GABA inactivation at the crayfish neuromuscular junction

Changes in the effective membrane resistance of the abductor muscle of the dactylopodite of the crayfish were used to indicate changes in the GABA concentration in the synaptic cleft. Following bath application of GABA (10^?5 to 5 × 10^?5M), the muscle membrane resistance decreased and then increased slowly over the next few minutes. Renewing the solution or stirring the bath restored the GABA effect. Higher GABA concentrations produced a large stable decrease in membrane resistance. An active uptake system for GABA in the junctional region is suggested by the observation that the slow increase in membrane resistance following GABA application was decreased by cooling to 2°C or by the addition of known GABA uptake blockers such as L -DABA, β-guanidinopropionic acid, or nipecotic acid. The transport inhibitors, PCMBS and chlorpromazine, produced irreversible decreases in muscle membrane resistance, which precluded examining their effects on GABA inactivation. The decrease in GABA effect was not dependent on the external sodium concentration or on the degree of receptor activation. Nipecotic acid, which blocked GABA inactivation, did not affect the decay of the neurally evoked inhibitory junctional potential.     

Effect of Homo-β-Proline and Other Heterocyclic GAB A Analogues on GABA Uptake in Neurons and Astroglial Cells and on GABA Receptor Binding

Abstract: Two groups of GABA (γ-aminobutyric acid) analogues, one comprising derivatives of β-proline and the other compounds structurally related to nipecotic acid, were investigated as potential inhibitors of high-affinity GABA transport in neurons and glial cells, as well as displacers of GABA receptor binding. In addition to cis -4-hydroxynipecotic acid, which is known as a potent inhibitor of GABA uptake, homo-β-proline was the only compound which proved to be a potent inhibitor of glial as well as neuronal GABA uptake. IC₅₀ values for GABA uptake into glial cells and brain cortex "prisms" were 20 and 75 μM, respectively, and the IC₅₀ value obtained for GABA uptake into cultured neurons was 10 μM. A kinetic analysis of the action of homo-β-proline on GABA uptake into cultured astrocytes and neurons showed that this compound acts as a competitive inhibitor of GABA uptake in both cell types. From the apparent K _m values, K _i values for homo-β-proline of 16 and 6 μM could be calculated for glial and neuronal uptake, respectively. This mechanism of action strongly suggests that homo-β-proline interacts with the GABA carriers. Furthermore, homo-β-proline also displaced GABA from its receptor with an IC₅₀ value of 0.3 μM. The cis -4-hydroxynipecotic acid analogues, cis- and trans-4-mercaptonipecotic acid, had no inhibitory effect on glial or neuronal GABA uptake. Other SH reagents, PCMB, NEM and DTNB, were shown to be relatively weak inhibitors of GABA uptake into cultured astrocytes, suggesting that SH groups are not directly involved in the interaction between GABA and its transport carrier.     

INHIBITION OF GABA UPTAKE IN RAT BRAIN SLICES BY NIPECOTIC ACID, VARIOUS ISOXAZOLES AND RELATED COMPOUNDS

—A variety of isoxazoles structurally related to muscimol (3-hydroxy-5-aminomethylisoxazole) were tested as inhibitors of the uptake of GABA and some other amino acids in rat brain slices, and of the activity of the GABA-metabolizing enzymes l -glutamate 1-carboxylyase and GABA:2-oxo-glutarate aminotransferase. A bicyclic derivative, 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol, proved to be a more potent inhibitor of GABA uptake than muscimol. Structure-activity studies on this derivative, which appeared to be a competitive inhibitor of GABA uptake, led to the findings that nipecotic acid (piperidine-3-carboxylic acid) is a powerful non-competitive inhibitor of GABA uptake, and that perhydro-1,2-oxazine-6-carboxylic acid is a relatively weak competitive inhibitor of GABA uptake.     

