Amplification by glycine of the effect of the GABA transport inhibitor THPO on synaptosomal GABA level

Mice were injected intramuscularly (2 mmol/kg) with the glia-selective GABA uptake inhibitor 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol (THPO) 60 min prior to sacrifice, or with glycine (10 mmol/kg) 45 min before death, or with a combination of both. After decapitation of the animals, the brains were removed and synaptosomes prepared and analyzed for content of GABA, taurine, glutamine, serine, glutamate and aspartate. While no differences as compared with control animals were found for aspartate, serine and glutamine, synaptosomal GABA levels were increased significantly after injections with either THPO or glycine. The individual effects of THPO and glycine were found to be additive. Taurine levels were decreased to a similar extent in animals which had received either THPO alone or THPO in conjunction with glycine. Treatment with THPO and glycine in combination led to a decrease in the synaptosomal glutamate content. The findings are consistent with the previously observed synergistic anticonvulsant actions of THPO and glycine being mediated via the GABA neurotransmitter system.     

Changes in the Amino Acid Content of Nerve Endings (Synaptosomes) Induced by Drugs that Alter the Metabolism of Glutamate and γ-Aminobutyric Acid

The study was centered on the changes in the amino acid content of nerve endings (synaptosomes) induced by drugs that alter the metabolism of glutamate or gamma-aminobutyric acid (GABA), and that possess convulsant or anticonvulsant properties. The onset of seizures induced by various convulsant agents was associated with a decreased content of GABA and an increased content of glutamate in synaptosomes. The concurrent administration of pyridoxine prevented both the biochemical changes and the convulsions. The administration of gabaculine to mice resulted in large increases in the GABA content of synaptosomes that were counteracted by decreases in glutamate, glutamine, and aspartate levels such that the total content of the four amino acids remained unchanged. The administration of aminooxyacetic acid (0.91 mmol/kg) resulted initially in seizure activity, but subsequently in an anticonvulsant action. No simple relationship existed between the excitable state of the brain induced by aminooxyacetic acid and the changes in the synaptosomal levels of any of the amino acid transmitters. A hypothesis was, however, formulated that explained the convulsant-cum-anticonvulsant action of aminooxyacetic acid on the basis of compartmentation of GABA within the nerve endings.     

Hormonal control of metabolism of gamma-aminobutyric acid in the rat hypothalamus and hippocampus

It has been established that hydrocortisone administration increased the amount of total, free, bound and synaptosomal GABA in the hypothalamus, glutamate decarboxylase activity in the homogenate and synaptosomes and time of the mediator turnover. ACTH administration increased the GABA content and glutamate decarboxylase activity in synaptosomes. The total amino acid content and time of its turnover got higher only with single hormone administration. In the hippocamp hydrocortisone administration increased the total and free GABA contents, its turnover time, glutamate decarboxylase activity in the homogenate and decreased GABA-aminotransferase activity in the homogenate and synaptosomes. The GABA level in synaptosomes grew only with multiple hormone administration. Single administration of ACTH decreased the total GABA content, glutamate decarboxylase activity in the homogenate, while its multiple administration increased the GABA level in synaptosomes followed by a decrease of GABA-aminotransferase activity in the homogenate and synaptosomes. The GABA turnover time fell with single hormone administration and grew with the multiple one. Adrenalectomy induced no changes in the GABA content and activity of its metabolism enzymes in the hypothalamus, however the bound GABA level decreased, while the turnover time increased. In the hippocamp adrenalectomy decreased total, free and synaptosomal GABA contents, glutamate decarboxylase activity in a homogenate and turnover time. Subsequent hydrocortisone administration only partly normalized the revealed changes of the GABA metabolism in the brain structures under adrenalectomy.     

