Evidence for a cytosolic-dependent light induction of chloroplastic glutamine synthetase during greening of etiolated rice leaves

During the greening of etiolated rice leaves, total glutamine synthetase activity increases about twofold, and after 48 h the level of activity usually observed in green leaves is obtained. A density-labeling experiment with deuterium demonstrates that the increase in enzyme activity is due to a synthesis of the enzyme. The enhanced activity obtained upon greening is the result of two different phenomena: there is a fivefold increase of chloroplastic glutamine synthetase content accompanied by a concommitant decrease (twofold) of the cytosolic glutamine synthetase. The increase of chloroplastic glutamine synthetase (GS₂) is only inhibited by cycloheximide and not by lincomycin. This result indicates a cytosolic synthesis of GS₂. The synthesis of GS₂ was confirmed by a quantification of the protein by an immunochemical method. It was demonstrated that GS₂ protein content in green leaves is fivefold higher than in etiolated leaves.Abbreviations AbH heavy chain of antibodies - AbL light chain of antibodies - AP acid phosphatase - CH cycloheximide - G6PDH glucose-6-phosphate dehydrogenase - GS glutamine synthetase - GS₁ cytosolic glutamine synthetase - GS₂ chloroplastic glutamine synthetase - LC lincomycin - NAD-MDH NAD malate dehydrogenase - NADP-G3PDH NADP glyceraldehyde-3-phosphate dehydrogenase     

Early events in the induction of glutamine synthetase activity by hydrocortisone in embryonic chick neural retina

Summary Primary cultures of 10-day embryonic chick neural retinas were used to investigate early aspects of the mechanism of hydrocortisone action on glutamine synthetase activity. As little as 2 hr of hydrocortisone exposure served to initiate significant increases in the glutamine synthetase activity levels assayed after 24 hr culture. Time course studies indicated that the increase in glutamine synthetase activity observed after 24 hr in culture resulted from a two-phase rise in activity and that cycloheximide was effective in suppressing the second-phase rise. Additional inhibition studies demonstrated that the second-phase increase in enzyme activity required continuous protein synthesis during the initial 6 hr. The evidence suggests a mechanism of hydrocortisone action involving the production of a protein which is important for the induction of glutamine synthetase activity by hydrocortisone. This work was supported by a National Science Foundation (U.S.A.) Training Grant.     

Modulation of Glutamine Synthesis in Cultured Astrocytes by Nitric Oxide

1. Previous results suggest that glutamine synthesis in brain could be modulated by nitrix oxide. The aim of this work was to assess this possibility.2. As glutamine synthetase in brain is located mainly in astrocytes, we used primary cultures of astrocytes to assess the effects of increasing or decreasing nitrix oxide levels on glutamine synthesis in intact astrocytes.3. Nitric oxide levels were decreased by adding nitroarginine, an inhibitor of nitric oxide synthase. To increase nitric oxide we used S-nitroso-N-acetylpenicillamine, a nitric oxide generating agent.4. It is shown that S-nitroso-N-acetylpenicillamine decreases glutamine synthesis in intact astrocytes by 40–50%. Nitroarginine increases glutamine synthesis slightly in intact astrocytes.5. These results indicate that brain glutamine synthesis may be modulated in vivo by nitric oxide.     

Airborne manganese exposure differentially affects end points of oxidative stress in an age-and sex-dependent manner

Juvenile female and male (young) and 16-mo-old male (old) rats inhaled manganese in the form of manganese sulfate (MnSO₄) at 0, 0.01, 0.1, and 0.5 mg Mn/m³ or manganese phosphate at 0.1 mg Mn/m³ in exposures of 6h/d, 5d/wk for 13 wk. 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. Although most brain regions in the three groups of animals were unaffected by manganese exposure in terms of GS protein levels, there was significantly increased protein (p<0.05) in the hippocampus and decreased protein in the hypothalamus of young male rats exposed to manganese phosphate as well as in the aged rats exposed to 0.1 mg/m³ MnSO₄. Conversely, GS protein was elevated in the olfactory bulb of females exposed to the high dose of MnSO₄. Statistically significant decreases (p<0.05) in MT and GS mRNA as a result, of manganese exposure were observed in the cerebellum, olfactory bulb, and hippocampus in the young male rats, in the hypothalamus in the young female rats, and in the hippocampus in the senescent males. Total GSH levels significantly (p<0.05) decreased in the olfactory bulb of manganese exposed young male rats and increased in the olfactory bulb of female rats exposed to manganese. Both the aged and young female rats had significantly decreased (p<0.05) GSH in the striatum resulting from manganese inhalation. The old male rats also had depleted GSH levels in the cerebellum and hypothalamus as a result, of the 0.1-mg/m³ manganese phosphate exposure. These results demonstrate that age and sex are variables that must be considered whenassessing the neurotoxicity of manganese.     

