共查询到20条相似文献,搜索用时 15 毫秒
1.
L. Bettendorff F. Sluse †G. Goessens P. Wins T. Grisar 《Journal of neurochemistry》1995,65(5):2178-2184
Abstract: Culture of neuroblastoma cells in a medium of low-thiamine concentration (6 n M ) and in the presence of the transport inhibitor amprolium leads to the appearance of overt signs of necrosis; i.e., the chromatin condenses in dark patches, the oxygen consumption decreases, mitochondria are uncoupled, and their cristae are disorganized. Glutamate formed from glutamine is no longer oxidized and accumulates, suggesting that the thiamine diphosphate-dependent α-ketoglutarate dehydrogenase activity is impaired. When thiamine (10 µ M ) is added to the cells, the O2 consumption increases, respiratory control is restored, and normal cell and mitochondrial morphology is recovered within 1 h. Succinate, which is oxidized via the thiamine diphosphate-independent succinate dehydrogenase, is also able to restore a normal O2 consumption (with respiratory control) in digitonin-permeabilized thiamine-deficient cells. Our results therefore suggest that the slowing of the citric acid cycle is the main cause of the biochemical lesion induced by thiamine deficiency as observed in Wernicke's encephalopathy. 相似文献
2.
Effect of Pyrithiamine Treatment and Subsequent Thiamine Rehabilitation on Regional Cerebral Amino Acids and Thiamine-Dependent Enzymes 总被引:5,自引:2,他引:3
Pyrithiamine-induced thiamine-deficiency encephalopathy in the rat shows many neuropathological and biochemical similarities to Wernicke's encephalopathy in humans. Treatment of rats with pyrithiamine resulted in moderate reductions of glutamate in thalamus and pons and in generalized severe reductions of aspartate in pons (by 89%, p less than 0.01), thalamus (by 83%, p less than 0.01), cerebellum (by 53%, p less than 0.01), and cerebral cortex (by 33%, p less than 0.05). Alanine concentrations were concomitantly increased. Activities of the thiamine-dependent enzyme alpha-ketoglutarate dehydrogenase (alpha KGDH) were decreased in parallel with the aspartate decreases; pyruvate dehydrogenase complex activities were unchanged in all brain regions. Following thiamine administration to symptomatic pyrithiamine-treated rats, neurological symptoms were reversed and concentrations of glutamate, aspartate, and alanine, as well as alpha KGDH activities, were restored to normal in cerebral cortex and pons. Aspartate levels and alpha KGDH activities remained below normal values, however, in thalamus. Thus, pyrithiamine treatment leads to reductions of cerebral alpha KGDH and (1) decreased glucose (pyruvate) oxidation resulting in accumulation of alanine and (2) decreased brain content of glutamate and aspartate. Such changes may be of key significance in the pathophysiology of the reversible and irreversible signs of Wernicke's encephalopathy in humans. 相似文献
3.
Extracellular Glutamate During Focal Cerebral Ischaemia in Rats: Time Course and Calcium Dependency 总被引:8,自引:1,他引:8
Florence Wahl Tihomir P. Obrenovitch Aidan M. Hardy Michel Plotkine Roger Boulu Lindsay Symon 《Journal of neurochemistry》1994,63(3):1003-1011
Abstract: The time course of changes in extracellular glutamic acid levels and their Ca2+ dependency were studied in the rat striatum during focal cerebral ischaemia, using microdialysis. Ischaemia-induced changes were compared with those produced by high K+-evoked local depolarization. To optimize time resolution, glutamate was analysed continuously as the dialysate emerged from the microdialysis probe by either enzyme fluorimetry or biosensor. The Ca2+ dependency of glutamate changes was examined by perfusing the probe with Ca2+-free medium. With normal artificial CSF, ischaemia produced a biphasic increase in extracellular glutamate, which started from the onset of ischaemia. During the first phase lasting ~10 min, dialysate glutamate level increased from 5.8 ± 0.9 µM· min?1 to 35.8 ± 6.2 µM where it stabilized for ~3 min. During the second phase dialysate glutamate increased progressively to its maximum (82 ± 8 µM), reached after 55 min of ischaemia, where it remained for as long as it was recorded (3 h). The overall changes in extracellular glutamate were similar when Ca2+ was omitted from the perfusion medium, except that the first phase was no longer detectable and, early in ischaemia, extracellular glutamate increased at a significantly slower rate than in the control group (2.2 ± 1 µM· min?1; p < 0.05). On the basis of these data, we propose that most of the glutamate released in the extracellular space in severe ischaemia is of metabolic origin, probably originating from both neurons and glia, and caused by altered glutamate uptake mechanisms. Comparison with high K+-induced glutamate release did not suggest that glutamate “exocytosis,” early after middle cerebral artery occlusion, was markedly limited by deficient ATP levels. 相似文献
4.