A Comparative Study and Partial Characterization of Multi-Uptake Systems for γ-Aminobutyric Acid

Previous work by the authors had indicated that synaptosome-enriched preparations from the cerebral cortex of the rat contained a high-, a medium-, and a low-affinity uptake system for gamma-aminobutyric acid (GABA). The present study demonstrated that this phenomenon also prevailed in synaptosomes from rat diencephalon, mesencephalon, and cerebellum, although the Vmax values for the high- and medium-affinity systems in the cerebellum were very low relative to those of the other regions. When a different type of preparation containing nerve endings (glomeruli) was obtained from the cerebellum, it possessed a Vmax value for the high-affinity system that was more similar to that for the corresponding system in synaptosomes from the other brain regions. In contrast to the above situation, synaptosomes from rat olfactory bulb lacked the low-affinity uptake system, as did synaptosomes from dog olfactory bulb. The aspartate/glutamate uptake systems, as measured with D-aspartate, provided a regional pattern quite different from those of GABA uptake. Only two uptake systems, a high- and low-affinity system, were observed in all regions tested. All three GABA uptake systems were present in cortical synaptosomes from the mouse, hamster, and guinea pig, and all three systems were sodium dependent, energy dependent, temperature sensitive, and totally inhibited by nipecotic acid.     

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.     

Lipid Peroxidation-Induced Inhibition of γ-Aminobutyric Acid Uptake in Rat Brain Synaptosomes: Protection by Glucocorticoids

Incubation of rat brain synaptosomes with xanthine and xanthine oxidase (X/XO) resulted in an inhibition of gamma-aminobutyric acid (GABA) uptake. The inhibitory effects of X/XO were temperature- and time-dependent, and were characterized by an increased Km for GABA and a decreased Vmax. Inhibition of GABA uptake by X/XO was associated with both the formation of malonyldialdehyde (MDA) and conjugated dienes, indicating that lipid peroxidation was involved. Studies with catalase, superoxide dismutase (SOD), mannitol, and chelated iron suggested that hydroxyl radical (OH X) was probably responsible for the initiation of lipid peroxidation. Both the peroxidation of synaptosomal membranes and the inhibition of GABA uptake by X/XO were enhanced by the addition of ADP and FeCl2. The X/XO-induced inhibition of GABA uptake by synaptosomes could be prevented by preincubation of synaptosomes with certain glucocorticoids prior to X/XO exposure. Methylprednisolone sodium succinate (MPSS), dexamethasone sodium phosphate (DMSP), and prednisolone sodium succinate (PSS) all prevented the inhibition of GABA uptake by X/XO. MPSS was most effective at concentrations around 100 microM, DMSP was slightly more potent, and PSS was optimal at around 300 microM. On the other hand, hydrocortisone sodium succinate (HCSS) was ineffective at preventing X/XO-induced inhibition of GABA uptake at concentrations up to 3 mM. The steroids are presumed to work through a mechanism that blocked the formation of lipid peroxides, as MPSS inhibited the formation of conjugated dienes in synaptosomes exposed to X/XO at a concentration that also protected GABA uptake.     

Interactions of Di-n-Propylacetate, Gabaculine, and Aminooxyacetic Acid: Anticonvulsant Activity and the γ-Aminobutyrate System

Abstract: Di-n-propylacetate (DPA), aminooxyacetic acid (AOAA), and gabaculine were administered alone or in combination to Swiss mice. Six hours after administration of the drugs the anticonvulsant action (against isonicotinic acid hydrazide-induced seizures) of AOAA and DPA combined was less than that of AOAA alone. The cause of this phenomenon appeared to be an interaction between DPA and AOAA with respect to inhibition of GABA-T activity, resulting in a long-term diminished inhibition by AOAA, which in turn led to a lessening of the AOAA-induced elevation in the GABA content of nerve endings (synaptosomes). An excellent correlation was observed between the delay in onset of seizures and the elevation of synaptosomal GABA content.     

