Evidence that glutaric acid reduces glutamate uptake by cerebral cortex of infant rats

The role of excitotoxicity in the cerebral damage of glutaryl-CoA dehydrogenase deficiency (GDD) is under intense debate. We therefore investigated the in vitro effect of glutaric (GA) and 3-hydroxyglutaric (3-OHGA) acids, which accumulate in GDD, on [(3)H]glutamate uptake by slices and synaptosomal preparations from cerebral cortex and striatum of rats aged 7, 15 and 30 days. Glutamate uptake was significantly decreased by high concentrations of GA in cortical slices of 7-day-old rats, but not in cerebral cortex from 15- and 30-day-old rats and in striatum from all studied ages. Furthermore, this effect was not due to cellular death and was prevented by N-acetylcysteine preadministration, suggesting the involvement of oxidative damage. In contrast, glutamate uptake by brain slices was not affected by 3-OHGA exposure. Immunoblot analysis revealed that GLAST transporters were more abundant in the cerebral cortex compared to the striatum of 7-day-old rats. Moreover, the simultaneous addition of GA and dihydrokainate (DHK), a specific inhibitor of GLT1, resulted in a significantly higher inhibition of [(3)H]glutamate uptake by cortical slices of 7-day-old rats than that induced by the sole presence of DHK. We also observed that both GA and 3-OHGA exposure did not alter the incorporation of glutamate into synaptosomal preparations from cerebral cortex and striatum of rats aged 7, 15 and 30 days. Finally, GA in vivo administration did not alter glutamate uptake into cortical slices from 7-day-old rats. Our findings may explain at least in part why cortical neurons are more vulnerable to damage at birth as evidenced by the frontotemporal cortical atrophy observed in newborns affected by GDD.     

Evidence that Hyperprolinemia Alters Glutamatergic Homeostasis in Rat Brain: Neuroprotector Effect of Guanosine

This study investigated the effects of acute and chronic hyperprolinemia on glutamate uptake, as well as some mechanisms underlying the proline effects on glutamatergic system in rat cerebral cortex. The protective role of guanosine on effects mediated by proline was also evaluated. Results showed that acute and chronic hyperprolinemia reduced glutamate uptake, Na(+), K(+)-ATPase activity, ATP levels and increased lipoperoxidation. GLAST and GLT-1 immunocontent were increased in acute, but not in chronic hyperprolinemic rats. Our data suggest that the effects of proline on glutamate uptake may be mediated by lipid peroxidation and disruption of Na(+), K(+)-ATPase activity, but not by decreasing in glutamate transporters. This probably induces excitotoxicity and subsequent energy deficit. Guanosine was effective to prevent most of the effects promoted by proline, reinforcing its modulator role in counteracting the glutamate toxicity. However, further studies are needed to assess the modulatory effects of guanosine on experimental hyperprolinemia.     

Reduction of Glutamate Uptake into Cerebral Cortex of Developing Rats by the Branched-Chain Alpha-Keto Acids Accumulating in Maple Syrup Urine Disease

In the current study we investigated the effect of the branched-chain alpha-keto acids (BCKA) co-ketoisocaproic (KIC), alpha-keto-beta-methylvaleric (KMV), and alpha-ketoisovaleric (KIV) acids, which accumulate in maple syrup urine disease (MSUD), on the in vitro uptake of [3H]glutamate by cerebral cortical slices from rats aged 9, 21, and 60 days of life. We initially observed that glutamate uptake into cerebral cortex of 9- and 21-day-old rats was significantly higher, as compared to that of 60-day-old rats. Furthermore, KIC inhibited this uptake by tissue slices at all ages studied, whereas KMV and KIV produced the same effect only in cortical slices of 21- and 60-day-old rats. Kinetic assays showed that KIC significantly inhibited glutamate uptake in the presence of high glutamate concentrations (50 microM and greater). We also verified that the reduction of glutamate uptake was not due to cellular death, as evidenced by tetrazolium salt and lactate dehydrogenase viability tests of cortical slices in the presence of the BCKA. It is therefore presumed that the reduced glutamate uptake caused by the BCKA accumulating in MSUD may lead to higher extracellular glutamate levels and potentially to excitotoxicity, which may contribute to the neurological dysfunction of the affected individuals.     

