Glutamine Formation Research Articles

On the Function of Mitochondrial Metabolism during Photosynthesis in Spinach (Spinacia oleracea L.) Leaves (Partitioning between Respiration and Export of Redox Equivalents and Precursors for Nitrate Assimilation Products).

The functioning of isolated spinach (Spinacia oleracea L.) leaf mitochondria has been studied in the presence of metabolite concentrations similar to those that occur in the cytosol in vivo. From measurements of the concentration dependence of the oxidation of the main substrates, glycine and malate, we have concluded that the state 3 oxidation rate of these substrates in vivo is less than half of the maximal rates due to substrate limitation. Analogously, we conclude that under steady-state conditions of photosynthesis, the oxidation of cytosolic NADH by the mitochondria does not contribute to mitochondrial respiration. Measurements of mitochondrial respiration with glycine and malate as substrates and in the presence of a defined malate:oxaloacetate ratio indicated that about 25% of the NADH formed in vivo during the oxidation of these metabolites inside the mitochondria is oxidized by a malate-oxaloacetate shuttle to serve extramitochondrial processes, e.g. reduction of nitrate in the cytosol or of hydroxypyruvate in the peroxisomes. The analysis of the products of the oxidation of malate indicates that in the steady state of photosynthesis the activity of the tricarboxylic acid cycle is very low. Therefore, we have concluded that the mitochondrial oxidation of malate in illuminated leaves produces mainly citrate, which is converted via cytosolic aconitase and NADP-isocitrate dehydrogenase to yield 2-oxoglutarate as the precursor for the formation of glutamate and glutamine, which are the main products of photosynthetic nitrate assimilation.

Extracellular potassium regulates the glutamine content of astrocytes: mediation by intracellular pH

Based upon previous evidence that glutamine formation in astrocytes is pH-sensitive and that raised extracellular K+ alkalinizes astrocytic cytoplasm, it was hypothesized that extracellular K+ might regulate glutamine formation. In this study, the free glutamine content of mouse cerebral astrocytes incubated with 0.1 mM glutamate and 0.1 mM ammonium increased by 80-90% when the extracellular K+ concentration was raised from 3 to 12 mM. The corresponding K(+)-induced intracellular alkalinization of +0.13 pH units only partially reversed a glutamate-induced intracellular acidification of -0.24 pH units. By comparison, adjustment of extracellular pH from 7.4 to 7.8 shifted intracellular pH by +0.25 pH units, fully reversing the glutamate-induced acidification and increasing glutamine content by 120-180%. The effect of K+ on intracellular pH increased to +0.25 pH units in bicarbonate-buffered solution, suggesting that the regulation of glutamine formation by extracellular K+ is enhanced in the presence of bicarbonate.

Interaction between the glutamine cycle and the uptake of large neutral amino acids in astrocytes.

When astrocyte cultures are incubated with glutamate and ammonium, the clearance of these substrates followed by the formation and export of glutamine simulates the action of the "glutamine cycle" that is believed to function in the CNS. In the present study this process was found to increase the uptake of large neutral amino acids (LNAAs), namely, histidine, kynurenine, leucine, phenylalanine, and tryptophan, by two- to threefold in mouse cerebral astrocytes. The enhancement of kynurenine uptake was shown to depend on the formation of glutamine and to saturate at low levels of glutamine. LNAAs transiently lowered the glutamine content of astrocytes that were incubated with glutamate and ammonium, but they did not affect net export of glutamine to the solution at normal physiological pH. However, on adjustment of the pH of the solution to 7.8, which causes a large increase in glutamine content without affecting export, kynurenine now significantly increased net glutamine export. These findings relate to proposed mechanisms of cerebral dysfunction in hyperammonemia.

