Induction by Hydrocortisone of Glutamine Synthetase in Mouse Primary Astrocyte Cultures

Glutamine synthetase activity was investigated in developing primary astroglial cultures established from newborn mouse cerebral hemispheres. Between the 2nd and 4th week of culture there was little change in activity under our standard culturing conditions; however, when hydrocortisone (10 microM) was added to the cultures for 48 h, the enzyme activity increased two- to fourfold, depending upon the age of the culture, with maximum response in 2-week-old cultures. The addition of dibutyryl cyclic AMP (dBcAMP) to the culture medium caused morphological differentiation of the astroglial cells but eliminated the response of the cells to hydrocortisone. Culturing in elevated serum levels, which delays morphological differentiation and inhibits astroglial cytodifferentiation after exposure to dBcAMP, shifted the time of maximal response to hydrocortisone from 2 to 3 weeks and prevented the abolishment of glutamine synthetase induction by dBcAMP. The induction of glutamine synthetase by hydrocortisone was prevented by actinomycin D (0.5 microgram/ml), indicating its dependence upon RNA and protein synthesis. The present work thus confirms reports in the literature that hydrocortisone induces glutamine synthetase in neural tissues, but differs from the findings of Moscona and co-workers in the chick retina that intact tissues are required for the induction to occur.     

Role of glutamine synthetase in the initiation of sporulation in Bacillus polymyxa

The activity of glutamine synthetase (GS) was investigated during culture development of Bacillus polymyxa CN 2219 and its asporogenous mutant deficient in protease production. At 28°C, temperature permissive for sporulation, the glutamine synthetase activity was found to decline in the wild type cells which acquire the competence for sporulation. This decline was not observed in the asporogenous mutant. Incubation at 37°C (temperature non permissive) suppressed sporulation in the wild type and maintained glutamine synthetase activity. The involvement of glutamine synthetase in the repression of sporulation was further confirmied by the action of l-methionine sulfoximine a specific inhibitor of glutamine synthetase, which overcomes the catabolite repression by ammonium and induces sporulation. Intracellular proteases were measured as early markers of the initiation of sporulation and were found to be induced during sporulation.Abbreviations GS glutamine synthetase - MSO l-methionine sulfoximine - GYS glucose-yeast extract-salts - GT -glutamyltransferase - PMSF phenylmethylsulfonylfluoride     

Hormonal induction of glutamine synthetase in cultures of embryonic retina cells: requirement for neuron-glia contact interactions

Cortisol induces glutamine synthetase (GS) in gliocytes of chick embryo neural retina. Using adherent cultures of retina cells we have demonstrated that responsiveness of the gliocytes to GS induction by the hormone requires contact with neurons. GS is not inducible in high-density cultures depleted of neurons and consisting only of gliocytes. In neuron-containing cultures, induced GS was detected immunohistochemically only in those gliocytes that were closely juxtaposed with clusters of neurons. Unlike the induction of GS, the expression of carbonic anhydrase-C (which does not require cortisol) persisted in these glia cells also in the absence of neurons. The nature and role of glia-neuron interactions in the hormonal induction of GS are briefly discussed.     

Cell density-dependent stimulation of glutamine synthetase activity in cultured mouse teratoma cells

A density dependent stimulation of glutamine synthetase (GS) activity has been observed in cultures of mouse teratoma cells. GS specific activity increased as cultures approached confluency to a level greater than 2-fold over the basal level found in sparse cultures. After confluency the GS specific activity returned to the basal level found in sparse cultures. The enzyme increase could not be attributed to age of cultures, medium or glutamine depletion, cell leakage of GS, or change in the amount of cellular protein. Dibutyryl cyclic AMP (db-cAMP) plus theophylline lowered GS specific activity both in cultured teratoma and in teratoma obtained from ascites grown tumors. The enzyme increase observed in cultured teratoma cells could be prevented by cycloheximide, and enhanced by hydrocortisone or actinomycin D.     

