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1.
1. One binding component with aK d value of 200×10–9 M and half-life of the ligand binding component of 30 min was found. 2. Chloride ions produced a significant increase ofl-[3H]aspartate andl-[3H]glutamate binding. 3.l-Glutamate,l-ibotenate,l-quisqualate, anddl-homocysteic acid were potent inhibitors ofl-[3H]aspartate binding. 4. In all brain regions major increases of binding were observed during the third week of the in ovo period of life.  相似文献   

2.
Primary neuronal cultures were made from eight-day-old embryonic chick telencephalon. Ten-day-old cultures were used to study the release ofd-[3H]aspartate andl-[3H]glutamate. Thed-[3H]aspartate release was stimulated by increasing potassium concentrations, but it was not calcium dependent. In contrast, the potassium dependentl-[3H]glutamate release was calcium dependent, and furthermorel-[3H]glutamate release was optimal at potassium concentrations<30 mM. The inhibitors of glutamate uptake, dihydrokainate and 1-aminocyclobutane-trans-1,3-dicarboxylic acid (CACB), also referred to as cis-1-aminocyclobutane-1,3-dicarboxylate, were used in the release experiments. Dihydrokainate had no effect on aspartate release, whereas CACB increased both the basal efflux ofd-[3H]aspartate and the potassium evoked release. CACB had no effect on the potassium stimulatedl-glutamate release. We believe thatl-glutamate is released mainly by a vesicular mechanism from the presumably glutamatergic neurons present in our culture.d-aspartate release observed by us, could be mediated by a transporter protein. The cellular origin of this release remains to be assessed.  相似文献   

3.
Culture-grown astrocytes derived from 3-day-old rat brain were incubated in the presence of [3H]guanosine and of the convulsant agentl-methionine-dl-sulfoximine (MSO). The resulting [3H]tRNA was purified from control and MSO-exposed cells at several time points during the incubation and was hydrolyzed to [3H]guanine and four [3H]methyl guanines which were separated by high pressure liquid chromatography. Three of the four [3H]methyl guanines were more highly labeled in the [3H]tRNA of the MSO-exposed cells, relative to that of the control cells throughout the entire incubation period. The findings extend to cultured astrocytes, the stimulatory effect of MSO on the methylation of neural tRNA guanines, previouly observed both in vitro using [14C]S-adenosyl-l-methionine and in vivo using [methyl 3-H]l-methionine.  相似文献   

4.
Metabolism ofl-[U-14C]lysine was studied in the human autopsy tissues and the intact monkeys through intracerebroventricular and intravenous injections. The human tissues were more active in the metabolism ofl-[14C]lysine to [14C]pipecolate than the rat tissues previously reported. This metabolism was equally active in the phosphate (pH 7) and the glycyl-glycine (pH 8.6) buffers with the brain and the kidney having higher activity than the liver. Besides [14C]pipecolate, traces of [14C]saccharopine and -[14C]aminoadipate were also detected in the liver incubation. Twenty-four hr after intraventricular injection ofl-[14C]lysine to the monkey, substantial labeling of pipecolate and -aminoadipate was observed in the brain and spinal cord, with the kidney, liver and the plasma having much reduced levels. Radioactivity levels of these two compounds were found low in the organs and plasma of the intravenously injected monkey. The urine of both monkeys contained only traces of [14C]pipecolate, even though it contained high levels ofl-[14C]lysine and -[14C]aminoadipate. It was concluded thatl-lysine is actively metabolized to pipecolate and -aminoadipate in the human and the monkey, that this reaction is most active in the brain whenl-lysine is intraventricularly administered, and that in contrast to the rat, the monkey may have an effective renal reabsorption for pipecolate which is similar to the human.  相似文献   

