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.     

Effects of Cyclic AMP Analogues and Phosphodiesterase Inhibitors on K+-Induced [3H]Noradrenaline Release from Rat Brain Slices and on Its Presynaptic α-Adrenergic Modulation

The possible role of cyclic AMP in the presynaptic alpha-adrenoceptor-mediated modulation of [3H]noradrenaline (NA) release induced by 13 mM K+ from superfused rat cerebral cortex slices was investigated. Both dibutyryl-cyclic AMP (db-cAMP) and 8-bromo-cyclic AMP (8-Br-cAMP) dose-dependently (10(-4) - 10(-2) M) enhanced K+-induced (3H]NA release, maximally to about 160% of control. In contrast, db-cAMP had no effect on calcium-induced [3H]NA release in the presence of the calcium ionophore A 23187. Surprisingly, the phosphodiesterase (PDE) inhibitors 3-isobutyl-1-methylxanthine (IBMX). 7-benzyl-IBMX, 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (ZK 62771), and 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724) appeared to inhibit K+-induced [3H]NA release in a dose-dependent (10(-5) - 10(-3) M) manner. At a concentration of 10(-4) M, AK 62771 caused an inhibition of [3H]NA release by 30%, and this inhibitory effect was not affected by 10(-6) M phentolamine nor by 10(-3) M db-cAMP or 10(-4) M theophylline. Theophylline by itself enhanced [3H]NA release to about 135% of control. The inhibitor effect of the alpha-adrenoceptor agonist oxymetazoline (1 micro M) and the enhancing effect of the antagonist phentolamine (1 micro M) on [3H]NA release were significantly decreased in the presence of 10(-3) M db-cAMP or 8-Br-cAMP, whereas 10(-4) M ZK 62771 had no effect. In the presence of 10(-2) M NaF, a potent activator of adenylate cyclase, the inhibitory effect of oxymetazoline (1 micro M) on [3H]NA release was significantly decreased. The data obtained with the cyclic AMP analogues support the hypothesis that activation of presynaptic alpha-receptors modulating NA release results in an inhibition of a presynaptic adenylate cyclase. Possible causes for the anomalous effects of th PDE inhibitors are discussed.     

Dopamine Inhibits Calcium-Independent γ-[3H]Aminobutyric Acid Release Induced by Kainate and High K+ in the Fish Retina

Kainic acid (KA) at micromolar concentrations stimulated the release of gamma-[3H]aminobutyric acid [( 3H]GABA) from a particulate fraction of the carp (Cyprinus carpio) retina. The KA action was dose-dependent but Ca2+-independent. A similar response was elicited by another glutamate receptor agonist, quisqualic acid, and high K+, but not by an aspartate agonist, N-methyl-D-aspartic acid. The stimulatory action of KA on the [3H]GABA release was selectively blocked by the KA blockers gamma-D-glutamylglycine and cis-2,3-piperidine dicarboxylic acid. Dopamine (DA), which is contained in DA interplexiform cells in the carp retina, inhibited the [3H]GABA release induced by KA and high K+ in a dose-dependent manner. 5-Hydroxytryptamine and two well-known GABA antagonists, bicuculline (Bic) and picrotoxin (Pic), also mimicked the DA effect on the GABA release at a comparable concentration. This inhibitory effect of DA as well as Bic and Pic on the [3H]GABA release evoked by KA was clearly antagonized by a DA blocker, haloperidol. The action of these agents (KA, DA, GABA antagonist) belonging to three different receptor categories on the GABAergic neurons (possibly external horizontal cells; H1 cells) is discussed in relation to other electrophysiological studies on the lateral spread of S-potentials between H1 cells.     

Spontaneous Release of γ-Aminobutyric Acid Formed from Putrescine and Its Enhanced Ca2+-Dependent Release by High K+ Stimulation in the Brains of Freely Moving Rats

The spontaneous release of [3H] gamma-aminobutyric acid ([3H]GABA) in various areas of rat brain injected with [3H]putrescine was examined using a push-pull perfusion technique. The release in a 25-min perfusate was highest in the caudate-putamen. The effect of high K+ stimulation on the release of [3H]GABA formed from [3H]putrescine was examined in the caudate-putamen. The release was enhanced by high K+ solution in a Ca2+-dependent manner.     