Interaction of kainic acid with Na(+)-dependent glutamate binding and uptake in the cerebral cortex of the developing mouse.

The effect of kainic acid, a structural analogue and specific agonist of glutamate, was studied on the Na(+)-dependent binding and uptake of this amino acid in cerebral cortex preparations from 7-day-old and 30-day-old mice. The specific binding of glutamate to a crude synaptic membrane fraction and uptake into cortical slices increased several fold during this period. Kainic acid (0.5 mM or 5 mM) significantly reduced glutamate binding and this effect was more pronounced in membrane fractions from older animals. In contrast to this, the inhibitory action of kainic acid on glutamate uptake was twofold more potent in 7-day-old mice. The results are discussed from the viewpoint of the relationship between the Na(+)-dependent binding of glutamate and its uptake.     

Diethylmaleate decreased ascorbic acid release induced by cerebral ischemia in cerebral cortex of the anesthetized rat

The effect of diethylmaleate administration on ascorbic acid release following cerebral ischemia was investigated in anesthetized rat brain cortex. Cerebral ischemia, induced by ligating bilateral common carotid arteries and unilateral middle cerebral artery, significantly increased the extracellular ascorbic acid levels. Diethylmaleate (4 mmoles/kg, i.p.), which has been shown in earlier studies to decrease the ischemia-induced glutamate release, significantly reduced the ischemia-induced ascorbic acid release. The ischemia-induced ascorbic acid release was unaffected by perfusing NMDA receptor antagonist MK 801 (75 microM). Additionally, elevated extracellular glutamate levels, achieved by either externally applied glutamate solutions or by perfusing L-trans-pyrrolidine-2,4-dicarboxylate (PDC) (31.4 mM and 15.7 mM) to inhibit the glutamate uptake transporter, also significantly increased the extracellular ascorbic acid levels. These results suggested that ascorbic acid release in cerebral ischemia might be related to the elevated extracellular glutamate levels, which occurs following cerebral ischemia.     

Guanosine Exerts Neuroprotective Effect in an Experimental Model of Acute Ammonia Intoxication

The nucleoside guanosine (GUO) increases glutamate uptake by astrocytes and acts as antioxidant, thereby providing neuroprotection against glutamatergic excitotoxicity, as we have recently demonstrated in an animal model of chronic hepatic encephalopathy. Here, we investigated the neuroprotective effect of GUO in an acute ammonia intoxication model. Adult male Wistar rats received an intraperitoneal (i.p.) injection of vehicle or GUO 60 mg/kg, followed 20 min later by an i.p. injection of vehicle or 550 mg/kg of ammonium acetate. Afterwards, animals were observed for 45 min, being evaluated as normal, coma (i.e., absence of corneal reflex), or death status. In a second cohort of rats, video-electroencephalogram (EEG) recordings were performed. In a third cohort of rats, the following were measured: (i) plasma levels of glucose, transaminases, and urea; (ii) cerebrospinal fluid (CSF) levels of ammonia, glutamine, glutamate, and alanine; (iii) glutamate uptake in brain slices; and (iv) brain redox status and glutamine synthetase activity in cerebral cortex. GUO drastically reduced the lethality rate and the duration of coma. Animals treated with GUO had improved EEG traces, decreased CSF levels of glutamate and alanine, lowered oxidative stress in the cerebral cortex, and increased glutamate uptake by astrocytes in brain slices compared with animals that received vehicle prior to ammonium acetate administration. This study provides new evidence on mechanisms of guanine-derived purines in their potential modulation of glutamatergic system, contributing to GUO neuroprotective effects in a rodent model of by acute ammonia intoxication.     

