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.     

Does extracellular calcium determine what pool of GABA is the target for alpha-latrotoxin?

Presynaptic neurotoxin alpha-latrotoxin, from the venom of Latrodectus mactans tredecimguttatus, causes massive [(3)H]GABA release from rat brain synaptosomes, irrespective of calcium presence in the extracellular medium. Whether the binding of alpha-latrotoxin to Ca(2+)-dependent (neurexin 1 alpha) or to Ca(2+)-independent (latrophilin) receptor triggers [(3)H]GABA release by the same mechanisms or different ones, inducing either exocytotic process or outflow by mobile membrane GABA transporter, is unknown. We examined alpha-latrotoxin-evoked [(3)H]GABA release from synaptosomes which cytosolic [(3)H]GABA pool was depleted either by applying competitive inhibitors of the GABA transporter, nipecotic acid and 2,4-diaminobutyric acid, or by permeation with digitonin. We also compared the effect of the GABA transporter inhibitors on depolarisation-evoked and alpha-latrotoxin-evoked [(3)H]GABA release using as depolarising agents 4-aminopyridine and high KCl in the Ca(2+)-containing and in Ca(2+)-free medium, respectively. Incubation of synaptosomes with nipecotic acid induced the essential acceleration of unstimulated [(3)H]GABA release and deep inhibition of high KCl-evoked Ca(2+)-independent [(3)H]GABA release. In contrast, at the similar conditions the effect of alpha-latrotoxin was greatly augmented with respect to the control response. Another way to assay what GABA pool was involved in alpha-latrotoxin-induced release lays in an analysis of the effects of depolarisation and alpha-latrotoxin in consecutive order. The preliminary 4-aminopyridine-stimulated [(3)H]GABA release attenuated the toxin effect. But when depolarisation occurred in Ca(2+)-free medium, no influence on alpha-latrotoxin effect was revealed. Employing digitonin-permeated synaptosomes, we have shown that alpha-latrotoxin could stimulate [3H]GABA release in the medium with 1mM EGTA, this effect of the toxin was blocked by concanavalin A and was ATP-dependent. The latter suggests that alpha-latrotoxin-released neurotransmitter has the vesicular nature. We assume that the type of the toxin membrane receptor does not determine the mechanisms of [(3)H]GABA release evoked by alpha-latrotoxin.     

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

The ability of gamma-aminobutyric acid (GABA) and glycine (Gly) to modulate each other's release was studied in synaptosomes from rat spinal cord, cerebellum, cerebral cortex, or hippocampus, prelabeled with [3H]GABA or [3H]Gly and exposed in superfusion to Gly or to GABA, respectively. GABA increased the spontaneous outflow of [3H]Gly (EC50, 20.8 microM) from spinal cord synaptosomes. Neither muscimol nor (-)-baclofen, up to 300 microM, mimicked the effect of GABA, which was not antagonized by either bicuculline or picrotoxin. However, the effect of GABA was counteracted by the GABA uptake inhibitors nipecotic acid and N-(4,4-diphenyl-3-butenyl)nipecotic acid. Moreover, the GABA-induced [3H]Gly release was Na+ dependent and disappeared when the medium contained 23 mM Na+. The effect of GABA was Ca2+ independent and tetrodotoxin insensitive. Conversely, Gly enhanced the outflow of [3H]GABA from rat spinal cord synaptosomes (EC50, 100.9 microM). This effect was insensitive to both strychnine and 7-chlorokynurenic acid, antagonists at Gly receptors, but it was strongly Na+ dependent. Also, the Gly-evoked [3H]GABA release was Ca2+ independent and tetrodotoxin insensitive. GABA increased the outflow of [3H]Gly (EC50, 11.1 microM) from cerebellar synaptosomes; the effect was not mimicked by either muscimol or (-)-baclofen nor was it prevented by bicuculline or picrotoxin. The GABA effect was, however, blocked by GABA uptake inhibitors and was Na+ dependent. Gly increased [3H]GABA release from cerebellar synaptosomes (EC50, 110.7 microM) in a strychnine- and 7-chlorokynurenic acid-insensitive manner. This effect was Na+ dependent. The effects of GABA on [3H]Gly release seen in spinal cord and cerebellum could be reproduced also with cerebrocortical synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serotonin-1B receptor-mediated inhibition of [3H]GABA release from rat ventral tegmental area slices