Quantification of Rat Cerebral Cortex Na+,K+-ATPase: Effect of Age and Potassium Depletion

Na+,K(+)-ATPase concentration in rat cerebral cortex was studied by vanadate-facilitated [3H]ouabain binding to intact samples and by K(+)-dependent 3-O-methylfluorescein phosphatase activity determinations in crude homogenates. Methodological errors of both methods were evaluated. [3H]Ouabain binding to cerebral cortex obtained from 12-week-old rats measured incubating samples in buffer containing [3H]ouabain, and ouabain at a final concentration of 1 x 10(-6) mol/L gave a value of 11,351 +/- 177 (n = 5) pmol/g wet weight (mean +/- SEM) without any significant variation between the lobes. Evaluation of affinity for ouabain was in agreement with a heterogeneous population of [3H]ouabain binding sites. K(+)-dependent 3-O-methylfluorescein phosphatase activity in crude cerebral homogenates of age-matched rats was 7.24 +/- 0.14 (n = 5) mumol/min/g wet weight, corresponding to a Na+,K(+)-ATPase concentration of 12,209 +/- 236 pmol/g wet weight. It was concluded that the present methods were suitable for quantitative studies of cerebral cortex Na+,K(+)-ATPase. The concentration of rat cerebral cortex Na+,K(+)-ATPase showed approximately 10-fold increase within the first 4 weeks of life to reach a plateau of approximately 11,000-12,000 pmol/g wet weight, indicating a larger synthesis of Na+,K+ pumps than tissue mass in rat cerebral cortex during the first 4 weeks of development. K+ depletion induced by K(+)-deficient fodder for 2 weeks resulted in a slight tendency toward a reduction in K+ content (6%, p > 0.5) and Na+,K(+)-ATPase concentration (3%, p > 0.4) in cerebral cortex, whereas soleus muscle K+ content and Na+,K(+)-ATPase concentration were decreased by 30 (p < 0.02) and 32% (p < 0.001), respectively. Hence, during K+ depletion, cerebral cortex can maintain almost normal K+ homeostasis, whereas K+ as well as Na+,K+ pumps are lost from skeletal muscles.     

Effects of Increased γ-Aminobutyric Acid Levels on GAD67 Protein and mRNA Levels in Rat Cerebral Cortex

Abstract: Rats were injected with saline or the γ-aminobutyric acid (GABA) transaminase inhibitor γ-vinyl-GABA for 7 days and the effects on GABA content and glutamic acid decarboxylase (GAD) activity, and the protein and mRNA levels of the two forms of GAD (GAD₆₇ and GAD₆₅) in the cerebral cortex were studied. γ-Vinyl-GABA induced a 2.3-fold increase in GABA content, whereas total GAD activity decreased by 30%. Quantitative immunoblotting showed that the decline in GAD activity was attributable to a 75–80% decrease in GAD₆₇ levels, whereas the levels of GAD₆₅ remained unchanged. RNA slot-blotting with a ³²P-labeled GAD₆₇ cDNA probe demonstrated that the change in GAD₆₇ protein content was not associated with a change in GAD₆₇ mRNA levels. Our results suggest that GABA specifically controls the level of GAD₆₇ protein. This effect may be mediated by a decreased translation of the GAD₆₇ mRNA and/or a change in the stability of the GAD₆₇ protein.     