Proline Reduces Creatine Kinase Activity in the Brain Cortex of Rats

Type II hyperprolinemia is an inherited disorder caused by a deficiency of ¹-pyrroline-5-carboxilic acid dehydrogenase, whose biochemical hallmark is proline accumulation in plasma and tissues. Although neurological symptoms occur in most patients, the neurotoxicity of proline is still controversial. The main objective of the present study was to investigate the effect of acute and chronic administration of proline on creatine kinase activity of brain cortex of Wistar rats. Acute treatment was performed by subcutaneous administration of one injection of proline to 22-day-old rats. For chronic treatment, proline was administered twice a day from the 6th to the 21st postpartum day. The results showed that creatine kinase activity was significantly inhibited in the brain cortex of rats subjected to acute proline administration. In contrast, this activity was increased in animals subjected to chronic administration. We also measured the in vitro effect of proline on creatine kinase activity in cerebral cortex of 22-day-old nontreated rats. Proline significantly inhibited creatine kinase activity. Considering the importance of creatine kinase forthe maintenance of energy homeostasis in the brain, it is conceivable that an alteration of this enzyme activity in the brain may be one of the mechanisms by which proline might be neurotoxic.     

Proline Alters Antioxidant Enzyme Defenses and Lipoperoxidation in the Erythrocytes and Plasma of Rats: In Vitro and In Vivo Studies

In the present study, we investigated, in vivo (acute and chronic) and in vitro, the effects of proline on the activities of antioxidant enzymes such as catalase (CAT), glutathione peroxidase and superoxide dismutase (SOD) in erythrocytes and also investigated the effect on thiobarbituric acid-reactive substances (TBARS) in the plasma of rats. For the experiments, the number of animals per group ranged from eight to ten. For acute administration, 29-day-old rats received one subcutaneous injection of proline (18.2 μmol/g body weight) or an equivalent volume of 0.9% saline solution (control) and were killed 1 h later. For chronic treatment, buffered proline was injected subcutaneously into rats twice a day at 10 h intervals from the 6th to the 28th day of age. Rats were killed 12 h after the last injection. For in vitro studies, proline (30.0 μM to 1.0 mM) was added to the incubation medium. Results showed that acute administration of proline reduced CAT and increased SOD activities, while chronic treatment increased the activities of CAT and SOD in erythrocytes and TBARS in the plasma of rats. Furthermore, in vitro studies showed that proline increased TBARS in the plasma (0.5 and 1.0 mM) and CAT activity (1.0 mM) in the erythrocytes of rats. The influence of the antioxidants (α-tocopherol plus ascorbic acid) on the effects elicited by proline was also studied. Treatment with antioxidants for 1 week or from the 6th to the 28th day of age prevented the alterations caused by acute and chronic, respectively, proline administration on the oxidative parameters evaluated. Data indicate that proline alters antioxidant defenses and induces lipid peroxidation in the blood of rats.     

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.     

Release of Glutamate from Striatum of Freely Moving Rats by pros-Methylimidazoleacetic Acid

Abstract: The effect of pros -methylimidazoleacetic acid (p-MIAA) was measured on the release of glutamate and aspartate from cerebral cortex, hippocampus, and striatum of freely moving rats, and on the uptake of ¹⁴C by striatal slices incubated in the presence of l -[¹⁴C]-glutamate. Twenty-four hours after implantation of a dialysis fiber, striatum, hippocampus, or cerebral cortex spontaneously released both glutamate and aspartate in the micromolar range. p-MIAA (1 µ M to 1 m M ), added to the dialysis perfusate, elicited a concentration-dependent increase of glutamate release from striatum with a maximal increase of about threefold. This effect did not occur in hippocampus or cortex. In none of these regions did p-MIAA increase aspartate release significantly. The p-MIAA effect was not mimicked by its isomer tele -methylimidazoleacetic acid. p-MIAA did not influence the uptake of glutamate by striatal slices. The glutamate-releasing action of p-MIAA may affect striatal function and explain the positive correlation between levels of p-MIAA in CSF and the severity of Parkinson's disease.     