Abstract: Previous studies have shown that complete blockade of metabolism in embryonic chick retina causes a time-dependent increase in the release of glutamate into the extracellular space. The present study examined the cellular source of this glutamate, i.e., neuronal and/or glial. Pure cultures of retinal neurons or glia were labeled for 10 min at 37°C with [3H]acetate. Retinal glia, but not retinal neurons, were found to selectively and preferentially metabolize acetate, thus producing 3H-labeled amino acids in the glial compartment. This finding provides direct evidence to substantiate findings from several other laboratories that have indirectly determined the preferential metabolism of acetate by glia by using mixed neuronal/glial populations. To study the cellular source of glutamate released during total metabolic blockade, whole retina were prelabeled with [3H]acetate plus [U-14C]glucose (to label the neuronal compartment). Total metabolic blockade was instituted with a combination of iodoacetate (IOA) plus KCN, and the release of glutamate into the medium was followed at 5, 15, and 30 min. During total energy blockade, net extracellular glutamate was not elevated at 5 min [0.17 ± 0.02 vs. 0.12 ± 0.01 µM for treated vs. control retina (means ± SEM), respectively], but was increased significantly at 15 (1.2 ± 0.26 µM) and 30 min (2.6 ± 0.22 µM). Total [3H]glutamate in the medium during IOA/KCN treatment was unchanged at 5 min, but was increased 1.5- and threefold above basal levels at 15 and 30 min, respectively. During the time when extracellular glutamate increased, the specific activity of [3H]glutamate remained fairly constant, 731 ± 134 and 517 ± 82 dpm/nmol (means ± SEM) at 15 and 30 min, respectively. In contrast, 14C-labeled glutamate in the medium did not increase during IOA/KCN treatment and paralleled basal levels. Thus, the specific activity of 14C-labeled extracellular glutamate decreased from 309 ± 87 dpm/nmol at 15 min to 42 ± 8 dpm/nmol at 30 min. Prior loading of the tissue with 0.5 mM trans-pyrrolidine-2,4-dicarboxylate (t-PDC), a glutamate transport inhibitor, blocked 57% of the glutamate released at 30 min of IOA/KCN exposure, suggesting that reversal of an Na+-dependent glutamate transporter was a key contributor to the appearance of extracellular glutamate during energy deprivation. The increase in extracellular [3H]glutamate, constancy of the specific activity of extracellular [3H]glutamate, decrease in the specific activity of extracellular [14C]glutamate, and attenuation of release by prior loading with t-PDC indicate that glial pools of glutamate released via reversal of the transporter contribute significantly to the rise in extracellular glutamate after metabolic inhibition in this preparation. 相似文献
5.