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.     

Effect of manganese on the development of glial cells cultured from prenatally alcohol exposed rats

Maternal alcohol abuse is known to produce retardation in brain maturation and brain functions. Using cultured glial cells as a model system to study these effects of alcohol we found an alcohol antagonizing property for manganese (Mn). Mn was added to the alcohol diet (MnCl₂ 25 mg/l of 20% v/v ethanol) of pregnant rats. Glial cells were cultured during 4 weeks from cortical brain cells of pups born to these mothers. Several biochemical parameters were examined: protein levels, enzymatic markers of glial cell maturation (enolase and glutamine synthetase), superoxide dismutase a scavenger of free radicals produced during alcohol degradation. The results were compared to appropriate controls. A beneficent effect of Mn was observed for the pups weight which was no more significantly different from the control values. Protein levels, enolase and glutamine synthetase activities were increased mainly during the proliferative period when Mn was added to the alcohol diet compared to the only alcohol treated animals. This Mn effect was not found for superoxide dismutase in cultured glial cells but exists in the total brain of the 2 week-old offspring. In the total 2 and 4 week-old brain the alcohol induced decrease of enolase and glutamine synthetase was also antagonized by the Mn suplementation. Our data suggest that Mn may act as a factor overcoming at least partially some aspects of alcohol induced retardation of nerve cell development.     

Glutamine synthetase activity and glutamine content in brain: modulation by NMDA receptors and nitric oxide

Acute intoxication with large doses of ammonia leads to rapid death. The main mechanism for ammonia elimination in brain is its reaction with glutamate to form glutamine. This reaction is catalyzed by glutamine synthetase and consumes ATP. In the course of studies on the molecular mechanism of acute ammonia toxicity, we have found that glutamine synthetase activity and glutamine content in brain are modulated by NMDA receptors and nitric oxide. The main findings can be summarized as follows.Blocking NMDA receptors prevents ammonia-induced depletion of brain ATP and death of rats but not the increase in brain glutamine, indicating that ammonia toxicity is not due to increased activity of glutamine synthetase or formation of glutamine but to excessive activation of NMDA receptors.Blocking NMDA receptors in vivo increases glutamine synthetase activity and glutamine content in brain, indicating that tonic activation of NMDA receptors maintains a tonic inhibition of glutamine synthetase.Blocking NMDA receptors in vivo increases the activity of glutamine synthetase assayed in vitro, indicating that increased activity is due to a covalent modification of the enzyme. Nitric oxide inhibits glutamine synthetase, indicating that the covalent modification that inhibits glutamine synthetase is a nitrosylation or a nitration.Inhibition of nitric oxide synthase increases the activity of glutamine synthetase, indicating that the covalent modification is reversible and it must be an enzyme that denitrosylate or denitrate glutamine synthetase.NMDA mediated activation of nitric oxide synthase is responsible only for part of the tonic inhibition of glutamine synthetase. Other sources of nitric oxide are also contributing to this tonic inhibition.Glutamine synthetase is not working at maximum rate in brain and its activity may be increased pharmacologically by manipulating NMDA receptors or nitric oxide content. This may be useful, for example, to increase ammonia detoxification in brain in hyperammonemic situations.     

Effect of Reducing Brain Glutamine Synthesis on Metabolic Symptoms of Hepatic Encephalopathy

Abstract: Liver failure, or shunting of intestinal blood around the liver, results in hyperammonemia and cerebral dysfunction. Recently it was shown that ammonia caused some of the metabolic signs of hepatic encephalopathy only after it was metabolized by glutamine synthetase in the brain. In the present study, small doses of methionine sulfoximine, an inhibitor of cerebral glutamine synthetase, were given to rats either at the time of portacaval shunting or 3–4 weeks later. The effects on several characteristic cerebral metabolic abnormalities produced by portacaval shunting were measured 1–3 days after injection of the inhibitor. All untreated portacaval-shunted rats had elevated plasma and brain ammonia concentrations, increased brain glutamine and tryptophan content, decreased brain glucose consumption, and increased permeability of the blood–brain barrier to tryptophan. All treated rats had high ammonia concentrations, but the brain glutamine content was normal, indicating inhibition of glutamine synthesis. One day after shunting and methionine sulfoximine administration, glucose consumption, tryptophan transport, and tryptophan brain content remained near control values. In the 3–4-week-shunted rats, which were studied 1–3 days after methionine sulfoximine administration, the effect was less pronounced. Brain glucose consumption and tryptophan content were partially normalized, but tryptophan transport was unaffected. The results agree with our earlier conclusion that glutamine synthesis is an essential step in the development of cerebral metabolic abnormalities in hyperammonemic states.     

