Excitatory Amino Acid-Evoked Release of γ-[3H]Aminobutyric Acid from Striatal Neurons in Primary Culture

The actions of excitatory amino acids on the release of previously incorporated gamma-[3H]aminobutyric acid ([3H]GABA) were examined in purified (greater than 93%) striatal neurons derived from the fetal mouse brain and differentiated in primary culture. Glutamate, KCl, and veratrine evoked a dose-dependent, saturable, and reversible release of [3H]GABA from striatal neurons. Glutamate actions were not reduced in the absence of calcium, and were insensitive to tetrodotoxin. The dose-response relationships of excitatory amino acids demonstrated the following rank order of potency: glutamate greater than aspartate = N-methyl-D-aspartate greater than kainate much greater than quisqualate. Kainate, however, was the most effective agonist, evoking an eightfold increase over baseline levels of [3H]GABA release. Aspartate- and N-methyl-D-aspartate-evoked release was abolished in the presence of either 2-aminophosphonovaleric acid or gamma-D-glutamylglycine. Release due to glutamate and kainate was partially or ineffectively attenuated by these agents. Glutamate-, aspartate-, and N-methyl-D-aspartate-evoked GABA releases were augmented when calcium was omitted from the bathing medium and reduced when sodium was replaced with choline or lithium. Kainate-evoked release was unaffected when calcium was omitted, virtually unchanged when choline replaced sodium, and markedly potentiated when lithium was substituted for sodium. These findings suggest that at least two distinct receptor systems for excitatory amino acids mediate the evoked release of [3H]GABA from striatal neurons in primary culture. These two systems, aspartate/N-methyl-D-aspartate- and kainate-preferring, are distinguishable on the basis of their pharmacological and ionic properties.     

Modulation of [3H]Acetylcholine Release from Cultured Amacrine-Like Neurons by Adenosine A1 Receptors

Abstract: We have investigated the effect of endogenous adenosine on the release of [³H]acetylcholine ([³H]ACh) in cultured chick amacrine-like neurons. The release of [³H]ACh evoked by 50 m M KCl was mostly Ca²⁺ dependent, and it was increased in the presence of adenosine deaminase and in the presence of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), an adenosine A₁ receptor antagonist. The effect of adenosine on [³H]ACh release was sensitive to pertussis toxin (PTX) and was due to a selective inhibition of N-type Ca²⁺ channels. Ligand binding studies using [³H]DPCPX confirmed the presence of adenosine A₁ receptors in the preparation. Using specific inhibitors of the plasma membrane adenosine carriers and of the ectonucleotidases, we found that the extracellular accumulation of adenosine in response to KCl depolarization was due to the release of endogenous adenosine per se and to the extracellular conversion of released nucleotides into adenosine. Activation of adenosine A₁ receptors was without effect on the intracellular levels of cyclic AMP under depolarizing conditions, but it inhibited the accumulation of inositol phosphates. Our results indicate that in cultured amacrine-like neurons, the Ca²⁺-dependent release of [³H]ACh evoked by KCl is under tonic inhibition by adenosine, which activates A₁ receptors. The effect of adenosine on the [³H]ACh release may be due to a direct inhibition of N-type Ca²⁺ channels and/or secondary to the inhibition of phospholipase C and involves the activation of PTX-sensitive G proteins.     

Maitotoxin-Induced Release of γ-[3H]Aminobutyric Acid from Cultures of Striatal Neurons

The potent marine toxin, maitotoxin, induced the release of gamma-[3H]aminobutyric acid (GABA) from reaggregate cultures of striatal neurons in a dose-dependent manner. Maitotoxin-induced release occurred following a lag period of several minutes and was persistent. Release induced by 70 mM K+ on the other hand was immediate and transient in nature. Co2+ (3 mM) and Cd2+ (1 mM) inhibited maitotoxin-induced release of GABA as did removal of extracellular Ca2+. However, the organic calcium antagonists nisoldipine, nitrendipine, and D-600 at concentrations of 10(-6) M did not block maitotoxin-induced or 70 mM K+-induced release. High concentrations of D-600 (10(-4) M) partially blocked both maitotoxin- and 70 mM K+-induced release. The dihydropyridine calcium agonist BAY K8644 (10(-6) M) did not enhance maitotoxin-induced or 70 mM K+-induced release. Replacement of Na+ in the incubation medium with choline led to an increased basal output of GABA and an apparent inhibition of the effect of maitotoxin. These data are discussed with reference to the hypothesis that maitotoxin can directly activate voltage-sensitive calcium channels.     

