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.     

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

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

THE SPONTANEOUS AND ELECTRICALLY EVOKED RELEASE, FROM SLICES OF GUINEA-PIG CEREBRAL CORTEX, OF ENDOGENOUS AMINO ACIDS LABELLED VIA METABOLISM OF d-[U-14C]GLUCOSE

Abstract— In an effort to identify neurotransmitters in slices of guinea-pig cerebral cortex, a study was made of the release of endogenous amino acids which had become labelled via metabolism of d -[U-¹⁴C]glucose. While incorporation of ¹⁴C into endogenous glutamate, aspartate, GABA, alanine and threonine-serine-glutamine (unseparated) was large enough to permit measurement of their release, that into other amino acids was not. In parallel experiments, the release of exogeneous labelled glutamate, aspartate, GABA and α-aminoisobutyrate was examined. Electrical field stimulation evoked a transient increase in the release of all the adequately labelled endogenous amino acids and all the exogenous amino acids. The stimulated ‘increase’ in the release of each of the endogenous ¹⁴C-labelled transmitter candidates (glutamate, aspartate and GABA) was larger than that of any other amino acid (except that of exogenous GABA). When the experiments were performed without the glucose (5 mm ) usually present in the medium bathing the slices, larger amounts of each labelled amino acid were released from the slices than in the presence of glucose. Moreover, the pattern of selective release of the endogenous labelled transmitter candidates was much more pronounced in the absence of glucose. It is likely that in the absence of glucose, release from the tissue was larger because cells in the slice were relatively depolarized and uptake of amino acids into cells was impaired. Because previous evidence suggests that over 90% of glucose consumption occurs in the ‘large metabolic compartment’ which is thought to be composed of neuronal elements, neurons were probably the main site from which the larger release of endogenous ¹⁴C-labelled transmitter candidates was evoked. The exogenous amino acids were probably released from several cellular elements in the slices. It was concluded that the pattern of a selective release of the endogenous labelled transmitter candidates may have been indicative of a transmitter releasing mechanism in nerve terminals.     

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.     

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.     

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.     

BACLOFEN: EFFECTS ON AMINO ACID RELEASE AND METABOLISM IN SLICES OF GUINEA PIG CEREBRAL CORTEX

Slices of guinea-pig cerebral cortex were used to investigate the effects of the antispastic drug β-(p-chlorophenyl)-γ-aminobutyrate (Baclofen, Lioresal) on the release and metabolism of several amino acids. Electrical stimulation of slices evoked (1) a relatively large release, probably from nerve terminals, of ¹⁴C-labelled tissue glumate, aspartate and γ-aminobutyrate (GABA) synthesized via metabolism of D-[U-¹⁴C]glucose and (2) a relatively small release, probably not from nerve terminals, of ¹⁴C-labelled tissue alanine and threonine-serine-glutamine and of exogenous radiolabeled glutamate, aspartate, GABA and α-aminoisobutyrate that had been taken up from the medium. Baclofen (4μM) preferentially inhibited the release of ¹⁴C-labelled tissue glutamate and aspartate. It had no effect on the concentrations and specific radio-activities of most of the labelled tissue amino acids in the slices. However, it increased the turnover of ¹⁴C-labelled tissue glycine approx 4-fold and elevated the specific radio activity of tissue alanine by 40%. It was concluded that Baclofen affects transmission not by modulating the release of the inhibitory amino acid GABA, but by selectively suppressing the release of the excitatory amino acids glutamate and aspartate from nerve terminals. Provided that this action obtains in the spinal cord, it may at least partly underlie the antispastic action of Baclofen as glutamate and aspartate are presumed to be the transmitters released from terminals of non-nociceptive primary afferent fibers and excitatory interneurons, respectively. The Baclofen-induced increase in glycine turnover suggests an additional effect on inhibitory glycinergic interneurons in the spinal cord.     

4-Aminobutyrate Can Be Released Exocytotically from Guinea-Pig Cerebral Cortical Synaptosomes

Guinea-pig synaptosomes possess two functional pools of 4-aminobutyrate (GABA). One is rapidly labelled by added [14C]GABA, is steadily released in a Ca2+-independent manner when the Na+ electrochemical potential across the plasma membrane is collapsed, and is depleted by the GABA analogue 2,4-diaminobutyrate (DABA), all of which is consistent with a cytosolic location. A second, noncytosolic compartment only slowly equilibrates with exogenous [14C]GABA, is not depleted by DABA, but can release 350 pmol of endogenous GABA/mg of protein (8% of the total intrasynaptosomal GABA) within 15 s of depolarization in the presence of Ca2+. Ca2+-independent release occurs by thermodynamic reversal of the plasma membrane uptake pathway following artifactually prolonged depolarization, whereas Ca2+-dependent release is consistent with physiological exocytosis from vesicular stores.     

