Effect of dibutyryl cyclic AMP and dexamethasone on glutamine synthetase gene expression in rat astrocytes in culture

Astrocytes are the primary site of glutamate conversion to glutamine in the brain. We examined the effects of treatment with either dibutyryl cyclic AMP and/or the synthetic glucocorticoid dexamethasone on glutamine synthetase enzyme activity and steady-state mRNA levels in cultured neonatal rat astrocytes. Treatment of cultures with dibutyryl cyclic AMP alone (0.25 mM–1.0 mM) increased glutamine synthetase activity and steady state mRNA levels in a dose-dependent manner. Similarly, treatment with dexamethasone alone (10^–7–10^–5 M) increased glutamine synthetase mRNA levels and enzyme activity. When astrocytes were treated with both effectors, additive increases in glutamine synthetase activity and mRNA were obtained. However, the additive effects were observed only when the effect of dibutyryl cyclic AMP alone was not maximal. These findings suggest that the actions of these effectors are mediated at the level of mRNA accumulation. The induction of glutamine synthetase mRNA by dibutyryl cyclic AMP was dependent on protein synthesis while the dexamethasone effect was not. Glucocorticoids and cyclic AMP are known to exert their effects on gene expression by different molecular mechanisms. Possible crosstalk between these effector pathways may occur in regulation of astrocyte glutamine synthetase expression.Abbreviations used GS glutamine synthetase - dbcAMP dibutyryl cyclic AMP - MEM minimal essential medium - cyx cycloheximide - GRE glucocorticoid response element - CRE cyclic AMP response element     

Induction of glutamine synthetase by 8-bromo cyclic AMP in primary cultures of rat brain astrocytes

Glutamine synthetase (GS) is the key enzyme in cerebral glutamine production. Understanding the regulation of the expression of GS is important for definition of the control of glutamine metabolism in brain. Therefore, we studied the control of GS expression by 8-bromo cyclic AMP in primary cultures of astrocytes prepared from brains of neonatal rats. GS activity was increased by 8-bromo cyclic AMP in a dose- and time-dependent manner. This increase was associated with a corresponding increase in the steady-state level of GS mRNA.     

Induction of astrocyte argininosuccinate synthetase and argininosuccinate lyase by dibutyryl cyclic AMP and dexamethasone

Arginine is an intermediate in the elimination of excess nitrogen and is the substrate for nitric oxide synthesis. Arginine synthesis has been reported in brain tissue. We have studied the activity of the arginine biosynthetic enzymes argininosuccinate synthetase and argininosuccinate lyase in dexamethasone and/or dibutyryl cyclic AMP treated rat astrocyte cultures. Argininosuccinate lyase activity was stimulated by treatment with either effector and an additive effect was obtained when both agents were added simultaneously. Argininosuccinate synthetase was also increased in dexamethasone treated astrocytes. The effect of dibutyryl cyclic AMP on argininosuccinate synthetase was variable, suggesting a role for additional factors in its regulation as compared to argininosuccinate lyase. Regulation of arginine synthesis in astrocytes may be important to insure that arginine is not limiting for nitric oxide synthesis in neural tissue.     

Effects of cyclic AMP and butyrate on cell cycle,DNA, RNA,and purine synthesis of cultured astrocytes

Dibutyryl cyclic monophosphate (dBcAMP) has been shown to inhibit growth, and alter the morphology of astrocytes. However, the potential contribution of its hydrolytic product, butyrate, in inducing some of the changes that have been attributed to dBcAMP, is not clear. DNA, RNA, and purine synthesis were therefore studied in primary astrocyte cultures after 24 hours of exposure to varying concentrations of butyrate, dBcAMP, and agents that increase intracellular cAMP levels. Progression of cells through cell cycle was also studied by flow cytometry. Dibutyryl cAMP partially arrested cells in Go/G1 phase of cell cycle while sodium butyrate increased the percentage population of cells in G2/M phase. DNA synthesis and de novo purine synthesis were inhibited after treatment with dBcAMP, sodium butyrate, and various drugs that increase intracellular cAMP levels. RNA synthesis was increased with cAMP but was not affected by sodium butyrate. Our study shows that at millimolar concentrations, butyrate is capable of altering the cell cycle and inhibiting DNA synthesis in primary astrocyte cultures, in a manner that is similar although not identical to the effects of dBcAMP.     

