Influence of Pathological Concentrations of Ammonia on Metabolic Fate of 14C-Labeled Glutamate in Astrocytes in Primary Cultures

Rates of glutamine formation and of carbon dioxide production (as an indication of oxidative deamination of glutamate) were determined in primary cultures of astrocytes exposed to 50 microM labeled glutamate in the absence or presence of added ammonia (0.1-3 mM). Glutamine formation (1.7 nmol/min/mg protein) was unaffected by all concentrations of added ammonia. This probably reflects the presence of a low content of ammonia (0.1-0.2 mM), originating from degradation of glutamine, in the cells even in the absence of added ammonia, and it shows that pathophysiological concentrations of ammonia do not increase the formation of glutamine from exogenous glutamate. The carbon dioxide production rate was 5.9 nmol/min/mg protein, i.e., three to four times higher than the rate of glutamine formation. It was significantly reduced (to 3.5 nmol/min/mg protein) in the presence of 1 mM or more of ammonia. This is in keeping with suggestions by others that toxic levels of ammonia affect oxidative metabolism.     

Exogenous Glutamate Is Metabolized to Glutamine and Exported by Rat Primary Astrocyte Cultures

Rat cortical astrocytes in primary culture were examined for their capacity to transport and metabolize exogenous L-[U-14C]glutamate. After incubation for time periods up to 120 min, cells and incubation media were analyzed for labelled and endogenous glutamate and its metabolic products by HPLC coupled with fluorescence detection and liquid scintillation counting. Glutamine was the major labelled metabolite after 120 min, accounted for 38% of the original glutamate label, and was found primarily in the incubation medium. A further 13.5% of the label was recovered in deaminated metabolites of glutamate, 1.2% was associated with aspartate, 23% remained in glutamate, and 10.2% was found in an acid-precipitated cell fraction. More than 84% of the label was recovered in these fraction. suggesting that the maximum possible formation and loss of 14CO2 was 16%. The rate of total glutamine synthesis was 1.1 nmol X mg protein-1 X min-1 when 9 microM exogenous glutamate was present. The total amount of glutamine synthesized greatly exceeded the consumption of glutamate, indicating that a substantial proportion of glutamine was synthesized from other carbon sources. Almost all of the newly formed glutamine was exported into the medium. These results indicate that astrocytes in primary culture, by accumulating glutamate, producing glutamine, and exporting it, are capable of carrying out the glial component of the glutamine cycle.     

Metabolic Fate of 14C-Labeled Glutamate in Astrocytes in Primary Cultures

The metabolic fate of L-[U-14C]- and L-[1-14C]glutamate was studied in primary cultures of mouse astrocytes. Conversion of the uniformly labeled compound to glutamine and aspartate was followed by determination of specific activities after dansylation with [3H]dansyl chloride and subsequent thin layer chromatography of the dansylated amino acids. Metabolic fluxes were calculated from the alterations of specific activities and the pool sizes, which were likewise measured by a dansylation method. Formation of 14CO2 from [1-14C]glutamate was determined by the trapping of CO2 in hyamine hydroxide in a gas-tight chamber, which is, in the known absence of glutamate decarboxylase activity in the cultured astrocytes, an unequivocal expression of the metabolic flux via alpha-ketoglutarate to CO2 and succinyl-CoA. The metabolic fluxes determined by these procedures amounted to 2.4 nmol/min/mg protein for glutamine synthesis, 1.1 nmol/min/mg protein for aspartate production, and 4.1 nmol/min/mg protein for formation and subsequent decarboxylation of alpha-ketoglutarate. The latter process was unaffected by virtually complete inhibition of glutamate-oxaloacetic transaminase with aminooxyacetic acid, indicating that the formation of alpha-ketoglutarate occurs as an oxidative deamination rather than as a transamination. This suggests that the formation of alpha-ketoglutarate from glutamate represents a net degradation, not an isotopic exchange.     

