Inhibition by Neurotoxic Phospholipases A2 of Synaptosomal Uptake of γ-Aminobutyric Acid

A comparison has been made of the abilities of several neurotoxic and nontoxic phospholipases A₂ from snake venoms to inhibit the intake of γ-aminobutyric acid into synaptosomes from rat cerebral cortex. The neurotoxic phospholipases A₂ inhibited GABA uptake more than the nontoxic enzymes did. However, there was a poor correlation between the measured specific enzyme activity of a phospholipase A₂ and its ability to inhibit the uptake of GABA.     

Mutual Inhibition Kinetic Analysis of γ-Aminobutyric Acid, Taurine, and β-Alanine High-Affinity Transport into Neurons and Astrocytes: Evidence for Similarity Between the Taurine and β-Alanine Carriers in Both Cell Types

The transport kinetics of gamma-aminobutyric acid (GABA), taurine, and beta-alanine in addition to the mutual inhibition patterns of these compounds were investigated in cultures of neurons and astrocytes derived from mouse cerebral cortex. A high-affinity uptake system for each amino acid was demonstrated both in neurons (Km GABA = 24.9 +/- 1.7 microM; Km Tau = 20.0 +/- 3.3 microM; Km beta-Ala = 73.0 +/- 3.6 microM) and astrocytes (Km GABA = 31.4 +/- 2.9 microM, Km Tau = 24.7 +/- 1.3 microM; Km beta-Ala = 70.8 +/- 3.6 microM). The maximal uptake rates (Vmax) determined were such that, in neurons, Vmax GABA greater than Vmax beta-Ala = Vmax Tau, whereas in astrocytes, Vmax beta-Ala greater than Vmax Tau = Vmax GABA. Taurine was found to inhibit beta-alanine uptake into neurons and astrocytes in a competitive manner, with Ki values of 217 microM in neurons and 24 microM in astrocytes. beta-Alanine was shown to inhibit taurine uptake in neurons and astrocytes, also in a competitive manner, with Ki values of 72 microM in neurons and 71 microM in astrocytes. However, beta-alanine was found to be a weak noncompetitive inhibitor of neuronal and astrocytic GABA uptake, whereas in reverse experiments, GABA displayed weak noncompetitive inhibition of neuronal and astrocytic uptake of beta-alanine. Likewise, taurine was a weak noncompetitive inhibitor of GABA uptake in neurons and similarly, GABA was a weak noncompetitive inhibitor of taurine uptake into neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Triethyllead Inhibits γ-Aminobutyric Acid Binding to Uptake Sites in Synaptosomal Membranes

Triethyllead (TEL), the active metabolite of tetraethyllead, was shown previously to inhibit selectively high-affinity Na+-dependent uptake of gamma-aminobutyric acid (GABA) into cerebrocortical synaptosomes. Such inhibition was not related to the Na+ gradient, Na+,K+-ATPase activity, [Cl-], or energy charge. We report here that TEL inhibits GABA binding to the presynaptic transporter involved in Na+-dependent uptake. Scatchard plot analysis of Na+-dependent [3H]GABA binding to a highly purified synaptic plasma membrane preparation revealed that 25 microM TEL reduced the Bmax by 44%, leaving the KD unchanged. This binding was reversible and predominantly involved membrane uptake sites, as characterized by pharmacological specificity to GABA ligands. Approximately 85% of specific GABA binding was considered membrane uptake site binding, as indicated by sensitivity to nipecotic acid and diaminobutyric acid, with relative insensitivity to muscimol, bicuculline methiodide, baclofen, and beta-alanine. With respect to previous data, these finding suggest that TEL inhibits Na+-sensitive high-affinity GABA uptake by interfering with GABA binding to its presynaptic transporter.     

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.     