Inhibitory Effect of Several Nitric Oxide-Generating Compounds on Purified Na+,K+-ATPase Activity from Porcine Cerebral Cortex

Abstract: Nitric oxide (NO)-generating compounds (NO donors) such as sodium nitroprusside, S-nitroso-N-acetylpenicillamine, S-nitroso-l -glutathione, 3-morpholinosyndnonimine (SIN-1), (dl )-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide, and 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene inhibited the Na⁺,K⁺-ATPase activity purified from porcine cerebral cortex. NO-reducing or -scavenging agents, such as superoxide dismutase or N-(dithiocarbamate)-N-methyl-d -glucamine sodium salt, l -ascorbic acid, and sulfhydryl (SH) compounds, such as dithiothreitol or the reduced form of glutathione, but not α-tocopherol, prevented the inhibition of the enzyme activity by all NO donors except sodium nitroprusside. Enzyme inhibition could also be reversed by these SH compounds, but not by superoxide dismutase, l -ascorbic acid, and α-tocopherol. 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazolin-1-oxyl 3-oxide (PTIO), which is able to scavenge NO radicals and generate nitrogen dioxide radicals (^?NO₂), potentiated the inhibition of this enzyme activity induced by all NO donors (except SIN-1). PTIO did not potentiate, but rather attenuated, the SIN-1-induced inhibition. SIN-1 has been reported to release both NO and superoxide and thereby to rapidly form peroxynitrite (ONOO^?). These potentiated and attenuated inhibitions of the enzyme activity induced by PTIO plus all of the NO donors except sodium nitroprusside were prevented by SH compounds, but not by superoxide dismutase, l -ascorbic acid, and α-tocopherol. These results suggest that NO donors may release NO or NO-derived products, presumably ^?NO₂ and ONOO^?, and may inhibit the Na⁺,K⁺-ATPase activity by interacting with a SH group at the active site of the enzyme.     

Reductions of Γ-Aminobutyric Acid and Glutamate Uptake and (Na++ K+)-ATPase Activity in Brain Slices and Synaptosomes by Arachidonic Acid

Arachidonic acid, a major polyunsaturated fatty acid of membrane phospholipids in the CNS, reduced the high-affinity uptake of glutamate and gamma-aminobutyric acid (GABA) in both rat brain cortical slices and synaptosomes. alpha-Aminoisobutyric acid uptake was not affected. Intrasynaptosomal sodium was increased concomitant with decreased (Na+ + K+)-ATPase activity in synaptosomal membranes. The reduction of GABA uptake in synaptosomes could be partially reversed by alpha-tocopherol. The inhibition of membrane-bound (Na+ + K+)-ATPase by arachidonic acid was not due to a simple detergent-like action on membranes, since sodium dodecyl sulfate stimulated the sodium pump activity in synaptosomes. These data indicate that arachidonic acid selectively modifies membrane stability and integrity associated with reductions of GABA and glutamate uptake and of (Na+ + K+)-ATPase activity.     

The Effect of Antibodies to Gangliosides on Ca2+ Channel-Linked Release of γ-Aminobutyric Acid in Rat Brain Slices

Antibodies to GM1 ganglioside enhance the release of gamma-aminobutyric acid (GABA) from rat brain slices induced by depolarization with either 40 mM K+ or 200 microM veratrine. Three new observations are now reported. (a) GABA release induced by the Ca2+ ionophore A23187 was not affected by these antibodies. Because this Ca2+ ionophore causes transmitter release by bypassing depolarization-induced opening of Ca2+ channels, this result suggests that gangliosides participate either in the functioning of such Ca2+ channels or in the Na+ channels involved in depolarization. (b) The enhancement (by antibodies to GM1 ganglioside) of GABA release induced by high K+ levels occurred in the presence of tetrodotoxin (0.01 microM). (c) GABA release induced by veratrine in the absence of Ca2+ was not affected by the antibodies. These latter two observations indicate that Na+ channels are not involved in the action of the antibodies. We conclude that this evidence points to the participation of gangliosides in Ca2+ channel functions involved in GABA release in rat brain slices.     

