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1.
Exogenous Glutamate Concentration Regulates the Metabolic Fate of Glutamate in Astrocytes 总被引:7,自引:4,他引:3
Mary C. McKenna Ursula Sonnewald Xueli Huang Joseph Stevenson H. Ronald Zielke 《Journal of neurochemistry》1996,66(1):386-393
Abstract: The metabolic fate of glutamate in astrocytes has been controversial since several studies reported >80% of glutamate was metabolized to glutamine; however, other studies have shown that half of the glutamate was metabolized via the tricarboxylic acid (TCA) cycle and half converted to glutamine. Studies were initiated to determine the metabolic fate of increasing concentrations of [U-13 C]glutamate in primary cultures of cerebral cortical astrocytes from rat brain. When astrocytes from rat brain were incubated with 0.1 m M [U-13 C]glutamate 85% of the 13 C metabolized was converted to glutamine. The formation of [1,2,3-13 C3 ]glutamate demonstrated metabolism of the labeled glutamate via the TCA cycle. When astrocytes were incubated with 0.2–0.5 m M glutamate, 13 C from glutamate was also incorporated into intracellular aspartate and into lactate that was released into the media. The amount of [13 C]lactate was essentially unchanged within the range of 0.2–0.5 m M glutamate, whereas the amount of [13 C]aspartate continued to increase in parallel with the increase in glutamate concentration. The amount of glutamate metabolized via the TCA cycle progressively increased from 15.3 to 42.7% as the extracellular glutamate concentration increased from 0.1 to 0.5 m M , suggesting that the concentration of glutamate is a major factor determining the metabolic fate of glutamate in astrocytes. Previous studies using glutamate concentrations from 0.01 to 0.5 m M and astrocytes from both rat and mouse brain are consistent with these findings. 相似文献
2.
Metabolism of Glycine in Primary Astroglial Cells: Synthesis of Creatine, Serine, and Glutathione 总被引:5,自引:1,他引:4
Ralf Dringen Stephan Verleysdonk Bernd Hamprecht Wieland Willker Dieter Leibfritz Annette Brand 《Journal of neurochemistry》1998,70(2):835-840
Abstract: The metabolism of [2-13 C]glycine in astrogliarich primary cultures obtained from brains of neonatal Wistar rats was investigated using 13 C NMR spectroscopy. After a 24-h incubation of the cells in a medium containing glucose, glutamate, cysteine, and [2-13 C]glycine, cell extracts and incubation media were analyzed for 13 C-labeled compounds. Labeled creatine, serine, and glutathione were identified in the cell extracts. If arginine and methionine were present during the incubation with [2-13 C]glycine, the amount of de novo synthesized [2-13 C]creatine was two-fold increased, and in addition, 13 C-labeled guanidinoacetate was found in cell extracts and in the media after 24 h of incubation. A major part of the [2-13 C]glycine was utilized for the synthesis of glutathione in astroglial cells. 13 C-labeled glutathione was found in the cell extracts as well as in the incubation medium. The presence of newly synthesized [2-13 C]serine, [3-13 C]serine, and [2,3-13 C]serine in the cell extracts and the incubation medium proves the capability of astroglial cells to synthesize serine out of glycine and to release serine. Therefore, astroglial cells are able to utilize glycine as a precursor for the synthesis of creatine and serine. This proves that at least one cell type of the brain is able to synthesize creatine. In addition, guanidinoacetate, the intermediate of creatine synthesis, is released by astrocytes and may be used for creatine synthesis by other cells, i.e., neurons. 相似文献
3.
