Excitatory amino acid-evoked release of [H]GABA from hippocampal neurons in primary culture

The novel glutamate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) inhibited glutamate stimulated [³H]GABA release from cortical neurons in vitro. Kainate-induced release was blocked in a competitive fashion butN-methyl-d-aspartate (NMDA)-induced release was blocked non-competitively by CNQX. 7-Chlorokynurenate (7-CK) also inhibited NMDA evoked [³H]GABA release non-competitively, but had no effect on kainate induced release. The effects of both CNQX and 7-CK on NMDA-induced release were reversed by addition of exogenous glycine but the effects of CNQX on kainate-induced release were not altered by glycine. This suggests that both CNQX and 7-CK may interact with the glycine regulatory site of the NMDA receptor.     

Ca-dependent release of [H]GABA in cultured chick retina cells

Depolarization by K+ (50 mM) of cultured chick retina cells released 1.14 +/- 0.28% of the accumulated [3H] gamma-aminobutyric acid (GABA) in the absence of Ca2+, but when 1.0 mM Ca2+ was present, the internal free calcium ion concentration [Ca2+]i rose by about 750 nM and the [3H]GABA release about doubled to a value of 2.22 +/- 0.2% of the total [3H]GABA. Nitrendipine (0.1 microM), a blocker of the L-type Ca2+ channels, blocked the [Ca2+]i response to K+ depolarization by about 65%, and the omega-Conotoxin GVIA (omega-CgTx) (0.5 microM), a blocker of the N-type of Ca2+ channels, inhibited by 27% the [Ca2+]i rise due to K+ depolarization. Parallel experiments showed that nitrendipine inhibits [3H]GABA release to the level observed in the absence of Ca2+, whereas omega-CgTx did not inhibit significantly the release of [3H]GABA. The results also show that the release of [3H]GABA due to K(+)-depolarization in the absence of Ca2+ can be totally blocked by 1-(2-(((Diphenylmethylene) amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid hydrochloride (NNC-711), an inhibitor of the GABA carrier. However, in the presence of Ca2+, NNC-711 blocks the release only by about 66%, corresponding to the Ca(2+)-independent release. Thus, it is concluded that [3H]GABA is released in chick retina cells by the exocytotic mechanism, which is Ca(2+)-dependent, and by reversal of the carrier, which is Ca(2+)-independent, in much the same way as has been found for other GABAergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of oxidative stress on the release of [H]GABA in cultured chick retina cells

The effect of ascorbate (1.5 mM)/Fe²⁺ (7.5 μM)-induced oxidative stress on the release of pre-accumulated [³H]γ-aminobutyric acid ([³H]GABA) from cultured chick retina cells was studied. Depolarization of control cells with 50 mM K⁺ increased the release of [³H]GABA by 1.01 ± 0.16% and 2.5 ± 0.3% of the total, in the absence and in the presence of Ca²⁺, respectively. Lipid peroxidation increased the release of [³H]GABA to 2.07 ± 0.31% and 3.6 ± 0.39% of the total, in Ca²⁺-free or in Ca²⁺-containing media, respectively. The inhibitor of the GABA carrier, 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid hydrochloride (NNC-711) blocked almost completely the release of [³H]GABA due to K⁺-depolarization in the absence of Ca²⁺, but only 65% of the release occurring in the presence of Ca²⁺ in control and peroxidized cells. Under oxidative stress retina cells release more [³H]GABA than control cells, being the Ca²⁺-independent mechanism, mediated by the reversal of the Na⁺/GABA carrier, the most affected. MK-801 (1 μM), a non-competitive antagonist of the NMDA receptor-channel complex, blocked by 80% the release of [³H]GABA in peroxidized cells, whereas in control cells the inhibitory effect was of 40%. The non-selective blocker of the non-NMDA glutamate receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), inhibited the release of [³H]GABA by 30% and 70% in control and peroxidized cells, respectively. Glycine (5 μM) stimulated [³H]GABA release evoked by 50 mM K⁺-depolarization in control but not in peroxidized cells. The release of -[³H]aspartate (a non-metabolized analog of -glutamate) evoked by 50 mM K⁺, in the absence of Ca²⁺, was significantly higher in peroxidized cells (6.76 ± 0.64% of the total) than in control cells (3.79 ± 0.27% of the total). The results suggest that oxidative stress induced by ascorbate/Fe²⁺ causes an excessive release of endogenous excitatory amino acids upon K⁺-depolarization. The glutamate released may activate NMDA and non-NMDA receptors, raising the intracellular Na⁺ concentration and consequently stimulating the release of [³H]GABA by reversal of the Na⁺/GABA carrier.     