In order to assess a role of 5-HT(1B) receptors for regulation of GABA transmission in the ventral tegmental area (VTA), VTA slices from the rat were incubated with [(3)H]GABA and beta-alanine, and superfused in the presence of nipecotic acid and aminooxyacetic acid. [(3)H]GABA release was induced by exposures to the medium containing 30 mM potassium for 2 min. The results showed that high potassium-evoked [(3)H]GABA release was sensitive to calcium withdrawal or blockade of sodium channels by tetrodotoxin, suggesting that tritium overflow induced by high potassium derived largely from neuronal stores. Administration of CP 93129 (0.15 and 0.45 microM), a 5-HT(1B) receptor agonist, or RU 24969 (0.15 and 0.45 microM), a 5-HT(1B/1A) receptor agonist, but not 8-OH-DPAT (0.45 microM), a 5-HT(1A) receptor agonist, inhibited high potassium-evoked [(3)H]GABA release in a concentration-related manner. The RU 24969-induced inhibition of [(3)H]GABA release was antagonized by either SB 216641, a 5-H(1B) receptor antagonist, or cyanopindolol, a 5-HT(1B/1A) receptor antagonist, but not by WAY 100635, a 5-HT(1A) receptor antagonist. Pre-treatment with SB 216641 also antagonized CP 93129-induced inhibition of [(3)H]GABA release. The results support the hypothesis that 5-HT(1B) receptors within the VTA can function as heteroreceptors to inhibit GABA release.     

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)     

Pharmacological heterogeneity of release-regulating presynaptic AMPA/kainate receptors in the rat brain: study with receptor antagonists

Presynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainate receptors mediating hippocampal [(3)H]noradrenaline or [(3)H]serotonin release, striatal [(3)H]dopamine release and cortical [(3)H]acetylcholine release were pharmacologically characterized using several AMPA/kainate receptor antagonists. The releases of the four transmitters elicited by exposing synaptosomes to AMPA were antagonized by NBQX, indicating that they reflect AMPA/kainate receptor activation. GYKI52466 did not inhibit the AMPA-induced release of [(3)H]noradrenaline, [(3)H]dopamine or [(3)H]serotonin, while it weakly affected the AMPA-mediated release of [(3)H]acetylcholine. On the contrary, LY300164 and LY303070 were potent antagonists able to discriminate among AMPA/kainate receptor subtypes. Both compounds blocked the AMPA receptors mediating [(3)H]dopamine and [(3)H]acetylcholine release. However, LY303070, but not LY300164, inhibited the AMPA-induced release of [(3)H]noradrenaline, while the AMPA-mediated [(3)H]serotonin release was sensitive to LY300164 but not to LY303070. SYM2206 mimicked LY300164 and prevented the AMPA-induced release of [(3)H]dopamine, [(3)H]acetylcholine and [(3)H]serotonin, but not that of [(3)H]noradrenaline. NS102 failed to antagonize the AMPA-induced release of all four transmitters. LY293558 prevented the AMPA-mediated release of [(3)H]noradrenaline, [(3)H]dopamine, [(3)H]acetylcholine or [(3)H]serotonin. Differently, LY377770 did not inhibit the AMPA-mediated release of [(3)H]noradrenaline and [(3)H]acetylcholine, but it potently blocked the AMPA-induced release of [(3)H]serotonin and, less so, of [(3)H]dopamine. AMOA inhibited the AMPA-induced release of [(3)H]serotonin or [(3)H]acetylcholine, but not that of [(3)H]noradrenaline or [(3)H]dopamine. GAMS prevented the AMPA-mediated release of [(3)H]acetylcholine and, more weakly, that of [(3)H]dopamine, but it failed to inhibit the release of [(3)H]noradrenaline or [(3)H]serotonin elicited by AMPA. gamma-DGG did not affect the AMPA-mediated release of any of the four transmitters studied. In conclusion, based on the antagonist profiles obtained, the four receptors here analyzed all belong to the AMPA-preferring subclass of glutamate receptors; however, they appear to differ from each other, probably due to differences in subunit composition. The compounds LY300164, LY303070, LY377770, AMOA and GAMS may be useful to discriminate among AMPA-preferring receptor subtypes.     

