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.     

Tetanus toxin treatment in vitro inhibits the release of GABA from rat cerebral cortex slices evoked by high K+ and Na+-free media

The influence of tetanus toxin in vitro on the release of exogenous [³H]GABA was studied with rat cerebral cortex slices. The influx, long-term accumulation and spontaneous efflux of GABA were not modified by the toxin. The release induced by high K⁺ (50 mM) medium from the superfused slices pretreated with the toxin was significantly inhibited in a time- and dose-dependent fashion. This release was attenuated during superfusion with Ca²⁺-free medium and the toxin no longer affected the remaining Ca²⁺-independent release. The release induced by Na⁺-free media did not require extracellular Ca²⁺ ions, and the toxin inhibited the release both with and without Ca²⁺. The toxin treatment had no marked influence on the ouabain (20 μM) or veratrine (25–50 μM)-induced release of GABA. The toxin treatment in vitro appears to modify some step(s) in the stimulated release of GABA without affecting its unstimulated membrane transport. Tetanus toxin may thus prove a valuable tool in studying the mechanisms of the release of GABA and possibly other inhibitory transmitters in synapses of the central nervous system.     

Effect of Nipecotic Acid, a γ-Aminobutyric Acid Transport Inhibitor, on the Turnover and Release of γ-Aminobutyric Acid in Rat Cortical Slices

Abstract: To see the effect of a γ-aminobutyric acid GABA uptake inhibitor on the efflux and content of endogenous and labeled GABA, rat cortical slices were first labeled with [³H]GABA and then superfused in the absence or presence of 1 mM nipecotic acid. Endogenous GABA released or remaining in the slices was measured with high performance liquid chromatography, which was also used to separate [³H]GABA from its metabolites. In the presence of 3 mM K⁺, nipecotic acid released both endogenous and [³H]GABA, with a specific activity four to five times as high as that present in the slices. The release of labeled metabolite(s) of [³H]GABA was also increased by nipecotic acid. The release of endogenous GABA evoked by 50 mM K⁺ was enhanced fourfold by nipecotic acid but that of [³H]GABA was only doubled when expressed as fractional release. In a medium containing no Ca²⁺ and 10 mM Mg²⁺, the release evoked by 50 mMK⁺ was nearly suppressed in either the absence or the presence of nipecotic acid. In the absence of nipecotic acid electrical stimulation (bursts of 64 Hz) was ineffective in evoking release of either endogenous or [³H]GABA, but in the presence of nipecotic acid it increased the efflux of endogenous GABA threefold, while having much less effect on that of [³H]GABA. Tetrodotoxin (TTX) abolished the effect of electrical stimulation. Both high K⁺ and electrical stimulation increased the amount of endogenous GABA remaining in the slices, and this increase was reduced by omission of Ca²⁺ or by TTX. The results suggest that uptake of GABA released through depolarization is of major importance in removing GABA from extracellular spaces, but the enhancement of spontaneous release by nipecotic acid may involve intracellular heteroexchange. Depolarization in the presence of Ca²⁺ leads to an increased synthesis of GABA, in excess of its release, but the role of this excess GABA remains to be established.     

Sedimentation and release properties of P2 fractions derived from rat cerebral cortex slices incubated with radiolabeled GABA for a short or long time period

On homogenization of rat cerebral cortex slices previously incubated with [³H] GABA or [¹⁴C]GABA for 5 or 30 min, respectively, particles were recovered in P₂ fractions which exhibited similar buoyant density, but different sedimentation velocity on linear sucrose density gradient centrifugation. The K⁺-evoked release of [³H]GABA from particles isolated from slices previously incubated for 5 min with [³H]GABA was increased in the presence of exogenous Ca²⁺. In contrast, the K⁺-evoked release from particles isolated from slices previously incubated for 30 min with [³H]GABA, was not influenced by the presence of exogenous Ca²⁺.These results suggest that, depending on the incubation time of slices, exogenously applied GABA can be detected in differnnt pools. These pools not only seem to differ in their Ca²⁺ dependency of K⁺-evoked release but also in their subcellular localization.     