Stimulation of γ-[3H]Aminobutyric Acid Release from Cultured Mouse Cerebral Cortex Neurons by Sulphur-Containing Excitatory Amino Acid Transmitter Candidates: Receptor Activation Mediates Two Distinct Mechanisms of Release

In primary cultures of mouse cerebral cortex neurons, sulphur-containing excitatory amino acids (SAAs; namely, L-cysteine sulphinate, L-cysteate, L-homocysteine sulphinate, L-homocysteate, S-sulphocysteine) at concentrations ranging from 0.1 microM to 1 mM evoked a saturable release of gamma-[3H]aminobutyric acid ([3H]GABA) in the absence of any other depolarizing agent. All SAAs exhibited essentially similar potency (EC50, 100-150 microM) in releasing [3H]GABA although a variable profile of maximal stimulatory effect was observed when compared with basal release. The intracellular accumulation of the lipophilic cation, [3H]tetraphenylphosphonium, was significantly reduced in the presence of all SAAs, thus verifying a depolarization of the neuronal plasma membrane. SAA-stimulated release of [3H]GABA was shown to comprise two distinct components, calcium-dependent and calcium-independent, which occur after activation of N-methyl-D-aspartate (NMDA) and non-NMDA receptors. Thus, all SAA-evoked responses were antagonized by the selective, competitive NMDA-receptor antagonist, 3-[(+/-)-2-carboxypiperazin-4-yl]propyl-1-phosphonic acid (IC50 range, greater than 50 microM) and the non-NMDA-receptor antagonist, 6,7-dinitroquinoxalinedione (IC50 range, 5-50 microM). Removal of magnesium ions from the superfusion medium caused a significant potentiation of SAA-evoked responses without having any effect on basal levels of [3H]GABA efflux, a result consistent with an involvement of NMDA-receptor activation. Calcium-independent release (i.e., that release remaining in the presence of 1 mM cobalt ions) was a distinct component but of smaller magnitude. Using 500 microM excitatory amino acid agonist concentrations, this component of release was (1) markedly attenuated by 15 microM SKF-89976-A, a non-transportable inhibitor of the GABA carrier, and (2) abolished when choline ions replaced sodium ions in the superfusion medium or when in the presence of excitatory amino acid receptor antagonists. These observations are clearly consistent with a receptor-mediated, depolarization-induced reversal of the GABA carrier.     

Presence of Radiolabelled Metabolites in Release Studies Using [3H]γ-Aminobutyric Acid

Abstract: Most studies on γ-aminobutyric acid (GABA) release from nervous tissue have been conducted using radiolabelled GABA in the presence of aminooxyacetic acid (AOAA) to inhibit GABA: 2-oxoglutarate aminotransferase (GABA-T) to prevent conversion of labelled GABA to labeled catabolites. Here we present data showing that even in the presence of 10 μM-AOAA the spontaneous release of tritium from rat cortical synaptosomes prelabelled with 2,3-[³H]GABA is mainly in the form of tritiated water but that the increase in tritium release in the presence of unlabelled GABA or high potassium-ion concentrations is in the form of authentic [³H]GABA. Interpretation of results should take these facts into account.     

Nitric Oxide-Induced [3H]GABA Release from Cerebral Cortical Neurons Is Mediated by Peroxynitrite

Abstract: The functional significance of peroxynitrite in the release of [³H]GABA induced by nitric oxide (NO) liberated from NO generators was investigated using cerebral cortical neurons in primary culture. NO generators such as sodium nitroprusside (SNP) and S -nitroso- N -acetylpenicillamine (SNAP) increased [³H]GABA release in a dose-dependent manner. These increases in [³H]GABA release were significantly inhibited by hemoglobin, indicating that those NO generators evoke the release of [³H]GABA by the formation of NO. Two types of superoxide scavengers, Cu²⁺/Zn²⁺ superoxide dismutase and ceruloplasmin, significantly reduced the increase in [³H]GABA release induced by both SNP and SNAP, which assumes that NO requires superoxide to induce [³H]GABA release from the neurons. In addition, synthesized peroxynitrite induced a dose-dependent increase in [³H]GABA release from the neurons. These results indicate that NO-induced [³H]GABA release is mediated by peroxynitrite formed by the reaction of NO with superoxide.     

