KINETICS OF IN VIVO CONVERSION OF γ-[3H]AMINOBUTYRIC ACID TO γ-[3H]HYDROXYBUTYRIC ACID BY RAT BRAIN1,2

Abstract— The appearance of γ-[³H]hydroxybutyric acid ([³H]GHB) in rat brain at various times after the intraventricular administration of [³H]GABA was determined. Radioactivity recovered as [³H]GHB was maximal 30 s after [³H]GABA administration and declined exponentially thereafter. From a linear transformation of the disappearance with time of [³H]GHB formed from [³H]GABA, the fractional rate of disappearance and turnover time of GHB were calculated. Administration of amino-oxyacetic acid (50 mg/kg i.p.) 1 h before [³H]GABA, reduced [³H]GHB formation, measured 4 min after [³H]GABA, to 28% of that found in control animals. This strongly suggests that GABA-transaminase catalyzes at least one step in the conversion pathway. [³H]GHB recoverable 4 min after [³H]GABA was unchanged when animals were pretreated with pyrazole (1.25–5.0 mmol/kg), diphenyl-hydantoin (25 and 75 mg/kg), phenobarbital (7.5–60 mg/kg), ethanol (1.25–5.0 g/kg), or morphine (2.5–10 mg/kg). Significantly more [³H]GHB could be recovered at several time points from animals which had been pretreated with 50 mg/kg i.p. of the convulsant 3-mercaptopropionic acid.     

L-lysine is a barbiturate-like anticonvulsant and modulator of the benzodiazepine receptor

Our earlier observations showed thatl-lysine enhanced the activity of diazepam against seizures induced by pentylenetetrazol (PTZ), and increased the affinity of benzodiazepine receptor binding in a manner additive to that caused by -aminobutyric acid (GABA). The present paper provides additional evidence to show thatl-lysine has central nervous system depressant-like characteristics.l-lysine enhanced [³H]flunitrazepam (FTZ) binding in brain membranes was dose-dependent and stimulated by chloride, bromide and iodide, but not fluoride. Enhancement of [³H]FTZ binding byl-lysine at a fixed concentration was increased by GABA but inhibited by pentobarbital between 10^–7 to 10^–3M. While GABA enhancement of [³H]FTZ binding was inhibited by the GABA mimetics imidazole acetic acid and tetrahydroisoxazol pyridinol, the enhancement by pentobarbital andl-lysine of [³H]FTZ binding was dose-dependently increased by these two GABA mimetics. The above results suggest thatl-lysine and pentobarbital acted at the same site of the GABA/benzodiazepine receptor complex which was different from the GABA binding site. The benzodiazepine receptor antagonist imidazodiazepine Ro15-1788 blocked the antiseizure activity of diazepam against PTZ. Similar to pentobarbital, the anti-PTZ effect ofl-lysine was not blocked by Ro15-1788. Picrotoxinin and the GABA, receptor antagonist bicuculline partially inhibitedl-lysine's enhancement of [³H]FTZ binding with the IC₅₀s of 2 M and 0.1 M, respectively. The convulsant benzodiazepine Ro5-3663 dose-dependently inhibited the enhancement of [³H]FTZ binding byl-lysine. This article shows the basic amino acidl-lysine to have a central nervous system depressant characteristics with an anti-PTZ seizure activity and an enhancement of [³H]FTZ binding similar to that of barbiturates but different from GABA.     

Influence of Sodium-Independent γ-Aminobutyric Acid Binding on the Assay of Sodium-Dependent γ-Aminobutyric Acid Binding in a Membrane Preparation of Rat Brain

A large amount of [³H]GABA was bound to crude synaptic membrane fractions of rat. by sodium-independent process in a medium that contained 100 μM [³H]GABA used for assaying GABA uptake site. This [³H]-GABA binding was different from receptor binding of GABA. It was confirmed that this sodium-independent [²H]GABA binding scarcely occurred in the presence of a physiological concentration of sodium chloride, and that sodium-independent GABA binding had a negligible influence on sodium-dependent GABA binding.     