Metabolism and Brain Uptake of γ-Aminobutyric Acid in Galactosamine-Induced Hepatic Encephalopathy in Rats

Abstract: Kinetic studies of [³H]γ-aminobutyric acid ([³H]GABA) after an intravenous injection were performed in normal rats and in rats with severe degree of hepatic encephalopathy due to fulminant hepatic failure induced by galactosamine. Moreover, plasma and brain GABA levels, and GABA and glutamic acid decarboxylase activity were studied in some brain areas. After intravenous injection, [3H]GABA disappeared very rapidly in the blood of normal rats, with a prompt increase of ³H metabolites. In comatose rats, a delayed disappearance of [3H]GABA.as parallelled by a lower amount of metabolites, indirectly indicating a peripheral decrease of GABA-transaminase activity. The amount of [³H]GABA in brain was lightly but constantly lower in comatose rats than in controls, indicating that the change in permeability of the blood-brain barrier in hepatic encephalopathy does not affect the [3H]GABA uptake of the brain. Furthermore, the assay of endogenous GABA in blood, whole brain, and brain areas did not show any significant difference in any of the two groups. The finding that glutamic acid decarboxylase activity in brain was reduced, together with the indirect evidence of a reduction in GABA-transaminase, may account for the steady state of GABA in hepatic encephalopathy. However, the reduction in glutamic acid decarboxylase activity is in favor of a functional derangement at the GABA-ergic nerve terminals in this pathological condition.     

GABA in brain tissue of paroxysmal (px) chick

An investigation was made of GABA in whole brain and brain regions of paroxysmal (px) chicks. Whole brain levels of GABA were higher (P less than 0.05) in px chicks than in normal siblings by day 10 post-hatching. GABA in brain areas containing degeneration of auditory and vestibular nuclei and tracts was also found to be higher (P less than 0.05) in px chicks. These data indicate that a dysfunction of the GABA pathway occurs early in the px syndrome, possibly as a causative factor.     

Amino Acid Content of Nerve Endings (Synaptosomes) in Different Regions of Brain: Effects of Gabaculine and Isonicotinic Acid Hydrazide

Abstract: The amino acid content of synaptosomes was determined in six regions of rat brain, and in all regions the five predominant amino acids were glutamate, glutamine, aspartate, taurine, and GABA (γ-aminobutyrate). However, the proportions of the individual amino acids varied considerably from one region to another, the GABA content being particularly high and the taurine content low in synaptosomes from the diencephalon and mesencephalon. Administration of isonicotinic acid hydrazide to rats lowered the synaptosomal GABA level by similar amounts in all brain regions, but the administration of gabaculine resulted in a particularly long-acting elevation in GABA levels in the nerve endings of the diencephalon and mesencephalon. The possibility is raised that the high GABA levels in the nerve terminals of the diencephalon may be involved in the gabaculine-induced lowering of the body temperature of the rats. A constancy in the amount of the synaptosomal pool of "aspartate + glutamate + glutamine + GABA" was observed despite large changes in the relative amounts of the four amino acids brought about by gabaculine.     

γ-Hydroxybutyric Acid in Subcellular Fractions of Rat Brain

The presence of gamma-hydroxybutyric acid (GHB) in synaptosome-enriched fractions of rat brain was ascertained using a GLC technique. The stability of GHB in synaptosomes was evaluated by addition of various gamma-aminobutyric acid (GABA) transaminase (GABA-T) inhibitors, GHB, or ethosuximide to the homogenizing medium. Furthermore, changes in whole brain GHB levels were compared with those in the synaptosomal fraction in animals treated with GABA-T inhibitors, GABA, or ethosuximide. GHB was present in synaptosome-enriched fractions in concentrations ranging from 40 to 70 pmol/mg of protein. There was no evidence for redistribution, leakage, or metabolism of GHB during the preparation of synaptosomes. The elevations of whole brain GHB level associated with GABA-T or ethosuximide treatment were reflected by a parallel increase in synaptosomal GHB content. These data add to the growing evidence that GHB may have neurotransmitter or neuromodulator function.     