Effects of Creatine on Synthesis and Release of γ-[3H]Aminobutyric Acid

Creatine has been used previously to alter the energy balance of neurons in brain slices. In the present experiments, it was found to reduce the accumulation of gamma-[3H]aminobutyric acid ([3H]GABA) as synthesized from [3H]glutamine or [3H]glutamic acid in slices of rat neostriatum. The lowest effective concentration was 5 mM. Creatine (25 mM) was also effective when the degrading enzyme of GABA, i.e., GABA-alpha-oxoglutarate transaminase, was blocked by gabaculine. Creatine (25 mM) did not inhibit the uptake and subsequent accumulation of [3H]GABA. Thus, indirect evidence was obtained that creatine decreased the activity of the synthesizing enzyme of GABA, i.e., glutamate decarboxylase. When the direct effect of creatine (25 mM) on glutamate decarboxylase was studied in vitro, the agent indeed decreased the activity of the enzyme. Creatine (25 mM) also diminished the release of [3H]GABA (expressed as dpm/mg wet weight) from rat neostriatal slices, probably by reducing its synthesis and thus its readily releasable pool. These data are of importance for studies with creatine in complex neuronal systems, because they show that the agent changes not only neuronal energy balance, but also synthesis and release of the ubiquitous transmitter GABA.     

Release of Previously Incorporated γ-[3H]Aminobutyric Acid in Rabbit Caudate Nucleus Slices

The release of gamma-aminobutyric acid (GABA) was studied in slices of the head of the rabbit caudate nucleus. The slices were preincubated with [3H]GABA and then superfused. Aminooxyacetic acid was present throughout. Both the tritium in the slices and that in the superfusate consisted practically entirely of [3H]GABA. Stimulation for 2 min by electrical field pulses of 3 ms width and 9 V/cm voltage drop (36 mA current strength) at 5 or 20 Hz elicited an overflow of [3H]GABA that amounted to 0.23 or 0.47% of the tritium content of the tissue, respectively, and was diminished by 85% in the presence of tetrodotoxin. At higher current strength, less of the stimulation-evoked overflow was tetrodotoxin-sensitive. cis-1,3-Aminocyclohexane carboxylic acid diminished the uptake of [3H]GABA into the tissue but did not change the percentage released by electrical stimulation. Ca2+ withdrawal greatly accelerated basal [3H]GABA efflux and almost abolished the response to stimulation. Nipecotic acid 10-1,000 microM enhanced both the basal and (up to eightfold) the stimulation-evoked overflow. The method described allows us to elicit electrically a quasiphysiological, i.e., Ca2+-dependent and tetrodotoxin-sensitive, neuronal release of [3H]GABA. Nipecotic acid diverts released [3H]GABA from reuptake to overflow.     

Regional Selectivity of a γ-Aminobutyric Acid-Induced [3H]Acetylcholine Release Sensitive to Inhibitors of γ-Aminobutyric Acid Uptake

The effects of gamma-aminobutyric acid (GABA) on the release of [3H]acetylcholine ([3H]ACh) were studied in synaptosomes prepared from rat hippocampus, cerebral cortex, hypothalamus, and striatum and prelabelled with [3H]choline. When synaptosomes were exposed in superfusion to exogenous GABA (0.01-0.3 mM) the basal release of newly synthesized [3H]ACh was increased in a concentration-dependent way in hippocampus, cortex, and hypothalamus nerve endings. In contrast, the release of [3H]ACh was not significantly affected by GABA in striatal synaptosomes. The effect of GABA was not antagonized significantly by bicuculline or picrotoxin. Muscimol caused only a slight not significant increase of [3H]ACh release when tested at 0.3 mM whereas, at this concentration, (-)-baclofen was totally inactive. The GABA-induced release of [3H]ACh was counteracted by SKF 89976A, SKF 100561, and SKF 100330A, three strong and selective GABA uptake inhibitors. The data suggest that, in selective areas of the rat brain, GABA causes release of [3H]ACh following penetration into cholinergic nerve terminals through a GABA transport system.     