The fate of newly absorbed ammonium and nitrate ions in roots of jack pine seedlings

To examine the metabolic basis of the inability of boreal conifers to use nitrate efficiently, the assimilation and partitioning of 15N from 15NO3− and 15NH4+ was investigated in young jack pine seedlings (Pinus banksiana Lamb.) grown with either ammonium or nitrate nitrogen. 15N partitioning was also followed after exposure to enzymatic inhibitors for GS, GOGAT and aminotransferases. The accumulation patterns of 15N in roots indicated that newly absorbed ammonium ions were assimilated by the action of GS, and that GDH played only a very minor role. Results also demonstrated that ammonium incorporated into glutamine by GS was subsequently transf erred to glutamate by the activity of GOGAT. When GOGAT was inhibited, glutamine formation by GS also decreased and 15N accumulated in ammonium. Rapid accumulation of 15N in asparagine suggested that AS may play a role in the assimilation of free ammonium via glutamine. Rates of label incorporation were lower in nitrate-fed plants, and the inhibitors further reduced the amount of 15N in roots relative to the controls. The assimilation kinetics of 15N from 15NO3− showed that at least part of the incorporated 15N flowed through the GS/GOGAT cycle, as in ammonium-fed plants. This indicated that nitrate reduction takes place in root cells of jack pine. However, the response of nitrate-fed plants to GS and GOGAT inhibitors is atypical of nitrophilous plants. Exposure of nitrate-fed plants to MSO and azaserine resulted in a decrease in overall recovery of label in Gln, Glu, Asp and Asn with no significant modification of the distribution pattern. These results suggest either that: (1) part of the incorporated 15N-NO3 was assimilated through an alternative route, (2) nitrate absorption or nitrate reduction were inhibited by GS or GOGAT inhibitors or (3) the GS inhibitor did not inhibit GS activity at the site where nitrate was reduced.

The nutritive function of glia in a crystal-like nervous tissue: the retina of the honeybee drone.

Among the variety of roles and diverse possible functions that have been attributed to glial cells, the nutritive function is strongly supported by direct experimental evidence obtained in a model of the honeybee drone retina. We have shown that in this nervous tissue, with crystal-like structure, in which glial cells and photoreceptor neurons constitute two distinct metabolic compartments, glial cells transform glucose to alanine and, with proline, fuel the mitochondria of the photoreceptors. Proline supplies the Krebs cycle by making glutamate. The use of proline implies high ammonia production. Pyruvate transamination in the glia fixes ammonia at a rate exceeding glutamine formation. We favor the hypothesis that ammonia rather than K+ is the metabolic signal trafficking between neuron and glial cells.

Light and electron microscopic immunocytochemical localization of glutamine synthetase in the superficial pineal gland of the rat.

Glutamine synthetase (L-glutamate:ammonia ligase; EC 6.3.1.2), an enzyme catalysing the ATP-dependent formation of glutamine from glutamate and ammonia, was detected immunocytochemically only in glial (interstitial) cells of the superficial pineal gland of the rat. The results show the important role of pineal glial cells in the metabolism of the presumptive neurotransmitters, glutamate and gamma-aminobutyric acid (GABA) as well as in detoxification of ammonia.

Amino Acid Metabolism, Muscular Fatigue and Muscle Wasting

Recent investigations from our and other laboratories indicate that glycogen is a carbon-chain precursor in muscle for the synthesis of TCA cycle intermediates and glutamine. During intense exercise and in conditions of a relative lack of energy (hypoxia, trauma, sepsis) the metabolism of branched-chain amino acids (BCAA) is accelerated in muscle. In the primary BCAA aminotransferase reaction 2-oxoglutarate is used as amino-group acceptor (putting a carbon-drain on the TCA cycle) under formation of glutamate. Glutamate will subsequently react with ammonia, generated in the AMP deaminase reaction or by deamination of amino acids, under formation of glutamine in a reaction catalysed by glutamine synthetase (glutamate + ammonia + ATP--> glutamine + ADP). Muscle glycogen stores may be smaller or less available at high altitude. It is hypothesized that this will lead to premature fatigue (due to both a lack of fuel and of TCA cycle carbon-precursor) and to a reduction in the synthesis rate of glutamine. A chronic reduction in the synthesis rate of glutamine during a long term stay at high altitude on its turn may lead to gut atrophy, bacterial translocation, endotoxemia, muscle protein catabolism and a weakened immune status.