Annette Brand Dieter Leibfritz Bernd Hamprecht Ralf Dringen 《Journal of neurochemistry》1998,71(2):827-832
Abstract: The synthesis of hypotaurine and taurine was investigated in astroglia-rich primary cultures obtained from brains of neonatal Wistar rats using 1 H and 13 C nuclear magnetic resonance (NMR) spectroscopy. Cell extracts of astroglial cultures analyzed by 1 H NMR spectroscopy show prominent signals of hypotaurine. To identify cysteine as precursor for hypotaurine and taurine synthesis in astroglial cells, primary cultures were incubated with [3-13 C]cysteine for 24 or 72 h. Cell extracts and incubation media were then analyzed with 13 C NMR spectroscopy. Labeled hypotaurine, taurine, glutathione, and lactate were identified in the cell extracts. Within 72 h, 35.0% of the total intracellular hypotaurine and 22.5% of taurine were newly synthesized from [3-13 C]cysteine. The presence of [1-13 C]hypotaurine and [1-13 C]taurine in the incubation medium proves the release of those products of cysteine metabolism into the medium. Minor amounts of the [3-13 C]cysteine were used for the synthesis of glutathione in astroglial cells or metabolized to [3-13 C]lactate, which was found in cell extracts and media. These results indicate that the formation of hypotaurine and taurine is a major pathway of cysteine metabolism in astroglial cells. 相似文献
4.
Abstract: 13 C-NMR spectroscopy was used to evaluate the dynamic consequences of portacaval anastomosis on neuronal and astrocytic metabolism and metabolic trafficking between neurons and astrocytes. Glutamate is predominantly labeled from [1-13 C]glucose, whereas [2-13 C]acetate is more efficient in labeling glutamine, in accordance with its primary metabolism in astrocytes. Alanine and succinate labeling was only observed with [1-13 C]glucose as precursor. Brain [1-13 C]glucose metabolism in portacaval-shunted rats was similar to that in sham-operated controls with the exception of labeled glutamine and succinate formation, which was increased in shunted rats. The 13 C enrichment was, however, decreased owing to an increase in total glutamine and succinate. Using [2-13 C]acetate, on the other hand, flux of astrocytic label to neurons was severely decreased because label incorporation into glutamate, aspartate, and GABA was decreased following portacaval shunting. The latter amino acids are predominantly localized in neurons. These findings demonstrate that metabolic trafficking of amino acids from astrocytes to neurons is impaired in portacaval-shunted rats. 相似文献
5.
Role of Carnitine Palmitoyltransferase I in the Control of Ketogenesis in Primary Cultures of Rat Astrocytes 总被引:3,自引:2,他引:1
Cristina Blázquez Cristina Sánchez Guillermo Velasco Manuel Guzmán 《Journal of neurochemistry》1998,71(4):1597-1606
Abstract: The role of carnitine palmitoyltransferase I (CPT-I) in the control of ketogenesis was studied in primary cultures of rat astrocytes. Ketone bodies were the major product of [14 C]palmitate oxidation by cultured astrocytes, whereas CO2 made a minor contribution to the total oxidation products. Using tetradecylglycidate as a specific, cell-permeable inhibitor of CPT-I, a flux control coefficient of 0.77 ± 0.07 was calculated for CPT-I over the flux of [14 C]palmitate to ketone bodies. CPT-I from astrocytes was sensitive to malonyl-CoA (IC50 = 3.4 ± 0.8 µ M ) and cross-reacted on western blots with an antibody raised against liver CPT-I. On the other hand, astrocytes expressed significant acetyl-CoA carboxylase (ACC) activity, and consequently they contained considerable amounts of malonyl-CoA. Western blot analysis of ACC isoforms showed that ACC in astrocytes—like in neurons, liver, and white adipose tissue—mostly comprised the 265-kDa isoform, whereas the 280-kDa isoform—which was highly expressed in skeletal muscle—showed much lower abundance. Forskolin was used as a tool to study the modulation of the ketogenic pathway in astrocytes. Thus, forskolin decreased in parallel ACC activity and intracellular malonyl-CoA levels, whereas it stimulated CPT-I activity and [14 C]palmitate oxidation to both ketone bodies and CO2 . Results show that in cultured astrocytes (a) CPT-I exerts a very high degree of control over ketogenesis from palmitate, (b) the ACC/malonyl-CoA/CPT-I system is similar to that of liver, and (c) the ACC/malonyl-CoA/CPT-I system is subject to regulation by cyclic AMP. 相似文献
6.
Abstract: The activity of the pyruvate carboxylase was determined in brains of newborn and adult mice as well as primary cultures of astrocytes, of cerebral cortex neurons, and of cerebellar granule cells. The activity was found to be 0.25 ± 0.14, 1.24 ± 0.07, and 1.75 ± 0.13 nmol · min−1 · mg−1 protein in, respectively, neonatal brain, adult brain, and astrocytes. Neither of the two types of neurons showed any detectable enzyme activity (i.e., < 0.05 nmol · min−1 · mg−1 ). It is therefore concluded that pyruvate carboxylase is an astrocytic enzyme. 相似文献
7.
