Glutamine Synthetase Protein Levels Research Articles

Ursolic acid inhibits the synaptic release of glutamate and prevents glutamate excitotoxicity in rats

The present study evaluated the effect of ursolic acid, a natural pentacyclic triterpenoid, on glutamate release in rat cortical nerve terminals (synaptosomes) and its neuroprotection in a kainic acid-induced excitotoxicity rat model. In cortical synaptosomes, ursolic acid produced a concentration-dependent inhibition of evoked glutamate release with a half-maximum inhibition of release value of 9.5 μM, and calcium-free medium and the P/Q -type Ca2+ channel blocker, ω-agatoxin IVA, but not ω-conotoxin GVIA, an N-type Ca2+ channel blocker, prevented the ursoloic acid effect. The molecular docking study indicated that ursolic acid interacted with P/Q-type Ca2+ channels. Ursolic acid also significantly decreased the depolarization-induced activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and the subsequent phosphorylation of synapsin I, and the ursolic acid effect on evoked glutamate release was inhibited by the CaMKII inhibitor KN 62 in synaptosomes. In addition, in rats that were intraperitoneally injected with ursolic acid 30 min before kainic acid intraperitoneal injection, cortical neuronal degeneration was attenuated. This effect of ursolic acid in the improvement of kainic acid-induced neuronal damage was associated with the reduction of kainic acid-induced glutamate increase in the cortex of rats; this was characterized by the reduction of glutamate and glutaminase levels and elevation of glutamate dehydrogenase, glutamate transporter 1, glutamate-aspartate transporter, and glutamine synthetase protein levels. These results suggest that ursolic acid inhibits glutamate release from cortical synaptosomes by decreasing P/Q-type Ca2+ channel activity and subsequently suppressing CaMKII and exerts a preventive effect against glutamate neurotoxicity by controlling glutamate levels.

Exercise increases striatal Glu reuptake and improves motor dysfunction in 6-OHDA-induced Parkinson’s disease rats

The purpose of this study is to clarify that exercise may improve the motor dysfunction of Parkinson's disease (PD) model rats by increasing the reuptake of glutamate (Glu) in the striatum. The neurotoxin 6-hydroxydopamine (6-OHDA) was injected into the medial forebrain bundle (MFB) of the rats' right brain to establish PD model rats with unilateral injury, and the sham operation group was given the same dose of normal saline at the same site as the control group. The reliability of the model was evaluated by apomorphine (APO)-induced rotation test combined with tyrosine hydroxylase (TH) immunohistochemical staining in the substantia nigra and striatum. The exercise group started treadmill training intervention (11m/min, 30min/day, 5d/week, and 4weeks in total) 1 week after the operation. The balance bar test, suspension test, and the tail-lifting handstand test were used to evaluate exercise performance of rats; RT-PCR and western blotting were used to detect protein and mRNA expression of glutamate transporter-1 (GLT-1) and glutamine synthetase (GS) in the striatum; and isotope labeling was used to detect the ability of Glu reuptake in the striatum. (1) Compared with PD group, the number of TH immunoreactive cells in the substantia nigra and the content of TH immunoreactive fibers in the striatum did not change significantly in PD + Ex group. (2) Compared with PD group, the latency and total time of crossing the balance beam were significantly shorter (P < 0.01), the retention time of two forepaws on the metal wire was significantly longer (P < 0.01), the maximum lifting of head and trunk was significantly increased (P < 0.01) in PD + Ex group. (3) Compared with PD group, the ability of Glu reuptake in the striatum was significantly increased (P < 0.05), the expression levels of GLT-1 and GS mRNA in the striatum were significantly increased (P < 0.05), the protein expression of GLT-1 and GS in the striatum was significantly upregulated (P < 0.05) in PD + Ex group. Exercise intervention can significantly improve the motor dysfunction of PD model rats, increase the ability of striatal Glu reuptake significantly, and upregulate the expression levels of GLT-1 and GS protein and GS mRNA significantly. Exercise intervention may increase the protein expression level of GLT-1 and increase the reuptake ability of Glu in the striatum, thereby reducing the excitotoxic effect of excessive Glu on the postsynaptic membrane, and ultimately alleviate the motor dysfunction in PD model rats.