Involvement of GABAergic and glutamatergic systems in the anticonvulsant activity of 3-alkynyl selenophene in 21?day-old rats

In this study, we investigated the role of GABAergic and glutamatergic systems in the anticonvulsant action of 3-alkynyl selenophene (3-ASP) in a pilocarpine (PC) model of seizures. To this purpose, 21 day-old rats were administered with an anticonvulsant dose of 3-ASP (50 mg/kg, per oral, p.o.), and [(3)H]γ-aminobutyric acid (GABA) and [(3)H]glutamate uptakes were carried out in slices of cerebral cortex and hippocampus. [(3)H]GABA uptake was decreased in cerebral cortex (64%) and hippocampus (58%) slices of 21 day-old rats treated with 3-ASP. In contrast, no alteration was observed in [(3)H]glutamate uptake in cerebral cortex and hippocampus slices of 21 day-old rats that received 3-ASP. Considering the drugs that increase synaptic GABA levels, by inhibiting its uptake or catabolism, are effective anticonvulsants, we further investigated the possible interaction between sub-effective doses of 3-ASP and GABA uptake or GABA transaminase (GABA-T) inhibitors in PC-induced seizures in 21 day-old rats. For this end, sub-effective doses of 3-ASP (10 mg/kg, p.o.) and DL-2,4-diamino-n-butyric acid hydrochloride (DABA, an inhibitor of GABA uptake--2 mg/kg, intraperitoneally; i.p.) or aminooxyacetic acid hemihydrochloride (AOAA; a GABA-T inhibitor--10 mg/kg, i.p.) were co-administrated to 21 day-old rats before PC (400 mg/kg; i.p.) treatment, and the appearance of seizures was recorded. Results demonstrated that treatment with AOAA and 3-ASP or DABA and 3-ASP significantly abolished the number of convulsing animals induced by PC. The present study indicates that 3-ASP reduced [(3)H]GABA uptake, suggesting that its anticonvulsant action is related to an increase in inhibitory tonus.     

Age-related changes in the glutamate metabolism of cerebral cortical slices from rats

The glutamate metabolism in cerebral cortical slices from young (12 weeks) and aged (100 weeks) rats was studied. A highly significant reduction of low affinity glutamate uptake and its metabolism in the aged rats by 14.5% of the amount of the young rats was observed (Pº0.001). When the ratio of the radioactivity of respective metabolite divided by the sum total radioactivity of overall glutamate metabolites was compared, the incorporation to aspartate was significantly small (P<0.01), while that of the CO₂ liberated and the GABA synthesized were increased (P<0.05). These results support the hypothesis that in cerebral cortical slices from aged rats the transamination of glutamate is suppressed and its decarboxylation is enhanced despite markedly reduced low affinity glutamate uptake into the cerebral cortex. The difference could be explained by the fact that cerebral cortical slices from aged rats are more vulnerable to anoxia than those from young rats when exposed during slice preparation.     

Abnormalities of retinal metabolism in diabetes or experimental galactosemia. VI. Comparison of retinal and cerebral cortex metabolism,and effects of antioxidant therapy

Metabolic abnormalities observed in retina and in cerebral cortex were compared in diabetic rats and experimentally galactosemic rats. Diabetes or experimental galactosemia of 2 months duration significantly increased oxidative stress in retina, as shown by elevation of retinal thiobarbituric acid reactive substances (TBARS) and subnormal activities of antioxidant defense enzymes, but had no such effect in the cerebral cortex. Activities of sodium potassium adenosine triphosphatase [(Na,K)-ATPase] and calcium ATPase became subnormal in retina as well as in cerebral cortex. In contrast, protein kinase C (PKC) activity was elevated in retina but not in cerebral cortex in the same hyperglycemic rats. Dietary supplementation with an antioxidant mixture (containing ascorbic acid, Trolox, α-tocopherol acetate, N-acetyl cysteine, β-carotene, and selenium) prevented the diabetes- induced and galactosemia-induced elevation of retinal oxidative stress, the elevation of retinal PKC activity and the decrease of retinal ATPases. In cerebral cortex, administration of the antioxidant diet also prevented the diabetes-induced decreases in (Na,K)-ATPase and calcium ATPases, but had no effect on TBARS and activities of PKC and antioxidant-defense enzymes. The results indicate that retina and cerebral cortex differ distinctly in their response to elevation of tissue hexose, and that cerebral cortex is more resistant than retina to diabetes-induced oxidative stress. The greater resistance to oxidative stress in cerebral cortex, as compared to retina, is consistent with the resistance of cerebral cortex to microvascular disease in diabetes, and with a hypothesis that oxidative stress contributes to microvascular disease in diabetes. Dietary supplementation with these antioxidants offers a means to inhibit multiple hyperglycemia-induced retinal metabolic abnormalities.     