Glutamine synthetase induction in chick embryo retina monolayers

Induction of glutamine synthetase (GS) by cortisol has been shown to occur in monolayer cultures of cells obtained by enzymatic dissociation of retinas from 8- and 12-day-old chick embryos with papain (0.1%) or trypsin (0.25%). Although essentially single cells when plated, monolayers obtained by enzymatic dissociation show significant aggregation by 4--6 h. Monolayers prepared by mechanical dispersion (cells forced through successively smaller gage needles) are minimally inducible, perhaps owing to poor viability in such cultures. Storage at 4 degrees C for 24 h prior to treatment with cortisol significantly elevated both basal GS activity and inducibility in whole (but not in monolayer) retina cultures.     

Regulation of urease and ammonia assimilatory enzymes in Selenomonas ruminantium.

Urease and glutamine synthetase activities in Selenomonas ruminantium strain D were highest in cells grown in ammonia-limited, linear-growth cultures or when certain compounds other than ammonia served as the nitrogen source and limited the growth rate in batch cultures. Glutamate dehydrogenase activity was highest during glucose (energy)-limited growth or when ammonia was not growth limiting. A positive correlation (R = 0.96) between glutamine synthetase and urease activities was observed for a variety of growth conditions, and both enzyme activities were simultaneously repressed when excess ammonia was added to ammonia-limited, linear-growth cultures. The glutamate analog methionine sulfoximine (MSX), inhibited glutamine synthetase activity in vitro, but glutamate dehydrogenase, glutamate synthase, and urease activities were not affected. The addition of MSX (0.1 to 100 mM) to cultures growing with 20 mM ammonia resulted in growth rate inhibition that was dependent upon the concentration of MSX and was overcome by glutamine addition. Urease activity in MSX-inhibited cultures was increased significantly, suggesting that ammonia was not the direct repressor of urease activity. In ammonia-limited, linear-growth cultures, MSX addition resulted in growth inhibition, a decrease in GS activity, and an increase in urease activity. These results are discussed with respect to the importance of glutamine synthetase and glutamate dehydrogenase for ammonia assimilation under different growth conditions and the relationship of these enzymes to urease.     

Glutamine synthetase induction in chick embryo retina monolayers

Induction of glutamine synthetase (GS) by cortisol has been shown to occur in monolayer cultures of cells obtained by enzymatic dissociation of retinas from 8- and 12-day-old chick embryos with papain (0.1%) or trypsin (0.25%). Although essentially sigle cells when plated, monolayers obtained by enzymatic dissociation show significant aggregation by 4–6 h. Monolayers prepared by mechanical dispersion (cells forced through successively smaller gage needles) are minimally inducible, perhaps owing to poor viability in such cultures. Storage at 4°C for 24 h prior to treatment with cortisol significantly elevated both basal GS activity and inducibility in whole (but not in monolayer) retina cultures.     

Separation of two L-glutamine-hydroxylamine glutamyltransferases from rat liver.

The purification of glutamine synthetase (GS) from rat liver demonstrates that a small portion of glutamine-hydroxylamine-glutamyltransferase activity (GT) remains associated with GS activity (GT(S)). As GS is purified from the water extract, the ratio between GT(S) activity (GT(T)) is left to be extracted by KC1 from the pellet and, on further purification, appears to be independent of GS activity. Subtle differences in pH optimum, substrate requirement and reaction rates on addition of cofactors and amino acids in vitro and in responses to hormonal stimuli in vivo indicate that the glutamine transfer reaction may be catalyzed by two distinguishable proteins; only the minor component may be identical to GS.     

Effects of medium glutamine,glutamate, and ammonia on glutamine synthetase activity in cultured mouse astroglial cells

Mouse astroglial cells were grown during the last week of culture in either glutamine-free or glutamine-containing medium. The addition of cortisol to the glutamine-containing medium resulted in a doubling of astroglial glutamine synthetase (GS) activity. Withdrawal of glutamine from the medium resulted in a 50% elevation of GS and addition of cortisol to such a medium resulted in a further increase in GS which was not additive to glutamine withdrawal. Both in glutamine-free and glutamine-containing medium, the addition of glutamate resulted in a depression of both basal and cortisol induced GS activity. The simultaneous addition of ammonia plus glutamate to the culture medium ameliorated the glutamate mediated depressive effects on cortisol induced but not basal GS activity. Glutamine withdrawal from the culture medium resulted in an astroglial protein deficit. The addition of ammonia to the medium considerably reduced this deficit and the addition of glutamate completely eliminated this protein deficit.     

