Mechanism of conversion of aspartate into glutamate in cerebral-cortex slices.
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ABSTRACT:
SUBMITTER: Simon G
PROVIDER: S-EPMC1270222 | biostudies-other | 1967 Jan
REPOSITORIES: biostudies-other
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Project description:1. When guinea-pig cerebral-cortex slices were incubated with [U-(14)C]glutamate as substrate, the specific radioactivities of the citric acid-cycle intermediates were lower than that of the aspartate isolated from the same vessels. 2. Aspartate was significantly labelled when [5-(14)C]glutamate was used as substrate and the aspartate contained almost no label when [1-(14)C]glutamate was present as substrate. 3. When specifically labelled glutamate was used as substrate, the label was found in the isolated aspartate in the position that would be predicted by citric acid-cycle mechanisms. 4. The results are consistent with the theory of ;compartmentation' of amino acid metabolism.
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Project description:1. Cerebral-cortex slices from rat brain, loaded with labelled l-glutamate as a result of aerobic incubation with labelled glucose, lost less than 15% of this glutamate on subsequent incubation in the presence of unlabelled glucose and l-glutamate. This indicates that very little exchange occurs between extracellular l-glutamate and glutamate accumulated in the neurons as a result of glucose metabolism. 2. Slices, loaded with labelled l-glutamate as a result of aerobic incubation in a medium containing unlabelled glucose and labelled l-glutamate, lost more than half of this glutamate on subsequent incubation in the presence of unlabelled l-glutamate. This indicates that exchange occurs between extracellular glutamate and glutamate accumulated in brain slices as a result of its uptake from the incubation medium. 3. Evidence was obtained suggesting that only a part of the glutamate, accumulated in brain slices as a result of its uptake from an incubation medium containing both glucose and l-glutamate, entered the neurons; apparently almost all the rest entered the glia. 4. It is concluded that the slices contain a pool of glutamate, derived from glucose and located in the neurons, which is poorly exchangeable with extracellular glutamate, and another pool of glutamate, derived from extracellular glutamate and located in the glia, which is freely exchangeable with extracellular glutamate.
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Project description:1. Intracellular concentrations of intermediates and cofactors of glycolysis were measured in guinea-pig cerebral cortex slices incubated under varying conditions. 2. Comparison of mass-action ratios with apparent equilibrium constants for the reactions of glycolysis showed that hexokinase, phosphofructokinase and pyruvate kinase catalyse reactions generally far from equilibrium, whereas phosphoglucose isomerase, aldolase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, adenlyate kinase and creatine phosphokinase are generally close to equilibrium. The possibility that glyceraldehyde 3-phosphate dehydrogenase may catalyse a ;non-equilibrium' reaction is discussed. 3. Correlation of changes in concentrations of substrates for enzymes catalysing ;non-equilibrium' reactions with changes in rates of glycolysis caused by alteration of the conditions of incubation showed that hexokinase, phosphofructokinase, pyruvate kinase and possibly glyceraldehyde 3-phosphate dehydrogenase are subject to metabolic control in cerebral cortex slices. 4. It is suggested that the glycolysis is controlled by two regulatory systems, the hexokinase-phosphofructokinase system and the glyceraldehyde 3-phosphate dehydrogenase-pyruvate kinase system. These are discussed. 5. It is concluded that the rate of glycolysis in guinea-pig cerebral cortex slices is limited either by the rate of glucose entry into the slices or by the hexokinase-phosphofructokinase system. 6. It is concluded that addition of 0.1mm-ouabain to guinea-pig cerebral cortex slices causes inhibition of either glyceraldehyde 3-phosphate dehydrogenase or phosphoglycerate kinase or both, in a manner independent of the known action of ouabain on the sodium- and potassium-activated adenosine triphosphatase.
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Project description:By the use of 1mm-iodoacetate to inhibit glycolysis in guinea-pig cerebral tissue slices, the kinetics of the uptake of monosaccharides on transfer of tissue from 0 degrees to 37 degrees were studied. d-Ribose, d-galactose, d-mannose, l-sorbose, and d-fructose showed diffusion kinetics, whereas 2-deoxy-d-glucose, d-glucose, d-arabinose and d-xylose showed saturation kinetics.
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Project description:Aberrant function of glutamatergic pathways is likely to underlie the pathology of schizophrenia. Evidence of oxidative stress in the disease pathology has also been reported. N-Acetylaspartate (NAA) is metabolically linked to both cascades and may be a key marker in exploring the interconnection of glutamatergic pathways and oxidative stress. Several studies have reported positive correlation between the levels of NAA and Glx (the sum of glutamate and glutamine) in several brain regions in healthy subjects, by using proton magnetic resonance spectroscopy ([(1)H]MRS). Interestingly, one research group recently reported decoupling of the relationship between NAA and Glx in the hippocampus of patients with schizophrenia. Here we report levels of NAA and Glx measured using [(1)H]MRS, relative to the level of creatine (Cr) as an internal control. The dorsolateral prefrontal cortex (DLPFC) and anterior cingulate cortex (ACC) in 25 patients with schizophrenia and 17 matched healthy controls were studied. In DLPFC, NAA/Cr and Glx/Cr were significantly positively correlated in healthy controls after correction for the effect of age and smoking status and after correction for multiple comparisons (r= 0.627, P= 0.017). However, in patients with schizophrenia, the positive correlation between NAA/Cr and Glx/Cr was not observed even after correcting for these two variables (r= -0.330, P= 0.124). Positive correlation between NAA/Cr and Glx/Cr was not observed in the ACC in both groups. Decoupling of NAA and Glx in the DLPFC may reflect the interconnection of glutamatergic pathways and oxidative stress in the pathology of schizophrenia, and may possibly be a biomarker of the disease.
