Inhibition Of Glutamine Synthetase Activity Research Articles

MPP+ toxicity in rat striatal slices: relationship between non-selective effects and free radical production.

Incubations of rat striatal slices have been used to assay MPP+ neurotoxicity. MPP+, at concentrations of 1 mM or higher, caused a marked increase in hydroxyl radicals, measured as malondialdehyde (MDA) accumulation, but not in nitric oxide production. At these doses, MPP+ showed an effect on dopamine terminals, causing a massive dopamine decrease, and on non-neuronal glial cells, where a marked reduction in glutamine synthetase activity was detected. At lower concentrations (25 mu M), the toxic effect on dopaminergic endings was maintained without increasing malondialdehyde concentrations or inhibiting glutamine synthetase activity. The effect on glutamine synthetase was prevented by the addition to the medium of 0.5% dimethyl sulfoxide, a hydroxyl-radical scavenger, but this did not protect the effect of dopamine depletion. We propose that non-selective effects of MPP+, at doses of 1 mM or higher, are mediated by extracellular overproduction of hydroxyl radicals. The main factor responsible for this overproduction would not be the released dopamine but rather the MPP+ itself through non selective inhibition of the mitochondrial respiratory chain or through a redox cycling that can trigger oxygen radical production.

Ammonium assimilation in rice based on the occurrence of15N and inhibition of glutamine synthetase activity

Abstract Assimilation of ammonium (NH4) into free amino acids and total reduced nitrogen (N) was monitored in both roots and shoots of two‐week old rice seedlings supplied with 5 mM 99% (15NH4)2SO4 in aerated hydroponic culture with or without a 2 h preincubation with 1 mM methionine sulfoximine (MSX), an inhibitor of glutamine synthetase (GS) activity. 15NH4 was not assimilated into amino acids when the GS/GOGAT (glutamate synthase) cycle was inhibited by MSX. Inhibition of glutamine synthetase (GS) activity in roots with MSX increased both the amount of NH4 and the abundance of 15N labeled NH4. In contrast, the amount of Gln and Glu, and their proportions as 15N, decreased in roots when GS activity was inhibited. This research confirms the importance of GS/GOGAT in NH4 assimilation in rice roots. 15N‐labeled studies indicate that NH4 ions incorporated by roots of rice are transformed primarily into glutamine (Gln) and glutamic acid (Glu) before being converted to other amino acids through transamination...

Active glutamine synthetase is required for ammonium‐ or glutamine‐promoted prevention of nitrate and nitrite reduction in the cyanobacterium Phormidium laminosum

The filamentous non‐N2‐fixing cyanobacterium Phormidium laminosum (strain OH‐1‐p.Cl1) was able to utilize glutamine as the sole nitrogen source. The addition to ammonium‐grown cultures of the irreversible inhibitor of glutamine synthetase activity L‐methionine‐D, L‐sulfoximine (MSX) inhibited cell growth. Supplying glutamine to the culture restored cell growth. This re‐established growth was not due to interference by glutamine of MSX uptake by the cells, since glutamine synthetase (GS, EC 6.3.1.2) activity remained completely inhibited by MSX even when glutamine was simultaneously present. Both glutamine and ammonium exerted a negative effect on nitrate reductase (NR. EC 1.7.7.2) and nitrite reductase (NiR, EC 1.7.7.1) in vivo. This negative effect was reversed by MSX. When glutamine was added to MSX‐treated cells, intracellular glutamine level was high, but the activity of both reductases remained at a high level. These results suggest that the presence of the active form of glutamine synthetase is required for the in vivo prevention of nitrate assimilation caused by ammonium and glutamine.

