RESUMO
Traumatic brain injury (TBI) is a devastating neurological disorder that usually presents in acute and chronic forms. Brain edema and associated increased intracranial pressure in the early phase following TBI are major consequences of acute trauma. On the other hand, neuronal injury, leading to neurobehavioral and cognitive impairments, that usually develop months to years after single or repetitive episodes of head trauma, are major consequences of chronic TBI. The molecular mechanisms responsible for TBI-induced injury, however, are unclear. Recent studies have suggested that early mitochondrial dysfunction and subsequent energy failure play a role in the pathogenesis of TBI. We therefore examined whether oxidative metabolism of (13)C-labeled glucose, lactate or glutamine is altered early following in vitro mechanical percussion-induced trauma (5 atm) to neurons (4-24 h), and whether such events contribute to the development of neuronal injury. Cell viability was assayed using the release of the cytoplasmic enzyme lactate dehydrogenase (LDH), together with fluorescence-based cell staining (calcein and ethidium homodimer-1 for live and dead cells, respectively). Trauma had no effect on the LDH release in neurons from 1 to 18 h. However, a significant increase in LDH release was detected at 24 h after trauma. Similar findings were identified when traumatized neurons were stained with fluorescent markers. Additionally (13)C-labeling of glutamate showed a small, but statistically significant decrease at 14 h after trauma. However, trauma had no effect on the cycling ratio of the TCA cycle at any time-period examined. These findings indicate that trauma does not cause a disturbance in oxidative metabolism of any of the substrates used for neurons. Accordingly, such metabolic disturbance does not appear to contribute to the neuronal death in the early stages following trauma.
Assuntos
Morte Celular , Glucose/metabolismo , Glutamina/metabolismo , Ácido Láctico/metabolismo , Neurônios/metabolismo , Percussão , Animais , Células Cultivadas , Neurônios/citologia , Ratos , Ratos Sprague-DawleyRESUMO
Glutamatergic neurotransmission accounts for a considerable part of energy consumption related to signaling in the brain. Chemical energy is provided by adenosine triphosphate (ATP) formed in glycolysis and tricarboxylic acid (TCA) cycle combined with oxidative phosphorylation. It is not clear whether ATP generated in these pathways is equivalent in relation to fueling of the energy-requiring processes, i.e., vesicle filling, transport, and enzymatic processing in the glutamatergic tripartite synapse (the astrocyte and pre- and postsynapse). The role of astrocytic glycogenolysis in maintaining theses processes also has not been fully elucidated. Cultured astrocytes and neurons were utilized to monitor these processes related to glutamatergic neurotransmission. Inhibitors of glycolysis and TCA cycle in combination with pathway-selective substrates were used to study glutamate uptake and release monitored with D-aspartate. Western blotting of glyceraldehyde-3-P dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK) was performed to determine whether these enzymes are associated with the cell membrane. We show that ATP formed in glycolysis is superior to that generated by oxidative phosphorylation in providing energy for glutamate uptake both in astrocytes and in neurons. The neuronal vesicular glutamate release was less dependent on glycolytic ATP. Dependence of glutamate uptake on glycolytic ATP may be at least partially explained by a close association in the membrane of GAPDH and PGK and the glutamate transporters. It may be suggested that these enzymes form a complex with the transporters and the Na(+) /K(+) -ATPase, the latter providing the sodium gradient required for the transport process.
Assuntos
Trifosfato de Adenosina/metabolismo , Comunicação Celular/fisiologia , Ácido Glutâmico/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Transmissão Sináptica/fisiologia , Animais , Metabolismo Energético/fisiologia , HumanosRESUMO
Cultures of dissociated cerebellum from 7-day-old mice were used to investigate the mechanism involved in synthesis and cellular redistribution of GABA in these cultures consisting primarily of glutamatergic granule neurons and a smaller population of GABAergic Golgi and stellate neurons. The distribution of GAD, GABA and the vesicular glutamate transporter VGlut-1 was assessed using specific antibodies combined with immunofluorescence microscopy. Additionally, tiagabine, SKF 89976-A, betaine, beta-alanine, nipecotic acid and guvacine were used to inhibit the GAT1, betaine/GABA (BGT1), GAT2 and GAT3 transporters. Only a small population of cells were immuno-stained for GAD while many cells exhibited VGlut-1 like immuno-reactivity which, however, never co-localized with GAD positive neurons. This likely reflects the small number of GABAergic neurons compared to the glutamatergic granule neurons constituting the majority of the cells. GABA uptake exhibited the kinetics of high affinity transport and could be partly (20%) inhibited by betaine (IC(50) 142 microM), beta-alanine (30%) and almost fully (90%) inhibited by SKF 89976-A (IC(50) 0.8 microM) or nipecotic acid and guvacine at 1 mM concentrations (95%). Essentially all neurons showed GABA like immunostaining albeit with differences in intensity. The results indicate that GABA which is synthesized in a small population of GAD-positive neurons is redistributed to essentially all neurons including the glutamatergic granule cells. GAT1 is not likely involved in this redistribution since addition of 15 microM tiagabine (GAT1 inhibitor) to the culture medium had no effect on the overall GABA content of the cells. Likewise the BGT1 transporter cannot alone account for the redistribution since inclusion of 3 mM betaine in the culture medium had no effect on the overall GABA content. The inhibitory action of beta-alanine and high concentrations of nipecotic acid and guvacine on GABA transport strongly suggests that also GAT2 or GAT3 (HUGO nomenclature) could play a role.
