Synaptic vesicles from hog brain—their isolation and the coupling between synthesis and uptake of γ-aminobutyrate by glutamate decarboxylase

Purified synaptic vesicles were isolated from hog cerebral cortex by a rapid procedure consisting of homogenization of cerebral cortex slices in iso-osmotic sucrose, differential centrifugation and sucrose density-gradient centrifugation. The purity of the vesicles was evaluated both biochemically and morphologically. The vesicles contained high amounts of γ-aminobutyrate (GABA) and acetylcholine at specific concentrations of 390 nmol/mg protein and 7.2 nmol/mg protein respectively.

Glutamate decarboxylase, the enzyme which catalyses GABA formation, binds to the synaptic vesicles in a calcium-dependent manner. The percentage of glutamate decarboxylase bound to the vesicles increases from about 5% without calcium, reaching a plateau of about 60% at 4 mM Ca²⁺. Magnesium in concentrations 0.2–10 mM has no significant effect on glutamate decarboxylase binding. Also in phospholipid vesicles (small unilamellar phosphatidylserine-phosphatidylcholine. 2:1 liposomes) Ca²⁺, but not Mg²⁺, induced the binding of glutamate decarboxylase, reaching a plateau of 50% at 2 mM Ca²⁺. Both in synaptic vesicles and in phospholipid vesicles the calcium-dependent glutamate decarboxylase binding seems to be specific, and not caused by unspecific association of proteins, since the specific binding (bound enzyme activity/mg bound protein) increases 3-fold from 0 to 4 mM Ca²⁺.

The functional role of this binding was studied in GAD containing vesicles by measuring the relationship between the accumulation of [³H]GABA, newly synthetized from [³H]glutamate, and the uptake of added [¹⁴C]GABA. No significant uptake of [¹⁴C]GABA was found under the experimental conditions used, whereas large amounts of [³H]GABA were found within the vesicles. It appears that the [³H]GABA accumulation process is functionally linked to [³H]GABA synthesis and is mediated by the membrane-bound glutamate decarboxylase. This synthesis-coupled uptake of GABA into synaptic vesicles possibly serves to bring about a plasticity effect in previously stimulated GABAergic nerve endings.     

The release of [3H]GABA formed from [3H]glutamate in rat hippocampal slices

to compare the storage and release of endogenous GABA, of [3H]GABA formed endogenously from glutamate, and of exogenous [14C]GABA, hippocampal slices were incubated with 5 microCi/ml [3,4-3H]1-glutamate and 0.5 microCi/ml [U-14C]GABA and then were superfused in the presence or absence of Ca+ with either 50 mM K+ or 50 microM veratridine. Endogenous GABA was determined by high performance liquid chromatography which separated labeled GABA from its precursors and metabolites. Exogenous [14C]GABA content of the slices declined spontaneously while endogenous GABA and endogenously formed [3H]GABA stayed constant over a 48 min period. In the presence of Ca+ 50 mM K+ and in the presence or absence of Ca2+ veratridine released exogenous [14C]GABA more rapidly than endogenous or endogenously formed [3H]GABA, the release of the latter two occurring always in parallel. The initial specific activity of released exogenous [14C]GABA was three times, while that of endogenously formed [3H]GABA was only 50% higher than that in the slices. There was an excess of endogenous GABA content following superfusion with 50 mM K+ and Ca2+, which did not occur in the absence of Ca2+ or after veratridine. The observation that endogenous GABA and [3H]GABA formed endogenously from glutamate are stored and released in parallel but differently from exogenous labelled GABA, suggests that exogenous [3H] glutamate can enter a glutamate pool that normally serves as precursor of GABA.     

β-N-Oxalylamino-l-Alanine: Action on High-Affinity Transport of Neurotransmitters in Rat Brain and Spinal Cord Synaptosomes