Robert A. Waniewski 《Journal of neurochemistry》1992,58(1):167-174
The effect of ammonia on glutamate accumulation and metabolism was examined in astrocyte cultures prepared from neonatal rat cortices. Intact astrocytes were incubated with 70 microM L-[14C(U)]glutamate and varying amounts of ammonium chloride. The media and cells were analyzed separately by HPLC for amino acids and labelled metabolites. Extracellular glutamate was reduced to 8 microM by 60 min. Removal of glutamate from the extracellular space was not altered by addition of ammonia. The rate of glutamine synthesis was increased from 3.6 to 9.3 nmol/mg of protein/min by addition of 100 microM ammonia, and intracellular glutamate was reduced from 262 to 86 nmol/mg of protein after 30 min. The metabolism of accumulated glutamate was matched nearly perfectly by the synthesis of glutamine, and both processes were proportional to the amount of added ammonia. The transamination and deamination products of glutamate were minor metabolites that either decreased or remained unchanged with increasing ammonia. Thus, ammonia addition stimulates the conversion of glutamate to glutamine in intact astrocyte cultures. At physiological concentrations of ammonia, glutamine synthesis appears to be limited by the rate of glutamate accumulation and the activity of competing reactions and not by the activity of glutamine synthetase. 相似文献
6.
Exogenous Glutamate Is Metabolized to Glutamine and Exported by Rat Primary Astrocyte Cultures 总被引:13,自引:13,他引:0
Rat cortical astrocytes in primary culture were examined for their capacity to transport and metabolize exogenous L-[U-14C]glutamate. After incubation for time periods up to 120 min, cells and incubation media were analyzed for labelled and endogenous glutamate and its metabolic products by HPLC coupled with fluorescence detection and liquid scintillation counting. Glutamine was the major labelled metabolite after 120 min, accounted for 38% of the original glutamate label, and was found primarily in the incubation medium. A further 13.5% of the label was recovered in deaminated metabolites of glutamate, 1.2% was associated with aspartate, 23% remained in glutamate, and 10.2% was found in an acid-precipitated cell fraction. More than 84% of the label was recovered in these fraction. suggesting that the maximum possible formation and loss of 14CO2 was 16%. The rate of total glutamine synthesis was 1.1 nmol X mg protein-1 X min-1 when 9 microM exogenous glutamate was present. The total amount of glutamine synthesized greatly exceeded the consumption of glutamate, indicating that a substantial proportion of glutamine was synthesized from other carbon sources. Almost all of the newly formed glutamine was exported into the medium. These results indicate that astrocytes in primary culture, by accumulating glutamate, producing glutamine, and exporting it, are capable of carrying out the glial component of the glutamine cycle. 相似文献
7.
Lourdes Massieu Alberto Morales-Villagrán Ricardo Tapia 《Journal of neurochemistry》1995,64(5):2262-2272
Abstract: It is well documented that neurons exposed to high concentrations of excitatory amino acids, such as glutamate and aspartate, degenerate and die. The clearance of these amino acids from the synaptic cleft depends mainly on their transport by high-affinity sodium-dependent carriers. Using microdialysis in vivo and HPLC analysis, we have studied the effect of the administration of inhibitors of the glutamate transporter (l -trans-pyrrolidine-2,4-dicarboxylate and dihydrokainate) on the extracellular concentration of endogenous amino acids in the rat striatum. In addition, we have analyzed whether the changes observed in the concentration of glutamate and aspartate were injurious to striatal cells. Neuronal damage was assessed by biochemical determination of choline acetyltransferase and glutamate decarboxylase activities, 7 days after the microdialysis procedure. In other experiments, pyrrolidine dicarboxylate and dihydrokainate, as well as two other inhibitors of the glutamate carrier, dl -threo-β-hydroxyaspartate and l -aspartate-β-hydroxamate, were microinjected into the striatum, and neuronal damage was assessed, both biochemically and histologically, 7 or 14 days after the injection. Dihydrokainate and pyrrolidine dicarboxylate produced a similar remarkable increase in the concentration of extracellular aspartate and glutamate. However, the former induced also notable elevations in the concentration of other amino acids. Clear neuronal damage was observed only after dihydrokainate administration, which was partially prevented by intraperitoneal injection of (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate or by intrastriatal coinjection of 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline. No cell damage was observed with the other three glutamate carrier inhibitors used. It is concluded that an increased extracellular glutamate level in vivo due to dysfunction of its transporter is not sufficient for inducing neuronal damage. The neurotoxic effects of dihydrokainate could be explained by direct activation of glutamate postsynaptic receptors, an effect not shared by the other inhibitors used. 相似文献
8.