Assuntos
Cerebelo/citologia , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Ácido Glutâmico/metabolismo , Neurônios/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Betaína/farmacologia , Células Cultivadas , GABAérgicos/farmacologia , Agonistas GABAérgicos/farmacologia , Glutamato Descarboxilase/metabolismo , Lipotrópicos/farmacologia , Camundongos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Ácidos Nipecóticos/farmacologia , Tiagabina , Proteína Vesicular 1 de Transporte de Glutamato/metabolismoRESUMO
The significance and functional roles of glycogen shunt activity in the brain are largely unknown. It represents the fraction of metabolized glucose that passes through glycogen molecules prior to entering the glycolytic pathway. The present study was aimed at elucidating this pathway in cultured astrocytes from mouse exposed to agents such as a high [K+], D-aspartate and norepinephrine (NE) known to affect energy metabolism in response to neurotransmission. Glycogen shunt activity was assessed employing [1,6-13C]glucose, and the glycogen phosphorylase inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) to block glycogen degradation. The label intensity in lactate, reflecting glycolytic activity, was determined by mass spectrometry. In the presence of NE a substantial glycogen shunt activity was observed, accounting for almost 40% of overall glucose metabolism. Moreover, when no metabolic stimulant was applied, a compensatory increase in glycolytic activity was seen when the shunt was inhibited by DAB. Actually the labeling in lactate exceeded that obtained when glycolysis and glycogen shunt both were operational, i.e. supercompensation. A similar phenomenon was seen when astrocytes were exposed to D-aspartate. In addition to glycolysis, tricarboxylic acid (TCA) cycle activity was monitored, analyzing labeling by mass spectrometry in glutamate which equilibrates with alpha-ketoglutarate. Both an elevated [K+] and D-aspartate induced an increased TCA cycle activity, which was altered when glycogen degradation was inhibited. Thus, the present study provides evidence that manipulation of glycogen metabolism affects both glycolysis and TCA cycle metabolism. Altogether, the results reveal a highly complex interaction between glycogenolysis and glycolysis, with the glycogen shunt playing a significant role in astrocytic energy metabolism.
Assuntos
Astrócitos/metabolismo , Encéfalo/metabolismo , Catecolaminas/metabolismo , Ácido Glutâmico/metabolismo , Glicogênio/metabolismo , Glicólise/fisiologia , Agonistas Adrenérgicos/farmacologia , Animais , Animais Recém-Nascidos , Ácido Aspártico/metabolismo , Ácido Aspártico/farmacologia , Astrócitos/efeitos dos fármacos , Encéfalo/ultraestrutura , Células Cultivadas , Ciclo do Ácido Cítrico/efeitos dos fármacos , Ciclo do Ácido Cítrico/fisiologia , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/fisiologia , Inibidores Enzimáticos/farmacologia , Glicogênio Fosforilase/antagonistas & inibidores , Glicogênio Fosforilase/metabolismo , Glicogenólise/efeitos dos fármacos , Glicogenólise/fisiologia , Camundongos , Norepinefrina/metabolismo , Norepinefrina/farmacologiaRESUMO
The fine tuning of both glutamatergic and GABAergic neurotransmission is to a large extent dependent upon optimal function of astrocytic transport processes. Thus, glutamate transport in astrocytes is mandatory to maintain extrasynaptic glutamate levels sufficiently low to prevent excitotoxic neuronal damage. In GABA synapses hyperactivity of astroglial GABA uptake may lead to diminished GABAergic inhibitory activity resulting in seizures. As a consequence of this the expression and functional activity of astrocytic glutamate and GABA transport is regulated in a number of ways at transcriptional, translational and post-translational levels. This opens for a number of therapeutic strategies by which the efficacy of excitatory and inhibitory neurotransmission may be manipulated.