Effect of Inhibitors of GABA Transaminase on the Synthesis, Binding, Uptake and Metabolism of GABA

Abstract: Four catalytic inhibitors of GABA aminotransferase (gabaculine, γ-acetylenic GABA, γ-vinyl GABA, ethanolamine O -sulphate) as well as aminooxyacetic acid and valproate were studied for effects on neurochemical assays for GABA synthesis, receptor binding, uptake and metabolism in mouse and rat brain preparations. Gabaculine did not interfere with GABA synthesis as reflected by the activity of glutamate decarboxylase (GAD), it was only a weak inhibitor (IC₅₀= 0.94 mM) of GABA receptor binding sites but was a moderately potent inhibitor of GABA uptake (IC₅₀= 81 μM) and very potent (IC₅₀= 1.8 μM) with respect to inhibition of the GABA-metabolizing enzyme GABA aminotransferase (GABA-T). γ-Acetylenic GABA was a weak inhibitor of GAD and GABA binding (IC₅₀ > 1 mM), but virtually equipotent to inhibit uptake and metabolism of GABA (IC₅₀ 560 and 150 μM, respectively). This was very similar to γ-vinyl GABA, except that this drug did not decrease GAD activity. Ethanolamine O -sulphate was found to show virtually no inhibition of GAD and GABA uptake, but was a fairly potent inhibitor of GABA binding (IC₅₀= 67 μM) and in this respect, 500 times more potent than as an inhibitor of GABA-T. Aminooxyacetic acid was a powerful inhibitor of both GAD and GABA-T (IC₅₀ 14 and 2.7 μM, respectively), but had very little affinity to receptor and uptake sites for GABA. Valproate showed no effects on GABA neurochemical assays which could be related to anticonvulsant action. The present results suggest that the anticonvulsant properties of the four catalytic inhibitors of GABA-T tested are at least in part mediated through a direct influence on GABA receptors and uptake sites.     

Inhibitors of the GABA uptake systems

Summary This review describes a novel class of heterocyclic GABA uptake inhibitor with no affinity for the GABA receptors. The parent compound nipecotic acid is a potent inhibitor of neuronal and glial GABA uptake, and nipecotic acid is a substrate for the transport carriers concerned. The structurally related cyclic amino acids guvacine and cis-4-hydroxynipecotic acid are also potent inhibitors of both GABA transport systems. Even minor structural alterations of these compounds result in considerable or complete loss of activity. Whereas homonipecotic acid is a weak but selective inhibitor of glial GABA uptake, homoguvacine is virtually inactive. Similarly the lower homologues of nipecotic acid and guvacine, -proline and 3-pyrroline-3-carboxylic acid, respectively, show some selectivity with respect to inhibition of glial GABA uptake, but these compounds are much weaker than the parent compounds. The bicyclic compounds THPO and THAO, in which the carboxyl groups of nipecotic acid and homonipecotic acid have been replaced by 3-isoxazolol units are moderately potent and practically specific inhibitors of glial GABA uptake. cis-4-Mercaptonipecotic acid is considerably weaker than the closely related analogue cis-4-hydroxynipecotic acid, but the former compound may interact irreversibly with the GABA transport carriers.The results demonstrate a pronounced substrate specificity of the glial and in particular the neuronal GABA transport system. It is evident that the GABA molecule is transported in a conformation different from that, in which it activates its receptors. These findings are of importance for the development of drugs for selective pharmacological regulation of the functions of central GABA-mediated synapses in certain neurological diseases.     

The metabolism of gamma-aminobutyric acid (GABA) in the lobster nervous system--uptake of GABA in the nerve-muscle preparations

The lack of information on the mechanism of inactivation of the crustacean neuromuscular inhibitory transmitter compound prompted a study of the disposition of radioactive γ-aminobutyric acid (GABA) in lobster nerve-muscle preparations. A specific GABA transport system was found. Radioactive GABA was concentrated by the tissues to levels several times those in the medium, and net uptake could be demonstrated. The process was dependent on sodium ions in the medium; neither lithium nor choline could substitute for sodium. Incubations with increasing GABA concentrations indicated that uptake was a saturable mechanism with an apparent K_m of 5.8 × 10⁻⁵m . Of many compounds tested, only desmethylimipramine, chlopromazine (and several related compounds), and certain close structural analogues (guanidinoacetic acid, β-guani-dinopropionic acid and,β-hydroxy-GAB A) were effective inhibitors of uptake. The inhibition with all these compounds, however, was at high concentrations (5 × 10⁻⁴ to 10⁻³m ) which limited their usefulness for physiological studies. A separate uptake mechanism for glutamate was found in the lobster nerve-muscle preparations. This process was not described in detail, but certain properties are similar to those of the GABA transport system. The cellular location of the GABA uptake system remains unknown. By analogy with noradrenaline inactivation, however, it is postulated that uptake could serve to terminate the physiological actions of GABA by rapidly removing it from its sites of action in synaptic clefts.     