Muscarinic receptors and second-messenger responses of neurons in primary culture

The coupling of muscarinic receptors to second messenger responses was investigated in primary cultures of neurons from the fetal mouse brain. Neurons were maintained in monolayer culture, in serum-free medium; immunocytochemical studies found these cultures to be nearly exclusively neuronal. In striatal cultures, [3H]N-methylscopolamine (NMS) bound specifically and with high affinity (Kd = 70 pM) to a homogeneous population of receptors on intact neurons (320 fmol/mg cellular protein). Displacement of the binding of [3H]NMS by pirenzepine indicated the presence of heterogeneous sites (81% high affinity sites, Kh = 51 nM, K1 = 1.5 microM); AF-DX 116 showed the opposite selectivity (15% high affinity sites, Kh = 56 nM, K1 = 1.3 microM). The dopamine agonist SKF-38393 (1 microM) enhanced the accumulation of cyclic adenosine monophosphate (AMP) in these cultures 2.5-fold; addition of carbachol reduced cyclic AMP levels by 30% (EC50, 1.7 microM). In the presence of 1 mM lithium, carbachol stimulated the accumulation of inositol monophosphate 5-fold (EC50, 61 microM). Both responses were antagonized by pirenzepine (apparent Ki of 23 nM for the phosphoinositide response and 200 nM for the cyclic AMP response) and AF-DX 116 (apparent Ki 540 nM and 160 nM, respectively). In binding studies on brainstem cultures, AF-DX 116 indicated the presence of two sites of approximately equal abundance (Kh = 170 nM, K1 = 2.9 microM); data for pirenzepine were adequately fit by a one-site model (Kd = 630 nM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Glutamate increases the [Ca]i but stimulates Ca-independent release of [H]GABA in cultured chick retina cells

The effect of glutamate of [Ca²⁺]_i and on [³H]γ-aminobutyric acid (GABA) release was studied on cultured chick embryonic retina cells. It was observed that glutamate (100 μM) increases the [Ca²⁺]_i by Ca²⁺ influx through Ca²⁺ channels sensitive to nitrendipine, but not to ω-conotoxin GVIA (ω-Cg Tx) (50%), and by other channels insensitive to either Ca²⁺ channel blocker. Mobilization of Ca²⁺ by glutamate required the presence of external Na⁺, suggesting that Na⁺ mobilization through the ionotropic glutamate receptors is necessary for the Ca²⁺ channels to open. The increase in [Ca²⁺]_i was not related to the release of [³H]GABA induced by glutamate, suggesting that the pathway for the entry of Ca²⁺ triggered by glutamate does not lead to exocytosis. In fact, the glutamate-induced release of [³H]GABA was significantly depressed by Ca_o²⁺, but it was dependent on Na_o⁺, just as was observed for the [³H]GABA release induced by veratridine (50 μM). The veratridine-induced release could be fully inhibited by TTX, but this toxin had no effect on the glutamate-induced [³H]GABA release. Both veratridine- and glutamate-induced [³H]GABA release were inhibited by 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid (NNC-711), a blocker of the GABA carrier. Blockade of the NMDA and non-NMDA glutamate receptors with MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively, almost completely blocked the release of [³H]GABA evoked by glutamate. Continuous depolarization with 50 mM K⁺ induced maximal release of [³H]GABA of about 1.5%, which is much smaller than the release evoked by glutamate under the same conditions (6.0–6.5%). Glycine (3 μM) stimulated [³H]GABA release induced by 50 mM K⁺, and this effect was blocked by MK-801, suggesting that the effect of K⁺ on [³H]GABA release was partially mediated through the NMDA receptor which probably was stimulated by glutamate released by K⁺ depolarization. We conclude that glutamate induces Ca²⁺-independent release of [³H]GABA through reversal of the GABA carrier due to Na⁺ entry through the NMDA and non-NMDA, TTX-insensitive, channels. Furthermore the GABA carrier seems to be inhibited by Ca²⁺ entering by the pathways open by glutamate. This Ca²⁺ does not lead to exocytosis, probably because the Ca²⁺ channels used are located at sites far from the active zones.     