Assuntos
Astrócitos/fisiologia , Glutamatos/fisiologia , Proteínas de Membrana Transportadoras , Transmissão Sináptica/fisiologia , Ácido gama-Aminobutírico/fisiologia , Sistema X-AG de Transporte de Aminoácidos/biossíntese , Sistema X-AG de Transporte de Aminoácidos/fisiologia , Animais , Astrócitos/efeitos dos fármacos , Proteínas de Transporte/biossíntese , Proteínas de Transporte/fisiologia , Proteínas da Membrana Plasmática de Transporte de GABA , Humanos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/fisiologia , Transmissão Sináptica/efeitos dos fármacosRESUMO
Rat cerebral nonsynaptic mitochondria were incubated in medium containing 2 mM glutamine (Gln) or 2 mM glutamate (Glu), in the presence of a Gln uptake inhibitor histidine (His) as well as other basic amino acids, lysine and arginine (Lys, Arg) not inhibiting Gln uptake. Subsequently, the mitochondrial contents of Glu and Gln were determined by HPLC. Incubation in the presence of Glu alone increased the Glu content from approximately 3.5 to 15 nmol/mg protein, without affecting the Gln content. On the other hand, incubation with Gln increased the content of Gln from approximately 1.5 to approximately 12 nmol/mg, and that of Glu to 10 nmol/mg. As expected, addition of His did not alter the Glu and Gln content resulting from incubation with Glu. However, His significantly decreased to almost the preincubation level the content of Glu in mitochondria incubated with Gln, without affecting the content of Gln. No other amino acid had any effect on these parameters. The results point to the existence of distinct Gln pools, one of which is accessible to external Gln via a His-sensitive transporter and is accessible for deamidation in the mitochondria.
Assuntos
Amidas/metabolismo , Química Encefálica/efeitos dos fármacos , Glutamina/metabolismo , Histidina/farmacologia , Mitocôndrias/metabolismo , Animais , Arginina/metabolismo , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Ácido Glutâmico/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Lisina/metabolismo , Masculino , Mitocôndrias/efeitos dos fármacos , RatosRESUMO
Pyruvate carboxylation was studied in cerebellar astrocytes and granule neurons. The cells were incubated in medium containing [U-(13)C]glucose (2.5 mM) and [U-(13)C]lactate (1 mM) and varying amounts of 3-nitropropionic acid (3-NPA) plus/minus aspartate. 3-NPA alone clearly stopped tricarboxylic acid (TCA) cycle activity at the succinate dehydrogenase step in both culture types as evidenced by a buildup of succinate. Labeling of aspartate and glutamate was abolished in neurons in the presence of 3-NPA. In astrocytes, however, labeled glutamate and glutamine derived from pyruvate carboxylation was detected. Unchanged glucose and lactate metabolism in the absence of a functioning malate aspartate shuttle indicates the importance of the glycerol-3-phosphate shuttle in brain cells. To compensate for the loss of oxaloacetate in the presence of 3-NPA, unlabeled aspartate (0.25 mM) was added. In this case [1,2-(13)C] and [3,4-(13)C]aspartate were observed in neurons but not in astrocytes. This labeling pattern in aspartate occurs after a full turn of the TCA cycle and thus indicates only partial inhibition by 3-NPA in the neurons when aspartate is present. In astrocytes, however, aspartate derived from uniformly labeled pyruvate was observed clearly indicating pyruvate carboxylation. The present study has unequivocally demonstrated a quantitatively important pyruvate carboxylation in astrocytes but it was not possible to demonstrate the presence of such carboxylation in neurons. Based on the present results it may be safely concluded that neuronal pyruvate carboxylation is unlikely to be of quantitative significance.
Assuntos
Astrócitos/metabolismo , Encéfalo/metabolismo , Dióxido de Carbono/metabolismo , Ciclo do Ácido Cítrico/fisiologia , Neurônios/metabolismo , Fosforilação Oxidativa , Ácido Pirúvico/metabolismo , Aminoácidos/metabolismo , Animais , Ácido Aspártico/metabolismo , Radioisótopos de Carbono , Células Cultivadas , Cerebelo , Convulsivantes/farmacologia , Glucose/metabolismo , Ácido Láctico/metabolismo , Camundongos , Nitrocompostos , Propionatos/farmacologiaRESUMO
Glutamate dehydrogenase (GDH) specific activities, kinetic properties and allosteric regulation were studied in extracts from cultured neurons and astrocytes prepared from mouse cerebral cortex and cerebellum. Considerable differences were observed in the specific activity of the enzyme among the different cell types with astrocytes expressing the highest GDH activity. This may reflect the functional importance of these cells in glutamate uptake and metabolism. Among the neurons, the glutamatergic cerebellar granule cells showed a GDH specific activity that was 60% higher (P < 0.01) than that of the GABAergic cerebral cortical neurons. Also, the K(m) for ammonia was 1.7-fold higher in the cortical neurons than in the other cell types. These findings may reflect a particular need for the glutamatergic granule cells to synthesize glutamate via the GDH pathway. No differences were observed among the different cell types with regard to the allosteric properties of GDH expressed by these cells.