† D. Hantaz-Ambroise ‡D. Cambier A. Aït-Ikhlef §Ph. Parvy M. Murawsky F. Rieger 《Journal of neurochemistry》1995,65(3):1199-1204
Abstract: The wobbler mouse develops an inherited motoneuronal degeneration of unknown origin in the spinal cord. Primary cultures of adult wobbler spinal cord astrocytes display abnormal morphological characteristics with fewer processes and paucity of cell-cell contacts. We have searched for a possible involvement of glutamate and glutamine intra- and extracellular accumulations in vitro in the abnormal differentiation of mutant astrocytes. We have found significantly higher glutamate and glutamine concentrations in the culture media of mutant astrocytes over a 3-day period compared with normal control astrocytes. Moreover, intracellular glutamate concentrations decreased substantially in mutant astrocytes, but intracellular glutamine concentrations remained unchanged. Furthermore, decreasing initial glutamine concentrations in the culture medium (glutamine-depleted medium) led to the recovery of normal extra- and intracellular concentrations of glutamate and recovery of quasi-normal morphological differentiation and increased cell-cell contacts, leading to an essentially normal looking astrocyte network after 3 days of culture. Under these conditions, which lead to recovery, the only remaining abnormality was the higher glutamine extracellular concentration attained in the originally depleted glutamine media. These findings suggest that mechanisms regulating glutamate/glutamine synthesis and/or influx/efflux are defective in wobbler astrocytes, leading to metabolic imbalance and possible cytotoxic effects characterized by disturbed intercellular networks and poor differentiation. 相似文献
9.
Abstract: The specific binding of L-[3 H]glutamate to its receptors was investigated on crude membrane preparations from different brain regions of pentylenetetrazole-kindled rats using a binding assay technique. Pentylenetetrazole kindling induced by 10 intraperitoneal applications of 45 mg/kg over a period of 20 days resulted in a significant increase of both the convulsive susceptibility of animals to the convulsant and the specific L-[3 H]glutamate binding in hippocampus and in motor, frontal, and inferiotemporal (acoustic) cortex tested with a L-[3 H]glutamate concentration of 50 n M . No differences were observed in the other brain structures studied. Kinetic studies indicated that the enhanced L-[3 H]glutamate binding to hippocampal membranes from kindled rats reflects changes in the density of the glutamate binding sites rather than an increase in receptor affinity. To study the effect of acute generalized convulsions on L-[3 H]glutamate binding to synaptosomal membranes of hippocampus and visual cortex, rats were treated 24 h before the experiment with 60 mg/kg of pentylenetetrazole, i.p. Under these conditions, no differences between treated and control rats were observed. From these findings, it is concluded that the increase in glutamate receptor density demonstrated in hippocampus and several neocortical brain structures of pentylenetetrazole-kindled rats may be the expression of a specific enhancement of susceptibility of glutamatergic systems to this excitatory amino acid developing in the course of formation of pentylenetetrazole-induced kindling. 相似文献
10.
Inhibition of Glutamate Uptake with l-trans-Pyrrolidine-2,4-Dicarboxylate Potentiates Glutamate Toxicity in Primary Hippocampal Cultures 总被引:2,自引:1,他引:1
Michael B. Robinson Sina Djali Jeffrey R. Buchhalter 《Journal of neurochemistry》1993,61(6):2099-2103
Sodium-dependent, high-affinity glutamate transport is generally assumed to limit the toxicity of glutamate in vivo and in vitro, but there is very little direct evidence to support this hypothesis. In the present study, the effects of the specific uptake inhibitor l -trans-pyrrolidine-2,4-dicarboxylate on the toxicity and clearance of glutamate were examined in hippocampal neuronal cultures. At a concentration that was not toxic by itself, l -trans-pyrrolidine-2,4-dicarboxylate increased the toxicity of glutamate approximately fivefold and slowed the clearance of glutamate from the extracellular space. This toxicity was almost completely blocked by the N-methyl-d -aspartate receptor antagonist, d -2-amino-5-phosphonopentanoate. These studies provide direct evidence that sodium-dependent, high-affinity glutamate transport limits glutamate toxicity in vitro. 相似文献
11.