The role of glutamine synthetase in regulation of nitrogen metabolism within the soil microbial community

Recent advances in our understanding of the enzymology and regulatory systems involved in microbial metabolism of N hold promise to elucidate some of the underlying factors controlling metabolism of N in soil ecosystems. A review of recent work is used to construct a paradigm for N metabolism regulation in soil based on the central role of glutamine synthetase (GS) in such regulation within the soil microbial community. The studies involved use of GS inhibitors to elucidate the role of GS activity in regulation of soil N metabolism. Such studies have shown that the glutamine formed by microbial assimilation of NH 4 + via GS activity influences the regulatory mechanisms controlling both the production and activity of enzymes involved in N metabolism. For example, these studies showed that the inhibition of GS activity within the soil microbial community relieved the repression of urease production caused by microbial assimilation of inorganic N and blocked the short-term regulation of assimilatory nitrate reductase (ANR) by NH 4 + assimilation. Other studies have indicated that common environmental factors in soil may influence GS activity in microorganisms and thereby may influence metabolism of N within the soil microbial community. The paradigm for N metabolism regulation in soil that has emerged from such studies should lead to a better understanding of the mechanisms controlling fate of N in soil ecosystems

Phosphinothricin Reverts the Ammonia-DependentEnhancement of Phosphoenolpyruvate Carboxylase Activity

Summary The effect of ammonium assimilation on phosphoenolpyruvate carboxylase (PEPCase) activity was investigatedin detached leaves from N-limited barley plants. The time-course induction of PEPCase was dependent on the rate of ammonia assimilation and not on ammonia uptake or accumulation. Treatment of N-deprived leaves with phosphinothricin (an inhibitor of glutamine synthetase activity) caused inhibition of ammonia assimilation, resulting in the reversion of ammonia-dependent enhancement of PEP-Case. The results indicate that glutamine level controls phosphoenolpyruvate carboxylase activation; consequently, if glutamine synthesis is inhibited, PEPCase activity is not enhanced. Determination of malate content showed that as PEPCase activity increased in response to increasingammonia assimilation, there was a linear decline in the level of that metabolite. We have also analyzed the in vivo effect of malate on ammonium-dependent activation of PEPCase.High malate uptake partially abolished PEPCase activation, but the induced PEPCase activity seems to be less sensitive to malate than the control one.

Ammonia-induced brain edema and intracranial hypertension in rats after portacaval anastomosis

Brain edema, leading to intracranial hypertension and brain herniation, is a major cause of death in fulminant liver failure. Astrocyte swelling is a prominent neuropathological feature in experimental fulminant liver failure. It has been postulated that the osmotic effects of glutamine, generated in astrocytes from ammonia and glutamate in a reaction catalyzed by glutamine synthetase, could mediate brain swelling. Normal rats and rats that received a portacaval anastomosis were infused with ammonium acetate or a sodium acetate control; brain water in cerebral cortex was measured with the gravimetry method, intracranial pressure by means of a cisterna magna catheter and cortical amino acids using high-performance liquid chromatography. Although brain edema was detected in both groups receiving ammonia, it was of a greater magnitude in portacaval anastomosis rats (80.94% + 0.17% vs. 80.24% + 0.09%, p < 0.01), resulting in the development of intracranial hypertension. When portacaval anastomosis rats were infused with ammonium acetate and pretreated with 150 mg/kg methionine-sulfoximine, an inhibitor of glutamine synthetase activity, brain edema was ameliorated and intracranial pressure did not rise. A dose-dependent reduction in brain glutamine levels was seen with increasing doses of methionine-sulfoximine; however, brain edema did not decrease beyond the 150 mg/kg dose, suggesting that the increase in brain water was not solely a result of glutamine accumulation. We conclude that brain edema of a magnitude that results in intracranial hypertension is more likely to develop in rats after portacaval anastomosis receiving a continuous ammonia infusion. The osmotic effects of glutamine appear to mediate, but only in part, the increase in brain water seen in this preparation.(ABSTRACT TRUNCATED AT 250 WORDS)

Glufosinate ammonium—Some aspects of its mode of action in mammals

The broad-spectrum herbicide glufosinate ammonium is a structural analogue of glutamate and acts in plants by inhibition of glutamine synthetase leading to a complete breakdown of ammonia metabolism. Owing to the structural analogy of glufosinate ammonium to glutamate, its effect on various glutamate-utilizing systems needed to be investigated in mammals. Although in laboratory animals glufosinate ammonium causes an inhibition of glutamine synthetase activity in different tissues, this inhibition led to slight increases of glutamate and ammonia levels at high sublethal and lethal doses only. After oral administration for 28 days, glufosinate ammonium had no effect on glutathione and carbohydrate metabolism and no effect on biosynthesis of non-essential amino acids in rats and dogs. Glufosinate ammonium does not interfere with various neurotransmitter receptors in vitro and does not influence the catecholamine neurotransmitter tissue concentrations after iv application. The results of these studies show that—in contrast to the plant metabolism—in mammals the inhibition of glutamine synthetase activity in various tissues does not lead to a breakdown of ammonia metabolism. The mammalian metabolism obviously compensates for this inhibition of glutamine synthetase activity by various other metabolic pathways. It is concluded that under the conditions of recommended use of glufosinate ammonium as an active ingredient in herbicides, a detrimental effect on the health of both users and consumers is extremely unlikely.