5.
Four amphipathic molecules with known local anesthetic activity, dibucaine, tetracaine, chlorpomazine, and quinacrine, inhibited the binding ofl-[3H]glutamic acid to rat brain synaptic plasma membranes and to the purified glutamate binding protein. Neither haloperidol nor diphenylhydantoin had significant inhibitory effects on the glutamate binding activity of the membranes or of the purified protein. The amphipathic drugs apparently inhibitedl-[3H]glutamate binding to synaptic membranes by a mixed type of inhibition. The inhibitory activity of quinacrine on glutamate binding to the synaptic membranes was greater in a low ionic strength, Ca2+-free buffer medium, than in a physiologic medium (Krebs-Henseleit buffer). Removal of Ca2+ from the Krebs solution enhanced quinacrine's inhibition of glutamate binding. Quinacrine up to 1 mM concentration did not inhibit the high affinity Na+-dependentl-glutamate transport in these membrane preparations. The importance of Ca2+ in the expression of quinacrine's effects on the glutamate binding activity of synaptic membranes and the observed tetracaine and chlorpromazine-induced increases in the transition temperature for the glutamate binding process of these membranes, were indicative of an interaction of the local anesthetics with the lipid environment of the glutamate binding sites.  相似文献   

6.
Brains from human alcoholics and non-alcoholics were obtained shortly after death. The hippocampus was dissected, homogenized, and processed for the isolation of a synaptic membraneenriched fraction and the study ofl-[3H]glutamic acid and 3-((±)-2-carboxypiperazin-4-yl)-[1,23H]propyl-l-phosphonic acid ([3H]CPP) binding sites. The pharmacological characteristics ofl-[3H]glutamic acid binding to synaptic membranes isolated from hippocampus corresponded to the labeling of a mixture of N-methyl-d-aspartate (NMDA), kainate and quisqualic acid receptor sites. Synaptic membranes prepared from the hippocampus of individuals classified as alcoholics had significantly higher density of glutamate binding sites than identically prepared membranes from non-alcoholic individuals. In addition, there was a clear definition of a population ofl-glutamate binding sites (approx. 10% of total) in the membranes from alcoholics that had a higher affinity for the ligand than the major set of sites labeled in membranes from both alcoholics and non-alcoholics. Neither the age of the individuals at the time of death nor the time that elapsed between death and processing of brain tissue were significant factors in determining either recovery of purified synaptic membranes from brain homogenates orl-[3H]glutamate binding to synaptic membranes. In order to determine whether some of the changes inl-[3H]glutamic acid binding were due to alterations in binding at the NMDA receptor subtype, we also measured binding of [3H]CPP to extensively washed crude synaptosomal membranes. Membranes from brains of alcoholics had higher affinity (3-fold) for [3H]CPP but lower binding capacity (3-fold) when compared with those of non-alcoholics. These observations suggest selective changes among different glutamate receptor subtypes in human brain under conditions of chronic alcohol intake.  相似文献   

7.
Our earlier observations showed thatl-lysine enhanced the activity of diazepam against seizures induced by pentylenetetrazol (PTZ), and increased the affinity of benzodiazepine receptor binding in a manner additive to that caused by -aminobutyric acid (GABA). The present paper provides additional evidence to show thatl-lysine has central nervous system depressant-like characteristics.l-lysine enhanced [3H]flunitrazepam (FTZ) binding in brain membranes was dose-dependent and stimulated by chloride, bromide and iodide, but not fluoride. Enhancement of [3H]FTZ binding byl-lysine at a fixed concentration was increased by GABA but inhibited by pentobarbital between 10–7 to 10–3M. While GABA enhancement of [3H]FTZ binding was inhibited by the GABA mimetics imidazole acetic acid and tetrahydroisoxazol pyridinol, the enhancement by pentobarbital andl-lysine of [3H]FTZ binding was dose-dependently increased by these two GABA mimetics. The above results suggest thatl-lysine and pentobarbital acted at the same site of the GABA/benzodiazepine receptor complex which was different from the GABA binding site. The benzodiazepine receptor antagonist imidazodiazepine Ro15-1788 blocked the antiseizure activity of diazepam against PTZ. Similar to pentobarbital, the anti-PTZ effect ofl-lysine was not blocked by Ro15-1788. Picrotoxinin and the GABA, receptor antagonist bicuculline partially inhibitedl-lysine's enhancement of [3H]FTZ binding with the IC50s of 2 M and 0.1 M, respectively. The convulsant benzodiazepine Ro5-3663 dose-dependently inhibited the enhancement of [3H]FTZ binding byl-lysine. This article shows the basic amino acidl-lysine to have a central nervous system depressant characteristics with an anti-PTZ seizure activity and an enhancement of [3H]FTZ binding similar to that of barbiturates but different from GABA.  相似文献   

