Glutamine Synthase Activity Research Articles

Carbon, Nitrogen, and Nutrient Interactions in Beta vulgaris L. as Influenced by Nitrogen Source, NO3- versus NH4+

Sugar beets (Beta vulgaris L. cv F58-554H1) were grown hydroponically in a 16-h light, 8-h dark period (photosynthetic photon flux density of 0.5 mmol m-2 s-1) for 4 weeks from sowing in half-strength Hoagland nutrient solution containing 7.5 mM nitrate. Half of the plants were then transferred to 7.5 mM ammonium N; the rest remained in solution with 7.5 mM nitrate N. Upon transfer from nitrate to ammonium, the total N concentration decreased sharply in the fibrous roots and petiole/midribs and increased substantially in the leaf blades. This was because of the decreased nitrate concentrations in fibrous roots and petioles and a concomitant increase in amino acid/amide-N and protein N in leaf blades. Sugar beets acclimated to ammonium partly by a 2.5-fold increase in glutamine synthase activity in fibrous roots and a 1.7-fold increase in leaf blades. Rapid ammonium assimilation into glutamine consumed carbon skeletons, leading to a depletion of foliar starch, sucrose, and maltose. Ammonium treatment stimulated activities of some glycolytic/Krebs cycle enzymes, e.g. pyruvate dehydrogenase. Nitrate-fed leaf blades contained substantially larger concentrations of osmolytes (i.e. nitrate, cations, and sucrose), which may have contributed to the faster rates of leaf expansion in nitrate-fed compared to ammonium-fed plants.

Oxidative Damage to Lipids and DNA Concurrent with Decrease of Antioxidants in Rat Testes After Acute Iron Intoxication

The effect of acute iron overload was studied in rat testes 20 h after a single administration of iron-dextran (500 mg/kg body wt, ip). Total testes iron content was 6.1-fold higher in iron-treated rats compared to controls. The endogenous level of lipid peroxidation was evaluated as 2-thiobarbituric acid-reactive substances (TBARS). Testes iron concentration (0.12-2.67 μmol/g of tissue) was positively correlated (r = 0.86; P < 0.01) with testes TBARS (26.2-77.5 nmol/g tissue). Testes content of lipid-soluble antioxidants, α-tocopherol, ubiquinol-9, and ubiquinol-10, were inversely correlated with testes iron content. The steady-state level of 8-oxo-7,8-dihydro-2′-deoxyguanosine in testes DNA was 25% higher (P < 0.01) in iron-treated rats compared to controls (2.4 ± 0.2 oxo8dG/105dG). The content of protein carbonyl groups (1.45 ± 0.13 nmol/mg protein) and the activity of glutamine synthase (1.32 ± 0.07 units/mg protein) were similar for the iron-treated and control rats. Fe treatment did not affect superoxide dismutase, catalase, and glutathione peroxidase activities. The results indicate that acute iron overload causes iron accumulation in rat testes, which is associated with increased lipid and DNA oxidative damage and depletion of lipid-soluble antioxidants.

Lobular patterns of expression and enzyme activities of glutamine synthase, carbamoylphosphate synthase and glutamate dehydrogenase during postnatal development of the porcine liver