INHIBITION OF THE UPTAKE OF GABA AND RELATED AMINO ACIDS IN RAT BRAIN SLICES BY THE OPTICAL ISOMERS OF NIPECOTIC ACID

R(-)-Nipecotic acid was a more potent inhibitor than the S(+)-isomer of the uptake of GABA, (+)-nipecotic acid, and β-alanine in rat brain slices. (-)-Nipecotic acid was an order of magnitude more potent as an inhibitor of GABA uptake than as an inhibitor of β-alanine uptake, whereas the (+)-isomer was less selective. (–)-Nipecotic acid was a weak inhibitor of L-proline uptake and of rat brain acetylcholinesterase activity. Kinetic studies showed that both isomers of nipecotic acid were competitive inhibitors of GABA uptake when added at the same time as GABA, but non-competitive inhibitors when preincubated with the tissue for 15 min before addition of GABA. The apparent slope inhibition constants, which were not influenced by preincubation, indicated that (–)-nipecotic acid has an affinity for the carrier some 5 times higher than that for (+)-nipecotic acid. (–)-Nipecotic acid stimulated the release of preloaded radioactive GABA from rat brain slices. These observations indicate that (–)-nipecotic acid is a substrate-competitive inhibitor of GABA which combines with the GABA carrier and is taken up. (?)-Nipecotic acid and (+)-2,4-diaminobutyric acid, on the basis of their absolute structures and inhibition kinetics, are proposed to interact in a similar way with the GABA transport system.     

STRUCTURE-ACTIVITY STUDIES ON THE INHIBITION OF GABA BINDING TO RAT BRAIN MEMBRANES BY MUSCIMOL AND RELATED COMPOUNDS

Abstract— A series of compounds structurally related to muscimol (5-aminomethyl-3-isoxazolol) was tested as inhibitors of the sodium-independent binding of GABA to membranes from rat brain. Muscimol, 5-(l-aminoethyl)-3-isoxazolol, 5-(2-aminoethyl)-3-isoxazolol (homomuscimol), and the bicyclic derivative 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) were relatively potent inhibitors of GABA binding. THIP is an analogue of muscimol locked in a folded conformation. The structurally related compound 1,2,3,6-tetrahydropyridine-4-carboxylic acid (isoguvacine), a semirigid analogue of trans-4-aminocrotonic acid, was also a potent inhibitor of GABA binding. Apart from muscimol, these inhibitors of GABA binding did not influence the sodium-dependent,'high-affinity' uptake of GABA in rat brain slices, whereas the potent GABA uptake inhibitors guvacine and nipecotic acid did not influence GABA binding. The present results support previous findings that different conformational modes of GABA interact with GABA postsynaptic receptors and the neuronal GABA transport system in rat brain, and indicate that the 'active conformation' of GABA with respect to the receptors is partially folded and almost planar. Based on a comparison of the present results with previous in vivo studies the structural requirements for GABA-like activity in rat cerebral cortex and cat spinal cord seem to be somewhat different.     

The Uptake of GABA in Trypanosoma cruzi

Gamma aminobutyric acid (GABA) is widely known as a neurotransmitter and signal transduction molecule found in vertebrates, plants, and some protozoan organisms. However, the presence of GABA and its role in trypanosomatids is unknown. Here, we report the presence of intracellular GABA and the biochemical characterization of its uptake in Trypanosoma cruzi, the etiological agent of Chagas' disease. Kinetic parameters indicated that GABA is taken up by a single transport system in pathogenic and nonpathogenic forms. Temperature dependence assays showed a profile similar to glutamate transport, but the effect of extracellular cations Na⁺, K⁺, and H⁺ on GABA uptake differed, suggesting a different uptake mechanism. In contrast to reports for other amino acid transporters in T. cruzi, GABA uptake was Na⁺ dependent and increased with pH, with a maximum activity at pH 8.5. The sensitivity to oligomycin showed that GABA uptake is dependent on ATP synthesis. These data point to a secondary active Na⁺/GABA symporter energized by Na⁺‐exporting ATPase. Finally, we show that GABA occurs in the parasite's cytoplasm under normal culture conditions, indicating that it is regularly taken up from the culture medium or synthesized through an still undescribed metabolic pathway.     