Andrea Volterra† Davide Trotti Paola Cassutti Cinzia Tromba Antonino Salvaggio Roberto C. Melcangi ‡ Giorgio Racagni† 《Journal of neurochemistry》1992,59(2):600-606
By using both synaptosomes and cultured astrocytes from rat cerebral cortex, we have investigated the inhibitory action of arachidonic acid on the high-affinity glutamate uptake systems, focusing on the possible physiological significance of this mechanism. Application of arachidonic acid (1-100 microM) to either preparation leads to fast (within 30 s) and largely reversible reduction in the uptake rate. When either melittin (0.2-1 microgram/ml), a phospholipase A2 activator, or thimerosal (50-200 microM), which inhibits fatty acid reacylation in phospholipids, is applied to astrocytes, both an enhancement in extracellular free arachidonate and a reduction in glutamate uptake are seen. The two effects display similar dose dependency and time course. In particular, 10% uptake inhibition correlates with 30% elevation in free arachidonate, whereas inhibition greater than or equal to 60% is paralleled by threefold stimulation of arachidonate release. In the presence of albumin (1-10 mg/ml), a free fatty acid-binding protein, inhibition by either melittin, thimerosal, or arachidonic acid is prevented and an enhancement of glutamate uptake above the control levels is observed. Our data show that neuronal and glial glutamate transport systems are highly sensitive to changes in extracellular free arachidonate levels and suggest that uptake inhibition may be a relevant mechanism in the action of arachidonic acid at glutamatergic synapses. 相似文献
12.
Sex Differences in Acute Swim Stress-Induced Changes in the Binding of MK-801 to the NMDA Subclass of Glutamate Receptors in Mouse Forebrain 总被引:1,自引:0,他引:1
Acute swim stress (3 min at 32°C) in mice produces increases in the binding of MK-801 to the NMDA subclass of glutamate receptors to forebrain membranes prepared from male mice. Scatchard analyses indicate that the observed increases in the binding of MK-801 in membranes from male mice are the result of changes in the affinity and density of low-affinity binding sites and in the density of high-affinity binding sites. In female mice, any changes in the binding of MK-801 appear to be much less pronounced and restricted to the low-affinity binding sites. These results are in contrast to the situation with binding to GABA receptors where acute swim stress increases GABA binding in forebrain membranes much more in female than in male mice. This indicates significant sex differences in the responses of receptors for the major excitatory and inhibitory transmitters to acute swim stress. These rapid changes in MK-801 binding may result from changes in endogenous modulators as appears to be the case in the acute swim stress-induced changes in GABA binding. As with GABA binding, the endogenous modulators are likely to include steroids, the sex differences reflecting differences in modulation by gonadal steroids and the stress-induced changes reflecting differences in modulation by adrenal steroids. Estradiol, progesterone, and corticosterone treatments have been reported by other workers to influence the properties of glutamate receptors. 相似文献
13.
Net Glutamate Release from Astrocytes Is Not Induced by Extracellular Potassium Concentrations Attainable in Brain 总被引:2,自引:0,他引:2
Abstract: Elevated extracellular potassium concentration ([K+ ]e ) has been shown to induce reversal of glial Na+ -dependent glutamate uptake in whole-cell patch clamp preparations. It is uncertain, however, whether elevated [K+ ]e similarly induces a net glutamate efflux from intact cells with a physiological intracellular milieu. To answer this question, astrocyte cultures prepared from rat and mouse cortices were incubated in medium with elevated [K+ ]e (by equimolar substitution of K+ for Na+ ), and glutamate accumulation was measured by HPLC. With [K+ ]e elevations to 60 m M , medium glutamate concentrations did not increase during incubation periods of 5–120 min. By contrast, 45 min of combined inhibition of glycolytic and oxidative ATP production increased medium glutamate concentrations 50–100-fold. Similar results were obtained in both rat and mouse cultures. Studies were also performed using astrocytes loaded with the nonmetabolized glutamate tracer d -aspartate, and parallel results were obtained; no increase in medium d -aspartate content resulted from [K+ ]e elevation up to 90 m M , whereas a large increase occurred during inhibition of energy metabolism. These results suggest that a net efflux of glutamate from intact astrocytes is not induced by any [K+ ]e attainable in brain. 相似文献
14.