Potentiation by γ-Aminobutyric Acid of α1-Agonist-Induced Accumulation of Inositol Phosphates in Slices of Rat Cerebral Cortex

Noradrenaline-induced accumulation of 3H-labeled inositol mono-, bis-, and trisphosphate (IP1, IP2, and IP3, respectively) in lithium-treated slices of rat cerebral cortex preincubated with [3H]inositol was potentiated by gamma-aminobutyric acid (GABA). However, the effect on [3H]IP2 accumulation was much greater than that on [3H]IP1 or [3H]IP3 accumulation. The principal effect of GABA on noradrenaline concentration-response curves for both [3H]IP1 and [3H]IP2 was to cause an increase in the maximal response attainable. However, whereas the EC50 for GABA potentiation of [3H]IP1 formation was 0.5 mM, the curve for the potentiation of [3H]IP2 formation showed a marked upturn at GABA concentrations of greater than 1 mM. Prazosin (1 microM) blocked the noradrenaline-induced formation of all three inositol phosphates (IPs), in both the presence and the absence of 2 mM GABA. 3H-IP formation induced by phenylephrine and methoxamine was also potentiated by GABA, and again the greatest effect was on [3H]IP2 accumulation. The ratio of [3H]IP2/[3H]IP1 formed in response to 100 microM noradrenaline was increased by 2 mM GABA at all times from 10 to 60 min, whereas the ratio of [3H]IP3/[3H]IP1 was little altered. The effect of GABA was not mimicked by the GABAA agonists isoguvacine and 3-aminopropanesulphonic acid and was not blocked by bicuculline methiodide. (-)-Baclofen, a GABAB agonist, did produce some stimulation of the response to noradrenaline, but to a much lesser extent than GABA. Of the agents tested, nipecotic acid came nearest to reproducing the effect of GABA, in that the major effect was on [3H]IP2 accumulation. The effects of 2 mM GABA and 2 mM nipecotic acid were not additive. GABA potentiation of noradrenaline-induced 3H-IP formation was still apparent in the absence of Li+, but the increase of [3H]IP2 content was less than that of [3H]IP1 content.     

Acetylcholine Release in the Rat Prefrontal Cortex In Vivo: Modulation by α2-Adrenoceptor Agonists and Antagonists

Abstract: We have previously shown that the release of acetylcholine (ACh) in the medial prefrontal cortex of the conscious rat, as measured by microdialysis, is increased following intraperitoneal injection of the selective α₂-adrenoceptor antagonist (+)-efaroxan. To characterize further the receptor pharmacology of this response, the effects of other selective α₂-adrenoceptor ligands were examined. The α₂-adrenoceptor antagonists idazoxan (2.5 and 20 mg/kg), atipamezole (2.5 mg/kg), and fluparoxan (10 mg/kg) increased ACh outflow by up to 250–325% of basal levels over a 3-h period following intraperitoneal injection. The α₂-adrenoceptor agonists UK-14304 (2.5 mg/kg) and guanabenz (2.5 mg/kg) reduced ACh outflow by 80 and 60%, respectively. Clonidine (0.00063–0.16 mg/kg) had no significant depressant effect and at 2.5 mg/kg increased ACh outflow to 233% of basal levels. These results indicate a modulatory role for α₂-adrenoceptors on the release of ACh in the rat prefrontal cortex in vivo. Based on the facilitatory effects produced by the antagonists alone, this α₂-adrenoceptor modulation appears to be tonic and inhibitory. The ability of α₂-adrenoceptor antagonists to enhance ACh outflow suggests a therapeutic usefulness in disorders where cortical ACh release deficits have been implicated.     

Opioid Effects on 45Ca2+ Uptake and Glutamate Release in Rat Cerebral Cortex in Primary Culture

Abstract: Primary cultures of rat cortex, conveniently prepared from newborn animals, were used to study opioid effects on ⁴⁵Ca²⁺ uptake and glutamate release. ⁴⁵Ca²⁺ uptake, induced by treatment with glutamate or NMDA, was largely blocked by the NMDA antagonist MK-801. K⁺ depolarization-induced ⁴⁵Ca²⁺ uptake was also reduced by MK-801, indicating that the effect was mediated by glutamate release. Direct analysis verified that glutamate, and aspartate, were indeed released. Opioid peptides of the prodynorphin system were also released and these, or other peptides, were functionally active, because naloxone treatment increased glutamate release, as well as the ⁴⁵Ca²⁺ uptake induced by depolarization. Opioid agonists, selective for μ-, κ-, and δ-receptors, inhibited the ⁴⁵Ca²⁺ uptake induced by K⁺ depolarization. The combination of low concentrations of MK-801 and opioid agonists resulted in additive inhibition of K⁺- induced ⁴⁵Ca²⁺ uptake. The results indicate that this system may be useful as an in vitro CNS model for studying modulation by opioids of glutamate release and Ca²⁺ uptake under acute, and perhaps also chronic, opiate treatment.     