Superoxide Radical-Mediated Alteration of Synaptosome Membrane Structure and High-Affinity γ-[14C]Aminobutyric Acid Uptake

Mouse cortical synaptosomal structure and function are altered when exposed to hypoxanthine/xanthine oxidase (HPX/XOD)-generated active oxygen/free radical species. The structure of both the synaptic vesicle and plasma membrane systems are altered by HPX/XOD treatment. The alteration of synaptic vesicle structure is exhibited by a significant increase in the cumulative length of nonsynaptic vesicle membrane per nerve terminal. With respect to the nerve terminal plasma membrane, the length of the perimeter of the synaptosome is increased as the membrane pulls away from portions of the terminal in blebs. The functional lesion generated by HPX/XOD treatment results in a reduction in selective high-affinity gamma-[14C]aminobutyric acid (GABA) uptake. Kinetic analysis of the reduction in high-affinity uptake reveals that the Vmax is significantly altered whereas the Km is not. Preincubation with specific active oxygen/free radical scavengers indicates that the super-oxide radical is directly involved. This radical, most probably in the protonated perhydroxyl form, initiates lipid peroxidative damage of the synaptosomal membrane systems. Low-affinity [14C]GABA transport is unaltered by the HPX/XOD treatment. The apparent ineffectiveness of free radical exposure on low-affinity [14C]GABA transport coupled with its effectiveness in reducing high-affinity transport supports the idea that two separate and different amino acid uptake systems exist in CNS tissue, with the high-affinity being more sensitive (lipid-dependent) and/or more energy-dependent (Na+,K+-ATPase) than the low-affinity system.     

Release of Endogenous Taurine and γ-Aminobutyric Acid from Brain Slices from the Adult and Developing Mouse

The spontaneous and potassium-stimulated release of endogenous taurine and gamma-aminobutyric acid (GABA) from cerebral cortex and cerebellum slices from adult and developing mice was studied in a superfusion system. The spontaneous release of GABA was of the same magnitude in slices from adult and developing mice, but the spontaneous release of taurine was considerably greater in the adults. The potassium-stimulated release of GABA from cerebral cortex slices was about five times greater in adult than in 3-day-old mice, but the potassium-stimulated release of taurine was more than six times greater in 3-day-old than in adult mice. In cerebellar slices from 7-day-old mice, potassium stimulation also evoked a massive release of taurine, whereas the evoked release from slices from adult mice was rather negligible. Also in cerebellar slices the potassium-stimulated release of GABA exhibited the opposite quantitative pattern. The stimulated release of both GABA and taurine was partially calcium dependent. The results suggest that taurine may be an important regulator of excitability in the developing brain.     

Antibodies to the Human γ2 Subunit of the γ-Aminobutyric AcidA/Benzodiazepine Receptor

Abstract: A γ-aminobutyric acid_A (GABA_A) receptor (GABA_AR) γ₂ subunit (short form) was cloned from an adult human cerebral cortex cDNA library in bacteriophage λgt11. The 261-bp intracellular loop (IL) located between M3 and M4 was amplified using the polymerase chain reaction and inserted into the expression vectors λgt11 and pGEX-3X. Both γ-galactosidase (LacZ) and glutathione-S-transferase (GST) fusion proteins containing the γ₂IL were purified, and a rabbit antibody to the LacZ–γ₂IL was made. The antibody reacted with the γ₂IL of both LacZ and GST fusion proteins and immunoprecipitated the GABA_AR/ benzodiazepine receptor (GABA_AR/BZDR) from bovine and rat brain. The antibody reacted in affinity-purified GABA_AR/BZDR immunoblots with a wide peptide band of 44,000–49,000 M_r. Immunoprecipitation studies with the anti-γ₂IL antibody suggest that in the cerebral cortex, 87% of the GABA_ARs with high affinity for benzodiazepines and 70% of the GABA_ARs with high affinity for muscimol contain at least a γ subunit, probably a γ₂. These results indicate that there are [³H]muscimol binding GABA_ARs that do not bind [³H]flunitrazepam with high affinity. Immunoprecipitations with this and other anti-GABA_AR/BZDR antibodies indicate that the most abundant combination of GABA_AR subunits in the cerebral cortex involves α₁, γ₂ (or other γ), and β₂ and/or β₃ subunits. These subunits coexist in >60% of the GABA_AR/BZDRs in the cerebral cortex. The results also show that a considerable proportion (20–25%) of the cerebellar GABA_AR/BZDRs is clonazepam insensitive. At least 74% of these cerebellar receptors, which likely contain α₆, also contain γ₂ (or other γ) subunit(s). The α₁ and β₂ or β₃ subunits are also frequently associated with γ₂ (or other γ) and α₆ in these cerebellar receptors.     