Role of glutamine synthetase in citric acid fermentation byAspergillus niger

The activity of glutamine synthetase fromAspergillus niger was significantly lowered under conditions of citric acid fermentation. The intracellular pH of the organism as determined by bromophenol blue dye distribution and fluorescein diacetate uptake methods was relatively constant between 6·0–6·5, when the pH of the external medium was varied between 2·3–7·0.Aspergillus niger glutamine synthetase was rapidly inactivated under acidic pH conditions and Mn²⁺ ions partially protected the enzyme against this inactivation. Mn²⁺-dependent glutamine synthetase activity was higher at acidic pH (6·0) compared to Mg²⁺-supported activity. While the concentration of Mg²⁺ required to optimally activate glutamine synthetase at pH 6·0 was very high (≥ 50 mM), Mn²⁺ was effective at 4 mM. Higher concentrations of Mn²⁺ were inhibitory. The inhibition of both Mn²⁺ and Mg²⁺-dependent reactions by citrate, 2-oxoglutarate and ATP were probably due to their ability to chelate divalent ions rather than as regulatory molecules. This suggestion was supported by the observation that a metal ion chelator, EDTA also produced similar effects. Of the end-products of the pathway, only histidine, carbamyl phosphate, AMP and ADP inhibitedAspergillus niger glutamine synthetase. The inhibitions were more pronounced when Mn²⁺ was the metal ion activator and greater inhibition was observed at lower pH values. These results permit us to postulate that glutamine synthesis may be markedly inhibited when the fungus is grown under conditions suitable for citric acid production and this block may result in delinking carbon and nitrogen metabolism leading to acidogenesis     

In vitro inhibition of brain phosphate-activated glutaminase by ammonia and manganese

IntroductionAs a consequence of the loss of liver function in chronic liver disease, increased levels of ammonia, manganese, and glutamine have been observed in the brain of hepatic encephalopathy patients.ObjectiveIn the present study, we explored phosphate activated glutaminase (PAG) activity in mitochondrial enriched fractions under treatment with ammonia and manganese.MethodsWe dissected out the brain cortex, striatum, and cerebellum of male Wistar rats 250−280 g weight; brain sections were pooled to obtain enriched mitochondrial fractions by differential centrifugation. Aliquots equivalent to 200 μg of protein were incubated with semi-log increasing concentrations of ammonia and/or manganese both as chloride salts (from 0 to 10 000 μM) and glutamine (4 mM) for 30 min. Then, the glutamate produced by the reaction was determined by HPLC coupled with fluorescence detection.Results and discussionBoth manganese and ammonia inhibited PAG in a concentration-dependent manner. Non-linear modeling was used to determine IC50 and IC20 for ammonia (120 μM) and manganese (2 mM). We found that PAG activity under the combination of IC20 of ammonia and manganese was equivalent to the sum of the effects of both substances, being PAG inhibition more pronounced in mitochondrial fractions from cerebellum. The PAG inhibition observed here could potentially explain a pathway for glutamine accumulation, by means of the inhibition of PAG activity as a consequence of increased concentrations of manganese and ammonia in the brain under liver damage conditions.     

Induction of glutamine synthetase by 8-bromo cyclic AMP in primary cultures of rat brain astrocytes

Glutamine synthetase (GS) is the key enzyme in cerebral glutamine production. Understanding the regulation of the expression of GS is important for definition of the control of glutamine metabolism in brain. Therefore, we studied the control of GS expression by 8-bromo cyclic AMP in primary cultures of astrocytes prepared from brains of neonatal rats. GS activity was increased by 8-bromo cyclic AMP in a dose- and time-dependent manner. This increase was associated with a corresponding increase in the steady-state level of GS mRNA.     

Glutamate and glutamine in the brain of the neonatal rat during hypercapnia.