Binding sites for [3H]GABA, [3H]flunitrazepam and [35S]TBPS in insect CNS

The CNS of the cockroach Periplaneta americana contains saturable, specific binding sites for [³H]GABA, [³H]flunitrazepam and [³⁵S]TBPS. The [³H]GABA binding site exhibits a pharmacological profile distinct from that reported for mammalian GABA_A and GABA_B receptors. The most potent inhibitors of [³H]GABA binding were GABA and muscimol, whereas isoguvacine, thiomuscimol and 3-aminopropane sulphonic acid were less effective. Bicuculline methiodide and baclofen were ineffective. Binding of [³⁵S]TBPS was partially inhibited by 1.0 × 10⁻⁶ M GABA, whilst binding of [³H]flunitrazepam was enhanced by 1.0 × 10⁻⁷ M GABA. The pharmacological profile of the [³H]flunitrazepam binding site showed some similarities with the peripheral benzodiazepine binding sites of vertebrates, with Ro-5-4864 being a far more effective inhibitor of binding than clonazepam. Thus a class of GABA receptors with pharmacological properties distinct from mammalian GABA receptor subtypes is present in insect CNS.     

Interaction of barbiturates with dihydropicrotoxinin binding sites related to the GABA receptor-ionophore system.

Barbiturate drugs of diverse chemical structure inhibited the binding of [³H] α-dihydropicrotoxinin to rat brain membranes. This biologically active analoque of picrotoxin labels membrane sites related to the convulsant action of these drugs in inhibiting GABA postsynaptic receptor-ionophore function at a site distinct from the GABA receptor. Depressant barbiturates such as pentobarbital inhibited dihydropicrotoxinin binding competitively at therapeutic concentrations (IC₅₀ = 50 μM) whereas the drug does not alter GABA receptors, uptake, or release at this concentration. Antiepileptics such as phenobarbital (IC₅₀=400 μM), were weaker inhibitors of binding. Convulsant barbiturates, however, such as dimethylbutylbarbiturate (IC₅₀=0.05 μM) and cyclohexylidene-ethyl barbiturate (IC₅₀=0.7 μM), were potent inhibitors. The displacement of radioactive dihydropicrotoxinin binding by the convulsant barbiturates had different slopes and Hill numnbers (0.4) compared to displacement by depressant barbiturates and picrotoxinin itself (Hill numbers = 1.0), indicating heterogeneity of binding sites or negative cooperativity. These potent intractions of barbiturates with dihydropicrotoxinin binding sites are consistent with neurophysiological evidence that depressant or convulsant action of barbiturates may involve modulation of CNS inhibitory synaptic transmission at the level of the postsynaptic GABA receptor-ionophores.     

The convulsant drugs 3-mercaptopropionate and methionine sulfoximine inhibitl-glutamate andl-aspartate binding to a hydrophobic protein fraction from rat cerebral cortex

The action of the convulsant drugs, methionine sulfoximine (MSO), 3-mercaptopropionate (3-MP), megimide (MG), and allylglycine on the binding ofl-[¹⁴C]aspartate,l-[¹⁴C]glutamate and [¹⁴C]GABA to a hydrophobic protein fraction isolated from rat cerebral cortex was studied. Using the convulsant at 10^–4 M concentration and the radioactive ligands at 10⁶ M the binding ofl-[¹⁴C]glutamate was inhibited 60% by 3-MP and 40% by MSO, while MG and allylglycine had no effect. The binding ofl-[¹⁴C]aspartate was inhibited 55%, and 10–20% by 3-MP and MSO, respectively, while MG and allylglycine had not effect. None of the drugs mentioned, except for a minimal inhibition by MG, altered the binding of [¹⁴C]GABA. Neither MSO nor 3-MP affected the high-affinity sites forl-[¹⁴C]glutamate orl-[¹⁴C]aspartate, but they had a strong inhibitory action on the medium affinity site. These results are discussed in relation to the possible mechanism of action of these drugs onl-glutamate andl-aspartate receptors.     

