Rejane G. Tavares

Quinolinic acid stimulates synaptosomal glutamate release and inhibits glutamate uptake into astrocytes

Quinolinic acid (QA) is an endogenous neurotoxin involved in various neurological diseases, whose action seems to be exerted via glutamatergic receptors. However, the exact mechanism responsible for the neurotoxicity of QA is far from being understood. We have previously reported that QA inhibits vesicular glutamate uptake. In this work, investigating the effects of QA on the glutamatergic system from rat brain, we have demonstrated that QA (from 0.1 to 10mM) had no effect on synaptosomal L-[3H]glutamate uptake. The effect of QA on glutamate release in basal (physiological K+ concentration) or depolarized (40 mM KCl) conditions was evaluated. QA did not alter K+-stimulated glutamate release, but 5 and 10mM QA significantly increased basal glutamate release. The effect of dizolcipine (MK-801), a noncompetitive antagonist of N-methyl-D-aspartate (NMDA) receptor on glutamate release was investigated. MK-801 (5 microM) did not alter glutamate release per se, but completely abolished the QA-induced glutamate release. NMDA (50 microM) also stimulated glutamate release, without altering QA-induced glutamate release, suggesting that QA effects were exerted via NMDA receptors. QA (5 and 10mM) decreased glutamate uptake into astrocyte cell cultures. Enhanced synaptosomal glutamate release, associated with inhibition of glutamate uptake into astrocytes induced by QA could contribute to increase extracellular glutamate concentrations which ultimately lead to overstimulation of the glutamatergic system. These data provide additional evidence that neurotoxicity of QA may be also related to disturbances on the glutamatergic transport system, which could result in the neurological manifestations observed when this organic acid accumulates in the brain.

Quinolinic acid inhibits glutamate uptake into synaptic vesicles from rat brain.

Quinolinic acid (QA) is an endogenous and potent neurotoxin associated with the neurotoxicity of various common diseases. The uptake of neurotransmitters into synaptic vesicles is an important event involved in the storage and release of neurotransmitters by vesicles. The influence of QA on the uptake of glutamate, GABA and glycine into rat brain synaptic vesicles was investigated. QA (0.3-10 mM) significantly inhibited (>50%) the uptake of glutamate into synaptic vesicles, whereas QA at concentrations up to 10 mM had no significant effect on GABA or glycine uptake. Such results indicate that QA is able to selectively inhibit the vesicular uptake of glutamate, without interfering with the uptake of the inhibitory neurotransmitters GABA and glycine. These findings might be related to the neurotoxic effects of QA in the brain.

Inhibition of glutamate uptake into synaptic vesicles of rat brain by the metabolites accumulating in maple syrup urine disease

Maple syrup urine disease is an inherited metabolic disorder characterized by tissue accumulation of branched-chain amino acids and their corresponding keto acids in the affected children. Although this disorder is predominantly characterized by neurological symptoms, only few studies were carried out to investigate its neuropathology. In this study we investigated the effect of the metabolites accumulating in maple syrup urine disease on the in vitro uptake of [3H]glutamate by synaptic vesicles of rat brain. Synaptic vesicle preparations from whole brain of male adult Wistar rats (200-250 g) were incubated with the branched-chain amino acids and their corresponding keto acids at final concentrations ranging from 0.25 to 10 mM for the determination of glutamate uptake. Glutamate uptake was significantly inhibited by L-leucine, L-isoleucine, L-2-ketoisocaproic acid and L-2-keto-3-methylvaleric acid by approximately 60%, whereas L-valine and L-2-ketoisovaleric acid showed no effect. We also verified that the metabolites probably act by competitive inhibition. Therefore, it is possible that extracellular glutamate levels may be increased in maple syrup urine disease and that excitotoxicity may be involved in the neuropathology of this disorder.

In vivo quinolinic acid increases synaptosomal glutamate release in rats: reversal by guanosine.