Neurotoxic potential of reactive astrocytes in canine distemper demyelinating leukoencephalitis

Canine distemper virus (CDV) causes a fatal demyelinating leukoencephalitis in young dogs resembling human multiple sclerosis. Astrocytes are the main cellular target of CDV and undergo reactive changes already in pre-demyelinating brain lesions. Based on their broad range of beneficial and detrimental effects in the injured brain reactive astrogliosis is in need of intensive investigation. The aim of the study was to characterize astrocyte plasticity during the course of CDV-induced demyelinating leukoencephalitis by the aid of immunohistochemistry, immunofluorescence and gene expression analysis. Immunohistochemistry revealed the presence of reactive glial fibrillary acidic protein (GFAP)+ astrocytes with increased survivin and reduced aquaporin 4, and glutamine synthetase protein levels, indicating disturbed blood brain barrier function, glutamate homeostasis and astrocyte maladaptation, respectively. Gene expression analysis revealed 81 differentially expressed astrocyte-related genes with a dominance of genes associated with neurotoxic A1-polarized astrocytes. Accordingly, acyl-coA synthetase long-chain family member 5+/GFAP+, and serglycin+/GFAP+ cells, characteristic of A1-astrocytes, were found in demyelinating lesions by immunofluorescence. In addition, gene expression revealed a dysregulation of astrocytic function including disturbed glutamate homeostasis and altered immune function. Observed findings indicate an astrocyte polarization towards a neurotoxic phenotype likely contributing to lesion initiation and progression in canine distemper leukoencephalitis.

Persistent increase in ventral hippocampal long‐term potentiation by juvenile stress: A role for astrocytic glutamine synthetase

A traumatic childhood is among the most important risk factors for developing stress-related psychopathologies such as posttraumatic stress disorder or depression later in life. However, despite the proven role of astrocytes in regulating transmitter release and synaptic plasticity, the contribution of astrocytic transmitter metabolism to such stress-induced psychopathologies is currently not understood. In rodents, childhood adversity can be modeled by juvenile stress exposure, resulting in increased anxiety, and impaired coping with stress in adulthood. We describe that such juvenile stress in rats, regardless of additional stress in adulthood, leads to reduced synaptic efficacy in the ventral CA1 (vCA1) Schaffer collaterals, but increased long-term potentiation (LTP) of synaptic transmission after high-frequency stimulation. We tested whether the glutamate-glutamine-cycle guides the lasting changes on plasticity observed after juvenile stress by blocking the astrocytic glutamate-degrading enzyme, glutamine synthetase (GS). Indeed, the pharmacological inhibition of GS by methionine sulfoximine in slices from naïve rats mimics the effect of juvenile stress on vCA1-LTP, while supplying glutamine is sufficient to normalize the LTP. Assessing steady-state mRNA levels in the vCA1 stratum radiatum reveals distinct shifts in the expression of GS, astrocytic glutamate, and glutamine transporters after stress in juvenility, adulthood, or combined juvenile/adult stress. While GS mRNA expression levels are lastingly reduced after juvenile stress, GS protein levels are maintained stable. Together our results suggest a critical role for astrocytes and the glutamate-glutamine cycle in mediating long-term effects of juvenile stress on plasticity in the vCA1, a region associated with anxiety and emotional memory processing.