beta-N-Oxalylamino-L-alanine (BOAA) is a dicarboxylic diamino acid present in Lathyrus sativus (chickling pea). Excessive oral intake of this legume in remote areas of the world causes humans and animals to develop a type of spastic paraparesis known as lathyrism. BOAA is one of several neuroactive glutamate analogs reported to stimulate excitatory receptors and, in high concentrations, cause neuronal vacuolation and necrosis. The present study investigates the action of BOAA in vitro on CNS high-affinity transport systems for glutamate, gamma-aminobutyric acid (GABA), aspartate, glycine, and choline and in the activity of glutamate decarboxylase (GAD), the rate-limiting enzyme in the decarboxylation of glutamate to GABA. Crude synaptosomal fractions (P2) from rat brain and spinal cord were used for all studies. [3H]Aspartate transport in brain and spinal cord synaptosomes was reduced as a function of BOAA concentration, with reductions to 40 and 30% of control values, respectively, after 15-min preincubation with 1 mM BOAA. Under similar conditions, transport of [3H]glutamate was reduced to 74% (brain) and 60% (spinal cord) of control values. High-affinity transport of [3H]GABA, [3H]glycine, and [3H]choline, and the enzyme activity of GAD, were unaffected by 1 mM BOAA. While these data are consistent with the excitotoxic (convulsant) activity of BOAA, their relationship to the pathogenesis of lathyrism is unknown.     

The Role of GlycineB Binding Site and Glycine Transporter (GlyT1) in the Regulation of [3H]GABA and [3H]Glycine Release in the Rat Brain

The effect of N-methyl-D-aspartic acid (NMDA), a selective glutamate receptor agonist, on the release of previously incorporated [³H]-aminobutyric acid(GABA) was examined in superfused striatal slices of the rat. NMDA (0.01 to 1.0 mM) increased [³H]GABA overflow with an EC₅₀ value of 0.09 mM. The [³H]GABA releasing effect of NMDA was an external Ca²⁺-dependent process and the GABA uptake inhibitor nipecotic acid (0.1 mM) potentiated this effect. These findings support the view that NMDA evokes GABA release from vesicular pool in striatal GABAergic neurons. Addition of glycine (1 mM), a cotransmitter for NMDA receptor, did not influence the NMDA-induced [³H]GABA overflow. Kynurenic acid (1 mM), an antagonist of glycine_B site, decreased the [³H]GABA-releasing effect of NMDA and this reduction was suspended by addition of 1 mM glycine. Neither glycine nor kynurenic acid exerted effects on resting [³H]GABA outflow. These data suggest that glycine_B binding site at NMDA receptor may be saturated by glycine released from neighboring cells. Glycyldodecylamide (GDA) and N-dodecylsarcosine, inhibitors of glycineT1 transporter, inhibited the uptake of [³H]glycine (IC₅₀ 33 and 16 M) in synaptosomes prepared from rat hippocampus. When hippocampal slices were loaded with [³H]glycine, resting efflux was detected whereas electrical stimulation failed to evoke [³H]glycine overflow. Neither GDA (0.1 mM) nor N-dodecylsarcosine (0.3 mM) influenced [³H]glycine efflux. Using Krebs-bicarbonate buffer with reduced Na⁺ for superfusion of hippocampal slices produced an increased [³H]glycine outflow and electrical stimulation further enhanced this release. These experiments speak for glial and neuronal [³H]glycine release in hippocampus with a dominant role of the former one. GDA, however, did not influence resting or stimulated [³H]glycine efflux even when buffer with low Na⁺ concentration was applied.     

Release of γ-[3H]Aminobutyric Acid from Rat Olfactory Bulb and Substantia Nigra: Differential Modulation by Glutamic Acid

We have studied the glutamate modulation of gamma-[3H]aminobutyric acid ([3H]GABA) release from GABAergic dendrites of the external plexiform layer of the olfactory bulb and from GABAergic axons of the substantia nigra. In the olfactory bulb, [3H]GABA release was induced by high K+ and kainate, and not by aspartate and glutamate alone. However, when the tissue was conditioned by a previous K+ depolarization, glutamate and aspartate caused [3H]GABA release. The effect of glutamate was significantly enhanced when the GABA uptake mechanism was blocked by nipecotic acid. N-Methyl-D-aspartate and quisqualate did not cause [3H]GABA release under the same conditions. The acidic amino acid receptor antagonist 2-amino-4-phosphonobutyric acid and the N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonovaleric acid significantly inhibited the K+-glutamate- and the kainate-induced [3H]GABA release. Mg2+ (5 mM), which blocks the N-methyl-D-aspartate receptors, significantly inhibited the K+-glutamate-induced but not the kainic acid-induced [3H]GABA release. The K+-glutamate-stimulated release, but not the K+-stimulated [3H]GABA release, was strongly inhibited by Na+-free solutions or by 300 nM tetrodotoxin. Apparently the glutamate-induced release of [3H]GABA occurs through an interneuron because it is dependent on the presence of nerve conduction. In the substantia nigra no [3H]GABA release was elicited by any of the glutamate agonists tested. The present results clearly differentiate between the effects of glutamate on the release of [3H]GABA from the substantia nigra and from the olfactory bulb.(ABSTRACT TRUNCATED AT 250 WORDS)     