Carbamoylphosphate synthase and glutamine synthase show a complementary distribution in the liver lobule of the rat. In the human liver lobule, which is approximately 2-fold larger than that of the rat, an intermediate, ‘empty’ zone is present between the periportal carbamoylphosphate synthase-positive and the pericentral glutamine synthase-positive zone. To investigate whether these differences in gene expression can be attributed to the size of the liver lobule, we investigated the patterns of expression of carbamoylphosphate synthase, glutamine synthase and glutamate dehydrogenase during postnatal development of the pig, a species in which the total number of lobules does not increase after birth. We demonstrate that lobular size increases 3-fold between 1 week and 8 months after birth. In the same developmental period the number of hepatocytes on the porto-central axis increases 2-fold, resulting in a 3-fold increase in cellular volume. However, the lobular patterns of expression of carbamoylphosphate synthase, glutamate dehydrogenase and glutamine synthase do not change anymore after 1 month, i.e., when lobular diameter is comparible to that in rat liver, showing that lobular size is not a major determinant of the heterogeneous patterns of expression of these enzymes. The adult patterns of expression of glutamine synthase, glutamate dehydrogenase and, in particular carbamoylphosphate synthase in the porcine liver resemble those of man. Changes in the enzyme activities fo glutamate dehydrogenase and carbamoylphosphate synthase are not related to the lobular size. However, the 70% decrease of GS activity in the 8-month-old pigs corresponds with the gradual 2–3-fold decrease in the size of the GS-positive compartment during postnatal developmental. During adulthood GS activity increases again to values observed 1 week after birth demonstrating a 2-fold increase in cellular glutamine synthase content. The present data show that the pig is an excellent model to study the regulation and functional implication of zonation of gene expresion in the human liver.

Distribution and activity of glutamine synthase and carbamoylphosphate synthase upon enlargement of the liver lobule by repeated partial hepatectomies

Glutamine synthase and carbamoylphosphate synthase show a strikingly heterogeneous and fully complementary distribution in the rat liver. In the human liver, however, there is a midlobular zone where both enzymes are absent. The diameter of the human liver lobule is approximately twice the size of the rat lobule. To investigate whether lobule size is a major determinant for the expression patterns of glutamine synthase and carbamoylphosphate synthase, Wistar strain rats were partially hepatectomized 3 times, at weekly or monthly intervals. Due to hepatic regeneration the cross-sectional area of the liver lobules increased twofold. However, a midlobular zone which lacked expression of both glutamine synthase and carbamoylphosphate synthase did not develop in these livers, thus showing that lobular size is not a major determinant for the distribution patterns of glutamine and carbamoylphosphate synthase. The twofold increase in the cross-sectional area of the liver lobule was associated with a similar reduction in the relative number of glutamine synthase-positive cells and in the enzyme content of the liver, indicating that the regeneration process does not affect the pericentral pattern of glutamine synthase expression. After regeneration was complete, the glutamine synthase content in the liver was restored to its original value, demonstrating a twofold increase in the cellular concentration of glutamine synthase-positive hepatocytes. An increase in the diameter of the liver lobule was only seen after the first partial hepatectomy. Liver growth following subsequent partial hepatectomies can be explained by an increase in the length of the liver lobule and/or by splitting of liver lobules. The zonal distribution of DNA replication, which is characteristic of the first partial hepatectomy, is lost after repeated partial hepatectomies. Furthermore, evidence was obtained that the signal for inducing DNA synthesis may originate at the level of single liver units.

Cerebral metabolism of [1,2-13C2]acetate as detected by in vivo and in vitro 13C NMR.

The metabolism of [1,2-13C2]acetate in rat brain was studied by in vivo and in vitro 13C NMR spectroscopy, in particular by taking advantage of the homonuclear 13C-13C spin coupling patterns. Well nourished rats were infused with [1,2-13C2]acetate or [1-13C]acetate in the jugular vein, and the in situ kinetics of 13C labeling during the infusion period was followed by 13C NMR techniques. The in vivo 13C NMR spectra showed signals from (i) the C-1 carbon of [1,2-13C2] acetate or [1-13C]acetate, (ii) 13CO3H-, and (iii) the natural abundance 13C carbons of sufficiently mobile fatty acids. Methanol/HCl/perchloric acid extracts of the brains were prepared and were further analyzed by high resolution 13C NMR. The homonuclear 13C-13C spin coupling patterns after infusion of [1,2-13C2]acetate showed very different isotopomer populations in glutamate, glutamine, and gamma-aminobutyric acid. Analyzing the relative proportions of these isotopomers revealed (i) two different glutamate compartments in the rat brain characterized by the presence and absence, respectively, of glutamine synthase activity, (ii) two different tricarboxylic acid cycles, one preferentially metabolizing [(1,2-13C2]acetate, the other mainly using unlabeled acetyl-coenzyme A, (iii) a hitherto unknown cerebral pyruvate recycling system associated with the tricarboxylic acid cycle, metabolizing primarily unlabeled acetyl-coenzyme A, and (iv) a predominant production of gamma-aminobutyric acid in the glutamate compartment lacking glutamine synthase.

