Increase In Glutaminase Activity Research Articles

Impact of hypoxic preconditioning on the alterations in phosphate-activated glutaminase activity in the brains of offspring

The hypoxic preconditioning model is widely used in experimental studies to identify mechanisms for increasing the tolerance of organisms to subsequent hypoxic exposure. Glutaminase is the main enzyme that synthesizes glutamate, which has two important physiological roles: in the postnatal period, it acts as an excitatory neurotransmitter, and during the prenatal period, it regulates neurogenesis, synaptogenesis, and the survival of nerve cells. In our work, in 4 experimental groups, we investigated the effect of hypoxic preconditioning performed during days 16–21 of pregnancy on glutamate synthesis in the brains of 17-day-old and 6-month-old offspring. It was found that prenatal hypoxia led to a pronounced increase in the enzyme activity in various brain structures in early postnatal ontogenesis, while a decrease was observed in adult animals. In contrast, exposure to acute hypoxia resulted in a more significant increase in glutaminase activity in the brains of adult animals. Prenatal fetal hypoxic preconditioning caused a weakening effect on the increase in enzyme activity in 17-day-old rat offspring and a down-regulation in 6-month-olds, compared to the group that suffered acute hypoxia in the postnatal period. Thus, a neuroprotective adaptive-compensatory effect of prenatal preconditioning has been demonstrated, which can be associated with both the physiological and excitotoxic effects of glutamate.

Engineering and Expression Strategies for Optimization of L-Asparaginase Development and Production.

Genetic engineering for heterologous expression has advanced in recent years. Model systems such as Escherichia coli, Bacillus subtilis and Pichia pastoris are often used as host microorganisms for the enzymatic production of L-asparaginase, an enzyme widely used in the clinic for the treatment of leukemia and in bakeries for the reduction of acrylamide. Newly developed recombinant L-asparaginase (L-ASNase) may have a low affinity for asparagine, reduced catalytic activity, low stability, and increased glutaminase activity or immunogenicity. Some successful commercial preparations of L-ASNase are now available. Therefore, obtaining novel L-ASNases with improved properties suitable for food or clinical applications remains a challenge. The combination of rational design and/or directed evolution and heterologous expression has been used to create enzymes with desired characteristics. Computer design, combined with other methods, could make it possible to generate mutant libraries of novel L-ASNases without costly and time-consuming efforts. In this review, we summarize the strategies and approaches for obtaining and developing L-ASNase with improved properties.

Bisphenol-A Neurotoxic Effects on Basal Forebrain Cholinergic Neurons In Vitro and In Vivo.

The widely used plasticizer bisphenol-A (BPA) is well-known for producing neurodegeneration and cognitive disorders, following acute and long-term exposure. Although some of the BPA actions involved in these effects have been unraveled, they are still incompletely known. Basal forebrain cholinergic neurons (BFCN) regulate memory and learning processes and their selective loss, as observed in Alzheimer's disease and other neurodegenerative diseases, leads to cognitive decline. In order to study the BPA neurotoxic effects on BFCN and the mechanisms through which they are induced, 60-day old Wistar rats were used, and a neuroblastoma cholinergic cell line from the basal forebrain (SN56) was used as a basal forebrain cholinergic neuron model. Acute treatment of rats with BPA (40 µg/kg) induced a more pronounced basal forebrain cholinergic neuronal loss. Exposure to BPA, following 1- or 14-days, produced postsynaptic-density-protein-95 (PSD95), synaptophysin, spinophilin, and N-methyl-D-aspartate-receptor-subunit-1 (NMDAR1) synaptic proteins downregulation, an increase in glutamate content through an increase in glutaminase activity, a downregulation in the vesicular-glutamate-transporter-2 (VGLUT2) and in the WNT/β-Catenin pathway, and cell death in SN56 cells. These toxic effects observed in SN56 cells were mediated by overexpression of histone-deacetylase-2 (HDAC2). These results may help to explain the synaptic plasticity, cognitive dysfunction, and neurodegeneration induced by the plasticizer BPA, which could contribute to their prevention.

Understanding gut-liver axis nitrogen metabolism in Fatty Liver Disease.

