Interaction Between the Glutamine Cycle and the Uptake of Large Neutral Amino Acids in Astrocytes

Abstract: When astrocyte cultures are incubated with glutamate and ammonium, the clearance of these substrates followed by the formation and export of glutamine simulates the action of the "glutamine cycle" that is believed to function in the CNS. In the present study this process was found to increase the uptake of large neutral amino acids (LNAAs), namely, histidine, kynurenine, leucine, phenylalanine, and tryptophan, by two-to threefold in mouse cerebral astrocytes. The enhancement of kynurenine uptake was shown to depend on the formation of glutamine and to saturate at low levels of glutamine. LNAAs transiently lowered the glutamine content of astrocytes that were incubated with glutamate and ammonium, but they did not affect net export of glutamine to the solution at normal physiological pH. However, on adjustment of the pH of the solution to 7.8, which causes a large increase in glutamine content without affecting export, kynurenine now significantly increased net glutamine export. These findings relate to proposed mechanisms of cerebral dysfunction in hyperammonemia.     

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.     

Resveratrol Increases Glutamate Uptake, Glutathione Content, and S100B Secretion in Cortical Astrocyte Cultures

Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a polyphenol present in grapes and red wine, which has antioxidant properties and a wide range of other biological effects. In this study, we investigated the effect of resveratrol, in a concentration range of 10–250 μM, on primary cortical astrocytes; evaluating cell morphology, parameters of glutamate metabolism such as glutamate uptake, glutamine synthetase activity and glutathione total content, and S100B secretion. Astrocyte cultures were prepared of cerebral cortex from neonate Wistar rats. Morphology was evaluated by phase-contrast microscopy and immunocytochemistry for glial fibrillary acidic protein (GFAP). Glutamate uptake was measured using l-[2,3-³H]glutamate. Glutamine synthetase and content of glutathione were measured by enzymatic colorimetric assays. S100B content was determined by ELISA. Typical polygonal morphology becomes stellated when astrocyte cultures were exposed to 250 μM resveratrol for 24 h. At concentration of 25 μM, resveratrol was able to increase glutamate uptake and glutathione content. Conversely, at 250 μM, resveratrol decreased glutamate uptake. Unexpectedly, resveratrol at this high concentration increased glutamine synthetase activity. Extracellular S100B increased from 50 μM upwards. Our findings reinforce the protective role of this compound in some brain disorders, particularly those involving glutamate toxicity. However, the underlying mechanisms of these changes are not clear at the moment and it is necessary caution with its administration because elevated levels of this compound could contribute to aggravate these conditions.     

Effect of 8-bromo-cAMP and dexamethasone on glutamate metabolism in rat astrocytes

Glutamine synthetase (GS) activity in cultured rat astrocytes was measured in extracts and compared to the intracellular rate of glutamine synthesis by intact control astrocytes or astrocytes exposed to 1 mM 8-bromo-cAMP (8Br-cAMP)+1 M dexamethasone (DEX) for 4 days. GS activity in extracts of astrocytes treated with 8Br-cAMP+DEX was 7.5 times greater than the activity in extracts of control astrocytes. In contrast, the intracellular rate of glutamine synthesis by intact cells increased only 2-fold, suggesting that additional intracellular effectors regulate the expression of GS activity inside the intact cell. The rate of glutamine synthesis by astrocytes was 4.3 times greater in MEM than in HEPES buffered Hank's salts. Synthesis of glutamine by intact astrocytes cultured in MEM was independent of the external glutamine or ammonia concentrations but was increased by higher extracellular glutamate concentrations. In studies with intact astrocytes 80% of the original [U-¹⁴C]glutamate was recovered in the medium as radioactive glutamine, 2–3% as aspartate, and 7% as glutamate after 2 hours for both control and treated astrocytes. The results suggest: (1) astrocytes are highly efficient in the conversion of glutamate to glutamine; (2) induction of GS activity increases the rate of glutamate conversion to glutamine by astrocytes and the rate of glutamine release into the medium; (3) endogenous intracellular regulators of GS activity control the flux of glutamate through this enzymatic reaction; and, (4) the composition of the medium alters the rate of glutamine synthesis from external glutamate.     