Assuntos
Astrócitos/enzimologia , Cerebelo/enzimologia , Córtex Cerebral/enzimologia , Glutamato Desidrogenase/metabolismo , Ácido Glutâmico/metabolismo , Neurônios/enzimologia , Difosfato de Adenosina/metabolismo , Difosfato de Adenosina/farmacologia , Animais , Animais Recém-Nascidos , Astrócitos/citologia , Células Cultivadas , Cerebelo/citologia , Córtex Cerebral/citologia , Glutamato Desidrogenase/antagonistas & inibidores , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacologia , Cinética , Camundongos , Neurônios/citologiaRESUMO
The metabolism of glucose and lactate was investigated in cultured mouse cerebellar astrocytes, a culture preparation consisting of a homogeneous population of cells, by incubating the cells in a medium containing either [U-(13)C]glucose or [U-(13)C]lactate in combination with unlabeled lactate and glucose, respectively. After the incubation, cell extracts and media were analyzed by GC/MS (gas chromatography/mass spectrometry) for labeling patterns in aspartate, glutamate, and glutamine, as well as the tricarboxylic acid (TCA) cycle constituents citrate and fumarate. Triple labeling of extracellular citrate exceeded that of double labeling from [U-(13)C]glucose. This was not the case when lactate was the labeled precursor. These results indicate that pyruvate carboxylation in biosynthesis of releasable citrate was less prominent from lactate compared with glucose. As observed in the case of extracellular citrate, triple labeling of intracellular aspartate was higher than double labeling when [U-(13)C]glucose was the precursor, but not with [U-(13)C]lactate as precursor. The pattern of labeling in citrate was different intra- and extracellularly and the extent of labeling extracellularly was 10 times higher using [U-(13)C]glucose compared with [U-(13)C]lactate. However, the intracellular citrate labeling from [U-(13)C]glucose only exceeded that originating from labeled lactate by a factor of two. This is in contrast to the labeling pattern obtained for glutamine, since intracellularly this was equally prominent using [U-(13)C]glucose and [U-(13)C]lactate as substrates. Moreover, extracellularly the labeling was only slightly higher when using [U-(13)C]glucose compared with [U-(13)C]lactate. Intracellular glutamate and extracellular glutamine exhibited similar incorporation patterns with regard to double compared with triple labeling and the extent of incorporation of label from [U-(13)C]lactate compared with [U-(13)C]glucose. It should be noted that the main intracellular pools of citrate and glutamine were compartmentalized; i.e., releasable citrate and glutamine exhibited a labeling pattern distinctly different from that of their intracellular pools. Moreover, carboxylation of pyruvate using glucose as the precursor was more important for biosynthesis of releasable glutamine and citrate, compared with their intracellular pools. Based on the results a model of multiple compartments is suggested. The compartments differ with regard to utilization of lactate and glucose, synthetic pathways for TCA cycle intermediates and amino acids, particularly citrate and glutamine, as well as the contents of different metabolites.
Assuntos
Aminoácidos/biossíntese , Astrócitos/metabolismo , Compartimento Celular/fisiologia , Ácido Cítrico/metabolismo , Metabolismo Energético/fisiologia , Glutamina/biossíntese , Líquido Intracelular/metabolismo , Animais , Astrócitos/citologia , Radioisótopos de Carbono/farmacocinética , Células Cultivadas/citologia , Células Cultivadas/metabolismo , Cerebelo/citologia , Cerebelo/metabolismo , Ciclo do Ácido Cítrico/fisiologia , Glucose/metabolismo , Glucose/farmacocinética , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Ácido Láctico/metabolismo , Ácido Láctico/farmacocinética , Camundongos , Ácido Pirúvico/metabolismoRESUMO
Uptake and release processes in cerebellar astrocytes and granule neurons (glutamatergic) for glutamate were investigated by the use of [3H]D-aspartate, a non-metabolizable glutamate analog. The effects of DL-threo-beta-benzyloxyaspartate (DL-TBOA) and L-trans-pyrrolidine-2,4-dicarboxylate (t-2,4-PDC) on uptake and release of [3H]D-aspartate were studied. Both compounds inhibited potently uptake of [3H]D-aspartate in neurons and astrocytes (IC50 values 10-100 microM), DL-TBOA being slightly more potent than t-2,4-PDC. Release of preloaded [3H]D-aspartate from neurons or astrocytes could be stimulated by addition of excess t-2,4-PDC whereas addition of DL-TBOA had no effect on [3H]D-aspartate efflux. Moreover, DL-TBOA inhibited significantly the depolarization-induced (55 mM KCI) release of preloaded [3H]D-aspartate in the neurons. The results reflect the fact that DL-TBOA is not transported by the glutamate carriers while t-2,4-PDC is a substrate which may heteroexchange with [3H]D-aspartate. It is suggested that DL-TBOA may be used to selectively inhibit depolarization coupled glutamate release mediated by reversal of the carriers.