The homeostasis of the most important nitrogen-containing intermediates, ammonia and glutamine, is a tightly regulated process in which the gut-liver axis plays a central role. Several studies revealed that nitrogen metabolism is altered in Metabolic Dysfunction-Associated Fatty Liver Disease (MAFLD), a consensus-driven novel nomenclature for Non-Alcoholic Fatty Liver Disease (NAFLD), the most common chronic liver disease worldwide. Both increased ammonia production by gut microbiota and decreased ammonia hepatic removal due to impaired hepatic urea cycle activity or disrupted glutamine synthetase activity may contribute to hepatic ammonia accumulation underlying steatosis, which can eventually progress to hyperammonemia in more advanced stages of steatohepatitis and overt liver fibrosis. Furthermore, our group recently showed that augmented hepatic ammoniagenesis via increased glutaminase activity and overexpression of the high activity glutaminase 1 isoenzyme occurs in Fatty Liver Disease. Overall, the improved knowledge of disrupted nitrogen metabolism and metabolic miscommunication between the gut and the liver suggests that the reestablishment of altered gut-liver axis nitrogenous balance is an appealing and attractive therapeutic approach to tackle Fatty Liver Disease, a growing and unmet health problem.

Blocking glycine receptors reduces neuroinflammation and restores neurotransmission in cerebellum through ADAM17-TNFR1-NF-\u03ba\u03b2 pathway

BackgroundChronic hyperammonemia induces neuroinflammation in cerebellum, with glial activation and enhanced activation of the TNFR1-NF-kB-glutaminase-glutamate-GABA pathway. Hyperammonemia also increases glycinergic neurotransmission. These alterations contribute to cognitive and motor impairment. Activation of glycine receptors is reduced by extracellular cGMP, which levels are reduced in cerebellum of hyperammonemic rats in vivo.We hypothesized that enhanced glycinergic neurotransmission in hyperammonemic rats (1) contributes to induce neuroinflammation and glutamatergic and GABAergic neurotransmission alterations; (2) is a consequence of the reduced extracellular cGMP levels. The aims were to assess, in cerebellum of hyperammonemic rats, (a) whether blocking glycine receptors with the antagonist strychnine reduces neuroinflammation; (b) the cellular localization of glycine receptor; (c) the effects of blocking glycine receptors on the TNFR1-NF-kB-glutaminase-glutamate-GABA pathway and microglia activation; (d) whether adding extracellular cGMP reproduces the effects of strychnine.MethodsWe analyzed in freshly isolated cerebellar slices from control or hyperammonemic rats the effects of strychnine on activation of microglia and astrocytes, the content of TNFa and IL1b, the surface expression of ADAM17, TNFR1 and transporters, the phosphorylation levels of ERK, p38 and ADAM17. The cellular localization of glycine receptor was assessed by immunofluorescence. We analyzed the content of TNFa, IL1b, HMGB1, glutaminase, and the level of TNF-a mRNA and NF-κB in Purkinje neurons. Extracellular concentrations of glutamate and GABA were performed by in vivo microdialysis in cerebellum. We tested whether extracellular cGMP reproduces the effects of strychnine in ex vivo cerebellar slices.ResultsGlycine receptors are expressed mainly in Purkinje cells. In hyperammonemic rats, enhanced glycinergic neurotransmission leads to reduced membrane expression of ADAM17, resulting in increased surface expression and activation of TNFR1 and of the associated NF-kB pathway. This increases the expression in Purkinje neurons of TNFa, IL-1b, HMGB1, and glutaminase. Increased glutaminase activity leads to increased extracellular glutamate, which increases extracellular GABA. Increased extracellular glutamate and HMGB1 potentiate microglial activation. Blocking glycine receptors with strychnine or extracellular cGMP completely prevents the above pathway in hyperammonemic rats.ConclusionsGlycinergic neurotransmission modulates neuroinflammation. Enhanced glycinergic neurotransmission in hyperammonemia would be due to reduced extracellular cGMP. These results shed some light on possible new therapeutic target pathways for pathologies associated to neuroinflammation.

Lamotrigine reverses masseter overactivity caused by stress maybe via Glu suppression