Release of endogenous dopamine, 3,4-dihydroxyphenylacetic acid,and amino acid transmitters from rat striatal slices

The release of endogenous dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) was measured in superfused striatal slices of the rat and the results compared with data obtained for the release of endogenous (a) DA and DOPAC in the cerebral cortex, nucleus accumbens and thalamus; (b) 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), GABA, and glutamate in the striatum; and (c) GABA, glutamate and 5-HT in the cerebral cortex. In superfused slices of all four CNS regions, there appeared to be a Ca²⁺-dependent, K⁺-stimulated release of endogenous DA. In addition, in slices of the striatum and nucleus accumbens there also appeared to be a Ca²⁺-dependent, 60 mM K⁺ stimulated release of endogenous DOPAC. In the striatum, 16 mM Mg²⁺ was as effective as 2.5 mM Ca²⁺ in promoting the 60 mM K⁺-stimulated release of DOPAC. In addition, 16 mM Mg²⁺ appeared to function as a weak Ca²⁺ agonist since it also promoted the release of DA to approximately 40% of the level attained with Ca²⁺ in the presence of 60 mM K⁺. On the other hand, in the striatum, 16 mM Mg²⁺ inhibited the Ca²⁺-dependent, 60 mM K⁺-stimulated release of GABA and glutamate. Similar Mg²⁺-inhibition was observed in the cerebral cortex not only for GABA and glutamate but also for DA and 5-HT. With the use of -methyl -tyrosine (tyrosine hydroxylase inhibitor), cocaine (uptake inhibitor) and pargyline (monoamine oxidase inhibitor), it was determined that (a) most of the released DA and DOPAC was synthesized in the slices during the superfusion; (b) DOPAC was not formed from DA which had been released and taken up; and (c) DA and DOPAC were released from DA nerve terminals. In addition, the data indicate a difference in the release process between the amino acids and the monoamines from striatal slices since Mg²⁺ inhibited the Ca²⁺-dependent, K⁺-stimulated release of GABA and glutamate and appeared to promote the release of DA and 5-HT.     

ELECTRICALLY INDUCED RELEASE OF [3H]GABA FROM NEOCORTICAL THIN SLICES. EFFECTS OF STIMULUS WAVEFORM AND OF AMINO-OXYACETIC ACID

The efflux of [³H]GABA or [¹⁴C]GABA from superfused neocortical thin slices, held on quick transfer electrodes, has been compared with that of the non-transmitter amino acid model [¹⁴C]α- amino-isobutyrate (AIB), and, to a lesser extent, with [³H]norepinephrine. Electrical stimulation of the slices with sine-wave current (50 Hz); rectangular, biphasic pulses, (80/s, 3 ms); or rectangular, monophasic pulses (100/s, 5 ms), was unable to release GABA at stimulating potentials that are able to release known transmitter substances. Release of GABA and AIB was only seen with higher applied potentials, when also non-transmitter amino acids were released. It was also found that amino-oxyacetic acid(10^-5 M and 5 × 10^-5 M) increased the excitability of the slices, and allowed the release of both GABA and AIB to occur with weaker stimuli. This effect was independent of extracellular calcium.     

Influence of aminooxyacetic acid on the potassium-evoked release of [3H]γ-aminobutyric acid from slices of rat cerebral cortex

The release of [³H]GABA from superfused slices of rat cerebral cortex was investigated in the presence and absence of the GABA-transaminase inhibitor aminooxyacetic acid (AOAA). In the latter case, an ion-exchange column chromatographic technique was used to separate [³H]GABA from tritiated metabolites released with it into the superfusate. In the absence of AOAA, omission of Ca²⁺ from the superfusion medium reduced the release of [³H]GABA evoked by a 30 mM K⁺ pulse by 81.6%, whereas in comparable experiments carried out in the presence of AOAA omission of Ca²⁺ reduced the K⁺-evoked release by only 23.5%. Similar results were obtained when a 50 mM K⁺ pulse was used, where-upon omission of Ca²⁺ reduced [³H]GABA release by 78.7% in the absence of AOAA as compared with a reduction of only 47.9% when AOAA was present. It is concluded that the presence of AOAA decreases the Ca²⁺-dependence of K⁺-evoked [³H]GABA release in this system.     

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.     