Implication of ionotropic glutamate receptors in the release of noradrenaline in hippocampal CA1 and CA3 subregions under oxygen and glucose deprivation

A strong linkage between adrenergic and glutamatergic systems exists in the CNS but it is still unclear whether the excessive release of noradrenaline under ischemic conditions is modulated by excitatory amino acids. We studied the effect of selective glutamate receptor antagonists on the release of [3H]noradrenaline evoked by glucose and oxygen deprivation in hippocampal CA1, CA3 and dentate gyrus subregions. The release of glutamate, aspartate and GABA was measured by HPLC. Omission of oxygen and glucose increased the release of [3H]noradrenaline as well as the release of amino acids. Maximum effect on noradrenaline release was observed in CA1 region. The relative increase of the release after 30 min energy deprivation (R(2)) versus the basal release under normal conditions (R(1)), i.e. the R(2)/R(1) ratio was 7.1+/-1.0, 3.87+/-0.4 and 3.26+/-0.27 for CA1, CA3 and dentate gyrus, respectively. The [3H]noradrenaline outflow in response to glucose and oxygen deprivation was abolished at low temperature, but not by Ca(2+) removal, suggesting a cytoplasmic release process. In CA1 and CA3 [3H]noradrenaline release was significantly attenuated by MK-801, an NMDA receptor antagonist. The AMPA receptor antagonist GYKI-53784 had no effect in CA3, but partly reduced noradrenaline release in CA1.Our results suggest that ionotropic glutamate receptors seem to be implicated in the massive cytoplasmic release of noradrenaline in CA1 what may contribute to its selective vulnerability.     

N-methyl-D-aspartate receptors mediating hippocampal noradrenaline and striatal dopamine release display differential sensitivity to quinolinic acid, the HIV-1 envelope protein gp120, external pH and protein kinase C inhibition

NMDA receptors regulating hippocampal noradrenaline (NA) and striatal dopamine (DA) release have been compared using superfused synaptosomes prelabelled with the [(3)H]catecholamines. Both receptors mediated release augmentation when exposed to NMDA plus glycine. Quinolinic acid (100 microM or 1 mM) plus glycine (1 microM)-elicited [(3)H]NA, but not [(3)H]DA release. The NMDA (100 microM)-evoked release of [(3)H]NA and [(3)H]DA was similar and concentration-dependently enhanced by glycine or D-serine (0.1-1 microM); in contrast, the HIV-1 envelope protein gp120 potently (30-100 pM) enhanced the NMDA-evoked release of [(3)H]NA, but not that of [(3)H]DA. Gp120 also potentiated quinolinate-evoked [(3)H]NA release. Ifenprodil (0.1-0.5 microM) or CP-101,606 (0.1-10 microM) inhibited the NMDA plus glycine-evoked release of both [(3)H]catecholamines. Zinc (0.1-1 microM) was ineffective. Lowering external pH from 7.4 to 6.6 strongly inhibited the release of [(3)H]NA elicited by NMDA plus glycine, whereas the release of [(3)H]DA was unaffected. The protein kinase C inhibitors GF 109203X (0.1 microM) or H7 (10 microM) selectively prevented the effect of NMDA plus glycine on the release of [(3)H]NA. GF 109203X also blocked the release of [(3)H]NA induced by NMDA or quinolinate plus gp120. It is concluded that the hippocampal NMDA receptor and the striatal NMDA receptor are pharmacologically distinct native subtypes, possibly containing NR2B subunits but different splice variants of the NR1 subunit.     