Negative allosteric modulators of AMPA-preferring receptors inhibit [(3)H]GABA release in rat striatum

The effect of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), a selective glutamate receptor agonist, on the release of previously incorporated [(3)H]GABA was examined in superfused striatal slices of the rat. The slices were loaded with [(3)H]GABA in the presence of beta-alanine (1 mM) and superfused with Krebs-bicarbonate buffer containing nipecotic acid (0.1 mM) and aminooxyacetic acid (0.1 mM) to inhibit GABA uptake and metabolism. AMPA (0.01 to 3 mM) increased basal [(3)H]GABA outflow and nipecotic acid potentiated this effect. The [(3)H]GABA releasing effect of AMPA was an external Ca(2+)-dependent process in the absence but not in the presence of nipecotic acid. Cyclothiazide (0.03 mM), a positive modulator of AMPA receptors, failed to evoke [(3)H]GABA release by itself, but it dose-dependently potentiated the [(3)H]GABA releasing effect of AMPA. The AMPA (0.3 mM)-induced [(3)H]GABA release was antagonized by NBQX (0.01 mM) in a competitive fashion (pA(2) 5.08). The negative modulator of AMPA receptors, GYKI-53784 (0.01 mM) reversed the AMPA-induced [(3)H]GABA release by a non-competitive manner (pD'(2) 5.44). GYKI-53784 (0. 01-0.1 mM) also decreased striatal [(3)H]GABA outflow on its own right, this effect was stereoselective and was not influenced by concomitant administration of 0.03 mM cyclothiazide. GYKI-52466 (0. 03-0.3 mM), another negative modulator at AMPA receptors, also inhibited basal [(3)H]GABA efflux whereas NBQX (0.1 mM) by itself was ineffective in alteration of [(3)H]GABA outflow.The present data indicate that AMPA evokes GABA release from the vesicular pool in neostriatal GABAergic neurons. They also confirm that multiple interactions may exist between the agonist binding sites and the positive and negative modulatory sites but no such interaction was detected between the positive and negative allosteric modulators. Since GYKI-53784, but not NBQX, inhibited [(3)H]GABA release by itself, AMPA receptors located on striatal GABAergic neurons may be in sensitized state and phasically controlled by endogenous glutamate. It is also postulated that these AMPA receptors are located extrasynaptically on GABAergic striatal neurons.     

Effects of thiopental on transport and metabolism of glutamate in cultured cerebellar granule neurons

This study was performed to analyze the effects of the barbiturate thiopental on neuronal glutamate uptake, release and metabolism. Since barbiturates are known to bind to the GABA(A) receptor, some experiments were carried out in the presence of GABA. Cerebellar granule neurons were incubated for 2 h in medium containing 0.25 mM [U-(13)C]glutamate, 3 mM glucose, 50 microM GABA and 0.1 or 1 mM thiopental when indicated. When analyzing cell extracts, it was surprisingly found that in addition to glutamate, aspartate and glutathione, GABA was also labeled. In the medium, label was observed in glutamate, aspartate and lactate. Glutamate exhibited different labeling patterns, indicating metabolism in the tricarboxylic acid cycle, and subsequent release. A net uptake of [U-(13)C]glutamate and unlabeled glucose was seen under all conditions. The amounts of most metabolites synthesized from [U-(13)C]glutamate were unchanged in the presence of GABA with or without 0.1 mM thiopental. In the presence of 1 mM thiopental, regardless of the presence of GABA, decreased amounts of [1,2, 3-(13)C]glutamate and [U-(13)C]aspartate were found in the medium. In the cell extracts increased [U-(13)C]glutamate, [1,2, 3-(13)C]glutamate, labeled glutathione and [U-(13)C]aspartate were observed in the 1 mM thiopental groups. Glutamate efflux and uptake were studied using [(3)H]D-aspartate. While efflux was substantially reduced in the presence of 1 mM thiopental, this barbiturate only marginally inhibited uptake even at 3 mM. These results may suggest that the previously demonstrated neuroprotective action of thiopental could be related to its ability to reduce excessive glutamate outflow. Additionally, thiopental decreased the oxidative metabolism of [U-(13)C]glutamate but at the same time increased the detectable metabolites derived from the TCA cycle. These latter effects were also exerted by GABA.     

