Kainic Acid Differentially Affects the Synaptosomal Release of Endogenous and Exogenous Amino Acidic Neurotransmitters

Presynaptic actions of kainic acid have been tested on uptake and release mechanisms in synaptosome-enriched preparations from rat hippocampus and goldfish brain. Kainic acid increased in a Ca2+-dependent way the basal release of endogenous glutamate and aspartate from both synaptosomal preparations, with the maximum effect (40-80%) being reached at the highest concentration tested (1 mM). In addition, kainic acid potentiated, in an additive or synergic way, the release of excitatory amino acids stimulated by high K+ concentrations. Kainic acid at 1 mM showed a completely opposite effect on the release of exogenously accumulated D-[3H]aspartate. The drug, in fact, caused a marked inhibition of both the basal and the high K+-stimulated release. Kainic acid at 0.1 mM had no clear-cut effect, whereas at 0.01 mM it caused a small stimulation of the basal release. The present results suggest that kainic acid differentially affects two neurotransmitter pools that are not readily miscible in the synaptic terminals. The release from an endogenous, possibly vesiculate, pool of excitatory amino acids is stimulated, whereas the release from an exogenously accumulated, possibly cytoplasmic and carrier-mediated, pool is inhibited or slightly stimulated, depending on the external concentration of kainic acid. Kainic acid, in addition, strongly inhibits the high-affinity uptake of L-glutamate and D-aspartate in synaptic terminals. All these effects appear specific for excitatory amino acids, making it likely that they are mediated through specific recognition sites present on the membranes of glutamatergic and aspartatergic terminals. The relevance of the present findings to the mechanism of excitotoxicity of kainic acid is discussed.     

Amino acid neurotransmitters in the CNS. Characteristics of the acidic amino acid exchange

D-Aspartate exchange, defined as amino acid-stimulated D-[3H]aspartate efflux, was investigated in a preparation of rat brain synaptosomes. The efflux of radiolabelled D-aspartate was found to be enhanced by micromolar concentrations of externally added D- and L-aspartate, L-glutamate, L-cysteate and L-cysteinesulphinate. The stimulation of release by external amino acids followed Michaelis-Menten kinetics; the apparent Km values (in microM) were: 14.65 +/- 0.98 for D-aspartate; 8.00 +/- 1.5 for L-aspartate; 22.31 +/- 1.62 for L-glutamate; 6.76 +/- 0.3 for L-cysteate and 7.89 +/- 1.23 for L-cysteinesulphinate. The Vmax values for efflux were 2.16-4.06 nmol/min per mg protein. The exchange process was found to require external NaCl but was very little affected by increase in the external [K+]. The demonstration of exchange as a part of the transport process provides support for the suggestion that in synaptosomal preparations a substantial portion of influx and efflux of amino acid neurotransmitters occurs via a reversible membrane carrier.     

Decreased Uptake and Release of D-Aspartate in the Guinea Pig Spinal Cord After Partial Cordotomy

This study attempts to determine if L-glutamate and/or L-aspartate may be transmitters of neural tracts descending from the brain to the spinal cord. The uptake and electrically evoked release of D-[3H]aspartate, a putative marker for L-glutamate and L-aspartate, were measured in the cervical enlargement of the guinea pig spinal cord. These activities were compared using unlesioned animals and others with a lesion on the right side of the spinal cord. Partial cordotomy (segment C5) produced a heavy loss of descending fibers, a small loss of primary sensory fibers, and a depression of the uptake and the Ca2+ -dependent, electrically evoked release of D-aspartate ipsilateral and caudal to the lesion. Contralaterally, there was a moderate loss of corticospinal fibers, some loss of other descending axons, and a depression of D-aspartate release. Dorsal rhizotomy (segments C4-T1) produced a heavy loss of primary sensory fibers ipsilateral to the lesion. Ipsilaterally, but not contralaterally, the uptake and release of D-aspartate were depressed. Degeneration after partial cordotomy in combination with dorsal rhizotomy was assumed to be the sum of that produced by each lesion separately. This combined lesion depressed D-aspartate uptake ipsilaterally and depressed D-aspartate release on both sides of the cervical enlargement. None of the lesions altered the uptake and the evoked release of [3H]GABA. These findings support the hypothesis that the synaptic endings of one or more neural tracts descending from the brain to the spinal cord mediate the uptake and release of D-aspartate and, therefore, may use L-glutamate or L-aspartate as a transmitter.     