8.
Membranes prepared from cerebellar granule cells and cortical astrocytes exhibited specific, saturable binding ofl-[3H]glutamate. The apparent binding constant K d was 135 nM and 393 nM and the maximal binding capacity Bmax 42 and 34 mol/kg in granule cells and astrocytes, respectively. In granule cells the binding was strongly inhibited by the glutamate receptor agonists kainate, quisqualate, N-methyl-d-aspartate (NMDA),l-homocysteate and ibotenate, and the antagonistdl-5-aminophosphonovalerate. In astrocytes, only quisqualate among these was effective.l-Aspartate,l-cysteate,l-cysteinesulphinate and -d-glutamylglycine were inhibitors in both cell types. The binding was totally displaced in both cell types byl-cysteinesulphinate with IC50 in the micromolar range. In astrocytes the binding was also totally displaced by quisqualate, but in granule cells only partially by NMDA, kainate and quisqualate in turn. It is concluded from the relative potencies of agonists and antagonists in [3H]glutamate binding that cerebellar granule cells express the NMDA, kainate and quisqualate types of the glutamate receptor, while only the quisqualate-sensitive binding seems to be present in cortical astrocytes.  相似文献   

9.
A subconvulsant dose of sodium fluoroacetate inhibited the metabolic utilization of intracerebrally-administered N-acetyl-l -[U-14C]asparticacid and the labelling of glutamine from this precursor in mouse brain, but not the labelling of glutamate or aspartate. A convulsant dose also inhibited the utilization of l -[U-14C]aspartic acid. When intraperitoneal injection of a convulsant dose of sodium fluoroacetate was followed by intracerebral injection of N-acetyl-l -[U-14C]asparticacid, the levels of N-acetylaspartate, aspartate and glutamate in brain were lowered, while the glutamine content was increased. The specific radioactivity of glutamine relative to that of glutamate was much lower when these compounds were labelled from l -[U-14C]aspartic acid than when N-acetyl-l -[U-14C]aspartic acid was used as the precursor. Intracerebral injection of tracer amounts of l -[U-14C]aspartic acid reduced the content of N-acetylaspartate in brain and raised the glutamine content. Sodium fluoroacetate had no additional effect on the relative specific radioactivity of glutamine or the content of N-acetylaspartate, aspartate, glutamate or glutamine when l -[U-14C]aspartic acid was the precursor. We consider the results to be consistent with a selective inhibition both by sodium fluoroacetate and by exogenous aspartic acid of the tricarboxylic acid cycle in brain associated with the biosynthesis of glutamine. We suggest that the activity of this pathway may regulate the metabolism of N-acetylaspartate and aspartate.  相似文献   

10.
Evoked release of glutamate and aspartate from cultured cerebellar granule cells was studied after preincubation of the cells in tissue culture medium with glucose (6.5 mM), glutamine (1.0 mM),d[3H] aspartate and in some cases aminooxyacetate (5.0 mM) or phenylsuccinate (5.0 mM). The release of endogenous amino acids and ofd-[3H] aspartate was measured under physiological and depolarizing (56 mM KCl) conditions both in the presence and absence of calcium (1.0 mM), glutamine (1.0 mM), aminooxyacetate (5.0 mM) and phenylsuccinate (5.0 mM). The cellular content of glutamate and aspartate was also determined. Of the endogenous amino acids only glutamate was released in a transmitter fashion and newly synthesized glutamate was released preferentially to exogenously suppliedd-[3H] aspartate, a marker for exogenous glutamate. Evoked release of endogenous glutamate was reduced or completely abolished by respectively, aminooxyacetate and phenylsuccinate. In contrast, the release ofd-[3H] aspartate was increased reflecting an unaffected release of exogenous glutamate and an increased psuedospecific radioactivity of the glutamate transmitter pool. Since aminooxyacetate and phenylsuccinate inhibit respectively aspartate aminotransferase and mitochondrial keto-dicarboxylic acid transport it is concluded that replenishment of the glutamate transmitter pool from glutamine, formed in the mitochondrial compartment by the action of glutaminase requires the simultaneous operation of mitochondrial keto-dicarboxylic acid transport and aspartate aminotransferase which is localized both intra- and extra-mitochondrially. The purpose of the latter enzyme apparently is to catalyze both intra- and extra-mitochondrial transamination of -ketoglutarate which is formed intramitochondrially from the glutamate carbon skeleton and transferred across the mitochondrial membrane to the cytosol where transmitter glutamate is formed. This cytoplasmic origin of transmitter glutamate is in aggreement with the finding thatd-[3H] aspartate readily labels the transmitter pool even when synthesis of endogenous transmitter is impaired in the presence of AOAA or phenylsuccinate.Special issue dedicated to Dr Elling Kvamme  相似文献   

