Glutamine Synthetase Protein Levels Research Articles

Molecular regulation of lung endothelial glutamine synthetase expression.

The lungs play a crucial role in maintaining amino acid homeostasis by exporting glutamine. Lung glutamine release is increased markedly in patients with sepsis, and in rat models injection of endotoxin causes up-regulation of glutamine synthetase (GS), the principal enzyme of glutamine synthesis. To investigate the molecular regulation of this response in the lung microvasculature we studied the effects of several hormones and cytokines that mediate the septic response on the expression of GS in rat microvascular pulmonary endothelial cells (MPECs). MPECs were grown to confluence and incubated with the synthetic glucocorticoid dexamethasone, prostaglandins, cytokines, or activated complement C5a. Cellular lysates were prepared and total cellular RNA was extracted, hybridized with a GS complementary DNA derived probe, and normalized to reduced glyceraldehyde-phosphate dehydrogenase. GS protein content was determined by Western blotting with a GS antibody. Of the compounds tested, only dexamethasone caused a marked increase (tenfold or greater) of GS messenger RNA and protein levels in MPECs. Dexamethasone-induced accumulation of GS messenger RNA was rapid, dose-dependent, and maximal after 4 hours of exposure. GS protein levels were maximal after 8 hours and remained elevated for at least 48 hours. The dose of dexamethasone sufficient to induce 50% of maximal GS messenger RNA and protein level increase was approximately 10 nmol/L. The dexamethasone-induce increase of GS messenger RNA level was completely blocked by the glucocorticoid receptor antagonist RU38486 and by the transcriptional inhibitor actinomycin D but was not inhibited by the translational inhibitor cycloheximide. Glucocorticoids augment GS expression in rat lung microvascular endothelial cells in a manner consistent with a direct transcriptional response via glucocorticoid receptors. Other septic response mediators had minimal effect on GS expression. Induction of GS expression by adrenocorticoids is likely to contribute to the marked ability of the lungs to augment glutamine production during septic states.

Glutamine synthetase (GS) expression is reduced in senile dementia of the Alzheimer type.

Glutamine synthetase (GS), a metabolic marker of the mature astrocyte, was investigated in the temporal neocortex of postmortem brain samples of 8 cases, either not demented or affected by senile dementia of the Alzheimer type. A negative correlation between the GS protein level and the density of both classical beta A4 deposits and senile plaques was evidenced. Such a correlation for GS underlies a dysfunction of the astroglial metabolism and particularly of the glutamate and ammonia neutralization. Since GS is sensitive to oxidative lesioning, the changes in GS level that were observed, occurring at the posttranslational stage, might reflect oxidative damage and have severe consequences on the pathological cascade of events.

Glucocorticoids Regulate Rat Glutamine Synthetase Expression in a Tissue-Specific Manner

During stress states, organismal glutamine production is augmented secondary to an increase in the activity of glutamine synthetase (GS) in the lung and skeletal muscle. Because glucocorticoids are key regulators of the metabolic response to stress, we undertook a survey of glucocorticoid induction of GS expression in rat organs in response to dexamethasone. Male adult rats were injected with glucocorticoid or vehicle and 4 hr later, 10 organs were assayed for GS messenger RNA and protein contents by Northern and Western blotting. We observed a 20-fold range of GS mRNA levels in organs of control animals. Blotting detected two GS RNA species of approximately 2.8- and 1.4-kb sizes in all tissue except testis, where an additional 2-kb RNA species was observed. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mBNA levels were also assayed and used as a normalization factor. An approximately 10-fold range of GAPDH mRNA levels was observed. Four hours after dexamethasone injection, a nearly a 5-fold increase in glutamine synthetase mRNA levels in lung and muscle, as well as an approximately 2-fold increase in heart were observed. Relative to GAPDH mRNA, a significant decrease in GS mRNA levels was observed in the liver. A wide range of glutamine synthetase protein contents were observed in rat organs. Comparison of Northern and Western blotting results revealed a dichotomy in the ratio of relative GS mRNA and protein level in rat organs, suggesting that tissue-specific posttranscriptional processes determine GS protein levels. Four hours after dexamethasone injection, an apparent increase in GS protein was observed in the lung, muscle, and thymus; however, significant induction of GS protein was demonstrated only in lung. The differential response of GS mRNA and protein levels to dexamethasone suggests that GS expression is induced by glucocorticoids in a tissue-specific manner.

Effects of modafinil-induced wakefulness on glutamine synthetase regulation in the rat brain

Changes in the level of glutamine synthetase (GS), an enzyme chiefly found in glial cells, were investigated in the brains of rats treated with modafinil, an awakening drug interfering with central catecholamine function. Two hours (waking period) and 7 h (recovery period) after intra-peritoneal injection of 128 mg/kg modafinil, a significant increase in the level of GS protein was observed by immunotitration in both the locus coeruleus (+30%) and in the frontoparietal cortex (+50%). No changes were observed with 64 mg/kg of modafinil. Gs mRNA was quantified in the entire cortex by Northern blot hybridization using an oligonucleotidic GS cDNA probe. A significant increase in the GS-mRNA level (+70%) was observed in the CX of rats 2 h after injection of 128 mg/kg modafinil; the level tended to return to control values 7 h later during the recovery period. The level of glial acid fibrillary protein (GFAP), an astroglial marker, was unchanged after modafinil treatment. These changes in GS levels after modafinil treatment are discussed in terms of neuron-glia interactions in the regulation of brain metabolism during pharmacologically induced wakefulness, excluding possible stress effects.