Stimulation of Synaptosomal γ-Aminobutyric Acid Synthesis by Glutamate and Glutamine

gamma-Aminobutyric acid (GABA) synthesis was studied in rat brain synaptosomes by measuring the increase of GABA level in the presence of the GABA-transaminase inhibitor gabaculine. The basal rate of synaptosomal GABA synthesis in glucose-containing medium (25.9 nmol/h/mg of protein) was only 3% of the maximal activity of glutamate decarboxylase (GAD; 804 +/- 83 nmol/h/mg of protein), a result indicating that synaptosomal GAD operates at only a small fraction of its catalytic capacity. Synaptosomal GABA synthesis was stimulated more than threefold by adding 500 microM glutamine. Glutamate also stimulated GABA synthesis, but the effect was smaller (1.5-fold). These results indicate that synaptosomal GAD is not saturated by endogenous levels of its substrate, glutamate, and account for part of the unused catalytic capacity. The greater stimulation of GABA synthesis by glutamine indicates that the GAD-containing compartment is more accessible to extrasynaptosomal glutamine than glutamate. The strong stimulation by glutamine also shows that the rates of uptake of glutamine and its conversion to glutamate can be sufficiently rapid to support GABA synthesis in nerve terminals. Synaptosomes carried out a slow net synthesis of aspartate in glucose-containing medium (7.7 nmol/h/mg of protein). Aspartate synthesis was strongly stimulated by glutamate and glutamine, but in this case the stimulation by glutamate was greater. Thus, the larger part of synaptosomal aspartate synthesis occurs in a different compartment than does GABA synthesis.     

Glutamate Metabolism in Rat Cortical Astrocyte Cultures

Glutamate metabolism in rat cortical astrocyte cultures was studied to evaluate the relative rates of flux of glutamate carbon through oxidative pathways and through glutamine synthetase (GS). Rates of 14CO2 production from [1-14C]glutamate were determined, as was the metabolic fate of [14C(U)]glutamate in the presence and absence of the transaminase inhibitor aminooxyacetic acid and of methionine sulfoximine, an irreversible inhibitor of GS. The effects of subculturing and dibutyryl cyclic AMP treatment of astrocytes on these parameters were also examined. The vast majority of exogenously added glutamate was converted to glutamine and exported into the extracellular medium. Inhibition of GS led to a sustained and greatly elevated intracellular glutamate level, thereby demonstrating the predominance of this pathway in the astrocytic metabolism of glutamate. Nevertheless, there was some glutamate oxidation in the astrocyte culture, as evidenced by aspartate production and labeling of intracellular aspartate pools. Inhibition of aspartate aminotransferase caused a greater than 70% decrease in 14CO2 production from [1-14C]glutamate. Inhibition of GS caused an increase in aspartate production. It is concluded that transamination of glutamate rather than oxidative deamination catalyzed by glutamate dehydrogenase is the first step in the entry of glutamate carbon into the citric acid cycle in cultured astrocytes. This scheme of glutamate metabolism was not qualitatively altered by subculturing or by treatment of the cultures with dibutyryl cyclic AMP.     

The pathway of glutamate metabolism in rat brain mitochondria

1. The pathway of glutamate metabolism in non-synaptic rat brain mitochondria was investigated by measuring glutamate, aspartate and ammonia concentrations and oxygen uptakes in mitochondria metabolizing glutamate or glutamine under various conditions. 2. Brain mitochondria metabolizing 10mm-glutamate in the absence of malate produce aspartate at 15nmol/min per mg of protein, but no detectable ammonia. If amino-oxyacetate is added, the aspartate production is decreased by 80% and ammonia production is now observed at a rate of 6.3nmol/min per mg of protein. 3. Brain mitochondria metabolizing glutamate at various concentrations (0-10mm) in the presence of 2.5mm-malate produce aspartate at rates that are almost stoicheiometric with glutamate disappearance, with no detectable ammonia production. In the presence of amino-oxyacetate, although the rate of aspartate production is decreased by 75%, ammonia production is only just detectable (0.3nmol/min per mg of protein). 4. Brain mitochondria metabolizing 10mm-glutamine and 2.5mm-malate in States 3 and 4 were studied by using glutamine as a source of intramitochondrial glutamate without the involvement of mitochondrial translocases. The ammonia production due to the oxidative deamination of glutamate produced from the glutamine was estimated as 1nmol/min per mg of protein in State 3 and 3nmol/min per mg of protein in State 4. 5. Brain mitochondria metabolizing 10mm-glutamine in the presence of 1mm-amino-oxyacetate under State-3 conditions in the presence or absence of 2.5mm-malate showed no detectable aspartate production. In both cases, however, over the first 5min, ammonia production from the oxidative deamination of glutamate was 21-27nmol/min per mg of protein, but then decreased to approx. 1-1.5nmol/min per mg. 6. It is concluded that the oxidative deamination of glutamate by glutamate dehydrogenase is not a major route of metabolism of glutamate from either exogenous or endogenous (glutamine) sources in rat brain mitochondria.     