Estrogen (17β-Estradiol) Enhances Glutamine Synthetase Activity in C6-Glioma Cells

Glutamine synthetase (GS) is the major glutamine-forming enzyme of vertebrates and is accepted to be a marker of astroglial cells. Maturation of astroglial cells is characterized by an increase of GS activity, and the regulation of this enzyme is the topic of many publications. Because of the fundamental role of the GS in controlling brain glutamate and glutamine level, it is essential to understand the mechanism of expression of this enzyme. To our knowledge, the effect of estrogen (17β-estradiol) on GS activity in glial cells has not been reported. We examined the effect of treatment with estrogen on glutamine synthetase enzyme activity in glial cells. C6-glioma cells in later passage have many astrocytic characteristics and provided a convenient and well-established model system. We adapted a colorimetric method to measure GS-catalyzed γ-glutamyltransferase (GT) activity in C6-glioma cells. The assay monitors GT activity of glutamine synthetase by following the absorbance of the product γ-glutamyl hydroxamate at 540 nm. We observed that, the absorbance of γ-glutamyl hydroxamate significantly increased in estrogen treated cells (0.13±0.03), as compared to untreated cells (0.058±0.015). Estrogen also significantly increased concentration of glutamine in C6-glioma cells as measured by fluorometric assay. In addition, western blot analysis showed that estrogen significantly increased the amount of glutamine synthetase compared to control. This estrogen effect could have important physiological implications on cerebral glutamate and glutamine metabolism.     

Air-breathing catfish, Clarias batrachus upregulates glutamine synthetase and carbamyl phosphate synthetase III during exposure to high external ammonia

We assessed the possible upregulation of glutamine synthetase (GS) and typical 'fish type' carbamyl phosphate synthetase III (CPS III) in detoxification of ammonia in different tissues of the walking catfish (Clarias batrachus) during exposure to 25 mM NH(4)Cl for 7 days. Exogenous ammonia led to an increase in ammonia and urea concentrations in different tissues. The results revealed the presence of relatively high levels of GS activity in the brain, liver and kidney, unexpectedly, also in the muscle, and even higher levels in the intestine and stomach. Exposure to high external ammonia (HEA) caused significant increase of activities of GS, CPS III and CPS I-like enzymes, accompanied with the upregulation of GS and CPS III enzyme proteins in different tissues. Exposure to HEA also led to a sharp rise of plasma cortisol level, suggesting being one of the primary causes of upregulation of GS and CPS III enzymes activity. Liver perfusion experiments further revealed that exposure to HEA enhances the capacity of trapping ammonia to glutamine and urea by the liver of walking catfish. These results suggest that the upregulation of GS and CPS III activity in walking catfish during exposure to HEA plays critical roles to ameliorate the toxic ammonia to glutamine, and also to urea via the induced ornithine-urea cycle possibly through the involvement of cortisol.     

Multiple mechanisms by which glutamine synthetase levels are controlled in murine tissue culture cells

We report the isolation of a complimentary DNA (cDNA) clone encoding glutamine synthetase, derived from a population of methionine sulfoxime-resistant mouse GF1 fibroblasts. When GF1 cells are incubated for 48 h in the presence of the glucocorticoid hormone dexamethasone, the specific activity of glutamine synthetase (GS), assayed as glutamyltransferase activity, increases by threefold. Based on dot hybridization analysis, hormonal treatment also produces a similar increase in the level of GS mRNA. When GF1 cells or mouse Neuro 2A neuroblastoma cells are transferred from medium containing 4 mM glutamine to glutamine-free medium, glutamyltransferase activity increases by at least fivefold. However, the presence or absence or glutamine in the medium does not affect the relative level of glutamine synthetase mRNA in either cell line. With both GF1 and Neuro 2A cells, the half-time for the decline in glutamine synthetase enzyme activity on addition of glutamine to the medium is approximately 1.5 h. This rapid decline, coupled with the lack of effect of glutamine on the level of GS messenger RNA in Neuro 2A cells, renders it unlikely that neural cells alter glutamine synthetase levels in response to glutamine by a biosynthetic mechanism, as suggested by previous authors [L. Lacoste, K.D. Chaudhary, and J. Lapointe (1982) J. Neurochem. 39, 78-85].     