Effects of 3-hydroxy-4-amino-4,5,6,7-tetrahydro-1,2-benzisoxazol (exo-THPO) and its N-substituted analogs on GABA transport in cultured neurons and astrocytes and by the four cloned mouse GABA transporters

The system of GABA transporters in neural cells constitutes an efficient mechanism for terminating inhibitory GABAergic neurotransmission. As such these transporter are important therapeutical targets in epilepsy and potentially other neurological diseases related to the GABA system. In this study a number of analogs of 3-hydroxy-4-amino-4,5,6,7-tetrahydro-1,2-benzisoxazol (exo-THPO), a promising lead structure for inhibitors of GABA uptake were investigated. It was found that the selectivity of N-acetyloxyethyl-exo-THPO for inhibition of the astroglial GABA uptake system was 10-fold as compared to inhibition of the neuronal GABA uptake system. Selectivity in this magnitude may provide potent anti-convulsant activity as has recently been demonstrated with the likewise glia-selective GABA uptake inhibitor, N-methyl-exo-THPO. In contrast to the competitive inhibition of GABA uptake exhibited by N-substituted analogs of 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol (THPO), nipecotic acid, and guvacine, N-4,4-diphenyl-3-butenyl(DPB)-N-methyl-exo-THPO and 4-phenylbutyl-exo-THPO exhibited non-competitive type inhibition kinetics. The lipophilic character of a number of GABA analogs was concluded by far to constitute the determining factor for the potency of these compounds as inhibitors of GAT1-mediated uptake of GABA. This finding underscores the complexity of the pharmacology of the GABA transport system, since these non-competitive inhibitors are structurally very similar to some competitive GABA uptake inhibitors. Whether these structure-activity relationships for inhibition of GABA uptake may provide sufficient information for the development of new structural leads and to what extent these compounds may be efficient as therapeutical anti-convulsant agents remain to be elucidated.     

Developmental changes in high-affinity uptake of GABA by cultured neurons

High-affinity uptake of [³H]-aminobutyric acid (GABA) was studied in cultures of neonatal rat cortical neurons grown on pre-formed monolayers of non-neuronal (glial) cells. Both the maximum rate (V _max) and, to a smaller extent, theK _m of [³H]GABA uptake increased with time. In addition, in parallel with these changes, 2,4-diaminobutyric acid and cis-3-aminocyclohexane-1-carboxylic acid (ACHC), compounds which are considered typical substrate/inhibitors of GABA uptake in neurons, became progressively stronger inhibitors of [³H]GABA uptake. Consequently, the present results may mean that the studies using uptake, of [³H]GABA, [³H]ACHC, or [³H]DABA as a specific marker for GABAergic neurons differentiating during the ontogenetic development of the central nervous system may have to be interpreted with caution.     

COMPETITIVE INHIBITION OF GABA UPTAKE IN RAT BRAIN SLICES BY SOME GABA ANALOGUES OF RESTRICTED CONFORMATION

Abstract— trans -4-aminocrotonic acid, dl - cis -aminocyclohexane-ltarboxylic acid and 4-aminotetrolic acid were found to be competitive inhibitors of GABA uptake in rat brain slices. These inhibitors are analogues of extended conformations of GABA, which indicates that these conformations are important in the initial binding of this inhibitory transmitter to its transport carrier.     