Potentiation by γ-Aminobutyric Acid of α1-Agonist-Induced Accumulation of Inositol Phosphates in Slices of Rat Cerebral Cortex

Noradrenaline-induced accumulation of 3H-labeled inositol mono-, bis-, and trisphosphate (IP1, IP2, and IP3, respectively) in lithium-treated slices of rat cerebral cortex preincubated with [3H]inositol was potentiated by gamma-aminobutyric acid (GABA). However, the effect on [3H]IP2 accumulation was much greater than that on [3H]IP1 or [3H]IP3 accumulation. The principal effect of GABA on noradrenaline concentration-response curves for both [3H]IP1 and [3H]IP2 was to cause an increase in the maximal response attainable. However, whereas the EC50 for GABA potentiation of [3H]IP1 formation was 0.5 mM, the curve for the potentiation of [3H]IP2 formation showed a marked upturn at GABA concentrations of greater than 1 mM. Prazosin (1 microM) blocked the noradrenaline-induced formation of all three inositol phosphates (IPs), in both the presence and the absence of 2 mM GABA. 3H-IP formation induced by phenylephrine and methoxamine was also potentiated by GABA, and again the greatest effect was on [3H]IP2 accumulation. The ratio of [3H]IP2/[3H]IP1 formed in response to 100 microM noradrenaline was increased by 2 mM GABA at all times from 10 to 60 min, whereas the ratio of [3H]IP3/[3H]IP1 was little altered. The effect of GABA was not mimicked by the GABAA agonists isoguvacine and 3-aminopropanesulphonic acid and was not blocked by bicuculline methiodide. (-)-Baclofen, a GABAB agonist, did produce some stimulation of the response to noradrenaline, but to a much lesser extent than GABA. Of the agents tested, nipecotic acid came nearest to reproducing the effect of GABA, in that the major effect was on [3H]IP2 accumulation. The effects of 2 mM GABA and 2 mM nipecotic acid were not additive. GABA potentiation of noradrenaline-induced 3H-IP formation was still apparent in the absence of Li+, but the increase of [3H]IP2 content was less than that of [3H]IP1 content.     

POTASSIUM-INDUCED RELEASE OF [3H]GABA AND OF [3H]NORADRENALINE FROM NORMAL AND RESERPINIZED RAT BRAIN CORTEX SLICES. DIFFERENCES IN CALCIUMDEPENDENCY, AND IN SENSITIVITY TO POTASSIUM IONS

The release of [³H]GABA induced by elevated extracellular potassium (K)_o, from thin rat brain cortex slices, has been compared with that of [³H]noradrenaline ([³H]NA), released by the same procedures, both from normal slices, and from slices pre-treated with reserpine and nialamide, [³H]NA being predominantly a vesicular component in the former situation, and a soluble substance in the latter one. 46 mM-(K)_o released considerably more [³H]NA from normal than from drug-treated slices, while the release of GABA was about two thirds of the latter. When 4min ‘pulses’ of increasing concentrations of potassium were applied, it was observed that the release of GABA and of [³H]NA from drug-treated slices increased in proportion to (K)_o, up to 36-46 mM and then declined considerably with higher (K)_o. The dependency of potassium-induced release on the concentration of calcium in the medium, indicated that release of [³H]NA from normal slices was proportional to calcium up to 1.5-2 mM, while that of [³H]NA from drug-treated slices increased up to 0.5 mM-Calcium, and then declined with higher concentrations. GABA release also increased up to 0.5 mM-calcium, but no further changes were observed at higher concentrations. The calcium antagonist D-600 inhibited high (K)_o-induced release of [³H]NA from normal slices to a greater extent than that of [³H]GABA or of [³H]NA from drug-treated slices. These results, in which elevated (K)_o-induced release of [³H]GABA resembles considerably that of soluble NA, but differs from that of NA present in synaptic vesicles, suggest that release of [³H]GABA also occurs from the soluble cytoplasmic compartment, and that the partial calcium requirement that is found is unrelated to that of transmitter secretion. These findings are also a further indication of the lack of specificity of elevated (K)_o as a stimulus for inducing transmitter secretions.     