Guido Maura Stefano Thellung Gian Carlo Andrioli Antonio Ruelle Maurizio Raiteri 《Journal of neurochemistry》1993,60(3):1179-1182
Abstract: Primary cultures of cerebral cortical astrocytes were incubated with [U-13 C]glutamate (0.5 m M ) in modified Dulbecco's medium for 2 h. Perchloric acid (PCA) extracts of the cells as well as redissolved lyophilized media were subjected to NMR spectroscopy to identify 13 C-labeled metabolites. NMR spectra of the PCA extracts exhibited distinct multiplets for glutamate, aspartate, glutamine, and malate. The culture medium showed peaks for a multitude of compounds released from the astrocytes, among which lactate, glutamine, alanine, and citrate were readily identifiable. For the first time incorporation of label into lactate from glutamate was clearly demonstrated by doublet formation in the C-3 position and two doublets in the C-2 position of lactate. This labeling pattern can only occur by incorporation from glutamate, because natural abundance will only produce singlets in proton-decoupled 13 C spectra. Glutamine, released into the medium, was labeled uniformly to a large extent, but the C-3 position not only showed the expected apparent triplet but also a doublet due to 13 C incorporation into the C-4 position of glutamine. The doublet accounted for 11% of the total label in the glutamine synthesized and released within the incubation period. The corresponding labeling pattern of [13 C]glutamate in the PCA extracts showed that 19% of the glutamate contained 12 C. Labeling of lactate, citrate, malate, and aspartate as well as incorporation of 12 C into uniformly labeled glutamate and glutamine could only arise via the tricarboxylic acid cycle. The relative amount of glutamate metabolized via this route is at least 70% as calculated from the areas of the C-3 resonances of these compounds. Only a maximum of 30% was converted to glutamine directly. 相似文献
8.
S. C. Cun-nane S. C. R. Williams J. D. Bell S. Brookes† K. Craig† R. A. Iles‡ M. A. Crawford§ 《Journal of neurochemistry》1994,62(6):2429-2436
Abstract: Polyunsaturated fatty acids are needed for normal neonatal brain development, but the degree of conversion of the 18-carbon polyunsaturated fatty acid precursors consumed in the diet to their respective 20-and 22-carbon polyunsaturates accumulating in the brain is not well known. In the present study, in vivo 13 C nuclear magnetic resonance spectroscopy was used to monitor noninvasively the brain uptake and metabolism of a mixture of uniformly 13 C-enriched 16-and 18-carbon polyunsaturated fatty acid methyl esters injected intragastrically into neonatal rats. In vivo NMR spectra of the rat brain at postnatal days 10 and 17 had larger fatty acid signals than in uninjected controls, but changes in levels of individual fatty acids could not be distinguished. One day after injection of the U-13 C-polyunsaturated fatty acid mixture, 13 C enrichment (measured by isotope ratio mass spectrometry) was similar in brain phospholipids, free fatty acids, free cholesterol, and brain aqueous extract; 13 C enrichment remained high in the phospholipids and cholesterol for 15 days. 13 C enrichment was similar in the main fatty acids of the brain within 1 day of injection but 15 days later had declined in all except arachidonic acid while continuing to increase in docosahexaenoic acid. These changes in 13 C enrichment in brain fatty acids paralleled the developmental changes in brain fatty acid composition. We conclude that, in the neonatal rat brain, dietary 16-and 18-carbon polyunsaturates are not only elongated and desaturated but are also utilized for de novo synthesis of long-chain saturated and monounsaturated fatty acids and cholesterol. 相似文献
9.