The metabolic role of isoleucine in detoxification of ammonia in cultured mouse neurons and astrocytes

Cerebral hyperammonemia is a hallmark of hepatic encephalopathy, a debilitating condition arising secondary to liver disease. Pyruvate oxidation including tricarboxylic acid (TCA) cycle metabolism has been suggested to be inhibited by hyperammonemia at the pyruvate and -ketoglutarate dehydrogenase steps. Catabolism of the branched-chain amino acid isoleucine provides both acetyl-CoA and succinyl-CoA, thus by-passing both the pyruvate dehydrogenase and the -ketoglutarate dehydrogenase steps. Potentially, this will enable the TCA cycle to work in the face of ammonium-induced inhibition. In addition, this will provide the -ketoglutarate carbon skeleton for glutamate and glutamine synthesis by glutamate dehydrogenase and glutamine synthetase (astrocytes only), respectively, both reactions fixing ammonium. Cultured cerebellar neurons (primarily glutamatergic) or astrocytes were incubated in the presence of either [U-¹³C]glucose (2.5 mM) and isoleucine (1 mM) or [U-¹³C]isoleucine and glucose. Cell cultures were treated with an acute ammonium chloride load of 2 (astrocytes) or 5 mM (neurons and astrocytes) and incorporation of ¹³C-label into glutamate, aspartate, glutamine and alanine was determined employing mass spectrometry. Labeling from [U-¹³C]glucose in glutamate and aspartate increased as a result of ammonium-treatment in both neurons and astrocytes, suggesting that the TCA cycle was not inhibited. Labeling in alanine increased in neurons but not in astrocytes, indicating elevated glycolysis in neurons. For both neurons and astrocytes, labeling from [U-¹³C]isoleucine entered glutamate and aspartate albeit to a lower extent than from [U-¹³C]glucose. Labeling in glutamate and aspartate from [U-¹³C]isoleucine was decreased by ammonium treatment in neurons but not in astrocytes, the former probably reflecting increased metabolism of unlabeled glucose. In astrocytes, ammonia treatment resulted in glutamine production and release to the medium, partially supported by catabolism of [U-¹³C]isoleucine. In conclusion, i) neuronal and astrocytic TCA cycle metabolism was not inhibited by ammonium and ii) isoleucine may provide the carbon skeleton for synthesis of glutamate/glutamine in the detoxification of ammonium.     

Glutathione Turnover in Cultured Astrocytes: Studies with [15N]Glutamate

The incorporation of [15N]glutamic acid into glutathione was studied in primary cultures of astrocytes. Turnover of the intracellular glutathione pool was rapid, attaining a steady state value of 30.0 atom% excess in 180 min. The intracellular glutathione concentration was high (20-40 nmol/mg protein) and the tripeptide was released rapidly into the incubation medium. Although labeling of glutathione (atom% excess) with [15N]glutamate occurred rapidly, little accumulation of 15N in glutathione was noted during the incubation compared with 15N in aspartate, glutamine, and alanine. Glutathione turnover was stimulated by incubating the astrocytes with diethylmaleate, an electrophile that caused a partial depletion of the glutathione pool(s). Diethylmaleate treatment also was associated with significant reductions of intraastrocytic glutamate, glycine, and cysteine, i.e., the constituents of glutathione. Glutathione synthesis could be stimulated by supplementing the steady-state incubation medium with 0.05 mM L-cysteine, such treatment again partially depleting intraastrocytic glutamate and causing significant reductions of 15N labeling of both alanine and glutamine, suggesting that glutamate had been diverted from the synthesis of these amino acids and toward the formation of glutathione. The current study underscores both the intensity of glutathione turnover in astrocytes and the relationship of this turnover to the metabolism of glutamate and other amino acids.     