Ligand Binding to CNS Muscarinic Receptor Is Transiently Modified by Convulsant 3-Mercaptopropionic Acid Administration

The administration of convulsant drugs has proven a powerful tool to study experimental epilepsy. We have already reported that the administration of convulsant 3-mercaptopropionic acid (mp) at 150 mg/kg enhances binding affinity of muscarinic antagonist [³H]quinuclidinyl benzilate ([³H]QNB) to certain rat CNS membranes during seizure and postseizure without affecting site number. Results obtained with a 100-mg/kg dose of mp have shown reversible increases in [³H]QNB binding to cerebellum and hippocampus, whereas a delayed response has been found in striatum. Neither a subconvulsant dose nor in vitro addition modifies binding. In order to evaluate preseizure, seizure as well as early (30 min) and late (24 h) postseizure stages, we employed a 50 mg/kg dose and tested [³H]QNB binding to CNS membranes. Changes in binding were as follows (in %): in cerebellum, +37, +86, and +40 at preseizure, seizure and early postseizure stages, respectively, but there was a decrease at late postseizure; in hippocampus, +27 at pre- and seizure stages, but a decrease at early and late postseizure. No changes were found in striatum or cerebral cortex membranes at any stage studied. Saturation curves analysed by Scatchard plots indicated that changes in [³H]QNB binding to cerebellar membranes are attributable to an increase in ligand affinity at seizure, followed by a decrease in binding site number at postseizure. A similar profile was observed for hippocampus except that the decrease in binding site number, though lower than at postseizure, was already evident at seizure stage. Results confirm a region-specific response to the convulsant and transient changes provide an example of neuronal plasticity.     

The Binding of the GAB A Agonist [3H]THIP to Rat Brain Synaptic Membranes

Abstract: THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) is a specific GABA agonist with potent analgesic properties. The binding of radioactive THIP to thoroughly washed, frozen, and thawed membranes isolated from rat brains has been studied at 2°C under sodium ion-free conditions and compared with the binding of [³H]GABA and [³H]piperidine-4-sulphonic acid ([³H]P4S). The best computer fits to the experimental data were in all cases attained with a receptor model based on three independent binding sites, of which only the high- and medium-affinity sites could be characterised satisfactorily. While the K_D values were found to be comparable for all three ligands employed, the density of the high-affinity binding site (B_M1) was, with the exception of the membranes from the cerebellum, considerably lower for [³H]THIP than for [³H]GABA and [³ H]P4S. The regional distribution of the GABA receptors, which bind [³H]THIP, was different from those recognizing [³H]GABA and [³H]P4S. A number of analogues, including asymmetric compounds with known configuration, were tested as inhibitors of the binding of [³H]GABA, [³H]muscimol, [³H]THIP, [³H]isoguvacine, and [³H]P4S. The concentrations of the asymmetric compounds required for the inhibition of [³H]P4S binding were much higher than those required for the displacement of [³H]GABA, [³H]muscimol, [³H]THIP, and [³H]isoguvacine. The comparable relative potencies of inhibitors do, however, indicate that all of the ligands bind to the GABA receptors.     

In vitro neuropharmacological evaluation of penitrem-induced tremorgenic syndromes: importance of the GABAergic system