Assuntos
Astrócitos/metabolismo , Ácido D-Aspártico/metabolismo , Ácido D-Aspártico/farmacocinética , Ácidos Dicarboxílicos/farmacologia , Ácido Glutâmico/metabolismo , Neurônios/metabolismo , Inibidores da Captação de Neurotransmissores/farmacologia , Pirrolidinas/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Células Cultivadas , Cerebelo/citologia , Camundongos , Neurônios/efeitos dos fármacos , TrítioRESUMO
GABA exists in at least two different intracellular pools, i.e., a cytoplasmic or metabolic pool and a vesicular pool. This study was performed to gain information about the quantitative role of the tricarboxylic acid (TCA) cycle in biosynthesis of GABA from glutamine when GABA was selectively released from either one of these two pools. Cultured cerebral cortical neurons (GABAergic) were incubated in a medium containing 0.5 mM [U-13C]glutamine and subsequently depolarized for release of GABA from either the vesicular or the cytoplasmic pool. The vesicular release was induced by 55 mM K+ in the presence of tiagabine, a nontransportable inhibitor of the plasma membrane GABA carriers, whereas the cytoplasmic release via a reversal of the GABA carrier was induced by exposure to N-methyl-D-aspartate (NMDA; 50 microM) in the presence of (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionate (AMPA; 50 microM). Cell extracts were analyzed by 13C magnetic resonance spectroscopy subsequent to the incubation or depolarization. The percentage of GABA generated from glutamine via the TCA cycle decreased from 60% to 46% during depolarization, inducing GABA release from the cytoplasmic pool, whereas a significant change in this parameter was not observed after release from the vesicular pool. These observations indicate that, during release from the cytoplasmic pool, the fraction of GABA synthesized directly from glutamine without involvement of the TCA cycle is more pronounced than that occurring during resting conditions and when release occurs from the vesicular pool. This might be explained by differences in the regulation of the two isoforms of glutamate decarboxylase (GAD(65) and GAD(67)), which presumably play different roles in the maintenance of GABA in the two pools. Both isoforms were found in the cultured cerebral cortical neurons, as shown by Western blotting employing an antibody recognizing GAD(65) as well as GAD(67).
Assuntos
Citosol/enzimologia , Neurônios/metabolismo , Vesículas Transportadoras/enzimologia , Ácido gama-Aminobutírico/metabolismo , Animais , Isótopos de Carbono , Compartimento Celular/fisiologia , Células Cultivadas , Córtex Cerebral/citologia , Ciclo do Ácido Cítrico/fisiologia , Feminino , Glutamato Descarboxilase/metabolismo , Glutamina/metabolismo , Isoenzimas/metabolismo , Espectroscopia de Ressonância Magnética , Potenciais da Membrana/fisiologia , Camundongos , Mitocôndrias/metabolismo , Neurônios/citologia , GravidezRESUMO
The metabolism of [U-(13)C]lactate (1 mM) in the presence of unlabeled glucose (2.5 mM) was investigated in glutamatergic cerebellar granule cells, cerebellar astrocytes, and corresponding co-cultures. It was evident that lactate is primarily a neuronal substrate and that lactate produced glycolytically from glucose in astrocytes serves as a substrate in neurons. Alanine was highly enriched with (13)C in the neurons, whereas this was not the case in the astrocytes. Moreover, the cellular content and the amount of alanine released into the medium were higher in neurons than astrocytes. On incubation of the different cell types in medium containing alanine (1 mM), the astrocytes exhibited the highest level of accumulation. Altogether, these results indicate a preferential synthesis and release of alanine in glutamatergic neurons and uptake in cerebellar astrocytes. A new functional role of alanine may be suggested as a carrier of nitrogen from glutamatergic neurons to astrocytes, a transport that may operate to provide ammonia for glutamine synthesis in astrocytes and dispose of ammonia generated by the glutaminase reaction in glutamatergic neurons. Hence, a model of a glutamate-glutamine/lactate-alanine shuttle is presented. To elucidate if this hypothesis is compatible with the pattern of alanine metabolism observed in the astrocytes and neurons from cerebellum, the cells were incubated in a medium containing [(15)N]alanine (1 mM) and [5-(15)N]glutamine (0.5 mM), respectively. Additionally, neurons were incubated with [U-(13)C]glutamine to estimate the magnitude of glutamine conversion to glutamate. Alanine was labeled from [5-(15)N]glutamine to 3.3% and [U-(13)C]glutamate generated from [U-(13)C]glutamine was labeled to 16%. In spite of the modest labeling in alanine, it is clear that nitrogen from ammonia is transferred to alanine via transamination with glutamate formed by reductive amination of alpha-ketoglutarate. With regard to the astrocytic part of the shuttle, glutamine was labeled to 22% in one nitrogen atom whereas 3.2% was labeled in two when astrocytes were incubated in [(15)N]alanine. Moreover, in co-cultures, [U-(13)C]alanine labeled glutamate and glutamine equally, whereas [U-(13)C]lactate preferentially labeled glutamate. Altogether, these results support the role proposed above of alanine as a possible ammonia nitrogen carrier between glutamatergic neurons and surrounding astrocytes and they show that lactate is preferentially metabolized in neurons and alanine in astrocytes.