The Caenorhabditis elegans snf-11 gene encodes a sodium-dependent GABA transporter required for clearance of synaptic GABA

Sodium-dependent neurotransmitter transporters participate in the clearance and/or recycling of neurotransmitters from synaptic clefts. The snf-11 gene in Caenorhabditis elegans encodes a protein of high similarity to mammalian GABA transporters (GATs). We show here that snf-11 encodes a functional GABA transporter; SNF-11-mediated GABA transport is Na+ and Cl- dependent, has an EC50 value of 168 microM, and is blocked by the GAT1 inhibitor SKF89976A. The SNF-11 protein is expressed in seven GABAergic neurons, several additional neurons in the head and retrovesicular ganglion, and three groups of muscle cells. Therefore, all GABAergic synapses are associated with either presynaptic or postsynaptic (or both) expression of SNF-11. Although a snf-11 null mutation has no obvious effects on GABAergic behaviors, it leads to resistance to inhibitors of acetylcholinesterase. In vivo, a snf-11 null mutation blocks GABA uptake in at least a subset of GABAergic cells; in a cell culture system, all GABA uptake is abolished by the snf-11 mutation. We conclude that GABA transport activity is not essential for normal GABAergic function in C. elegans and that the localization of SNF-11 is consistent with a GABA clearance function rather than recycling.     

Development of a high-affinity GABA uptake system in embryonic amphibian spinal neurons

High-affinity uptake systems for amino acid neurotransmitter precursors have been highly correlated with the use of the particular amino acid or its derivative as a transmitter. We have found interneurons in the Xenopus embryo spinal cord which accumulate GABA by a high-affinity uptake system. They originate near the end of gastrulation and their ability to accumulate GABA first appears at the early tail bud stage. By position and appearance they are comparable to some of the embryonic interneurons described by A. Roberts and J. D. W. Clarke (1982, Phil. Trans. R. Soc. London Ser. B 296, 195-212). GABA-accumulating neurons also develop in dissociated cell cultures made from the presumptive spinal cord of neural plate stage Xenopus embryos. GABA accumulation in cultured neurons, as in cells in vivo, occurs via a high-affinity uptake system; GABA-accumulating cells have the same time of origin as the cells in vivo, and the ability to accumulate GABA in the population of cultured neurons appears at a time equivalent to that observed in intact sibling embryos. Thus it seems likely that the population of GABA-accumulating neurons developing in cell culture corresponds to the GABA-accumulating interneurons in vivo. The development of these neurons in dissociated cell cultures permits perturbation experiments that would be difficult to perform in vivo. We have examined the development of high-affinity GABA uptake in conditions that permit no electrical impulse activity in the cultures. The onset and extent of development of GABA accumulation in the neuronal population are normal under these conditions.     

A unique type of GABA binding by Mycobacterium leprae.

Neurotropism is one of the unusual properties of Mycobacterium leprae. The organism contains glutamic acid decarboxylase that generates gamma-amino-butyric acid (GABA) which is an inhibitory neurotransmitter. The binding of GABA by M. leprae in vitro was studied by using 3H-GABA as substrate. The bacteria had high-affinity binding sites for the amino acid. The uptake was a specific saturable process with a Km of 66.7 pM, pH optimum of 7.3 and a temperature optimum of 37 degrees C. The binding did not seem to be time-dependent, being complete in about 5 min. None of the known antagonists and agonists of GABA uptake by neurons, showed any significant effect on M. leprae; the receptors in the bacteria are apparently of a non-neuronal type, and different from those reported in spermatozoa and Pseudomonas.     

EFFECTS OF AMINO-OXYACETIC ACID ON [3H]GABA UPTAKE BY RAT BRAIN SLICES

Abstract— The effect of amino-oxyacetic acid on the uptake of [³H]GABA by rat brain slices was studied. When added simultaneously with [³H]GABA, amino-oxyacetic acid had no significant effect on [³H]GABA uptake. However, preincubation of brain slices with amino-oxyacetic acid prior to addition of [³H]GABA produced inhibition of uptake, which increased with longer duration of preincubation. The inhibitory effect of amino-oxyacetic acid was maximal at 2 mM concentration and concentrations sufficient to inhibit significantly GABA:glutamate transaminase (10^--⁶ M) had no effect on [³H]GABA uptake. D-Cycloserine and β-hydrazino-propionic acid also inhibited [³H]GABA uptake, but the amounts required were considerably in excess of those needed to inhibit GABA:glutamate transaminase. 4-Deoxypyridoxine inhibited [³H]GABA uptake, whether given in vivo or in vitro , and the inhibitory effect of amino-oxyacetic acid was reversed with pyridoxine. GABA transport appears to be dependent on pyridoxal phosphate and interference with this function of the vitamin is suggested as the basis for the inhibitory effect of amino-oxyacetic acid on [³H]GABA uptake.     