Functional integration of the transport of ammonium, glutamate and glutamine in astrocytes

This short review surveys the effects of extracellular potassium, released by neuronal activity, on the fluxes of ammonium, glutamate and glutamine in astrocytes. There is evidence that each of these fluxes is modulated by potassium-induced changes in astrocytic pH. The result is viewed as an integrated response to neuronal activity. The unusually high permeability of astrocyte cell membrane to ammonium ions, together with the normal transmembrane gradient of pH, enables astrocytes to accumulate ammonium appreciably. However, at loci of neuronal activity, effective ammonium ion permeability is diminished and the cytosol is alkalinized, resulting in a local decline in intracellular ammonium concentration. Intracellular potassium concentration rises at these same loci, creating the conditions for a 'potassium-ammonium countercurrent' in which ammonium ions migrate intracellularly towards sites of neuronal activity as potassium ions diffuse away.Physiologic elevations of extracellular potassium evoke a marked 'paradoxical' increase in the velocity of glutamate uptake in astrocytes. This increase correlates well with the extent of potassium-induced alkalinization. Further, recent evidence identifies a major transporter of glutamine in astrocytes (System N) as a glutamine/proton exchanger. Potassium can reverse the transmembrane gradient of protons in astrocytes, and increase intracellular glutamine concentration, creating the conditions for a reversal of glutamine flux via System N from uptake to export. These flux changes, evoked by potassium released from active neurons, combine to accelerate glutamate-glutamine cycling.     

siRNA knock down of glutamate dehydrogenase in astrocytes affects glutamate metabolism leading to extensive accumulation of the neuroactive amino acids glutamate and aspartate

Glutamate is the most abundant excitatory neurotransmitter in the brain and astrocytes are key players in sustaining glutamate homeostasis. Astrocytes take up the predominant part of glutamate after neurotransmission and metabolism of glutamate is necessary for a continuous efficient removal of glutamate from the synaptic area. Glutamate may either be amidated by glutamine synthetase or oxidatively metabolized in the mitochondria, the latter being at least to some extent initiated by oxidative deamination by glutamate dehydrogenase (GDH). To explore the particular importance of GDH for astrocyte metabolism we have knocked down GDH in cultured cortical astrocytes employing small interfering RNA (siRNA) achieving a reduction of the enzyme activity by approximately 44%. The astrocytes were incubated for 2h in medium containing either 1.0mM [(15)NH(4)(+)] or 100μM [(15)N]glutamate. For those exposed to [(15)N]glutamate an additional 100μM was added after 1h. Metabolic mapping was performed from isotope incorporation measured by mass spectrometry into relevant amino acids of cell extracts and media. The contents of the amino acids were measured by HPLC. The (15)N incorporation from [(15)NH(4)(+)] into glutamate, aspartate and alanine was decreased in astrocytes exhibiting reduced GDH activity. However, the reduced GDH activity had no effect on the cellular contents of these amino acids. This supports existing in vivo and in vitro studies that GDH is predominantly working in the direction of oxidative deamination and not reductive amination. In contrast, when exposing the astrocytes to [(15)N]glutamate, the reduced GDH activity led to an increased (15)N incorporation into glutamate, aspartate and alanine and a large increase in the content of glutamate and aspartate. Surprisingly, this accumulation of glutamate and net-synthesis of aspartate were not reflected in any alterations in either the glutamine content or labeling, but a slight increase in mono labeling of glutamine in the medium. We suggest that this extensive net-synthesis of aspartate due to lack of GDH activity is occurring via the concerted action of AAT and the part of TCA cycle operating from α-ketoglutarate to oxaloacetate, i.e. the truncated TCA cycle.     

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.     