Succinic semialdehyde dehydrogenase (SSADH) catalyzes the NADP-dependent oxidation of succinic semialdehyde to succinate, the final step of the GABA shunt pathway. SSADH deficiency in humans is associated with excessive elevation of GABA and γ-hydroxybutyrate (GHB). Recent studies of SSADH-null mice show that elevated GABA and GHB are accompanied by reduced glutamine, a known precursor of the neurotransmitters glutamate and GABA. In this study, cerebral metabolism was investigated in urethane-anesthetized SSADH-null and wild-type 17-day-old mice by intraperitoneal infusion of [1,6-13 C2 ]glucose or [2-13 C]acetate for different periods. Cortical extracts were prepared and measured using high-resolution 1 H-[13 C] NMR spectroscopy. Compared with wild-type, levels of GABA, GHB, aspartate, and alanine were significantly higher in SSADH-null cortex, whereas glutamate, glutamine, and taurine were lower. 13 C Labeling from [1,6-13 C2 ]glucose, which is metabolized in neurons and glia, was significantly lower (expressed as μmol of 13 C incorporated per gram of brain tissue) for glutamate-(C4,C3), glutamine-C4, succinate-(C3/2), and aspartate-C3 in SSADH-null cortex, whereas Ala-C3 was higher and GABA-C2 unchanged. 13 C Labeling from [2-13 C]acetate, a glial substrate, was lower mainly in glutamine-C4 and glutamate-(C4,C3). GHB was labeled by both substrates in SSADH-null mice consistent with GABA as precursor. Our findings indicate that SSADH deficiency is associated with major alterations in glutamate and glutamine metabolism in glia and neurons with surprisingly lesser effects on GABA synthesis. 相似文献
10.
Quantification of the GABA Shunt and the Importance of the GABA Shunt Versus the 2-Oxoglutarate Dehydrogenase Pathway in GABAergic Neurons 总被引:1,自引:1,他引:0
Bjørnar Hassel Cecilie U. Johannessen Ursula Sonnewald Frode Fonnum 《Journal of neurochemistry》1998,71(4):1511-1518
Abstract: We investigated the activity of the cerebral GABA shunt relative to the overall cerebral tricarboxylic acid (TCA) cycle and the importance of the GABA shunt versus 2-oxoglutarate dehydrogenase for the conversion of 2-oxoglutarate into succinate in GABAergic neurons. Awake mice were dosed with [1-13 C]glucose, and brain extracts were analyzed by 13 C NMR spectroscopy. The percent enrichments of GABA C-2 and glutamate C-4 were the same: 5.0 ± 1.6 and 5.1 ± 0.2%, respectively (mean ± SD). This, together with previous data, indicates that the flux through the GABA shunt relative to the overall cerebral TCA cycle flux equals the GABA/glutamate pool size ratio, which in the mouse is 17%. It has previously been shown that under the experimental conditions used in this study, the 13 C labeling of aspartate from [1-13 C]glucose specifically reflects the metabolic activity of GABAergic neurons. In the present study, the reduction in the formation of [13 C]aspartate during inhibition of the GABA shunt by γ-vinyl-GABA indicated that not more than half the flux from 2-oxoglutarate to succinate in GABAergic neurons goes via the GABA shunt. Therefore, because fluxes through the GABA shunt and 2-oxoglutarate dehydrogenase in GABAergic neurons are approximately the same, the TCA cycle activity of GABAergic neurons could account for one-third of the overall cerebral TCA cycle activity in the mouse. Treatment with γ-vinyl-GABA, which increased GABA levels dramatically, caused changes in the 13 C labeling of glutamate and glutamine, which indicated a reduction in the transfer of glutamate from neurons to glia, implying reduced glutamatergic neurotransmission. In the most severely affected animals these alterations were associated with convulsions. 相似文献
11.
Metabolic Precursors and Compartmentation of Cerebral GABA in Vigabatrin-Treated Rats 总被引:1,自引:0,他引:1
Abstract: The metabolic precursors and cerebral compartmentation of the augmented GABA pool induced by vigabatrin, an irreversible inhibitor of GABA transaminase, have been investigated by 13 C NMR. Adult rats receiving rat chow ad libitum were given either drinking water only or drinking water containing 2.5 g/L vigabatrin for 7 days. Both groups of animals were infused either with [1,2-13 C2 ]acetate (15 µmol/min/100 g body weight), an exclusive precursor of GABA formation through the glial glutamine pathway, or with [1,2-13 C2 ]glucose (15 µmol/min/100 g body weight), a substrate that can produce GABA through the glial glutamine pathway or by direct metabolism in the neurons. The brains were frozen in situ, extracted with perchloric acid, and analyzed by 13 C NMR. In vigabatrin-treated animals [13 C]glutamine, a common intermediate for [13 C]GABA synthesis from glucose or acetate, was accumulated to similar amounts during infusions with [1,2-13 C2 ]glucose or [1,2-13 C2 ]acetate. However, [13 C]GABA accumulation was sevenfold higher during [1,2-13 C2 ]glucose infusions or twofold higher during [1,2-13 C2 ]acetate infusions. These results show that the direct pathway of GABA formation by neuronal metabolism of glucose predominates over the alternative pathway through glial glutamine. Near-equilibrium relationships of the aminotransferases of GABA and aspartate imply that the observed [13 C]GABA accumulation occurs initially in the neuronal compartment. 相似文献
12.