Purification and regulation of glutamine synthetase in a collagenolytic Vibrio alginolyticus strain

Glutamine synthetase (EC 6.3.1.2) has been purified from a collagenolytic Vibrio alginolyticus strain. The apparent molecular weight of the glutamine synthetase subunit was approximately 62,000. This indicates a particle weight for the undissociated enzyme of 744,000, assuming the enzyme is the typical dodecamer. The glutamine synthetase enzyme had a sedimentation coefficient of 25.9 S and seems to be regulated by a denylylation and deadenylylation. The pH profiles assayed by the -glutamyltransferase method were similar for NH₄-shocked and unshocked cell extracts and isoactivity point was not obtained from these eurves. The optimum pH for purified and crude cell extracts was 7.9. Cell-free glutamine synthetase was inhibited by some amino acids and AMP. The transferase activity of glutamine synthetase from mid-exponential phase cells varied greatly depending on the sources of nitrogen or carbon in the growth medium. Glutamine synthetase level was regulated by nitrogen catabolite repression by (NH₄)₂SO₄ and glutamine, but cells grown, in the presence of proline, leucine, isoleucine, tryptophan, histidine, glutamic acid, glycine and arginine had enhanced levels of transferase activity. Glutamine synthetase was not subject to glucose, sucrose, fructose, glycerol or maltose catabolite repression and these sugars had the opposite effect and markedly enhanced glutamine synthetase activity.Abbreviations GS glutamine synthetase - SMM succinate minimal medium - ASMM ammonium/succinate minimal medium - GT -glutamyl transferase - SVP snake venom phosphodiesterase     

Relationship of adhesiveness of cells in culture with specific enzyme activity.

l-Glutamine is required by mouse teratoma cells and other mouse ascites tumor cells in the synthesis of complex carbohydrates involved in intercellular adhesion. Since l-glutamine is synthesized by the enzyme glutamine synthetase (GS) (EC 6.3.1.2), these studies were undertaken to determine if a relationship exists between cellular adhesiveness and GS specific activity. Two types of experiment were performed to examine this relationship. Actinomycin D enhanced both teratoma cell GS specific activity and cellular adhesiveness over controls in batch cultures at confluency. Also, the relationship between cell adhesiveness and GS specific activity during the cell cycle was studied using cell populations synchronized with thymidine plus Colcemid. In these synchronized cultures, cellular adhesiveness displayed an oscillatory pattern with peaks of GS specific activity occurring just prior to peaks of adhesiveness. The levels of GS specific activity and intercellular adhesiveness were enhanced by the addition of hydrocortisone, a steroid known to induce GS specific activity in mouse teratoma cells. These results demonstrate a correlation between GS specific activity and cellular adhesiveness. Based upon previous work which implicates l-glutamine in intercellular adhesion, it is not unreasonable to speculate that GS specific activity and cellular adhesiveness may be causally related.     

Relationships between nitrogenase,glutamine synthetase,glutamine, and energy charge in Azotobacter vinelandii

When continuous cultures of Azotobacter vinelandii were supplied with ammonium or nitrate in amounts, which just repressed nitrogenase synthesis completely, both the intracellular glutamine level and the degree of adenylylation of the glutamine synthetase (GS) increased only slightly (from 0.45–0.50 mM and from 2 to 3 respectively), while the total GS level remained unaffected. Higher amounts of ammonium additionally inhibited the nitrogenase activity, caused a strong rise in the intracellular glutamine concentration and adenylylation of the GS, but caused no change in the ATP/ADP ratio. These results are considered as evidence that in A. vinelandii the regulation of nitrogenase synthesis is not linked to the adenylylation state of the GS and to the intracellular glutamine level, and that the inhibition of the nitrogenase activity as a consequence of a high extracellular ammonium level is not mediated via a change in the energy charge.Abbreviations GS glutamine synthetase - GS-S(Mg) Mg²⁺ dependent synthetic activity of GS - GS-T(Mn) Mn²⁺ dependent transferase activity of GS     

Glutamine synthetase, glutaminase and phosphodiesterase activities in brain under hypoxia: in vitro effect of cortisol, GABA and serotonin on glutamine synthetase.