Glutamate induces formation of free radicals in rat brain synaptosomes

The influence of glutamate and agonists of its ionotropic receptors on free radical formation in rat brain synaptosomes was investigated using the fluorescent dye DCFDA. Glutamate at concentrations of 100 μM and 1 mM increased the production of reactive oxygen species. This phenomenon was eliminated by removing calcium from the incubation medium. Addition of NMDA (100 μM) or kainate (100 μM) to a suspension of synaptosomes also led to free radical formation. The influence of glutamate receptor agonists was blocked by the specific antagonists MK-801 and NBQX. Thus, activation of NMDA and AMPA/kainate receptors can lead to oxidative stress in neuronal presynaptic endings.     

Glucocorticoid Stimulation of Glutamine Synthetase Production in Cultured Rat Glioma Cells

Abstract: A sensitive radioisotopic assay has been used to examine the kinetic properties and regulation of biosynthesis of glutamine synthetase in C-6 glioma cultures. The K_m values for glutamate, MgATP, and ammonium ion were 5mM, 14 mM, and 0.042 mM, respectively, when measured at the pH optimum of 7.2. There was an absolute requirement for a divalent metal ion, with 15 mM- Mg²⁺ being the preferred ion at pH 7.2. Activity was completely inhibited after 30 min with 8 mM-L-methionhe sulfoximine. The addition of 1 μM-cortisol to C-6 cultures caused a two to threefold increase in glutamine synthetase specific activity over a 96-h period, while dexamethasone at the same concentration elevated the level some 7-10-fold. This was specific for glucocorticoids, as other steroid hormones or catecholamines did not significantly affect glutamine synthetase specific activity. Cycloheximide (30 μM) or actinomycin D (0.01 μg/ml) blocked the hormone response. The continued presence of hormone was required in order to maintain an elevated enzyme level. The results suggest that glucocorticoids act to induce glutamine synthetase by stimulating new enzyme synthesis.     

Guanosine enhances glutamate uptake in brain cortical slices at normal and excitotoxic conditions

1. The effect of guanosine on L-[2,3-³H]glutamate uptake was investigated in brain cortical slices under normal or oxygen–glucose deprivation (OGD) conditions.2. In slices exposed to physiological conditions, guanosine (1–100 M) stimulated glutamate uptake (up to 100%) in a concentration-dependent manner when a high (100 M) but not a low (1 M) concentration of glutamate was used.3. In slices submitted to OGD, guanosine 1 and 100 M also increased 100 M glutamate uptake (38 and 70%, respectively).4. The increasing of glutamate and taurine released to the incubation medium in cortical slices submitted to OGD were significantly attenuated by the presence of guanosine in the incubation medium.5. Guanosine prevented the increase in propidium iodide incorporation into cortical slices induced by OGD, indicating a protective role against ischemic injury.6. These results support the hypothesis of a protective role for guanosine during brain ischemia, possibly by activating glutamate uptake into neural cells.     

Nickel-induced depression of nitrogen assimilation in wheat roots

The influence of 50 and 100 μM Ni on the activities of nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), glutamate synthase (GOGAT), glutamate dehydrogenase (GDH), alanine aminotransferase (AlaAT) and aspartate aminotransferase (AspAT) was studied in the wheat roots. Root fresh weight, tissue Ni, nitrate, ammonium, glutamate and protein concentrations were also determined. Exposure to Ni resulted in a marked reduction in fresh weight of the roots accompanied by a rapid accumulation of Ni in these organs. Both nitrate and ammonium contents in the root tissue were considerably enhanced by Ni stress. While protein content was not significantly influenced by Ni application, glutamate concentration was slightly reduced on the first day after treatment with the higher Ni dose. Treatment of the wheat seedlings with 100 μM Ni led to a decrease in NR activity; however, it did not alter the activation state of this enzyme. Decline in NiR activity observed after application of 100 μM Ni was more pronounced than that in NR. The activities of GS and NADH-GOGAT also showed substantial decreases in response to Ni stress with the latter being more susceptible to this metal. Starting from the fourth day, both aminating and deaminating GDH activities in the roots of the seedlings supplemented with Ni were lower in comparison to the control. While the activity of AspAT remained unaltered after Ni application that of AlaAT showed a considerable enhancement. The results indicate that exposure of the wheat seedlings to Ni resulted in a general depression of nitrogen assimilation in the roots. Increase in the glutamate-producing activity of AlaAT may suggest its involvement in supplying the wheat roots with this amino acid under Ni stress.     