Release of Endogenous and Accumulated Exogenous Amino Acids from Slices of Normal and Climbing Fibre-Deprived Rat Cerebellar Slices

Efflux of various amino acids from slices of rat cerebellar hemispheres was determined under resting or depolarizing conditions. It was increased under high K+(50 mM) as compared to low K+ (5 mM) conditions by 1258 pmol/mg protein for aspartate, 478 for gamma-aminobutyric acid (GABA), 44,693 for glutamate, and 615 for glycine. These were significantly higher than the corresponding values obtained under low-Ca2+ (0.1 mM), high-Mg2+ (12 mM) conditions, whereas for 11 other amino acids the K+-induced efflux was similar under normal and low-Ca2+ concentrations. The K+-induced efflux of exogenously accumulated L-[3H]aspartate, D-[3H]aspartate, and L-[3H]glutamate was higher by factors of 2, 5.8, and 6.3, respectively, under normal Ca2+ conditions, as compared with low-Ca2+, high-Mg2+ conditions. After climbing fibre degeneration induced by destruction of the inferior olive with 3-acetylpyridine, release of endogenous aspartate and exogenous L-[3H]glutamate and D-[3H]aspartate was significantly reduced, by 26%, 38%, and 27%, respectively. These results support the hypothesis that climbing fibres may use aspartate or a related compound as a neurotransmitter. In rat cerebellar tissue, L-[3H]glutamate and L-[3H]aspartate differ in several aspects: (1) L-[3H]glutamate uptake was 4 times higher than that of L-[3H]aspartate; (2) fractional rate constant of K+- evoked release of L-[3H]aspartate was 7% X 2.5 min-1, and of L-[3H]glutamate 36% X 2.5 min-1; and (3) specific activity of L-[3H]glutamate in the eluate collected during K+ stimulation was 3.5 times the value in the tissue, whereas for L-[3H]aspartate, specific activities in the eluate and tissue were similar.     

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

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

Compartmentation of citric acid cycle metabolism in brain: effect of aminooxyacetic acid, ouabain and Ca2+ on the labelling of glutamate, glutamine, aspartate and gaba by [1-14C]acetate, [U-14C]glutamate and [U-14C]asparate

—(1) The effects of aminooxyacetic acid, ouabain and Ca²⁺ on the compartmentation of amino acid metabolism have been studied in slices of brain incubated with sodium-[1-¹⁴C]acetate, l-[U-¹⁴C]glutamate and l-[U-¹⁴C]aspartate as tracer metabolites. (2) Aminooxyacetic acid (10^-3 m) inhibited the labelling of aspartate from [¹⁴C]acetate and [¹⁴C]glutamate, as well as the incorporation of label from [¹⁴C]aspartate into glutamate and glutamine. It also inhibited the labelling of GABA from all three radioactive precursors, as would be anticipated if there was inhibition of several transaminases as well as glutamate decarboxylase. The RSA of glutamine labelled from [1-¹⁴C]acetate was increased. This finding indicated that the glutamate pool which is utilized for glutamine formation is associated with glutamate dehydrogenase, and this enzyme appears to be related to the ‘synthetic tricarboxylic acid cycle’. AOAA exerted its major inhibitory effects on the citric acid‘energy cycle’with which transaminases are associated. (3) Ouabain (10^-5 m) inhibited the labelling of glutamine to a much greater extent than the labelling of glutamate from [1-¹⁴C]acetate. It also caused leakage of amino acids from the tissue into the medium. Its effect on the glutamate–glutamine system was interpreted to be a selective inhibition of the 'synthetic’citric acid cycle. (4) The omission of Ca²⁺ from the incubation medium was associated with formation of glutamine with RSA less than 1·0 when labelled from [U-¹⁴C]glutamate, [U-¹⁴C]aspartate and lower than normal when labelled from [1-¹⁴C]acetate.     