Role of Pyruvate Carboxylase in Facilitation of Synthesis of Glutamate and Glutamine in Cultured Astrocytes

Abstract: CO₂ fixation was measured in cultured astrocytes isolated from neonatal rat brain to test the hypothesis that the activity of pyruvate carboxylase influences the rate of de novo glutamate and glutamine synthesis in astrocytes. Astrocytes were incubated with ¹⁴CO₂ and the incorporation of ¹⁴C into medium or cell extract products was determined. After chromatographic separation of ¹⁴C-labelled products, the fractions of ¹⁴C cycled back to pyruvate, incorporated into citric acid cycle intermediates, and converted to the amino acids glutamate and glutamine were determined as a function of increasing pyruvate carboxylase flux. The consequences of increasing pyruvate, bicarbonate, and ammonia were investigated. Increasing extracellular pyruvate from 0 to 5 mM increased pyruvate carboxylase flux as observed by increases in the ¹⁴C incorporated into pyruvate and citric acid cycle intermediates, but incorporation into glutamate and glutamine, although relatively high at low pyruvate levels, did not increase as pyruvate carboxylase flux increased. Increasing added bicarbonate from 15 to 25 mM almost doubled CO₂ fixation. When 25 mM bicarbonate plus 0.5 mM pyruvate increased pyruvate carboxylase flux to approximately the same extent as 15 mM bicarbonate plus 5 mM pyruvate, the rate of appearance of [¹⁴C]glutamate and glutamine was higher with the lower level of pyruvate. The conclusion was drawn that, in addition to stimulating pyruvate carboxylase, added pyruvate (but not added bicarbonate) increases alanine aminotransferase flux in the direction of glutamate utilization, thereby decreasing glutamate as pyruvate + glutamate →α-ketoglutarate + alanine. In contrast to previous in vivo studies, the addition of ammonia (0.1 and 5 mM) had no effect on net ¹⁴CO₂ fixation, but did alter the distribution of ¹⁴C-labelled products by decreasing glutamate and increasing glutamine. Rather unexpectedly, ammonia did not increase the sum of glutamate plus glutamine (mass amounts or ¹⁴C incorporation). Low rates of conversion of α-[¹⁴C]ketoglutarate to [¹⁴C]glutamate, even in the presence of excess added ammonia, suggested that reductive amination of α-ketoglutarate is inactive under conditions studied in these cultured astrocytes. We conclude that pyruvate carboxylase is required for de novo synthesis of glutamate plus glutamine, but that conversion of α-ketoglutarate to glutamate may frequently be the rate-limiting step in this process of glutamate synthesis.     

Glutamine from Glial Cells Is Essential for the Maintenance of the Nerve Terminal Pool of Glutamate: Immunogold Evidence from Hippocampal Slice Cultures

Abstract: The immunogold labeling for glutamate and glutamine was studied at the electron microscopic level in hippocampal slice cultures following inhibition of l -glutamine synthetase [ l -glutamate:ammonia ligase (ADP-forming); EC 6.3.1.2]. In control cultures, glutamate-like immunoreactivity was highest in terminals, intermediate in pyramidal cell bodies, and low in glial cells. Glutamine-like immunoreactivity was high in glial cells, intermediate in pyramidal cell bodies, and low in terminals. After inhibition of glutamine synthetase with l -methionine sulfoximine, glutamate-like immunoreactivity was reduced by 52% in terminals and increased nearly fourfold in glia. Glutamine-like immunoreactivity was reduced by 66% in glia following l -methionine sulfoximine, but changed little in other compartments. In cultures that were treated with both l -methionine sulfoximine and glutamine (1.0 m M ), glutamate-like immunoreactivity was maintained at control levels in terminals, whereas in glia glutamate-like immunoreactivity was increased and glutamine-like immunoreactivity was decreased to a similar extent as in cultures treated with l -methionine sulfoximine alone. We conclude that (a) glutamate accumulates in glia when the flux through glutamine synthetase is blocked, emphasizing the importance of this pathway for the handling of glutamate; and (b) glutamine is necessary for the maintenance of a normal level of glutamate in terminals, and neither reuptake nor de novo synthesis through pathways other than the glutaminase reaction is sufficient.     