Aluminium Stress Affects Nitrogen Fixation and Assimilation in Soybean (Glycine max L.)

Nitrogen fixation and assimilation in nodules and roots were studied in soybean (Glycine max L.) exposed to different levels of aluminium (Al) stress (0, 50, 200 and 500 μM). Al at 500 μM induced oxidative stress, which became evident from an increase in lipid peroxidation accompanied by a concomitant decline in antioxidant enzyme activities and leghaemoglobin breakdown. Consequently, there was also a reduction in nitrogenase activity. However, the leghaemoglobin levels and nitrogenase activity were unexpectedly found to be higher in nodules when the plants were treated with 200 μM Al. Of the enzymes involved in nitrogen assimilation, the activity of glutamate dehydrogenase-NADH was reduced in nodules under Al stress, but it was significantly higher in roots at 500 μM Al as compared to that in the control. In nodules, the glutamine synthetase/glutamate synthase-NADH pathway, assayed in terms of activity and expression of both the enzymes, was inhibited at >50 μM Al; but in roots this inhibitory effect was apparent only at 500 μM Al. No significant changes in ammonium and protein contents were recorded in the nodules or roots when the plants were treated with 50 μM Al. However, Al at ≥200 μM significantly increased the ammonium levels and decreased the protein content in the nodules. But these contrasting effects on ammonium and protein contents due to Al stress were observed in the roots only at 500 μM Al. The results suggest that the effect of Al stress on nitrogen assimilation is more conspicuous in nodules than that in the roots of soybean plants.     

Exogenous Glutamate Concentration Regulates the Metabolic Fate of Glutamate in Astrocytes

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-¹³C]glutamate in primary cultures of cerebral cortical astrocytes from rat brain. When astrocytes from rat brain were incubated with 0.1 m M [U-¹³C]glutamate 85% of the ¹³C metabolized was converted to glutamine. The formation of [1,2,3-¹³C₃]glutamate demonstrated metabolism of the labeled glutamate via the TCA cycle. When astrocytes were incubated with 0.2–0.5 m M glutamate, ¹³C from glutamate was also incorporated into intracellular aspartate and into lactate that was released into the media. The amount of [¹³C]lactate was essentially unchanged within the range of 0.2–0.5 m M glutamate, whereas the amount of [¹³C]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.     

Enzymes involved in ammonia assimilation in the fungus Acremonium persicinum

Abstract Acremonium persicinum grown in batch culture with ammonium tartrate as the nitrogen source possessed an NADP⁺-dependent glutamate dehydrogenase and a glutamine synthetase. Glutamate synthase was not detected under the culture conditions used. Kinetic studies of the NADP⁺-dependent glutamate dehydrogenase at 25°C and pH 7.6 revealed an apparent K _m of 3.2 × 10⁻⁴ M for 2-oxoglutarate and an apparent K _m of 1.0 × 10⁻⁵ M for ammonium ions, with corresponding apparent V _max values of 0.089 and 0.13 μmol substrate converted/min/mg of protein, respectively. Glutamine synthetase was measured by the γ-glutamyl transferase reaction at 30°C and pH 7.55. This transferase reaction of glutamine synthetase had a higher rate at 30°C than at 25°C or 37°C.     

