EFFECTS OF AMINOOXYACETIC ACID AND l-GLUTAMIC ACID-γ-HYDRAZIDE ON GABA METABOLISM IN SPECIFIC BRAIN REGIONS

Abstract— Aminooxyacetic acid (AOAA) administration produced an increase in γ-aminobutyric acid (GABA) levels in regions of cerebral cortex, subcortex and cerebellum. In some cortical areas studied, the maximal effect was observed with 25 mg/kg AOAA; in other regions GABA levels were increased further with 50 and 75 mg/kg AOAA. Pretreatment with 25 mg/kg AOAA effectively inhibited GABA:2-oxoglutarate aminotransferase (GABA-T) and partially inhibited glutamic acid decarboxylase (GAD) activity in regions of cerebral cortex. However, this dose did not affect GAD activity in substantia nigra while GABA-T in the nigra and in the cerebellum was only partially inhibited. In both cortical and subcortical areas, the increase in GABA produced by 25 mg/kg of AOAA was linear. In contrast, l -glutamic acid-hydrazide (GAH) had no effect in the pyriform and cingulate cortex for the first 60 min after injection, and produced a biphasic GABA increase in caudate and substantia nigra over a 4 h period. Results suggest that GAH and AOAA affect regional GABA metabolism differentially and that there are several problems associated with estimating absolute GABA synthesis rates by measuring the rate or GABA accumulation after inhibition of GABA catabolism with these agents. This approach, however, may provide an easily obtainable indication of whether drugs or other manipulations are altering GABA synthesis in a given region.     

Effect of THIP and SL 76002, two clinically experimented GABA-mimetic compounds, on anterior pituitary GABA receptors and prolactin secretion in the rat

In the present study, the ability of three direct GABA agonists, muscimol, THIP and SL 76002 to displace 3H-GABA binding from anterior pituitary and medio-basal hypothalamus membranes was evaluated. Further, the effect of both THIP and SL 76002 on baseline prolactin levels or after stimulation of hormone release with haloperidol has been also studied. Either muscimol, THIP or SL 76002 have shown to posses 7-, 7- and 3-fold higher affinity, respectively, for the central nervous system than for the anterior pituitary 3H-GABA binding sites. Moreover, THIP and SL 76002 have demonstrated to be respectively, 25- and 1000- fold less potent than muscimol in inhibiting 3H- GABA binding at the level of the anterior pituitary and about 25- and 2700- fold less potent at the level of the medio-basal hypothalamus. Under basal conditions, either THIP or SL 76002 were ineffective to reduce prolactin release. However, after stimulation of prolactin secretion through blockade of the dopaminergic neurotransmission with haloperidol (0.1 mg/kg), both THIP (10 mg/kg) and SL 76002 (200 mg/kg) significantly counteracted the neuroleptic-induced prolactin rise with a potency which is in line with their ability to inhibit 3H-GABA binding in the anterior pituitary. The present results indicate that both compounds inhibit prolactin release under specific experimental situations probably through a GABAergic mechanism. In view of the endocrine effects of these GABA-mimetic compounds, the possibility arises for an application of these type of drugs in clinical neuroendocrinology.     

Effects of antidepressive agents on glutamatergic autoregulatory presynaptic mechanism in the rat cerebral cortex

Amitriptyline, imipramine, desipramine, viloxazine, moclobemide and its derivative, novel antidepressant befol (10(-6)-5 x 10(-4) M) decreased by 12-20% K(+)-stimulated 3H-D-asp release from perfused synaptosomes of rat brain cortex. Glutamic acid diethyl ester (GDEE) (10(-4) M) antagonized the effect of amitriptyline, imipramine, desipramine and befol and reversed the effect of moclobemide and viloxazine. Among neuroleptics studied, only carbidine, which possesses antidepressant activity together with antipsychotic one in clinics, decreased 3H-D-asp release by GDEE-sensitive mechanism. Effect of haloperidol and chlorpromazine was not affected by GDEE. It is concluded that autoregulatory mechanism on the terminals of glutamatergic neurons may be involved in the antidepressant action.     

