Effects of medium glutamine,glutamate, and ammonia on glutamine synthetase activity in cultured mouse astroglial cells

Mouse astroglial cells were grown during the last week of culture in either glutamine-free or glutamine-containing medium. The addition of cortisol to the glutamine-containing medium resulted in a doubling of astroglial glutamine synthetase (GS) activity. Withdrawal of glutamine from the medium resulted in a 50% elevation of GS and addition of cortisol to such a medium resulted in a further increase in GS which was not additive to glutamine withdrawal. Both in glutamine-free and glutamine-containing medium, the addition of glutamate resulted in a depression of both basal and cortisol induced GS activity. The simultaneous addition of ammonia plus glutamate to the culture medium ameliorated the glutamate mediated depressive effects on cortisol induced but not basal GS activity. Glutamine withdrawal from the culture medium resulted in an astroglial protein deficit. The addition of ammonia to the medium considerably reduced this deficit and the addition of glutamate completely eliminated this protein deficit.     

Hypoxia regulates glutamate metabolism and membrane transport in rat PC12 cells

We investigated the effect of hypoxia on glutamate metabolism and uptake in rat pheochromocytoma (PC12) cells. Various key enzymes relevant to glutamate production, metabolism and transport were coordinately regulated by hypoxia. PC12 cells express two glutamate-metabolizing enzymes, glutamine synthetase (GS) and glutamate decarboxylase (GAD), as well as the glutamate-producing enzyme, phosphate-activated glutaminase (PAG). Exposure to hypoxia (1% O(2)) for 6 h or longer increased expression of GS mRNA and protein and enhanced GS enzymatic activity. In contrast, hypoxia caused a significant decrease in expression of PAG mRNA and protein, and also decreased PAG activity. In addition, hypoxia led to an increase in GAD65 and GAD67 protein levels and GAD enzymatic activity. PC12 cells express three Na(+)-dependent glutamate transporters; EAAC1, GLT-1 and GLAST. Hypoxia increased EAAC1 and GLT-1 protein levels, but had no effect on GLAST. Chronic hypoxia significantly enhanced the Na(+)-dependent component of glutamate transport. Furthermore, chronic hypoxia decreased cellular content of glutamate, but increased that of glutamine. Taken together, the hypoxia-induced changes in enzymes related to glutamate metabolism and transport are consistent with a decrease in the extracellular concentration of glutamate. This may have a role in protecting PC12 cells from the cytotoxic effects of glutamate during chronic hypoxia.     

Activity profiles of enzymes involved in glutamine and glutamate metabolism in the apple during autumnal senescence

Seasonal changes in glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 2.6.1.53), and glutamate dehydrogenase (EC 1.4.1.3) were measured in both senescing leaf and bark tissues of ‘Golden Delicious’ apple trees (Malus domestica Borkh.). From the measured enzyme activities we attempted to estimate the in vivo catalytic potentials of the enzymes with special reference to nitrogen mobilization and conservation of senescing apple trees. The cumulative glutamine synthetase activity of leaf tissue was about three times higher than that of bark. The estimated catalytic potential of leaf glutamine synthetase was 800-fold higher than the actual protein nitrogen loss of senescing leaves. The cumulative glutamate synthase activity of bark was about six times higher than that of leaf. The estimated catalytic potential of bark glutamate synthase was 160-times higher than the actual protein nitrogen gain in that tissue. The cumulative glutamate dehydrogenase activities in leaf and bark tissue were approximately the same. However, the catalytic potential of leaf glutamate dehydrogenase was twice that of leaf glutamate synthase. It is thus concluded that the physiological role of glutamine synthetase in senescing leaf tissue is to furnish the amide(s) prior to mobilization of nitrogen to storage tissue. The higher activity of glutamate synthase in bark tissue could provide a mechanism to transform the imported amide nitrogen to amino nitrogen of glutamate for storage protein synthesis. The possible regulatory factors upon the activity of these enzymes in the tissues of senescing apple trees are discussed.     