Glutamate, the main excitatory neurotransmitter in the mammalian central nervous system (CNS), plays important role in brain physiological and pathological events. Quinolinic acid (QA) is a glutamatergic agent that induces seizures and is involved in the etiology of epilepsy. Guanine-based purines (GBPs) (guanosine and GMP) have been shown to exert neuroprotective effects against glutamatergic excitotoxic events. In this study, the influence of QA and GBPs on synaptosomal glutamate release and uptake in rats was investigated. We had previously demonstrated that QA “in vitro” stimulates synaptosomal L-[3H]glutamate release. In this work, we show that i.c.v. QA administration induced seizures in rats and was able to stimulatesynaptosomal L-[3H]glutamate release. This in vivo neurochemical effect was prevented by i.p. guanosine only when this nucleoside prevented QA-induced seizures. I.c.v. QA did not affect synaptosomal L-[3H]glutamate uptake. These data provided new evidence on the role of QA and GBPs on glutamatergic system in rat brain.

Glutaric acid stimulates glutamate binding and astrocytic uptake and inhibits vesicular glutamate uptake in forebrain from young rats

Glutaric acidemia type I (GA I) is an inherited neurometabolic disorder caused by glutaryl-CoA dehydrogenase deficiency, which leads to accumulation in body fluids and in brain of predominantly glutaric acid (GA), and to a lesser extent of 3-hydroxyglutaric and glutaconic acids. Neurological presentation is common in patients with GA I. Although the mechanisms underlying brain damage in this disorder are not yet well established, there is growing evidence that excitotoxicity may play a central role in the neuropathogenesis of this disease. In the present study, preparations of synaptosomes, synaptic plasma membranes and synaptic vesicles, as well as cultured astrocytes from rat forebrain were exposed to various concentrations of GA for the determination of the basal and potassium-induced release of [(3)H]glutamate by synaptosomes, Na(+)-independent glutamate binding to synaptic membranes and vesicular glutamate uptake and Na(+)-dependent glutamate uptake into astrocytes, respectively. GA (1-100 nM) significantly stimulated [(3)H]glutamate binding to brain plasma membranes (40-70%) in the absence of extracellular Na(+) concentrations, reflecting glutamate binding to receptors. Furthermore, this stimulatory effect was totally abolished by the metabotropic glutamate ligands DHPG, DCG-IV and l-AP4, attenuated by the ionotropic non-NMDA glutamate receptor agonist AMPA and had no interference of the NMDA receptor antagonist MK-801. Moreover, [(3)H]glutamate uptake into synaptic vesicles was inhibited by approximately 50% by 10 and 100 nM GA and Na(+)-dependent [(3)H]glutamate uptake by astrocytes was significantly increased (up to 50%) in a dose-dependent manner (maximal stimulation at 100 microM GA). In contrast, synaptosomal glutamate release was not affected by the acid at concentrations as high as 1 mM. These results indicate that the inhibition of glutamate uptake into synaptic vesicles by low concentrations GA may result in elevated concentrations of the excitatory neurotransmitter in the cytosol and the stimulatory effect of this organic acid on glutamate binding may potentially cause excitotoxicity to neural cells. Finally, taken together these results and previous findings showing that GA markedly decreases synaptosomal glutamate uptake, it is possible that the stimulatory effect of GA on astrocyte glutamate uptake might indicate that astrocytes may protect neurons from excitotoxic damage caused by GA by increasing glutamate uptake and therefore reducing the concentration of this excitatory neurotransmitter in the synaptic cleft.

Effects of methylmalonic and propionic acids on glutamate uptake by synaptosomes and synaptic vesicles and on glutamate release by synaptosomes from cerebral cortex of rats.

Neurological dysfunction is common in patients with methylmalonic and propionic acidemias. However, the mechanisms underlying the neuropathology of these disorders are far from understood. In the present study we investigated the in vitro effects of methylmalonic (MMA) and propionic (PA) acids at various concentrations (1 microM-5 mM) on three parameters of the glutamatergic system, namely the basal and potassium-induced release of L-[3H]glutamate by synaptosomes, Na+-dependent L-[3H]glutamate uptake by synaptosomes and Na+-independent L-[3H]glutamate uptake by synaptic vesicles from cerebral cortex of male adult Wistar rats. The results showed that MMA significantly increased potassium-induced but not basal L-[3H]glutamate release from synaptosomes with no alteration in synaptosomal L-[3H]glutamate uptake. A significant reduction of L-[3H]glutamate incorporation into vesicles caused by MMA was also detected. In contrast, PA had no effect on these parameters. These findings indicate that MMA alters the glutamatergic system. Although additional studies are necessary to evaluate the importance of these observations for the neuropathology of methylmalonic acidemia, it is possible that the effects elicited by MMA may lead to excessive glutamate concentrations at the synaptic cleft, a fact that may explain previous in vivo and in vitro findings associating MMA with excitotoxicity.