α2-Adrenergic receptors mediate inhibition of cyclic AMP production in neurons in primary culture

The actions of adrenergic agents on the intracellular production of cyclic adenosine monophosphate (AMP) was examined in intact cortical and striatal neurons in primary culture, generated from the fetal mouse brain. Exposure of striatal neurons to the β-adrenergic agonist isoproterenol (10 μM) resulted in a 5-fold increase in intraneuronal cyclic AMP; norepinephrine (100 μM), alone or in combination with isoproterenol, produced only a 3-fold increase in cyclic AMP levels. However, in the presence of yohimbine (10 μM), cyclic AMP productions due to norepinephrine or isoproterenol plus norepinephrine were identical to isoproterenol alone. When striatal or cortical neurons were exposed to pertussis toxin (100 ng/ml) overnight, there was no detectable difference between isoproterenol- and norepinephrine-stimulated cyclic AMP production. These data suggest thatα₂-adrenergic receptors mediate the attenuation of cyclic AMP production in neurons and do so via the inhibitory guanine nucleotide regulatory protein of adenylate cyclase.     

Regulation of NMDA-stimulated [C]GABA and [H]acetylcholine release by striatal glutamate and dopamine receptors

Striatal function is heavily influenced by glutamatergic and dopaminergic afferent input. To ultimately better understand how the N-methyl- -aspartate (NMDA) antagonist, phencyclidine (PCP), alters striatal function, we sought to determine how NMDA receptor function is influenced by activation of other glutamatergic receptors and by dopaminergic receptors. To this end, we used NMDA-stimulated efflux of [¹⁴C]GABA and [³H]acetylcholine (ACh) from striatal slices to assess the influence of these receptors on NMDA function. NMDA-stimulated [¹⁴C]GABA release was more sensitive to NMDA and glycine antagonists than was [³H]ACh release, suggesting that different NMDA receptors regulate the release of these neurotransmitters. Furthermore, NMDA-stimulated [³H]ACh release was inhibited by a D₂ receptor mechanism whereas NMDA-stimulated [¹⁴C]GABA release was enhanced by D₁ receptor activation. NMDA and (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrobromide (AMPA) interact additively to evoke [³H]ACh release, and synergistically to evoke [¹⁴C]GABA release. An additive effect of NMDA and kainate (KA) was found on [¹⁴C]GABA release, but NMDA and KA acted in a less than additive manner in evoking [³H]ACh release. KA-stimulated [³H]ACh release was largely blocked by NMDA antagonists, suggesting mediation through activation of NMDA receptors, probably secondary to KA-induced glutamate release. A selective group II metabotropic receptor agonist inhibited NMDA-stimulated [¹⁴C]GABA and [³H]ACh release. On the other hand, NMDA-stimulated [¹⁴C]GABA release was potentiated by activation of group I metabotropic receptors. Thus, in addition to the differential modulation by D₁- and D₂-like receptors, the release of striatal neurotransmitters by NMDA receptor activation depends on the extent to which the other glutamate receptors, both ionotropic and metabotropic, are activated.     