GABA synthesis in brain slices is dependent on glutamine produced in astrocytes

The rate of -aminobutyric acid (GABA) synthesis in rat-brain slices was determined by inhibiting GABA transaminase with 20-M gabaculine and measuring the increase of GABA. Added 500-M glutamine increased the rate of GABA synthesis by 50%, indicating that glutamate decarboxylase is not saturated in brain slices. The stimulation of GABA synthesis with added glutamine in brain slices was much less than that reported for synaptosomes. The lower stimulation in slices was attributable to astrocytic glutamine production, as the rate of GABA synthesis decreased by 44% when glutamine production was inhibited with methionine sulfoximine. Added glutamine restored the rate to the maximal value observed in brain slices. The rate of GABA synthesis was decreased by 65% in slices pretreated with an inhibitor of glutaminase, and added glutamine did not reverse this effect. These results suggest that glutamine produced by astrocytes is a quantitatively important precursor of GABA synthesis in cortical slices.     

Glutamine and Aspartate Loading of Synaptosomes: A Reevaluation of Effects on Calcium-Dependent Excitatory Amino Acid Release

Guinea-pig cerebral cortical synaptosomes were preincubated for 60 min with 100 microM D-aspartate, L-aspartate, or L-glutamate. The total D- plus L-aspartate content of the synaptosomal fraction increased to 235%, 195%, or 164%, respectively, of the control. Despite this no increase was seen in the very low KCl evoked, Ca2+-dependent release of aspartate. Preincubation with the three amino acids changed the synaptosomal glutamate content to 78% (D-aspartate), 149% (L-aspartate), or 168% (L-glutamate) of control. However there was no statistically significant effect of these preincubations on the extent of Ca2+-dependent glutamate release. Thus the Ca2+-dependent release of aspartate and glutamate is not determined by the total synaptosomal content of these amino acids. The addition of 0.1-0.5 mM glutamine to the incubation caused a massive appearance of glutamate in the extrasynaptosomal medium. Analysis of specific activities showed that glutamine was hydrolysed directly by an extrasynaptosomal glutaminase, and that intrasynaptosomal glutamate was predominantly labelled by uptake of this glutaminase-derived glutamate. No increase was seen in the extent of Ca2+-dependent release of glutamate (by fluorimetry) either after preincubation with glutamine or in the continued presence of glutamine. Thus we are unable to confirm reports that glutamine expands the transmitter pool of glutamate. The extrasynaptosomal glutaminase activity in the synaptosomal preparation was inhibited by Ca2+ and activated by phosphate. Identical kinetics were obtained with "free" brain mitochondria, confirming the origin of the glutamine-derived glutamate.     

Ammonium metabolism in the symbiotic sea anemone Anemonia viridis

The temperate sea anemone Anemonia viridis (Forskål) forms an endosymbiotic association with dinoflagellate algae commonly referred to as zooxanthellae. It is now well established that under appropriate environmental conditions, these associations can be autotrophic for carbon. Under such conditions, many of these symbioses, including A. viridis, not only retain excretory ammonium, but can take up ammonium added to the surrounding seawater. The flux from inorganic to organic nitrogen will be via the free amino acid pools and in A. viridis these were found to be markedly different between zooxanthellae and host with glycine and taurine dominant in the latter. When anemones were maintained with 20 M ammonium, the concentration of free amino groups increased in the zooxanthellae but appeared not to change in the host. There was no evidence that the ratio of glutamine – glutamate in zooxanthellae changed when anemones were maintained with 20 M ammonium for 47 days. These ratios imply that zooxanthellae from this temperate symbiosis may not be nitrogen-limited. GDH was detected in both zooxanthellae and host where it was most active with the coenzyme NADPH. In addition, GDH showed activity when glutamine replaced ammonium as the substrate, indicating that the host may have alternative means to assimilate ammonium. Zooxanthellae were shown to possess GOGAT activity in the presence of a ferredoxin analogue. This suggests that in vivo zooxanthellae could assimilate ammonium via the activity of GS linked with ferredoxin-dependent GOGAT. Given evidence from other studies of rapid ammonium assimilation and essential amino acid synthesis in symbiotic host tissue, it appears that the capacity of cnidarians to metabolise nitrogen may at present be underestimated.     