Wenzhang Wang Fan Zhang Li Li Fangqiang Tang Sandra L. Siedlak Hisashi Fujioka Yingchao Liu Bo Su Yan Pi Xinglong Wang 《The Journal of biological chemistry》2015,290(1):168-182
Mitochondrial dysfunction plays a central role in glutamate-evoked neuronal excitotoxicity, and mitochondrial fission/fusion dynamics are essential for mitochondrial morphology and function. Here, we establish a novel mechanistic linker among glutamate excitotoxicity, mitochondrial dynamics, and mitochondrial dysfunction in spinal cord motor neurons. Ca2+-dependent activation of the cysteine protease calpain in response to glutamate results in the degradation of a key mitochondrial outer membrane fusion regulator, mitofusin 2 (MFN2), and leads to MFN2-mediated mitochondrial fragmentation preceding glutamate-induced neuronal death. MFN2 deficiency impairs mitochondrial function, induces motor neuronal death, and renders motor neurons vulnerable to glutamate excitotoxicity. Conversely, MFN2 overexpression blocks glutamate-induced mitochondrial fragmentation, mitochondrial dysfunction, and/or neuronal death in spinal cord motor neurons both in vitro and in mice. The inhibition of calpain activation also alleviates glutamate-induced excitotoxicity of mitochondria and neurons. Overall, these results suggest that glutamate excitotoxicity causes mitochondrial dysfunction by impairing mitochondrial dynamics via calpain-mediated MFN2 degradation in motor neurons and thus present a molecular mechanism coupling glutamate excitotoxicity and mitochondrial dysfunction. 相似文献
15.
Cerebral Vulnerability Is Associated with Selective Increase in Extracellular Glutamate Concentration in Experimental Thiamine Deficiency 总被引:1,自引:0,他引:1
Alan S. Hazell Roger F. Butterworth Antoine M. Hakim 《Journal of neurochemistry》1993,61(3):1155-1158
Abstract: The concentration of apolipoprotein E (apoE), a high-affinity ligand for the low-density lipoprotein receptor, increases dramatically in peripheral nerve following injury. This endoneurial apoE is thought to play an important role in the redistribution of lipids from the degenerating axonal and myelin membranes to the regenerating axons and myelin sheaths. The importance of apoE in nerve repair was examined using mutant mice that lack apoE. We show that at 2 and 4 weeks following sciatic nerve crush, regenerating nerves in apoE-deficient mice were morphologically similar to regenerating nerves in control animals, indicating that apoE is not essential for peripheral nerve repair. Moreover, cholesterol synthesis was reduced in regenerating nerves of apoE-deficient mice as much as in regenerating nerves of control animals. These results suggest that the intraneural conservation and reutilization of cholesterol following nerve injury do not require apoE. 相似文献
16.