The effects of the fungal neurotoxin penitrem A on the GABAergic and glutamatergic systems in rat brain were evaluated. Penitrem A inhibited binding of the GABA_A-receptor ligand [³H]TBOB to rat forebrain and cerebellar membrane preparations with IC₅₀ (half maximal inhibitory concentration) values of 11 and 9 μM, respectively. Furthermore, penitrem A caused a concentration-dependent increase of [³H]flunitrazepam and [³H]muscimol binding in rat forebrain, but not in cerebellar preparations. The stimulation of [³H]flunitrazepam binding by penitrem A was abolished by the addition of GABA. In cerebellar preparations, a different pharmacological profile was found, with penitrem A allosterically inhibiting [³H]TBOB binding by interacting with a bicuculline-sensitive site. Moreover, penitrem A inhibited the high affinity uptake of GABA and glutamate into cerebellar synaptosomes with IC₅₀ values of 20 and 47 μM, respectively. The toxin showed no effect on NMDA or AMPA glutamate receptor binding. In conclusion, our results suggest that penitrem A exerts region-specific effects in the brain, leading to positive modulation of GABA_A-receptor function in forebrain. Conversely, penitrem A may act as a bicuculline-like convulsant in cerebellum.     

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.     

The GABA/benzodiazepine receptor complex in the nervous system of a hypertensive strain of rat

-Aminobutyric acid (GABA) has been implicated in the development of hypertension and in the regulation of blood pressure. The spontaneously hypertensive rat (SHR) offers an opportunity to explore the role of central GABA and other neurotransmitters in the genesis of high blood pressure. The receptor binding of [³H]GABA, [³H]flunitrazepam, and [³H]glutamate to synaptic membranes from the cerebral cortex and cerebellum of SHR rats were measured in animals of various ages. No significant differences between the SHR and a normotensive control strain of rats were found for any of the assays. The results indicate that in this model of hypertension, neither GABA nor glutamate function are involved, at least not in the cerebral cortex or cerebellum.     

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.     

RO5-4864 inhibits the binding of [35S]t-butylbicyclophosphorothionate to rat brain membranes

RO5-4864, a 1,4-benzodiazepine, has recently been shown to possess anticonvulsant, convulsant and anxiogenic properties and to inhibit Ca++-calmodulin-stimulated membrane phosphorylation. RO5-4864 inhibited the binding of [35S]t-butylbicyclophosphorothionate (TBPT) to cerebral cortex, cerebellar and hippocampus membranes, with an IC50 value of approximately 20 microM. TBPT binds apparently to the picrotoxinin site of the benzodiazepine-GABA receptor-ionophore complex and appears to be a site of action for several classes of convulsant, depressant and anxiolytic drugs that modulate GABAergic transmission. RO5-4864 inhibited [35S] TBPT binding in cerebral cortex, apparently competitively. Antagonists of GABA and central benzodiazepine sites did not interfere with the ability of RO5-4864 to inhibit [35S] TBPT binding. The properties of RO5-4864 to inhibit TBPT binding are similar to other convulsants and GABA antagonists (except bicuculline) which inhibit TBPT binding. These results suggest that RO5-4864 interacts with the TBPT binding sites of the oligomeric GABA receptor complex.     

[3H]Bicuculline Methochloride Binding to Low-Affinity γ-Aminobutyric Acid Receptor Sites

Abstract: The binding of [³H]bicuculline methochloride (BMC) to mammalian brain membranes was characterized and compared with that of [³H]γ-aminobutyric acid ([³H]GABA). The radiolabeled GABA receptor antagonist showed significant displaceable binding in Tris-citrate buffer that was improved by high concentrations of chloride, iodide, or thiocyanate, reaching >50% displacement in the presence of 0.1 M SCN^?. An apparent single class of binding sites for [³H]BMC (K_D= 30 nM) was observed in 0.1 M SCN^? for fresh or frozen rat cortex or several regions of frozen and thawed bovine brain. The B_max was about 2 pmol bound/mg of crude mitochondrial plus microsomal membranes from unfrozen washed and osmotically shocked rat cortex, similar to that for [³H]GABA. Frozen membranes, however, showed decreased levels of [³H]BMC binding with no decrease or an actual increase in [³H]GABA binding sites. [³H]BMC binding was inhibited by GABA receptor specific ligands, but showed a higher affinity for antagonists and lower affinity for agonists than did [³H]GABA binding. Kinetics experiments with [³H]GABA binding revealed that low- and high-affinity sites showed a similar pharmacological specificity for a series of GABA receptor ligands, but that whereas all agonists had a higher affinity for slowly dissociating high-affinity [³H]GABA sites, bicuculline had a higher affinity for rapidly dissociating low-affinity [³H]GABA sites. This reverse potency between agonists and antagonists during assay of radioactive antagonists or agonists supports the existence of agonist- and antagonist-preferring conformational states or subpopulations of GABA receptors. The differential affinities, as well as opposite effects on agonist and antagonist binding by anions, membrane freezing, and other treatments, suggest that [³H]BMC may relatively selectively label low-affinity GABA receptor agonist sites. This study, using a new commercially available preparation of [³H]bicuculline methochloride, confirms the report of bicuculline methiodide binding by Mohler and Okada (1978), and suggests that this radioactive GABA antagonist will be a valuable probe in analyzing various aspects of GABA receptors.     