Assuntos
Alanina/metabolismo , Amônia/metabolismo , Astrócitos/metabolismo , Cerebelo/metabolismo , Ácido Glutâmico/metabolismo , Lactatos/metabolismo , Neurônios/metabolismo , Animais , Astrócitos/citologia , Isótopos de Carbono , Células Cultivadas , Cerebelo/citologia , Técnicas de Cocultura , Cromatografia Gasosa-Espectrometria de Massas , Glucose/metabolismo , Glutamina/metabolismo , Cinética , Camundongos , Modelos Químicos , Modelos Neurológicos , Neurônios/citologiaRESUMO
This study was performed to analyze the effects of the barbiturate thiopental on neuronal glutamate uptake, release and metabolism. Since barbiturates are known to bind to the GABA(A) receptor, some experiments were carried out in the presence of GABA. Cerebellar granule neurons were incubated for 2 h in medium containing 0.25 mM [U-(13)C]glutamate, 3 mM glucose, 50 microM GABA and 0.1 or 1 mM thiopental when indicated. When analyzing cell extracts, it was surprisingly found that in addition to glutamate, aspartate and glutathione, GABA was also labeled. In the medium, label was observed in glutamate, aspartate and lactate. Glutamate exhibited different labeling patterns, indicating metabolism in the tricarboxylic acid cycle, and subsequent release. A net uptake of [U-(13)C]glutamate and unlabeled glucose was seen under all conditions. The amounts of most metabolites synthesized from [U-(13)C]glutamate were unchanged in the presence of GABA with or without 0.1 mM thiopental. In the presence of 1 mM thiopental, regardless of the presence of GABA, decreased amounts of [1,2, 3-(13)C]glutamate and [U-(13)C]aspartate were found in the medium. In the cell extracts increased [U-(13)C]glutamate, [1,2, 3-(13)C]glutamate, labeled glutathione and [U-(13)C]aspartate were observed in the 1 mM thiopental groups. Glutamate efflux and uptake were studied using [(3)H]D-aspartate. While efflux was substantially reduced in the presence of 1 mM thiopental, this barbiturate only marginally inhibited uptake even at 3 mM. These results may suggest that the previously demonstrated neuroprotective action of thiopental could be related to its ability to reduce excessive glutamate outflow. Additionally, thiopental decreased the oxidative metabolism of [U-(13)C]glutamate but at the same time increased the detectable metabolites derived from the TCA cycle. These latter effects were also exerted by GABA.
Assuntos
Cerebelo/metabolismo , Moduladores GABAérgicos/farmacologia , Tiopental/farmacologia , Animais , Ácido Aspártico/metabolismo , Transporte Biológico Ativo/efeitos dos fármacos , Células Cultivadas , Cerebelo/citologia , Cerebelo/efeitos dos fármacos , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/metabolismo , Metabolismo Energético/efeitos dos fármacos , Cromatografia Gasosa-Espectrometria de Massas , Espectroscopia de Ressonância Magnética , Camundongos , Ácido gama-Aminobutírico/farmacologiaRESUMO
Cultured neocortical neurons were incubated in medium containing [U-13C]glucose (0.5 mM) and in some cases unlabeled glutamine (0.5 mM). Subsequently the cells were "superfused" for investigation of the effect of depolarization by 55 mM K+. Cell extracts were analyzed by 13C magnetic resonance spectroscopy and gas chromatography/mass spectrometry to determine incorporation of 13C in glutamate, GABA, aspartate and fumarate. The importance of the tricarboxylic acid (TCA) cycle for conversion of the carbon skeleton of glutamine to GABA was evident from the effect of glutamine on the labeling pattern of GABA and glutamate. Moreover, analysis of the labeling patterns of glutamate in particular indicated a depolarization induced increased oxidative metabolism. This effect was only observed in glutamate and not in neurotransmitter GABA. Based on this a hypothesis of mitochondrial compartmentation may be proposed in which mitochondria associated with neurotransmitter synthesis are distinct from those aimed at energy production and influenced by depolarization. The hypothesis of mitochondrial compartmentation was further supported by the finding that the total percent labeling of fumarate and aspartate differed significantly from each other. This can only be explained by the existence of multiple TCA cycles with different turnover rates.