Glutamic Acid and γ-Aminobutyric Acid Modulate Each Other's Release Through Heterocarriers Sited on the Axon Terminals of Rat Brain

Abstract: The effects of γ-aminobutyric acid (GABA) on the spontaneous release of endogenous glutamic acid (Glu) or aspartic acid (Asp) and the effects of Glu on the release of endogenous GABA or [³H]GABA were studied in superfused rat cerebral cortex synaptosomes. GABA increased the outflow of Glu (EC₅₀17.2 μM) and Asp (EC₅₀ 18.4 μM). GABA was not antagonized by bicuculline or picrotoxin. Neither muscimol nor (-)-baclofen mimicked GABA. The effects of GABA were prevented by GABA uptake inhibitors and were Na⁺ dependent. Glu enhanced the release of [³H]GABA (EC₅₀ 11.5 μM) from cortical synaptosomes. Glu was not mimicked by the glutamate receptor agonists N-methyl-d -aspartic, kainic, or quisqualic acid. The Glu effect was decreased by the Glu uptake inhibitor D-threo-hydroxyaspartic acid (THA) and it was Na⁺ sensitive. Similarly to Glu, D-Asp increased [³H]GABA release (EC₅₀ 9.9 μM), an effect blocked by THA. Glu also increased the release of endogenous GABA from cortex synaptosomes. In this case the effect was in part blocked by the (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist 6-cyano-7-nitroquinoxaiine-2, 3-dione, whereas the 6-cyano-7-nitroquinoxaline- 2, 3-dione-insensitive portion of the effect was prevented by THA. GABA increased the [³H]D-Asp outflow (EC₅₀ 13.7 μM) from hippocampal synaptosomes in a muscimol-, (-)- baclofen-, bicuculline-, and picrotoxin-insensitive manner. The GABA effect was abolished by blocking GABA uptake and was Na⁺ dependent. Glu increased the release of [³H]- GABA from hippocampal synaptosomes (EC₅₀ 7.1 μM) in an N-methyl-d -aspartic acid-, kainic acid-, or quisqualic acid-insensitive way. The effect of Glu was prevented by THA and was Na⁺ dependent. As in the cortex, the effect of Glu was mimicked by D-Asp in a THA-sensitive manner. It is proposed that high-affinity GABA or Glu heterocarriers are sited respectively on glutamatergic or GA- BAergic nerve terminals in rat cerebral cortex and hippocampus. The uptake of GABA may modulate Glu and Asp release, whereas the uptake of Glu may modulate the release of GABA. The existence of these heterocarriers is in keeping with the reported colocalization of GABA and Glu in some cortical and hippocampal neurons. Preliminary data suggest that these mechanisms may also be present in rat cerebellum and spinal cord.     

Spontaneous and depolarization-induced efflux of hypotaurine from mouse cerebral cortex slices: Comparison with taurine and gaba

The spontaneous and potassium- or veratrine-stimulated efflux of [³⁵S]hypotaurine from superfused cerebral cortex slices of adult mice was compared with the release of [³H]taurine and [³H]GABA. Initially GABA was the fastest released. Hypotaurine was, however, eventually released fastest, since its spontaneous efflux did not slow down during superfusions as did taurine and GABA effluxes. More than 60 % of all preloaded labelled amino acids still remained in the slices after 80-min superfusions. The effluxes of all three amino acids were stimulated by potassium and veratrine depolarizations: GABA efflux most and hypotaurine efflux least. The veratrine-stimulated release of taurine was long-lasting, while all other responses started and ended abruptly. With respect to efflux properties hypotaurine resembled more GABA than taurine.     

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.     

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

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

Effect of aminooxyacetic acid on the release of preloaded [3H]GABA and radioactive metabolites from slices of developing mouse brain

The release of [³H]-aminobutyric acid (GABA) and its radioactive metabolites from slices of the cerebral cortex, cerebellum, striatum and brain stem of developing and adult mice was studied. The slices were incubated and superfused in the absence and presence of the GABA aminotransferase (GABA-T) inhibitor aminooxyacetic acid (AOAA). Exposure to 100 M AOAA totally inhibited GABA-T and all radioactivity released from slices was in authentic GABA. In studies on developing brain the 10-M concentration was also effective enough, except in cerebellar slices. In the absence of AOAA the major part of radioactivity spontaneously released from slices of adult cerebral cortex and cerebellum was tritiated water and still about one third part in the presence of 10 M AOAA. Potassium stimulation induced only the release of radioactive GABA but not labeled metabolites in both presence and absence of AOAA. AOAA reduced the stimulation-induced release of GABA. It is recommended that the use of GABA-T inhibitors should be discontinued in release experiments. Then labeled GABA must be separated in the effluents from its radioactive breakdown products.     