A role for calcium/calmodulin kinase(s) in the regulation of GABA exocytosis

A possible role for protein kinases in the regulation of GABA exocytosis in nerve endings was investigated. The effect on the release of the radioactive neurotransmitter ([³H]GABA) from mouse brain synaptosomes of several protein kinase inhibitors was estimated after treatment with 37 mM K⁺ in the absence of external Na⁺, a condition under which [³H]GABA release is completely Ca²⁺ dependent. Among the inhibitors one group inhibit the kinases by binding to the catalytic site (i.e. staurosporine and H7) and others (TFP, sphingosine and W7) act on the regulatory site of protein kinases. The compounds of the second group, which are reported to inhibit calmodulin dependent events and the increase in cytosolic Ca²⁺ (Ca _i ) induced by high K⁺ depolarization, were the most efficient inhibitors of [³H]GABA release. The selective inhibitor of CaMPK II, KN-62, also markedly diminished [³H]GABA release as well as the increase in Ca _i induced by high K⁺. The kinase inhibitors from the first group that are unable to diminish the increase in Ca _i induced by high K⁺ were also less efficient inhibitors of [³H]GABA release even at high concentrations. The present results indicate that at the doses tested all the drugs inhibit to some extent the release of the Ca²⁺ dependent fraction of [³H]GABA perhaps by inhibiting a CaMPK II mediated phosphorylation step triggered by depolarization and facilitated by the elevation of Ca _i . In addition, the second group of antagonists and KN-62 inhibit the elevation of Ca _i to high K⁺ thus exhibiting a higher efficiency on [³H]GABA release than the first group of antagonists.     

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

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

Neuronal and Glial Release of [3H]GABA from the Rat Olfactory Bulb

Abstract: GABA uptake and release mechanisms have been shown for neuronal as well as glial cells. To explore further neuronal versus glial components of the [³H]-γ-aminobutyric acid ([³H]GABA) release studies were performed with two different microdissected layers of the olfactory bulb of the rat: the olfactory nerve layer (ONL), consisting mainly of glial cells, and the external plexiform layer (EPL) with a high density of GABAergic dendritic terminals. In some experiments substantia nigra was used as a GABAergic axonal system and the trigeminal ganglia as a peripheral glial model. Spontaneous release of [³H]GABA was always lower in neuronal elements as compared with glial cells. A veratridine-evoked release was observed from the ONL but not from the trigeminal ganglia. Tetrodotoxin (TTX) abolished the veratridine-evoked release from the ONL, which also showed a partial inhibition when high magnesium concentrations were used in a Ca²⁺-free solution. β-Alanine was strongly exchanged with [³H]GABA from the ONL of animals with the olfactory nerve lesioned and from animals with no lesion; but only a small heteroexchange was found from the external plexiform layer. The β-alanine heteroexchange was able to deplete the releasable GABA store from the ONL of lesioned animals. In nonlesioned animals and the external plexiform layer, the veratridine-stimulated release of [³H]GABA was not significantly reduced after the β-alanine heteroexchange. Stimulation of the [³H]GABA release by high concentrations of potassium elicited a higher release rate from axonal terminals than from dendrites or glia. Neurones and glia showed a similar inhibition of [³H]GABA release when a high magnesium concentration was added to a calcium-free solution. When D-600 was used as a calcium-flux blocker no inhibition of the release was observed in glial cells, whereas an almost complete blockage was found in both neuronal preparations (substantia nigra and EPL). These results provide further evidence for differential release mechanisms of GABA from CNS neurones and glial cells.     

