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

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

Respective effects of glucose and glutamine on the glutamine synthetase activity of human skin fibroblasts

Human A431 carcinoma cell line is known to have 30 fold amplified epidermal growth factor receptor (EGF-R) gene. We have studied the effect of steroid hormone dexamethasone (DEX) and protein synthesis inhibitor cycloheximide (CHX) on the expression of EGF-R gene in this cell line. DEX treatment and protein synthesis inhibition by CHX treatment cause a rapid 3 to 4 fold increase in the level of EGF-R mRNA and combined treatment of the above two agents have less than additive effect. It appears that mRNA for EGF-R accumulate within the cell during protein synthesis inhibition and upon removal of CHX, gets translated into EGF-R specific protein as judged by immuno-dot assay. We did not observe the phenomenon of super induction nor much of an additive effect under condition of combined DEX and CHX treatment.Abbreviations EGF-R Epidermal Growth Factor Receptor - DEX Dexamethasone - CHX Cycloheximide     

Proline accumulation and glutamine synthetase activity are increased by salt-induced proteolysis in cashew leaves

In this study cashew (Anacardium occidentale) plants were exposed to a short- and long-term exposure to NaCl in order to establish the importance of the salt-induced proteolysis and the glutamine synthetase activity on the proline accumulation. The cashew leaf showed a prominent proline accumulation in response to salt stress. In contrast, the root tissue had no significant changes in proline content even after the drastic injury caused by salinity on the whole plant. The leaf proline accumulation was correlated to protease activity, accumulation of free amino acid and ammonia, and decrease of both total protein and chlorophyll contents. The leaf GS activity was increased by the salt stress whereas in the roots it was slightly lowered. Although the several amino acids in the soluble pool of leaf tissue have showed an intense increment in its concentrations in the salt-treated plants, proline was the unique to show a proportional increment from 50 to 100 mol m-3 NaCl exposure (16.37 to 34.35 mmol kg-1 DM, respectively). Although the leaf glutamate concentration increased in the leaves of the salt-stressed cashew plants, as compared to control, its relative contribution to the total amino acid decreased significantly in stressed leaves when compared to other amino acids. In addition, when the leaf discs were incubated with NaCl in the presence of exogenous precursors (Glu, Gln, Orn or Arg) involved in the proline synthesis pathways, the glutamate was unique in inducing a significant enhancement of the proline accumulation compared to those discs with precursor in the absence of NaCl. These results, together with the salt-induced increase in the GS activity, suggest an increase in the de novo synthesis of proline probably associated with the increase of the concentration of glutamate. Moreover, the prominent salt-induced proline accumulation in the leaves was associated with the higher salt-sensitivity in terms of proteolysis and salt-induced senescence as compared to the roots. In conclusion, the leaf-proline accumulation was due, at least in part, to the increase in the salt-induced proteolysis associated with the increments in the GS activity and hence the increase in the concentration of glutamate precursor in the soluble amino acid pool.     

Sequence of glutamine synthetase from Salmonella typhimurium and implications for the protein structure

To aid in the interpretation of the 3.5 A resolution electron density map of glutamine synthetase (GS) from Salmonella typhimurium, the nucleotide sequence of the gene coding for this enzyme has been determined. The predicted sequence of 468 amino acids (Mr = 51,628) has been compared to the sequence and sequence fragments reported by others for GS of Anabaena and Escherichia coli. The homology between the pairs of sequences is sufficiently strong to suggest that the overall three-dimensional structures of the three GS are similar. The predicted positions of alpha helices are in moderately good agreement with the electron-density map.     

New crystal forms of glutamine synthetase and implications for the molecular structure.

New crystal forms of glutamine synthetase from Escherichia coli are reported. Two of these (II A and II B) demand that the dodecameric molecule contains a 2-fold axis of symmetry perpendicular to the apparent hexagonal face.Whereas forms II A and II B and others reported previously (I and III A) were grown from enzyme containing covalently bound AMP groups, a third new form (III C) was grown from enzyme lacking covalently bound AMP groups. Form III C is isomorphous with form III A. This demonstrates that the addition of AMP groups, which profoundly affect the catalytic and regulatory properties of glutamine synthetase, does not alter the dimensions of the molecular envelope. Thus adenylylation of the enzyme does not seem to cause a quaternary structural transition, though small changes of intensities suggest that there may be tertiary structural changes within the subunits.Other new forms include form III B, a low symmetry polymorph, closely related to form III A, and form IV, a trigonal polymorph with large asymmetric unit. All crystal forms are consistent with a symmetry of 622 for the glutamine synthetase molecule.     