Ontogenetic Changes in Glial Fibrillary Acid Protein Phosphorylation,Glutamate Uptake and Glutamine Synthetase Activity in Olfactory Bulb of Rats

Phosphorylation of the glial fibrillary acidic protein (GFAP) in hippocampal and cerebellar slices from immature rats is stimulated by glutamate. This effect occurs via a group II metabotropic glutamate receptor in the hippocampus and an NMDA ionotropic receptor in the cerebellum. We investigated the glutamate modulation of GFAP phosphorylation in the olfactory bulb slices of Wistar rats of different ages (post-natal day 15 = P15, post-natal day 21 = P21 and post-natal day 60 = P60). Our results showed that glutamate stimulates GFAP phosphorylation in young animals and this is mediated by NMDA receptors. We also observed a decrease in glutamate uptake at P60 compared to P15, a finding similar to that found in the hippocampus. The activity of glutamine synthetase was elevated after birth, but was found to decrease with development from P21 to P60. Together, these data confirm the importance of glutamatergic transmission in the olfactory bulb, its developmental regulation in this brain structure and extends the concept of glial involvement in glutamatergic neuron-glial communication.     

Ammonia assimilation in Corynebacterium glutamicum and a glutamate dehydrogenase-deficient mutant

In the wild-type of Corynebacterium glutamicum, the specific activity of glutamate dehydrogenase (GDH) remained constant at 1.3 U (mg protein)–1 when raising the ammonia (NH4) concentration in the growth medium from 1 to 90 mM. In contrast, the glutamine synthetase (GS) and glutamate synthase (GOGAT) activities decreased from 1.1 U (mg protein)–1 and 42 mU (mg protein)–1, respectively, to less than 10 % of these values at NH4 concentrations > 10 mM suggesting that under these conditions the GDH reaction is the primary NH4 assimilation pathway. Consistent with this suggestion, a GDH-deficient C. glutamicum mutant showed slower growth at NH4 concentrations 10 mM and, in contrast to the wild-type, did not grow in the presence of the GS inhibitor methionine sulfoximine. © Rapid Science Ltd. 1998     

Glutamine synthetase and glutamate dehydrogenase in cadmium-stressed triticale seedlings

The studies were performed on young triticale seedlings grown on a mineral medium containing 5 mM NO ₃ ⁻ as the nitrogen source, with the addition of 0.5 mM CdCl₂. It was determined that cadmium ions accumulated mainly in the plant roots. Decreases in nitrate concentrations both in the roots and shoots of seedlings, as well as decreases in soluble protein contents with simultaneous increases in endopeptidase activity were also observed. Both in roots and shoots significant decreases in glutamic acid were noted. Toxic cadmium ion accumulation in seedlings significantly modified activity of primary nitrogen assimilating enzymes, i.e. glutamine synthetase (GS, EC 6.3.1.2) and glutamate dehydrogenase (GDH, EC 1.4.1.2). There was a significant decrease in GS activity both in roots and in shoots of the stressed plants, in comparison to plants grown without cadmium. In shoots of the control plants and plants subjected to stress two GS isoforms were discovered: cytoplasmatic (GS₁) and chloroplastic (GS₂). Substantial decreases in total glutamine synthetase activity in green parts of seedlings, occurring under stress conditions, result from dramatic decrease in GS₂ activity (by 60 % in relation to the control plants); despite simultaneous increases in the cytoplasmatic isoform (GS₁) activity by approx. 96 %. Cadmium ions accumulating in roots and shoots of seedlings not only increased GDH activity, but also modified its coenzymatic specificity.     