Regulation of urea uptake in Pseudomonas aeruginosa.

The energy-dependent urea permease was studied in two strains of Pseudomonas aeruginosa, measuring the uptake (transport and metabolism) of 14C-urea. In both strains urea uptake in vivo and urease activity in vitro differed significantly with respect to kinetic parameters, temperature and pH dependence and response to metabolic inhibitors. Ammonium strongly interfered both with the expression of the urea uptake system and its activity. The inhibition of the uptake activity by ammonium was partially relieved by hydraziniumsulfate, which prevented the translocation of ammonium into the cell, and in a methylammonium/ammonium transport-defective mutant of strain DSM 50071. Furthermore, methionine-sulfoximine, which prevented the intracellular glutamine formation from ammonium via inhibition of glutamine synthetase, relieved the inhibition of urea uptake by ammonium. These findings suggested that urea uptake activity in P. aeruginosa is regulated by intracellular glutamine.

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.

Dissociation by NH4Cl treatment of the enzymic activities of glutamine synthetase II from Rhizobium leguminosarum biovar viceae.

Glutamine synthetase II (GSII) was purified to homogeneity from Rhizobium leguminosarum biovar viceae and characterized. The sequence of 26 amino acid residues from the amino-terminal end of the protein showed high similarity with the sequence of GSII from Bradyrhizobium japonicum or from Rhizobium meliloti. Non-denaturing PAGE showed that GSII, either in crude extracts or in the pure state, was a mixture of an octamer and a tetramer and that under specific conditions the octamer/tetramer ratio could be modified in either direction. The pure enzyme was used to raise an antiserum which was highly specific. Addition of NH4Cl to a bacterial culture derepressed for GSII caused a specific decrease in transferase activity, faster than the one observed when the amount of immunoreactive material was measured by different methods. On the other hand, biosynthetic activity, measured as the rate of ADP or glutamine formation, paralleled the rate of decrease in immunoreactive material. A partially purified enzyme preparation retained this dissociation of kinetic parameters, strongly suggesting a post-translational modification. These findings are discussed with respect to the possible role of GSII in the Rhizobium-legume symbiosis.

Selective inhibition of glial cell metabolism in vivo by fluorocitrate

The effect of fluorocitrate on glial and neuronal amino acid metabolism was studied. One nmol of fluorocitrate administered intrastriatally in the rat caused a 95% reduction of glutamine formation from [ 14C]acetate, a substrate which enters the glial cells selectively. The metabolism of [ 14C]glucose which enters neurons, was unaffected by fluorocitrate treatment except for the glutamine formation. This is evidence that fluorocitrate is a selective inhibitor of the glial Krebs' cycle. [ 14C]Citrate and 2-oxoglutarate labelled amino acids in a manner similar to [ 14C]acetate, which shows that these substrates are taken up and metabolized by glial cells. Differences in the labelling of γ-aminobutyric acid (GABA) from [ 14C]acetate and citrate suggest that astrocytes associated with GABAergic and glutamatergic nerve terminals may differ in their preference for amino acid precursors.

Chapter 18: Nitrogen metabolism: neuronal-astroglial relationships

The extracellular concentration of glutamic acid in the central nervous system (CNS) is much lower than that of peripheral tissues (2 - 3 pM vs - 50 pM). This is important for two reasons: (a) glutamate is the premier excitatory neurotransmitter, and a low level in the synaptic cleft amplifies the signal-to-noise ratio upon the release of this amino acid from pre-synaptic terminals; and (b) excessive stimulation of certain glutamatergic neurons, particularly those bearing N-methyl-Daspartate (NMDA) receptors, can lead to neuronal injury and/or death. Astrocytes are well equipped to metabolize the glutamate they transport since they are enriched with the glutamine synthetase pathway. The importation of glutamate into astrocytes and subsequent formation of glutamine constitutes one limb of the “glutamate-glutamine cycle,” which is completed by the export of glutamine to neurons, where it is hydrolyzed to glutamate via phosphate dependent glutaminase. The glutamate-glutamine cycle, thereby accomplishes the two major requirements of glutamatergic neurotransmission— that is, the rapid removal of glutamate from the synaptic cleft, and the restoration of glutamate to neurons in the form of glutamine, a non-neuroactive compound that can be shuttled safely between astrocytes and neurons.