High-affinity GABA uptake and GABA-metabolizing enzymes in pig forebrain white matter: a quantitative study

GABA receptor activation in central nervous white matter may be protective during white matter hypoxia in the adult, and it may modify axonal conduction, especially in the developing brain. GABA uptake is important for the shaping of the GABA signal, but quantitative data are lacking for GABA uptake and GABA-metabolizing enzymes in central nervous white matter. We report that high-affinity uptake of GABA in adult pig corpus callosum, fimbria, subcortical pyramidal tracts, and occipital white matter is approximately 20% of that in temporal cortex gray matter. Tiagabine (0.1 microM), an antiepileptic drug that specifically inhibits the GAT-1 GABA transporter inhibited GABA uptake 50% in temporal cortex and 60-68% in white structures. This finding indicates that GAT-1 is an important GABA transporter in white matter and suggests that white matter GABA uptake is inhibited during tiagabine therapy. GABA transaminase activity in white structures was approximately 20% of neocortical values. Glutamate decarboxylase (GAD) activity in white structures was only 4% of that in neocortex (7-12 pmol/mg tissue x min(-1) versus approximately 200 pmol/mg tissue x min(-1)). Since white matter activity of citrate synthase of the tricarboxylic acid cycle was approximately 25% of neocortical values ( approximately 0.4 nmol/mg tissue x min(-1) versus approximately 1.5 nmol/mg tissue x min(-1)), the low GAD activity suggests a slower metabolic turnover of GABA in white than in gray matter.     

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.     

Na+-independent binding of GABA to the triton X-100 treated synaptic membranes from cerebellum of rat brain

The treatment of the membranes from cerebellum of rat brain with 0.5% Triton X-100 increases both the affinity and the density of the Na⁺-independent binding sites for ³H-GABA (γ-aminobutyric acid) from the values obtained from membranes of rat brain after an extensive freezing and thawing treatment (Young $\text{et al.}$, 1976). Upon repeated washings of the Triton-treated membranes, the binding of ³H-GABA is further increased and follows biphasic kinetics which indicates two binding components having dissociation constants of 5.9 and 27 nM and densities of 1.35 and 3.9 pmole/mg protein, respectively. GABA agonist, imidazoleacetic acid, and the GABA antagonists, bicuculline and d-tubocurarine, inhibit 50% of ³H-GABA binding at 1, 47 and 85 μM concentrations (IC₅₀ values), respectively. The IC₅₀ values for these compounds are unchanged by Na⁺. Thus, the Na⁺-independent binding of ³H-GABA to the Triton-treated membranes may represent binding to the synaptic GABA receptors.     

NMDA receptor involvement in imipramine withdrawal-associated effects on swim stress,GABA levels and NMDA receptor binding in rat hippocampus

Abrupt antidepressant withdrawal after chronic treatment is associated with a stress response that may negatively affect the long-term outcome of depression, the neurochemical correlates, of which, remain undetermined. Prolonged depression involves the stress-related release of glucocorticoids and glutamate, while response to antidepressants involves gamma-amino butyric acid (GABA) and the glutamate N-methyl-D-aspartate (NMDA) receptor. Here, imipramine (IMI) was administered to rats for three weeks followed by acute withdrawal for seven days. Levels of GABA in the hippocampus (HC), and effects on swim stress immobility (SSI), were determined. Furthermore, glutamate/NMDA receptor binding properties were determined using [(3)H]-CGP-39653. Finally, the ability of dizocilpine (MK801), a glutamate NMDA antagonist, to reverse IMI withdrawal was determined. Chronic IMI (15 mg/kg ip) significantly reduced SSI together with a slight but insignificant decrease in HC GABA levels. However, IMI significantly reduced specific binding (B(max)) of [(3)H]-CGP-39653. Withdrawal of IMI for 7 days resulted in a loss of efficacy on SSI, a slight increase in GABA and a significant reversal of IMI effects on [(3)H]-CGP-39653 binding. MK801 (0.2 mg/kg ip) alone for seven days caused a significant decrease in SSI, a significant suppression of HC GABA, and significantly decreased [(3)H]-CGP-39653 B(max). MK801 during IMI-withdrawal significantly decreased GABA, prompted recovery on SSI, though not significantly, but significantly reversed withdrawal effects on [(3)H]-CGP-39653 B(max). In conclusion, acute antidepressant discontinuation is associated with subtle changes on HC GABA, a resurgence of NMDA receptor density and a loss of its anti-immobility response. These responses are reversed by a NMDA antagonist suggesting that abrupt antidepressant discontinuation mobilises glutamate activity.     