Cloning and nucleotide sequence of an archaebacterial glutamine synthetase gene: Phylogenetic implications

Summary The glnA gene of the thermophilic sulphur-dependent archaebacterium Sulfolobus solfataricus was identified by hybridization with the corresponding gene of the cyanobacterium Spirulina platensis and cloned in Escherichia coli. The nucleotide sequence of the 1696 bp DNA fragment containing the structural gene for glutamine synthetase was determined, and the derived amino acid sequence (471 residues) was compared to the sequences of glutamine synthetases from eubacteria and eukaryotes. The homology between the archaebacterial and the eubacterial enzymes is higher (42%–49%) than that found with the eukaryotic counterpart (less than 20%). This was true also when the five most conserved regions, which it is possible to identify in both eubacterial and eukaryotic glutamine synthetases, were analysed.     

Distribution of glutamine synthetase and an inverse relationship between glutamine synthetase expression and intramuscular glutamine concentration in the horse

Glutamine plays important roles in the interorgan transport of nitrogen, carbon and energy but little is known about glutamine metabolism in the horse. In this study we determined the tissue distribution of glutamine synthetase expression in three Standardbred mares. Expression of glutamine synthetase was highest in kidney and mammary gland, and relatively high in liver and adipose tissue. Expression was lower in gluteus muscle, thymus, colon and lung, and much lower in small intestine, pancreas and uterus. The pattern of glutamine synthetase expression in the horse is similar to that of other herbivores and it is likely that skeletal muscle, liver, adipose tissue and lungs are the major sites of net glutamine synthesis in this species. Expression did not differ between adipose tissue depots but did vary between different muscles. Expression was highest in gluteus and semimembranous muscles and much lower in diaphragm and heart muscles. The concentration of intramuscular free glutamine was inversely correlated with expression of glutamine synthetase (r=-0.81, p=0.0017). The concentration of free glutamine was much higher in heart muscle (21.6+/-0.9 micromol/g wet wt) than in gluteus muscle (4.19+0.33 micromol/g wet wt), which may indicate novel functions and/or regulatory mechanisms for glutamine in the equine heart.     

NMDA receptor modulation by the neuropeptide apelin: implications for excitotoxic injury

Excitotoxic neuronal damage via over-activation of the NMDA receptor has been implicated in many neurodegenerative diseases. In vitro modeling of excitotoxic injury has shown that activation of G-protein coupled receptors (GPCRs) counteracts such injury through modulation of neuronal pro-survival pathways and/or NMDA receptor signaling. We have previously demonstrated that the GPCR APJ and its endogenous neuropeptide ligand apelin can protect neurons against excitotoxicity, but the mechanism(s) of this neuroprotection remain incompletely understood. We hypothesized that apelin can promote neuronal survival by activating pro-survival signaling as well as inhibiting NMDA receptor-mediated excitotoxic signaling cascades. Our results demonstrate that (i) apelin activates pro-survival signaling via inositol trisphosphate (IP(3) ), protein kinase C (PKC), mitogen-activated protein kinase kinase 1/2 (MEK1/2), and extracellular signal-regulated kinase-1/2 (ERK1/2) to protect against excitotoxicity, and (ii) apelin inhibits excitotoxic signaling by attenuating NMDA receptor and calpain activity, and by modulating NMDA receptor subunit NR2B phosphorylation at serine 1480. These studies delineate a novel apelinergic signaling pathway that concurrently promotes survival and limits NMDA receptor-mediated injury to protect neurons against excitotoxicity. Defining apelin-mediated neuroprotection advances our understanding of neuroprotective pathways and will potentially improve our ability to develop therapeutics for excitotoxicity-associated neurodegenerative disorders.     

Identification and cellular location of glutamine synthetase in human sperm

Glutamine synthetase (GS) catalyzes the de novo synthesis of glutamine, an amino acid that has been shown to influence sperm motility in mammals. To date, no information is available about GS content in human sperm. In this study, we have characterized the presence and cellular location of GS in fresh human normozoospermic samples. We have detected a single band corresponding to GS by Western blot. Confocal analysis has revealed GS immunoreactivity in the post-acrosomal head region. Moreover, double-labeling experiments with either F-actin or calicin have demonstrated GS confinement in the post-acrosomal region of the perinuclear theca. These data have been validated by a post-embedding ultra-structural study. The presence of GS in the post-acrosomal region of the perinuclear theca suggests that human sperm can carry out in glutamine synthesis.     