GABAB Receptor-Mediated Inhibition of Histamine H1-Receptor-Induced Inositol Phosphate Formation in Slices of Rat Cerebral Cortex

Histamine-stimulated accumulation of [3H]inositol monophosphate ([3H]IP1) in lithium-treated slices of rat cerebral cortex was inhibited by gamma-aminobutyric acid (GABA) (IC50 0.30 +/- 0.03 mM). The maximum level of inhibition was 69 +/- 2%. GABA alone caused a small stimulation of basal accumulation of [3H]IP1. The inhibitory action of GABA on the response to histamine was mimicked by the GABAB agonist (-)-baclofen, IC50 0.69 +/- 0.04 microM, which was 430-fold more potent as an inhibitor than the (+)-isomer. (-)-Baclofen also inhibited histamine-induced formation of [3H]inositol bisphosphate ([3H]IP2) and [3H] inositol trisphosphate ([3H]IP3). Inhibition curves for GABA and for (-)-and and (+)-baclofen had Hill coefficients greater than unity. (-)-Baclofen, at concentrations that caused inhibition of histamine-induced [3H]IP1 accumulation, did not alter the basal level of [3H]IP1 or the incorporation of [3H]inositol into total inositol phospholipids. Isoguvacine, a GABAA agonist, had no effect on either the histamine-stimulated or basal accumulation of [3H]IP1. GABA had no effect on carbachol-stimulated [3H]IP1 formation.     

Release of γ-Amino[3H]butyric Acid from Cultured Amacrine-Like Neurons Mediated by Different Excitatory Amino Acid Receptors

The release of preaccumulated gamma-amino[3H]butyric acid ([3H]GABA) from putative GABAergic amacrine cells was studied in neuronal monolayer cultures made from embryonic chick retina. Release was specifically stimulated by excitatory amino acid agonists. N-Methyl-D-aspartate (NMDA; EC50, 19.1 +/- 5.0 microM), kainic acid (EC50, 15.6 +/- 2.3 microM), and the presumptive endogenous ligand glutamate (EC50, 3.6 +/- 0.5 microM) showed the same efficacy. Quisqualic acid, although the most potent agonist (EC50, 0.56 +/- 0.12 microM), was only half as efficacious. The time course of [3H]GABA release and autoradiographic visualization of responsive GABA-accumulating cells suggest that approximately 50% of the [3H]GABA-accumulating cells possess no or very low responsiveness to quisqualic acid. Depolarization (56 mM KCl)-induced release was fivefold lower than the maximal effect elicited by excitatory amino acids. Release of [3H]GABA and of endogenous GABA was entirely independent of extracellular Ca2+ but was completely abolished after replacement of Na+ by choline or Li+. The effects of NMDA and low concentrations of glutamate (up to 10 microM) were blocked by 2-amino-5-phosphonovaleric acid, by MK 801, and (in a voltage-dependent manner) by Mg2+. The reduction of NMDA responses by kynurenic acid was reversed by D-serine, and quisqualic acid competitively inhibited kainic acid-evoked release. Our results show that the cultured [3H]GABA-accumulating neurons, which probably represent the in vitro counterparts of GABAergic amacrine cells, express at least two types of excitatory amino acid receptors (of the NMDA and non-NMDA type), both of which can mediate a Ca2(+)-independent but Na2(+)-dependent release of GABA.     

Release of Previously Incorporated γ-[3H]Aminobutyric Acid in Rabbit Caudate Nucleus Slices

The release of gamma-aminobutyric acid (GABA) was studied in slices of the head of the rabbit caudate nucleus. The slices were preincubated with [3H]GABA and then superfused. Aminooxyacetic acid was present throughout. Both the tritium in the slices and that in the superfusate consisted practically entirely of [3H]GABA. Stimulation for 2 min by electrical field pulses of 3 ms width and 9 V/cm voltage drop (36 mA current strength) at 5 or 20 Hz elicited an overflow of [3H]GABA that amounted to 0.23 or 0.47% of the tritium content of the tissue, respectively, and was diminished by 85% in the presence of tetrodotoxin. At higher current strength, less of the stimulation-evoked overflow was tetrodotoxin-sensitive. cis-1,3-Aminocyclohexane carboxylic acid diminished the uptake of [3H]GABA into the tissue but did not change the percentage released by electrical stimulation. Ca2+ withdrawal greatly accelerated basal [3H]GABA efflux and almost abolished the response to stimulation. Nipecotic acid 10-1,000 microM enhanced both the basal and (up to eightfold) the stimulation-evoked overflow. The method described allows us to elicit electrically a quasiphysiological, i.e., Ca2+-dependent and tetrodotoxin-sensitive, neuronal release of [3H]GABA. Nipecotic acid diverts released [3H]GABA from reuptake to overflow.     