Anticonvulsant Activity of Muscimol and γ-Aminobutyric Acid Against Pyridoxal Phosphate-Induced Epileptic Seizures

The intracerebroventricular injection of pyridoxal phosphate (PLP, 0.125-1.25 μmol/rat) causes epileptic seizures (4 min → 1 min) that are preventable or reversible by GABA (1 μmol/rat), by muscimol (O.025 μmol/rat), or by diazepam (1.75 μmol/rat). At the peak of PLP-induced convulsions, the activities of GAD and GABA-T in 14 regions of rat brain remained unaltered, whereas the concentrations of PLP remained elevated. The PLP-induced convulsion was blocked by DABA (10 μmol/rat) but was not altered by β-alanine (50 μmol/rat). The previous in vitro studies have shown that PLP increases the uptake of [³H]GABA into synaptosomes and inhibits the binding of [³H]GABA to synaptic membranes. These data suggest that PLP-induced convulsion is due to reduced availability of GABA to its recognition sites, rather than to alteration in the activity of GABA metabolizing enzymes, or unavailability of PLP as a coenzyme for GAD and GABA-T. Since the duration of PLP-induced epileptic seizures is short and can be prevented by GABA agonists, PLP may be used as a tool to study the nature of GABA-mediated neuroinhibition and the properties of GABA receptor sites.     

γ-Aminobutyric Acid-Activated 36Cl- Influx: A Functional In Vitro Assay for CNS γ-Aminobutyric Acid Receptors of Insects

The effects of gamma-aminobutyric acid (GABA) on the uptake of 36Cl- into a membrane microsac preparation from isolated nerve cords of the cockroach Periplaneta americana was studied. On addition of 1 microM GABA (after 4-s incubation, then rapid quenching) the influx of 36Cl- was stimulated to a level 75% above that of the control value. This stimulation was reduced by picrotoxin (100 microM), but was not significantly affected by bicuculline (100 microM). Results of 36Cl- influx experiments are in agreement with data obtained from radiolabelled ligand binding assays and electrophysiological investigations on the same tissue. The method described represents a functional in vitro assay for CNS GABA receptors of insects.     

Stimulation of Synaptosomal γ-Aminobutyric Acid Synthesis by Glutamate and Glutamine

gamma-Aminobutyric acid (GABA) synthesis was studied in rat brain synaptosomes by measuring the increase of GABA level in the presence of the GABA-transaminase inhibitor gabaculine. The basal rate of synaptosomal GABA synthesis in glucose-containing medium (25.9 nmol/h/mg of protein) was only 3% of the maximal activity of glutamate decarboxylase (GAD; 804 +/- 83 nmol/h/mg of protein), a result indicating that synaptosomal GAD operates at only a small fraction of its catalytic capacity. Synaptosomal GABA synthesis was stimulated more than threefold by adding 500 microM glutamine. Glutamate also stimulated GABA synthesis, but the effect was smaller (1.5-fold). These results indicate that synaptosomal GAD is not saturated by endogenous levels of its substrate, glutamate, and account for part of the unused catalytic capacity. The greater stimulation of GABA synthesis by glutamine indicates that the GAD-containing compartment is more accessible to extrasynaptosomal glutamine than glutamate. The strong stimulation by glutamine also shows that the rates of uptake of glutamine and its conversion to glutamate can be sufficiently rapid to support GABA synthesis in nerve terminals. Synaptosomes carried out a slow net synthesis of aspartate in glucose-containing medium (7.7 nmol/h/mg of protein). Aspartate synthesis was strongly stimulated by glutamate and glutamine, but in this case the stimulation by glutamate was greater. Thus, the larger part of synaptosomal aspartate synthesis occurs in a different compartment than does GABA synthesis.     