11.
The metabolism ofl-proline toN-acetyl-d-glucosamine (GlcNAc) during germ tube formation ofCandida albicans (C. albicans) ATCC 1002 was studied. In uptake experiments, 6.9 nmol ofl-[14C]proline were taken up by 1×106 cells during 3 h of incubation at 37°C. The percentage of germ tube formation was 94 under the same condition. The presence of GlcNAc reduced the uptake ofl-proline to 3.0 nmol. The percentage of germ tube formation was 95 in the presence and absence of GlcNAc. The [3H]GlcNAc uptake was 3.0 nmol and was constant whetherl-proline was present or not. After the preparation of a chitin fraction from germ tubes that were labeled withl-[14C]proline, the radioactivity froml-proline was detected in the glucosamine (GlcN) fraction by thin-layer chromatography (TLC). The metabolism ofl-proline to GlcNAc in chitin during germ tube formation was confirmed in this experiment.  相似文献   

12.
To evaluate the hypothesis that glutamic acid may be the neurotransmitter of descending, excitatory supraspinal pathways, the uptake and release ofl-[3H] glutamate and the levels of endogenous glutamate were measured in preparations from rat lumbar spinal cord following complete mid-thoracic transection. Following transection, the activity of the synaptosomal high-affinty glutamate uptake process was increased in both dorsal and ventral halves of lumbar cord between 1 and 14 days after transection and returned to control levels by 21 days posttransection. At 7 days, the increased activity of the uptake process forl-[3H] glutamate resulted in elevation ofV max with no significant alteration inK t as compared to age-matched controls. Depolarization-induced release ofl-[3H]glutamate from prelabeled slices did not differ significantly from control in the lesioned rat except at 21 days after lesion when the amount of tritium release was significantly greater in the transected preparations than in control. Amino acid analysis of the lumbar cord from control and transected rats indicated only a 10% decrease in the level of endogenous glutamate and no alterations in the concentration of GABA and glycine 7 days after lesion. These findings do not support the hypothesis that glutamate serves as a major excitatory neurotransmitter in supraspinal pathways innervating the lumbar cord of the rat.  相似文献   

13.
Astrocytes have been proposed to regulate the extracellular space in the brain, even if rather little is known about their specific functions. One possibility for obtaining more knowledge on the functions of astroglial cells is to examine how they respond on exposure to pharmacological agents. Na+-valproate is an anticonvulsive drug which is used in the treatment of several types of epilepsy. The mechanisms of action of the drug are not fully understood, but the GABA-ergic system, both in neurons and astrocytes, has been shown to be affected. In the present study, the effects of valproate were investigated on astroglial cells in primary cultures from newborn rat cerebral cortex. The transport of the drug itself and its effects on the transport of the amino acid transmitters glutamate, aspartate and -aminobutyric acid (GABA) into astrocytes were examined. The [3H]valproate transport into the astrocytes was increased after exposure tol-glutamate but notl-aspartate. On the other hand, after acute exposure for the drug, the transport of [3H]l-glutamate and [3H]l-aspartate decreased, as also did the affinity but not the transport capacity for the [3H]GABA uptake. However, after 5 days chronic valproate exposure, no effects could be seen on the uptake kinetics ofl-glutamate orl-aspartate. For GABA, the affinity decreased, while the transport capacity remained unchanged compared with controls. The results showed that valproate, glutamate, aspartate and GABA were capable of interacting significantly with each others transport into the astrocytes.  相似文献   