Experimental and non-experimental stress significantly increase masseter muscle tone, which has been linked to the symptoms and pathogenesis of several stomatognathic system diseases. Until now, the mechanism underlying this phenomenon has remained unclear. The current study was performed to determine the mechanism of the stress-induced increase in masseter muscle tone and to investigate the effect of lamotrigine on this change. Animals challenged by repeated restraint stress received either saline as a vehicle or lamotrigine in doses of 20, 30 or 40mg/kg body weight, whereas control animals received saline without stress treatment. Masseter muscle tone was assessed using electromyography. The activity of glutamate-related metabolic enzymes (glutaminase and glutamine synthetase) in the trigeminal motor nucleus was also investigated. Our results showed an interesting phenomenon: masseter muscle activity increased concurrently with the upregulation of the glutamate concentration after stress treatment. The activities of glutaminase and glutamine synthetase in the trigeminal motor nucleus were also upregulated and downregulated, respectively, when the rats were challenged by prolonged stress. The animals treated with lamotrigine at moderate and high doses had significantly decreased masseter muscle tone compared with stressed animals treated with vehicle. These results suggested that increased glutaminase activity and decreased glutamine synthetase activity increased glutamate production and decreased glutamate decomposition, causing an increase in glutamate levels in the trigeminal motor nucleus and eventually increasing masseter muscle tone. The administration of lamotrigine at doses of 30 or 40mg/kg body weight effectively mitigated the adverse effects of stress on masseter muscle tone via inhibition of glutamate release.

Imbalance of system of glutamin - glutamic acid in the placenta and amniotic fluid at placental insufficiency

Metabolism of glutamine and glutamic acid has been investigated in the placenta and amniotic fluid under conditions of placental insufficiency. The development of placental insufficiency is characterized by the increased content of glutamic acid and a decrease of glutamine in both placenta and amniotic fluid. These changes changes were accompanied by changes in the activity of enzymes involved in the metabolism of these amino acids. There was a decrease in glutamate dehydrogenase activity and an increase in glutaminase activity with the simultaneous decrease of glutamine synthetase activity. The compensatory decrease in the activity of glutamine keto acid aminotransferase did not prevent a decrease in the glutamine level. The impairments in the system glutamic acid-glutamine were more pronounced during the development of premature labor.

Small molecule glutaminase inhibitors block glutamate release from stimulated microglia

Glutaminase plays a critical role in the generation of glutamate, a key excitatory neurotransmitter in the CNS. Excess glutamate release from activated macrophages and microglia correlates with upregulated glutaminase suggesting a pathogenic role for glutaminase. Both glutaminase siRNA and small molecule inhibitors have been shown to decrease excess glutamate and provide neuroprotection in multiple models of disease, including HIV-associated dementia (HAD), multiple sclerosis and ischemia. Consequently, inhibition of glutaminase could be of interest for treatment of these diseases. Bis-2-(5-phenylacetimido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) and 6-diazo-5-oxo-l-norleucine (DON), two most commonly used glutaminase inhibitors, are either poorly soluble or non-specific. Recently, several new BPTES analogs with improved physicochemical properties were reported. To evaluate these new inhibitors, we established a cell-based microglial activation assay measuring glutamate release. Microglia-mediated glutamate levels were significantly augmented by tumor necrosis factor (TNF)-α, phorbol 12-myristate 13-acetate (PMA) and Toll-like receptor (TLR) ligands coincident with increased glutaminase activity. While several potent glutaminase inhibitors abrogated the increase in glutamate, a structurally related analog devoid of glutaminase activity was unable to block the increase. In the absence of glutamine, glutamate levels were significantly attenuated. These data suggest that the in vitro microglia assay may be a useful tool in developing glutaminase inhibitors of therapeutic interest.

Primary Afferent Nociceptor as a Target for the Relief of Pain

Primary Afferent Nociceptor as a Target for the Relief of Pain

Targeting Mitochondrial Glutaminase Activity Inhibits Oncogenic Transformation

Rho GTPases impact a number of activities important for oncogenesis. We describe a small molecule inhibitor that blocks oncogenic transformation induced by various Rho GTPases in fibroblasts, and the growth of human breast cancer and B lymphoma cells, without affecting normal cells. We identify the target of this inhibitor to be the metabolic enzyme glutaminase, which catalyzes the hydrolysis of glutamine to glutamate. We show that transformed fibroblasts and breast cancer cells exhibit elevated glutaminase activity that is dependent on Rho GTPases and NF-κB activity, and is blocked by the small molecule inhibitor. These findings highlight a previously unappreciated connection between Rho GTPase activation and cellular metabolism and demonstrate that targeting glutaminase activity can inhibit oncogenic transformation.

P57 Administration of branched chain amino acids to hyperammonemic cirrhotic animals is associated with worsening of hyperammonemia and increase in brain oedema

IntroductionDespite branched chain amino acids (BCAA) being used for over 30 years, its efficacy in treating hyperammonemia remains controversial. Recent studies suggest that the use of BCAA in cirrhosis is...

