GABA transaminase inhibitors enhance the release of endogenous GABA but decrease the release of beta-alanine evoked by electrical stimulation of slices of the rat medulla oblongata

Slices of the rat medulla oblongata were superfused and electrically stimulated. The amount of endogenous GABA, beta-alanine and glutamate release from the slices was determined by high performance liquid chromatography with fluorometric detection. Inhibitors of GABA-transaminase (GABA-T), aminooxyacetic acid (10(-5) M), gamma-acetylenic GABA (10(-4) and 10(-3) M) and gabaculine (10(-5) M), enhanced the stimulus-evoked release of GABA and reduced that of beta-alanine, while no change was observed in the release of glutamate. These changes in amino acid release from the slices were accompanied by an increase in the content of GABA and a decrease in that of beta-alanine. The stimulus-evoked release of these amino acids was abolished by Ca2+-deprivation, in either the presence or absence of GABA-T inhibitors. These results suggest a modulatory role of GABA-T for synaptically releasable GABA and involvement of this enzyme in the synthesis of releasable beta-alanine.     

GABA transport by nerve ending fractions of cat brain

Abstract— Nerve ending and mitochondrial fractions were prepared from cat cerebral cortex by differential and sucrose density gradient centrifugation. The isolated fractions were characterized morphologically and enzymatically and the nerve-ending fraction was shown to be particularly rich in glutamate decarboxylase activity. The uptake of [³H] γ-aminobutyric acid (GABA) was studied by trapping the particles on a Millipore filter. GABA uptake was not saturable over the concentration range studied (0.018 to 8000 μM. Tissue/medium ratios were consistently greater than 2·5, and uptake was significantly reduced by ouabain and iodoacetate, or by replacing sodium with lithium or choline chloride. GABA uptake was significantly inhibited by desmethylimipramine (DMI) with an IC₅₀ of approx. 10–20 μM. GABA uptake was reduced by structurally similar amino acids lacking an α-amino group, and also by methionine and phenylalanine, but not by short chain neutral amino acids.     

Effects of Anoxia on the Stimulated Release of Amino Acid Neurotransmitters in the Cerebellum In Vitro

Abstract: The effect of anoxia and ischemia on the release of amino acid transmitters from cerebellar slices induced by veratridine or high [K⁺] was studied. Synaptic specificity was tested by examining the tetradotoxin (TTX)-sensitive and the Ca²⁺-dependent components of stimulated release. Evoked release of endogenous amino acids was investigated in addition to more detailed studies on the stimulated efflux of preloaded [¹⁴C]GABA and d -[³H]aspartate (a metabolically more stable anologue of acidic amino acids).[¹⁴C]GABA release evoked by either method of stimulation was unaffected by periods of up to 35 min of anoxia and declined moderately by 45 min. In contrast, induced release of d -[³H]Asp increased markedly during anoxia to a peak at about 25 min, followed by a decline when anoxia was prolonged to 45 min. Evidence was obtained that the increased evoked efflux of d -[³H]Asp from anoxic slices was not due to impaired reuptake of the released amino acid and that it was completely reversible by reoxygenation of the slices. Results of experiments examining the evoked release of endogenous amino acids in anoxia were consistent with those obtained with the exogenous amino acids. Only 4 of the 10 endogenous amino acids studied exhibited TTX-sensitive veratridine-induced release under aerobic conditions (glutamate, aspartate, GABA, and glycine). Anoxia for 25 min did not affect the stimulated efflux of these amino acids with the exception of glutamate, which showed a significant increase. Compared with anoxia, effects of ischemia on synaptic function appeared to be more severe. Veratridine-evoked release of [¹⁴C]GABA was already depressed by 10 min and that of d -[³H]Asp showed a modest elevation only at 5 min. Stimulated release of d -Asp and labelled GABA declined progressively after 5 min. These findings were compared with changes in tissue ATP concentrations and histology. The latter studies indicated that in anoxia the earliest alterations are detectable in glia and that nerve terminals were the structures by far the most resistant to anoxic damage. The results thus indicated that evoked release of amino acid transmitters in the cerebellum is compromised only by prolonged anoxia in vitro. In addition, it would appear that the stimulated release of glutamate is selectively accentuated during anoxia. This effect may have a bearing on some hypoxic behavioral changes and, perhaps, also on the well-known selective vulnerability of certain neurons during hypoxia.     