Comparative Studies of Glutamine Synthetase Levels in the Brains of Patients with Schizophrenia and Mentally Healthy People

In this study the authors compared the levels of glutamine synthetase (GS) enzymatic activities, as well as the levels of immunoreactive GS and GS-like protein (GSLP) in the readily soluble protein fraction of brain samples taken during autopsies of patients with schizophrenia and subjects from a control group. Enzymatic GS activity was measured and levels of immunoreactive GS and GSLP were determined in postmortem samples of the prefrontal, anterior, and posterior cingulate cortex (Brodmann areas 10, 24, and 23, respectively) and cerebellum cortex obtained from patients with schizophrenia (n = 8) and a control group (n = 9), matched according to age and postmortem interval. GS activity was evaluated in vitro in the “transferase” reaction by the formation of γ-glutamyl hydroxamate, and the levels of immunoreactive GS and GSLP were evaluated by ECL Western blotting using monoclonal and polyclonal antibodies. No differences in GS enzymatic activity were found in all of the studied brain structures. The level of immunoreactive GS in the prefrontal cortex was significantly decreased in patients with schizophrenia compared with the controls (at p < 0.001 according to the Mann–Whitney U-test); it was unchanged in the posterior cingulate cortex and significantly elevated in the anterior cingulate cortex and cerebellum (p < 0.001 and p < 0.004, respectively), whereas the level of immunoreactive GSLP was elevated in all studied brain areas (p < 0.004, p < 0.02, p < 0.04, and p < 0.006, respectively). The alteration of GS and GSLP levels in brains of patients with schizophrenia is one of the factors that disturb glutamate metabolism in these brain structures and is an important aspect of schizophrenia pathogenesis.

Glutamine Triggers Acetylation-Dependent Degradation of Glutamine Synthetase via the Thalidomide Receptor Cereblon

Cereblon (CRBN), a substrate receptor for the cullin-RING ubiquitin ligase 4 (CRL4) complex, is a direct protein target for thalidomide teratogenicity and antitumor activity of immunomodulatory drugs (IMiDs). Here we report that glutamine synthetase (GS) is an endogenous substrate of CRL4(CRBN). Upon exposing cells to high glutamine concentration, GS is acetylated at lysines 11 and 14, yielding a degron that is necessary and sufficient for binding and ubiquitylation by CRL4(CRBN) and degradation by the proteasome. Binding of acetylated degron peptides to CRBN depends on an intact thalidomide-binding pocket but is not competitive with IMiDs. These findings reveal a feedback loop involving CRL4(CRBN) that adjusts GS protein levels in response to glutamine and uncover a new function for lysine acetylation.

Temporospatial expression and cellular localization of glutamine synthetase following traumatic spinal cord injury in adult rats

Glutamine synthetase (GS) is an enzyme involved in an endogenous mechanism of protection against glutamate neurotoxicity and is important in the regulation of astrocyte migration. To date, limited information is available concerning the expression of GS in normal spinal cords and following injury. In the present study, GS expression was identified in astrocytes, oligodendrocytes and microglia in normal rat spinal cords. Following traumatic spinal cord injury (SCI), the glutamate concentration increased rapidly at 1h and returned to baseline rapidly. However, the GS activity and protein levels were found to decrease at 4h and then increase gradually from day3 following SCI. The quantification of astrocytes, oligodendrocytes and activated microglia/macrophages, as well as immunohistochemistry staining of day7 post‑injured spinal cords, indicated that the astrocytes and microglia/macrophages contributed to the increase in GS. Collectively, the results provided evidence for the temporospatial expression and location of GS following SCI and suggested that the changes in GS levels may contribute to glutamate neurotoxicity and glial cell response following SCI.

Abstract 5245: Adipocyte production of glutamine protects leukemia cells from L-asparaginase

