into endogenous amino acids. Calculations of the fraction of the tissue radioactivity released indicated that the selectivity was not an artifact due to differential incorporation. Because glucose in mammalian brain is metabolized almost entirely by the so-called 'large compartment', it is tentatively concluded that the releasable 'transmitter pool' of glutamate, aspartate, and GABA is located in this 'large compartment'.     

Depolarization-Induced Release of Amino Acids From the Vestibular Nuclear Complex

There is evidence from immunohistochemistry, quantitative microchemistry, and pharmacology for several amino acids as neurotransmitters in the vestibular nuclear complex (VNC), including glutamate, γ-aminobutyrate (GABA), and glycine. However, evidence from measurements of release has been limited. The purpose of this study was to measure depolarization-stimulated calcium-dependent release of amino acids from the VNC in brain slices. Coronal slices containing predominantly the VNC were prepared from rats and perfused with artificial cerebrospinal fluid (ACSF) in an interface chamber. Fluid was collected from the chamber just downstream from the VNC using a microsiphon. Depolarization was induced by 50 mM potassium in either control calcium and magnesium concentrations or reduced calcium and elevated magnesium. Amino acid concentrations in effluent fluid were measured by high performance liquid chromatography. Glutamate release increased fivefold during depolarization in control calcium concentration and twofold in low calcium/high magnesium. These same ratios were 6 and 1.5 for GABA, 2 and 1.3 for glycine, and 2 and 1.5 for aspartate. Differences between release in control and low calcium/high magnesium ACSF were statistically significant for glutamate, GABA, and glycine. Glutamine release decreased during and after depolarization, and taurine release slowly increased. No evidence for calcium-dependent release was found for serine, glutamine, alanine, threonine, arginine, taurine, or tyrosine. Our results support glutamate and GABA as major neurotransmitters in the VNC. They also support glycine as a neurotransmitter and some function for taurine.     

Glutamine and 2-oxoglutarate as metabolic precursors of the transmitter pools of glutamate and GABA: Correlation of regional uptake by rat brain synaptosomes

To more clearly define the roles of glutamine and 2-oxoglutarate as metabolic precursors of the transmitter pools of glutamate and GABA we have determined the relative rates at which these four substances, and adenosine and serotonin are accumulated by synaptosomes derived from twelve regions of the rat brain. Inital transport conditions and low substrate concentrations were used to maximize uptake by high-affinity systems, except the uptake of glutamine was determined at both low and high concentrations. Because the uptake of 2-oxoglutarate is markedly enhanced by glutamine, 2-oxoglutarate uptake was determined with and without glutamine (0.2 mM) added to the incubation medium. For each substrate, regional differences in uptake ranged from approximately two- to fourteen-fold. An analysis of uptake kinetics revealed that the regional differences were due primarily to differences in transport capacity rather than substrate affinities, at least for glutamate, GABA, and 2-oxoglutarate. Thirty-four correlation analyses of relative uptake values were performed. Strong correlations were found between 2-oxoglutarate and glutamate, and between glutamine and glutamate, whereas no strong correlations occurred between these substrates and GABA. Our results support the view that both glutamine and 2-oxoglutarate are major precursors of the transmitter pool of glutamate throughout the rat brain, but their relative contributions toward replenishing the transmitter pool of GABA are less certain.Special issue dedicated to Elling Kvamme.     

Release of D,L-threo-beta-hydroxyaspartate as a false transmitter from excitatory amino acid-releasing nerve terminals

This study examined whether preaccumulated D,L-threo-beta-hydroxyaspartate (tHA), a competitive substrate for the high-affinity excitatory amino acid (EAA) transporter, is released as a false transmitter from EAA-releasing nerve terminals. Potassium-stimulation (50 mM for 1 min) evoked significant release of the endogenous EAAs (aspartate and glutamate) from superfused neocortical minislices. Endogenous EAA release was largely calcium-dependent and was inhibited by tetanus toxin, a neurotoxin which specifically blocks vesicular exocytosis. In parallel experiments, minislices were pre-incubated with 500 microM tHA. Potassium (50 mM) evoked significant release of tHA and this release was also calcium-dependent and reduced by tetanus toxin. Pre-accumulation of tHA did not affect the release of endogenous glutamate whereas the release of endogenous aspartate was significantly attenuated. These data suggest that tHA selectively accumulates in a vesicular aspartate pool and is released upon depolarization as a false transmitter from EAA nerve terminals.     

Glutamine uptake and metabolism to N-acetylaspartylglutamate (NAAG) by crayfish axons and glia

We have proposed that N-acetylaspartylglutamate (NAAG) or its hydrolytic product glutamate, is a chemical signaling agent between axons and periaxonal glia at non-synaptic sites in crayfish nerves, and that glutamine is a probable precursor for replenishing the releasable pool of NAAG. We report here, that crayfish central nerve fibers synthesize NAAG from exogenous glutamine. Cellular accumulation of radiolabel during in vitro incubation of desheathed cephalothoracic nerve bundles with [3H]glutamine was 74% Na(+)-independent. The Na(+)-independent transport was temperature-sensitive, linear with time for at least 4 h, saturable between 2.5 and 10 mM L-glutamine, and blocked by neutral amino acids and analogs that inhibit mammalian glutamine transport. Radiolabeled glutamine was taken up and metabolized by both axons and glia to glutamate and NAAG, and a significant fraction of these products effluxed from the cells. Both the metabolism and release of radiolabeled glutamine was influenced by extracellular Na(+). The uptake and conversion of glutamine to glutamate and NAAG by axons provides a possible mechanism for recycling and formation of the axon-to-glia signaling agent(s).