A simple animal model of hyperammonemia.

Rats were fed a standard diet or the standard diet supplemented with ammonium acetate (20% w/w) for up to 100 days. The effect of the ingestion of the high-ammonium diet on some aspects of nitrogen metabolism in rats was studied. Ammonia levels in blood increased approximately 3-fold; in brain, liver and muscle the increases were 36, 34 and 50%, respectively. Urea levels in blood and urea excretion increased approximately 2-fold. There was no increase of carbamyl phosphate synthase. Liver glutamine synthase activity increased by 58% and glutamate dehydrogenase by 40%, whereas glutaminase was not affected. Glutamine content in brain was twice that of controls. This new animal model to study hyperammonemia offers several advantages over others: it is simpler, is bloodless, requires no animal manipulation and permits long-term studies.

Characteristics of rat hepatocytes sorted by fluorescence-activated flow cytometry: Effects of mixed function oxidase inducers

Fluorescence-activated flow cytometry has been used to separate rat liver parenchymal cells into five equal computer-generated regions on the basis of their mixed function activities towards diethoxyfluorescein. Enzymic activities in the sorted fractions showed 2.3–2.9-fold enrichment of 3-cyano-7-ethoxycoumarin O-deethylase, lactate dehydrogenase and pyruvate kinase activities in the cells of region 5. Cytochrome P-450 PBla and glutamine synthase activities were enriched 1.8- and 7-fold respectively in region 2 hepatocytes. Pretreatment of rats with phenobarbitone or 3-methylcholanthrene did not change the pattern of enrichment with respect to monooxygenase or lactate dehydrogenase activities. However, cytochrome P-450 PB3a and MC1a respectively were concentrated in the cells from region 5. In contrast, ethoxyquin caused enrichment of cytochrome P-450 in the hepatocytes in region 2. This fraction also contained the highest percentage of γ-glutamyltranspeptidase positive cells.

Changes in the activity of glutamate related enzymes in cerebral cortex, during insulin-induced seizures.

The activity of glutamate related enzymes and the concentration of glutamine, glutamate and gamma-amino n-butyric acid (GABA) were investigated in the cerebral cortex of rats, in different stages of insulin-induced hypoglycemia. Hypoglycemia was produced by intraperitoneal injection of insulin 0.05-100 units per kg body weight. The minimum required dose to produce irreversible severe hypoglycemia was 0.5 units/kg. In 85% of the cases an insulin induced hypoglycemic convulsion, was achieved 130-150 minutes after injection. Blood glucose levels during insulin induced seizures ranged between 8-15 mg%. In the range of 0.5-100 u insulin/kg the degree of hypoglycemia and the onset of convulsions were identical. The concentration of glutamine was significantly reduced during convulsive and postconvulsive stages. Glutamate and GABA concentrations were reduced significantly in all stages of insulin-induced hypoglycemia. The decrease in glutamine concentration was concurrent with an increase in the activity of its degradative enzyme, glutaminase. This was apparent at the preconvulsive, convulsive and postconvulsive stages. The activity of other enzymes related to energy production such as glutamate dehydrogenase (GDH), glutamate transaminase (GPT) and aspartate aminotransferase (AAT) were also increased. The activity of glutamine synthase (GS) was unaffected by hypoglycemia. Insulin induced changes in glutamine, glutamate and their related enzymes could not be attributed to convulsion since a similar pattern of changes was observed in the preconvulsive and postconvulsive stages, and no changes were detected following picrotoxin-induced seizures.