Abstract Obesity is associated with increased cancer incidence and mortality. Adipose tissue has been demonstrated to play a role in several types of cancer, including acute lymphoblastic leukemia (ALL). We have shown that obesity impairs the survival of mice transplanted with ALL and treated with the front-line chemotherapy, L-Asparaginase (ASNase), which acts through the deamination of asparagine (ASN) and glutamine (GLN). We have also shown that adipocytes protect ALL cells from ASNase in vitro. We hypothesized that adipocytes protect ALL cells from ASNase via production of GLN. Fat pads were excised from control and diet-induced obese mice. Glutamine synthetase, the rate-limiting enzyme for GLN synthesis, was analyzed by western blot. Amino acids secreted by fat pads were measured using HPLC. ALL cell sensitivity to ASN and/or GLN depletion, and ASNase, was determined by trypan blue exclusion in culture alone and in transwells over 3T3-L1 adipocytes. Mouse fat pads expressed high levels of glutamine synthetase protein, and this was not affected by obesity or 5 days of L-asparaginase treatment (3,000 IU/kg/day). Fat pad explants secreted GLN (251±50 nmoles/100 mg tissue), but not ASN, over 72 hours. Confirming that GLN secretion could protect ALL from ASNase, twice daily addition of 500 nmoles of GLN to murine ALL cells completely reversed ASNase cytotoxicity (64.0x10^4 vs. 7.9x10^4 viable cells after 72 hours of 1 IU/mL ASNase, p&amp;lt;0.01). Testing a panel of human ALL cell lines, we found that proliferation of 3 of 9 cell lines were decreased when ASN was removed from the media (RS4;11, SupB15, Molt4), while 8 of 9 cell lines were decreased when GLN was removed from the media (RS4;11, SupB15, Molt4, SEM, RCH-ACV, BV-173, SD-1, HL60). To test whether blocking adipocyte production of GLN would reverse their ability to protect ALL cells, we pretreated 3T3-L1 adipocytes with methionine sulfoximine (MSO), an irreversible inhibitor of glutamine synthetase. MSO pretreatment blocked GLN secretion by adipocytes and completely reversed adipocyte protection of ALL cells (with no adipocytes: 7.5x10^4; with untreated adipocytes: 14.8x10^4; with MSO treated adipocytes: 6.2x10^4, p=0.03 vs. untreated adipocytes). Finally, we tested whether an alternative preparation of ASNase with a higher glutaminase activity, ERWinase, could overcome adipocyte protection of three GLN dependent cell lines: SD-1, HL60, and Molt-4. Compared to E. coli ASNase, ERWinase treatment resulted in significantly decreased survival of all 3 cell lines in the presence of adipocytes (SD-1 4.2x10^4 vs. 6.7x10^4, p&amp;lt;0.05; HL60 4.1x10^4 vs. 8.8x10^4, p&amp;lt;0.05; Molt-4 16.9x10^4 vs. 26.4x10^4, p&amp;lt;0.05). These studies uncover a novel mechanism of adipocyte protection of leukemia cells from chemotherapy: adipocytes protect ALL cells from ASNase treatment via the production of GLN. Adipocyte production of GLN is both necessary and sufficient to protect ALL cells from ASNase treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5245. doi:1538-7445.AM2012-5245

Glutamine Acts as a Neuroprotectant against DNA Damage, Beta-Amyloid and H2O2-Induced Stress

Glutamine is the most abundant free amino acid in the human blood stream and is ‘conditionally essential’ to cells. Its intracellular levels are regulated both by the uptake of extracellular glutamine via specific transport systems and by its intracellular synthesis by glutamine synthetase (GS). Adding to the regulatory complexity, when extracellular glutamine is reduced GS protein levels rise. Unfortunately, this excess GS can be maladaptive. GS overexpression is neurotoxic especially if the cells are in a low-glutamine medium. Similarly, in low glutamine, the levels of multiple stress response proteins are reduced rendering cells hypersensitive to H2O2, zinc salts and DNA damage. These altered responses may have particular relevance to neurodegenerative diseases of aging. GS activity and glutamine levels are lower in the Alzheimer's disease (AD) brain, and a fraction of AD hippocampal neurons have dramatically increased GS levels compared with control subjects. We validated the importance of these observations by showing that raising glutamine levels in the medium protects cultured neuronal cells against the amyloid peptide, Aβ. Further, a 10-day course of dietary glutamine supplementation reduced inflammation-induced neuronal cell cycle activation, tau phosphorylation and ATM-activation in two different mouse models of familial AD while raising the levels of two synaptic proteins, VAMP2 and synaptophysin. Together, our observations suggest that healthy neuronal cells require both intracellular and extracellular glutamine, and that the neuroprotective effects of glutamine supplementation may prove beneficial in the treatment of AD.