Glutamine-dependent urea synthesis in elasmobranch fishes

Marine elasmobranchs (sharks, skates, and rays) synthesize and retain in their tissues high concentrations of urea (up to 0.4 M) and certain amines, such as trimethylamine oxide (up to 0.2 M), for the purpose of osmoregulation. Although the pathway of urea synthesis in ureosmotic elasmobranchs is similar to the classical urea cycle in ureotelic species, there are several properties that are unique. Indeed, this might be anticipated, since the primary function of urea synthesis in elasmobranchs is osmoregulation, rather than ammonia detoxification. The existence in different species of a major biosynthetic pathway resulting in the same end product, but which serves quite different physiological purposes in each species, can provide an opportunity for comparative investigations that contribute to our understanding of the regulatory properties and evolutionary development of the pathway. The purpose of this commentary is to consider the unique properties of urea synthesis in elasmobranchs and to comment on the comparative relationships to teleost fishes and mammalian species; most studies discussed here have been carried out with spiny dogfish shark, Squalus acanthias, a representative elasmobranch. Like ureotelic species, hepatic urea synthesis in ureosmotic elasmobranchs requires both mitochondrial and cytosolic enzymes, but there are notable differences (Fig. 1). The most significant difference is that the initial step of ammonia assimilation for urea synthesis in hepatic mitochondria of elasmobranchs is the formation of glutamine, which is subsequently utilized as the substrate for carbamoyl-phosphate synthesis (Anderson and Casey, J. Biol. Chem., 259, 456-462, 1984). This is due to the presence of high levels of glutamine synthetase (GSase) and a unique acetylglutamateand glutamine-dependent carbamoyl-phosphate synthetase (CPSase 111), both localized exclusively in the mitochondrial matrix (Casey and Anderson, J. Biol. Chem., 257,8449-8453, 1982, and Comp. Biochem. Physiol., 82B, 307-315,1985). GSase is a cytosolic enzyme in liver of mammalian species and in some, but not all, teleost fishes. In mammalian and amphibian species the first step of ammonia assimilation for urea synthesis in liver is direct conversion of ammonia to carbamoyl phosphate catalyzed by a corresponding mitochondrial acetylglutamateand ammoniadependent CPSase I (which cannot utilize glutamine as a substrate). The properties of elasmobranch mitochondrial CPSase I11 are similar to the mammalian mitochondrial CPSase I, except for the fact that glutamine rather than ammonia serves as the nitrogen-donating substrate (Anderson, J. Biol. Chem., 256, 12 228 12 238, 1981).

Cloning and functional characterization of the rat glutamine synthetase gene

Glutamine synthetase catalyzes the formation of glutamine from glutamate and ammonia. It plays a central role in both amino acid neurotransmitter metabolism and ammonia detoxification in the central nervous system. Glutamine synthetase expression is regulated in developmental, hormonal, and in tissue- and cell-specific manners. We have cloned a full-length cDNA coding for rat glutamine synthetase, and have found an AT-rich area of conservation in the 3′- untranslated regions between rat, mouse, and chicken, which may play a part in the regulation of the stability of the glutamine synthetase message. We have also cloned and mapped the gene coding for rat glutamine synthetase, and identified, by sequence analysis, areas potentially important for the regulation of glutamine synthetase transcription. Transient transfection of a variety of cell lines with deletion constructs of the glutamine synthetase promoter driving a chloramphenicol acetyltransferase reporter gene functionally demonstrates regions of the promoter containing elements important for transcriptional regulation.