Alterations in Uptake and Release Rates for GABA, Glutamate, and Glutamine During Biochemical Maturation of Highly Purified Cultures of Cerebral Cortical Neurons, a GABAergic Preparation

This study demonstrates that virtually homogenous cultures of mouse cerebral neurons, obtained from 15-day-old embryos, differentiate at least as well as cultures which in addition contain astrocytes. This was indicated by glutamate decarboxylase activity which within 2 weeks rose from a negligible value to twice the level in the adult mouse cerebral cortex, and by a gamma-aminobutyric acid (GABA) uptake rate which quadrupled during the second week in culture and reached higher values than in brain slices. Within the same period, the GABA content increased four to five times to 75 nmol/mg protein, and a potassium-induced increase in [14C]GABA efflux became apparent. Although the development was faster than in vivo, optimum differentiation required maintenance of the cultures beyond the age of 1 week. Uptake and release rates for glutamate and glutamine underwent much less developmental alteration. At no time was there any potassium-induced release of radioactivity after exposure to [14C]glutamate, and the glutamate uptake was only slightly increased during the period of GABAergic development. This indicates that exogenous glutamate is not an important GABA precursor. Similarly, glutamine uptake was unaltered between days 7 and 14, although a small potassium-induced release of radioactivity after loading with glutamine suggests a partial conversion to GABA.     

Excess Extracellular and Low Intracellular Glutamate in Poorly Differentiating Wobbler Astrocytes and Astrocyte Recovery in Glutamine-Depleted Culture Medium

Abstract: The wobbler mouse develops an inherited motoneuronal degeneration of unknown origin in the spinal cord. Primary cultures of adult wobbler spinal cord astrocytes display abnormal morphological characteristics with fewer processes and paucity of cell-cell contacts. We have searched for a possible involvement of glutamate and glutamine intra- and extracellular accumulations in vitro in the abnormal differentiation of mutant astrocytes. We have found significantly higher glutamate and glutamine concentrations in the culture media of mutant astrocytes over a 3-day period compared with normal control astrocytes. Moreover, intracellular glutamate concentrations decreased substantially in mutant astrocytes, but intracellular glutamine concentrations remained unchanged. Furthermore, decreasing initial glutamine concentrations in the culture medium (glutamine-depleted medium) led to the recovery of normal extra- and intracellular concentrations of glutamate and recovery of quasi-normal morphological differentiation and increased cell-cell contacts, leading to an essentially normal looking astrocyte network after 3 days of culture. Under these conditions, which lead to recovery, the only remaining abnormality was the higher glutamine extracellular concentration attained in the originally depleted glutamine media. These findings suggest that mechanisms regulating glutamate/glutamine synthesis and/or influx/efflux are defective in wobbler astrocytes, leading to metabolic imbalance and possible cytotoxic effects characterized by disturbed intercellular networks and poor differentiation.     

Metabolic characteristics of recombinant Chinese hamster ovary cells expressing glutamine synthetase in presence and absence of glutamine

To elucidate the metabolic characteristics of recombinant CHO cells expressing glutamine synthetase (GS) in the medium with or without glutamine, the concentrations of extra- and intracellular metabolites and the activities of key metabolic enzymes involved in glutamine metabolism pathway were determined. In the absence of glutamine, glutamate was utilized for glutamine synthesis, while the production of ammonia was greatly decreased. In addition, the expression of recombinant protein was increased by 18%. Interestingly, the intracellular glutamine maintained almost constant, independent of the presence of glutamine or not. Activities of glutamate-oxaloacetate aminotransferase (GOT), glutamate-pyruvate aminotransferase (GPT), and glutamate dehydrogenase (GDH) increased in the absence of glutamine. On the other hand, intracellular isocitrate and the activities of its downstream isocitrate dehydrogenase in the TCA cycle increased also. In combination with these two factors, a 8-fold increase in the intracellular α-ketoglutarate was observed in the culture of CHO-GS cells in the medium without glutamine.     