Inhibition of Na+,K+-ATPase by Ouabain Opens Calcium Channels Coupled to Acetylcholine Release in Guinea Pig Myenteric Plexus

Abstract: Ouabain, an Na⁺,K⁺-ATPase inhibitor, increases the release of acetylcholine (ACh) from various preparations in a Ca²⁺-independent way. However, in other preparations the release of ACh evoked by ouabain is dependent on the presence of extracellular calcium. In the present study, we have labeled the ACh of myenteric plexus longitudinal muscles of guinea pig ileum and compared the effect of calcium channel blockers on ouabain-evoked release of [³H]ACh. Release of [³H]ACh evoked by ouabain is dose dependent and decreased markedly in the absence of calcium or in the presence of cadmium, a nonspecific calcium channel blocker. N-type calcium channel blockage by the ω-conotoxins GVIA (selective N-type calcium channel blocker) and MVIIC (a nonselective calcium channel blocker) inhibited by 45 and 55%, respectively, the release of [³H]ACh. L-type calcium channel suppression by low concentrations of verapamil, nifedipine, and diltiazem had no effect on the release of [³H]ACh. The release of transmitter was also not affected significantly by nickel, a T-type calcium channel blocker. In addition, ω-agatoxin-IVA, at concentrations that block P- and Q-type calcium channels, did not affect significantly the release of [³H]ACh. Thus, extracellular Ca²⁺ is essential for the release of ACh induced by ouabain from guinea pig ileum myenteric plexus. In this preparation, the N-type calcium channel plays a dominant role in transmitter release evoked by inhibition of Na⁺,K⁺-ATPase, but other routes of calcium entry in addition to these channels can also support the release of neurotransmitter induced by ouabain.     

Phorbol Ester Pretreatment Desensitizes the Inhibition of Ca2+ Channels Induced by k-Opiate, α2-Adrenergic, and Muscarinic Receptor Agonists

Acute treatment of rat spinal cord-dorsal root ganglion cocultured neurons with 12-O-tetradecanoylphorbol 13-acetate (TPA), a known activator of protein kinase C, inhibited the dihydropyridine-sensitive voltage-dependent 45Ca2+ influx measured in these cells (IC50 of approximately 100 nM, 66% inhibition at 1 microM TPA). However, prolonged preincubation (24 h) of the cells with 100 nM TPA followed by extensive washing completely abolished, i.e., desensitized, the capacity of a second application of TPA to inhibit the activity of the voltage-dependent Ca2+ channels. Moreover, this treatment also abolished the inhibition of Ca2+ influx produced by kappa-opiate as well as by alpha 2-adrenergic and muscarinic receptor agonists. Substantial desensitization was already observed following a 1-h pretreatment with 100 nM TPA. In contrast to TPA, an inactive phorbol ester (4 beta-phorbol 13-acetate) did not affect the inhibition of the voltage-dependent Ca2+ influx by these receptor agonists. These results suggest that protein kinase C may have a role in the modulation of Ca2+ channels by kappa-opiate, alpha 2-adrenergic, and muscarinic receptor agonists.     

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)     

Influence of Na+, K+, and Ca2+ on Glutamine Synthesis and Distribution in Rat Brain Cortex Slices: A Possible Linkage of Glutamine Synthetase with Cerebral Transport Processes and Energetics in the Astrocytes

The ouabain-induced suppression of glutamine synthesis and retention in incubating rat brain cortex slices was found to be mimicked by changes in the cationic content of the incubation medium, which cause an increase in the intracellular [Na+] and a decrease in the intracellular [K+]. The suppression of glutamine synthesis (and fixation of ammonia) was also found to take place when Ca2+ was omitted from the incubation medium. This occurred whether endogenous or exogenous glutamate was the substrate for glutamine synthesis. The suppressions cannot be due solely to an effect on glutamate uptake, because the uptake is not markedly affected by these conditions. The results show that Na+, K+, and Ca2+ influence the synthesis and distribution of glutamine in the brain. They suggest that Ca2+ and the Na+, K+ pump may serve a role in regulating the activity of ATP-dependent glutamine synthetase, a key enzyme of the glutamate-glutamine cycle, located in the astrocytes. This may be mediated via a direct effect on the enzyme or through an effect on the production of ATP.     