14.
The release ofd-[3H]aspartate (used as a tracer for endogenous glutamate and aspartate) was studied at high K+ (100 mM) and under ischemia in rats implanted with 0.3 mm diameter dialysis tubing through the hippocampus. The effect on thed-[3H]aspartate release of the two -aminobutyric acid (GABA) agonists 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol (THIP) and (±)--(p-chlorophenyl)GABA (baclofen), which specifically activate GABAA and GABAB receptors, respectively, was studied. Initial experiments employing HPLC analysis showed a coincident increase in the amounts of glutamate, aspartate and the amount of radioactivity following introduction of K+ (100 mM) or a period of ischemia suggesting that thed-[3H]aspartate labels the transmitter pools of the two amino acids under the present experimental conditions. The presence of 10 mM baclofen or 10 mM THIP in the perfusion medium did not inhibit ischemia inducedd-[3H]aspartate release. On the contrary, 10 mM baclofen alone (but not 0.1 or 1 mM) in the perfusion medium induced release ofd-[3H]aspartate in a calcium dependent manner, whereas 10 mM THIP had no significant releasing effect.Special issue dedicated to Dr. Elling Kvamme  相似文献   

15.
Radioligand binding of d-[3H]aspartic and l-[3H]glutamic acids to plasma membranes from rat Harderian gland was evaluated. Binding was optimal under physiological conditions of pH and temperature, and equilibrium was reached within 50 min. Specific binding for d-Asp and l-Glu was saturable, and Eadie–Hofstee analysis revealed interaction with a single population of binding sites (for d-Asp K d = 860 ± 28 nM, B max = 27.2 ± 0.5 pmol/mg protein; for l-Glu, K d = 580 ± 15 nM and B max = 51.3 ± 0.8 pmol/mg protein). l-[3H]glutamate had higher affinity and a greater percentage of specific binding than did d-[3H]aspartate. The pharmacological binding specificity of l-[3H]glutamate indicated an interaction with NMDA-type receptors. Specifically, the order of potency of the displacing compound tested was l-Glu > d-Asp > NMDA > MK801 > d-AP5 > glycine. For d-[3H]aspartate, the data revealed an interaction of d-Asp with either NMDA-type receptors or putative specific binding sites.  相似文献   

16.
The effect of the cholesterol-depleting agent methyl-β-cyclodextrin (MβCD) on exocytotic, transporter-mediated, tonic release, the ambient level and uptake of l-[14C]glutamate was assessed in rat brain synaptosomes using different methodological approaches of MβCD application. The addition of 15 mM MβCD to synaptosomes (the acute treatment, AT) immediately resulted in the extraction of cholesterol and in a two times increase in the extracellular l-[14C]glutamate level. When 15 mM MβCD was applied to synaptosomes for 35 min followed by washing of the acceptor (the long-term pretreatment, LP), this level was only one-third higher than in the control. The opposite effects of MβCD on tonic l-[14C]glutamate release and glutamate transporter reversal were found in AT and LP. Tonic release was dramatically enlarged in AT, but decreased after LP. Transporter-mediated release was increased several times in AT, but attenuated in LP. Depolarization-evoked exocytotic release of l-[14C]glutamate was completely lost in AT, whereas after LP, it was decreased by half in comparison with the control. Na+-dependent l-[14C]glutamate uptake was decreased by ~60% in AT, whereas in LP, it was lowered by ~40% only. The presence of MβCD in the incubation media during AT caused dramatic dissipation of the proton gradient of synaptic vesicles that was shown with the pH-sensitive dye acridine orange, whereas after LP, no statistically significant changes were registered in synaptic vesicle acidification. It was concluded that the diverse changes in glutamate transport in AT and LP were associated with the difference in the functional state of synaptic vesicles.  相似文献   