Effect of fructose-1,6-bisphosphate on glutamate uptake and glutamine synthetase activity in hypotix astrocyte cultures

Astrocytes are important in regulating the microencironment of neurons both by catabolic and synthetic pathways. The glutamine synthetase (GS) activity observed in astrocytes affects neurons by removing toxic substances, NH₃ and glutamate; and by providing an important neuronal substrate, glutamine. This glutamate cycle might play a critical role during periods of hypoxia and ischemia, when an increase in extracellular excitatory amino acids is observed. It was previously shown in our laboratory that fructose-1,6-bisphosphate (FBP) protected cortical astrocyte cultures from hypoxic insult and reduced ATP loss following a prolonged (18–30 hrs) hypoxia. In the present study we established the effects of FBP on the level of glutamate uptake and GS activity under normoxic and hypoxic conditions. Under normoxic conditions, [U-¹⁴C]glutamate uptake and glutamine production were independent of FBP treatment; whereas under hypoxic conditions, the initial increase in glutamate uptake and an overall increase in glutamine production in astrocytes were FBP-dependent. Glutamine synthetase activity was dependent on FBP added during the 22 hours of either normoxic- or hypoxic-treatment, hence significant increases in activity were observed due to FBP regardless of the oxygen/ATP levels in situ. These studies suggest that activation of GS by FBP may provide astrocytic protection against hypoxic injury.     

Activation of P2X(7) receptors decreases glutamate uptake and glutamine synthetase activity in RBA-2 astrocytes via distinct mechanisms

Glutamate clearance by astrocytes is critical for controlling excitatory neurotransmission and ATP is an important mediator for neuron-astrocyte interaction. However, the effect of ATP on glutamate clearance has never been examined. Here we report that treatment of RBA-2 cells, a type-2-like astrocyte cell line, with ATP and the P2X(7) receptor selective agonist 3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (BzATP) decreased the Na+-dependent [3H]glutamate uptake within minutes. Mechanistic studies revealed that the decreases were augmented by removal of extracellular Mg2+ or Ca2+, and was restored by P2X7 selective antagonist , periodate-oxidized 2',3'-dialdehyde ATP (oATP), indicating that the decreases were mediated through P2X(7) receptors. Furthermore, stimulation of P2X7 receptors for 2 h inhibited both activity and protein expression of glutamine synthetase (GS), and oATP abolished the inhibition. In addition, removal of extracellular Ca(2+) and inhibition of protein kinase C (PKC) restored the ATP-decreased GS expression but failed to restore the P2X(7)-decreased [3H]glutamate uptake. Therefore, P2X7-mediated intracellular signals play a role in the down-regulation of GS activity/expression. Activation of P2X7 receptors stimulated increases in intracellular Na+ concentration ([Na+](i)) suggesting that the P2X(7)-induced increases in [Na+](i) may affect the local Na+ gradient and decrease the Na+-dependent [3H]glutamate uptake. These findings demonstrate that the P2X7-mediated decreases in glutamate uptake and glutamine synthesis were mediated through distinct mechanisms in these cells.     

Some properties of glutamine synthetase and glutamate synthase from Derxia gummosa

The incorporation of ¹⁵N into washed cells of Derxia gummosa from labelled-(NH₄)₂SO₄ and -KNO₃ respectively was inhibited by both L-methionine-DL-sulphoximine and azaserine. Glutamine synthetase purified to homogeneity from this bacterium had a molecular weight of 708 000 and was composed of 12 similar subunits each of 59 000. The enzyme assayed by γ-glutamyltransferase method had K_m values for L-glutamine and hydroxylamine of 12.5 and 1.2 mM, respectively. Optimal pH values for adenylylated and deadenylylated forms were pH 7.0 and pH 8.0, respectively. The adenylylated enzyme was deadenylylated by treatment with snake venom phosphodiesterase. The inhibitions by both glutamate and ammonia were competitive. The activity was markedly inhibited by L-methionine-DL-sulphoximine, alanine, glycine and serine and to a lesser extent by aspartate, phenylalanine and lysine. Various tri-, di- and mono-phosphate nucleotides, organic acids (pyruvate, oxalate and oxaloacetate) were also inhibitory. Glutamate synthase purified 167-fold had specific requirements for NADH, L-glutamine and 2-ketoglutarate. The K_m values for NADH, glutamine and 2-ketoglutarate were 9.6, 270 and 24 μM respectively. Optimal pH range was 7.2–8.2. The enzyme was inhibited by azaserine, methionine, aspartate, AMP, ADP and ATP.     