Effects of Glutamine Synthetase Inhibitors on Rice Sterility

Bialaphos, a glutamine synthetase (GS) inhibitor, is an effective inducer of rice sterility, l-Phosphinothricin (l-PTC), a metabolite of bialaphos, is the real inhibitor of GS from the results of an enzyme assay of GS from rice plants. Bialaphos and l-PTC induced a high level of sterility in rice at a concentration of 0.1 mm at the meiosis stage. The complete inhibition of GS activity was induced by 24-h treatments with 0.1 mm of bialaphos and l-PTC, respectively. These inhibitions continued until anthesis.In proportion to the inhibition of GS activity, the free ammonia content was increased in the panicles after treating with 0.1 mm of bialaphos and l-PTC. This accumulation of ammonia continued until anthesis.These results suggest that ammonia accumulating in the panicles as a result of GS inhibition directly caused sterility in rice.

Postharvest inhibition of glutamine synthetase activity with phosphinothricin reduces the shelf-life of asparagus

Harvested spears of asparagus ( Asparagus officinalis L.) accumulate ammonia in their tips towards the end of shelf-life. However, the accumulation of ammonia is not due to limitations in the activity of the ammonia assimilating enzyme, glutamine synthetase (GS). When GS was inhibited by postharvest treatment with phosphinothricin (PPT), the ammonia content of the tips increased almost immediately and the shelf-life of the spears was severely reduced with symptoms of deterioration identical to those commonly ascribed to chilling injury. PPT treatment also inhibited the pattern of glutamine and asparagine accumulation seen during shelf-life. These observations emphasize that during the postharvest life of asparagus GS has a pivotal role in reassimilating considerable amounts (3% of dry weight) of ammonia in the tips.

The effect of 4 days methionine sulfoximine administration on net muscle protein breakdown in rats

It has been suggested that the amino acid glutamine is essential for the regulation of protein turnover in muscle, but in vivo data are lacking. Therefore, administration of methionine sulfoximine (MSO) was used to inhibit glutamine synthetase activity and to induce in vivo muscle glutamine depletion. Glutamine metabolism of the hindquarter was measured by determining fluxes and intracellular concentrations after an overnight fast in ether anaesthetized normal rats, MSO treated rats and their pairfed controls. Fluxes and intracellular concentrations of several other amino-acids including 3-methylhistidine and ammonia were also determined. MSO treatment resulted in a 50% decrease in arterial glutamine concentration, a 55% reduction in intracellular muscle glutamine and a 50% increase in muscle ammonia. Four day MSO treatment significantly increased hindquarter muscle plasma flow. Precursors of muscle glutamine synthesis seem to be preferentially used for increased production of alanine. Ammonia was found to be released in increased amounts. The increased efflux of amino-acids including phenylalanine and tyrosine indicate that MSO treatment induces net muscle protein catabolism. These results suggest that, in vivo, reduced intramuscular glutamine concentrations correlate with increased net muscle protein breakdown.

Restoration of cerebrovascular CO2 responsivity by glutamine synthesis inhibition in hyperammonemic rats.