Reynold Spector 《Journal of neurochemistry》1981,36(3):1186-1191
Abstract: The transport of the lipid-soluble sugarless flavins, [14 C]lumiflavin and [14 C]lumichrome, into and from the isolated choroid plexus and brain slices was studied in vitro. The isolated choroid plexus accumulated both [14 C] flavins by a saturable, energy-requiring process that did not depend on binding or intracellular metabolism of the [14 C] flavins. Both sugar-containing and sugarless flavins, as well as cyclic organic acids, significantly inhibited [14 C]lumiflavin and [14 C]Iumichrome uptake by the isolated choroid plexus. Within 2.5 min, 75% of the [14 C]lumiflavin accumulated by the isolated choroid plexus was released into the medium. Brain slices accumulated [14 C]lumiflavin by a saturable process that did not meet all the criteria for active transport. Ninety-five percent of the [14 C]lumiflavin accumulated by brain slices was released into the medium within 7.5 min. In vivo , 2 h after the intraventricular injection of 6.5 nmol [14 C]lumiflavin, almost all of the [14 C]flavin was cleared from the CNS. Addition of 3.5 μmol FMN to the intraventricular injectate significantly decreased the clearance of [14 C]lumiflavin from the CNS. These studies document that the sugarless flavins are transported by the flavin transport systems in the CNS. 相似文献
13.
The magnitude of metabolic activation is greatly underestimated in autoradiographic studies using [1- or 6-14 C]glucose compared to parallel assays with [14 C]deoxyglucose indicating that most of the label corresponding to the additional [14 C]glucose consumed during activation compared to rest is quickly released from activated structures. Label could be lost by net release of [14 C]lactate from brain or via lactate exchange between blood and brain. These possibilities were distinguished by comparison of glucose and lactate specific activities in arterial blood and brain before, during, and after generalized sensory stimulation and during spreading cortical depression. Over a wide range of brain lactate concentrations, lactate specific activity was close to the theoretical maximum, i.e. half that of [6-14 C]glucose, indicating that exchange-mediated dilution of lactate is negligible and that efflux of [14 C]lactate probably accounts for most of the label loss. Low lactate dilution also indicates that dilution of glutamate C4 fractional enrichment in [13 C]glucose studies, currently ascribed predominantly to lactate exchange, arises from other unidentified pathways or factors. Alternative explanations for glutamate dilution (presented in Supporting Information) include poorly labeled amino acid pools and oxidative metabolism of minor substrates in astrocytes to first dilute the astrocytic glutamine pool, followed by dilution of glutamate via glutamate–glutamine cycling. 相似文献
14.
Trine M. Lund Øystein Risa† Ursula Sonnewald† Arne Schousboe Helle S. Waagepetersen 《Journal of neurochemistry》2009,110(1):80-91
Ketone bodies serve as alternative energy substrates for the brain in cases of low glucose availability such as during starvation or in patients treated with a ketogenic diet. The ketone bodies are metabolized via a distinct pathway confined to the mitochondria. We have compared metabolism of [2,4-13 C]β-hydroxybutyrate to that of [1,6-13 C]glucose in cultured glutamatergic neurons and investigated the effect of neuronal activity focusing on the aspartate–glutamate homeostasis, an essential component of the excitatory activity in the brain. The amount of 13 C incorporation and cellular content was lower for glutamate and higher for aspartate in the presence of [2,4-13 C]β-hydroxybutyrate as opposed to [1,6-13 C]glucose. Our results suggest that the change in aspartate–glutamate homeostasis is due to a decreased availability of NADH for cytosolic malate dehydrogenase and thus reduced malate–aspartate shuttle activity in neurons using β-hydroxybutyrate. In the presence of glucose, the glutamate content decreased significantly upon activation of neurotransmitter release, whereas in the presence of only β-hydroxybutyrate, no decrease in the glutamate content was observed. Thus, the fraction of the glutamate pool available for transmitter release was diminished when metabolizing β-hydroxybutyrate, which is in line with the hypothesis of formation of transmitter glutamate via an obligatory involvement of the malate–aspartate shuttle. 相似文献
15.