Hypoxia induced metabolism dysfunction of rat astrocytes in primary cell cultures

In order to study the astroglial contribution to hypoxic injury on brain tissue metabolism, modifications of glutamine synthetase (GS) lactate dehydrogenase (LDH) enolase and malate dehydrogenase activity produced by reduced oxygen supply have been determined in primary cultures of astrocytes prepared from newborn rat cerebral cortex. Enzymatic activities were measured immediately after the hypoxic treatment (9 h) and during post injury recovery. GS level is significantly decreased in response to low oxygen pressure and increased above control value during the post hypoxic recovery period. The magnitude of GS reduction by hypoxia depends on the age of the cells in culture. Lactate dehydrogenase and enolase levels were significantly enhanced during the two periods considered. No modification of the MDH level was observed. The synthesis of LDH isoenzymes containing mainly M subunits is specifically induced by hypoxia. Our results suggest that astroglial cells may represent a particularly sensitive target toward hypoxia injury in brain tissue. Low oxygen pressure available may modify some fundamental metabolical functions of these cells such as glutamate turnover and lactic acid accumulation.     

Inhibition of pea leaf glutamine synthetase by methionine sulphoximine,phosphinothricin and other glutamate analogues

The kinetics of the inhibition of glutamine synthetase from Pisum sativum leaves by l-methionine sulphoximine and dl-phosphinothricin were determined. Inhibition by both compounds was mixed-competitive, and apparent K_i values of 0.16 mM and 0.073 mM respectively were determined. dl-5-Hydroxylysine, dl-glutamate-4-tetrazole and l-4-methyleneglutamic acid were also strong inhibitors. Analogues of methionine sulphoximine, dl-ethionine sulphoximine and dl-prothionine sulphoximine were poor inhibitors of glutamine synthetase. Other glutamine and glutamate analogues e.g. azaserine, albizziine, asparagine and kainic acid had no inhibitory action.     

Effect of Azotobacter strains on sugar beet callus proliferation and nitrogen metabolism enzymes

The effect of five Azotobacter chroococcum strains and nitrogen content in nutrient media on callus growth of two Beta vulgaris L. cultivars were investigated, as well as the activity of nitrate reductase (NR), glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in inoculated callus tissue. On medium with full nitrogen content (1 N) the inoculation with A. chroococcum strain A2 resulted in the highest calli mass, while strains A8 and A14 maximally increased NR activity. On media with 1/8 N the highest effect on calli growth, GS and GDH activity had the strain A8. The strain A2/1 significantly increased callus proliferation on medium without N. Asymbiotic association between sugar beet calli and Azotobacter depended on genotype/strain interaction and was realised in presence of different nitrogen levels. This revised version was published online in July 2006 with corrections to the Cover Date.     

Light-dependent reduction of pyruvate by pea chloroplasts in the presence of glutamate dehydrogenase and C5-dicarboxylic acids

Illuminated pea chloroplasts supported (glutamine plus α-oxoglutarate (α-OG)) and (NH₃ plus α-OG)-dependent O₂ evolution. The properties of these reactions were consistent with light-coupled glutamate synthase and glutamine synthetase activities. In the presence of a glutamate-oxidizing system (component C) comprised of NAD-specific glutamate dehydrogenase (NAD-GDH), lactate dehydrogenase (LDH), 4 mM pyruvate and 0.2 mM NAD, illuminated chloroplasts supported O₂ evolution in the presence of glutamine. The reaction did not proceed in the absence of any one of the constituents of component C and the properties of O₂ evolution were consistent with light-coupled glutamate synthase activity. In the presence of component C, chloroplasts also catalysed O₂ evolution in the presence of catalytic concentrations of glutamate. Studies of O₂ evolution and metabolism of [¹⁴C]-glutamate in the presence of the inhibitors methionine sulphoximine (MSO) and azaserine suggest that O₂ evolution was dependent on the synthesis of glutamine from the products of glutamate oxidation. This was supported by polarographic studies using α-OG and NH₃ instead of glutamate.The results are consistent with a C₅-dicarboxylic acid shuttlemechanism for the export of reducing equivalents from illuminated chloroplasts (glutamate) and recycling of the oxidation products (α-OG and NH₃).     