A Search for Receptors Modulating the Release of γ-[3H]Aminobutyric Acid in Rabbit Caudate Nucleus Slices

Various putative striatal transmitters and related compounds were studied for their effects on the release of gamma-aminobutyric acid (GABA) from slices of the head of the rabbit caudate nucleus. The slices were preincubated with [3H]GABA and then superfused and stimulated electrically at 5 or 20 Hz. Aminooxyacetic acid was present throughout. The main changes observed were the following. The basal and, less consistently, the electrically evoked overflow of [3H]GABA were enhanced by 3,4-dihydroxyphenylethylamine (dopamine), an effect not blocked by cis-flupentixol or domperidone and not mimicked by apomorphine and D1-selective agonists. The electrically evoked overflow was diminished by 5-hydroxytryptamine (serotonin); the inhibition was prevented by methiothepin. The basal but not the electrically evoked overflow was enhanced by carbachol; acetylcholine and nicotine also accelerated the basal outflow whereas oxotremorine caused no consistent change; the effect of carbachol and acetylcholine were blocked by hexamethonium but not by atropine or by tetrodotoxin. These findings indicate that the GABA neurons in the caudate nucleus may be stimulated by dopamine, although the receptor type involved remains unclear; inhibited by serotonin; and stimulated by acetylcholine acting via a nicotine receptor. However, all drug effects observed were relatively small. No evidence was obtained for autoreceptors, alpha 2-adrenoceptors or receptors for opioids, adenosine or substance P at the GABA neurons.     

Stimulation of [3H]GABA and β-[3H]Alanine Release from Rat Brain Slices by cis-4-Aminocrotonic Acid

Abstract: cis -4-Aminocrotonic acid (CACA; 100 µ M ), an analogue of GABA in a folded conformation, stimulated the passive release of [³H]GABA from slices of rat cerebellum, cerebral cortex, retina, and spinal cord and of β-[³H]alanine from slices of cerebellum and spinal cord without influencing potassium-evoked release. In contrast, CACA (100 µ M ) did not stimulate the passive release of [³H]taurine from slices of cerebellum and spinal cord or of d -[³H]aspartate from slices of cerebellum and did not influence potassium-evoked release of [³H]taurine from the cerebellum and spinal cord and d -[³H]aspartate from the cerebellum. These results suggest that the effects of CACA on GABA and β-alanine release are due to CACA acting as a substrate for a β-alanine-sensitive GABA transport system, consistent with CACA inhibiting the uptake of β-[³H]alanine into slices of rat cerebellum and cerebral cortex. The observed K _i for CACA against β-[³H]alanine uptake in the cerebellum was 750 ± 60 µ M . CACA appears to be 10-fold weaker as a substrate for the transporter system than as an agonist for the GABA_c receptor. The effects of CACA on GABA and β-alanine release provide indirect evidence for a GABA transporter in cerebellum, cerebral cortex, retina, and spinal cord that transports GABA, β-alanine, CACA, and nipecotic acid that has a similar pharmacological profile to that of the GABA transporter, GAT-3, cloned from rat CNS. The structural similarities of GABA, β-alanine, CACA, and nipecotic acid are demonstrated by computer-aided molecular modeling, providing information on the possible conformations of these substances being transported by a common carrier protein.     

[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.     

Early Undernutrition and [3H]γ-Aminobutyric Acid Binding in Rat Brain

The effect of early undernutrition and dietary rehabilitation on [3H]gamma-aminobutyric acid ([3H]GABA) binding in rat brain cerebral cortex and hippocampus was examined. Undernourished animals were obtained by exposing their mothers to a protein-deficient diet during both gestation and lactation. Saturation analysis of [3H]GABA binding in the cerebral cortex and hippocampus revealed high- and low-affinity components in the undernourished group, whereas control animals possessed only a low-affinity site. The concentration of low-affinity binding sites was greater in the undernourished animals. Rehabilitation of undernourished animals completely abolished the binding site differences. Treatment of brain membranes with Triton X-100 yielded two binding components in both the undernourished and control animals, although the concentration of lower affinity sites was still greater in the undernourished group. Neither the efficacy nor the potency of GABA to activate benzodiazepine binding in cerebral cortex was modified by undernutrition. These data suggest that early undernourishment modifies the characteristics of [3H]GABA binding, perhaps by reducing the brain content of endogenous inhibitors of the higher affinity binding site. The lack of effect on GABA-activated benzodiazepine binding suggests the possibility that neither the high- nor the low-affinity GABA binding sites are coupled to this receptor component.     

Phosphatidylserine Enhancement of [3H]γ-Aminobutyric Acid Uptake by Rat Whole Brain Synaptosomes

Abstract: [³H] γ -Aminobutyric acid ([³H]GABA) binding to purified lipids was examined in an organic solvent-aqueous partition system. In addition, the [³H]GABA binding capacity in the partition system was compared with the capacity of lipids to alter sodium-dependent [³H]GABA uptake into synaptosomes isolated from rat whole brains. [³H]GABA was found to bind to all of the lipids studied in the organic solvent-aqueous partition system [phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), gangliosides, and sulfatide], although PS exhibited the greatest binding capacity. [³H]GABA uptake into synaptosomes was enhanced by PS (48.0%) but was not altered by any other lipid. PS enhancement of [³H]GABA uptake required the presence of sodium and was blocked by nipecotic acid (10 μ m ). These results suggest that PS may play a role in the sodium-dependent GABA reuptake process in the presynaptic nerve end.     