Assuntos
Ciclo do Ácido Cítrico/fisiologia , Neocórtex/metabolismo , Neurônios/metabolismo , Aminoácidos/metabolismo , Animais , Isótopos de Carbono , Células Cultivadas , Eletrofisiologia , Fumaratos/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Glucose/farmacologia , Glutamina/farmacologia , Espectroscopia de Ressonância Magnética , Camundongos , Neocórtex/citologia , Neocórtex/efeitos dos fármacos , Neocórtex/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologiaRESUMO
A novel inhibitor of liver glycogen phosphorylase, isofagomine, was investigated as a possible inhibitor of the enzyme in the brain and in cultured astrocytes. Additionally, the effect of the drug on norepinephrine (NE) induced glycogen degradation in astrocytes was studied. Astrocytes were cultured from mouse cerebral cortex and homogenates were prepared from the cells as well as from mouse brain. Isofagomine dose-dependently inhibited glycogen phosphorylase when measured in the direction of glycogen degradation in both preparations with IC50 values (mean +/- SEM) of 1.0 +/- 0.1 microM and 3.3 +/- 0.5 microM in brain and astrocyte homogenates, respectively. Moreover, isofagomine at a concentration of 400 microM completely prevented NE induced depletion of glycogen stores and the concomitant lactate production in intact astrocytes. It is suggested that this novel glycogen phosphorylase inhibitor may be a valuable tool to investigate the functional importance of glycogen in astrocytes and in the brain.
Assuntos
Astrócitos/metabolismo , Córtex Cerebral/metabolismo , Inibidores Enzimáticos/farmacologia , Glicogênio/metabolismo , Piperidinas/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Relação Dose-Resposta a Droga , Imino Piranoses , Camundongos , Norepinefrina/farmacologia , Fosforilases/antagonistas & inibidoresRESUMO
GABA, which is present in the brain in large amounts, is distributed among distinctly different cellular pools, possibly reflecting its multiple functions as metabolite, neurotransmitter, and neurotrophin. Its metabolic enzymes also exhibit heterogeneity, because glutamate decarboxylase exists in two isoforms with different subcellular distribution and regulatory properties. Moreover, recent evidence points to a more pronounced regulatory role of the tricarboxylic acid cycle than hitherto anticipated in the biosynthetic machinery responsible for formation of GABA from glutamine. Additionally, GABAergic neurons may contain distinct populations of mitochondria having different turnover rates of the tricarboxylic acid cycle with different levels of association with GABA synthesis from 2-oxoglutarate via glutamate. These aspects are discussed in relation to the different functional roles of GABA and its prominent involvement in epileptogenic activity.
Assuntos
Encéfalo/fisiologia , Neurônios/fisiologia , Ácido gama-Aminobutírico/fisiologia , Animais , Encéfalo/metabolismo , Diferenciação Celular , Epilepsia/fisiopatologia , Glutamato Descarboxilase/metabolismo , Glutamina/metabolismo , Humanos , Isoenzimas/metabolismo , Neurônios/citologia , Transmissão Sináptica , Ácido gama-Aminobutírico/metabolismoRESUMO
In contrast to the classic concept of direct conversion of glutamine to gamma-aminobutyric acid (GABA; via glutamate), this process may involve alpha-ketoglutarate as an intermediary metabolite and tricarboxylic acid (TCA) cycle activity. To obtain information about a possible differential role of these pathways for the synthesis of cytosolic and vesicular GABA, cultured neocortical neurons were incubated in medium containing [U-(13)C]glucose (0.5 mM) and in some cases unlabeled glutamine (0.5 mM). Subsequently, the cells were "superfused" for investigation of the effect of depolarization by 55 mM K+. To make sure that depolarization by 55 mM K+ released only vesicular GABA, tiagabin, a nontransportable inhibitor of the plasma membrane GABA carriers, was included in the medium to prevent GABA release from the cytoplasmic pool by reversal of the carriers. The importance of the TCA cycle for conversion of the carbon skeleton of glutamine to GABA was evident from the effect of glutamine on the labeling pattern of GABA. Percentage of labeling by GABA released into the depolarizing medium was the same as that in the corresponding cell extracts and was unaffected by the presence of glutamine during incubation. Despite the existence of multiple forms of glutamate decarboxylase, compartmentation of glutamate pools, and functionally different compartments within neurons, there appears to be full equilibration between the vesicular and cytosolic pools of GABA. However, during depolarization, the newly synthesized pool of GABA from glutamine does not rapidly equilibrate with the vesicular pool.