Hypo-osmotic swelling modifies glutamate-glutamine cycle in the cerebral cortex and in astrocyte cultures

In our previous work, we found that perfusion of the rat cerebral cortex with hypo-osmotic medium triggers massive release of the excitatory amino acid L-glutamate but decreases extracellular levels of L-glutamine (R. E. Haskew-Layton et al., PLoS ONE, 3: e3543). The release of glutamate was linked to activation of volume-regulated anion channels, whereas mechanism(s) responsible for alterations in extracellular glutamine remained unclear. When mannitol was added to the hypo-osmotic medium to reverse reductions in osmolarity, changes in microdialysate levels of glutamine were prevented, indicating an involvement of cellular swelling. As the main source of brain glutamine is astrocytic synthesis and export, we explored the impact of hypo-osmotic medium on glutamine synthesis and transport in rat primary astrocyte cultures. In astrocytes, a 40% reduction in medium osmolarity moderately stimulated the release of L-[(3) H]glutamine by ～twofold and produced no changes in L-[(3) H]glutamine uptake. In comparison, hypo-osmotic medium stimulated the release of glutamate (traced with D-[(3) H]aspartate) by more than 20-fold. In whole-cell enzymatic assays, we discovered that hypo-osmotic medium caused a 20% inhibition of astrocytic conversion of L-[(3) H]glutamate into L-[(3) H]glutamine by glutamine synthetase. Using an HPLC assay, we further found a 35% reduction in intracellular levels of endogenous glutamine. Overall, our findings suggest that cellular swelling (i) inhibits astrocytic glutamine synthetase activity, and (ii) reduces substrate availability for this enzyme because of the activation of volume-regulated anion channels. These combined effects likely lead to reductions in astrocytic glutamine export in vivo and may partially explain occurrence of hyperexcitability and seizures in human hyponatremia.     

Metabotropic Glutamate Receptors in Glial Cells

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system (CNS) and exerts its actions via a number of ionotropic glutamate receptors/channels and metabotropic glutamate (mGlu) receptors. In addition to being expressed in neurons, glutamate receptors are expressed in different types of glial cells including astrocytes, oligodendrocytes, and microglia. Astrocytes are now recognized as dynamic signaling elements actively integrating neuronal inputs. Synaptic activity can evoke calcium signals in astrocytes, resulting in the release of gliotransmitters, such as glutamate, ATP, and d-serine, which in turn modulate neuronal excitability and synaptic transmission. In addition, astrocytes, and microglia may play an important role in pathology such as brain trauma and neurodegeneration, limiting or amplifying the pathologic process leading to neuronal death. The present review will focus on recent advances on the role of mGlu receptors expressed in glial cells under physiologic and pathologic conditions. Special issue article in honor of Dr. Anna Maria Giuffrida-Stella.     

Pathway of ammonia assimilation in illuminated and darkened Chlamydomonas reinhardii

Evidence is presented which shows that NH₃ assimilation in Chlamydomonas occurs exclusively via the glutamate synthase cycle in illuminated and darkened cells and those in which the internal level of NH₃ is elevated. This result indicates that glutamate dehydrogenase probably plays a catabolic rather than anabolic role in the N nutrition of the alga. Glutamine synthetase and glutamate dehydrogenase were characterized and their kinetic properties shown to be consistent with these proposals. It is suggested that reversible activity modulations of glutamine synthetase regulate the operation of the glutamate synthase cycle in the light but the availability of reductant and ATP limits its activity in darkened cells. The possible involvement of the two glutamate synthase enzymes in both light and dark assimilation is discussed.