17.
Glutamine-free culture of Vero cells has previously been shown to cause higher cell yield and lower ammonia accumulation than that in glutamine-containing culture. Nitrogen metabolism of asparagine and glutamate as glutamine replacer was studied here using nuclear magnetic resonance (NMR) spectroscopy. 15N-labelled glutamate or asparagine was added and their incorporation into nitrogenous metabolites was monitored by heteronuclear multiple bond coherence (HMBC) NMR spectroscopy. In cells incubated with l-[15N]glutamate, the 15N label was subsequently found in a number of metabolites including alanine, aspartate, proline, and an unidentified compound. No detectable signal occurred, indicating that glutamate was utilized by transamination rather than by oxidative deamination. In cells incubated with l-[2-15N]asparagine, the 15N label was subsequently found in aspartate, the amine group of glutamate/glutamine, and in two unidentified compounds. Incubation of cells with l-[4-15N]asparagine showed that the amide nitrogen of asparagine was predominantly transferred to glutamine amide. There was no detectable production of , showing that most of the asparagine amide was transaminated by asparagine synthetase rather than deaminated by asparaginase. Comparing with a glutamine-containing culture, the activities of phosphate-activated glutaminase (PAG), glutamate dehydrogenase (GDH) and alanine aminotransferase (ALT) decreased significantly and the activity of aspartate aminotransferase (AST) decreased slightly.  相似文献   

18.
Binding ofl-[3H]glutamate to membranes from whole chick retina and from subcellular fractions enriched with photoreceptor terminals (P1), or terminals from the inner plexiform layer (P2) was studied. Na+-dependent and Na+-independent binding to these membranes was demonstrated. Na+-independent binding was stereospecific. Kinetic analysis of the binding process indicated a single high-affinity system (K B=0.55 M) with a capacity of approximately 20 pmoles/mg protein in all the membrane fractions. [3H]Glutamate binding to P1 and P2 fractions was effectively displaced by several structural analogues of glutamate. Glutamate diethyl-ester appreciably displaced binding, whereas kainic acid did not displace bound glutamate. Data indicate the binding of [3H]glutamate to physiologically relevant receptors in the chick retina.  相似文献   

19.
Rat hippocampal slices preloaded withd-[3H]aspartate, a non metabolizable analogue ofl-glutamate, were superfused with artifical CSF. Depolarization was induced by 53.5 mM K+, in the presence of Ca2+ (1.3 mM) or Mg2+ (5 mM) to determine the Ca2+ dependent release. Haloperidol added in the superfusion medium at 100 M reduced by about 60% the Ca2+ dependent release ofd-[3H]aspartate. This drug at 20 M or 100 M inhibited the non-activated glutamate dehydrogenase (GDH) but had no effect on GDH activated by ADP (2 mM) or leucine (5 mM). In addition no effect was observed on phosphate activated glutaminase (PAG) in the presence either of 20 mM or 5 mM phosphate. These results indicate that the effect of haloperidol is exerted on presynaptic mechanisms regulating neurotransmitter release.  相似文献   

20.
Astrocytes possess a concentrativel-ascorbate (vitamin C) uptake mechanism involving a Na+-dependentl-ascorbate transporter located in the plasma membrane. The present experiments examined the effects of deprivation and supplementation of extracellularl-ascorbate on the activity of this transport system. Initial rates ofl-ascorbate uptake were measured by incubating primary cultures of rat astrocytes withl-[14C]ascorbate for 1 min at 37°C. We observed that the apparent maximal rate of uptake (V max) increased rapidly (<1 h) when cultured cells were deprived ofl-ascorbate. In contrast, there was no change in the apparent affinity of the transport system forl-[14C]ascorbate. The increase inV max was reversed by addition ofl-ascorbate, but notD-isoascorbate, to the medium. The effects of external ascorbate on ascorbate transport activity were specific in that preincubation of cultures withl-ascorbate did not affect uptake of 2-deoxy-D-[3H(G)]glucose. We conclude that the astroglial ascorbate transport system is modulated by changes in substrate availability. Regulation of transport activity may play a role in intracellular ascorbate homeostasis by compensating for regional differences and temporal fluctuations in external ascorbate levels.  相似文献   

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