Microdialysis with High NaCl Causes Central Release of Amino Acids and Dopamine

Abstract: Recent studies have shown that the neuropeptides arginine-8-vasopressin (AVP) and oxytocin (OXT) are released within the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus in response to microdialysis of these nuclei with high-NaCl perfusion media. These results suggest an inherent osmosensitivity of SON and PVN neurons. To investigate whether the observed release of AVP/OXT is a unique phenomenon to these neuropeptides, several brain regions were examined for the release of amino acids or dopamine in response to high- or low-NaCl stimulation. Urethane-anesthetized male Sprague-Dawley rats were perfused with five-ion solution using U-shaped microdialysis probes. Samples were collected at 30-min intervals and analyzed for amino acids and dopamine by HPLC. In the dialysates of all perfusion areas, including the SON, PVN, hippocampus, and striatum, concentrations of Asp, Glu, Ser, Gln, Gly, taurine (Tau), and γ-aminobutyric acid (GABA) were significantly increased during perfusion with high-NaCl medium. This release was found to be dose dependent when tested in the hippocampus and striatum with perfusion medium containing 0.5 or 1.0 M NaCl. However, only the release of Glu and Ser was found to be Ca²⁺ dependent. In contrast, the use of mannitol, a nonionic osmolyte, for perfusions in the striatum in concentrations of 0.5 and 1 M resulted in reduced levels of amino acids in the dialysates (Glu, Ser, Gln, and Tau). Low-NaCl perfusion medium (0.01 M) resulted in significantly increased Glu, Tau, Gly, and GABA levels in the striatum. In addition, dopamine levels in striatal dialysates were significantly increased during stimulation with 1 M NaCl. These results indicate that stimulation with high NaCl concentrations affects the release of several neurotransmitters and is not specific for AVP and OXT. The described phenomenon of the release of amino acids in response to this stimulation seems to be a response to the changed ionic concentration rather than to the osmolality. In light of these findings shown for amino acids and dopamine as well as those previously reported for AVP, OXT, and angiotensin, it would appear that sensitivity to tonicity changes brought about by microdialysis may be a feature of many transmitter systems.     

Transport of GABA from the perfused ventricular system of the cat

Abstract— The transport of GABA was studied in anaesthetized cats undergoing ventriculo-cisternal perfusion with radioactive GABA. Steady-state clearance of GABA from the CSF was greater than that of other amino acids previously studied, and was saturated at lower substrate concentrations, with an apparent K_t of 5·4 × 10^-5 M, after correcting for non-saturable transport. GABA clearance was inhibited by the inclusion of taurine or β-alanine in the perfusion fluid, but not by a number of the common neutral and acidic amino acids. Study of punch biopsies of brain tissue taken adjacent to the venticular system, at the completion of perfusions, showed accumulation of radioactive GABA in the tissue to values four times higher than those found in the perfusion fluid. Of the radioactivity which had been removed from the ventricular system, only 11 per cent remained in the brain at the completion of the perfusion. Excised cat choroid plexus showed a saturable uptake of GABA which was inhibited by inclusion of taurine, β-alanine, or β-guanidino propionic acid in the incubation medium.     

Effect of α-Amino-4-Phosphonobutyrate on the Release of Endogenous Glutamate and Aspartate from Cortical Synaptosomes of Epileptic Rats

The effect of the glutamate antagonist alpha-amino-4-phosphonobutyrate (APBA) on the release of endogenous amino acids from sensorimotor cortical synaptosomes of rats with a cortical cobalt focus and from non-epileptic rats was studied: (1) The release of endogenous glutamate, aspartate, and gamma-aminobutyric acid (GABA) from synaptosomal preparations of cobalt-induced epileptogenic tissues was increased compared with the release from the contralateral (sensorimotor) region or the sensorimotor cortex of normal animals. The intrasynaptosomal content of these amino acids was reduced in proportion to the amount released. The levels of other amino acids were unaffected or showed much smaller changes. (2) APBA (0.5-1 mM) decreased significantly the spontaneous release of aspartate and glutamate from the epileptic foci without affecting GABA or any other amino acid. (3) APBA produced no effect whatsoever on the release of any amino acid from synaptosomal preparations of nonepileptic focus.     

Ca2+-dependence of the evoked release from guinea pig cerebral cortex slices of endogenous 14C-labelled amino acids, labelled via D-[U-14C]glucose.