Bidirectional Movement of γ-Aminobutyric Acid in Rat Spinal Cord Slices

Abstract: The bidirectional movement of GABA (γ-aminobutyric acid) was studied in slices of rat spinal cord which were incubated in small volumes of medium. The appearance in the medium of endogenous GABA and the disappearance from the medium of [¹⁴C]GABA were used to calculate the rates of unidirectional uptake and unidirectional release of GABA. Under these conditions, no net uptake of GABA was observed when slices were incubated in media containing concentrations of GABA as high as 25 μm . Elevated potassium (60 mm ) stimulated the unidirectional release of endogenous GABA from spinal cord slices by a calcium-dependent process. Ouabain (0.1 mm ) more than doubled the unidirectional release of endogenous GABA in a calcium-independent manner, while unidirectional uptake was inhibited by 44%. Nipecotic acid (1.0 mm ) stimulated unidirectional release and inhibited unidirectional uptake of GABA.     

RELATIONSHIP BETWEEN Ca2+-DEPENDENT AND INDEPENDENT RELEASE OF [3H]GABA EVOKED BY HIGH K+, VERATRIDINE OR ELECTRICAL STIMULATION FROM RAT CORTICAL SLICES

Abstract— It has been reported that the release of GABA by high K⁺ is Ca²⁺-dependent while release induced by veratridine or electrical stimulation has been frequently found to be independent of Ca²⁺. To see the source of Ca²⁺-dependent and independent release of GABA, cortical slices which had accumulated [³H]GABA were exposed to 50 mm -K⁺ or 50 μm -veratridine for 48min. In the presence of Ca²⁺ the 2 agents released approx the same amount of [³H]GABA but tetrodotoxin (TTX) abolished release induced only by veratridine, while omission of Ca²⁺ reduced release induced only by 50mm -K⁺. Pre-exposure of the slices for 48min to 50mm -K⁺ in the presence of Ca²⁺ reduced the second release by 50mm -K⁺ by 77% and that by veratridine by 74%, suggesting that in the presence of Ca²⁺ the 2 depolarizing agents release [³H]GABA from the same pool. Pre-exposure to 50mm -K⁺ in the absence of Ca²⁺ reduced the second release by 50mm -K⁺ or by veratridine only by 37 and 27% respectively, indicating that most of the reduction in release was the result of a depletion of releasable [³H]GABA stores. The second exposure to 50mm -K⁺ in the absence of Ca²⁺ reduced the evoked release further, while exposure to veratridine in the absence of Ca²⁺, after depletion of the stores, enhanced release 2.7 times. Electrical stimulation (64 Hz, 2 ms, 40 mA, alternating polarity) during 24min in the presence of Ca” caused an initial 5-fold increase in efflux, which declined subsequently. In the absence of Ca²⁺, instead of a rapid increase, a slow but smaller increase in the efflux of [³H]GABA was found. TTX almost completely abolished the electrically evoked increase in release. Pre-treatment with 50mm -K⁺ reduced the electrically evoked release by 94% but electrical stimulation in the absence of Ca²⁺ after depletion of releasable stores doubled this release. Results suggest that in the presence of Ca²⁺, high K⁺, veratridine and electrical stimulation release [³H]GABA from the same Ca²⁺-dependent store, but in the absence of Ca²⁺ veratridine and electrical stimulation enhance the release from a Ca²⁺-independent store, probably as a result of an increased influx of Na⁺.     

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.     

Characteristics of GABA Release in Mouse Brain Stem Slices under Normal and Ischemic Conditions