The γ-Aminobutyrate Content of Nerve Endings (Synaptosomes) in Mice After the Intramuscular Injection of γ-Aminobutyrate-Elevating Agents: A Possible Role in Anticonvulsant Activity

Abstract: The intramuscular administration of L-cycloserine, gabaculine, and aminooxyacetic acid caused significant, time-dependent increases in the γ-aminobutyric acid (GABA) content of both whole brain and synaptosomalenriched preparations obtained from the tissue, a linear relationship being observed between the two parameters. In contrast, the administration of hydrazine resulted in a large increase in whole brain GABA level, with little change in the synaptosomal GABA content. The key factor in these different responses appeared to be the degree of inhibition of glutamic acid decarboxylase by the drugs. Pretreatment of mice with the GABA-elevating agents resulted in a delay in the onset of seizures, which was related directly to the increase in synaptosomal GABA content. Although the seizures were delayed, they occurred while the GABA content of nerve endings (synaptosomes) was above that in preparations from untreated animals. The decrease in GABA content at the onset of seizures, expressed as a percentage of the level at the time of injection of the convulsant agent, was, however, reasonably constant. A hypothesis to explain these results is proposed.     

Effect of neuroleptics and antidepressants on the process of gamma-aminobutyric acid-H3 uptake by isolated rat brain nerve endings]

The effects of phenothiazine neuropleptics--chlorpromazine, trifluoperazine, fluphenazine and of antidepressants-imipramine and phthoracizine on the GABA-H3 accumulation by synaptosomes of the rat cerebral cortex were studied. All neuroleptics were found to inhibit the process of neurotransmitter uptake by the brain synaptosomes. Antidepressants were less potent. Chlorpromazine had the highest inhibitory effect on GABA uptake and phthoracizine--the lowest. It is suggested that the influence of neurolptics on GABA uptake could play a certain role in the mode of a synaptic action of these drugs.     

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.     

Effect of Nitric Oxide Donors on GABA Uptake by Rat Brain Synaptosomes

The effect of nitric oxide donors and L-arginine on the uptake of GABA was studied in synaptosomes purified from rat brain. The neurotransmitter uptake was significantly reduced by S-nitrosoacetylpenicillamine and by sodium nitroprusside, although in this case to a lesser extent. A slight inhibitory effect was found preincubating rat brain synaptosomes with 1 mM L-arginine as well. The S-nitrosoacetylpenicillamine effect gradually disappeared with decomposition of the substance by exposure to light. The nitric oxide effect appears to be mainly due to a decrease in the V for synaptosomal GABA uptake and seems to be related to a partial collapse of nerve endings ionic gradients. Functionally, it could result over time in a reduced availability of GABA at the synapses involved.     

The Differential Effect of Ischemia on the Active Uptake of Dopamine, γ-Aminobutyric Acid, and Glutamate by Brain Synap to somes

Abstract: Ischemic stroke was induced in the Mongolian gerbil by left common carotid ligation. No change in uptake of [³H]dopamine, [³H]γ-aminobutyric acid ([³H]GABA), or [¹⁴C]glutamate in synaptosomes obtained from the ischemic hemisphere was observed for up to 8 h. At 16 h after ligation, marked decrements in uptake were observed in animals showing hemiparesis: Uptake values expressed as a percent of the corresponding control hemisphere were 15.2% for dopamine, 28.0% for GABA, and 47.5% for glutamate. The differential sensitivity of dopamine terminals compared with glutamate terminals was highly significant. Separate experiments performed with synaptosomes isolated from the corpus striatum showed that the greater sensitivity to damage was intrinsic to the dopamine nerve terminal and not the result of regional variations in ischemic damage in brain. No bilateral effect of ischemia on dopamine uptake was evident. In animals exhibiting milder behavioral deficits (circling), a smaller and comparable decrement in uptake of dopamine, GABA, and glutamate was evident at 16 h, whereas animals not affected behaviorally showed no decrement at 16 h. Following uptake, the subsequent fractional release of neurotransmitter stimulated by 60 mM-potassium ions was not affected at any time point studied. Therefore, the loss in uptake at 16 h probably represents overt destruction of nerve terminals. Experiments with urethane used in place of pentobarbital for anesthesia during carotid occlusion showed that "protection" by pentobarbital was not a factor in the delayed response to ischemia. These results show that damage or destruction of nerve terminals is a delayed event following ischemia and that dopamine terminals are intrinsically more sensitive than glutamate terminals.     