Decreased Uptake and Release of D-Aspartate in the Guinea Pig Spinal Cord After Dorsal Root Section

This study attempts to determine if L-glutamate and/or L-aspartate may be transmitters of dorsal sensory neurons. The uptake and the electrically evoked release of D-[3H]aspartate, a putative marker for L-glutamate and L-aspartate, were measured in the cervical enlargement (segments C4-T1) of the guinea pig spinal cord before and after cutting dorsal roots C5-T1 on the right side. The uptake and the release of gamma-aminobutyric acid (GABA) also were measured as indices of the integrity of GABAergic neurons in the spinal cord. The cervical enlargement was excised and divided into left and right halves, then into dorsal and ventral quadrants. Quadrants from unlesioned animals took up D-aspartate and GABA, achieving concentrations in the tissues which were 14-25 times that in the medium. Subsequently, electrical stimulation evoked a Ca2+-dependent release of D-aspartate and of GABA. The uptake and release of D-aspartate and GABA were similar in tissues taken from intact and sham-operated animals. However, dorsal rhizotomy, without damage to dorsal radicular or spinal blood vessels, depressed the uptake (by 22-29%) and the release (by 50%) of D-aspartate only in quadrants ipsilateral to the lesion. The uptake and the release of GABA were unchanged. In transverse sections of the cervical enlargement, stained to reveal degenerating fibers, by far the heaviest loss of axons occurred in the cuneate fasciculus and in the gray matter ipsilateral to the cut dorsal roots. These findings suggest that the synaptic endings of dorsal sensory neurons probably mediate the uptake and the release of D-aspartate and, therefore, may use L-glutamate or L-aspartate as a transmitter. When spinal blood vessels were damaged during dorsal rhizotomy, the deficits in D-aspartate uptake and release were larger than those in the absence of vascular damage and were accompanied by deficits in GABA uptake and release. These findings imply that vascular damage results in the loss of intraspinal neurons, some of which probably mediate the uptake and release of D-aspartate and, therefore, may use L-glutamate and/or L-aspartate as a transmitter.     

Pharmacologically distinct glutamate receptors on cerebellar granule cells

Cultured cerebellar granule cells were found to exhibit calcium-dependent release of 3H-D-aspartate when stimulated with excitatory amino acids. L-glutamate and L-aspartate were found to be potent stimulators of 3H-D-aspartate release, D-aspartate was weaker and only minor effects were seen with D-glutamate, quisqualate, kainate, N-methyl-D-aspartate (NMDA) and L-alpha-aminoadipate (L-alpha AA). It was also found that only L-glutamate and L-aspartate showed high affinity for the 3H-L-glutamate binding sites on granule cell membranes. Stimulation by L-glutamate of 3H-D-aspartate release could be blocked by various excitatory amino acid antagonists. From the relative potencies of agonists and antagonists on D-aspartate release it is suggested that cerebellar granule cells express functionally active glutamate receptors with pharmacological characteristics different from all known excitatory amino acid receptors.     

Na(+)-dependent transport of anionic amino acids by preimplantation mouse blastocysts.

Negatively charged amino acids, such as aspartate and glutamate, were selected as substrates by low- and high-Km components of mediated Na(+)-dependent transport in preimplantation mouse blastocysts. These and other relatively small anionic amino acids with two carbon atoms between the negatively charged groups (or up to three carbon atoms when the groups were both carboxyl groups) interacted strongly with the low-Km component of transport, whereas larger anionic amino acids interacted weakly or not at all. The low-Km system was also stereoselective except in the case of aspartate. Moreover, transport was Cl(-)-dependent and slower at pH values outside the range 5.6-7.4. L-Aspartate, D-aspartate and L-glutamate each interacted strongly with the low-Km component of transport with Km values for transport nearly equal to their Ki values for inhibition of transport of one of the other amino acids. By these criteria, the low-Km component of transport of anionic amino acids in blastocysts appears to be the same as the familiar system X-AG that is present in other types of mammalian cells. In contrast, the high-Km component of transport in blastocysts preferred L-aspartate to L-glutamate, whereas the reverse is true for fibroblasts. Therefore, transport of anionic amino acids in blastocysts may occur via at least one process that has not been described in other types of cells. Roughly half of mediated glutamate and aspartate transport in blastocysts may occur via the high-Km component of transport at the concentrations of these amino acids that may be present in uterine secretions.     