Hyperammonemia increases brain glutamine levels, causes astrocytic swelling, and depresses cerebral blood flow (CBF) responsivity to CO2. Methionine sulfoximine (MSO) inhibition of glutamine synthetase activity, known to be enriched in astrocytes, prevents ammonia-induced increases in brain glutamine and water content. We tested the hypothesis that inhibition of glutamine accumulation restores CBF responsivity to CO2 during acute hyperammonemia. Pentobarbital-anesthetized rats treated with either vehicle or MSO (150 mg/kg i.p.) received a 6-hour intravenous infusion of either sodium or ammonium acetate. With subsequent induction of hypercapnia, CBF increased from 113 +/- 14 (mean +/- SEM) to 194 +/- 9 ml/min per 100 g in control rats but was unchanged from 107 +/- 13 to 79 +/- 10 ml/min per 100 g in hyperammonemic rats. Treatment with MSO in hyperammonemic rats restored the CBF response to hypercapnia (from 73 +/- 8 to 141 +/- 14 ml/min per 100 g). With induction of hypocapnia, CBF decreased from 114 +/- 11 to 88 +/- 11 ml/min per 100 g in control rats but increased from 112 +/- 13 to 142 +/- 19 ml/min per 100 g in hyperammonemic rats. Treatment with MSO in hyperammonemic rats did not fully restore the response to hypocapnia but prevented the paradoxical increase in CBF (from 80 +/- 8 to 80 +/- 8 ml/min per 100 g). In control rats, MSO did not affect CO2 responsivity. Treatment with MSO prevented ammonia-induced increases in intracranial pressure. Hyposmotic-induced increases in brain water content and intracranial pressure attenuated the CBF response to hypercapnia but, unlike hyperammonemia, did not attenuate the response to hypocapnia. In contrast to hypercapnia, vasodilation in response to arterial hypotension was intact in hyperammonemic rats. We conclude that the grossly abnormal CBF responsivity to CO2 alterations during hyperammonemia is linked to glutamine accumulation rather than ammonia per se. Cerebral edema secondary to glutamine accumulation may contribute in part to abnormal CBF responses, although other aspects of astrocyte dysfunction are likely to be important.

Effect of pH on glutamine content derived from exogenous glutamate in astrocytes.

A shift in pH from 7.4 to 7.8 in the incubation solution caused a 3.4-fold increase in the free glutamine content of mouse cerebral astrocytes that were incubated with glutamate (100 microM) and ammonium (100 microM). This large and reversible steady-state increase in glutamine content was accompanied by smaller transient increases in the following: (a) net formation of glutamine; (b) clearance of glutamate from the incubation solution; and (c) glutamate content. The content of glutamine was reduced markedly by omission of either glutamate or ammonium from the incubation solution, or by inhibition of glutamine synthetase activity with methionine sulfoximine. The rate at which glutamine was exported from the astrocytes was unaffected by the pH change. The effects of pH on the concentration of free ammonia or on glutamate uptake do not appear to mediate the increase in glutamine content. Uptake of exogenous glutamine was little affected by the pH change. Therefore, possible mediation of the effect by an increase in intracellular pH must be considered. The response to altered pH described here may provide a cellular basis for the increased level of brain glutamine observed in hyperammonemia.

A macromolecular inhibitor of glutamine synthetase activity in tomato root extracts

In vitro glutamine synthetase (GS; EC 6.3.1.2) activity is very low in the roots of tomato plants ( Lycopersicon esculentum). Extracts prepared from roots substantially reduced the GS activity in a preparation from tomato leaves. The GS inhibitory activity could be separated from GS activity by gel filtration chromatography with a parallel recovery of enzyme activity. The results obtained indicate that the GS inhibitory activity detected in tomato roots is due to a protein factor with an apparent molecular mass of 110 kD. The protein was inhibitory to both cytosolic (GS1) and chloroplastic (GS2) glutamine synthetases from green leaves of different plant species. The GS inhibitor was found to competitively inhibit GS activity with respect to ATP. The physiological relevance of this protein in the regulation of glutamine synthesis, remains to be determined.

Glufosinate (Phosphinothricin) Inhibition of Nitrogen Metabolism in Barley and Green Foxtail Plants