In order to study the influence of hypercapnia on the content of glutamate and glutamine in the developing brain, pregnant rats and their offspring were kept in CO2 rich (6-10%) atmosphere and the litters were killed at different ages between 4 and 28 days. In the hypercapnic rats the content of both amino acids in the brain increases with age with almost the same time course as in normocapnic rats. At any age the glutamate content is lower in the hypercapnic animals than in control rats, whereas the glutamine content, beyond the first 8 days of life is increased. Both effects are rapidly reversible on return to air breathing. Although the glutamate-glutamine system is in full development, the influence of hypercapnia can be compared to that observed in adult rats. Hypercapnia did not change the glutaminase and the glutamine synthetase activity of the brain.     

Establishment of Namalva cell lines which grow continuously in glutamine-free medium

Glutamine has been shown to be a preferred energy source for some established cell lines and cancer cells in culture (Kovacevic, 1971; Kovacevic, 1972; Lavietes, 1974). Empirically, glutamine is the most abundant amino acid in most of the culture media developed. The major end product of glutamine metabolism is ammonia. Ammonia build up is one of the limiting factors in the proliferation of mammalian cells in higher density culture and is directly related to the initial glutamine concentration. The susceptibility of glutamine to thermodecomposition prevents the heat sterilization of glutamine-enriched media and this significantly increases the cost of medium preparation at the industrial scale. In an attempt to overcome these drawbacks, a population of Namalva cells capable of growing in glutamine-free media was established. The adapted cells were found to contain a higher level of glutamine synthetase activity which enable them to synthesize sufficient amounts of glutamine for their growth.Abbreviations GS glutamine synthetase - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid     

Glutamine-requiring mutants of Bacillus subtilis.

Two glutamine-requiring (Gln^?) mutants of $\text{Bacillus subtilis}$ SMY were deficient in glutamine synthetase activity $\text{in vitro}$. The Gln^? mutants sporulated poorly unless glutamine was provided at high concentrations. The differential rate of histidase synthesis following induction was 4- to 6-fold higher in the Gln^? mutants than in wild-type cells. In addition, glucose repression of utilization of alternative carbohydrates appeared to be partially relieved in the Gln^? mutants.     

Response of Zea mays to the Inoculation with Azospirillum on Nitrogen Metabolism under Greenhouse Conditions

The maize (Zea mays L.) plants inoculated by N₂-fixing bacterium Azospirillum showed increased activity of glutamate dehydrogenase (GDH) and glutamine synthetase (GS) in root cells free extracts over uninoculated control plants. Maximum differences in NADH-GDH activity were observed during the second and third weeks after sowing. The specific activity of GS showed a greater increase at the end of the assay. The percentage of nitrogen in leaves, root and foliage length, total fresh mass and nitrogenase activity were higher in inoculated plants than in the control ones.     

Starvation alters the activity and mRNA level of glutaminase and glutamine synthetase in the rat intestine

The metabolism of glutamine, the main respiratory fuel of enterocytes, is governed by the activity of glutaminase and glutamine synthetase. Because starvation induces intestinal atrophy, it might alter the rate of intestinal glutamine utilization. This study examined the effect of starvation on the activity, level of mRNA, and distribution of mRNA of glutaminase and glutamine synthetase in the rat intestine. Rats were randomized into groups and were either: (1) fed for 2 days with rat food ad libitum or (2) starved for 2 days. Standardized segments of jejunum and ileum were removed for the estimation of enzyme activity, level of mRNA, and in situ hybridization analysis. The jejunum of the fed rats had a greater activity of both enzymes per centimeter of intestine (P < 0.01), a greater glutaminase specific activity (1.97 +/- 0.45 vs. 1.09 +/- 0.34 micromol/hr/mg protein, P < 0.01), and a lower level of glutaminase and glutamine synthetase mRNA. The ileum of the fed rats had a greater activity of glutamine synthetase per centimeter of intestine (162.9 +/- 50.6 vs. 91.0 +/- 23.1 nmol/hr/cm bowel, P < 0.01), a lower level of glutaminase mRNA, and a greater level of glutamine synthetase mRNA. In situ hybridization analysis showed that starvation does not alter the distribution of glutaminase and glutamine synthetase mRNA in the intestinal mucosa. This study confirms that starvation decreases the total intestinal activity per centimeter of both glutaminase and glutamine synthetase. More importantly, the results indicate that the intestine adapts to starvation by accumulating glutaminase mRNA. This process prepares the intestine for a restoration of intake.     