Turnover and release of GABA in rat cortical slices: Effect of a GABA-T inhibitor,gabaculine

The turnover and release of endogenous and labeled GABA were followed in rat cortical slices after incubation with [³H]GABA. High performance liquid chromatography was used to measure endogenous GABA and to separate [³H]GABA from its metabolites. During superfusion with 3 mM K⁺ the slices rapidly lost their [³H]GABA content while maintaining constant GABA levels. Exposure to 50 mM K⁺ for 25 min caused an initial rapid rise in the release of both endogenous and [³H]GABA followed by a more rapid decline in the release of the latter. The specific activity of released GABA was two to four times higher than that in the slices. Depolarization lead to a net synthesis of GABA. The GABA-T inhibitor, gabaculine, (5 M) in vitro arrested the metabolism of [³H]GABA and rapidly doubled the GABA content but did not significantly increase the high K⁺ evoked release of endogenous GABA. In vivo pretreatment with 0.5 mM/kg gabaculine quadrupled GABA content and increased both the spontaneous and evoked release of endogenous GABA but while its Ca²⁺-dependent release increased by 50%, the Ca²⁺-independent release was enhanced sevenfold. This large Ca²⁺-independent release of GABA is likely to have different functional significance from the normal Ca²⁺-dependent release.     

RELEASE OF [3H]GABA FROM IN VITRO PREPARATIONS: COMPARISON OF THE EFFECT OF DABA AND β-ALANINE ON THE K+ AND PROTOVERATRINE STIMULATED RELEASE OF [3H]GABA FROM BRAIN SLICES AND SYNAPTOSOMES1

It has been proposed that the major portion of [³H]GABA released from rat cortical slices upon exposure to high K⁺ comes from a neuronal pool. Using carrier mediated exchange diffusion of DABA or β-alanine in the superfusion medium for GABA in the slice as a technique for manipulating neuronal and glial pools of GABA, it was found that DABA but not β-alanine substantially reduced the K⁺ stimulated release of [³H]GABA. The present study using synaptosomes as an in vitro model of the nerve ending was undertaken to ascertain whether this neuronal pool of releasable [³H]GABA was associated with a specific transmitter pool in nerve endings. A continuous superfusion system employing a Ca²⁺ pulse to produce a calcium coupled release (Levy et al, 1973) was used to study the effect of two concentrations (20 μm , 1 mm ) of DABA and β-alanine on the release of [³H]GABA from synaptosomes. In contrast to the results in slices, DABA at both concentrations had no effect on the release of [³H]GABA from synaptosomes in spite of evidence that exchange diffusion was occurring. With protoveratrine as the releasing agent there was no effect of DABA on the release of [³H]GABA from either slices or synaptosomes. The results suggest that the major portion of [³H]GABA released from cortical slices by high K⁺ comes from a non-transmitter pool in the neuron. Use of K⁺ stimulated release of amino acids from cortical slices as a criterion for neurotransmitter function must be viewed with caution.     

Stimulation of γ-[3H]Aminobutyric Acid Release from Cultured Mouse Cerebral Cortex Neurons by Sulphur-Containing Excitatory Amino Acid Transmitter Candidates: Receptor Activation Mediates Two Distinct Mechanisms of Release

In primary cultures of mouse cerebral cortex neurons, sulphur-containing excitatory amino acids (SAAs; namely, L-cysteine sulphinate, L-cysteate, L-homocysteine sulphinate, L-homocysteate, S-sulphocysteine) at concentrations ranging from 0.1 microM to 1 mM evoked a saturable release of gamma-[3H]aminobutyric acid ([3H]GABA) in the absence of any other depolarizing agent. All SAAs exhibited essentially similar potency (EC50, 100-150 microM) in releasing [3H]GABA although a variable profile of maximal stimulatory effect was observed when compared with basal release. The intracellular accumulation of the lipophilic cation, [3H]tetraphenylphosphonium, was significantly reduced in the presence of all SAAs, thus verifying a depolarization of the neuronal plasma membrane. SAA-stimulated release of [3H]GABA was shown to comprise two distinct components, calcium-dependent and calcium-independent, which occur after activation of N-methyl-D-aspartate (NMDA) and non-NMDA receptors. Thus, all SAA-evoked responses were antagonized by the selective, competitive NMDA-receptor antagonist, 3-[(+/-)-2-carboxypiperazin-4-yl]propyl-1-phosphonic acid (IC50 range, greater than 50 microM) and the non-NMDA-receptor antagonist, 6,7-dinitroquinoxalinedione (IC50 range, 5-50 microM). Removal of magnesium ions from the superfusion medium caused a significant potentiation of SAA-evoked responses without having any effect on basal levels of [3H]GABA efflux, a result consistent with an involvement of NMDA-receptor activation. Calcium-independent release (i.e., that release remaining in the presence of 1 mM cobalt ions) was a distinct component but of smaller magnitude. Using 500 microM excitatory amino acid agonist concentrations, this component of release was (1) markedly attenuated by 15 microM SKF-89976-A, a non-transportable inhibitor of the GABA carrier, and (2) abolished when choline ions replaced sodium ions in the superfusion medium or when in the presence of excitatory amino acid receptor antagonists. These observations are clearly consistent with a receptor-mediated, depolarization-induced reversal of the GABA carrier.     