Summary Evidence has shown that barley (Hordeum vulgare L. cv. Sampson) and green foxtail (Setaria viridis (L.) Beauv.) are differentially sensitive to foliar application of the glutamine synthetase inhibitor, glufosinate. Here, we investigated the influence of glufosinate on the nitrogen metabolism of the two species as a function of the time (0 to 72 h) after spray application at doses ranging from zero to 80 mg m−2. In both species ammonia accumulation was accompanied by an overall accumulation of amino acids and by declines in the mol percent glutamine-histidine, glutamate, asparagine, aspartate, and alanine. Also, the mol percent serine and glycine declined in barley. However, there were several-fold accumulations in aromatic and branched chain amino acids and amino acids derived from glutamate (e.g. arginine). Therefore, the inhibition of ammonia assimilation is accompanied by a reduction in the photorespiratory glyoxylate to glycine conversion (in barley only) and an increase in protein turnover. Glutamine synthetase activity, assayed in vitro under optimal conditions, was decreased in both species. The magnitude and the rapidity of (i) ammonia accumulation, (ii) changes in amino acid accumulation and composition, and (iii) inhibition of glutamine synthetase activity were different in both species and correlated with the lower sensitivity of barley than green foxtail to the herbicide.

Inhibition of brain glutamine accumulation prevents cerebral edema in hyperammonemic rats

The mechanism of brain swelling during hyperammonemia is not understood, but glutamine accumulation is consistently observed. We tested the hypothesis that brain swelling associated with hyperammonemia is a consequence of the osmotic effect of intracellular glutamine accumulation in brain. Increases in plasma ammonium levels from 31 +/- 3 to 601 +/- 38 mumol/l (+/- SE) were produced by 6 h of infusion of ammonium acetate in anesthetized rats. Hyperammonemia resulted in increased brain water content accompanied by more than a tripling of brain glutamine concentration compared with control rats receiving sodium acetate (5.6 +/- 0.4 vs. 18.8 +/- 0.4 mmol/kg). Inhibition of glutamine synthetase activity by pretreatment with L-methionine sulfoximine prevented both the increase in brain glutamine levels and the increase in brain water content despite elevated plasma ammonium levels (908 +/- 196 mumol/l). Thus cerebral edema during hyperammonemia is associated with glutamine accumulation. We suggest that accumulated glutamine may serve as an idiogenic osmole causing swelling. Because brain swelling eventually leads to increased intracranial pressure and tissue hypoxia, these data suggest a unifying mechanism to account for the many pathophysiological abnormalities found during coma associated with various forms of liver disease, inborn errors of metabolism, and Reye's syndrome.

Reduction of glutamine synthetase activity in astroglia exposed in culture to low levels of inorganic lead

Astroglia serve as a site of lead (Pb) deposition in Pb-exposed animals. Thus, the potential exists for astroglial function to be impaired by elevated intracellular Pb levels. We previously showed that the specific activity of glutamine synthetase (GS), an astroglial enzyme with a key role in glutamate and ammonia metabolism in the brain, is reduced in fetal guinea pigs exposed to low levels of lead. This observation indicates either a direct effect of Pb on astroglia or an indirect systemic effect. The purpose of the present study was to test the hypothesis that Pb directly reduces GS activity in astroglia. Cultured rat astroglia were fed three times per week with medium containing 0, 0.25, 0.5, or 1 μM Pb acetate for 7, 14, or 21 days. Trypan blue dye exclusion, cell number, and GS activity were measured. Lead treatment reduced the ability of cells to exclude trypan blue in a dose- and time-dependent manner, with the greatest reduction (28%) on day 21 in the group treated with 1 μM Pb. Cell numbers were reduced a maximum of 15% on day 15, but were unaffected on days 7 and 21. The effects of Pb exposure on GS activity were much more pronounced. For example, cultures exposed to 0.25 of 1 μM Pb for 7 days showed, respectively, 17 and 52% reduction in activity from the control value. Slightly greater reductions were measured on days 14 and 21. The much greater sensitivity in vitro of GS activity than dye exclusion or cell numbers to low level lead supports a specific enzymatic inhibition. In order to test the possibility that Pb might directly inhibit GS activity Pb-treated cells, we also measured the effect of Pb on GS activity in cytosolic extracts of the cells. Pb inhibited GS activity in a dose-dependent manner ranging from 27% inhibition at 0.1 nM to 67% at 0.1 μM and 100% at 10 μM Pb. These results indicate that astroglial function is vulnerable to Pb exposure, even at low lead levels.