Spontaneous and electrically evoked release of exogenous labelled amino acids and endogenous amino acids labelled from D-[U-14C]glucose were compared in control and Ca2+-free medium using guinea pig cerebral cortex slices. Spontaneous release of all labelled amino acids, except that of endogenous 14C-labelled threonine-serine-glutamine (unseparated) and exogenous [14C]aspartate, was doubled in Ca2+-free medium. The major portion of the electrically evoked release of endogenous [14C]glutamate, [14C]aspartate, gamma-amino[14C]butyrate (14C-labelled GABA) and exogenous 3H-labelled GABA was Ca2+-inpendent. More than half of the evoked release of the other labelled amino acids was Ca2+-independent. As the pattern of Ca2+-dependence of the evoked release concurred with the selectivity of the evoked release for endogenous [14C]-glutamate, [14C]aspartate, and 14C-labelled GABA, it was concluded that these labelled amino acids were probably released from the amino acid 'transmitter pool'.     

Evidence using in vivo microdialysis that aminotransferase activities are important in the regulation of the pools of transmitter amino acids

The effect of aminooxyacetic acid (AOAA), an inhibitor of pyridoxal phosphate-dependent enzymes (including the aminotransferases), on the K⁺-evoked release of amino acids was studied during microdialysis of neostriatum in anesthetized rats. K⁺-evoked (100 mM) release of asparatate, glutamate, and GABA was inhibited by 74%, 70%, and 63%, respectively, by 20 mM Mg²⁺ and are therefore reflecting release from the transmitter pools of these amino acids. Treatment with AOAA decreased the K⁺-evoked release of aspartate, glutamate, and GABA instantly, with a delayed decrease in the efflux of glutamine and alanine, arguing that the synthesis of transmitter amino acids in particular is sensitive to the activity of pyridoxal phosphate-dependent enzymes. Interestingly, GABA release increased severalfold following the initial decrease, probably reflecting inhibition by AOAA on GABA aminotransferase, the enzyme most sensitive to inhibition by AOAA, and responsible for enzymatic inactivation of transmitter GABA.Special issue dedicated to Dr. Claude Baxter.     

Biphasic and Opposite Effects of Dopamine and Apomorphine on Endogenous GABA Release in the Rat Substantia Nigra

Abstract: A push-pull cannula technique was used to study the in vivo release of endogenous GABA in the rat substantia nigra. Intranigral application of both dopamine (DA) and apomorphine produced biphasic changes in the rate of endogenous GABA release. The presence of 10 μM-DA in the perfusion medium increased GABA release (140%). At 25 μM-DA, both stimulation and inhibition of the nigral GABA release were observed. Higher concentrations of DA produced a decrease of the GABA release (50%). A small amount of apomorphine (10 μM in the perfusion medium) resulted in a decrease in GABA release (75%). Application of 25 μM-apomorphine produces opposite effects, similar to those observed after addition of 25 μM-DA. We observed an enhanced GABA release from the substantia nigra at 100 μM-apomorphine in the perfusion medium (360%). The presence of 5 μM-haloperidol produced a small decrease in the rate of GABA release (80%). Both the inhibitory effect of 25 μM-DA and the excitatory effect of 100 μM-apomorphine could be blocked by haloperidol added to the perfusion medium. Dibutyryl cyclic AMP (1.5 mM) and 2-amino-6, 7-dihydroxyl(1, 2, 3, 4) tetrahydronapthalene (ADTN) (50 μM) added to the perfusion medium produced an inhibition of nigral GABA release (55% and 35% respectively) similar to that observed after addition of 50 μM-DA. The amounts of lysine and ethanolamine (measured with GABA concurrently) released into the perfusion medium did not change in most of the experiments. The changes in the rates of release of these compounds that were observed in some experiments were either in the same or in the opposite direction of the change in GABA release. These results suggest that dopaminergic processes within the substantia nigra affect GABA-ergic neurotransmission and that DA and apomorphine have different effects on GABA release.     

Spontaneous release of endogenous aspartate and glutamate from rat striatal slices is increased following destruction of local neurons by ibotenic acid