Calcium-Independent γ-Aminobutyric Acid Release from Growth Cones: Role of γ-Aminobutyric Acid Transport

Neuronal growth cones isolated in bulk from neonatal rat forebrain have uptake and K(+)-stimulated release mechanisms for gamma-aminobutyric acid (GABA). Up to and including postnatal day 5, the K(+)-stimulated release of [3H]GABA and endogenous GABA is Ca2+ independent. At these ages, isolated growth cones neither contain synaptic vesicles nor stain for synaptic vesicle antigens. Here we examined the possibility that the release mechanism underlying Ca2(+)-independent GABA release from isolated growth cones is by reversal of the plasma membrane GABA transporter. The effects of two GABA transporter inhibitors, nipecotic acid and an analogue of nipecotic acid, SKF 89976-A, on K(+)-stimulated release of [3H]GABA from superfused growth cones were examined. Nipecotic acid both stimulated basal [3H]GABA release and enhanced K(+)-stimulated release of [3H]GABA, which indicates that this agent can stimulate GABA release and is, therefore, not a useful inhibitor with which to test the role of the GABA transporter in K(+)-stimulated GABA release from growth cones. In contrast, SKF 89976-A profoundly depressed both basal and K(+)-stimulated [3H]GABA release. This occurred at similar concentrations at which uptake was blocked. These observations provide evidence for a major role of the GABA transporter in GABA release from neuronal growth cones.     

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.     

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.     

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.     

Involvement of P2X7 receptors in the regulation of neurotransmitter release in the rat hippocampus

Although originally cloned from rat brain, the P2X7 receptor has only recently been localized in neurones, and functional responses mediated by these neuronal P2X7 receptors (P2X7 R) are largely unknown. Here we studied the effect of P2X7 R activation on the release of neurotransmitters from superfused rat hippocampal slices. ATP (1-30 mm) and other ATP analogues elicited concentration-dependent [3 H]GABA outflow, with the following rank order of potency: benzoylbenzoylATP (BzATP) > ATP > ADP. PPADS, the non-selective P2-receptor antagonist (3-30 microm), Brilliant blue G (1-100 nm) the P2X7 -selective antagonist and Zn2+ (0.1-30 microm) inhibited, whereas lack of Mg2+ potentiated the response by ATP. In situ hybridization revealed that P2X7 R mRNA is expressed in the neurones of the cell body layers in the hippocampus. P2X7 R immunoreactivity was found in excitatory synaptic terminals in CA1 and CA3 region targeting the dendrites of pyramidal cells and parvalbumin labelled structures. ATP (3-30 microm) and BzATP (0.6-6 microm) elicited concentration-dependent [14 C]glutamate efflux, and blockade of the kainate receptor-mediated transmission by CNQX (10-100 microm) and gadolinium (100 microm), decreased ATP evoked [3 H]GABA efflux. The Na+ channel blocker TTX (1 microm), low temperature (12 degrees C), and the GABA uptake blocker nipecotic acid (1 mm) prevented ATP-induced [3 H]GABA efflux. Brilliant blue G and PPADS also reduced electrical field stimulation-induced [3 H]GABA efflux. In conclusion, P2X7 Rs are localized to the excitatory terminals in the hippocampus, and their activation regulates the release of glutamate and GABA from themselves and from their target cells.     

NMDA and Non-NMDA Receptor-Mediated Release of [3H]GABA from Granule Cell Dendrites of Rat Olfactory Bulb