Overexpression of glutamine synthetase in human primary liver cancer

Background/Aims: We have identified several clones specifically expressed during malignant cell proliferation by screening a complementary DNA library constructed from a human primary liver cancer with subtractive probes. One clone was identified as the glutamine synthetase (GS) transcript. Its expression is tightly regulated during development, especially in the hepatic lobule. Because this enzyme is involved in nitrogen homeostasis, it might contribute to tumor development/progression in primary liver cancer. Methods: We compared the expression of GS messenger RNA (mRNA) and protein in tumorous and nontumorous liver from 34 patients with primary liver cancers, using a combination of Northern blot, dot blot, western blot, and determination of GS enzyme activity. Results: GS mRNA was higher in tumors versus nontumors in 23 of 34 primary liver cancer samples. GS activity was higher in 6 of 8 selected primary liver cancer samples with high RNA levels. GS protein levels were proportional to enzyme activity. A major GS transcript of 2.8 kilobase was detected by Northern blotting and sequencing. This comprised the minor 1.8-kb transcript and a long 3′ untranslated region; the latter contained an AT-rich zone, fully conserved in the chicken, mouse, and rat, which might be important for stability. Conclusions: Our results show an overexpression of GS in human primary liver cancers and, thus, point to its potential involvement in hepatocyte transformation.

Glutamine synthetase modulation in the brain of rats subjected to deprivation of paradoxical sleep

Changes in the level of glutamine synthetase (GS), an enzyme mainly located in astrocytes, were investigated in rat brain after deprivation of paradoxical sleep (PSD) and during recovery. An immunotitration method was used to evaluate the relative level of GS in brain tissue. At the end of a 24 h PSD, a significant increase in GS protein was observed both in the frontoparietal cortex (CX) and in the locus coeruleus area (LC). Four hours later during recovery, the level of GS protein returned to normal level in the CX but fell below control levels in the LC. In contrast, in the CX, the level of glial fibrillary acidic protein, an astroglial marker, did not change after PSD or during recovery. GS mRNA was quantified in the entire cortex by northern blot hybridization using of an oligonucleotidic GS-cDNA probe. We observed an increase in the GS mRNA level in the cortex of PSD rats of the same magnitude as the increase in GS protein. Both GS mRNA and GS protein tended to return to control values 4 h later during recovery. These results are discussed with particular attention to stress effects and possible physiological mechanisms regarding the regulation of amino acid levels by neurotransmitters during prolonged waking or neuronal excitation.

Nitrogen Metabolism in the Stalk Tissue of Maize

During ear development in maize (Zea mays L.), nitrogenous compounds are translocated from vegetative organs to the kernels. At anthesis, the stalk contains approximately 40% of the total plant N, and contributes 45% of the N remobilized to the ear. Therefore, the stalk has an important function as a temporary reservoir for N. Little is known of the metabolism of maize stalks, and this paper describes initial studies of enzymes of N metabolism. High in vitro activity of glutamine synthetase (GS) in maize stalk samples throughout ear development contrasted with a peak in activity of glutamate synthase soon after anthesis and negligible nitrate reductase. With fresh sections of stalk tissue collected at anthesis, (15)N-feeding experiments confirmed high GS and low nitrate reductase activities. Two isoforms of GS were separated from extracts from stalk tissue: GS1, the cytoplasmic form, increased to maximum levels at 2 weeks postanthesis and remained fairly high thereafter; whereas the plastidic form, GS2, declined progressively during kernel development. Western blot analysis confirmed the presence of constantly high levels of GS protein after anthesis. The levels of GS proteins decreased after transfer of N-starved, hydroponically grown plants to N-rich conditions in order to restrict remobilization of N. In contrast, transfer of plants grown under abundant N conditions to N-free medium, which encourages N remobilization, resulted in a relative increase in GS protein. Because glutamine is the major form of N transported in maize, the results indicate that GS, specifically the GS1 isoform, has a central role in the remobilization on nitrogenous compounds from the stalk to the ear.

Glutamine synthetase modulation in astrocyte cultures of different mouse brain areas

Astroglial cells from mouse cerebral hemispheres, cerebellum, olfactory bulbs, and medulla oblongata were grown in the presence of either hormones (hydrocortisone, insulin) or cell second messengers (dBcAMP, dBcGMP). Glutamine synthetase (GS) specific activity, GS protein level, and GS translation were investigated under the effect of these factors. Hydrocortisone produced a simultaneous increase in GS translation, GS level, and activity. This increase was observed in the astrocytes cultured from the four brain areas but at a variable magnitude depending on the area. The hydrocortisone effect appeared at the transcriptional level. Inversely, insulin decreased both the GS activity and the in vitro translated GS. This effect was seen only in the olfactory bulbs and the medulla. DBcAMP increased the GS biological activity only in the cerebral hemisphere cultures. It raised, however, the level of translated GS and GS protein in astrocytes from all the areas, suggesting a post-translational effect for intracellular cAMP. DBcGMP only affected GS in the astrocytes from cerebral hemispheres and the medulla modulating either the GS transcription or the messenger RNA stability. These results suggest specific regulation for GS expression, depending on the brain area from which the cells were dissociated or on the astroglial cell population present in these cultures affecting either the transcription, the mRNA stability, or the biological activity of the protein.