Electrical Stimulation of the Stratum Radiatum Increases the Release and Neosynthesis of Aspartate, Glutamate, and γ-Aminobutyric Acid in Rat Hippocampal Slices

The release of endogenous aspartic, glutamic, and gamma-aminobutyric acids (Asp, Glu, GABA, respectively) was measured in the effluent from superfused hippocampal slices using a new and sensitive mass spectrometric method. The stimulation of the stratum radiatum of the rat dorsal hippocampus caused a Ca2+-dependent increase in the release of these amino acids. This release was accompanied by an increase in the incorporation of [13C2] from [13C]glucose into Asp, Glu, and GABA, suggesting an increase in their neosynthesis. The removal of Ca2+ from the superfusion fluid brought about a marked decrease in Asp and Glu release at rest, and prevented their stimulation-evoked release and the appearance of population spikes. The results support the hypothesis that Asp and Glu are excitatory neurotransmitters in intrinsic hippocampal circuits and are possibly released from the Schaffer collaterals and commissural fibres. The increase in GABA release and neosynthesis during stimulation of the stratum radiatum could be related to recurrent inhibition evoked by transsynaptic stimulation of the pyramidal cells.     

Participation of endogenous fatty acids in the secretory activity of the pancreatic B-cell.

The pancreatic B-cell may represent a fuel-sensor organ, the release of insulin evoked by nutrient secretagogues being attributable to an increased oxidation of exogenous and/or endogenous substrates. The participation of endogenous fatty acids in the secretory response of isolated rat pancreatic islets was investigated. Methyl palmoxirate (McN-3716, 0.1 mM), an inhibitor of long-chain-fatty-acid oxidation, suppressed the oxidation of exogenous [U-14C]palmitate and inhibited 14CO2 output from islets prelabelled with [U-14C]palmitate. Methyl palmoxirate failed to affect the oxidation of exogenous D-[U-14C]glucose or L-[U-14C]glutamine, the production of NH4+ and the output of 14CO2 from islets prelabelled with L-[U-14C]glutamine. In the absence of exogenous nutrient and after a lag period of about 60 min, methyl palmoxirate decreased O2 uptake to 69% of the control value. Methyl palmoxirate inhibited insulin release evoked by D-glucose, D-glyceraldehyde, 2-oxoisohexanoate, L-leucine, 2-aminobicyclo[2.2.1]heptane-2-carboxylate or 3-phenylpyruvate. However, methyl palmoxirate failed to affect insulin release when the oxidation of endogenous fatty acids was already suppressed, e.g. in the presence of pyruvate or L-glutamine. These findings support the view that insulin release evoked by nutrient secretagogues tightly depends on the overall rate of nutrient oxidation, including that of endogenous fatty acids.     

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)     

Uptake of [14C]GABA by rat brain slices; the effect of Ca2+

The [14C]GABA uptake by slices (0.3 mm thick) of Wistar rat brain cortex was studied for its dependence on the GABA concentration in the medium, time of incubation and the presence of Ca2+. This process is characterized by the absence of saturation; the uptake by slices increases sharply when the concentration of exogenous [14C]GABA reaches 200 microM. Bicucullin (10(-4) M), an antagonist of GABA, inhibits the accumulation of GABA in the concentration of 0.2 microM by 60%, that evidences for a considerable contribution of the receptor binding to this process. The [14C]GABA uptake when Ca2+ is absent in the incubation medium and when its concentration is 10(-3) M is practically the same and comparatively low concentrations of Ca2+ (10(-6)-10(-4] decrease the GABA uptake.     

Relationship between synaptosomal uptake and release of [14C]gaba, [14C]diaminobutyric acid and [14C]beta-alanine.

[¹⁴C]GABA is taken up by rat brain synaptosomes via a high affinity, Na⁺-dependent process. Subsequent addition of depolarizing levels of potassium (56.2 MM) or veratridine (100 μM) stimulates the release of synaptosomal [¹⁴C]GABA by a process which is sensitive to the external concentration of divalent cations such as Ca²⁺, Mg²⁺, and Mn²⁺. However, the relatively smaller amount of [¹⁴C]GABA taken up by synaptosomes in the absence of Na⁺ is not released from synaptosomes by Ca²⁺ -dependent, K ⁺-stimulation. [¹⁴C]DABA, a competitive inhibitor of synaptosomal uptake of GABA (Iversen & Johnson , 1971) is also taken up by synaptosomal fractions via a Na ⁺ -dependent process; and is subsequently released by Ca²⁺ -dependent, K⁺-stimulation. On the other hand, [¹⁴C]β-alanine, a purported blocker of glial uptake systems for GABA (Schon & Kelly , 1974) is a poor competitor of GABA uptake into synaptosomes. Comparatively small amounts of [¹⁴C] β-alanine are taken up by synaptosomes and no significant amount is released by Ca²⁺ -dependent, K⁺-stimulation. These data suggest that entry of [¹⁴C]GABA into a releasable pool requires external Na⁺ ions and maximal evoked release of [¹⁴C]GABA from the synaptosomal pool requires external Ca²⁺ ions. The GABA analogue, DABA, is apparently successful in entering the same or similar synaptosomal pool. The GABA analogue, β-alanine, is not. None of the compounds or conditions studied were found to simultaneously affect both uptake and release processes. Compounds which stimulated release (veratridine) or inhibited release (magnesium) were found to have minimal effect on synaptosomal uptake. Likewise compounds (DABA) or conditions (Na⁺-free medium) which inhibited uptake, had little effect on release.     