Abstract: We have studied the effect of glutamate and the glutamatergic agonists N-methyl-d -aspartate (NMDA), kainate, and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) on [³H]GABA release from the external plexiform layer of the olfactory bulb. The GABA uptake blocker nipecotic acid significantly increased the basal [³H]GABA release and the release evoked by a high K⁺ concentration, glutamate, and kainate. The glutamate uptake blocker pyrrolidine-2,4-dicarboxylate (2,4-PDC) inhibited by 50% the glutamate-induced [³H]GABA release with no change in the basal GABA release. The glutamatergic agonists NMDA, kainate, and AMPA also induced a significant [³H]GABA release. The presence of glycine and the absence of Mg²⁺ have no potentiating effect on NMDA-stimulated release; however, when the tissue was previously depolarized with a high K⁺ concentration, a significant increase in the NMDA response was observed that was potentiated by glycine and inhibited by the NMDA receptor antagonist 2-amino-5-phosphonoheptanoic acid (AP-7). The kainate and AMPA effects were antagonized by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) but not by AP-7. The glutamate effect was also inhibited by CNQX but not by the NMDA antagonist 2-amino-5-phosphonopentanoic acid (AP-5); nevertheless, in the presence of glycine, [³H]GABA release evoked by glutamate was potentiated, and this response was significantly antagonized by AP-5. Tetrodotoxin inhibited glutamate- and kainate-stimulated [³H]GABA release but not the NMDA-stimulated release. The present results show that in the external plexiform layer of the olfactory bulb, glutamate is stimulating GABA release through a presynaptic, receptor-mediated mechanism as a mixed agonist on NMDA and non-NMDA receptors; glutamate is apparently also able to induce GABA release through heteroexchange.     

Presynaptic Mechanisms Underlying the γ-Aminobutyric Acid-Evoked Receptor-Independent Release of [3H]Norepinephrine in Rat Hippocampus

The effects of gamma-aminobutyric acid (GABA) on the spontaneous efflux of [3H]norepinephrine ([3H]NE) were studied in synaptosomes prepared from rat hippocampus and prelabelled with [3H]NE. It had been observed previously that, when synaptosomes were exposed in superfusion to GABA, the basal release of the tritiated catecholamine was enhanced, apparently with no involvement of the known GABA receptors. The mechanisms underlying this effect have now been investigated. The potency of GABA as a releaser of [3H]NE was decreased by lowering the Na+ content of the superfusion medium, and its effect disappeared at 23 mM Na+. The GABA-induced [3H]NE release was counteracted by the GABA uptake inhibitor N-(4,4-diphenyl-3-butenyl)nipecotic acid (SKF 89976A), but it was unaffected by the NE uptake blockers desmethylimipramine and nisoxetine. The GABA-induced release of [3H]NE was Ca2+-dependent and tetrodotoxin-sensitive. The data support the hypothesis that GABA provoked [3H]NE release by a novel mechanism which involves penetration into the noradrenergic nerve terminals through a GABA carrier located on the NE terminals themselves. This uptake process might be electrogenic and provoke depolarization of the nerve terminals, causing an exocytotic release of [3H]NE.     

Release of Purines, Noradrenaline, and GABA from Rat Hippocampal Slices by Field Stimulation

Labelled adenine, noradrenaline (NA), and gamma-aminobutyric acid (GABA) were taken up by the transversely cut hippocampal slice. [3H]NA and [14C]GABA were retained as such, [3H]- (or [14C]-) adenine mainly as adenine nucleotides. There was a spontaneous overflow of all three types of compounds ranging from 0.1 (GABA) to 0.21 (NA) %/min. The rate of [3H]NA overflow increased rapidly during electrical field stimulation. The release rate was well maintained over a 15-min period. The rate of [14C]GABA release also increased rapidly but it was not maintained over a 15-min period even if uptake and/or metabolism was inhibited by nipecotic acid (1 mM) and aminooxyacetic acid (AOAA, 0.1 mM). The bulk of the purines was released after the stimulation period. For all compounds the amounts released were frequency- and calcium-dependent. At a frequency of 3 Hz a 10 V stimulation was sufficient to cause a maximal [3H]NA release and 20 V to cause maximal [14C]GABA release, but 14C-purine release was increased further by increasing the voltage to 40 V. The evoked purine release was inhibited by a nucleoside uptake inhibitor (dipyridamole). On stimulation of [3H]NA-labelled slices the released radioactivity was composed of greater than 95% unchanged NA. The specific activities of NA in the slice and in the superfusate were practically identical. In [3H]adenine-labelled slices the released radioactivity was composed of adenosine, inosine, and hypoxanthine, but the activity in the slice of ATP, ADP, and AMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional significance of nitric oxide in ionomycin-evoked [3H]GABA release from mouse cerebral cortical neurons