P51 Inadequate compensation by glutamine synthetase and increased glutaminase activity contributes to hyperammonaemia in cirrhosis

IntroductionIn cirrhosis, the function of the urea cycle is compromised which leads to accumulation of ammonia. In this situation, ammonia metabolism is regulated by glutamine synthetase (GS) and glutaminase (GA)...

Gut ammonia production and its modulation

Systemic hyperammonemia has been largely found in patients with cirrhosis and hepatic encephalopathy, and ammonia plays a major role in the pathogenesis of hepatic encephalopathy. However, controversial points remain: a) the correlation between plasma ammonia levels and neurophysiological impairment. The lack of correlation between ammonia levels and grade of hepatic encephalopathy in some cases has been considered a weakness of the ammonia hypothesis, but new methods for ammonia measurements and the implication of systemic inflammation in the modulation of ammonia neurotoxicity could explain this gap; b) the source of ammonia production. Hyperammonemia has been considered as derived from urea breakdown by intestinal bacteria and the majority of treatments were targeted against bacteria-derived ammonia from the colon. However, some data suggest an important role for small intestine ammonia production: 1) the hyperammonemia after porto-caval shunted rats has been found similar in germ-free than in non-germ-free animals. 2) In cirrhotic patients the greatest hyperammonemia was found in portal drained viscera and derived mainly from glutamine deamination. 3) The amount of time required to increase of ammonia (less than one hour) after oral glutamine challenge supports a small intestine origin of the hyperammonemia. As the main source of ammonia in cirrhotics derives from portal drained viscera owing to glutamine deamidation, increased glutaminase activity in the intestine seems to be responsible for systemic hyperammonemia. Lastly, some genetic alterations in the glutaminase gene such as the haplotype TACC could modulate intestinal ammonia production and the risk of overt hepatic encephalopathy in cirrhotics.

Bcl-2 and Mn-SOD Antisense Oligodeoxynucleotides and a Glutamine-enriched Diet Facilitate Elimination of Highly Resistant B16 Melanoma Cells by Tumor Necrosis Factor-α and Chemotherapy

Mitochondrial glutathione (mtGSH) depletion increases sensitivity of Bcl-2-overexpressing B16 melanoma (B16M)-F10 cells (high metastatic potential) to tumor necrosis factor-alpha (TNF-alpha)-induced oxidative stress and death in vitro. In vivo, mtGSH depletion in B16M-F10 cells was achieved by feeding mice (where the B16M-F10 grew as a solid tumor in the footpad) with an L-glutamine (L-Gln)-enriched diet, which promoted in the tumor cells an increase in glutaminase activity, accumulation of cytosolic L-glutamate, and competitive inhibition of GSH transport into mitochondria. L-Gln-adapted B16M-F10 cells, isolated using anti-Met-72 monoclonal antibodies and flow cytometry-coupled cell sorting, were injected into the portal vein to produce hepatic metastases. In l-Gln-adapted invasive (iB16M-Gln+) cells, isolated from the liver by the same methodology and treated with TNF-alpha and an antisense Bcl-2 oligodeoxynucleotide, viability decreased to approximately 12%. iB16M-Gln+ cell death associated with increased generation of O2*- and H2O2, opening of the mitochondrial permeability transition pore complex, and release of proapoptotic molecular signals. Activation of cell death mechanisms was prevented by GSH ester-induced mtGSH replenishment. The oxidative stress-resistant survivors showed an adaptive response that includes overexpression of manganese-containing superoxide dismutase (Mn-SOD) and catalase activities. By treating iB16M-Gln+ cells with a double anti- antisense therapy (Bcl-2 and SOD2 antisense oligodeoxynucleotides) and TNF-alpha, metastatic cell survival decreased to approximately 1%. Chemotherapy (taxol plus daunorubicin) easily removed this minimum percentage of survivors. This contribution identifies critical molecules that can be sequentially targeted to facilitate elimination of highly resistant metastatic cells.