Isolation and characterization of a Neurospora crassa mutant altered in the alpha polypeptide of glutamine synthetase

We report the isolation and characterization of a Neurospora crassa glutamine synthetase (GS) mutant altered in one of the two polypeptides (GS alpha) of this enzyme. We used the gln-1bR8 mutant strain that synthesizes only the GS alpha monomer and lacks the GS beta monomer and selected for growth in minimal medium in the presence of alpha-methyl-DL-methionine-SR-sulfoximine (alpha-me-MSO), an inhibitor of GS activity. The GS activity of the gln-1bR8;alpha-me-MSOR strain drastically reduced its transferase activity and only slightly reduced its synthetase activity, and it was resistant to inhibition by alpha-me-MSO and L-methionine-DL-sulfoximine. The mutation that overcame the inhibitory effect of alpha-me-MSO also altered the antigenic, kinetic, and physical properties of GS alpha. The low GS activity of the alpha-me-MSO-resistant strain was compensated for by a higher glutamate/glutamine ratio and a lower glutamate synthase activity, allowing this strain to grow as well as the parental strain. The mutation that conferred resistance to alpha-me-MSO was not linked to the gln-1bR8 mutation, providing direct evidence of the existence of two genes involved with the structure of the two polypeptides of N. crassa GS.

Effect of L-methionine sulphoximine on the enzymes of nitrogen metabolism in barley leaves

L-methionine sulphoximine, a potent inhibitor of glutamine synthetase decreased nitrate reductase activity by 50% at the end of 12h of treatment while nitrite reductase was insignificantly affected. By 3 h the inhibition of glutamine synthetase activity was complete. Elevated levels of ammonia induced by L-methionine sulphoximine did not influence glutamate dehydrogenase. It is inferred that ammonia accumulation does not affect photosynthetic electron transport which supplies reducing power to nitrite reductase. The failure of glutamate dehydrogenase activity to be induced by high ammonium levels shows that it is not involved in the process of ammonia assimilation in the leaves.

Sustained photoproduction of ammonia from nitrate or nitrite by permeabilized cells of the cyanobacterium Phormidium laminosum

The addition of micromolar amounts of the glutamate analogue l-methionine- d, l-sulphoximine (MSX) to cell suspensions of the filamentous non N 2-fixing cyanobacterium Phormidium laminosum rapidly caused the complete inhibition of glutamine synthetase activity and prevented cell growth. Such MSX-treated cells were able to photoproduce ammonia from nitrate or nitrite at about 2.3 μmol (mg chlorophyll) −1 h −1. This value could be significantly increased after cell permeabilization by repeated freeze—thaw (F/T) cycles or by storing cells frozen at −20 °C for several days. Photoproduction of ammonia showed an optimum pH value of 8.5 and was saturated by 10 mM nitrate or nitrite. However, the ratio ammonia released/nitrate disappearance was higher at low nitrate concentrations. Although free F/T cells showed higher initial rates of ammonia production, when such cells were immobilized in calcium alginate beads and packed into a continuous flow reactor they photoproduced ammonia for 100 h at a maximal rate of about 10 μmol (mg chlorophyll) −1 h −1.

Age-dependent variation in the sensitivity of rat brain glutamine synthetase to l-methionine-dl-sulfoximine

Effects of a subacute dose (150 mg/kg body wt) of methionine sulfoximine was studied on the behavioural changes and glutamine synthetase activity in cerebral cortex, cerebellum and brain stem of rats of four different age groups. Animals of 10 days and 90 days age groups were less vulnerable to the toxic effects of methionine sulfoximine. Rats of 180 days age exhibited various behavioural changes upon the administration of methionine sulfoximine and entered into convulsions at the end of 17 hr. In rats of 360 days age group these changes appeared quite early and the animals convulsed at the end of 13 hr. Animals of these two age groups failed to recover from toxic effects of methionine sulfoximine. Methionine sulfoximine inhibited glutamine synthetase activity in the brains of rats of all age groups. Maximum inhibition of activity was noticed at the end of 12 hr in 10-day-old rats while in the animals of other age groups it was seen at the end of 3 hr. In animals of all age groups the enzyme activity was inhibited by 60–70% except in 360-day-old rats where the inhibition was 95%. Behavioural changes and inhibition of glutamine synthetase were out of phase with each other. It is suggested that the toxicity of methionine sulfoximine may not be due to its effect on glutamine synthetase alone. Further, these results also suggest that the toxic effects of methionine sulfoximine vary with the age of the animal.