We investigated a role of nitric oxide (NO) on ionomycin-evoked [3H]GABA release using mouse cerebral cortical neurons. lonomycin dose-dependently released [3H]GABA up to 1 microM. The extent of the release by 0.1 microM ionomycin was in a range similar to that by 30 mM KCl. The ionomycin (0.1 microM)-evoked [3H]GABA release was dose-dependently inhibited by NO synthase inhibitors and hemoglobin, indicating that the ionomycin-evoked [3H]GABA release is mediated through NO formation. The inhibition of cGMP formation by 1H-[1,2,4] oxodizao [4,3-a] quinoxalin-1-one (ODQ), a selective inhibitor for NO-sensitive guanylate cyclase, showed no affects on the ionomycin-evoked [3H]GABA release. Tetrodotoxin and dibucaine significantly suppressed the ionomycin-evoked [3H]GABA release and ionomycin increased fluorescence intensity of bis-oxonol, suggesting the involvement of membrane depolarization in this release. The ionomycin-evoked [3H]GABA release was maximally reduced by about 50% by GABA uptake inhibitors. The concomitant presence of nifedipine and omega-agatoxin VIA (omega-ATX), inhibitors for L- and P/Q-type voltage-dependent calcium channels, respectively, caused the reduction in the ionomycin-evoked release by about 50%. The simultaneous addition of nifedipine, omega-ATX and nipecotic acid completely abolished the release. Although ionomycin released glutamate, (+)-5-methyl-1-,11-dihydro-5H-dibenzo-[a,d]cycloheptan-5,10-imine (MK-801) and 6,7-dinitroquinoxaline-2,3-dione (DNQX) showed no effects on the ionomycin-induced [3H]GABA release. Based on these results, it is concluded that NO formed by ionomycin plays a critical role in ionomycin-evoked [3H]GABA release from the neurons.     

Phenylarsine oxide inhibits alpha-latrotoxin-stimulated [3H]GABA release from rat brain synaptosomes

Phosphatidylinositol 4,5-biphosphate has been implicated in a variety of membrane-trafficking processes, including exocytosis of neurotransmitters. However, there are contradictory findings concerned ability of phenylarsine oxide (PAO), an inhibitor of phosphatidylinositol 4-kinase, to affect exocytotic release of different types of neurotransmitters. We bent our efforts to a detailed analysis of action of PAO on Ca(2+)-dependent and Ca(2+)-independent [3H]GABA release produced by exposure of rat brain synaptosomes to different concentrations of alpha-latrotoxin. We also compared PAO action on alpha-latrotoxin- and 4-aminopyridine (4-AP)-evoked [3H]GABA release. The experiments have shown that release of [3H]GABA evoked by the depolarization with 4-AP was decreased by 80% as a result of action of 3 microM PAO and the complete inhibition of release was observed with 10 microM PAO. When alpha-latrotoxin as a stimulant was applied, release of [3H]GABA was increased as toxin concentration used was elevated from 0.5 to 3.0 nM, however, concomitantly, the response of the toxin-induced [3H]GABA release to PAO became attenuated: 10 microM PAO led to almost complete inhibition of the effect of 0.5 nM alpha-latrotoxin and only partly decreased (by 40%) the response to 3.0 nM alpha-latrotoxin. To test whether the efficacy of PAO depended on the toxin-induced outflow of cytosolic [3H]GABA, synaptosomes with depleted cytosolic [3H]GABA pool were also exploited. Depletion was performed by means of heteroexchange of cytosolic [3H]GABA with nipecotic acid. The experiments have shown that treatment of loaded synaptosomes with nipecotic acid resulted in some increase of [3H]GABA release evoked by 0.5 nM alpha-latrotoxin, but in the two-fold decrease of the response to 3.0 nM alpha-latrotoxin. PAO essentially inhibited [3H]GABA release from depleted synaptosomes irrespective of alpha-latrotoxin concentration used. Therefore, the amount of [3H]GABA released from cytosolic pool determined, in considerable degree, the insensitivity of alpha-latrotoxin action to PAO. Thus, our data show that subnanomolar concentrations of alpha-latrotoxin may be used for stimulation of exocytotic release of [3H]GABA. Exposure of synaptosomes with nanomolar toxin concentrations leads not only to stimulation of exocytosis, but also to leakage of [3H]GABA from cytosolic pool. PAO potently inhibits exocytotic release of [3H]GABA and its inhibitory effectiveness is diminished as far as the outflow of [3H]GABA is elevated.     

