Glutamine Synthetase mRNA Levels Research Articles

Reciprocal regulation of glutamine synthetase and carbamoylphosphate synthetase levels in rat liver

In glucocorticosteroid-treated diabetic rats, glutamine synthetase enzyme levels in the liver are decreased 3-fold, whereas carbamoylphosphate synthetase enzyme levels are increased 2.3-fold. In addition, immunohistochemistry shows that under these conditions the distribution of carbamoylphosphate synthetase is expanded over the entire liver acinus, whereas that of glutamine synthetase is reduced to very few cells bordering the central (terminal hepatic) veins. Using a newly isolated cDNA complementary to rat liver glutamine synthetase mRNA, we show that this regulation is primarily effected at a pretranslational level. (For data on carbamoylphosphate synthetase mRNA levels, see De Groot et al. (1986) Biochim. Biophys. Acta 866, 61–67.) Furthermore, hybridization studies show stimulatory effects of both glucocorticosteroids and thyroid hormone on the glutamine synthetase mRNA level. Attempts to localize glutamine synthetase mRNA within the liver acinus by selective destruction of the pericentral zone failed because of generally low levels of liver mRNAs after CCl 4 poisoning. In contrast to the situation after birth, significantly higher glutamine synthetase mRNA/enzyme activity ratios in fetal rat liver point to the presence of additional post-transcriptional control mechanisms before birth. These findings complement similar observations on carbamoylphosphate synthetase gene expression (De Groot et al. (1986) Biochim. Biophys. Acta 866, 61–67).

Glucocorticoid receptor-mediated induction of glutamine synthetase in skeletal muscle cells in vitro.

We studied the regulation by glucocorticoids of glutamine synthetase in L6 muscle cells in culture. Glutamine synthetase activity was strikingly enhanced by dexamethasone. The dexamethasone-mediated induction of glutamine synthetase activity was blocked by RU38486 [11 beta-(4-dimethylaminophenyl)17 beta-hydroxy-17 alpha-(prop-1-ynyl)estra-4,9-dien-3-one], a glucocorticoid antagonist, indicating the involvement of intracellular glucocorticoid receptors in the induction process. RU38486 alone was without effect. Northern blot analysis revealed that dexamethasone-mediated enhancement of glutamine synthetase activity involves increased levels of glutamine synthetase mRNA. Increased enzyme activity was specific for glucocorticoids; other steroid hormones were essentially without effect. The induction of glutamine synthetase was selective, in that glutaminase activity was not induced by dexamethasone treatment of L6 cells. Thus, glucocorticoids regulate the expression of glutamine synthetase mRNA in cultured muscle cells via interaction with intracellular receptors. Such regulation may be relevant to control of glutamine production by muscle.

Developmental regulation of glutamine synthetase and carbonic anhydrase II in neural retina.

Glutamine synthetase (GS) is expressed in the neural retina only in Muller glia cells and is inducible with cortisol. A chicken genomic clone that contains at least part of the coding region for the GS enzyme was used to investigate developmental changes in the level of GS mRNA in embryonic chicken retina. A major GS transcript (approximately equal to 3 kilobases) detected by the probe begins to accumulate sharply on day 15 of embryonic development. When cortisol is prematurely supplied to early embryonic retina, it induces precocious accumulation of GS mRNA and of the GS enzyme. At later ages, these effects of cortisol are significantly greater, which suggests that competence to transcribe or stabilize GS mRNA in response to stimulation with cortisol increases with development. Carbonic anhydrase II (CA-II) is expressed in early retina in all the cells, but it becomes later restricted to Muller glia. Using cloned CA-II cDNA, we detected a high level of CA-II mRNA in early retina, followed by a decline due to arrest of CA-II mRNA accumulation in differentiated neurons. As glia cells mature, CA-II mRNA and the enzyme increase to a new high level. Therefore, changes in CA-II gene expression during retina development reflect differentiation-dependent cell-type-specific control of CA-II mRNA accumulation.