Fenibut binding with bicuculline-insensitive GABA receptors in the rat brain

(+/-) Fenibut beta-phenyl-GABA) was not able to displace 3H-GABA in Na+ independent GABA binding (IC50 greater than 250 microM). Nevertheless, (+/-) fenibut and (+/-) baclofen effectively displaced 3H-GABA in Ca2+ dependent GABA binding in the presence of 50 microM (+) bicuculline. (+/-) Fenibut was less potent in this respect. It is suggested that fenibut may act via bicuculline-insensitive GABA receptors.     

Effect of chronic neuroleptic or L-DOPA administration on GABA levels in the rat substantia nigra

Male albino rats were administered daily with haloperidol, clozapine or L-DOPA and sacrificed 18 hours after the last dose of the drug. Acutely haloperidol (5mg/kg, i.p.) greatly lowered nigral GABA levels whereas after 167 daily doses the nigral GABA levels were not significantly different from controls, but were significantly increased as compared with the acutely treated animals. In contrast, acute L-DOPA (2 × 100mg, p.o.) greatly raised nigral GABA levels whereas after chronic L-DOPA (167 days) nigral GABA levels were not significantly different from controls and were significantly lower as compared with the animals receiving the acute treatment. Clozapine (20 mg/kg, i.p. either acutely or chronically) did not have as marked an effect on nigral GABA levels as did haloperidol. Of these various drug regimens only chronic L-DOPA significantly affected nigral GAD activity, producing a moderate decrease.     

Non-opioid analgesia of the neuropeptide, neo-kyotorphin and possible mediation by inhibition of GABA release in the mouse brain

The novel neuropeptide, neo-kyotorphin, produced a naloxone-resistant analgesia in the tail pinch test when given (IC) to mice. Pretreatments with implantation of a morphine pellet or with phentolamine (10 micrograms IT) or with reserpine (10 mg/kg SC) did not attenuate this analgesia, yet the analgesia was antagonized by GABA mimetics, such as muscimol (0.1 microgram IC), nipecotic acid (100 mg/kg IP). Neo-kyotorphin inhibited the Ca2+-dependent and depolarization-evoked release of 3H-GABA, from crude synaptosomes of the lower brain stem of rats. These findings suggest that inhibition of GABA in the brain may in part be involved in neo-kyotorphin-induced analgesia.     

Effects of chronic administration of antipsychotic drugs on GABA and other amino acids in the mesolimbic area of rat brain.

Albino rats were injected once daily with chlorpromazine (20 mg/kg) or haloperidol (3 mg/kg) for 100 days, and were sacrificed 24 hours or 35 days after the last injection. Brains were frozen rapidly in liquid nitrogen, after which the mesolimbic area (containing nucleus accumbens and olfactory tubercle) and striatum were dissected from each frozen brain. Amino acids were quantitated in the mesolimbic area, and GAD enzyme activity was measured in the striatum. Chronic administration of chlorpromazine and haloperidol did not alter brain GABA content or GAD activity in the treated rats, as compared to saline-injected controls, either 1 or 35 days after injections ended. However, a significant elevation of aspartate content and reduction of glycine content was found after chronic administration of haloperidol. These data suggest that alterations which may be found in the GABA system in autopsied brain from psychotic patients do not result from previous antipsychotic drug therapy.     