Inhibition of glutamate uptake into synaptic vesicles from rat brain by 3-nitropropionic acid in vitro.

The exact mechanisms by which 3-nitropropionic acid (3-NP), a naturally occurring plant and fungal neurotoxin, exerts its neurotoxic effects are not fully understood. However, blockage of ATP synthesis by the irreversible inhibition of succinate dehydrogenase activity, increased production of free radicals, and secondary excitotoxicity have been implicated in its actions. In the present study, synaptic vesicle preparations from brain of adult rats were incubated with 3-NP at final concentrations ranging from 0.01 to 10 mM for the determination of glutamate uptake. The effect of 3-NP on gamma-aminobutyric acid (GABA) and glycine uptake was also studied. Glutamate incorporation into vesicles was inhibited by 3-NP in a dose-dependent manner, whereas doses of up to 10 mM neurotoxin did not affect GABA or glycine uptake. Moreover, 3-NP did not inhibit the ATPase activity of synaptic vesicles. These findings indicate that low concentrations of 3-NP are able to selectively prevent vesicular glutamate storage, and this may represent at least one of the mechanisms responsible for the neurotoxic effects of 3-NP.

The effects of ebselen on [3H]glutamate uptake by synaptic vesicles from rat brain

Ebselen is a selenium organic compound, which has been shown to be a neuroprotective agent in brain disorders involving glutamate receptors. However, the effects of ebselen on the functionality of the glutamatergic system are still poorly investigated. In this study, by using synaptic vesicle preparation, we investigated the effects of ebselen (0.3 to 10 microM) on (i) vesicular glutamate uptake, (ii) bafilomycin-sensitive H+ -ATPase activity, and (iii) proton gradient formation (DeltapH). Ebselen presented a dual effect on glutamate uptake: the 1 microM concentration resulted in a 60% increase of the uptake, while the 10 microM concentration resulted in 60% inhibition. We also observed that ebselen (10 microM) inhibited the H+ -ATPase activity and dissipated the DeltapH. The inhibitory effects of ebselen were prevented by dithiothreitol (DTT). These findings suggest that high concentrations of ebselen may oxidize the essential thiol groups of the H+ -ATPase, which in turn affect its activity and compromise the vesicular glutamate uptake, and consequently lead to an impairment of the neural homeostasis.

BAL modulates glutamate transport in synaptosomes and synaptic vesicles from rat brain.

The therapeutic use of BAL (2,3-dimercaptopropanol) as treatment for poisoning has been halted by data suggesting serious neurotoxicity. This article is a report on the effects of BAL and other dithiols, DMSA (meso-2,3-dimercaptosuccinic acid) and DMPS (2,3-dimercaptopropane-1-sulfonic acid), on [3H]glutamate release and uptake by rat brain synaptosomes and [3H]glutamate uptake by synaptic vesicles. BAL (100 μM) inhibited glutamate uptake (30%) and stimulated its basal release (30%) in synaptosomes, without affecting K+-stimulated release. BAL also inhibited glutamate uptake by synaptic vesicles (up to 60%). DMPS and DMSA (100 μM) had no significant effects on these parameters. The data reported here provide some evidence of glutamate involvement in BAL-induced neurotoxicity by demonstrating direct effects of BAL on glutamatergic system modulation.

Anticonvulsant Effect of Alternanthera Brasiliana Extract On Pentylenetetrazole-induced Seizures in Rats

Epilepsy is a disorder that affects 1-2% of the population and a significant percentage of these patients do not respond to anticonvulsant drugs available in the market suggesting the need to investigate new pharmacological treatments. Numerous substances have been tested for potential anticonvulsant activity, but only a few generated anticonvulsant drugs. In this study, the potential anticonvulsant effect of Alternanthera brasiliana extract was investigated using an animal model of acute epilepsy induced by pentylenetetrazol (PTZ). The animals received injections of A. brasiliana extract (20, 100 or 500 mg/kg) or vehicle; 30 minutes later, they received an injection of PTZ, and were then observed for 30 minutes. Seizure latency and duration were recorded. The administration of 20 mg/kg of A. brasiliana extract had an anticonvulsant effect when compared with the control group. Thus, further studies using other seizure models as well as the investigation of isolated fractions of the extract are needed to elucidate the mechanisms of action of A. brasiliana.