Effect of methylmercury on glutamate metabolism in cerebellar astrocytes in culture

The effect of methylmercury (MeHg) on [U-13C]glutamate metabolism was studied in cerebellar astrocytes using 13C nuclear magnetic resonance spectroscopy. The cells were preincubated in medium containing 25 or 50 microM MeHg and 10% fetal calf serum for 4h and then in medium with [U-13C]glutamate (0.5mM) for 2h. Labeled glutamate, glutamine and aspartate were observed both in the cell extracts and media, labeled glutathione in the cell extracts and labeled lactate and alanine in the media. The amount of glutamate removed from the media was decreased in the 50 microM MeHg group, furthermore, the levels of both labeled and unlabeled glutamine were decreased. This might indicate a decreased synthesis and/or increased degradation. An increase was observed for glutathione in the 25 microM group, which might be due to an upregulated synthesis of glutathione in response to the toxic effects of MeHg. The percentage of [U-13C]glutamate used for the synthesis of metabolites via the tricarboxylic acid cycle was increased in the presence of 50 microM MeHg. However, the percentage used for energy production was decreased in both groups, indicating selective mitochondrial vulnerability due to the inhibitory effect of MeHg.     

Nitrate metabolism in the cyanobacterium Anabaena cycadeae: Regulation of nitrate uptake and reductase by ammonia

Nitrogen regulation of nitrate uptake and nitrate reductase (EC 1.7.99.4) was studied in the cyanobacterium Anabaena cycadeae Reinke and its glutamine auxotroph. Development of the nitrate uptake system preceded, and was independent of, the development of the nitrate reductase system. The levels of both systems were several-fold higher in the glutamine auxotroph lacking glutamine synthetase (EC 6.3.1.2) than in the wild type strain having normal glutamine synthetase activity. The nitrate uptake system was found to be NH4-repressible and the nitrate reductase system NO₃⁻-inducible. NH₄⁺ was the initial repressor signal for the uptake process which was involved in the control of the NO₃⁻inducible reductase system.     

Hypoxia regulates glutamate metabolism and membrane transport in rat PC12 cells

We investigated the effect of hypoxia on glutamate metabolism and uptake in rat pheochromocytoma (PC12) cells. Various key enzymes relevant to glutamate production, metabolism and transport were coordinately regulated by hypoxia. PC12 cells express two glutamate-metabolizing enzymes, glutamine synthetase (GS) and glutamate decarboxylase (GAD), as well as the glutamate-producing enzyme, phosphate-activated glutaminase (PAG). Exposure to hypoxia (1% O(2)) for 6 h or longer increased expression of GS mRNA and protein and enhanced GS enzymatic activity. In contrast, hypoxia caused a significant decrease in expression of PAG mRNA and protein, and also decreased PAG activity. In addition, hypoxia led to an increase in GAD65 and GAD67 protein levels and GAD enzymatic activity. PC12 cells express three Na(+)-dependent glutamate transporters; EAAC1, GLT-1 and GLAST. Hypoxia increased EAAC1 and GLT-1 protein levels, but had no effect on GLAST. Chronic hypoxia significantly enhanced the Na(+)-dependent component of glutamate transport. Furthermore, chronic hypoxia decreased cellular content of glutamate, but increased that of glutamine. Taken together, the hypoxia-induced changes in enzymes related to glutamate metabolism and transport are consistent with a decrease in the extracellular concentration of glutamate. This may have a role in protecting PC12 cells from the cytotoxic effects of glutamate during chronic hypoxia.     

Hypoxia induced metabolism dysfunction of rat astrocytes in primary cell cultures

In order to study the astroglial contribution to hypoxic injury on brain tissue metabolism, modifications of glutamine synthetase (GS) lactate dehydrogenase (LDH) enolase and malate dehydrogenase activity produced by reduced oxygen supply have been determined in primary cultures of astrocytes prepared from newborn rat cerebral cortex. Enzymatic activities were measured immediately after the hypoxic treatment (9 h) and during post injury recovery. GS level is significantly decreased in response to low oxygen pressure and increased above control value during the post hypoxic recovery period. The magnitude of GS reduction by hypoxia depends on the age of the cells in culture. Lactate dehydrogenase and enolase levels were significantly enhanced during the two periods considered. No modification of the MDH level was observed. The synthesis of LDH isoenzymes containing mainly M subunits is specifically induced by hypoxia. Our results suggest that astroglial cells may represent a particularly sensitive target toward hypoxia injury in brain tissue. Low oxygen pressure available may modify some fundamental metabolical functions of these cells such as glutamate turnover and lactic acid accumulation.     