A radiochemical assay for glutamine synthetase, and activity of the enzyme in rat tissues

1. A radiochemical assay for glutamine synthetase has been developed in which an ATP-regenerating system is incorporated to prevent accumulation of inhibitory amounts of ADP. It is particularly suitable for assay of the enzyme in crude tissue extracts containing high adenosine triphosphatase activity. 2. A survey of the distribution of the enzyme in tissues from normal male rats showed that activity is present in liver, brain cortex, kidney cortex, spleen, testis and retina. 3. The K(m) of the enzyme for l-glutamate is approx. 1.5x10(-2)m.     

Changes of NMDA receptor binding in spontaneously epileptic rat and parent strains

We measured the binding of [³H]3-[(±)2-carboxypiperazin-4-yl]propyl-1-phosphonic acid ([³H]CPP), a competitive ligand forN-methyl-d-aspartate (NMDA) receptors, in double mutant spontaneously epileptic rats (SER:zi/zi, tm/tm) and their parent strains, zitter rats, and tremor rats, and WTC rats (control rats from tremor rats derived from Kyoto:Wistar rats) before and after the onset of seizures in tremor rats and SER. Significantly lower [³H]CPP binding receptor density (B_max) was found in the cortex of SER and zitter rats at 12–15 weeks of age than in that of WTC rats and tremor rats, and at 4 weeks of age the Bmax in zitter rats was lower than that in the other strains. The reduction of Bmax in SER at 12–15 weeks of age may reflect a down regulation of NMDA receptors due to repetitive tonic seizures in SER.     

Enhancement of asynchronous release from fast-spiking interneuron in human and rat epileptic neocortex

Down-regulation of GABAergic inhibition may result in the generation of epileptiform activities. Besides spike-triggered synchronous GABA release, changes in asynchronous release (AR) following high-frequency discharges may further regulate epileptiform activities. In brain slices obtained from surgically removed human neocortical tissues of patients with intractable epilepsy and brain tumor, we found that AR occurred at GABAergic output synapses of fast-spiking (FS) neurons and its strength depended on the type of connections, with FS autapses showing the strongest AR. In addition, we found that AR depended on residual Ca²⁺ at presynaptic terminals but was independent of postsynaptic firing. Furthermore, AR at FS autapses was markedly elevated in human epileptic tissue as compared to non-epileptic tissue. In a rat model of epilepsy, we found similar elevation of AR at both FS autapses and synapses onto excitatory neurons. Further experiments and analysis showed that AR elevation in epileptic tissue may result from an increase in action potential amplitude in the FS neurons and elevation of residual Ca²⁺ concentration. Together, these results revealed that GABAergic AR occurred at both human and rat neocortex, and its elevation in epileptic tissue may contribute to the regulation of epileptiform activities.     

Influence of human low density and high density lipoprotein cholesterol on the in vitro prostaglandin I2 synthetase activity

We investigated in vitro the influence of low density lipoprotein (LDL) cholesterol and high density lioprotein (HDL) cholesterol separated from human serum on prostaglandin I2 synthetase activity studied by the conversion of prostaglandin H2 to prostaglandin I2 by the microsomal fraction of pig aorta. 6-Oxo-prostaglandin F1 alpha was analyzed by gas-liquid chromatography using prostaglandin F1 alpha as internal standard. We found a linear negative correlation (P less than 0.001) between the amount of LDL cholesterol in the incubation solution and prostaglandin I2 synthetase activity, whereas there was a positive correlation (P less than 0.01) between HDL cholesterol and prostaglandin I2 synthesis. A very low concentration of LDL cholesterol and a high concentration of HDL cholesterol stimulated prostaglandin I2 synthesis, whereas a high LDL cholesterol concentration inhibited prostaglandin I2 biosynthesis by 64%. The concentration range of LDL and HDL cholesterol was representative of physiologically low, normal or elevated levels of lipoproteins.     

Subcellular localization of glutamine synthetase activity in carp muscle tissue and liver

Subcellular distribution of the glutaminesynthetase activity (EC 6.3.1.2) is studied in muscle tissue and liver of carp. It is established that this activity is distributed by subcellular fractions analogously to the glutamatedehydrogenase activity--enzyme-marker activity of mitochondria. An additional evidence for the association of the two given enzymes is obtained during equilibrium density-gradient centrifugation of mitochondria on 30-60% gradient of sorbitol. Comparison of the obtained results with data from literature testifies to a coincidence of the subcellular localization of the processes of ammonia formation and binding into glutamine, thus confirming a view of the leading role of glutaminesynthetase in ammonia detoxication in carps.