Expression of glutamine synthetase in mutant strains of the cyanobacterium Anabaena variabilis which liberate ammonia

Levels of glutamine synthetase (GS) biosynthetic activity, GS protein and GS mRNA were compared in the parent strain of Anabaena variabilis and in ammonia-liberating mutant strains (ED81 and ED92) which are resistant to ethylenediamine (EDA). GS activity, protein and mRNA levels were all reduced in ED92 whereas ED81 had levels of GS protein and mRNA similar to those of the parent strain but had much reduced GS biosynthetic activity. These findings are discussed in relation to the type of mutation occurring within in each mutant strain and in relation to the reduced GS activity in the Anabaena-Azolla symbiotic association.

Expression of glutamine synthetase in mutant strains of the cyanobacteriumAnabaena variabiliswhich liberate ammonia

Levels of glutamine synthetase (GS) biosynthetic activity, GS protein and GS mRNA were compared in the parent strain of Anabaena variabilis and in ammonia-liberating mutant strains (ED81 and ED92) which are resistant to ethylenediamine (EDA). GS activity, protein and mRNA levels were all reduced in ED92 whereas ED81 had levels of GS protein and mRNA similar to those of the parent strain but had much reduced GS biosynthetic activity. These findings are discussed in relation to the type of mutation occurring within in each mutant strain and in relation to the reduced GS activity in the Anabaena-Azolla symbiotic association.

Metabolic changes associated with cyanophage N-1 infection of the cyanobacterium Nostoc muscorum

The development of cyanophage N-1 in the N2-fixing cyanobacterium Nostoc muscorum is dependent on light. The redox state of thioredoxin m was altered in phage infected cells, with the proportion of reduced thioredoxin increasing during the eclipse period. In one step growth experiments, the specific activity of glucose-6-phosphate dehydrogenase increased transiently during the eclipse period, whereas that of glutamine synthetase increased towards the end of the eclipse period (2–4h after infection) then remained high until the end of the latent period (about 7 h after infection). The rate of respiratory O2 uptake was maintained until the end of the latent period. In contrast, the specific activity of phosphoribulokinase and the rate of photosynthetic O2 evolution began to decrease towards the end of the eclipse period and later than the level of extractable protein began to decrease. Nitrogenase activity remained high throughout the eclipse period then decreased rapidly after 5 h. The level of glutamine synthetase protein decreased in parallel with the decrease in total extractable protein, whereas the level of thioredoxin m protein decreased more slowly.

Photoproduction of ammonium by immobilized mutant strains of Anabaena variabilis

Ethylenediamine (EDA) is toxic to the cyanobacterium Anabaena variabilis and inhibits nitrogenase activity. The inhibition of nitrogenase was prevented by pretreatment of cells with l-methionine-d,l-sulphoximine (MSX). Mutant strains of Anabaena variabilis (ED81, ED92), resistant to EDA, had low levels of glutamine synthetase (GS) biosynthetic activity compared with the wild type strain. ED92 had a low level of GS protein whereas ED81 had a similar level to that of the parent strain as estimated using antibodies against GS. Both strains fixed N2 and liberated NH4+ into the media. Following immobilization of the mutant strains, sustained photoproduction of NH4+ was obtained in air-lift reactors at rates of up to 50 μmol NH4+ mg chl a−1 h−1, which were comparable to the rates obtained when immobilized cyanobacteria were treated with MSX.

Glutamine synthetase: activity and localization in cyanobacteria of the cycadsCycas revoluta andZamia skinneri

Nitrogen-fixing cyanobacteria inhabit the zone between the inner and outer cortex of cycad coralloid roots. In the growing tip of such roots the cyanobacterial heterocyst frequency, nitrogenase activity (C2H2-reduction) and glutamine synthetase activity (both transferase and biosynthetic) were comparable to those found in freeliving cyanobacteria. The relative level of glutamine synthetase protein and its pattern of cellular/subcellular localization in heterocysts and vegetative cells were also similar to those of free-living cyanobacteria. However, there was a progressive decline in nitrogenase activity along the coralloid root with maximum reduction occurring in the regions farthest from the growing tip. A similar but less pronounced pattern was observed for glutamine synthetase activity. Distribution of glutamine synthetase protein in cyanobacteria in the first 2-3 mm of the root tip indicated a slight decrease in the heterocysts and vegetative cells. However, the overall level of cyanobacterial glutamine synthetase protein did not change because of a drastic increase in the numbers of heterocysts, which contain a proportionally higher level of glutamine synthetase than the vegetative cells.