A study of the role of glutamate dehydrogenase in the nitrogen metabolism of Arabidopsis thaliana

Glutamate-dehydrogenase (GDH, EC 1.4.1.2) activity and isoenzyme patterns were investigated in Arabidopsis thaliana plantlets, and parallel studies were carried out on glutamine synthetase (GS, EC 6.3.1.2). Both NADH-GDH and NAD-GDH activities increased during plant development whereas GS activity declined. Leaves deprived of light showed a considerable enhancement of NADH-GDH activity. In roots, both GDH activities were induced by ammonia whereas in leaves nitrogen assimilation was less important. It was demonstrated that the increase in GDH activity was the result of de-novo protein synthesis. High nitrogen levels were first assimilated by NADH-GDH, while GS was actively involved in nitrogen metabolism only when the enzyme was stimulated by a supply of energy, generated by NAD-GDH or by feeding sucrose. When methionine sulfoximine, an inhibitor of GS, was added to the feeding solution, NADH-GDH activity remained unaffected in leaves whereas NAD-GDH was induced. In roots, however, there was a marked activation of GDH and no inactivation of GS. It was concluded that NADH-GDH was involved in the detoxification of high nitrogen levels while NAD-GDH was mainly responsible for the supply of energy to the cell during active assimilation. Glutamine synthetase, on the other hand was involved in the assimilation of physiological amounts of nitrogen. A study of the isoenzyme pattern of GDH indicated that a good correlation existed between the relative activity of the isoenzymes and the ratio of aminating to deaminating enzyme activities. The NADH-GDH activity corresponded to the more anodal isoenzymes while the NAD-GDH activity corresponded to the cathodal ones. The results indicate that the two genes involved in the formation of GDH control the expression of enzymes with different metabolic functions.Abbreviations GDH glutamate dehydrogenase - GS glutamine synthetase - MSO methionine sulfoximine     

Effects of choleragen on hormonal responsiveness of adenylate cyclase in human fibroblasts and rat fat cells

Choleragen increases cyclic AMP content of confluent human fibroblasts. Maximally effective concentrations of isoproterenol and prostaglandin E₁ also induce large increases in cyclic AMP content of human fibroblasts and in confluent cultures the effect of prostaglandin E₁ is much greater than that of isoproterenol. After incubation with choleragen, the increment in cyclic AMP produced by 2 μM isoproterenol is increased and approaches that produced by 5.6 μM prostaglandin E₁. Although the concentration of isoproterenol which produces a maximal increase in cyclic AMP is similar in both control and choleragen-treated cells, lower concentrations of isoproterenol are more effective in the choleragen-treated cells. In choleragen-treated cells, although the response to 5.6 μM prostaglandin E₁ is reduced by as much as 50%, the concentration of prostaglandin E₁ required to induce a maximal increase in cyclic AMP is $\text{1}\text{10}$ that required in control cells. Thus the capacities of intact human fibroblasts to respond to isoproterenol and prostaglandin E₁ can be altered independently during incubation of intact cells with choleragen. Differences in responsiveness to the two agonists were not demonstrable in adenylate cyclase preparations from control or choleragen-treated cells.In rat fat cells, the effects of choleragen on cyclic AMP content were much smaller than those in fibroblasts. In contrast to its effect on intact fibroblasts, choleragen treatment of rat fat cells did not alter the accumulation of cyclic AMP in response to a maximally effective concentration of isoproterenol. The responsiveness of adenylate cyclase preparations to isoproterenol was also not altered by exposure of fat cells to choleragen.     

Conditioned medium from cultured rat hepatocytes completely blocks induction of glutamine synthetase by dexamethasone in several rat liver epithelial cell lines

A variant RL-ET-1G of a rat liver epithelial cell line (RL-ET-1) characterized by a very high inducibility for glutamine synthetase (GS) in response to dexamethasone was established by cultivation in glutamine-free, glutamate-supplemented culture medium. Using this cell line, conditioned medium produced by periportal hepatocytes in primary culture was found to suppress this induction, acting with a lag-phase of about 8 h irrespective whether the GS activity was basal or preinduced. Analysis of the response of several epithelial cell lines to the conditioned medium showed a reciprocal relationship between the dexamethasone-dependent induction and the residual activity after exposure to the conditioned medium, indicating that a hypothetical factor in the conditioned medium was interfering with the induction process but not with the basal GS level of these cells. Careful analysis revealed that the effect of the conditioned medium was neither due to deficiency of a component used up by the hepatocytes, nor due to glutamine or ammonia, both of which affected GS activity at concentrations above 0.5 mmol/L. The hypothetical factor was found to be quite small (molecular mass range 100–500 Da), heat and acid stable, as well as highly water soluble. Most interestingly, the conditioned medium did not suppress GS induction in astroglial cells and in the two hepatoma cell lines C2 and FAO, but strongly diminished the spontaneous induction of GS in cocultured pig hepatocytes, suggesting that the hypothetical factor acts primarily on normal nontransformed liver-derived cell populations.     