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

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

Effect of Pressure on the Release of Radioactive Glycine and γ-Aminobutyric Acid from Spinal Cord Synaptosomes

Exposure to high hydrostatic pressure produces neurological changes referred to as the high-pressure nervous syndrome (HPNS). Manifestations of HPNS include tremor, EEG changes, and convulsions. These symptoms suggest an alteration in synaptic transmission, particularly with inhibitory neural pathways. Because spinal cord transmission has been implicated in HPNS, this study investigated inhibitory neurotransmitter function in the cord at high pressure. Guinea pig spinal cord synaptosome preparations were used to study the effect of compression to 67.7 atmospheres absolute on [3H]glycine and [3H]gamma-aminobutyric acid ([3H]GABA) release. Pressure was found to exert a significant suppressive effect on the depolarization-induced calcium-dependent release of glycine and GABA by these spinal cord presynaptic nerve terminals. This study suggests that decreased tonic inhibitory regulation at the level of the spinal cord contributes to the hyperexcitability observed in animals with compression to high pressure.     

In Vivo Changes in γ-Aminobutyric Acid Output from the Cerebral Cortex Induced by Inhibitors of γ-Aminobutyric Acid Uptake and Metabolism

Abstract: The effects of inhibitors of γ-aminobutyric acid (GABA) metabolism or uptake on GABA output from the cerebral cortex was studied by means of a collecting cup placed on the exposed cortex of rats anaesthetized with urethane. GABA was identified and quantified by a mass-fragmentographic method. Ethanolamine-O-sulphate (10⁻² M ) applied directly on the cerebral cortex caused a long-lasting twofold increase in GABA output, whereas dl -2, 4-diaminobutyric acid (5 × 10⁻³ M ) caused a sevenfold increase and β -alanine was inactive. The results indicate that glial uptake has little effect on GABA inactivation in the cerebral cortex. The inhibition of neuronal uptake seems a more effective tool to increase GABA concentration in the synaptic cleft, and consequently also in GABA output, than the inhibition of GABA metabolism.     

Sulfur Amino Acid Metabolism in the Developing Rhesus Monkey Brain: Subcellular Studies of Taurine, Cysteinesulfinic Acid Decarboxylase, γ-Aminobutyric Acid, and Glutamic Acid Decarboxylase

Abstract: Taurine, cysteinesulfinic acid decarboxylase (CSAD), glutamate, γ-aminobutyric acid (GABA), and glutamic acid decarboxylase (GAD) were measured in subcellular fractions prepared from occipital lobe of fetal and neonatal rhesus monkeys. In addition, the distribution of [³⁵S]taurine in subcellular fractions was determined after administration to the fetus via the mother, to the neonate via administration to the mother prior to birth, and directly to the neonate at various times after birth. CSAD, glutamate, GABA, and GAD all were found to be low or unmeasurable in early fetal life and to increase during late fetal and early neonatal life to reach values found in the mother. Taurine was present in large amounts in early fetal life and decreased slowly during neonatal life, arriving at amounts found in the mother not until after 150 days of age. Significant amounts of taurine, CSAD, GABA, and GAD were associated with nerve ending components with some indication that the proportion of brain taurine found in these organelles increases during development. All subcellular pools of taurine were rapidly labeled by exogenously administered [³⁵S]taurine. The subcellular distribution of all the components measured was compatible with the neurotransmitter or putative neuro-transmitter functions of glutamate, GABA, and taurine. The large amount of these three amino acids exceeds that required for such function. The excess of glutamate and GABA may be used as a source of energy. The function of the excess of taurine is still not clear, although circumstantial evidence favors an important role in the development and maturation of the CNS.     

Quantitative Immunoprecipitation Studies with Anti-γ-Aminobutyric AcidA Receptor γ2 1–15 Cys Antibodies

Antibodies raised against the synthetic peptide NH2-QKSDDDYEDYASNKTC-COOH (gamma 2 1-15 Cys), which corresponds to the N-terminal amino acid sequence with a C-terminal cysteine of the human gamma 2 subunit of the gamma-aminobutyric acidA (GABAA) receptor, were used to study the quantitative immunoprecipitation of agonist benzodiazepine binding sites from bovine brain. Anti-gamma 2 1-15 Cys antibodies were found to immunoprecipitate specifically in parallel [3H]flunitrazepam- and [3H]muscimol-reversible binding sites in a dose-dependent manner. The maximum percentages of [3H]flunitrazepam binding sites immunoprecipitated from detergent extracts of bovine cerebral cortex, cerebellum, and hippocampus were 68, 77, and 83%, respectively. Immunoprecipitation studies with anti-alpha 1 324-341 antibodies carried out in parallel with anti-gamma 2 1-15 Cys antibodies provided evidence for the promiscuity of the gamma 2 subunit within native GABAA receptors. These results substantiate the association of the gamma 2 polypeptide with native GABAA receptors.     