ZNRF1 regulates glutamine synthetase protein levels in Schwann cells by the ubiquitin-proteasome pathway

ZNRF1 regulates glutamine synthetase protein levels in Schwann cells by the ubiquitin-proteasome pathway

Cellular concentrations of glutamine synthetase in murine organs

Glutamine synthetase (GS) is the only enzyme that can synthesize glutamine, but it also functions to detoxify glutamate and ammonia. Organs with high cellular concentrations of GS appear to function primarily to remove glutamate or ammonia, whereas those with a low cellular concentration appear to primarily produce glutamine. To validate this apparent dichotomy and to clarify its regulation, we determined the GS concentrations in 18 organs of the mouse. There was a >100-fold difference in GS mRNA, protein, and enzyme-activity levels among organs, whereas there was only a 20-fold difference in the GS protein:mRNA ratio, suggesting extensive transcriptional and posttranscriptional regulation. In contrast, only small differences in the GS enzyme activity : protein ratio were found, indicating that posttranslational regulation is of minor importance. The cellular concentration of GS was determined by relating the relative differences in cellular GS concentration, detected using image analysis of immunohistochemically stained tissue sections, to the biochemical data. There was a >1000-fold difference in cellular concentrations of GS between GS-positive cells in different organs, and cellular concentrations were up to 20x higher in subpopulations of cells within organs than in whole organs. GS activity was highest in pericentral hepatocytes (approximately 485 micromol.g(-1).min-(1), followed in descending order by epithelial cells in the epididymal head, Leydig cells in the testicular interstitium, epithelial cells of the uterine tube, acid-producing parietal cells in the stomach, epithelial cells of the S3 segment of the proximal convoluted tubule of the kidney, astrocytes of the central nervous tissue, and adipose tissue. GS activity in muscle amounted to only 0.4 micromol.g(-1).min(-1). Our findings confirmed the postulated dichotomy between cellular concentration and GS function.

Alterations of Oxidative Stress Biomarkers Due to In Utero and Neonatal Exposures of Airborne Manganese

Neonatal rats were exposed to airborne manganese sulfate (MnSO4) (0, 0.05, 0.5, or 1.0 mg Mn/m3) during gestation (d 0-19) and postnatal days (PNDs) 1-18. On PND 19, rats were killed, and we assessed biochemical end points indicative of oxidative stress in five brain regions: cerebellum, hippocampus, hypothalamus, olfactory bulb, and striatum. Glutamine synthetase (GS) and tyrosine hydroxylase (TH) protein levels, metallothionein (MT), TH and GS mRNA levels, and reduced and oxidized glutathione (GSH and GSSG, respectively) levels were determined for all five regions. Mn exposure (all three doses) significantly (p = 0.0021) decreased GS protein levels in the cerebellum, and GS mRNA levels were significantly (p = 0.0008) decreased in the striatum. Both the median and high dose of Mn significantly (p = 0.0114) decreased MT mRNA in the striatum. Mn exposure had no effect on TH protein levels, but it significantly lowered TH mRNA levels in the olfactory bulb (p = 0.0402) and in the striatum (p = 0.0493). Mn exposure significantly lowered GSH levels at the median dose in the olfactory bulb (p = 0.0032) and at the median and high dose in the striatum (p = 0.0346). Significantly elevated (p = 0.0247) GSSG, which can be indicative of oxidative stress, was observed in the cerebellum of pups exposed to the high dose of Mn. These data reveal that alterations of oxidative stress biomarkers resulting from in utero and neonatal exposures of airborne Mn exist. Coupled with our previous study in which similarly exposed rats were allowed to recover from Mn exposure for 3 wk, it appears that many of these changes are reversible. It is important to note that the doses of Mn utilized represent levels that are a hundred- to a thousand-fold higher than the inhalation reference concentration set by the United States Environmental Protection Agency.