GABA is known to be the inhibitory neurotransmitter in the majority of brain stem nuclei. The release of GABA has been extensively studied both in vivo and in vitro in higher brain areas, whereas the mechanisms of release in the brain stem have not been systemically characterized. The properties of preloaded [³H]GABA were now investigated in mouse brain stem slices, using a superfusion system. The basal release was enhanced by K⁺ stimulation (50 mM K⁺) and under various cell-damaging conditions (ischemia, hypoglycemia, the presence of free radicals and metabolic poisons). No K⁺-stimulated release was discernible in the absence of Ca²⁺, indicating that the release was at least partly Ca²⁺-dependent. Moreover, the release was increased when Na⁺ or Cl⁻ was omitted from the superfusion medium. GABA and β-alanine stimulated the release, confirming the involvement of the reversed function of GABA transporters. Incubation of the slices with the anion channel inhibitors diisothiocyanostilbene and 4-acetamido-4′-isothiocyanostilbene-2,2′-disulphonate and with the Cl⁻ uptake inhibitor 9-anthracenecarboxylic acid also reduced GABA release, demonstrating that a part of it comprises leakage through anion channels. All these mechanisms were involved in the ischemia-induced GABA release, which was over 4-fold greater than the release in normoxia. Contrary to the other brain areas, GABA release in the brain stem was not affected by ionotropic glutamate receptors but may be modulated by metabotropic receptors. This ischemia-induced GABA release might constitute an important mechanism against excitotoxicity, protecting the brain stem under cell-damaging conditions. Special issue dedicated to Dr. Simo S. Oja     

UPTAKE AND RELEASE OF NIPECOTIC ACID BY RAT BRAIN SLICES

—Nipecotic acid, a potent inhibitor of GABA uptake, is taken up by slices of rat cerebral cortex by a sodium-dependent, ‘high affinity’ system (K_m 11 μM), and can be released from these slices by an increased potassium ion concentration in a calcium-dependent manner. Nipecotic acid and GABA appear to be taken up by the same osmotically-sensitive structures. GABA and substances which inhibit GABA uptake also inhibit the uptake of nipecotic acid. GABA can release preloaded nipecotic acid from brain slices, and nipecotic acid can release preloaded GABA. This indicates that GABA and nipecotic acid can be counter-transported using the same mobile carrier. Nipecotic acid appears to have a higher affinity than GABA for this carrier.     

Calcium-Dependent Release of Tyrosine in Brain Elicited by Stimulation of Neuropeptide Receptors

We sought to establish whether the endogenous opiate-receptor agonist Met-enkephalin (m-ENK) selectively modulates the release of endogenous tyrosine (Tyr) from brain slices prepared from the corpus striatum (CS). Amino acids (AAs) released from slices of CS and, for comparison, cerebral cortex (Cx) were measured by HPLC. Incubation of slices with m-ENK (1-10 microM) increased the basal release of Tyr (up to 293% of control) from CS, but not Cx, whereas other nonneurotransmitter AAs, phenylalanine (Phe) and valine (Val), were unchanged. The release of the putative neurotransmitter AAs glutamate (Glu), taurine (Tau), and glycine (Gly) were similarly increased by 50-150% with m-ENK in slices of CS, but not Cx. The enhanced release of AAs by m-ENK was prevented by removal of extracellular Ca2+ or by preincubation with the opiate receptor antagonist naloxone. Neuronal depolarization by potassium (5-55 mM) in the presence of Ca2+ did not affect the release of Tyr, whereas release of neurotransmitter AAs such as gamma-aminobutyric acid (GABA) were markedly increased. The increase in basal Tyr release by m-ENK was not the result of a decreased uptake of Tyr. Relative to slices, the basal release of Tyr, Phe, and Val from a synaptosomal (P2) preparation of CS was small (8-51%) compared to that of GABA, Gly, Glu, and Tau (49-123%). Nonetheless, m-ENK (10 microM) markedly increased the release of Tyr (to 833%), but not Glu, Gly, and Tau from the P2 fraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of pipecolic acid on the release and uptake of [3H]GABA from brain slices of mouse cerebral cortex

Recently, pipecolic acid (PA) has been involved in the functioning of the GABAergic system. In the present work we have studied the effect of PA on GABA uptake and release in cerebral cortex slices. PA (100 M) was able to increase the release of [³H]GABA (90%) stimulated by mild depolarization with 15 mM potassium. If during the labeling of the tissue with [³H]GABA, -alanine was present, PA also enhanced the release (42%). However, when nipecotic acid was present instead -alanine, no stimulation of [³H]GABA release by potassium was observed neither in the control nor in the presence of PA. Spontaneous release was not affected by PA in any of the experimental conditions tested. In uptake experiments, only when -alanine was present in the medium PA significantly diminished the uptake (36%) of [³H]GABA. These results suggest that the effect of PA is mostly at the presynaptic level, inhibiting the neuronal GABA uptake and/or enhancing its release.