Glutamine metabolism in skeletal muscle of septic rats

The metabolism of skeletal muscle glutamine was studied in rats made septic by cecal ligation and puncture technique. Blood glucose was not significantly different in septic rats, but lactate, pyruvate, glutamine, and alanine were markedly increased. Conversely, blood ketone body concentrations were markedly decreased in septic rats. Both plasma insulin and glucagon were markedly elevated in septic rats. Sepsis increased the rates of glutamine production in muscle, but without marked effects on skin and adipose tissue preparations, with muscle production accounting for over 87% of total glutamine produced by the hindlimb. Sepsis produced decreases in the concentrations of skeletal muscle glutamine, glutamate, 2-oxoglutarate, and adenosine monophosphate (AMP). The concentrations of ammonia, pyruvate, and inosine monophosphate (IMP) were increased. Hindlimb blood flow showed no marked change in response to sepsis, but was accompanied by an enhanced net release of glutamine and alanine. The maximal activity of glutamine synthetase was increased only in quadriceps muscles of septic rats, whereas that of glutaminase was decreased in all muscles studied. Tyrosine release from incubated muscle preparation was markedly increased in septic rats; however, its rate of incorporation was markedly decreased. It is concluded that there is an enhanced rate of production of glutamine from skeletal muscle of septic rats. This may be due to changes in efflux and/or increased intracellular formation of glutamine; these suggestions are discussed.

The methionine analog S-methylthiocysteine stimulates neutral amino acid transport by isolated brain microvessels

Abstract As an extension of the hypothesis suggesting that the role of hyperammonemia and of plasma amino acid imbalance in the pathogenesis of hepatic encephalopathy is mediated by glutamine formation at the level of the blood-brain barrier, the possible effects of mercaptans on neutral amino acid transport by isolated brain microvessels were investigated. In this experimental system, methanethiol as such had no effect on any of the neutral amino acid transport systems. If the isolated brain microvessels were preloaded in the presence of both methanethiol and cystine (or cysteine), there was a marked stimulation of the L-system-mediated uptake of hydrophobic (branched or aromatic) neutral amino acids, this enhancement being similar to that caused by glutamine preloading. Cystine or cysteine alone were ineffective, nor could they be replaced by other non-sulfur-containing amino acids. The stimulation of the L-system could be attributed to the formation of S -methylthiocysteine, a mixed disulfide whose chemical structure is similar to that of methionine. S -methylthiocysteine was found to be formed spontaneously, upon preincubation with methanethiol plus cystine or cysteine, within the endothelial cells of isolated brain microvessels. Methanethiol, via S -methylthiocysteine formation, appears therefore to deserve a place, synergistically with the ammonia-glutamine system, among the precipitating causes of hepatic encephalopathy.

Effect of Ascaris lumbricoides Infection on Brain Enzymes

Glutamine synthetase (EC 6.3.1.2) catalyses the formation of glutamine from glutamate and ammonia coupled with the breakdown of ATP to ADP and phosphate. In the presence of ATP, glutamate and Mg, but in the absence of ammonia the enzyme forms a complex containing glutamate, ADP and phosphate; with ammonia, this complex forms glutamine, ADP, phosphate and free enzyme. The presence of ATP is necessary for the enzyme to bind glutamate, and for the reverse reaction ADP is required for the binding glutamine (7):

Glutamine Synthetase is Expressed by Primary Sensory Neurons from Chick Embryos In Vitro but not In Vivo: Influence of Skeletal Muscle Extract.