Persistent Alterations in Biomarkers of Oxidative Stress Resulting from Combined In Utero and Neonatal Manganese Inhalation

Neonatal female and male rats were exposed to airborne manganese sulfate (MnSO4) during gestation and postnatal d 1-18. Three weeks postexposure, rats were killed and we assessed biochemical end points indicative of oxidative stress in five brain regions: cerebellum, hippocampus, hypothalamus, olfactory bulb, and striatum. Glutamine synthetase (GS) protein levels, metallothionein (MT) and GS mRNA levels, and total glutathione (GSH) levels were determined for all five regions. Overall, there was a statistically significant effect of manganese exposure on decreasing brain GS protein levels (p=0.0061), although only the highest dose of manganese (1 mg Mn/m3) caused a significant increase in GS messenger RNA (mRNA) in both the hypothalamus and olfactory bulb of male rats and a significant decrease in GS mRNA in the striatum of female rats. This highest dose of manganese had no effect on MT mRNA in either males or females; however, the lowest dose (0.05 mg Mn/m3) decreased MT mRNA in the hippocampus, hypothalamus, and striatum in males. The median dose (0.5 mg Mn/m3) led to decreased MT mRNA in the hippocampus and hypothalamus of the males and olfactory bulb of the females. Overall, manganese exposure did not affect total GSH levels, a finding that is contrary to those in our previous studies. Only the cerebellum of manganese-exposed young male rats showed a significant reduction (p<0.05) in total GSH levels compared to control levels. These data reveal that alterations in biomarkers of oxidative stress resulting from in utero and neonatal exposures of airborne manganese remain despite 3 wk of recovery; however, it is important to note that the doses of manganese utilized represent levels that are 100-fold to a 1000-fold higher than the inhalation reference concentration set by the US Environmental Protection Agency.

Effects of Manganese (Mn) on the Developing Rat Brain: Oxidative-Stress Related Endpoints

We evaluated biochemical endpoints related to oxidative stress in brains of neonatal rats exposed to manganese (Mn). Oral Mn chloride (MnCl 2) (0, 25, or 50 mg Mn chloride kg −1 body weight per day) was given daily to neonatal rats throughout lactation (i.e. from postnatal day (PND) 1 to 21). As previously reported by [J. Appl. Toxicol. 20 (2000) 179], this treatment paradigm results in increased cerebral cortex (CTX) Mn concentrations in PND 21 rats from both Mn treatment groups. High dose Mn exposure also results in increased cerebellar Mn concentrations. This preliminary study determined whether this exposure paradigm also affects cerebrocortical or cerebellar metallothionein (MT) mRNA levels, glutamine synthetase (GS) activity, GS protein levels, as well as total glutathione (GSH) levels. High dose Mn exposure significantly increased ( P<0.05) total cerebrocortical GSH without accompanying changes in any of the other measured parameters. Therefore, it is unlikely that high dose Mn exposure is associated with oxidative stress in this experimental paradigm.

Mercuric chloride, but not methylmercury, inhibits glutamine synthetase activity in primary cultures of cortical astrocytes