Preferential release of newly synthesized [3H]GABA from striatal slices to preloaded [3H]GABA

The release of [³H]GABA which is newly synthesized from [³H]l-glutamic acid (GLU) has been examined using striatal slices obtained from the rat brain. It was found that 8–10% of [³H]GLU transported was converted to [³H]GABA during the incubation of striatal slices in the presence of nipecotic acid (5 × 10^?5 M). Nipecotic acid was added to the medium in order to prevent possible reuptake of [³H]GABA released during its synthesis, and it was found to have no significant effect on the formation of [³H]GABA from [³H]GLU as well as on the uptake of [³H]GLU. The application of high potassium (60 mM) stimulation exhibited a significant enhancement of the release of this newly synthesized [³H]GABA in a Ca²⁺ dependent manner. Kinetic analysis revealed that the evoked release of newly synthesized [³H]GABA was approximately two times greater than that of previously-loaded [³H]GABA, whereas no significant difference was observed in the spontaneous release. An immobilization stress in water failed to affect the release of newly synthesized [³H]GABA from striatal slices despite the occurrence of a significant enhancement of GABA formation in this structure.These results suggest that newly synthesized GABA may be preferentially released from its nerve terminals in response to the excitation of neurons at least in the striatum as compared with previously accumulated GABA.     

Effect of Taurine on Neurotransmitter Release from Insect Synaptosomes

The effect of taurine on the release of [3H]acetylcholine ([3H]ACH) and [3H]gamma-aminobutyric acid ([3H]GABA) from preloaded locust synaptosomes has been studied. Veratridine (100 microM) and K+ (100 mM) both evoked [3H]ACh release and this was reduced in a concentration-dependent manner by taurine (5, 10, and 20 mM). In contrast to this, veratridine induced no observable release of [3H]GABA, and the response to K+ was slight. In the presence of taurine, however, a concentration-dependent enhancement of [3H]GABA release was observed. Since nipecotic acid (1 mM), an inhibitor of neuronal GABA uptake, also revealed [3H]GABA release induced by veratridine, it is suggested that both this effect and that of taurine are due to prevention of GABA reuptake. These results suggest that taurine may act as a neuromodulator in insects.     

Release of d-[3H]aspartic acid from the rat substantia nigra: effect of veratridine-evoked depolarization and cortical ablation

The spontaneous and veratridine-evoked release of radioactive d-aspartic acid, previously taken up by rat substantia nigra slices, was studied by using a superfusion system. Veratridine (25 μM, 1 min) markedly produced a 14-fold increase in d-[³H]aspartic acid release from nigral slices. Omission of Ca²⁺ and increasing Mg²⁺ concentration to 12 mM in the superfusion medium did substantially block d-[³H]aspartate release induced by veratridine depolarization. Nevertheless, veratridine was able to evoke [³H]amino acid release which seemed to be, at least, 30% Ca²⁺-independent. Additional experiments showed that tetrodotoxin (0.01–0.1 μM), a blocker of voltage-dependent Na⁺ channels, totally abolished veratridine-evoked release of d-[³H]aspartate from nigral slices.Lesion studies were performed in order to learn about the nature of the neuronal compartment in the substantia nigra upon which veratridine-depolarization acted to induce d-[³H]aspartate release. Unilateral ablation of the fronto-parietal cortex was accompanied by a significant decrease in the accumulation of nigral d-[³H]aspartate and by a large loss from ipsilateral nigral slices in d-[³H]aspartate release evoked by veratridine. In contrast, both the accumulation and veratridine-evoked release of [³H]dopamine, remained unchanged in the ipsilateral substantia nigra slices to the lesion.The findings reported suggest that d-[³H]aspartic acid may be taken up and then released, in a Ca²⁺-dependent manner, by nerve terminals located in the substantia nigra. In addition, the results shown provide support to the view that l-glutamate and/or l-aspartate may act as neurotransmitters at the cortico-nigral neuronal pathway.     