Endogenous GABA release from rat striatal slices: Effects of the GABAB receptor antagonist 2-hydroxy-saclofen

The reproducibility of endogenous GABA release evoked by multiple periods of electrical field stimulation was examined in rat striatal slices. In these experiments, NO-328 was used to block GABA uptake, and evoked GABA release (overflow) was completely Ca²⁺ dependent. A seemingly invariant observation in these experiments was that spontaneous GABA release (outflow) progressively decreased as a function of superfusion time and that GABA overflow decreased 25–30% in response to the second of two periods of stimulation (S2/S1 ratios = 0.70 to 0.75). The attenuation of GABA release was not explained by the amount of GABA lost to the superfusion buffer (fractional release), direct depletion of releasable pools of GABA, or slice viability. Furthermore, the decreases in GABA release were not dependent on stimulation frequency (5–15 Hz) or the absolute amount of GABA evoked by electrical stimulation. However, the GABA_B receptor antagonist 2-hydroxy-saclofen (2-OH-saclofen; 316 μM) not only enhanced GABA overflow, when superfused throughout both periods of stimulation, but also resulted in S2/S1 ratios of unity. When 2-OH-saclofen was superfused throughout the second stimulation period only, GABA overflow was almost two-fold greater than that evoked by the initial period of stimulation (2-OH-saclofen-free). In addition, these S2 responses were ～30% greater than S1 responses that were observed when 2-OH-saclofen was present throughout the entire superfusion period. These results indicate that activation of GABA_B receptors was involved in the progressive attenuation of GABA release and further emphasize that GABA_B receptors play an important role in modulating endogenous GABA release from striatal slices. © Wiley-Liss, Inc.     

Involvement of protein kinase C in the Ca-dependent vesicular release of GABA from central and enteric neurons of the guinea pig

The involvement of protein kinase C (PKC) in the release of endogenous γ-aminobutyric acid (GABA) was studied using slices of deep cerebellar nucleus and strips of small intestine from the guinea pig.12-O-tetradecanoylphorbol 13-acetate (TPA), but not 4α-phorbol-12,13-dedecanoate (4α-PDD), potentiated the high K⁺-evoked release of GABA from both preparations in the presence of tetrodotoxin. Ouabain evoked the release of GABA from both preparations, and this release was not altered by TPA. Therefore, the activation of protein kinase C potentiates the Ca²⁺-dependent vesicular release of GABA from nerve terminals of the central and enteric GABAergic neurons of the guinea pig.     

Development of changes in endogenous GABA release during kindling epileptogenesis in rat hippocampus

The calcium-dependent gamma-aminobutyric acid (GABA) and glutamate release from rat hippocampal CA1 slices, evoked by a 1-min depolarization with 50 mM K+, was investigated in different stages of kindling epileptogenesis. Kindling was induced by tetanic stimulation of the Schaffer collateral/commissural pathway. In agreement with our previous results, we found a significantly increased calcium-dependent GABA release compared to that of implanted controls, in a group of fully kindled animals 1 day after the last seizure and also 25-36 days after the last seizure. In addition, we found that the increase in GABA release was associated with late phases of kindling epileptogenesis since no significant alterations were found in partly kindled animals that had received only 6 kindling stimulations while a significant increase was apparent in animals that had received 14 tetanic stimuli. When the release protocol was carried out in the presence of SK&F 89776-A, a blocker of the GABA uptake carrier, an additional amount of GABA was found after depolarization. This additional amount of GABA, reflecting the amount of GABA taken up under conditions without blocker, was in kindled animals not different from controls which demonstrates that a reduced GABA uptake does not account for the observed enhanced release in kindled animals. The calcium-dependent release of glutamate evoked by 1 min of high potassium depolarization was not significantly changed in the kindled groups. Only after prolonged depolarization during 4 subsequent minutes a significant increase in animals of the fully kindled group and at long-term after kindling was observed. The threshold K+ concentration for eliciting a calcium-dependent release of GABA and glutamate, was not changed in the kindled animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

[H]Nipecotic acid binding to GABA uptake sites in human brain

The binding of [³H]nipecotic acid to frozen post-mortem human brain tissue has been characterized. Competition experiments with γ-aminobutyric acid (GABA), GABA uptake inhibitors, ligands active at post-synaptic GABA receptors and receptors for other neurotransmitter systems, suggest that [³H]nipecotic acid binds to the neuronal (but not glial) GABA uptake site. Competition and kinetic experiments suggest that 85% of the binding is to high affinity site. The dissaciation constants (K_d) measured in kinetic and equilibrium experiments were in the same range (0.5–0.6 μM). The regional distribution was studied in 19 brain regions and the binding was relatively homogenous. It is concluded that [³H]nipecotic acid binding can be used as a marker for neuronal GABA uptake sites in post-mortem human brain tissue.     