Messenger RNA for glutamine synthetase. Review article.

Of the various eucaryotic tissues, where glutamine synthetase (GS) mRNA and its regulation have been investigated, the induction of GS by glucocorticoids in the embryonic chick retina represents one of the systems most extensively studied. GS mRNA was first identified at the polysomal level by immunochemical precipitation of fractionated polysomes containing nascent GS chains with anti-GS gamma-globulin. The mRNA has been shown to be polyadenylated at the 3' end; on this basis, it has been partially purified from embryonic chick retina as well as from N. Crassa by chromatography on oligo(dT)-cellulose or poly(U)-sepharose and translated in cell-free protein synthesizing systems derived from wheat germ. Hormonal regulation of GS activity studied in the embryonic retina, hepatoma tissue culture cells, or in other tissues is always shown to be mediated by GS mRNA. In the retina, hydrocortisone (HC) elicits an age-related and transcription-dependent induction of GS by enhancing the level of GS mRNA in the polysomes through an increased supply of this mRNA from the nucleus. Comparative studies of three inhibitors of transcription, viz. actinomycin D, leucanthone and proflavine on the induction of GS by HC indicate that the latter inhibits GS mRNA selectively and reversibly with minimal effects on other RNA synthesis. Since proflavine acts by competing with HC-receptor binding sites in the nuclei, further studies on its interaction with the retina genome are likely to help identify the DNA sequences involved in the GS induction. In bacteria, studies on the genetics and physiology of various mutants with lesions in the structural gene for GS show that the transcription of the GS gene (gln A) is regulated both positively and negatively by GS and the product of another gene gln F. Purification of GS mRNA to homogeneity cloning of its cDNA and development of assay systems for cell-free transcription of GS are other studies likely to advance our knowledge on GS mRNA and its regulation.

Control of glutamine synthetase messenger RNA by hydrocortisone in the embryonic chick retina

Hydrocortisone (HC) and related glucocorticoids elicit an age-dependent induction of glutamine synthetase (GS) in the embryonic chick retina. We show here that HC has no effect either on the rate of synthesis of GS or on the low intracellular level of GS mRNA in the younger preinducible (7-day) retina. In the inducible (12-day) retina, GS induction appears to be a primary effect of HC since an increase in the rate of synthesis of GS is detectable as early as 45–60 min after exposure to HC. When these retinas are cultured with HC, the rate of GS synthesis as well as the amount of GS mRNA in the polysomal and in the nonpolysomal cytoplasmic fractions increased progressively up to 10 hr in culture and declined subsequently. The increase in the level of polysomal GS mRNA is not due to loading of new GS mRNA into polysomes from the nonpolysomal fraction and is thus a result of an enhanced supply of GS mRNA from nuclei. These data are compatible with the hypothesis that, in the inducible retina, HC induces GS by initiating or accelerating the transcription of structural genes for GS and/or increasing the stability of GS mRNA in the nuclei. We conclude that the development of the age-related inducibility of GS in the embryonic retina is due to the ability of HC to evoke elevated levels of GS mRNA in the polysomes of the inducible retina, resulting from an increased supply of GS mRNA from the nucleus. The preinducible retina is insensitive to this particular effect.

Messenger RNA for glutamine synthetase: Its partial purification, translation in a cell-free system and its regulation by hydrocortisone

Messenger RNA for glutamine synthetase (GS), extracted from polysomes of embryonic chick retina cells, was partially purified by chromatography on oligo (dT)-cellulose on the basis of its poly (A)-content and translated in a cell-free protein synthesizing system derived from wheat germ. Identification and quantitation of GS in the products of cell-free synthesis was achieved by immunoprecipitation of the enzyme and electrophoresis in sodium-dodecyl-sulfate polyacrylamide gels. Comparison of the amount of GS synthesized in vitro by equal amounts of mRNA purified from polysomes of control or hydrocortisone induced retina indicate that the hormone elevates the level of GS mRNA on the polysomes from induced cells.