Inhibition of GABA system involved in cyclosporine-induced convulsions.

In this study, we attempted to clarify the mechanisms mediating cyclosporine-evoked convulsions. Cyclosporine (50 mg/kg, i.p.) significantly enhanced the intensity of convulsions induced by bicuculline (GABA receptor antagonist), but not those induced by strychnine (glycine receptor antagonist), N-methyl-D-aspartic acid, quisqualic acid or kainic acid (glutamate receptor agonists). Bicuculline plus cyclosporine-induced convulsions were significantly suppressed by an activation of GABAergic transmission with diazepam, phenobarbital and valproate. The GABA turnover estimated by measuring aminooxyacetic acid-induced GABA accumulation in the mouse brain was significantly inhibited by cyclosporine (50 mg/kg, i.p.). When cultured rat cerebellar granule cells were exposed to 1 microM cyclosporine for 24 hr, the specific [3H]muscimol (10 nM) binding to intact granule cells decreased to 53% of vehicle controls. The present study provides the first evidence suggesting that cyclosporine inhibits GABAergic neural activity and binding properties of the GABAA receptor. These events are closely related to the occurrence of adverse central effects including tremors, convulsions, coma and encephalopathy under cyclosporine therapy.     

Development of the γ-Aminobutyric Acid Neurotransmitter System in the Rat Cerebral Cortex During Repeated Administration of the GABA-Transaminase Inhibitor Ethanolamine O-Sulphate

Ethanolamine O-sulphate (400 mg/kg, i.p.) was administered to rat pups at 9 days of age and on alternate days up to 17 days of age. At 18 days of age, gamma-aminobutyric acid (GABA) concentration was increased (three- to fourfold), glutamic acid decarboxylase (GAD) activity reduced to 55% of control, and the number of GABAA and GABAB binding sites increased in the cerebral cortex. This is the same pattern of change as seen previously with oral administration of ethanolamine O-sulphate to the adult rat but the changes occur more rapidly in the developing rat. A lower dose of ethanolamine O-sulphate (100 mg/kg, i.p.), administered according to the same schedule, caused a twofold increase in cortical GABA at 18 days of age whereas GAD activity and GABAA binding were not significantly altered.     

Changes in the characteristics of low affinity GABA binding sites elicited by Ro15-1788

3H-GABA binding was studied in cortical membranes from cerebral cortex of handling-habituated and naive rats after the in vitro addition of Ro15-1788. At low concentrations (10(-8), 10(-9) M) Ro15-1788 increased the total number of low affinity 3H-GABA binding sites in brain tissue from naive rats but failed to modify 3H-GABA binding in tissue from handling-habituated ones. On the contrary, Ro15-1788 at higher concentrations (10(-5), 10(-6)M) decreased the total number of low affinity 3H-GABA binding sites in tissue from handling-habituated rats but failed to modify 3H-GABA binding in tissue from naive animals. Ro15-1788 (10(-7)M) failed to modify significantly low affinity 3H-GABA binding in membranes from both naive and handling-habituated rats. However, this concentration abolished the effect of beta-carbolines and diazepam on 3H-GABA binding in membranes from naive and handling-habituated rats, respectively. The changes in the affinity of 3H-GABA binding were inversely related to the changes in the number. The results suggest that: a) the action "in vitro" of Ro15-1788 on low affinity 3H-GABA binding depends from its concentration at the benzodiazepine recognition sites; b) the benzodiazepine recognition site has a modulatory role in the control of the function of GABA-ergic receptor. Our data might explain the conflicting results obtained with this compound "in vivo".     