Mass Fragmentographic Determination of γ-Aminobutyric Acid and Glutamic Acid in Discrete Amygdaloid Nuclei of Rat Brain

A mass fragmentographic method for the simultaneous quantification of gamma-aminobutyric acid (GABA) and glutamic acid is described. In a convenient one-step reaction, the two amino acids were derivatized with pentafluoropropionic anhydride and pentafluoropropanol. The derivatization products were stable for several days. The technique has been applied to the assay of GABA and Glu in five amygdaloid nuclei of the rat brain. The GABA level was high in the central and medial nuclei, whereas the Glu level was high in the lateral and basal nuclei. The regional distribution of GABA was different from that of Glu within the amygdaloid nuclei.     

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.     

Generation of Two Forms of the γ-Aminobutyric AcidA Receptor γ-2-Subunit in Mice by Alternative Splicing

gamma-Aminobutyric acidA (GABAA) receptors are multisubunit ligand-gated ion channels which mediate neuronal inhibition by GABA and are composed of at least four subunit types (alpha, beta, gamma, and delta). The gamma 2-subunit appears to be essential for benzodiazepine modulation of GABAA receptor function. In cloning murine gamma 2-subunits, we isolated cDNAs encoding forms of the subunit that differ by the insertion of eight amino acids. LLRMFSFK, in the major intracellular loop between proposed transmembrane domains M3 and M4. The two forms of the gamma 2-subunit are generated by alternative splicing, as demonstrated by cloning and partial sequencing of the corresponding gene. The eight-amino-acid insertion encodes a potential consensus serine phosphorylation site for protein kinase C. These results suggest a novel mechanism for the regulation of the GABAA receptor by protein phosphorylation.     

Glutamic Acid Decarboxylase and γ-Aminobutyric Acid in Huntington''s Disease Fibroblasts and Other Cultured Cells, Determined by a [3H]Muscimol Radioreceptor Assay

A sensitive and reproducible [3H]muscimol radioreceptor assay was developed for measuring low levels of both glutamic acid decarboxylase activity and gamma-aminobutyric acid. By using this technique, endogenous gamma-aminobutyric acid and glutamic acid decarboxylase activity were detected in two rat neuroblastomas, B35 and B50, a human medulloblastoma cell line, TE671, and cultured human skin fibroblasts. Glutamic acid decarboxylase activities and gamma-aminobutyric acid levels were compared for human skin fibroblasts obtained from patients with Huntington's disease and their controls in a well-controlled, blind study. However, no significant difference was found to either measure between Huntington and control cells. Glutamic acid decarboxylase activity was relatively low in all cell types examined except for the TE671 cells, which had more than four times the activity found in the other cells. This human medulloblastoma cell line appears to be a good model for studying gamma-aminobutyric acid metabolism and the control of glutamic acid decarboxylase expression.     

γ-Aminobutyric Acid: Temperature Dependence in Benzodiazepine Receptor Binding

The effects of muscimol and/or incubation temperature on the inhibition of [3H]flunitrazepam receptor binding by benzodiazepine receptor ligands were investigated. At 0 degree C muscimol decreased the Ki values for some ligands as displacers of [3H]flunitrazepam binding to brain-specific sites while increasing or having no effect on the Ki values for other ligands. The Ki values for some ligands are higher at 37 degrees C than at 0 degree C but are reduced by muscimol at both 0 degrees and 37 degrees C. In contrast, the ligands whose Ki values are increased by muscimol either decreased or did not alter the Ki values at 37 degrees C as compared to those at 0 degree C. Incubation of membranes at 37 degrees C for 30 min accelerated gamma-aminobutyric acid (GABA) release by 221% over that at 0 degree C. These results indicate that changes in incubation temperature alter benzodiazepine receptor affinity for ligands via GABA.