Evidence that [3H]Dopamine Is Taken Up and Released from Nondopaminergic Nerve Terminals in the Rat Substantia Nigra In Vitro

Potassium chloride (25 mM) and (+)-amphetamine (100 microM) both stimulated the release of radioactivity from slices of substantia nigra preincubated with [3H]3,4-dihydroxyphenylethylamine [( 3H]dopamine). Potassium chloride (25 mM) released radioactivity from slices of both zona compacta and zona reticulata. Prior 6-hydroxydopamine (6-OHDA) lesions of one nigrostriatal pathway did not reduce the spontaneous release of radioactivity, or the potassium chloride- or amphetamine-induced release of radioactivity from slices of nigra ipsilateral to the lesion after preincubation with [3H]dopamine. The accumulation of radioactivity following incubation of nigral slices from 6-OHDA-lesioned animals with [3H]dopamine was increased when compared to uptake into slices from intact tissue. In synaptosomal preparations of striatum, nomifensine but not desipramine or fluoxetine inhibited [3H]dopamine uptake. In contrast, nomifensine, desipramine, and fluoxetine all inhibited [3H]dopamine uptake in nigral synaptosomal preparations. Following 6-OHDA lesions of one nigrostriatal pathway the uptake of [3H]dopamine into nigral synaptosomal preparations was unchanged but uptake into striatal preparations was substantially decreased. In contrast, bilateral electrolesions of the dorsal and medial raphe nuclei reduced [3H]dopamine uptake into nigral preparations but not into striatal synaptosomes. The uptake of [3H]5-hydroxytryptamine ([3H]5-HT) into synaptosomal preparations of substantia nigra was abolished by fluoxetine and reduced by desipramine, but was unaffected by nomifensine. In contrast, fluoxetine, desipramine, and nomifensine all inhibited [3H]5-HT uptake into striatal synaptosomal preparations. Following 6-OHDA lesions of one nigro-striatal pathway the uptake of [3H]5-HT into nigral synaptosomal preparations was unchanged but uptake into striatal preparations was reduced.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Effects of Volatile Anesthetics on the Extracellular Accumulation of [3H]GABA in Rat Brain Cortical Slices

GABA is an inhibitory neurotransmitter that appears to be associated with the action of volatile anesthetics. These anesthetics potentiate GABA-induced postsynaptic currents by synaptic GABA_A receptors, although recent evidence suggests that these agents also significantly affect extrasynaptic GABA receptors. However, the effect of volatile anesthetics on the extracellular concentration of GABA in the central nervous system has not been fully established. In the present study, rat brain cortical slices loaded with [³H]GABA were used to investigate the effect of halothane and sevoflurane on the extracellular accumulation of this neurotransmitter. The accumulation of [³H]GABA was significantly increased by sevoflurane (0.058, 0.11, 0.23, 0.46, and 0.93 mM) and halothane (0.006, 0.012, 0.024, 0.048, 0072, and 0.096 mM) with an EC₅₀ of 0.26 mM and 35 μM, respectively. TTX (blocker of voltage-dependent Na⁺ channels), EGTA (an extracellular Ca²⁺ chelator) and BAPTA-AM (an intracellular Ca²⁺ chelator) did not interfere with the accumulation of [³H]GABA induced by 0.23 mM sevoflurane and 0.048 mM halothane. SKF 89976A, a GABA transporter type 1 (GAT-1) inhibitor, reduced the sevoflurane- and halothane-induced increase in the accumulation of GABA by 57 and 63 %, respectively. Incubation of brain cortical slices at low temperature (17 °C), a condition that inhibits GAT function and reduces GABA release through reverse transport, reduced the sevoflurane- and halothane-induced increase in the accumulation of [³H]GABA by 82 and 75 %, respectively, relative to that at normal temperature (37 °C). Ouabain, a Na⁺/K⁺ ATPase pump inhibitor, which is known to induce GABA release through reverse transport, abolished the sevoflurane and halothane effects on the accumulation of [³H]GABA. The effect of sevoflurane and halothane did not involve glial transporters because β-alanine, a blocker of GAT-2 and GAT-3, did not inhibit the effect of the anesthetics. In conclusion, the present study suggests that sevoflurane and halothane increase the accumulation of GABA by inducing the reverse transport of this neurotransmitter. Therefore, volatile anesthetics could interfere with neuronal excitability by increasing the action of GABA on synaptic and extrasynaptic GABA receptors.