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.     

Modulation of the Electrically Evoked Release of 5-[3H]Hydroxytryptamine from Rat Cerebral Cortex: Effects of Alpidem, CL 218872, and Diazepam

The effect of omega (benzodiazepine)-receptor agonists, antagonists, and inverse agonists on the electrically evoked release of 5-[3H]hydroxytryptamine ([3H]5-HT) was studied in superfused slices of the rat frontal cerebral cortex. The electrically evoked release of [3H]5-HT was enhanced by nanomolar concentrations of diazepam and the selective omega 1-receptor agonists alpidem and CL 218872. The omega 1/omega 2- and omega 1-receptor antagonists flumazenil and CGS 8216, respectively, did not modify the electrically evoked release of [3H]5-HT. The omega 3-receptor agonist Ro 5-4864 and the omega 1-receptor inverse agonist ethyl-beta-carboline-3-carboxylate on their own did not affect the electrically evoked release of [3H]5-HT. On the other hand, the inverse agonist 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylic acid methyl ester (DMCM), at micromolar concentrations, inhibited both the spontaneous and the evoked release of [3H]5-HT. The facilitation of the electrically evoked release of [3H]5-HT by diazepam, alpidem, or CL 218872 was potentiated by gamma-aminobutyric acid (GABA). Exposure to flumazenil and CGS 8216 antagonized the facilitation by diazepam, alpidem, or CL 218872 of [3H]5-HT release. The inhibition of the release of [3H]5-HT by DMCM was not modified by exposure to either flumazenil, CGS 8216, or GABA. The inhibitory effect of DMCM was not observed when monoamine oxidase activity was inhibited by pargyline.(ABSTRACT TRUNCATED AT 250 WORDS)     

Foot-Shock Stress and Anxiogenic β-Carbolines Increase t-[35S]Butylbicyclophosphorothionate Binding in the Rat Cerebral Cortex, an Effect Opposite to Anxiolytics and γ-Aminobutyric Acid Mimetics

The effect of foot-shock stress on t-[35S]butylbicyclophosphorothionate [( 35S]TBPS) binding to fresh unwashed membrane preparations from rat cerebral cortex was studied and was compared to those of GABAA receptor agonists and antagonists and to positive and negative modulators of the GABAergic transmission. [35S]TBPS binding was increased in the cerebral cortex of rats exposed to foot shock compared to that of nonstressed rats. Scatchard analysis revealed that the effect of foot shock was due to an increase in the total number of [35S]TBPS binding sites. In contrast, the in vitro addition of muscimol or GABA induced a dose-dependent inhibition of [35S]TBPS binding, an effect abolished by the concomitant addition of the GABA receptor antagonist, bicuculline, which, per se, enhanced [35S]TBPS binding by 73%. Thus, bicuculline, similar to stress, increased [35S]TBPS binding in the same membrane preparation. In contrast to stress, the anxiolytic and positive modulators of the GABAergic transmission (ZK 93423, ZK 91296, and diazepam) inhibited the specific binding of [35S]TBPS in a concentration-dependent manner. The greatest inhibitory effect was produced by ZK 93423 at 30 microM (31% of control), followed by diazepam (54% of control) and by the partial agonist ZK 91296 (61% of control). Scatchard plot analysis indicated that the inhibition induced by ZK 93423 and diazepam was due to a decrease in the density of [35S]TBPS recognition sites. On the other hand, the anxiogenic beta-carbolines DMCM and FG 7142 mimicked the effect of stress. Thus, at a 10 microM concentration, DMCM and FG 7142 increased [35S]TBPS binding by 22% and 26%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)