We showed previously that amphetamine challenge produces a delayed increase in glutamate efflux in the ventral tegmental area of both naive and chronic amphetamine-treated rats. The present study examined the mechanisms underlying this response. The NMDA receptor antagonist MK-801 (0.1 mg/kg, i.p.) or the D1 dopamine receptor antagonist SCH 23390 (0.1 mg/kg, i.p.), given 30 min before acute amphetamine (5 mg/kg, i.p.), prevented amphetamine-induced glutamate efflux. Neither antagonist by itself altered glutamate efflux. Ibotenic acid lesions of the prefrontal cortex similarly prevented amphetamine-induced glutamate efflux, while producing a trend toward decreased basal glutamate levels (82.8% of sham group). Previous work has shown that the doses of NMDA and D1 receptor antagonists used in this study prevent the induction of behavioral sensitization when coadministered repeatedly with amphetamine, and that identical prefrontal cortex lesions performed before repeated amphetamine prevent the induction of ambulatory sensitization. Thus, treatments that prevent acute amphetamine from elevating glutamate efflux in the ventral tegmental area also prevent repeated amphetamine from eliciting behavioral sensitization. These findings suggest that repeated elevation of glutamate levels during a chronic amphetamine regimen may contribute to the cascade of neuroadaptations within the ventral tegmental area that enables the induction of sensitization. 相似文献
17.
The effect of the excitotoxin kainic acid on glutamate and glutamine metabolism was studied in cerebellar slices incubated with D-[2-14C]glucose, [U-14C]gamma-aminobutyric acid, [3H]acetate, [U-14C]glutamate, and [U-14C]glutamine as precursors. Kainic acid (1 mM) strongly inhibited the labeling of glutamine relative to that of glutamate from all precursors except [2-14C]glucose and [U-14C]glutamine. Kainic acid did not inhibit glutamine synthetase directly. The data indicate that in the cerebellum kainic acid inhibits the synthesis of glutamine from the small pool of glutamate that is thought to be associated with glial cells. Kainic acid also markedly stimulated the efflux of glutamate from cerebellar slices and this release was not sensitive to tetrodotoxin. Kainic acid stimulated efflux of both glucose- and acetate-labeled glutamate. In contrast, veratridine released glucose-labeled glutamate preferentially via a tetrodotoxin-sensitive mechanism. Kainic acid did not release [U-14C]glutamate from synaptosomal fractions. These results suggest that the bulk of the glutamate released from cerebellar slices by kainic acid comes from nonsynaptic pools. 相似文献
18.
Abstract: The uptake of a-ketoglutarate and malate by rat brain synaptosomal preparations was found to be affected by a variety of substances at physiologically relevant concentrations. Glutamine altered the uptake of γ-ketoglutarate by causing an apparent reduction in the substrate-carrier affinity and an increase in Vmax . In contrast, glutamine did not appear to affect the Vmax of malate uptake, but it did increase markedly the uptake velocity at low concentrations of malate. L-Glutamate and L-as-partate were comparatively strong inhibitors of γ-keto-glutarate and malate uptake. N-Acetylaspartate was a weak inhibitor of γ-ketoglutarate uptake, a finding that contrasts with our previous observation that this compound potently inhibited γ-ketoglutarate uptake by synaptosomes obtained from the cerebellum of 8- to 14-day-old mice. Ca2+ exhibited a variable effect but usually enhanced the uptake of γ-ketoglutarate. The addition of small amounts of postmicrosomal supernatant to the incubation medium enhanced the uptake of γ-ketoglutarate by low-density synaptosomes. By comparison, the uptake of glutamate, glutamine, γ-aminobutyric acid, and several other amino acids was not affected. The enhancement of γ-ketoglutarate uptake by the supernatant was due to a heat labile substance that was retained by dialysis tubing (MW cutoff = 8,000) and Amicon filter cones (CF 25), and was precipitated by ammonium sulfate at 60% saturation. In experiments in which the metabolic conversion of [U--14 C] γ-ketoglutarate to glutamate, as-partate, glutamine, and aminobutyric acid was determined, the presence of glutamine and glutamate in the incubation medium did not affect the pattern of labelling appreciably. 相似文献
19.