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Project description:The kinetics of the influx and efflux of radioactive l-glycine was studied in slices of rat cerebral cortex. The influx showed saturation kinetics and was inhibited by l-alanine. Influx was dependent on the presence of Na(+) ions and a metabolizable substrate. The efflux of glycine was accelerated by alanine. It was concluded that carrier-mediated facilitated diffusion was the mechanism of glycine uptake by, and efflux from, cerebral slices.
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Project description:Glutamate transporters catalyse the thermodynamically unfavourable transport of anionic amino acids across the cell membrane by coupling it to the downhill transport of cations. This coupling mechanism is still poorly understood, in part because the available crystal structures of these transporters are of relatively low resolution. Here we solve crystal structures of the archaeal transporter GltTk in the presence and absence of aspartate and use molecular dynamics simulations and binding assays to show how strict coupling between the binding of three sodium ions and aspartate takes place.
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Project description:Excitatory amino acid transporters (EAATs) are abundantly expressed by astrocytes, rapidly remove glutamate from the extracellular environment, and restrict the temporal and spatial extent of glutamate signaling. Studies probing EAAT function suggest that their capacity to remove glutamate is large and does not saturate, even with substantial glutamate challenges. In contrast, we report that neuronal activity rapidly and reversibly modulates EAAT-dependent glutamate transport. To date, no physiological manipulation has shown changes in functional glutamate uptake in a nonpathological state. Using iGluSnFr-based glutamate imaging and electrophysiology in the adult mouse cortex, we show that glutamate uptake is slowed up to threefold following bursts of neuronal activity. The slowing of glutamate uptake depends on the frequency and duration of presynaptic neuronal activity but is independent of the amount of glutamate released. The modulation of glutamate uptake is brief, returning to normal within 50 ms after stimulation ceases. Interestingly, the slowing of glutamate uptake is specific to activated synapses, even within the domain of an individual astrocyte. Activity-induced slowing of glutamate uptake, and the increased persistence of glutamate in the extracellular space, is reflected by increased decay times of neuronal NR2A-mediated NMDA currents. These results show that astrocytic clearance of extracellular glutamate is slowed in a temporally and spatially specific manner following bursts of neuronal activity ?30 Hz and that these changes affect the neuronal response to released glutamate. This suggests a previously unreported form of neuron-astrocyte interaction. SIGNIFICANCE STATEMENT:We report the first fast, physiological modulation of astrocyte glutamate clearance kinetics. We show that presynaptic activity in the cerebral cortex increases the persistence of glutamate in the extracellular space by slowing its clearance by astrocytes. Because of abundant EAAT expression, glutamate clearance from the extracellular space has been thought to have invariant kinetics. While multiple studies report experimental manipulations resulting in altered EAAT expression, our findings show that astrocytic glutamate uptake is dynamic on a fast time-scale. This shows rapid plasticity of glutamate clearance, which locally modulates synaptic signaling in the cortex. As astrocytic glutamate uptake is a fundamental and essential mechanism for neurotransmission, this work has implications for neurotransmission, extrasynaptic receptor activation, and synaptic plasticity.
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Project description:1. Sodium exchange was measured with (24)Na in incubated guinea-pig cerebral-cortex slices maintained under adequate metabolic conditions with a steady content of fluid and ions resembling that of brain in vivo. 2. Evidence was obtained indicating that Na(+) ions behaved in the inulin space as if they were extracellular, and that their entry into the non-inulin space of unstimulated tissue was about 10 times slower and could be separated, on the basis of complete exchangeability, into two components, a ;fast' one, which reacted to electrical stimulation, and a ;slow' one, exchanging at a rate of about 8muequiv./g./hr., which was not affected by stimulation. 3. The average rate of sodium turnover in unstimulated slices was 175-275muequiv./g./hr., whereas that for stimulated slices was approx. 4-6 times this, or 1050-1180muequiv./g./hr. The stimulated rate was equivalent to a turnover of 32% of the sodium in the non-inulin space/min., or 3mmuequiv./g./impulse. 4. Response to the onset of stimulation appeared to be immediate, but after cessation of stimulation increased sodium movements persisted for several minutes before return to unstimulated values. 5. Calculations based on electrochemical gradients suggested that about one-quarter of the energy available from respiration was required for sodium and potassium transport at maximal rates in both unstimulated and stimulated cerebral-cortex slices.
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Project description:1. Rat cerebral-cortex slices were incubated with (32)P(i), acetylcholine and eserine for periods of 10min and 2h. The specific radioactivity of phosphatidylinositol was elevated during these treatments by 36 and 106% respectively. 2. The specific radioactivities of the phosphatidylinositol in different cell structures were determined after subcellular fractionation. They were highest in the nuclear, microsomal and synaptic-vesicle fractions and lowest in myelin, both in the controls and in the acetylcholine-treated slices. 3. The stimulated labelling of phosphatidylinositol was relatively evenly distributed: no subcellular fraction showed a stimulation markedly higher than that in the homogenate. 4. Studies of the distributions and activities of marker enzymes indicated that the subcellular fractionation achieved was similar to that with fresh tissue. 5. The results are discussed in relation to the previous report that the stimulation is observed throughout the neuronal cell-bodies and in relation to the hypothesis that the labelled phosphatidylinositol produced by stimulation is a component of an acetylcholine-receptor proteolipid localized in the synaptic junction.
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