Methylmercury (MeHg) is highly neurotoxic with an apparent dose-related latency period between time of exposure and the appearance of symptoms. Astrocytes are known targets for MeHg toxicity and a site of mercury localization within the central nervous system (CNS). Glutamine synthetase (GS) is an enzyme localized predominately within astrocytes. GS converts two potentially toxic molecules, glutamate and ammonia, to the relatively non-toxic amino acid, glutamine. During prolonged exposure to MeHg, inorganic mercury (I-Hg) accumulates within the brain, suggesting in situ demethylation of MeHg to I-Hg. To determine if speciation of mercurials would differentially alter GS activity and expression, neonatal rat primary astrocyte cultures were exposed to MeHg or mercuric chloride (HgCl 2) for 1 or 6 h. MeHg produced no changes in GS activity, protein, or mRNA at any time or dose tested. In contrast, HgCl 2 produced a dose dependent decrease in astrocytic GS activity at both 1 and 6 h. There were no changes in GS protein or mRNA levels following HgCl 2 exposure. Additional studies were carried out to determine GS activity in cell lysates incubated with HgCl 2 or MeHg. In cell lysates, HgCl 2 was three-times more potent than MeHg in inhibiting GS activity. The inhibition of GS activity in cell lysates by HgCl 2 was reversed by the addition of dithiothreitol (DTT), while DTT did not restore GS activity following MeHg. These data suggest that astrocytic GS activity is not inhibited by physiologically relevant concentrations of MeHg, but is inhibited by I-Hg, which is present in CNS following chronic MeHg exposure.

Glutamine synthetase expression in muscle is regulated by transcriptional and posttranscriptional mechanisms.

Skeletal muscle exports glutamine (Gln) and increases the expression of the enzyme glutamine synthetase (GS) in response to physiological stress. Acute stress or direct glucocorticoid administration raises muscle GS mRNA levels dramatically without a parallel increase in GS protein levels. In the lung, this discrepancy is caused by feedback destabilization of the GS protein by its product Gln. It was hypothesized that muscle GS protein levels increase during stress only when the intracellular Gln pool has been depleted. Adult male rats were injected with the glucocorticoid hormone dexamethasone (DEX) to mimic the acute stress response and with the GS inhibitor methionine sulfoximine (MSO) to deplete muscle Gln stores. DEX increased GS mRNA levels by 2.8-fold but increased GS protein levels by an average of only 40%. MSO diminished muscle GLN levels by 68% and caused GS protein levels to rise in accordance with GS mRNA. Chronic stress was mimicked using 6 days of MSO treatment, which produced anorexia, 23% loss of body weight, and 64% decrease in muscle Gln levels, as well as pronounced increases in both muscle GS mRNA (26-fold) and protein levels (35-fold) without elevation of plasma glucocorticoid levels. Calorie-restricted pair-fed animals exhibited lesser increases in muscle GS mRNA (8-fold) and protein levels (5-fold) without a decline in muscle Gln content. Thus regulation of GS expression in both acute and chronic stress involved both transcriptional and posttranscriptional mechanisms, perhaps affected by muscle Gln content.

Regulation of Glutamine Synthetase from the White Button MushroomAgaricus bisporus

The regulation of glutamine synthetase (GS) fromAgaricus bisporuswas studied at the posttranscriptional level using a specific antibody fraction directed against purified GS. The cross-reactivity of the antiserum against variousAgaricusspecies and other fungi was tested and low reactivity with the Ascomycetes was found. GS protein and activity levels were measured in cell-free extracts of mycelium grown on different N sources. In mycelium grown on glutamine or ammonium as N source, the biosynthetic GS activity is higher than the transferase activity. Moreover, the results show a correlation between GS biosynthetic activity, GS protein, and previously reported mRNA levels. Also, after addition of ammonium or glutamine to glutamate-utilizing cultures, transferase activity decreased more rapidly than biosynthetic activity and GS protein level. This suggests a conformational modification which only affects transferase activity.

Glutamine synthetase gene expression in the lungs of endotoxin-treated and adrenalectomized rats.