Divalent cations reduce depolarization of primary afferent terminations by GABA

Divalent metal cations, including zinc, cadmium, cobalt, nickel, strontium, manganese, magnesium and calcium, reduced the depolarization by microelectrophoretic γ-aminobutyric acid (GABA) and piperidine-4-sulphonic acid of the central terminations of muscle group Ia primary afferent fibres in the cat spinal cord without affecting the inhibition by GABA of the firing of spinal interneurones. There thus appears to be a difference in either the interaction of GABA with recognition sites, or in the mechanism by which such interaction activates chloride ionophores, at GABA-mediated bicuculline-sensitive synapses on the central terminals of peripheral primary afferent neurones and those on neurones located within the central nervous system.     

Electrophysiological actions of norepinephrine in rat lateral hypothalamus. II. An in vitro study of the effects of iontophoretically applied norepinephrine on LH neuronal responses to γ-aminobutyric acid (GABA)

The preceding studies demonstrated that norepinephrine (NE) can consistently augment synaptically mediated (70%) and γ-aminobutyric acid (GABA)-induced (69%) inhibitory responses of lateral hypothalamic (LH) neurons in vivo. The present experiments further characterized the interactions of NE with LH neuronal responses to GABA in terms of α- and ß-receptor mechanisms and demonsrated the utility of the in vitro LH tissue slice preparation as a model for future extra- and intracellular studies of NE modulatory phenomena. Extracellular activity of LH cells was recorded from diencephalic slices (450 μm) incubated in artificial cerebrospinal fluid at 33 °C. Interactions between iontophoretically applied NE, isoproterenol (ISO) or phenylephrine (PE) and responses of LH neurons (n = 64) to GABA microiontophoresis were quantitated and characterized using computer-generated ratemeter and histogram records. This analysis revealed two distinct actions of NE on GABA-induced responses of LH neurons. In 8 of 32 cells tested (25%), locally applied NE markedly enhanced inhibitory responses to GABA iontophoresis in a manner identical to that observed in vivo. However, in 20 cells (62.5%), iontophoretic application of NE produced a clear antagonism of GABA responses. NE also exerted dual effects on the background firing rate of LH neurons, causing both inhibition and excitation. Overall, in those cells where NE administration increased spontaneous discharge, it either antagonized or had no effect on GABA-mediated inhibition. In contrast, spontaneous firing rate was never elevated above control levels in those cases where NE potentiated GABA responses. Additional experiments demonstrated that the GABA potentiating actions of the benzodiazepine, flurazepam, were preserved in LH tissue slice preparations. In addition, iontophoretic application of the ß-agonist, ISO, routinely suppressed the spontaneous activity of LH neurons and mimicked the facilitating action of NE on GABA. Likewise, microiontophoretic application of 8-bromo cyclic adenosine monophosphate (AMP) enhanced GABA-induced inhibition of LH firing rate in each of 11 cells tested. On the other hand, local administration of the alpha agonist, PE, routinely produced NE-like antagonism of GABA inhibition along with increases in spontaneous firing rate. Taken together these findings indicate that the commonly observed in vivo phenomena of NE augmentation of GABA and suppression of LH neuron spontaneous firing can be demonstrated in vitro, and most likely result from activation of beta adrenoceptors and subsequent elevation of cyclic AMP levels. As such these results suggest that the in vitro preparation will be a useful tool for further investigation of the transmembrane and intracellular events associated with noradrenergically mediated enhancement of GABA. However, in contrast to results obtained in vivo, NE antagonism of GABA inhibition and excitatory effects on spontaneous activity were more frequently observed in LH slices and appear to be mediated by alpha receptor activation. The reduced capacity of NE to augment GABA in vitro might be related to changes in the balance between α- and ß-mediated effects rather than deficits in GABA-facilitating mechanisms, since ISO, cyclic AMP and the benzodiazepine were all routinely capable of enhancing GABAergic responses.     