Involvement of membrane GABA transporter in alpha-latrotoxin-stimulated [3H]GABA release

Alpha-latrotoxin evokes massive [3H]GABA release from rat brain synaptosomes by stimulating exocytosis and outflow from non-vesicular pool. In the present study, GABA transporter-mediated [3H]GABA release was shown to be involved in alpha-latrotoxin-triggered release of [3H]GABA from non-vesicular pool. The following agents have been exploited as tools: (1) a protonophore carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazon (FCCP) and bafilomycin A1 for evoking depletion of synaptic vesicle [3H]GABA and enlargement of non-vesicular pool; (2) a non-substrate high-affinity GABA transport blocker NO-711 for determining participation of GABA carrier in the toxin-stimulated GABA release; (3) a competitive inhibitor of GABA reuptake nipecotic acid for heteroexchange [3H]GABA release. As shown by the experiments with nipecotic acid, FCCP and bafilomycin A1 considerably increase the content of non-vesicular [3H]GABA. The treatment of the synaptosomes with these agents modified the response to alpha-latrotoxin, particularly to its subnanomolar concentrations: the lack or substantial lowering of the toxin-evoked release during the first 2 min after the toxin addition and substantial enhancement of release up to the 5th minute were observed. Only the step of enhanced release was sensitive to GABA transporter blocker NO-711. Distinct sensitivity to NO-711 was shown to be characteristic for different steps of alpha-latrotoxin-stimulated [3H]GABA release from the control, untreated synaptosomes: lack of any effect of NO-711 during the first 2 min and powerful inhibition in 10 min after the toxin application. Taken together these data appear to indicate that the toxin non-simultaneously from vesicular and non-vesicular origins releases the neurotransmitter, the first rapid step reflects exocytosis stimulation, and the second tardy step is at least in part due to the release mediated by GABA transporters. The incomplete inhibition with NO-711 of the tardy step of the release evoked by nanomolar toxin concentrations suggests the participation not only of the GABA transporters.     

Stress-Induced Enhancement of Suppression of [3H]GABA Release from Striatal Slices by Presynaptic Autoreceptor

The effect of cold and immobilization stress on presynaptic GABAergic autoreceptors was examined using the release of [3H]GABA (gamma-aminobutyric acid) from slices of rat striatum. It was found that in vitro addition of delta-aminolevulinic acid, as well as GABA agonists such as muscimol and imidazoleacetic acid, exhibited a significant suppression of the striatal release of [3H]GABA evoked by the addition of high potassium, whereas delta-aminovaleric acid had no significant effects on the evoked release. These suppressive actions were antagonized invariably by the GABA antagonists, bicuculline and picrotoxin, but not by the glycine antagonist, strychnine. Cholinergic agonists, such as pilocarpine and tetramethylammonium, also attenuated significantly the evoked release of [3H]GABA from striatal slices, while none of its antagonists, including atropine, hexamethonium and d-tubocurarine, affected the release. On the other hand, in vitro addition of dopamine receptor agents such as dopamine, apomorphine, and haloperidol, or the inhibitory amino acids, glycine, beta-alanine, and taurine failed to influence the evoked release of [3H]GABA from striatal slices. Application of a cold and immobilization stress for 3 h was found to induce a significant enhancement of the suppressive effects by muscimol and delta-aminolevulinic acid on the evoked release of [3H]GABA, without affecting that by pilocarpine and tetramethylammonium. These results suggest that the release of GABA from striatal GABA neurons may be regulated by presynaptic autoreceptors for this neuroactive amino acid, and may play a significant functional role in the exhibition of various symptoms induced by stress.