Effects of chronic exposure to ammonia on glutamate and glutamine interconversion and compartmentation in homogeneous primary cultures of mouse astrocytes

Accumulation of radioactivity was studied in primary cultures of mouse astrocytes as a function of time of exposure (4–60 min) to 50 M glutamate and 200 M glutamine (initial concentrations), of whicheither glutamateor glutamine was¹⁴C-labeled. Both the glutamate pool and the glutamine pool were compartmentalized. Initially, by far the major intracellular glutamate pool (90%) was derived from extracellular glutamate and could be converted to glutamine. This allowed a rather accurate determination of metabolic flux from glutamate to glutamine, which under control conditions amounted to 2.0–2.2 nmol/min per mg protein. After chronic exposure to 3 mM ammonia for 3 days this flux was significantly increased to 3.1–3.6 nmol/min per mg protein. Acute exposure to ammonia caused a smaller, apparent increase, which was not statistically significant. The glutamine content was compartmentalized at all stages of the incubation. It consisted of at least two different pools. One of these was accessible to extracellular glutamine and could be converted to intracellular glutamate (constituting a sizeable fraction of the total glutamate pool after longer incubation), whereas the other constituted endogenously derived glutamine, formed from accumulated glutamate. The specific activity of the precursor pool for glutamate synthesis could not beaccurately determined and relatively exact fluxes therefore not be calculated. There was, however, no evidence that chronic exposure to ammonia decreases the rate of glutamine hydrolysis.     

Neuronal Glutamine Utilization: Pathways of Nitrogen Transfer tudied with [15N]Glutamine

Gas chromatography-mass spectrometry was used to evaluate the metabolism of [15N]glutamine in isolated rat brain synaptosomes. In the presence of 0.5 mM glutamine, synaptosomes accumulated this amino acid to a level of 25-35 nmol/mg protein at an initial rate greater than 9 nmol/min/mg of protein. The metabolism of [15N]glutamine generated 15N-labelled glutamate, aspartate, and gamma-aminobutyric acid (GABA). An efflux of both [15N]glutamate and [15N]aspartate from synaptosomes to the medium was observed. Enrichment of 15N in alanine could not be detected because of a limited pool size. Elimination of glucose from the incubation medium substantially increased the rate and amount of [15N]aspartate formed. It is concluded that: (1) With 0.5 mM external glutamine, the glutaminase reaction, and not glutamine transport, determines the rate of metabolism of this amino acid. (2) The primary route of glutamine catabolism involves aspartate aminotransferase which generates 2-oxoglutarate, a substrate for the tricarboxylic acid cycle. This reaction is greatly accelerated by the omission of glucose. (3) Glutamine has preferred access to a population of synaptosomes or to a synaptosomal compartment that generates GABA. (4) Synaptosomes maintain a constant internal level of glutamate plus aspartate of about 70-80 nmol/mg protein. As these amino acids are produced from glutamine in excess of this value, they are released into the medium. Hence synaptosomal glutamine and glutamate metabolism are tightly regulated in an interrelated manner.     

Nitrogen assimilation in the facultative methylotroph Hyphomicrobium X

A survey of the possible nitrogen assimilation pathways in Hyphomicrobium X showed that when the nitrogen source was satisfied by ammonium sulphate or methylamine and the supply was in excess, NADPH-dependent glutamate dehydrogenase was used to assimilate nitrogen. When the nitrogen supply was limited the cells expressed high levels of glutamine synthetase and NADH-dependent glutamine:2-oxoglutamate aminotransferase activity whilst the activity of the glutamate dehydrogenase was lower. When nitrate was the N-source, the glutamine synthetase/glutamine oxoglutamate aminotransferase pathway was utilised irrespective of the nitrogen concentration in the medium. Evidence was obtained to suggest that the glutamine synthetase activity was regulated by adenylylation/deadenylylation. Carbon-limited chemostat cultures showed low glutamine synthetase activity levels but the synthesis of the enzyme was derepressed when the cultures became N-limited.     

Astrocyte Leucine Metabolism: Significance of Branched-Chain Amino Acid Transamination