We sought to determine in rat striatum whether the release of neurotransmitter amino acids aspartate (Asp), glutamate (Glu) and gamma-aminobutyric acid (GABA) were affected by local neurons. To do so, unilateral microinjections of ibotenic acid, an excitotoxin that destroys local neurons without affecting fibers of passage, were made into the striatum. Release of endogenous amino acids from lesioned and intact striatal slices were measured by HPLC one week later. The effectiveness and specificity of the lesion were confirmed by measuring the enzyme activity associated with extrinsic dopamine neurons (tyrosine hydroxylase; 111±14%), intrinsic GABA neurons (glutamic acid decarboxylase; 19±7%) and intrinsic acetylcholine neurons (choline acetyltransferase; 37±10%). Destruction of local striatal neurons markedly attenuated the release of GABA (41±12% of control) elicited by depolarization with K⁺ (35 mM), but did not significantly reduce the K⁺-evoked release of Asp (80±17%) and Glu (92±8%). However, spontaneous release of Asp and Glu was significantly greater than that observed in unlesioned tissue (159±18% and 209±27%, respectively), while the spontaneous release of GABA was not significantly reduced (75±43%). Although release of the neurotransmitter amino acids Asp, Glu and GABA were affected by the lesion, the release of the non-neurotransmitter amino acid tyrosine was unaffected. These data are consistent with the hypotheses that: 1) the predominant source of releasable stores of endogenous Asp and Glu in the striatum arises from extinsic neurons, and 2) that the spontaneous release of Asp and Glu from axon terminals in the striatum may be regulated, at least in part, by local inhibitory neurons.     

In vivo release patterns and cardiovascular properties of inhibitory and excitatory amino acids in the hypothalamus

Summary The posterior hypothalamus of conscious, freely moving rats was superfused with artificial cerebrospinal fluid through a push-pull cannula and the release of amino acids was determined in the superfusate. Under basal conditions, the release rates of taurine, GABA and glutamate fluctuated according to ultradian rhythms with different frequencies. Hypothalamic superfusion with veratridine or high concentrations of potassium choride enhanced the release rates of taurine, GABA and glutamate in a concentration-dependent way. Tetrodotoxin decreased the basal release rates of the three amino acids. The release of arginine was not influenced significantly by these compounds. A fall of blood pressure elicited by intravenous infusion of nitroprusside decreased the release rates of GABA and taurine and enhanced the release of glutamate. Infusion of noradrenaline increased blood pressure and release rates of GABA and taurine, while the release of glutamate was not influenced. Neither the pressor, nor the depressor responses to drugs influenced the release of arginine in the hypothalamus. It is concluded that the inhibitory amino acids taurine and GABA released from hypothalamic neurons possess a tonic hypotensive function. The excitatory amino acid glutamate, released from glutamatergic neurons of the hypothalamus, seems to possess a hypertensive function in counteracting a fall of blood pressure.This work was supported by the Fonds zur Förderung der wissenschaftlichen Forschung. These results were presented at the Third International Congress on Amino Acids, Vienna, August 1993     

In Vivo Studies on the Extracellular, and Veratrine-Releasable, Pools of Endogenous Amino Acids in the Rat Striatum: Effects of Corticostriatal Deafferentation and Kainic Acid Lesion

The effects of corticostriatal deafferentation (decortication) and destruction of intrinsic neurons (intrastriatal kainate injection) on the extracellular concentration, and veratrine-releasable pools, of endogenous amino acids in the rat striatum were examined using the in vivo brain dialysis technique. Intracellular amino acid content was also determined. Decortication reduced selectively intra- and extracellular levels of glutamate (Glu) and aspartate (Asp). Extracellular changes were more pronounced than those in tissue content. gamma-Aminobutyric acid (GABA), taurine (Tau), and phosphoethanolamine (PEA) levels were not affected, whereas nonneuroactive amino acids were increased at 1 week but not at 1 month post-lesion. The intracellular pool of Glu and Asp was also reduced in kainate-lesioned striata. However, extracellular levels of these compounds were not affected significantly by this treatment. The tissue content of all other amino acids was decreased, the most prominent change being in the concentration of GABA. Extracellular GABA concentration was also reduced dramatically, whereas the concentrations of noneuroactive amino acids were increased to varying degrees. These data suggest that transmitter pools of neuroactive amino acids are an important supply for their extracellular pools. Lesion-induced alterations in nonneuroactive amino acids are discussed with regard to the loss of metabolic pools, glial reactivity, and changes in blood-brain barrier transport. Veratrine induced a massive release of neuroactive amino acids such as Glu, Asp, GABA, and Tau into the extracellular fluid, and a delayed increase in PEA. Extracellular levels of neuroactive amino acids were raised slightly. Decortication reduced, selectively, the amounts of Glu and Asp released by veratrine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Release of Endogenous Glutamate,Aspartate, GABA,and Taurine from Hippocampal Slices from Adult and Developing Mice under Cell-Damaging Conditions