Inhibition of Glutamate Transport in Synaptosomes by Dopamine Oxidation and Reactive Oxygen Species 总被引:6,自引:2,他引:6
Abstract: Dopamine can form reactive oxygen species and other reactive metabolites that can modify proteins and other cellular constituents. In this study, we tested the effect of dopamine oxidation products, other generators of reactive oxygen species, and a sulfhydryl modifier on the function of glutamate transporter proteins. We also compared any effects with those on the dopamine transporter, a protein whose function we had previously shown to be inhibited by dopamine oxidation. Preincubation with the generators of reactive oxygen species, ascorbate (0.85 m M ) or xanthine (500 µ M ) plus xanthine oxidase (25 mU/ml), inhibited the uptake of [3 H]glutamate (10 µ M ) into rat striatal synaptosomes (−54 and −74%, respectively). The sulfhydryl-modifying agent N -ethylmaleimide (50–500 µ M ) also led to a dose-dependent inhibition of [3 H]glutamate uptake. Preincubation with dopamine (100 µ M ) under oxidizing conditions inhibited [3 H]glutamate uptake by 25%. Exposure of synaptosomes to increasing amounts of dopamine quinone by enzymatically oxidizing dopamine with tyrosinase (2–50 U/ml) further inhibited [3 H]glutamate uptake, an effect prevented by the addition of glutathione. The effects of free radical generators and dopamine oxidation on [3 H]glutamate uptake were similar to the effects on [3 H]dopamine uptake (250 n M ). Our findings suggest that reactive oxygen species and dopamine oxidation products can modify glutamate transport function, which may have implications for neurodegenerative processes such as ischemia, methamphetamine-induced toxicity, and Parkinson's disease. 相似文献
20.
Role of Pyruvate Carboxylase in Facilitation of Synthesis of Glutamate and Glutamine in Cultured Astrocytes 总被引:1,自引:4,他引:1
William C. Gamberino Deborah A. Berkich Christopher J. Lynch Baiyang Xu Kathryn F. LaNoue 《Journal of neurochemistry》1997,69(6):2312-2325
Abstract: CO2 fixation was measured in cultured astrocytes isolated from neonatal rat brain to test the hypothesis that the activity of pyruvate carboxylase influences the rate of de novo glutamate and glutamine synthesis in astrocytes. Astrocytes were incubated with 14CO2 and the incorporation of 14C into medium or cell extract products was determined. After chromatographic separation of 14C-labelled products, the fractions of 14C cycled back to pyruvate, incorporated into citric acid cycle intermediates, and converted to the amino acids glutamate and glutamine were determined as a function of increasing pyruvate carboxylase flux. The consequences of increasing pyruvate, bicarbonate, and ammonia were investigated. Increasing extracellular pyruvate from 0 to 5 mM increased pyruvate carboxylase flux as observed by increases in the 14C incorporated into pyruvate and citric acid cycle intermediates, but incorporation into glutamate and glutamine, although relatively high at low pyruvate levels, did not increase as pyruvate carboxylase flux increased. Increasing added bicarbonate from 15 to 25 mM almost doubled CO2 fixation. When 25 mM bicarbonate plus 0.5 mM pyruvate increased pyruvate carboxylase flux to approximately the same extent as 15 mM bicarbonate plus 5 mM pyruvate, the rate of appearance of [14C]glutamate and glutamine was higher with the lower level of pyruvate. The conclusion was drawn that, in addition to stimulating pyruvate carboxylase, added pyruvate (but not added bicarbonate) increases alanine aminotransferase flux in the direction of glutamate utilization, thereby decreasing glutamate as pyruvate + glutamate →α-ketoglutarate + alanine. In contrast to previous in vivo studies, the addition of ammonia (0.1 and 5 mM) had no effect on net 14CO2 fixation, but did alter the distribution of 14C-labelled products by decreasing glutamate and increasing glutamine. Rather unexpectedly, ammonia did not increase the sum of glutamate plus glutamine (mass amounts or 14C incorporation). Low rates of conversion of α-[14C]ketoglutarate to [14C]glutamate, even in the presence of excess added ammonia, suggested that reductive amination of α-ketoglutarate is inactive under conditions studied in these cultured astrocytes. We conclude that pyruvate carboxylase is required for de novo synthesis of glutamate plus glutamine, but that conversion of α-ketoglutarate to glutamate may frequently be the rate-limiting step in this process of glutamate synthesis. 相似文献