Neuronal Glutamine Utilization: Glutamine/Glutamate Homeostasis in Synaptosomes

The synaptosomal metabolism of glutamine was studied under in vitro conditions that simulate depolarization in vivo. With [2-15N]glutamine as precursor, the [glutamine]i was diminished in the presence of veratridine or 50 mM KCl, but the total amounts of [15N]glutamate and [15N]aspartate formed were either equal to those of control incubations (veratridine) or higher (50 mM [KCl]). This suggests that depolarization decreases glutamine uptake and independently augments glutaminase activity. Omission of sodium from the medium was associated with low internal levels of glutamine which indicates that influx occurs as a charged Na(+)-amino acid complex. It is postulated that a reduction in membrane potential and a collapse of the Na+ gradient decrease the driving forces for glutamine accumulation and thus inhibit its uptake and enhance its release under depolarizing conditions. Inorganic phosphate stimulated glutaminase activity, particularly in the presence of calcium. At 2 mM or lower [phosphate] in the medium, calcium inhibited glutamine utilization and the production of glutamate, aspartate, and ammonia from glutamine. At a high (10 mM) medium [phosphate], calcium stimulated glutamine catabolism. It is suggested that a veratridine-induced increase in intrasynaptosomal inorganic phosphate is responsible for the enhancement of flux through glutaminase; calcium affects glutaminase indirectly by modulating the level of free intramitochondrial [phosphate]. Because phosphate also lowers the Km of glutaminase for glutamine, augmentation of the amino acid breakdown may occur even when depolarization lowers [glutamine]i. Reducing the intrasynaptosomal glutamate to 26 nmol/mg of protein had little effect on glutamine catabolism, but raising the pH to 7.9 markedly increased formation of glutamate and aspartate. It is concluded that phosphate and H+ are the major physiologic regulators of glutaminase activity.     

Glutamate Release from Guinea-Pig Synaptosomes: Stimulation by Reuptake-Induced Depolarization

Glutamate (10-100 microM) reversibly depolarizes guinea-pig cerebral cortical synaptosomes. This does not appear to be because of a conventional autoreceptor. Neither kainate at 1 mM, 100 microM N-methyl-D-aspartate (NMDA), 100 microM L-2-amino-4-phosphonobutanoate (APB), nor 100 microM quisqualate affects the Ca2+-dependent release of glutamate from suboptimally depolarized synaptosomes. However, kainate, quisqualate, and the quisqualate agonists beta-N-oxalylamino-L-alanine and alpha-amino-3-hydroxy-5-methylisoxazole propionate cause a slow Ca2+-independent release of glutamate from polarized synaptosomes. However, unlike kainate, quisqualate does not inhibit the acidic amino acid carrier. APB, NMDA, and the NMDA receptor-mediated neurotoxin beta-N-methylamino-L-alanine do not influence Ca2+-independent release at 100 microM. The depolarization of the plasma membrane by glutamate can be mimicked by D-aspartate, can be blocked by the transport inhibitor dihydrokainate, and is accompanied by the net uptake of acidic amino acids. L-Glutamate or D-aspartate at 100 microM increases the cytoplasmic free Ca2+ concentration. D-aspartate at 100 microM causes a Ca2+-dependent release of endogenous glutamate, superimposed on the Ca2+-independent heteroexchange with glutamate through the acidic amino acid carrier. The results suggest that the glutamatergic subpopulation of synaptosomes can be depolarized by exogenous glutamate.     