The releases of endogenous glutamate, aspartate, GABA and taurine from hippocampal slices from 7-day-, 3-, 12-, and 18-month-old mice were investigated under cell-damaging conditions using a superfusion system. The slices were superfused under hypoxic conditions in the presence and absence of glucose and exposed to hydrogen peroxide. In the adult hippocampus under normal conditions the basal release of taurine was highest, with a response only about 2-fold to potassium stimulation (50 mM). The low basal releases of glutamate, aspartate, and GABA were markedly potentiated by K⁺ ions. In general, the release of the four amino acids was enhanced under all above cell-damaging conditions. In hypoxia and ischemia (i.e., hypoxia in the absence of glucose) the release of glutamate, aspartate and GABA increased relatively more than that of taurine, and membrane depolarization by K⁺ markedly potentiated the release processes. Taurine release was doubled in hypoxia and tripled in ischemia but K⁺ stimulation was abolished. In both the mature and immature hippocampus the release of glutamate and aspartate was greatly enhanced in the presence of H₂O₂, that of aspartate particularly in developing mice. In the immature hippocampus the increase in taurine release was 10-fold in hypoxia and 30-fold in ischemia, and potassium stimulation was partly preserved. The release processes of the four amino acids in ischemia were all partially Ca²⁺-dependent. High concentrations of excitatory amino acids released under cell-damaging conditions are neurotoxic and contribute to neuronal death during ischemia. The substantial amounts of the inhibitory amino acids GABA and taurine released simultaneously may constitute an important protective mechanism against excitatory amino acids in excess, counteracting their harmful effects. In the immature hippocampus in particular, the massive release of taurine under cell-damaging conditions may have a significant function in protecting neural cells and aiding in preserving their viability.     

Endogenous Release of γ-Aminobutyric Acid from the Medial Preoptic Area Measured by Microdialysis in the Anaesthetised Rat

The characteristics of gamma-aminobutyric acid (GABA) release as monitored by microdialysis have been investigated in the chloral hydrate anaesthetised rat. The high outflow of GABA following insertion of the microdialysis probe (membrane 2 mm in length, 0.5 mm in diameter) into the medial preoptic area was found to decline to a stable baseline level after 2 h. After this time, perfusion with a medium containing 100 mM potassium ions evoked a 56-fold increase in GABA outflow. The addition of the calcium channel blocker verapamil (100 microM) to the perfusion medium induced significant 25 and 50% reductions in basal and potassium-stimulated GABA outflow, respectively. In the same animals, verapamil caused an 80% decrease in potassium-stimulated noradrenaline outflow. The glutamic acid decarboxylase inhibitors 3-mercaptopropionic acid and L-allylglycine added to the perfusion medium at a concentration of 10 mM reduced basal GABA release by approximately 50% with different time-courses of action. Ethanolamine-O-sulfate, a GABA-transaminase inhibitor, induced significant increases in basal GABA outflow 90 min after inclusion in the perfusion medium. These results demonstrate that microdialysis is a suitable technique with which to monitor extracellular levels of GABA and provide in vivo data on GABA release and degradation mechanisms.     

Spinal subarachnoid perfusion in the rat: glycine transport from spinal fluid

Abstract— A method was developed for perfusion of the spinal subarachnoid space in the rat. Bidirectional steady-state fluxes of [¹⁴C]glycine between spinal fluid and plasma were measured. [¹⁴C]glycine clearance from spinal fluid was 5-fold greater than its clearance from plasma. Glycine was transported out of spinal fluid by a saturable process, and the rate of transport was unaffected by the other depressant amino acids, GABA, β-alanine, and taurine. Perfused [¹⁴C]glycine and [³H]GABA distributed in an intracellular compartment in spinal cord. The preparation should be useful for study of the release of these inhibitory amino acids from the intact spinal cord.     

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.     

Effect of β-Bungarotoxin on the Release of Endogenous Amino Acids from the Sensorimotor Cortex

Abstract: β-Bungarotoxin, a snake neurotoxin purified from the venom of Bungarus multicinctus , caused a significant increase in the in vivo release of glutamate from the superfused sensorimotor cortex of awake animals. A smaller effect on GABA release was observed, but no change was detected in the release of six other amino acids measured. The effects on glutamate and GABA release were entirely blocked by tetrodotoxin (1 μM) and were reversible when the cortical tissue was washed with saline.     

The use of microdialysis for studying the regional effects of pharmacological manipulation on extracellular levels of amino acids--some methodological aspects.