CIS- AND TRANS-4-AMINOCROTONIC ACID AS GABA ANALOGUES OF RESTRICTED CONFORMATION

Cis-4-aminocrotonic acid, an analogue of GABA in a folded conformation, appears not to act as a GABA analogue with respect to bicuculline-sensitive postsynaptic receptors, ‘high affinity’ GABA uptake and GABA: 2-oxoglutarate aminotransferase in the mammalian central nervous system. On the other hand, trans-4-aminocrotonic acid, an analogue of GABA in an extended conformation, acts as efficiently as GABA with respect to each of the above systems, indicating that extended rather than folded conformations of GABA are likely to be important in the interaction of GABA with the specific macromolecules concerned.     

GABA concentrations in the striatum following repetitive cerebral ischemia

GABAergic neurons in the striatum are very sensitive to the effects of ischemia. The progressive decline in striatal GABA following transient forebrain ischemia in gerbils may be secondary to either a decreased production or an increase in reuptake mechanisms or both. The current experiment was designed to evaluate release of GABA by stimulation with K+ or inhibition of its uptake with nipecotic acid or their combination (K+ nipecotic) after repetitive forebrain ischemia in gerbils by in-vivo microdialysis on Days 1, 3, 5, and 14 following the insult. Infusion of nipecotic acid or potassium chloride, resulted in a significant increase in extracellular GABA. This response was significantly decreased in the post-ischemic animals. The synergistic effect of increased GABA concentrations by the infusion of nipecotic acid+potassium chloride seen in the controls was not evident in the post-ischemic animals. In conclusion, though there is a reduction in the extracellular GABA concentrations in the first week following an ischemic insult, restorative mechanisms are operative in the second week as seen by the increasing GABA concentrations.     

LOCALIZATION OF THE SITES OF γ-AMINOBUTYRIC ACID (GABA) UPTAKE IN LOBSTER NERVE-MUSCLE PREPARATIONS

The principal sites of γ-aminobutyric acid (GABA) uptake in lobster nerve-muscle preparations have been determined with radioautographic techniques after binding of the amino acid to proteins by aldehyde fixation. Semiquantitative studies showed that about 30% of the radioactive GABA taken into the tissue was bound to protein by fixation. Both light and electron micrographs showed dense accumulations of label over Schwann and connective tissue cell cytoplasm; muscle was lightly labeled, but axons and terminals were almost devoid of label. The possible role of Schwann and connective tissue cells in the inactivation of GABA released from inhibitory axons is discussed.     

NET UPTAKE OF L-GLUTAMATE AND GABA BY HIGH AFFINITY SYNAPTOSOMAL TRANSPORT SYSTEMS

Abstract— Reuptake of neuroactive amino acids by high affinity transport systems in the CNS is thought to terminate the neurotransmitter activity of these substances. This notion has been challenged since the homoexchange of synaptosomal and exogenous L-glutamate and the corresponding homoexchange of synaptosomal and exogenous GABA has been demonstrated. We reported that depolarizing media (56 mM-KCl, 1 mM-CaCl₂) lowers the GABA content of synaptosomes. In such synaptosomes, net and apparent (radioactive) GABA uptake are similar. When rat cortical synaptosomes (1 mg protein/ml) are incubated with 10μM-[¹⁴C] L-glutamate, net and apparent (radioactive) uptake are similar. When the synaptosome levels are decreased to 0.5 mg protein/ml or less, then net uptake becomes a fraction of radioactive uptake (exchange ensues). Net L-glutamate uptake is Na ⁺-dependent and temperature-dependent. Furthermore, a 1 mM concentration of KCl or RbCl supports net L-glutamate and GABA uptake. LiCl, NH₄Cl, CsCl and choline chloride are ineffective. In addition, diaminobutyric acid (but not β -alanine) inhibits net and apparent GABA uptake. The demonstration of net uptake of L-glutamate and GABA by their respective high affinity systems is consonant with the idea that these systems may play a role in neurotransmitter inactivation in the synaptic region.