Growth and metabolism of marine fish Chinook salmon embryo cells: response to lack of glucose and glutamine

A peculiar phenomenon, differing from the response of mammalian cells, occurred when Chinook salmon embryo (CHSE) cells were passaged in the medium lacking of both glucose and glutamine. To elucidate metabolic mechanism of CHSE cells, the metabolism parameters, key metabolic enzymes, and ATP levels were measured at different glucose and glutamine concentrations. In the glutamine-free culture, hexokinase activity kept constant, and lactate dehydrogenase (LDH) activity decreased. This indicated that lack of glutamine did not expedite glucose consumption but made it shift to lower lactate production and more efficient energy metabolism. The results coincided with the experimental results of unaltered specific glucose consumption rate and decreased yield coefficients of lactate to glucose. In the glucose-free culture, simultaneous increase of glutaminase activity and of specific ammonia production rate suggested an increased flux into the glutaminolysis pathway, and increases of both glutamate dehydrogenase activity and yield coefficient of ammonia to glutamine showed an increased flux into deamination pathway. However, when glucose and glutamine were both lacking, the specific consumption rates of most of amino acids increased markedly, together with decrease of LDH activity, indicating that pyruvate derived from amino acids, away from lactate production, remedied energy deficiency. When both glucose and glutamine were absent, intracellular ATP contents and the energy charge remained virtually unaltered.

Control of Puberty by Excitatory Amino Acid Neurotransmitters and its Clinical Implications

Excitatory amino acids, glutamate in particular, have a marked stimulatory effect on the reproductive axis, particularly at puberty. Glutamate, N-methyl-D-aspartate (NMDA), and kainate stimulate gonadotropin-releasing hormone (GnRH) secretion in immature mammals and NMDA receptor stimulation results in precocious puberty in rats and monkeys. Puberty is characterized by an increased sensitivity of GnRH to glutamate as well as an increase in glutaminase activity in the hypothalamus. Glutamatergic and GABAergic regulation of GnRH secretion seem strongly interdependent around puberty. In addition to the transsynaptic glutamatergic regulation of GnRH secretion, a coordinated activity of glutamatergic neurons and astroglial cells has been shown to play an active role in puberty. The participation of kainate receptors in the estradiol-induced advancement of puberty suggest that these receptors may be involved in the estradiol-mediated activation of GnRH secretion at puberty. A case of precocious puberty associated with hyperglycinemia illustrates the NMDA involvement in puberty in humans. In this patient, the occurrence of precocious puberty was thought to result from excessive stimulation by glycine of the NMDA receptors linked to the GnRH neurons. Glutamate plays several roles in the hypothalamic mechanism of puberty as it has been shown in animal models, but there are still few clinical data supporting the role of glutamate in human puberty.

Functional characterization of a salt- and thermotolerant glutaminase from Lactobacillus rhamnosus

The deamination of glutamine is a crucial step in the production of enzymatically hydrolyzed plant proteins to reach high glutamic acid yields. The required glutaminase activity usually is provided by addition of technical enzymes or by in situ generation from fungi, yeast or bacteria (i.e. Aspergillus oryzae in soy sauce production). We screened food-grade Lactobacilli for potential glutaminase activity and selected the enzyme found in Lactobacillus rhamnosus for further characterization. Glutaminase from L. rhamnosus was induced by growing the microorganism on hydrolyzed wheat gluten, a glutamine-rich protein source. Glutamine deaminating activity (glutaminase, EC 3.5.1.2) was found to be membrane-bound and lost its activity gradually upon solubilization. Functional studies of the glutaminase showed an optimal working pH of 7.0 and maximum activity at 50 °C. High salt-tolerance of the enzyme was observed, i.e. the presence of 5% (w/v) salt increased glutaminase activity almost two-fold and 90% of the initial activity still remained at 15% (w/v) salt. The glutaminase activity showed typical Michaelis–Menten behavior with an affinity constant K m of 4.8±0.4 mM for glutamine and a V max of 101±2 U/l.

PH-responsive stabilization of glutamate dehydrogenase mRNA in LLC-PK1-F+ cells.

During chronic metabolic acidosis, the adaptive increase in rat renal ammoniagenesis is sustained, in part, by increased expression of mitochondrial glutaminase (GA) and glutamate dehydrogenase (GDH) enzymes. The increase in GA activity results from the pH-responsive stabilization of GA mRNA. The 3'-untranslated region (3'-UTR) of GA mRNA contains a direct repeat of an eight-base AU-rich element (ARE) that binds zeta-crystallin/NADPH:quinone reductase (zeta-crystallin) with high affinity and functions as a pH-response element. RNA EMSAs established that zeta-crystallin also binds to the full-length 3'-UTR of GDH mRNA. This region contains four eight-base sequences that are 88% identical to one of the two GA AREs. Direct binding assays and competition studies indicate that the two individual eight-base AREs from GA mRNA and the four individual GDH sequences bind zeta-crystallin with different affinities. Insertion of the 3'-UTR of GDH cDNA into a beta-globin expression vector (pbetaG) produced a chimeric mRNA that was stabilized when LLC-PK1-F+ cells were transferred to acidic medium. A pH-responsive stabilization was also observed using a betaG construct that contained only the single GDH4 ARE and a destabilizing element from phosphoenolpyruvate carboxykinase mRNA. Therefore, during acidosis, the pH-responsive stabilization of GDH mRNA may be accomplished by the same mechanism that affects an increase in GA mRNA.