This report demonstrates that during the torpor phase of hibernation, hamsters utilize 14 C and 13 C glucose in torpor-specific brain metabolic pathways. Microdialysis of 14 C glucose into the striatum rapidly induced a steady state labeling of extracellular fluid (ECF) lactate and labeling of tissue GABA, glutamate, glutamine, and alanine in ipsilateral and contralateral striata. The same tissue metabolites were labeled in cortex, hypothalamus, and brainstem after microdialysis of 14 C lactate into the lateral ventricle. Serine, aspartate, glycine, taurine, tyrosine, and methionine were not synthesized from glucose or lactate during torpor. ECF levels of amino and organic acids were low and unchanging during torpor and increased late during arousal to cenothermia. Labeled intracellular 14 C GABA and glutamate were not communicated to the striatal ECF or ventricular space during torpor. 13 C NMR demonstrated rapid formation of lactate and functional tricarboxylic acid cycles in GABAergic and glutamatergic neurons, and enrichment of glutamine and alanine after i.v. 13 C glucose. Large changes in tissue levels of amino acids occur prior to or during entrance into torpor but not during torpor. It is proposed that cerebral intracellular dehydration, the enlargement of ECF and the biochemistries associated with brain water homeostasis may have a role in regulating hibernation. 相似文献
16.
Abstract. In the marine environment, the range of values of carbon isotope fractionation between particulate tissue of phytoplankton and inorganic carbon can be more than 20‰ (− 35‰ < δ13 C < − 14‰). This review considers the influence of seawater temperature, lipid content of phytoplanktonic cells, kinetic fractionation, and carbon pathway on δ13 C values observed at sea.
In order to study the contribution of carboxylases (RUBISCO and the β-carboxylases phosphoenolpyruvate carboxylase, phosphoenoplpyruvate carboxykinase and pyruvate carboxylase) to variations of particulate δ13 C values at sea, we present results obtained simultenously on carboxylase activities and δ13 C in various environmental conditions. The lowest δ13 C values are clearly associated with predominance of ribulose-1.5-bisphosphate carboxylase activity, but it was more difficult to explain the high δ13 C values. Different hypotheses are discussed. 相似文献
In order to study the contribution of carboxylases (RUBISCO and the β-carboxylases phosphoenolpyruvate carboxylase, phosphoenoplpyruvate carboxykinase and pyruvate carboxylase) to variations of particulate δ
17.
Excitatory Amino Acid Synthesis in Hypoxic Brain Slices: Does Alanine Act as a Substrate for Glutamate Production in Hypoxia? 总被引:4,自引:2,他引:2
J. L. Griffin C. Rae R. M. Dixon G. K. Radda P. M. Matthews 《Journal of neurochemistry》1998,71(6):2477-2486
Abstract: Excitatory amino acids are an important cause of cell death in the hypoxic and ischaemic brain. Neuronal glutamate stores are depleted rapidly in hypoxia, but alanine production rises under such conditions and has been suggested to be a potential precursor of glutamate. To test this hypothesis, we have investigated amino acid metabolism using 13 C NMR with superfused guinea pig cortical slices subjected to varying degrees of hypoxia. During severe hypoxia, brain slices metabolising 5 m M [2-13 C]pyruvate exported [2-13 C]alanine into the superfusion fluid. The metabolic fate of alanine during normoxia and hypoxia was tested by superfusion of brain slices with 10 m M glucose and 2 m M [2-13 C, 15 N]alanine. Metabolism of exogenous alanine leads to the release of aspartate into the superfusion fluid. The pattern of labelling of aspartate indicated that it was synthesised via the glial-specific enzyme pyruvate carboxylase. 13 C-labelled glutamate was produced with both normoxia and hypoxia, but concentrations were 30-fold lower than for labelled aspartate. Thus, although substantial amounts of glutamate are not synthesised from alanine in hypoxia, there is significant production of aspartate, which also may have deleterious effects as an excitatory amino acid. 相似文献
18.