Depression by Sodium Ions of Calcium Uptake Mediated by Non-N-Methyl-d-Aspartate Receptors in Cultured Cerebellar Neurons and Correlation with Evoked d-[3H]Aspartate Release

In a previous study we noted that the release of D-[3H]aspartate evoked by non-N-methyl-D-aspartate (non-NMDA) receptor agonists in cultured rat cerebellar granule cells was enhanced in the absence of extracellular Na+. To explain this apparent paradox, we tried in the present investigation to correlate the effect of Na+ removal on the kainate (KA)- and quisqualate (QA)-induced D-[3H]aspartate release with that on KA- and QA-induced 45Ca2+ accumulation. The releasing activity of KA, which was only partially Ca2+ dependent in the presence of Na+, became totally Ca2+ dependent in its absence. Moreover, the releasing activity of QA, which was Ca2+ independent in the presence of Na+, became 50% Ca2+ dependent in the absence of the monovalent cation. The releasing action of both agonists was in all cases antagonized by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and that induced by KA was also sensitive to kynurenic acid. When glutamate was tested as an agonist in the presence of Na+, it was found that its D-[3H]aspartate releasing action was Ca2+ independent and was largely due to heteroexchange. The evoked release was Ca2+ independent, scarcely sensitive to CNQX, and insensitive to NMDA antagonists. In Na(+)-free medium, the glutamate-evoked D-[3H]aspartate release was lower (due to the abolishment of heteroexchange), but was totally Ca2+ dependent and antagonized by CNQX and kynurenate. KA (30 microM-1 mM) stimulated the accumulation of 45Ca2+ in a dose-dependent and CNQX-sensitive way, the effect being progressively higher as the Na+ concentration in the medium was decreased. Li+ affected KA-induced 45Ca2+ accumulation in a way similar to Na+, although 45Ca2+ uptake was somewhat lower in Li(+)-containing medium. The voltage-activated calcium channel antagonists La3+ and (-)-202-791 caused only a limited inhibition of the KA-induced 45Ca2+ influx both in the presence and in the absence of Na+. Under all the conditions tested [presence and absence of Na+ and of (-)-202-791], the kainate-induced 45Ca2+ uptake was scarcely sensitive to the NMDA antagonist 2-amino-5-phosphonovalerate. QA and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid also stimulated 45Ca2+ influx in a CNQX-sensitive way, the effect being enhanced in Na(+)-free media. These agonists were, however, less effective than KA.(ABSTRACT TRUNCATED AT 400 WORDS)     

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)     

Inhibition by Diazepam and γ-Aminobutyric Acid of Depolarization-Induced Release of [14C]Cysteine Sulfinate and [3H]Glutamate in Rat Hippocampal Slices

Effects of diazepam and gamma-aminobutyric acid-related compounds on the release of [14C]cysteine sulfinate and [3H]glutamate from preloaded hippocampal slices of rat brain were examined by a superfusion method. Diazepam markedly inhibited the release of cysteine sulfinate and glutamate evoked either by high K+ or veratridine without affecting that of other neurotransmitter candidates, e.g., gamma-aminobutyric acid, acetylcholine, noradrenaline, and dopamine; IC50 values for the release of cysteine sulfinate and glutamate were about 20 and 7 microM, respectively. gamma-Aminobutyric acid (1 to 10 microM) and muscimol (100 microM) significantly reduced high K+-stimulated release of glutamate. Bicuculline, which had no effect on the release at a concentration of 50 microM by itself, antagonized the inhibitor effects of diazepam and gamma-aminobutyric acid on glutamate release. Similar results were obtained with the release of cysteine sulfinate except that a high concentration (100 microM) of gamma-aminobutyric acid was required for the inhibition. These results indicate the modulation by gamma-aminobutyric acid innervation of the release of excitatory amino acids in rat hippocampal formation, and also suggest that some of the pharmacological effects of diazepam may be a consequence of inhibition of excitatory amino acid transmission.     