2-phenylethynyl-butyltellurium improves memory in mice

The present study was conducted to evaluate the effect of 2-phenylethynyl-butyltellurium (PEBT), an organotellurium compound, at doses of 5 and 10 mg/kg on memory, employing the step-down inhibitory avoidance task in mice. Moreover, the involvement of glutamate uptake and release in cerebral cortex and hippocampus of mice was investigated. A single oral administration (p.o.) of PEBT at the dose of 10 mg/kg 1h before training (acquisition), immediately after training (consolidation) or 1 h before the test session (retrieval) of the step-down inhibitory avoidance task increased the step-through latency time in comparison to the control mice. In the open-field test, no significant differences in the number of crossings and rearings were observed among groups. The [(3)H]glutamate uptake by cerebral cortex and hippocampal slices of mice was significantly inhibited after 1h of treatment with PEBT. After 24h of PEBT exposure, only the hippocampal [(3)H]glutamate uptake was inhibited. The [(3)H]glutamate release by cerebral cortex and hippocampal synaptosomes of mice was not altered. These results suggest that PEBT improved memory stages (acquisition, consolidation and retrieval) in the step-down inhibitory avoidance task in mice. The improvement of memory by PEBT seems most likely to be mediated through an interaction with the amino acid transporters of the glutamatergic system.     

Arachidonic acid-induced inhibition of (Na+K+)-stimulated ATPase in the cerebral cortex and medulla oblongata of young and adult rats

The effect of arachidonic acid in 5.10(-4) and 5.10(-5) mol.l-1 concentration (as the Na salt, SIGMA) on ouabain-sensitive ATPase (E. C. 3.6.1.3) activity was studied in the cerebral cortex and medulla oblongata of 5-day-old and adult rats. In adult rats, arachidonic acid significantly inhibited ouabain-sensitive ATPase activity in both the cerebral cortex and the medulla oblongata. In 5-day-old rats, only the higher concentration (5.10(-4) mol.l-1) inhibited the enzyme statistically significantly; use of the lower concentration was not followed by any significant changes in Na+-K+-ATPase activity.     

Metabolic effects of hydrocortisone in mouse brain

The effect of intramuscular administration of hydrocortisone (10 mg/day per animal) for 5 days has been studied on the content of the amino acids belonging to the glutamate family, in the different regions of the mouse brain, along with the activities of glutamine synthetase, glutamate dehydrogenase, and aspartate, alanine, tyrosine, and ornithine aminotransferases. Further, since proline too is related to glutamate metabolism, the activity of proline oxidase was also studied in these regions. As hydrocortisone is known to influence the ionic fluxes in different tissues and the nitrogen metabolism, the activities of Na⁺,K⁺-ATPase together with the content of RNA and protein have also been estimated. A fall in the amino acids of the glutamate family in all three regions was observed with an increase in glutamate dehydrogenase activity in cerebral cortex. A significant fall in the protein content was also observed, mainly in the brain stem. A universal increase in Na⁺,K⁺-ATPase activity was observed in all three regions, with the highest in the cerebral cortex. The results indicate that hydrocortisone triggers increased utilization of glutamate in brain as an alternative to glucose, thereby shifting the nitrogen metabolism toward catabolism. The increased activity of Na⁺,K⁺-ATPase under these conditions would further aggravate the same and may lead to membrane stabilization.     