The metabolism of γ-aminobutyrate and glucose in potassium ion-stimulated brain tissue in vitro

1. The metabolism of gamma-aminobutyrate (GABA) was investigated in cerebral-cortex slices incubated in glucose-saline medium with [1-(14)C]GABA and [U-(14)C]-glucose as labelled substrates. 2. A rapid release of GABA from the tissue, amounting to 25-30% of the total, was observed on addition of 66m-equiv. of K(+)/1 to the medium; the liberation of other amino acids was relatively small. The effect was apparently specific for K(+); GABA was not released on addition of equivalent amounts of Na(+) or on increasing the respiration rate with 10mm-ammonium chloride. The results show that GABA behaves like the transmitter compounds (acetylcholine, catecholamines) on K(+) stimulation, and therefore now satisfies certain of the criteria required for a transmitter in mammalian brain. 3. The release of GABA from the tissue on addition of K(+) was followed by a slow re-uptake. The rate of uptake of GABA in a medium containing 5.9m-equiv. of K(+)/1 was more than four times that in a medium containing 66m-equiv. of K(+)/1. 4. The concentration of GABA in brain tissue incubated for 1h in a medium containing 66m-equiv. of K(+)/1 was about 50% higher than that observed under normal conditions. 5. There was evidence that exogenous [(14)C]GABA mixed with the endogenous pool(s), since the proportion of the total GABA released on K(+) stimulation was the same, and the specific radioactivity of the liberated GABA was close to that remaining in the tissue, whether the GABA was labelled by [1-(14)C]GABA from the medium or generated in the tissue from [(14)C]glucose. 6. On the basis of these findings and the observations outlined in the preceding papers it was possible to calculate the kinetic constants of GABA metabolism by computer simulation of the results. K(+) stimulation led to a 2.5-fold increase in the flux through the tricarboxylic acid cycle, whereas the flux in the GABA bypath was little affected; as a result the flux through the GABA bypath, which under normal conditions was 8% of that through the tricarboxylic acid cycle, decreased to 3-5%. 7. The metabolism of glutamine was greatly affected by K(+)-stimulation. The ratio of the concentration of glutamine in the slices to that in the medium, which under normal conditions was the smallest among the amino acids investigated, increased from about 17 to 63 in 1h. This effect was attributable partly to an uptake of glutamine from the medium (1.8mumol/h per g) and partly to a net increase in the total amount of glutamine (2.6mumol/h per g). At 1h after the addition of K(+) the net gain of glutamine could be accounted for by the decrease of glutamate. 8. Metabolic compartmentation was evident when brain-cortex slices were incubated in glucose-saline medium and the labelled substrate was [(14)C]GABA, since the specific radioactivity of glutamine exceeded that of glutamate. On addition of K(+) the signs of metabolic compartmentation promptly disappeared: this effect was apparently associated with an increase in the permeability of the compartments containing labelled metabolites derived from [(14)C]GABA. The change in the permeability, however, did not affect all the compartments; when the labelled substrate was [(14)C]glucose the equilibration of labelled amino acids between tissue and medium was similar under normal conditions and in the presence of high concentrations of K(+). 9. The metabolism of [(14)C]glucose was followed by measuring oxygen uptake, respiratory (14)CO(2), and incorporation of (14)C into amino acids. The results showed that K(+) stimulation increased the flux of glucose carbon, both in the glycolytic pathway and in the tricarboxylic acid cycle.     