Mechanism of increased renal gene expression during metabolic acidosis.

Increased renal catabolism of plasma glutamine during metabolic acidosis generates two ammonium ions that are predominantly excreted in the urine. They function as expendable cations that facilitate the excretion of acids. Further catabolism of alpha-ketoglutarate yields two bicarbonate ions that are transported into the venous blood to partially compensate for the acidosis. In rat kidney, this adaptation is sustained, in part, by the induction of multiple enzymes and various transport systems. The pH-responsive increases in glutaminase (GA) and phosphoenolpyruvate carboxykinase (PEPCK) mRNAs are reproduced in LLC-PK(1)-fructose 1,6-bisphosphatase (FBPase) cells. The increase in GA activity results from stabilization of the GA mRNA. The 3'-untranslated region of the GA mRNA contains a direct repeat of an eight-base AU sequence that functions as a pH-response element. This sequence binds zeta-crystallin/NADPH:quinone reductase with high affinity and specificity. Increased binding of this protein during acidosis may initiate the pH-responsive stabilization of the GA mRNA. In contrast, induction of PEPCK occurs at the transcriptional level. In LLC-PK(1)-FBPase(+) kidney cells, a decrease in intracellular pH leads to activation of the p38 stress-activated protein kinase and subsequent phosphorylation of transcription factor ATF-2. This transcription factor binds to cAMP-response element 1 within the PEPCK promoter and may enhance its transcription during metabolic acidosis.

Changes in the activity levels of glutamine synthetase, glutaminase and glycogen synthetase in rats subjected to hypoxic stress.

Exposure to high altitude causes loss of body mass and alterations in metabolic processes, especially carbohydrate and protein metabolism. The present study was conducted to elucidate the role of glutamine synthetase, glutaminase and glycogen synthetase under conditions of chronic intermittent hypoxia. Four groups, each consisting of 12 male albino rats (Wistar strain), were exposed to a simulated altitude of 7620 m in a hypobaric chamber for 6 h per day for 1, 7, 14 and 21 days, respectively. Blood haemoglobin, blood glucose, protein levels in the liver, muscle and plasma, glycogen content, and glutaminase, glutamine synthetase and glycogen synthetase activities in liver and muscle were determined in all groups of exposed and in a group of unexposed animals. Food intake and changes in body mass were also monitored. There was a significant reduction in body mass (28-30%) in hypoxia-exposed groups as compared to controls, with a corresponding decrease in food intake. There was rise in blood haemoglobin and plasma protein in response to acclimatization. Over a three-fold increase in liver glycogen content was observed following 1 day of hypoxic exposure (4.76 +/- 0.78 mg.g-1 wet tissue in normal unexposed rats; 15.82 +/- 2.30 mg.g-1 wet tissue in rats exposed to hypoxia for 1 day). This returned to normal in later stages of exposure. However, there was no change in glycogen synthetase activity except for a decrease in the 21-days hypoxia-exposed group. There was a slight increase in muscle glycogen content in the 1-day exposed group which declined significantly by 56.5, 50.6 and 42% following 7, 14, and 21 days of exposure, respectively. Muscle glycogen synthetase activity was also decreased following 21 days of exposure. There was an increase in glutaminase activity in the liver and muscle in the 7-, 14- and 21-day exposed groups. Glutamine synthetase activity was higher in the liver in 7- and 14-day exposed groups; this returned to normal following 21 days of exposure. Glutamine synthetase activity in muscle was significantly higher in the 14-day exposed group (4.32 mumol gamma-glutamyl hydroxamate formed.g protein-1.min-1) in comparison to normal (1.53 mumol gamma-glutamyl hydroxamate formed.g protein-1.min-1); this parameter had decreased by 40% following 21 days of exposure. These results suggest that since no dramatic changes in the levels of protein were observed in the muscle and liver, there is an alteration in glutaminase and glutamine synthetase activity in order to maintain nitrogen metabolism in the initial phase of hypoxic exposure.