Kainic Acid Inhibits the Synaptosomal Plasma Membrane Glutamate Carrier and Allows Glutamate Leakage from the Cytoplasm but Does Not Affect Glutamate Exocytosis

Kainate inhibits the exchange of D-aspartate into guinea-pig cerebrocortical synaptosomes. Kainate inhibits the Ca2+-independent efflux of endogenous glutamate in the presence of a trapping system for the released amino acid but potentiates a Ca2+-independent net efflux of endogenous and labelled glutamate and aspartate in the absence of the trap. Dihydrokainate has a similar effect. No discrepancy is seen between the release of endogenous and exogenously accumulated amino acid. These results are consistent with the presence of a slow leak of glutamate or aspartate from the cytoplasm independent of the kainate-sensitive Na+-cotransport pathway. In the presence of the trap, glutamate effluxes by both pathways, whereas in the absence of the trap, the Na+-cotransport pathway opposes the leak. Neither in the presence or absence of the glutamate trap does kainate induce, inhibit, or otherwise affect the Ca2+-dependent release of endogenous glutamate. The results enable many of the apparent complexities in the presynaptic actions of kainate to be resolved.     

Stimulation of Dopamine Release from Cultured Rat Mesencephalic Cells by Naturally Occurring Excitatory Amino Acids: Involvement of Both N-Methyl-D-Aspartate (NMDA) and Non-NMDA Receptor Subtypes

In rat mesencephalic cell cultures, L-glutamate at concentrations ranging from 100 microM to 1 mM stimulated release of [3H]dopamine that was attenuated by the non-N-methyl-D-aspartate (non-NMDA) receptor antagonist 6,7-dinitroquinoxalinedione, but not by the selective NMDA receptor antagonists (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801; 10 microM) and 3-(2-carboxypiperazine-4-yl)propyl-1-phosphonate (300 microM). Even at 1 mM glutamate, this release was Ca2+ dependent. These observations suggest that the release was mediated by a non-NMDA receptor. Only release stimulated by a lower concentration (10 microM) of glutamate was inhibited by MK-801 (10 microM), indicating that glutamate at this concentration activates the NMDA receptor. By contrast, L-aspartate at concentrations of 10 microM to 1 mM evoked [3H]dopamine release that was completely inhibited by MK-801 (10 microM) and was also Ca2+ dependent (tested at 1 and 10 mM aspartate). Thus, effects of aspartate involved activation of the NMDA receptor. Sulfur-containing amino acids (L-homocysteate, L-homocysteine sulfinate, L-cysteate, L-cysteine sulfinate) also evoked [3H]dopamine release. Release evoked by submillimolar concentrations of these amino acids was attenuated by MK-801 (10 microM), indicating involvement of the NMDA receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Neuronal Glutamine Utilization: Pathways of Nitrogen Transfer tudied with [15N]Glutamine

Gas chromatography-mass spectrometry was used to evaluate the metabolism of [15N]glutamine in isolated rat brain synaptosomes. In the presence of 0.5 mM glutamine, synaptosomes accumulated this amino acid to a level of 25-35 nmol/mg protein at an initial rate greater than 9 nmol/min/mg of protein. The metabolism of [15N]glutamine generated 15N-labelled glutamate, aspartate, and gamma-aminobutyric acid (GABA). An efflux of both [15N]glutamate and [15N]aspartate from synaptosomes to the medium was observed. Enrichment of 15N in alanine could not be detected because of a limited pool size. Elimination of glucose from the incubation medium substantially increased the rate and amount of [15N]aspartate formed. It is concluded that: (1) With 0.5 mM external glutamine, the glutaminase reaction, and not glutamine transport, determines the rate of metabolism of this amino acid. (2) The primary route of glutamine catabolism involves aspartate aminotransferase which generates 2-oxoglutarate, a substrate for the tricarboxylic acid cycle. This reaction is greatly accelerated by the omission of glucose. (3) Glutamine has preferred access to a population of synaptosomes or to a synaptosomal compartment that generates GABA. (4) Synaptosomes maintain a constant internal level of glutamate plus aspartate of about 70-80 nmol/mg protein. As these amino acids are produced from glutamine in excess of this value, they are released into the medium. Hence synaptosomal glutamine and glutamate metabolism are tightly regulated in an interrelated manner.     