During sepsis, the lung responds by exporting increased amounts of the amino acid glutamine. This response is accompanied by increased enzymatic activity of glutamine synthetase (GS), which catalyzes the synthesis of glutamine from glutamate and ammonia. It is also known that GS expression in the rat lung can be induced by glucocorticoid hormones. To determine whether the septic response and the response to glucocorticoids are related, we have characterized the induction of GS expression during lipopolysaccharide (LPS)-induced endotoxemia in normal, neutropenic, and adrenalectomized rats. Normal rats exhibited a time- and dose-dependent induction of GS mRNA levels after a single intraperitoneal dose of LPS. Responses to LPS were maximal at doses of 0.1 mg/kg body wt and above. A single 10 mg/kg body wt dose of LPS led to a rapid, transient sevenfold increase in GS mRNA (P < or = 0.1) and a twofold increase in GS protein level 8 h postinjection. Induction of lung GS mRNA 4 h after LPS injection was approximately fivefold in neutropenic (P < or = 0.1) and fourfold in nonneutropenic control rats (P < or = 0.1), suggesting that infiltrating neutrophils or neutrophil-derived factors are not required for GS induction. In response to high-dose, short-term endotoxemia, adrenalectomized rat lung GS mRNA increased twofold (P < or = 0.02) compared with sixfold in sham-operated control rats (P < or = 0.02). However, in response to low-dose, long-term endotoxemia, adrenalectomized rat lung GS mRNA increased threefold (P < or = 0.02) compared with fourfold in sham-operated control rats (P < or = 0.02). Adrenalectomy did not affect the elevation of lung GS mRNA levels in response to dexamethasone. In addition, GS mRNA was induced four- and sixfold in rat microvascular pulmonary endothelial cells exposed to plasma from control and septic rats, respectively. The addition of a glucocorticoid antagonist, RU-38486, completely blocked GS gene induction in the presence of control plasma but only attenuated the response to plasma from septic animals by 30%. These results suggest that GS gene induction during sepsis is only partially mediated by adrenal-derived glucocorticoid hormones.

Regulation of glutamine synthetase in human breast carcinoma cells and experimental tumors

Background. Acute deprivation of extracellular glutamine causes up-regulation of glutamine synthetase (GS) expression by a mechanism involving an increase in GS protein stability. This study examines GS expression in a highly glutamine-dependent and tumorigenic human breast cancer cell line, TSE cells, in response to acute and chronic glutamine deprivation in culture and during tumor formation. Methods. TSE cells were subjected to acute glutamine deprivation, adapted to growth in low glutamine concentrations, and subcutaneously injected into nude mice. GS protein and mRNA levels were assayed by Western and Northern blotting, and intracellular glutamine levels were evaluated by using a colorimetric assay. Results. GS protein levels increased, but GS mRNA levels were unchanged in response to acute glutamine deprivation. Chronic glutamine deprivation in vitro and tumor growth in vivo caused an increase in both GS protein and mRNA levels. Large tumors exhibited lower intracellular glutamine, higher GS protein, and relatively unchanged GS mRNA levels relative to small tumors. Conclusions. TSE tumors exhibit up-regulation of GS protein and mRNA levels and declines in intracellular glutamine content, suggesting that growth in vivo causes a chronic and progressive glutamine deprivation. Up-regulation of GS expression may contribute to adaptation to a nutrient-poor intratumor environment.

Differentiation of serum‐free mouse embryo cells into astrocytes is accompanied by induction of glutamine synthetase activity

Serum-free mouse embryo (SFME) cells derived in a defined serum-free medium have been cultured for more than 200 generations and display properties of neural progenitor cells. SFME cells express the neuroepithelial stem cell marker nestin in defined serum-free medium. Exposure of SFME cells to transforming growth factor beta (TGF-beta) or serum decreases nestin expression and induces the astrocyte marker glial fibrillary acidic protein, suggesting that SFME cells differentiate into astrocytes upon exposure to TGF-beta or serum. We examined the expression by SFME cells of the functional central nervous system (CNS) astrocyte marker glutamine synthetase (GS). GS activity is induced in SFME cells upon exposure to TFG-beta or serum. The induction of GS activity was dose- and time-dependent and was reversible. Retinoic acid, hydrocortisone, and dibutyryl cyclic AMP also induced GS expression. The induction of GS activity was accompanied by an increase in the level of GS mRNA and protein. This work provides further evidence that SFME cells represent neural progenitor cells which differentiate into functional astrocytes upon exposure to TGF-beta or serum.