Inhibition of transporter mediated γ-aminobutyric acid (GABA) release by SKF 89976-A,a GABA uptake inhibitor,studied in a primary neuronal culture from chicken

The effect of SKF 89976-A, a lipophilic non-substrate inhibitor of the -aminobutyric acid (GABA) transporter, on the release of radioactive GABA andd-aspartate has been studied. Neuronal cultures from 8 day old chick embryos, grown for six days, served as a model. The cultures were incubated with [³H]d-aspartate and [¹⁴C] GABA with the subsequent addition of high or low concentrations of SKF 89976-A. Finally the cultures were exposed to differently composed media for either 30 or 300 seconds. The release was quantified, using liquid scintillation counting. The efflux of [³H]d-aspartate and [¹⁴C] GABA was increased by [K⁺] and time, and a minimum value was obtained at [Ca²⁺] 1.05 mM. The release of both [³H]d-aspartate and [¹⁴C] GABA was inhibited by SKF 89976-A. The obtained results indicate that transporter mediated processes are the major mechanisms of transmitter release in the investigated model.     

Postnatal development and GABA allosteric modulation of benzodiazepine receptor binding in the vitamin B-6 deficient rat brain

We have measured the postnatal development and GABA modulation of benzodiazepine receptors in neuronal membranes from vitamin B-6 deficient and normal rats. In rats fed vitamin B-6 adequate and deficient diets there were age-dependent changes in [³H]flunitrazepam binding site affinity and in the number of binding sites. Vitamin B-6 deficiency produced a significant reduction in the potency of GABA to enhance [³H]flunitrazepam binding to cortical membranes prepared from 14 day old rats. These results suggests an uncoupling of the GABAa/benzodiazepine receptor at a developmental period when the animals are most susceptible to spontaneous seizures.     

Chronic exposure of cells expressing recombinant GABAA receptors to benzodiazepine antagonist flumazenil enhances the maximum number of benzodiazepine binding sites

The aim of this study was to better understand the mechanisms that underlie adaptive changes in GABAA receptors following their prolonged exposure to drugs. Exposure (48 h) of human embryonic kidney (HEK) 293 cells stably expressing recombinant alpha1beta2gamma2S GABAA receptors to flumazenil (1 or 5 microM) in the presence of GABA (1 microM) enhanced the maximum number (Bmax) of [3H]flunitrazepam binding sites without affecting their affinity (Kd). The flumazenil-induced enhancement in Bmax was not counteracted by diazepam (1 microM). GABA (1 nM-1 mM) enhanced [3H]flunitrazepam binding to membranes obtained from control and flumazenil-pretreated cells in a concentration-dependent manner. No significant differences were observed in either the potency (EC50) or efficacy (Emax) of GABA to potentiate [3H]flunitrazepam binding. However, in flumazenil pretreated cells the basal [3H]flunitrazepam and [3H]TBOB binding were markedly enhanced. GABA produced almost complete inhibition of [3H]TBOB binding to membranes obtained from control and flumazenil treated cells. The potencies of GABA to inhibit this binding, as shown by a lack of significant changes in the IC50 values, were not different between vehicle and drug treated cells. The results suggest that chronic exposure of HEK 293 cells stably expressing recombinant alpha1beta2gamma2S GABAA receptors to flumazenil (in the presence of GABA) up-regulates benzodiazepine and convulsant binding sites, but it does not affect the allosteric interactions between these sites and the GABA binding site. Further studies are needed to elucidate these phenomena.