Amino Acid Content of Nerve Endings (Synaptosomes) in Different Regions of Brain: Effects of Gabaculine and Isonicotinic Acid Hydrazide

Abstract: The amino acid content of synaptosomes was determined in six regions of rat brain, and in all regions the five predominant amino acids were glutamate, glutamine, aspartate, taurine, and GABA (γ-aminobutyrate). However, the proportions of the individual amino acids varied considerably from one region to another, the GABA content being particularly high and the taurine content low in synaptosomes from the diencephalon and mesencephalon. Administration of isonicotinic acid hydrazide to rats lowered the synaptosomal GABA level by similar amounts in all brain regions, but the administration of gabaculine resulted in a particularly long-acting elevation in GABA levels in the nerve endings of the diencephalon and mesencephalon. The possibility is raised that the high GABA levels in the nerve terminals of the diencephalon may be involved in the gabaculine-induced lowering of the body temperature of the rats. A constancy in the amount of the synaptosomal pool of "aspartate + glutamate + glutamine + GABA" was observed despite large changes in the relative amounts of the four amino acids brought about by gabaculine.     

In Vivo Release of Endogenous Amino Acids from the Rat Striatum: Further Evidence for a Role of Glutamate and Aspartate in Corticostriatal Neurotransmission

By means of the push-pull cannula method, the outflow of endogenous amino acids was studied in the striatum of halothane-anesthetized rats. Addition of K+ ions (30 mM for 4 min) to the superfusion fluid increased the release of aspartate (+116%), glutamate (+217%), taurine (+109%), and gamma-aminobutyric acid (GABA) (+429%) whereas a prolonged decrease in the outflow of glutamine (-28%) and a delayed reduction in the efflux of tyrosine (-25%) were observed. In the absence of Ca2+, the K+-induced release of aspartate, glutamate, and GABA was blocked whereas the K+-induced release of taurine was still present. Under these conditions, the decrease in glutamine efflux was reduced and that of tyrosine was abolished. Local application of tetrodotoxin (5 microM) decreased only the outflow of glutamate (-25%). One week following lesion of the ipsilateral sensorimotor cortex the spontaneous outflow of glutamine and of tyrosine was enhanced. Despite the lack of change in their spontaneous outflow, the K+-evoked release of aspartate and glutamate was less pronounced in lesioned than in control animals, whereas the K+-evoked changes in GABA and glutamine efflux were not modified. Our data indicate that the push-pull cannula method is a reliable approach for the study of the in vivo release of endogenous amino acids. In addition, they provide further evidence for a role for glutamate and aspartate as neurotransmitters of corticostriatal neurons.     

Release of Endogenous and Newly Synthesized Glutamate and of Other Amino Acids Induced by Non-N-Methyl-D-Aspartate Receptor Activation in Cerebellar Granule Cell Cultures

Amino acid release studies were performed by an HPLC procedure using differentiated rat cerebellar granule cell cultures. Kainic acid (KA; 50 microM) caused an increase (about threefold) in the release of endogenous glutamate and a lesser, but statistically significant, increase in the release of glutamine, glycine, threonine, taurine, and alanine. Quisqualic acid (QA) and, to a lesser degree, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) (both 50 microM) enhanced the release of the following amino acids in the order glutamate greater than aspartate greater than or equal to taurine, whereas the release of other amino acids was either unaffected or affected in a statistically nonsignificant way. The release of glutamate induced by KA was partially (43%) Ca2+ dependent. The other release-inducing effects of KA and QA were not Ca2+ dependent. In all cases, the evoked release could be prevented by the non-N-methyl-D-aspartate (non-NMDA) receptor antagonist 6-cyano-2,3-hydroxy-7-nitroquinoxaline, and thus appeared to be receptor mediated. NMDA (5 and 50 microM) had no release-inducing activity. The KA-, QA-, and AMPA-evoked release of newly synthesized [3H]glutamate and [3H]aspartate (formed in the cells exposed to [3H]glutamine) was very similar to the evoked release of endogenous glutamate and aspartate. On the other hand, the release of preloaded D-[3H]aspartate (purified by HPLC in the various fractions analyzed, before radioactivity determination) induced by 50 microM KA was twice as high as that of endogenous glutamate. In the case of high [K+] depolarization, in contrast, the release of preloaded D-[3H]aspartate was approximately 30% lower than that of endogenous glutamate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterogeneity of high affinity uptake of L-glutamate and L-aspartate in the mammalian central nervous system.