The purpose of this study was to examine and validate the use of microdialysis for sampling and pharmacologically manipulating extracellular amino acids in the brain. Repeated use of microdialysis probes in acute intracerebral experiments did not significantly alter the relative recovery in vitro for the amino acids quantitated (GABA, aspartate, glutamate, glycine, taurine, and alanine). Regional differences in basal levels of some of the amino acids were detected in dialysates collected from the dorsomedial hypothalamus, striatum, and frontal cortex. The percent in vitro recoveries for the amino acids from the probes used in the three regions were not significantly different suggesting that the regional differences in basal levels of amino acids were functionally derived and not a consequence of variations in probe recovery. Perfusion with nipecotic acid, an inhibitor of GABA uptake, resulted in selective elevations in extracellular GABA in the three regions studied. Conversely, perfusion with high-potassium, a depolarizing agent, resulted in significant elevations in not only extracellular GABA but also aspartate, glutamate, and taurine. Thus, microdialysis is a method which can be employed to assess and to pharmacologically manipulate extracellular amino acids in the rat brain.     

EFFECTS OF TETRODOTOXIN, CALCIUM AND MAGNESIUM ON THE RELEASE OF AMINO ACIDS FROM SLICES OF GUINEA-PIG CEREBRAL CORTEX

Abstract— Tetrodotoxin, Ca²⁺-deprivation and high-Mg²⁺ were used in an effort to identify the portion of the evoked release of endogenous amino acids, labelled via metabolism of [¹⁴C]-glucose, and several exogenous labelled amino acids, that came from nerve terminals when slices of guinea pig cerebral cortex were superfused with glucose-free solutions and stimulated electrically. With some exceptions, spontaneous release of labelled amino acids was decreased by 2 μm -tetrodotoxin but increased in Ca²⁺-free medium and in solutions containing an extra 24 mm -MgCl₂. Tetrodotoxin suppressed 85–90% of the stimulated release of almost all labelled amino acids, but had a smaller effect on the release of endogenous ¹⁴C-labelled threonine-serine-glutamine (unseparated). In Ca²⁺-free solution, the stimulated release of endogenous ¹⁴C-labelled glutamate, aspartate and GABA was suppressed by 80–90%, but that of endogenous ¹⁴C-labelled threonine-serine-glutamine was unaffected as was most of the release of the other labelled amino acids. In medium containing an extra 24mM-MgCl₂, the stimulated release of endogenous ¹⁴C-labelled glutamate, aspartate and GABA was suppressed by 75-85%, that of exogenous labelled aspartate and GABA by 50–65%, but the release of the other labelled amino acids was unaffected. The control stimulated releases of endogenous ¹⁴C-labelled glutamate, aspartate and GABA were much larger than those of other labelled amino acids but were reduced by tetrodotoxin, Ca²⁺-deprivation and high-Mg²⁺ to a level similar to that of the control stimulated releases of the other labelled amino acids. These results suggest that almost all of the stimulated release of endogenous ¹⁴C-labelled glutamate, aspartate and GABA came from nerve terminals while those of the other labelled amino acids came from other tissue elements. In addition, they are in accord with a transmitter role for glutamate, aspartate and GABA in cerebral cortex.     

Release of Endogenous Amino Acids from the Striatum from Developing and Adult Mice in Ischemia

In most other studies the release of amino acid neurotransmitters and modulators in vitro has been studied mostly using labeled preloaded compounds. For several reasons the estimated release may not reliably reflect the release of endogenous compounds. The magnitudes of the release cannot thus be quite correctly estimated using radioactive labels. The basal and K⁺-evoked release of the neuroactive endogenous amino acids γ-aminobutyrate (GABA), glycine, taurine, glutamate and aspartate was now studied in slices from the striatum from 7-day-old to 3-month-old mice under control (normoxic) and ischemic conditions. The release of alanine, threonine and serine was assessed as control. GABA and glutamate release was much greater in 3-month-old than in 7-day-old mice, whereas with taurine the situation was the opposite. Ischemia markedly enhanced the release of all these three amino acids. The release of aspartate and glycine was markedly enhanced as well whereas no effects were discernible in the release of glutamine, alanine, serine and threonine. K⁺ stimulation (50 mM) enhanced the release of GABA, glutamate, taurine, aspartate and glycine in most cases, except with taurine in 3-month-old mice under the ischemic conditions and with aspartate in 7-day-old mice under the control conditions. K⁺ stimulation did not affect the release of glutamine, alanine, serine or threonine. The results on endogenous amino acids are qualitatively similar to those obtained in our earlier experiments with labeled preloaded amino acids. In conclusion, in developing mice only inhibitory taurine is released in such amounts that may counteract the harmful effects of excitatory amino acids in ischemia.