Glutamine synthetase (GS) catalyses the ATP-dependent formation of glutamine from glutamate and ammonia. To determine whether dorsal root ganglion (DRG) cells from chick embryos express the enzyme in vivo or in vitro, GS was detected by immunocytochemical reaction either in vibratome sections of DRG or in dissociated DRG cell cultures. The immunocytochemical detection of GS showed that in vivo the DRG taken from chick embryos at day 10 (E10), E14, E18 or from chickens after hatching were free of any GS-positive ganglion cells; in contrast, in neuron-enriched cultures of DRG cells grown in vitro at E10, virtually all the neuronal cells (98.6 +/- 1.0%) express GS at 3, 5 or 7 days of culture. In mixed DRG cell cultures, only 83.6+/-4.6% of the neurons displayed a GS-immunoreactivity. In both culture conditions, neither the presence of horse serum nor the age of the culture appeared to affect the percentage of neurons which displayed a GS-immunoreactivity. After [3H]glutamine uptake, radioautographs revealed that only 80% of the neurons were labelled in neuron-enriched DRG cell cultures while 96% of the neurons were radioactive in mixed DRG cell cultures. Furthermore the most heavily [3H]glutamine-labelled neurons were exclusively found in mixed DRG cell cultures. Combination of both immunocytochemical detection of GS and radioautography after [3H]glutamine uptake showed that strongly GS-immunostained neurons corresponded to poorly radioactive ones and vice versa. When skeletal muscle extract (ME) was added to DRG cell cultures, the number of GS-positive neurons was reduced to 77.5 +/- 2.5% in neuron-enriched cultures or to 43.6 +/- 3.8% in mixed DRG cell cultures; in both types of culture, the intensity of the neuronal immunostaining was depressed. Furthermore, combined action of ME and non-neuronal cells potentiates the enzyme repression exerted separately by ME or non-neuronal cells. Since GS-immunoreactivity is expressed in DRG cells grown in vitro, but not in vivo, it is suggested that microenvironmental factors influence the expression of GS. More specifically, the repression of GS by primary sensory neurons grown in vitro may be strongly induced by soluble factors present in skeletal muscle, and to a lesser extent in brain, and potentiated by non-neuronal cells.

Genetic transformation of glutamine auxotrophy to prototrophy in the cyanobacterium Nostoc muscorum

Glutamine auxotrophic (Gln-) and L-methionine D,L-sulfoximine (MSX) resistant (MSXr) mutants of N. muscorum were isolated and characterized for nitrogen nutrition, nitrogenase activity, glutamine synthetase (GS) activity and glutamine amide, alpha-keto-glutarate amido transferase (GOGAT) activity. The glutamine auxotroph was found to the GOGAT-containing GS-defective, incapable of growth with N2 or NH4+ but capable of growth with glutamine as nitrogen source, thus, suggesting GS to be the primary enzyme of both ammonia assimilation and glutamine formation in the cyanobacterium. The results of transformation and reversion studies suggests that glutamine auxotrophy is the result of a mutation in the gln A gene and that gln A gene can be transferred from one strain to another by transformation.

Glutamine Metabolism in Skeletal Muscle of Glucocorticoid-Treated Rats

1. The effect of dexamethasone (30 micrograms day-1 100 g-1 body weight) on the regulation of glutamine metabolism was studied in skeletal muscles of rats after 9 days of treatment. 2. Dexamethasone resulted in negative nitrogen balance, and produced increases in the plasma concentrations of alanine (23.4%) and insulin (158%) but a decrease in the plasma concentration of glutamine (28.7%). 3. Dexamethasone treatment increased the rate of glutamine production in muscle, skin and adipose tissue preparations, with muscle production accounting for over 90% of total glutamine produced by the hindlimb. 4. Blood flow and arteriovenous concentration difference measurements across the hindlimb showed an increase in the net exchange rates of glutamine (25.3%) and alanine (90.5%) in dexamethasone-treated rats compared with corresponding controls. 5. Dexamethasone treatment produced significant decreases in the concentrations of skeletal muscle glutamine (51.8%) and 2-oxoglutarate (50.8%). The concentrations of alanine (16.2%), pyruvate (45.9%), ammonia (43.3%) and inosine 5'-phosphate (141.8%) were increased. 6. The maximal activity of glutamine synthetase was increased (21-34%), but there was no change in that of glutaminase, in muscles of dexamethasone-treated rats. 7. It is concluded that glucocorticoid administration enhances the rates of release of both glutamine and alanine from skeletal muscle of rats (both in vitro and in vivo). This may be due to changes in efflux and/or increased intracellular formation of glutamine and alanine.