Abstract: We studied astrocytic metabolism of leucine, which in brain is a major donor of nitrogen for the synthesis of glutamate and glutamine. The uptake of leucine into glia was rapid, with a V _max of 53.6 ± 3.2 nmol/mg of protein/min and a K _m of 449.2 ± 94.9 µ M . Virtually all leucine transport was found to be Na⁺ independent. Astrocytic accumulation of leucine was much greater (3×) in the presence of α-aminooxyacetic acid (5 m M ), an inhibitor of transamination reactions, suggesting that the glia rapidly transaminate leucine to α-ketoisocaproic acid (KIC), which they then release into the extracellular fluid. This inference was confirmed by the direct measurement of KIC release to the medium when astrocytes were incubated with leucine. Approximately 70% of the leucine that the glia cleared from the medium was released as the keto acid. The apparent K _m for leucine conversion to extracellular KIC was a medium [leucine] of 58 µ M with a V _max of ∼2.0 nmol/mg of protein/min. The transamination of leucine is bidirectional (leucine + α-ketoglutarate ↮ KIC + glutamate) in astrocytes, but flux from leucine → glutamate is more active than that from glutamate → leucine. These data underscore the significance of leucine handling to overall brain nitrogen metabolism. The release of KIC from glia to the extracellular fluid may afford a mechanism for the "buffering" of glutamate in neurons, which would consume this neurotransmitter in the course of reaminating KIC to leucine.     

Spontaneous Glutamate Release by a "Fibrous"-Like Cerebellar Astroglial Cell Clone

Abstract: To investigate the role of astrocytes in the metabolism of glutamate, the neurotransmitter of the granule cells of the cerebellar cortex, we have analyzed various parameters related to the synthesis of glutamate in astroglial cell clones that may be the in vitro counterparts of the cerebellar astrocytes. The "fibrous"-like clone spontaneously released large quantities of glutamate, even in the absence of glutamine in the culture medium, but did not release alanine. In contrast, the "Golgi-Bergmann"-like cells released alanine but not glutamate, whereas the "velate-protoplasmic"-like astrocytes released little glutamate and alanine. However, the glutamate oxaloacetate transaminase and glutamate pyruvate transaminase activities of the three astroglial cell lines, measured in the direction of glutamate synthesis, were comparable. In addition, the "velate protoplasmic" and "Golgi-Bergmann" clones did not consume glutamine present at 2 m M in the culture medium. These data suggest that the different types of in vivo cerebellar astrocytes may have distinct roles regarding glutamate-glutamine metabolism.     

Nitrogen control in Pseudomonas aeruginosa: A role for glutamine in the regulation of the synthesis of NADP-dependent glutamate dehydrogenase,urease and histidase

In Pseudomonas aeruginosa the formation of urease, histidase and some other enzymes involved in nitrogen assimilation is repressed by ammonia in the growth medium. The key metabolite in this process appears to be glutamine or a product derived from it, since ammonia and glutamate did not repress urease and histidase synthesis in a mutant lacking glutamine synthetase activity when growth was limited for glutamine. The synthesis of these enzymes was repressed in cells growing in the presence of excess glutamine. High levels of glutamine were also required for the derepression of NADP-dependent glutamate dehydrogenase formation in the glutamine synthetase-negative mutant.     

DL-7-azatryptophan and citrulline metabolism in the cyanobacterium Anabaena sp. strain 1F.

An alternative route for the primary assimilation of ammonia proceeds via glutamine synthetase-carbamyl phosphate synthetase and its inherent glutaminase activity in Anabaena sp. strain 1F, a marine filamentous, heterocystous cyanobacterium. Evidence for the presence of this possible alternative route to glutamate was provided by the use of amino acid analogs as specific enzyme inhibitors, enzymological studies, and radioistopic labeling experiments. The amino acid pool patterns of continuous cultures of Anabaena sp. strain 1F were markedly influenced by the nitrogen source. A relatively high concentration of glutamate was maintained in the amino acid pools of all cultures irrespective of the nitrogen source, reflecting the central role of glutamate in nitrogen metabolism. The addition of 1.0 microM azaserine increased the intracellular pools of glutamate and glutamine. All attempts to detect any enzymatic activity for glutamate synthase by measuring the formation of L-[14C]glutamate from 2-keto-[1-14C]glutarate and glutamine failed. The addition of 10 microM DL-7-azatryptophan caused a transient accumulation of intracellular citrulline and alanine which was not affected by the presence of chloramphenicol. The in vitro activity of carbamyl phosphate synthetase and glutaminase increased severalfold in the presence of azatryptophan. Results from radioisotopic labeling experiments with [14C]bicarbonate and L-[1-14C]ornithine also indicated that citrulline was formed via carbamyl phosphate synthetase and ornithine transcarbamylase. In addition to its effects on nitrogen metabolism, azatryptophan also affected carbon metabolism by inhibiting photosynthetic carbon assimilation and photosynthetic oxygen evolution.