Species dependent dual modulation of the benzodiazepine/GABA receptor chloride channel by dihydroergosine

Dihydroergosine (50 and 100 mg/kg) enhanced the incidence of bicuculline (3 mg/kg)-induced convulsions in female rats, while 100 mg/kg of dihydroergosine given to female mice made 45% convulsive dose of bicuculline (2.5 mg/kg) to be subconvulsive. The same dose of dihydroergosine enhanced in mice the latency of bicuculline (4 mg/kg)-induced convulsions. Although, in in vitro experiments dihydroergosine showed very weak ability to prevent the binding of 3H-muscimol, the drug was able to diminish and to augment the IC50 of bicuculline and GABA when added to crude synaptosomal pellet of the rat and mouse brain respectively. Lower concentrations of dihydroergosine stimulated and higher inhibited 3H-TBOB binding to the crude synaptosomal pellet of the rat brain. In the preparation of mouse brain dihydroergosine produced only inhibition of 3H-TBOB binding. Only slight quantitative differences were observed in bicuculline-induced stimulation and in GABA- and diazepam-induced inhibition of 3H-TBOB binding between the two species. The results suggest that the opposite species-dependent effects of dihydroergosine on bicuculline-induced convulsions are due to the ability of this drug to modulate species-dependently the benzodiazepine/GABA receptor chloride channel complex.     

Antiulcer activity of N-phthaloyl GABA--a new GABA mimetic agent

N-phthaloyl GABA (P. GABA) inhibited gastric ulceration induced by 3 hr restraint stress at 4 degrees C (CRS) in albino rats. Antiulcer activity of P. GABA was compared with sodium valproate and cimetidine. P. GABA, sodium valproate and cimetidine showed a dose dependent reduction of gastric ulceration. Pretreatment with GABA antagonists-bicuculline methiodide (0.5 mg/kg, im) or 3 mercaptopropionic acid (2 mg/kg, im) reversed the antiulcerogenic activity of both the drugs (P. GABA and sodium valproate). GABA antagonists as such did not induce gastric ulceration in normal rats.     

Chronic treatment with zinc and antidepressants induces enhancement of presynaptic/extracellular zinc concentration in the rat prefrontal cortex

Zinc exhibits antidepressant-like activity in preclinical tests/models. Moreover, zinc homeostasis is implicated in the pathophysiology of affective disorders. The aim of the present study was to examine the effect of chronic zinc, citalopram and imipramine intraperitoneal administration on the presynaptic and extracellular zinc concentration in the rat prefrontal cortex and hippocampus. We used two methods: zinc–selenium histochemistry (which images the pool of presynaptic-vesicle zinc) and anodic stripping voltammetry (ASV) for zinc determination in microdialysate (which assays the extracellular zinc concentration). We report that chronic (14×) zinc (hydroaspartate, 10 and 65 mg/kg) and citalopram (20 mg/kg) administration increased the pool of presynaptic zinc (by 34, 50 and 37%, respectively) in the rat prefrontal cortex. The 21% increase induced by imipramine (20 mg/kg) was marginally significant. Likewise, zinc (hydroaspartate, 65 mg/kg), citalopram and imipramine increased the extracellular zinc (although with a different pattern: time point, area under the curve and/or basal level) in this brain region. Furthermore, zinc induced an increase in presynaptic (by 65%) and extracellular zinc (by 90%) in the hippocampus, while both citalopram and imipramine did not. These results indicate that all of the treatments increase presynaptic/extracellular zinc concentrations in the rat prefrontal cortex, which may then contribute to their antidepressant mechanisms. Alterations induced by zinc (but not antidepressants) administration in the hippocampus may be related to specific zinc mechanisms. All the data (previous and present) on the effect of antidepressant treatments on the presynaptic/extracellular zinc concentrations suggest the involvement of this biometal presynaptic/synaptic homeostasis in the antidepressant mechanism(s).     