Effect of histamine on the development of astroglial cells in culture

The effect of histamine on different aspects of the growth of astrocytes was studied using primary cultures derived either from forebrain or from cerebellum of the rat. The influence on general growth and differentiation was monitored in terms of the activities of ornithine decarboxylase and glutamine synthetase enzymes, whereas [³H]thymidine incorporation into DNA was used as a specific index of cell proliferation. Treatment with 500 nM histamine of cells grown for 6 days in vitro, caused a time-dependent significant increase in ornithine decarboxylase activity of astrocytes from both sources. The maximum increase was observed at 4 h after histamine treatment, at that time the elevation in ornithine decarboxylase activity being about 80% and 300% over control values in the forebrain and the cerebellar astrocytes, respectively. Under similar experimental conditions, addition of histamine (500 nM) to medium resulted in a significant increase in [³H]thymidine incorporation into DNA in both types of cultures: in comparison with control, the elevation was about 45% at 48 h in forebrain astrocytes and at 24 h in cerebellar astrocytes. On the other hand, the specific activity of glutamine synthetase in cerebellar astrocytes was markedly enhanced (about 100%) by treatment with histamine (500 nM) for 4 days, but forebrain astrocytes were little affected. Addition of histamine to the culture medium produced no significant alteration in the activity of lactate dehydrogenase and protein content of either type of astroglial cells. The present findings, which support our earlier proposal that the biochemical properties of astrocytes differ between various brain regions, provide direct evidence for the involvement of histamine in the regulation of growth and development of astrocytes.     

Repeated Administration of γ-VinylGABA Reduces Rat Brain Glutamine Synthetase Activity

Abstract: The glutamine cycle has been proposed as a pathway in which glutamine synthesized in glia provides substrate for synthesis of the neurotransmitters glutamate and GABA as they are lost from neurons. To test whether GABA may regulate this pathway, the effect of elevated GABA on the glial enzyme glutamine synthetase was examined in rat brain. Repeated subcutaneous injections of the antiepileptic GABA transaminase inhibitor γ-vinylGABA at a dose of 150 mg/kg per day for 21 days reduced glutamine synthetase activity by 36% in the cortex and 22% in the cerebellum. At 30 mg/kg per day, glutamine synthetase activity was reduced by 9.5% in the cortex but unchanged in the cerebellum. The reductions were brain specific because the skeletal muscle and liver enzymes were unaffected by γ-vinylGABA administration. Amino acid analysis of the cortex from γ-vinylGABA-treated rats demonstrated a 270% increase in GABA levels after 150 mg/kg but no change after 30 mg/kg. GABA levels and glutamine synthetase activity were inversely correlated. The 150 mg/kg dose significantly lowered cortical glutamine and glutamate levels. The decline in brain glutamine synthetase activity with chronic γ-vinylGABA administration developed gradually over time and may be due to the slow turnover of this enzyme in vivo.     

Effect of dibutyryl cyclic AMP and dexamethasone on glutamine synthetase gene expression in rat astrocytes in culture

Astrocytes are the primary site of glutamate conversion to glutamine in the brain. We examined the effects of treatment with either dibutyryl cyclic AMP and/or the synthetic glucocorticoid dexamethasone on glutamine synthetase enzyme activity and steady-state mRNA levels in cultured neonatal rat astrocytes. Treatment of cultures with dibutyryl cyclic AMP alone (0.25 mM–1.0 mM) increased glutamine synthetase activity and steady state mRNA levels in a dose-dependent manner. Similarly, treatment with dexamethasone alone (10^–7–10^–5 M) increased glutamine synthetase mRNA levels and enzyme activity. When astrocytes were treated with both effectors, additive increases in glutamine synthetase activity and mRNA were obtained. However, the additive effects were observed only when the effect of dibutyryl cyclic AMP alone was not maximal. These findings suggest that the actions of these effectors are mediated at the level of mRNA accumulation. The induction of glutamine synthetase mRNA by dibutyryl cyclic AMP was dependent on protein synthesis while the dexamethasone effect was not. Glucocorticoids and cyclic AMP are known to exert their effects on gene expression by different molecular mechanisms. Possible crosstalk between these effector pathways may occur in regulation of astrocyte glutamine synthetase expression.Abbreviations used GS glutamine synthetase - dbcAMP dibutyryl cyclic AMP - MEM minimal essential medium - cyx cycloheximide - GRE glucocorticoid response element - CRE cyclic AMP response element