Acetylcholine attenuates synaptic GABA release to supraoptic neurons through presynaptic nicotinic receptors

Both inhibitory GABAergic and excitatory glutamatergic inputs to supraoptic nucleus (SON) neurons can influence the release of vasopressin and oxytocin. Acetylcholine is known to excite SON neurons and to increase vasopressin release. The functional significance of cholinergic receptors, located at the presynaptic nerve terminals, in the regulation of the excitability of SON neurons is not fully known. In this study, we determined the role of presynaptic cholinergic receptors in regulation of the inhibitory GABAergic inputs to the SON neurons. The magnocellular neurons in the rat hypothalamic slice were identified microscopically, and the spontaneous miniature inhibitory postsynaptic currents (mIPSCs) were recorded using the whole-cell voltage-clamp technique. The mIPSCs were abolished by the GABA_A receptor antagonist, bicuculline (10 μM). Acetylcholine (100 μM) significantly reduced the frequency of mIPSCs of SON neurons from 3.59±0.36 to 1.62±0.20 Hz (n=37), but did not alter the amplitude and the decay time constant of mIPSCs. Furthermore, the nicotinic receptor antagonist, mecamylamine (10 μM, n=13), eliminated the inhibitory effect of acetylcholine on mIPSCs of SON neurons. The muscarinic receptor antagonist, atropine (100 μM), did not alter significantly the effect of acetylcholine on mIPSCs in most of the 17 SON neurons studied. These results suggest that the excitatory effect of acetylcholine on the SON neurons is mediated, at least in part, by inhibition of presynaptic GABA release. Activation of presynaptic nicotinic receptors located in the GABAergic terminals plays a major role in the cholinergic regulation of the inhibitory GABAergic input to SON neurons.     

Effects of thiopental, halothane and isoflurane on the calcium-dependent and -independent release of GABA from striatal synaptosomes in the rat

The effects of the anesthetic agents thiopental, halothane and isoflurane on the release of GABA induced by depolarization and/or reversal of the GABA carrier were investigated in a synaptosomal preparation obtained from the rat striatum. Veratridine (1 μM) and KCl (9 mM) elicited a significant Ca²⁺-dependent release of [³H]GABA. The KCl-evoked release was not significantly modified in the presence of nipecotic acid (10⁻⁵ M), a selective blocker of the neuronal GABA carrier. The [³H]GABA release was significantly decreased by ω-conotoxin (10⁻⁷ M, a blocker of the N voltage-dependent Ca²⁺ channels, but was affected by neither nifedipine (10⁻⁴ M) nor ω-Aga-IVA (10⁻⁷ M) which block the L and Ca²⁺ channels, respectively. Thiopental application (10⁻⁵ to 10⁻³ M) was followed by a dose-related, significant, decrease in both the veratridine and KCl-induced releases, whether nipecotic acid was present or not. In contrast, halothane and isoflurane (1–3%) failed to alter [³H]GABA release. Altogether, these results suggest that reduction of the depolarization-evoked GABA release might contribute to thiopental anesthesia, but this seems unlikely for volatile anesthetics.     

Ovarian steroid modulation of [H]muscimol binding in the spinal cord of the rat

[³H]Muscimol binding was measured in the lumbar spinal cord of female rats by in vitro quantitative autoradiography. Ovariectomized rats were treated subcutaneously with either oil, estradiol benzoate (EB) or EB plus progesterone (P) in a regime known to reliably induce sexual receptivity. The level of [³H]muscimol binding was highest in laminae I–III and in the region around the central canal. Binding was lower in laminae IV–VI and was frequently undetectable in the ventral horn. There was a significant increase in the level of binding in laminae I–III after EB treatment. There was also a significant increase after treatment with EB+P in comparison to both the ovariectomized and EB-treated groups in this same region of the spinal cord.     