The effect of hypobaric hypoxia on the activities of glutamine synthetase, glutaminase and cyclic 3'5' AMP phosphodiesterase in rat brain was studied after exposure to 25,000' for 6 h. Glutamine synthetase activity was increased in all the regions of brain studied, and addition of gamma amino butyric acid, serotonin and cortisol in vitro produced a differential response. Glutaminase activity decreased in the whole brain. Cyclic 3'5' AMP phosphodiesterase activity decreased in cerebellum, medulla, hypothalamus and pituitary showing an accumulation of cyclic 3'5' AMP in these regions. The results suggest that glutamine synthesis and degradation are regulated in the central nervous system by cyclic AMP and cortisol: Gamma aminoburyric acid and other compounds can modulate the activity of glutamine synthetase and glutaminase.     

Factors affecting the production of glutamine in cultured mouse cells

Glutamine synthetase activity of NCTC clone 929 mouse cells (strain L) was studied as a function of the prior nutritional experience of the cells. Small enzyme increases were recorded in response to either glutamine depletion or chronic serum supplementation of the growth medium. Somewhat greater increases resulted from the administration of cortisol or certain other steroids, particularly if the hormone treatment was combined with glutamine withdrawal. High concentrations of glutamate in the medium did not augment the glutamine synthetase content of the cells and even caused an apparent decrease in it. The presence of glutamine in the culture medium resulted in a fairly rapid rate of disappearance of the glutamine synthetase of previously induced cells. The data suggest that glutamine and cortisol act independently on the cells in regulating the level of the enzyme.     

Ammonium assimilation in Rhizobium phaseoli by the glutamine synthetase-glutamate synthase pathway.

Evidence from in vitro and in vivo studies showed that in Rhizobium phaseoli ammonium is assimilated by the glutamine synthetase (GS)-glutamate synthase NADPH pathway. No glutamate dehydrogenase activity was detected. R. phaseoli has two GS enzymes, as do other rhizobia. The two GS activities are regulated on the basis of the requirement for low (GSI) or high (GSII) ammonium assimilation. When the 2-oxoglutarate/glutamine ratio decreases, GSI is adenylylated. When GSI is inactivated, GSII is induced. However, induction of GSII activity varied depending on the rate of change of this ratio. GSII was inactivated after the addition of high ammonium concentrations, when the 2-oxoglutarate/glutamine ratio decreased rapidly. Ammonium inactivation resulted in alteration of the catalytic and physical properties of GSII. GSII inactivation was not relieved by shifting of the cultures to glutamate. After GSII inactivation, ammonium was excreted into the medium. Glutamate synthase activity was inhibited by some organic acids and repressed when cells were grown with glutamate as the nitrogen source.     

浑球红假单胞菌氨同化途径的研究

本文测定了浑球红假单胞菌(Rhodobacter sphaeroides)菌株601谷氨酰胺合成酶(GS)、谷氨酸合酶(GOGAT)、谷氨酸脱氢酶(GDH)和丙氨酸脱氢酶(ADH)的活性。低氨时,GS/GOGAT活力高,GDH活力低,高氨时,GS/GOGAT活力低,GDH活力高。在以分子氮或低浓度氨为氮源的培养条件下,加入GS抑制刑MSX(L—methionine—DL—sulphoximine),细菌生长受到抑制。但是,生长在以谷氨酸为氮源的细菌则不受影响。上述结果表明,浑球红假单胞菌菌株601氨同化是通过GS/GOGAT途径和GDH途径。     

小麦开花后不同器官中硝酸还原酶和谷氨酰胺合成酶的活性比较

小麦开花后各器官的硝酸还原酶和谷氨酰胺合成酶均具有一定的活性,旗叶中硝酸还原酶和谷氨酰胺合成酶活性最高。开花后旗叶和根系硝酸还原酶和谷氨酰胺合成酶活性逐渐降低,颗壳和籽粒中硝酸还原酶和谷氨酰胺合成酶活性先升高,达到最大值后又降低。