Studies on the Role of α2-Adrenoceptors in the Control of Synaptosomal [3H]5-Hydroxytryptamine Release: Effects of Antidepressant Drugs

The sensitivity of alpha 2-adrenoceptors on 5-hydroxytryptamine (5-HT) nerve endings obtained from rat cerebral cortex was investigated following treatment with the antidepressant drugs desipramine (10 mg/kg/day for 21-28 days) or clorgyline (1 mg/kg/day for 21-28 days). [3H]5-HT (100 nM) was used to load cortical synaptosomes (P2) after experiments with uptake inhibitors confirmed that this concentration of amine ensured exclusive uptake into 5-HT nerve terminals. The sensitivity of K+-stimulated release of [3H]5-HT to alpha 2-adrenoceptor occupancy was assessed in a superfusion system by means of the dose-dependent inhibition of [3H]5-HT release by clonidine. This is blocked by yohimbine (1 microM), which, when administered alone, enhances release, suggesting that endogenous catecholamines released from other synaptosomes act on these alpha 2-heteroreceptors. The effect of addition of citalopram (1 microM) to superfusates suggests that some reuptake of [3H]5-HT occurs during superfusion. Of the tritium released into superfusates during "background" and K+-stimulated release, 17 and 90%, respectively is [3H]5-HT. The attenuation of K+-stimulated release by clonidine is apparently diminished by the chronic clorgyline regimen but not by desipramine. However, clorgyline elevates catecholamine levels, and this might increase endogenous noradrenaline (NA) efflux, which by competition with clonidine could appear to alter alpha 2-adrenoceptor sensitivity. This possibility was investigated by depleting NA with the neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4). These studies showed that the apparent effect of chronic clorgyline on alpha 2-adrenoceptor sensitivity to clonidine was due to competition with increased levels of endogenous NA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antinociceptive action of GABA-mimetic agent--N-phthaloyl GABA

N-phthaloyl GABA (P-GABA), a nonselective GABA-ergic drug, showed positive analgesic response in four different models in mice, viz-tail immersion, tail clip, hot plate and writhing-induced by acetic acid. Antinociceptive ED50 (ip in mice) of P-GABA was lowest in tail immersion method (ED50 = 24.27, mg/kg). Though pethidine (10 mg/kg, ip) significantly potentiated the antinociceptive action of P-GABA (20 mg/kg, ip), pretreatment of naloxone (5 mg/kg, im) did not influence the same. Pretreatment with atropine (10 mg/kg, im), picrotoxin (0.08 mg/kg) and 3-mercaptopropionic acid (2 mg/kg) reduced the antinociceptive action of P-GABA significantly. But pretreatment with bicuculline (0.4 mg/kg), a specific GABA antagonist, did not reduce the antinociceptive action of P-GABA.     

GABA interaction with lipids in organic medium

The interaction of 3H-GABA (gamma-aminobutyric acid and 14C-glutamate with lipids in an aqueous organic partition system was studied. With this partition system 3H-GABA and 14C-glutamate were able to interact with sphingomyelin, sulfatide, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine and phosphatidic acid but not with cholesterol or ceramide. In an homogeneous aqueous medium we could not demonstrate any interaction between 3H-GABA and lipids. The apparent dissociation constants (Kd) for 3H-GABA-lipids or 14C-glutamate-lipids interactions in organic medium were in the millimolar range and maximal charge (Bmax) between 3 and 7 moles of GABA or glutamate by mole of lipid. Amino acids such as glutamic acid, beta-alanine and glycine displaced 3H-GABA with the same potency as GABA itself; thus these results show that the interaction lacks pharmacological specificity. To detect this interaction lipid concentrations higher than 2 microM were required and in the partition system 3H-GABA and lipid phosphorus were both concentrated at the interface. Therefore lipids tested with a biphasic partition system do not fulfill the classical criteria for a neurotransmitter receptor at least not for GABA and glutamate.