[3H]Norepinephrine Release from Hippocampal Slices Is an In Vitro Biochemical Tool for Investigating the Pharmacological Properties of Excitatory Amino Acid Receptors

[3H]Norepinephrine ([3H]NE) efflux from preloaded rat hippocampal slices was increased in a dose-dependent manner by excitatory amino acids, with the following order of potencies: N-methyl-D-aspartate (NMDA) greater than kainic acid (KA) greater than L-glutamate greater than or equal to D,L-homocysteate greater than L-aspartate greater than quinolinic acid greater than quisqualic acid. The effect of the excitatory amino acids was blocked by physiological concentrations of Mg2+, with the exception of KA. D,L-2-Amino-7-phosphonoheptanoic acid dose-dependently inhibited the NMDA effect (ID50 = 69 microM), whereas at 1 mM it was ineffective versus KA. The release of [3H]-NE induced by quinolinic acid was blocked by 0.1 mM D,L-2-amino-7-phosphonohepatanoic acid. gamma-D-Glutamylglycine dose-dependently inhibited the KA effect with an ID50 of 1.15 mM. Tetrodotoxin (2 microM) reduced by 40 and 20% the NMDA and KA effects, respectively. The data indicate that [3H]NE release from hippocampal slices can be used as a biochemical marker for pharmacological investigations of excitatory amino acid receptors and their putative agonists and antagonists.     

[3H]Nipecotic Acid Binding to γ-Aminobutyric Acid Uptake Sites in Postmortem Human Brain

Binding of [3H]nipecotic acid, a proposed marker for GABAergic neurons, was investigated in postmortem human brain by use of a centrifugation assay. Binding was displaceable, apparently saturable, and to a single site, with typical KD and Bmax values of 1.85 microM and 124.2 pmol/mg of protein in the hippocampus. Regional distribution studies indicated a heterogeneous population of [3H]nipecotic acid binding sites with highest concentrations in the lateral globus pallidus. Putamen tissue from four cases of Huntington's disease showed a marked reduction in [3H]nipecotic acid binding. Binding correlated with both age and postmortem delay in the hippocampus. There was an effect of agonal state in which prolonged illness before death apparently caused a reduction in binding. Our results indicate that [3H]nipecotic acid may be used successfully as a marker for neuronal GABAergic uptake sites in human brain, but that the effects of variables such as age, postmortem delay, and agonal state must always be taken into account.     

Binding of [3H] γ-Aminobutyric Acid and [3H]Muscimol in Purified Rat Brain Synaptic Plasma Membranes and the Effects of Bicuculline

Abstract: The binding of [³H] γ-aminobutyric acid ([³H]GABA) and [³H]muscimol has been studied in purified synaptic plasma membrane (SPM) preparations from rat brain. Scatchard analysis of specific binding (defined as that displaced by 100 μMγ-aminobutyrate) indicated that the binding of both radiolabelled ligands was best described by a two component Langmuir adsorption isotherm. The apparent K_D and B_max values for [³H]GABA at 4°C were K_D1, 20 nM; K_D2,165 nM; B_max1, 0.48 pmol;B_max2, 6.0 pmol. mg^?1; for [³H]muscimol at 4°C they were: K_D1, 1.75 nM; K_D2, 17.5 nM; B_maxl, 0.84 pmol. mg^?1; B_max2, 4.8 pmol.mg^?1; and for [³H]muscimol at 37°C they were: K_D1, 7.0 nM; K_m, 60 nM; B_max], 0.5 pmol-mg^?1; B_max2, 7.2 pmol-mg¹. Under the experimental conditions used, the similar B_milx values for [³H]GABA and [³H]muscimol binding to the SPM preparations suggests that the high- and low-affinity components for the two radiolabeled ligands are identical. The effects of the GAB A antagonist bicuculline on the binding of [³H]muscimol at 4^CC and 37°C were studied. At 4°C, antagonism of muscimol binding appeared to be competitive at the high-affinity site but noncompetitive at the low-affinity site. At 37°C, antagonism was again competitive at the high-affinity site but was of a mixed competitive/noncompetitive nature at the low-affinity site. Assuming that binding to the high-affinity site is associated with the pharmacological actions of bicuculline, the apparent K_D values obtained suggest a pA₂ value of 5.3 against [³H]muscimol at 4°C and 37°C. This figure is in good agreement with several estimates of the potency of bicuculline based on pharmacological measurements. Results from displacement studies using [³H]GABA and [³H]muscimol suggest that [³H]GABA might be a more satisfactory ligand than [³H]muscimol in GABA radioreceptor assays.