The Effects of Scorpion Venom Toxin on the Release of Amino Acid Neurotransmitters from Cerebral Cortex In Vivo and In Vitro

Tityustoxin, the active component of the venom of the Brazilian yellow scorpion Tityus serrulatus, caused specific release of the neurotransmitter amino acids glutamate, aspartate and GABA in vivo from the superfused sensori-motor cortex of conscious unanesthetised rats and in vitro from rat cortical synaptosomes. The effects on synaptosomes appear to be due to a depolarising action. Synaptosomal potassium levels were depleted by the toxin. The action was also blocked both in vivo and in vitro by tetrodotoxin and was Ca2+-dependent. The uptake of [U-14C]GABA was inhibited by tityustoxin but this action was prevented by tetrodotoxin (1 microM). Since the release of [U-14C]GABA from synaptosomes due to the tityustoxin was also prevented by tetrodotoxin under identical circumstances, it is concluded that the tityustoxin has a primary action on release of neurotransmitters rather than on uptake.     

The Partitioning of Photosynthetically Assimilated 14C into Amino Acids in Wheat 1. Partitioning During Transport to the Grain

When ¹⁴CO₂ was fed to flag leaf laminae at 20 d post-anthesis,the transport organs between the leaf and the grains containedappreciable ¹⁴C in glutamine, glutamate, serine, alanine, threonineand glycine. Smaller amounts of ¹⁴C were present in gamma-aminobutyricacid (GABA), aspartate and cysteine. Other amino acids whichwere labelled in the source leaf were not labelled in the transportorgans. The export of labelled glutamine, serine, glycine andthreonine from the source leaf was favoured in comparison tothe other amino acids mentioned. Threonine accumulated, andwas subsequently metabolised, in the rachis. [¹⁴C]GABA alsoaccumulated in the rachis. In the grains, the relative amountof soluble [¹⁴C]alanine increased with chase time. This wasprobably due to de novo synthesis and reflected the specialrole of alanine in grain nitrogen metabolism. Wheat, Triticum aestivum, ¹⁴CO₂, amino acids, transport, carbon metabolism     

Decreased Release of D-Aspartate in the Guinea Pig Spinal Cord After Lesions of the Red Nucleus

This study attempts to determine if fibers that project from the guinea pig red nucleus to the spinal cord use L-glutamate and/or L-aspartate as transmitters. Unilateral injections of kainic acid were placed stereotaxically in the red nucleus to destroy the cells of origin of the rubrospinal tract. Six days after the injection, Nissl-stained sections through the lesion site showed that the majority of neurons in the red nucleus ipsilateral to the kainic acid injection were destroyed. In addition, the lesioned area included parts of the surrounding midbrain reticular formation. Silver-impregnated, transverse sections of the cervical spinal cord revealed the presence of degenerating fibers contralaterally in laminae IV-VII of the gray matter. Ipsilaterally, very sparse degeneration was evident in laminae VII and VIII of the gray matter. Two to six days after surgery, the electrically evoked, Ca2(+)-dependent release of both D-[3H]aspartate, a marker for glutamatergic/aspartatergic neurons, and gamma-amino[14C]-butyric acid ([14C]GABA) was measured in dissected quadrants of the spinal cervical enlargement. Lesions centered on the red nucleus depressed the release of D-[3H]aspartate by 25-45% in dorsal and ventral quadrants of the cervical enlargement contralaterally. The release of [14C]GABA was depressed by 27% in contralateral ventral quadrants. To assess the contribution of rubro- versus reticulospinal fibers to the deficits in amino acid release, unilateral injections of kainic acid were placed stereotaxically in the midbrain reticular formation lateral to the red nucleus. Nissl-stained sections through the midbrain revealed the presence of extensive neuronal loss in the midbrain and rostral pontine reticular formation, whereas neurons in the red nucleus remained undamaged. In the spinal cord, degenerating axons were present ipsilaterally in laminae VII and VIII of the gray matter. Some fiber degeneration was also evident contralaterally in laminae V and VI of the gray matter. This lesion did not affect the release of either D-[3H]aspartate or [14C]GABA in the spinal cord. The substantial decrements in D-[3H]aspartate release following red nucleus lesions suggests that the synaptic endings of rubrospinal fibers mediate the release of D-[3H]aspartate in the spinal cord. Therefore, these fibers may be glutamatergic and/or aspartatergic. Because other evidence suggests that rubrospinal neurons are probably not GABAergic, the depression of [14C]GABA release probably reflects changes in the activity of spinal interneurons following the loss of rubrospinal input.