Receptor regulation of the glutamate, GABA and taurine high-affinity uptake into astrocytes in primary culture

From experiments using dissociated primary astroglial cultures from newborn rat cerebral cortex, the stimulation of monoamine receptors (alpha, beta and 5HT) was shown to affect the high-affinity uptake kinetics of glutamate, GABA and taurine. In the presence of the alpha 1 agonist phenylephrine, there was an increased uptake (Vmax) of glutamate, while beta adrenoceptor activation slightly inhibited the glutamate uptake and stimulated the GABA and taurine uptakes. 5HT2 receptor stimulation caused a slight inhibition of the taurine uptake. The uptake rate of GABA was not affected by 5HT, alpha 1 or alpha 2 receptor agonists and the glutamate uptake was not affected by 5HT or alpha 2 receptor agonists. Nor was the taurine uptake affected by alpha 1 or alpha 2 receptor agonists. The active uptake of aspartate was unaffected by the presence of any of the monoamine receptor agonists used in this study. When the mechanisms behind these effects were studied, the GABA uptake seemed to be mediated via the G protein-adenylate cyclase complex in the receptor domain. Moreover, the K+ channels seemed to be involved. The taurine uptake, however, did not seem to be regulated by the same mechanism. It seems more probable that there is a direct interaction between the receptor and carrier of taurine at the membrane level. The mechanism underlying the receptor-regulated glutamate uptake is at present unclear, although it does not seem to involve protein kinase C.     

Adenosine receptor agonists inhibit the release of γ-aminobutyric acid (GABA) from the ischemic rat cerebral cortex

The effects of CPA (a selective A1 receptor agonist), NECA (a mixed A1 and A2 receptor agonist), and CGS 21680 (a selective A2 receptor agonist) on the ischemia-evoked release of gamma-aminobutyric acid (GABA) from rat cerebral cortex was investigated with the cortical cup technique. Cerebral ischemia (20 min) was elicited by four vessel occlusion. In control animals, superfusate GABA increased from a basal level of 206 +/- 26 nM (mean +/- S.E.M., n = 18) to 10,748 +/- 3,876 nM during the reperfusion period. Pretreatment with adenosine receptor agonists failed to affect basal levels of GABA release. However, CPA (10(-10) M), NECA (10(-9) M), and CGS 21680 (10(-8) M) significantly suppressed the ischemia-evoked release of GABA. The ability to block the ischemia-evoked release of GABA was not evident when the adenosine receptor agonists were administered at higher concentrations. Thus, the selective activation of either A1 or high-affinity A2a adenosine receptors results in an inhibition of ischemia-evoked GABA release.     

Kindling increases the K -evoked Ca -dependent release of endogenous GABA in area CA1 of rat hippocampus

The release of endogenous amino acids from hippocampal CA1 subslices under basal conditions and the release evoked by high potassium (50 mM K⁺) depolarization was studied during kindling epileptogenesis. Emphasis was put on the release of the amino acid neurotransmitters γ-aminobutyric acid (GABA) and glutamate. Kindling was induced by tetanic stimulation of the Schaffer-collaterals/commissural fibers of the dorsal hippocampus of the rat. The calcium-dependent GABA release in the presence of high K⁺ was significantly increased (40–46%) in fully kindled animals, 24 h after the last seizure, in comparison to controls. At long-term, 28 days after the last seizure, the calcium-dependent GABA release was still significantly increased (45–49%). An increased release of GABA in kindled animals was still found when GABA uptake was blocked by nipecotic acid. In contrast, no significant alterations were encountered in the basal or high potassium induced release of the excitatory amino acids aspartate and glutamate. These results suggest that kindling epileptogenesis is accompanied by a specific and long-lasting enhancement of GABA exocytosis which may lead to a desensitization of the GABA receptor, and thus determine the increase of seizure sensitivity.