The Interaction of Dithiothreitol and Acetyl Coenzyme A in a Radiochemical Assay for Rat Brain ATP:Citrate Oxaloacetate Lyase 总被引:1,自引:1,他引:0
J. Simpson 《Journal of neurochemistry》1981,37(1):100-106
Abstract: [14 C]Acetyl-CoA was found to react spontaneously with dithiothreitol to give a relatively apolar product which was readily extractable into a butanol-toluene scintillant. This technique was used in a rapid, reproducible assay for rat brain ATP:citrate lyase using [1,5-14 C]citrate as substrate. The tissue extract, a 14,000 g supernatant, exhibited a lyase activity of approximately 7 nmol acetyl-CoA produced/min per mg supernatant protein, and was inhibited ≥79% by α-ketoglutaric acid (10 m m ), Cu2+ (1 m m )and Zn2+ (1 m m ). [14 C]Oxaloacetate, [14 C]malate and endogenous citrate synthase were found not to interfere significantly with lyase estimations, but NADH was required in the reaction mixture to inhibit acetyl-CoA hydrolase activity. 相似文献
19.
[2-3 H]GLYCEROL AS A PRECURSOR OF PHOSPHOLIPIDS IN RAT BRAIN: EVIDENCE FOR LACK OF RECYCLING 总被引:1,自引:0,他引:1
Abstract— When [2-3 H]glycerol was injected intracranially into young rats, it was presented as a pulse label, leaving the brain rapidly and giving up much of its labelled hydrogen to water. [2-3 H]glycerol was efficiently incorporated into brain lipids, especially into choline and ethanolamine phospholipids. Following injection of a mixture of [3 H]- and [14 C]-labelled glycerol, the ratio of 3 H to 14 C in the phospholipids of both whole brain and the microsomal fraction decreased as a function of time after injection. This finding indicated less recycling of the tritium label. This lack of recycling was further indicated by the finding that 94 per cent of the tritium label of phosphatidyl choline was in the glycerol portion of the molecule rather than in the fatty acids. At 2 weeks following injection with [3 H]glycerol, 93 per cent of the total radioactivity in brain appeared in the lipid fraction. In contrast, following injection with [14 C]glycerol, only 57 per cent of the radioactivity appeared in lipid, with about 20 per cent in protein. 相似文献
20.
Abstract: Synaptoneurosomal and synaptosomal fractions from the brain cortex of adult (4-month-old) and aged (27-month-old) rats were used for studies on the uptake and subsequent release of [14 C]arachidonic acid ([14 C]AA) from brain lipids. The incorporation of AA and the pattern of its uptake into lipids of the aged brain cortex synapto-neurosomes and synaptosomes were not significantly different when compared with those in the adult brain cortex fractions. Serotonin (5-HT), at 10 μM to 1 μM in the presence of pargyline and the agonist of the 5-HT1A receptor, buspirone, stimulated AA uptake into membrane lipids, mainly into phosphatidylinositol, by about 40% exclusively in adult brain synaptoneurosomes. Aging significantly diminished the effect of 5-HT on AA uptake. Synaptoneurosomal and synaptosomal fractions prelabeled with [14 C]AA were used subsequently for investigation of voltage-dependent, muscarinic and 5-HT receptor-mediated AA release. Aging diminished markedly carbachol-stimulated Ca2+ -dependent AA liberation from membrane lipids of synaptoneurosomes and synaptosomes. Moreover, aging decreased voltage-dependent and 5-HT2 receptor-mediated AA release. These results show that aging affects receptor-dependent AA uptake and pre-and postsynaptic receptor-mediated AA release. These modulations of AA incorporation and release in aged brain may be of patho-physiological significance, in view of the importance of these processes for signal transmission in the brain. The changes of receptor-dependent processes of deacylation and reacylation may be responsible for alteration in the function of neuronal cells and may affect learning and memory ability and brain plasticity during aging. 相似文献