Interaction between GABA agonists and the cholinergic muscarinic system in rat corpus striatum

Chronic treatment with γ-acetylenic GABA(GAG), a GABA transaminase inhibitor, causes an increase in striatal dopamine receptor binding and function in rat brain suggesting that extrapyramidal side effects may accompany the use of these agents. In the present investigation it was found that chronic administration of THIP, a direct acting GABA receptor agonist, induced a similar increase in dopamine receptor binding. In addition, co-administration of atropine, a cholinergic muscarinic antagonist, was found to completely prevent the GABA-induced dopamine receptor increase. Furthermore, high affinity choline uptake, a measure of cholinergic activity, in striatal synaptosomes is enhanced after the acute administration of either GAG or THIP. Taken together these results support the notion of an interaction between dopaminergic, cholinergic and GABA-ergic neurons in the extrapyramidal system and indicate that co-administration of an anticholinergic agent may be of benefit in preventing the extrapyramidal side effects which may accompany the use of GABAergic agonists.     

Glutamate metabolism in the brain of young rats exposed to organic and inorganic lead

The synthesis of glutamate and its conversion to glutamine and GABA were studied using labelled glucose in cerebral cortex, cerebellum and brainstem of rats intoxicated acutely with tetraethyl lead and chronically with lead acetate. To assess the interconversion and the synaptosomal accumulation of these amino acids, the labelling of glutamate, glutamine and GABA were measured in whole tissue and synaptosomes after giving labelled glutamate. The radioactive carbon dioxide production from labelled glutamate by brain slices was measured to evaluate the oxidation of glutamate. The tissue levels of glutamate, glutamine and GABA and the activity of glutamate decarboxylase were also measured in both conditions.In inorganic lead toxicity, even though the glutamate pool size was reduced, the glutamate-glutamine cycling between synaptosomes and astrocytes was increased. The oxidation of glutamate and the glutamate-GABA cycling were reduced. These findings suggest that brain tries to maintain the endogenous glutamate levels by decreasing the oxidation of glutamate and increasing the uptake systems and the cycling through glutamine in inorganic lead toxicity. In organic lead toxicity, the glutamate pool as well as glutamate turnover was reduced markedly resulting in complete distortion of glutamate metabolism.     

Cerebral Glutamic Acid Decarboxylase Activity and γ-Aminobutyric Acid Concentrations in Mice Susceptible or Resistant to Audiogenic Seizures

Abstract: DBA/2 mice between 21 and 28 days of age are highly susceptible to sound-induced seizures. Drug studies suggest a possible deficit of γ-Aminobutyric acid (GABA)-mediated neurotransmission may be involved. We have measured the whole brain GABA concentration and glutamic acid decar-boxylase activity in DBA/2 mice at various ages before, during, and after the period of maximal susceptibility to audiogenic seizures. Corresponding determinations were carried out on age-matched TO mice, a strain much less susceptible to audiogenic seizures than DBA/2 mice at all ages. No significant differences in GABA concentration or glutamic acid decarboxylase activity were found between strains at any age. The susceptibility of DBA/2 mice to audiogenic seizures does not result from a gross inability to synthesise or store GABA.     

The effect of psychotropic substances on synaptosomal uptake of gamma-aminobutyric H3-acid and the activity of Na,K-ATPase]

The centrally acting drugs belonging to different groups--fluphenazine, trifluperidol, phthoracyzine, imipramine, diazepam, apomorphine, fentanyl, diphneylhydantoin, nonachlazine displayed in vitro an inhibitory effect on the uptake of gamma-aminobutyric acid by rat brain synaptosomes. A decrease in the activity of synaptosomal Na,K-ATPase was found in most cases. Drugs that failed to alter GABA uptake were as a rule found to be ineffective in relation to the enzyme activity (carbidine, morphine). GABA uptake was not affected by certain drugs inhibiting the Na,K-ATPase activity (azabuperon, tetrabenazine). It is supposed that the drugs used had at least two possible sites of action - Na,K-ATPase itself and hypothetic GABA transmembrane carrier.