Damage to Neurons in Culture Following Medium Change: Role of Glutamine and Extracellular Generation of Glutamate

Abstract— Changing the medium of primary cell cultures of CNS origin causes severe damage that is mediated via the N -methyl- d -aspartate (NMDA)-type of glutamate receptors and dependent on the presence of glutamine in the medium. Data presented here show that glutamine has two roles in culture damage: glutamine is contaminated with a small amount of glutamate, which is responsible for initiating culture damage, and glutamine is the source of the glutamate that is produced extracellularly in damaged cultures. The NMDA receptor plays a critical role minutes after medium change when the glutamate contaminating the glutamine binds to NMDA receptors; during this time, addition of a low level (10–20 μ M ) of 2-amino-5-phos-phonovaleric acid can block most culture damage and the appearance of extracellular glutamate. A higher level (300 μ M ) of 2-amino-5-phosphonovaleric acid can protect cultures when added at much later times (30–60 min). Between 3 and 6 h after medium change, the concentration of extracellular glutamate starts to rise and accumulates until the end of the culture period (20 h). Medium removed from cultures at 3 h or later after medium change and incubated alone (i.e., with no cells) also continues to generate glutamate; filtration (0.22 μrn pore size) or centrifugation (18,000 g) stops the appearance of this glutamate. 6-Diazo-5-oxo- l -norleucine, an inhibitor of the mitochondrial enzyme glutaminase, blocks the generation of glutamate. Mitochondria or mitochondrial fragments are probably released from the damaged cells and then convert extracellular glutamine to glutamate, resulting in generation of a high extracellular glutamate concentration.     

Regulation of ammonium and potassium uptake by glutamine and asparagine in Pisum arvense plants

Pisum arvense plants were subjected to 5 days of nitrogen deprivation. Then, in the conditions that increased or decreased the root glutamine and asparagine pools, the uptake rates of 0.5 mM NH₄ ⁺ and 0.5 mM K⁺ were examined. The plants supplied with 1 mM glutamine or asparagine took up ammonium and potassium at rates lower than those for the control plants. The uptake rates of NH₄ ⁺ and K⁺ were not affected by 1 mM glutamate. When the plants were pre-treated with 100 μM methionine sulphoximine, an inhibitor of glutamine synthesis, the efflux of NH₄ ⁺ from roots to ambient solution was enhanced. On the other hand, exposure of plants to methionine sulphoximine led to an increase in potassium uptake rate. The addition of asparagine, glutamine or glutamate into the incubation medium caused a decline in the rate of NH₄ ⁺ uptake by plasma membrane vesicles isolated from roots of Pisum arvense, whereas on addition of methionine sulphoximine increased ammonium uptake. The results indicate that both NH₄ ⁺ and K⁺ uptake appear to be similarly affected by glutamine and asparagine status in root cells. The research was supported by grant of KBN No. 6PO4C 068 08     

Elevation of glutathione levels by ammonium ions in primary cultures of rat astrocytes

It is well established that ammonia is detoxified in the brain to form glutamine and that astrocytes play a major role in this process. The synthesis of glutamine requires glutamate and ATP. Since glutamate and ATP are also required for the synthesis of glutathione (GSH), we examined the effect of pathophysiological concentrations of ammonia on levels of GSH in primary cultures of astrocytes. GSH content in the medium increased in a dose- and time-dependent manner in the presence of ammonia. After an initial decrease, cellular GSH content increased in a similar manner. The levels of glutathione disulfide (GSSG) were also increased. A linear relationship was observed between ammonia concentration and the increase in GSH levels. An increase in the efflux of GSH from cells into medium was also observed under these conditions. Buthionine sulfoximine and acivicin, but not methionine sulfoximine, blocked the ammonia induced increase in GSH levels. No, or minor, changes in the activities of enzymes (gamma-glutamyl transpeptidase, GSH reductase and GSH-peroxidase) that might influence GSH levels were identified and thus could not account for the ammonia induced increase in GSH levels in astrocytes. These findings indicate that pathophysiological concentrations of ammonium ions result in increased astroglial levels of GSH which may affect the metabolism and function of astrocytes.