Assuntos
Ciclo do Ácido Cítrico/fisiologia , Glutamina/metabolismo , Neocórtex/metabolismo , Neurônios/metabolismo , Vesículas Sinápticas/metabolismo , Ácido gama-Aminobutírico/biossíntese , Aminoácidos/análise , Animais , Células Cultivadas , Fumaratos/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Espectroscopia de Ressonância Magnética , Potenciais da Membrana/fisiologia , Camundongos , Mitocôndrias/metabolismo , Neocórtex/citologiaAssuntos
Astrócitos/metabolismo , Córtex Cerebral/metabolismo , Ciclo do Ácido Cítrico , Mitocôndrias/metabolismo , Neurônios/metabolismo , Acetatos/metabolismo , Animais , Ácido Aspártico/metabolismo , Astrócitos/citologia , Células Cultivadas , Glucose/metabolismo , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Mitocôndrias/ultraestrutura , Modelos Químicos , Neurônios/citologiaRESUMO
In cerebral cortical neurons, synthesis of the tricarboxylic acid (TCA) cycle-derived amino acids, glutamate and aspartate as well as the neurotransmitter of these neurons, gamma-aminobutyrate (GABA), was studied incubating the cells in media containing 0.5 mM [U-13C]glucose in the absence or presence of glutamine (0.5 mM). Lyophilized cell extracts were analyzed by 13C nuclear magnetic resonance (NMR) spectroscopy and HPLC. The present findings were compared to results previously obtained using 1.0 mM [U-13C]lactate as the labeled substrate for the neurons. Regardless of the amino acids studied, incubation periods of 1 and 4 h resulted in identical amounts of 13C incorporated. Furthermore, the metabolism of lactate was studied under analogous conditions in cultured cerebral cortical astrocytes. The incorporation of 13C from lactate into glutamate was much lower in the astrocytes than in the neurons. In cerebral cortical neurons the total amount of 13C in GABA, glutamate and aspartate was independent of the labeled substrate. The enrichment in glutamate and aspartate was, however, higher in neurons incubated with lactate. Thus, lactate appears to be equivalent to glucose with regard to its access to the TCA cycle and subsequent labeling of glutamate, aspartate and GABA. It should be noted, however, that incubation with lactate in place of glucose led to lower cellular contents of glutamate and aspartate. The presence of glutamine affected the metabolism of glucose and lactate differently, suggesting that the metabolism of these substrates may be compartmentalized.
Assuntos
Astrócitos/metabolismo , Córtex Cerebral/metabolismo , Glucose/metabolismo , Ácido Láctico/metabolismo , Neurônios/metabolismo , Animais , Ácido Aspártico/metabolismo , Isótopos de Carbono , Células Cultivadas , Córtex Cerebral/citologia , Cromatografia Líquida de Alta Pressão , Ciclo do Ácido Cítrico/fisiologia , Ácido Glutâmico/metabolismo , Espectroscopia de Ressonância Magnética , Camundongos , Ácido gama-Aminobutírico/metabolismoRESUMO
Primary cultures of mouse cerebral cortical neurons (GABAergic) were incubated for 4 hours in media without glucose containing 1.0 mmol/L [U-13C]lactate in the absence or presence of 0.5 mmol/L glutamine. Redissolved, lyophilized cell extracts were analyzed by 13C nuclear magnetic resonance spectroscopy to investigate neuronal metabolism of lactate and by HPLC for determination of the total amounts of glutamate (Glu), gamma-aminobutyric acid (GABA), and aspartate (Asp). The 13C nuclear magnetic resonance spectra of cell extracts exhibited multiplets for Glu, GABA, and Asp, indicating pronounced recycling of labeled tricarboxylic acid cycle constituents. There was extensive incorporation of 13C label into amino acids in neurons incubated without glutamine, with the percent enrichments being approximately 60% for Glu and Asp, and 27% for GABA. When 0.5 mmol/L glutamine was added to the incubation medium, the enrichments for Asp, Glu, and GABA were 25%, 35%, and 25%, respectively. This strongly suggests that glutamine is readily converted to Glu and Asp but that conversion to GABA may be complex. The observation that enrichment in GABA was identical in the absence and presence of glutamine whereas cycling was decreased in the presence of glutamine indicates that only C-2 units derived from glutamine are used for GABA synthesis, that is, that metabolism through the tricarboxylic acid cycle is a prerequisite for GABA synthesis from glutamine. The current study gives further support to the hypothesis that cellular metabolism is compartmentalized and that lactate is an important fuel for neurons in terms of energy metabolism and extensively labels amino acids synthesized from tricarboxylic acid cycle intermediates (Asp and Glu) as well as the neurotransmitter in these neurons (GABA).