Thyrotropin-releasing hormone (TRH) and its analog (DN-1417): interaction with pentobarbital in choline uptake and acetylcholine synthesis of rat brain slices

TRH or its analog DN-1417 (gamma-butyrolactone-gamma-carbonyl-L-histidyl-L-proliamide) given 15 min after intravenous (i.v.) administration of pentobarbital (30 mg/kg) markedly shortened the pentobarbital-induced sleeping time in rats. This effect was almost completely abolished by intracerebroventricular pretreatment with atropine methylbromide (20 micrograms/rat), thereby suggesting the involvement of cholinergic mechanism. The action mechanism was investigated using rat brain slices. TRH (10(-6)-10(-4)M) or DN-1417 (10(-7)-10(-5)M) caused significant increases in the uptake of [3H]-choline into striatal slices. TRH(10(-4)M) or DN-1417(10(-5)M) also stimulated the conversion of [3H]-choline to [3H]-acetylcholine in striatal slices. A 30% reduction of acetylcholine synthesis from [3H]-choline in hippocampal slices and a 40% reduction of [3H]-choline uptake in slices of cerebral cortex, hippocampus and hypothalamus were observed in rats pretreated with pentobarbital (60 mg/kg, i.v.). TRH or DN-1417 (20 mg/kg, i.v.) given 15 min after the administration of pentobarbital markedly reversed both of the pentobarbital effects. Direct application of pentobarbital (5 X 10(-4)M) to slices in vitro also caused a 20-40% reduction of [3H]-choline uptake of cerebral cortex, hippocampus and diencephalon. A concomitant application of TRH(10(-4)M) or DN-1417(10(-5)M) and pentobarbital abolished the pentobarbital effect. These results provide neurochemical evidence that the antagonistic effects of TRH and DN-1417 on pentobarbital-induced narcosis are closely related to alterations in the rat brain choline uptake and acetylcholine synthesis, which are considered to be measures of the activity of cholinergic neurons.     

POSTNATAL CHANGES IN THE HIGH AFFINITY UPTAKE OF GLYCINE and GABA IN THE RAT CENTRAL NERVOUS SYSTEM

Abstract— High affinity uptake systems for GABA into slices of cerebral cortex and for glycine into slices of spinal cord have been demonstrated in rats of 1 and 10 days postnatal age and compared with the systems in tissue slices from adult rats. For both systems there was an increase in the maximal rate of uptake of the substrate with development. For glycine uptake there was no significant change in apparent K_m during development, whereas there was a four-fold increase in the apparent K_m for GABA uptake. There were some changes with development in the apparent substrate specificity of the two systems suggesting increased specificity with maturation. Bicuculline and strychnine, antagonists of the postsynaptic inhibitory actions of GABA and glycine, produced convulsions in 1-, 2- and 10-day-old rats following intraperitoneal injection of doses somewhat lower than those required to convulse adult rats. These findings are consistent with other evidence that glycine and GABA are functioning as inhibitory transmitters at least as soon as 1 day after birth.     

Cortical Na+,K+-ATPase of immature rats following bicuculline-induced seizures

The Na+,K+-ATPase activity was investigated in cerebral cortex homogenates of 7-, 12- and 18-day-old rats in which seizures were induced by systemic (i.p.) administration of bicuculline. Na+,K+-ATPase activities in control animals increased during postnatal development, but they were not significantly influenced by seizure activity when determined under optimal conditions in vitro. Although the ratio of neuronal vs. non-neuronal cells in cortical samples of 7-, 12- and 18-day-old rats was different, there was a remarkable similarity in the activation curves for K+, obtained for Na+,K+-ATPase of all age groups under normal conditions; 50% of enzyme activities were attained at 1 mmol.l-1 K+ and the maximal activities were found around 10 mmol.l-1 K+. The activation curves for K+ in rats with bicuculline-induced seizures were not significantly different from those of the controls.     

Effect of hyperprolinemia on acetylcholinesterase and butyrylcholinesterase activities in rat

Summary. We observed here that acute proline (Pro) administration provoked a decrease (32%) of acetylcholinesterase (AChE) activity in cerebral cortex and an increase (22%) of butyrylcholinesterase (BuChE) activity in the serum of 29-day-old rats. In contrast, chronic administration of Pro did not alter AChE or BuChE activities. Furthermore, pretreatment of rats with vitamins E and C combined or alone, N-nitro-L-arginine methyl ester or melatonin prevented the reduction of AChE activity caused by acute Pro administration, suggesting the participation of oxidative stress in such effects.