Characteristics of high affinity uptake of L-glutamate are examined in order to evaluate the possible use of the uptake of [3H]L-glutamate, [3H]L-aspartate or any other suitable [3H]-labelled substrate as a marker for glutamatergic and aspartergic synapses in autoradiographic studies in the mammalian brain. Review of data on substrate specificity indicates the presence of at least two high affinity uptake systems specific for acidic amino acids in the central nervous tissue; one which takes up L-glutamate and L-aspartate and the other which is selective for L-glutamate only. Studies on ionic requirements, too, point to the existence of at least two distinct uptake systems with high affinity for L-glutamate. The Na(+)-dependent uptake system(s) handle(s) both L-glutamate and L-aspartate whereas the Na(+)-independent uptake system(s) show(s) selectivity for L-glutamate only. Available data do not favour the Na(+)-dependent binding of [3H]D-aspartate to thaw-mounted sections of frozen brain tissue as a suitable marker for glutamatergic/aspartergic synaptic nerve endings. However, there are reasons--such as the results of lesion studies and the existence of uptake sites which have a higher affinity for L-aspartate than for D-aspartate--to suggest that Na(+)-dependent binding of [3H]L-aspartate, rather than that of [3H]D-aspartate, should be further investigated as a possible marker for the glutamatergic/aspartergic synapses in the autoradiographic studies using sections of frozen brain.     

Solubilization of quisqualate-sensitive L-[3H]glutamate binding sites from guinea pig brain membranes using a zwitterionic detergent

L-[3H]Glutamate binding sites were solubilized with a zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate (CHAPS) plus ammonium thiocyanate from guinea pig synaptosomal membranes. The binding of L-[3H]glutamate to the solubilized binding sites was saturable and reversible. Scatchard analysis suggested the existence of two different classes of binding sites with KDs of 63.8 and 644 nM. The L-[3H]glutamate binding was displaced by excitatory amino acids with such an order of potency that L-glutamate much greater than D-glutamate congruent to L-aspartate greater than D-aspartate. Quisqualate effectively displaced the glutamate binding in biphasic manner. L-Glutamic acid diethyl ester, the quisqualate receptor antagonist, also showed a moderate displacing ability. Other neuroactive amino acid analogues displaced the glutamate binding only weakly, except for L- and D-homocysteic acids which had moderate potency. It is very likely from these results that the glutamate binding sites solubilized in this study are relevant to the physiological glutamate receptors especially of quisqualate-type.     

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.     

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⁺.     

Endogenous Amino Acid Release from Cultured Cerebellar Neuronal Cells: Effect of Tetanus Toxin on Glutamate Release

Endogenous amino acid release was measured in developing cerebellar neuronal cells in primary culture. In the presence of 25 mM K+ added to the culture medium, cerebellar cells survived more than 3 weeks and showed a high level of differentiation. These cultures are highly enriched in neurons, and electron-microscopic observation of these cells after 12 days in vitro (DIV) confirmed the presence of a very large proportion of cells with the morphological characteristics of granule cells, making synapses containing many synaptic vesicles. Synaptogenesis was also confirmed by immunostaining the cells with antisera against synapsin I and synaptophysin, two proteins associated with synaptic vesicles. From these cultures, endogenous glutamate release stimulated by 56 mM K+ was already detected after only a few days in culture, the maximal release value (1,579% increase over basal release) being reached after 10 DIV. In addition to that of glutamate, the release of aspartate, asparagine, alanine, and, particularly, gamma-aminobutyric acid (GABA) was stimulated by 56 mM K+ after 14 DIV, but to a lesser extent. No increase in serine, glutamine, taurine, or tyrosine release was observed during K+ depolarization. The effect of K+ on amino acid release was strictly Ca2+-dependent. Stimulation of the cells with veratridine resulted in a qualitatively similar effect on endogenous amino acid release. In the absence of Ca2+, 30% of the veratridine effect persisted. The Ca2+-dependent release was quantitatively similar after stimulation by veratridine and K+. Treatment of cerebellar cells with tetanus toxin (5 micrograms/ml) for 24 h resulted in a total inhibition of the Ca2+-dependent component of the glutamate release evoked by K+ or veratridine. It is concluded that glutamate is the main amino acid neurotransmitter of cerebellar cells developed in primary culture under the present conditions and that glutamate is probably mainly released through the exocytosis of synaptic vesicles.