Effect of bilateral adrenalectomy on VIP receptor/effector system in rat intestinal epithelial cells

The number of vasoactive intestinal peptide (VIP) receptors and the efficiency of VIP in the stimulation of cyclic AMP accumulation in rat jejunal epithelial cells increased after bilateral adrenalectomy. However, this condition increased neither receptor affinity nor VIP potency. In addition, jejunal VIP levels followed a parallel increase. These changes reversed to control conditions after glucocorticoid replacement with dexamethasone indicating that adrenalectomy modifies the intestinal VIP receptor/effector system and suggest a relationship between corticosteroids and VIP in the functions of intestinal epithelium.     

Effect of cyanophage N-1 development on nitrogen metabolism of cyanobacterium Nostoc muscorum

Abstract The impact of cyanophage N-1 development on nitrogenase, glutamine synthetase (GS) and aminotransferases activities in the diazotrophic cyanobacterium Nostoc muscorum was investigated during its latent period. The nitrogenase activity was inhibited after 2 h of infection, suggesting that phage development does not require the product of nitrogenase activity. GS activity was not inhibited until 4 h of infection; however, a decline in activity was subsequently observed. Glutamate oxaloacetate transaminase was inhibited after 1 h of infection and no activity was detectable during the entire latent period. In contrast, glutamate pyruvate transaminase activity increased 2-fold by 4 h of infection and remained higher than the background level until the end of the latent period. The results suggested that under nitrogen fixing conditions, N-1 multiplication proceeds in the absence of nitrogen fixation and that the metabolism of amino acids is altered in favour of phage multiplication.     

Glucagon-like peptide 1 modulates calcium responses to glutamate and membrane depolarization in hippocampal neurons

Glucagon-like peptide 1 (GLP-1) activates receptors coupled to cAMP production and calcium influx in pancreatic cells, resulting in enhanced glucose sensitivity and insulin secretion. Despite evidence that the GLP-1 receptor is present and active in neurons, little is known of the roles of GLP-1 in neuronal physiology. As GLP-1 modulates calcium homeostasis in pancreatic beta cells, and because calcium plays important roles in neuronal plasticity and neurodegenerative processes, we examined the effects of GLP-1 on calcium regulation in cultured rat hippocampal neurons. When neurons were pre-treated with GLP-1, calcium responses to glutamate and membrane depolarization were attenuated. Whole-cell patch clamp analyses showed that glutamate-induced currents and currents through voltage-dependent calcium channels were significantly decreased in neurons pre-treated with GLP-1. Pre-treatment of neurons with GLP-1 significantly decreased their vulnerability to death induced by glutamate. Acute application of GLP-1 resulted in a transient elevation of intracellular calcium levels, consistent with the established effects of GLP-1 on cAMP production and activation of cAMP response element-binding protein. Collectively, our findings suggest that, by modulating calcium responses to glutamate and membrane depolarization, GLP-1 may play important roles in regulating neuronal plasticity and cell survival.     

Induction of Glutamine Synthetase by Dibutyryl Cyclic AMP in C-6 Glioma Cells

Glutamine synthetase was found to be increased in C-6 glioma cells as a result of increasing culture passage and N-6,2'-O-dibutyryl cyclic AMP (dbcAMP) treatment. At low passage dbcAMP produced a 2.5-fold increase in glutamine synthetase activity per unit of cellular protein. At high passage control glutamine synthetase was approximately double that seen at low passage, but dbcAMP produced an additional 65% increase. Lactate dehydrogenase activity was also increased by dbcAMP treatment at both low and high passage, but culture passage produced no change in the lactate dehydrogenase. With increasing culture passage, the ratio of cellular protein to DNA doubled. Therefore, expression of data per unit of protein tended to minimize the apparent changes in activity. The maximum increase in glutamine synthetase activity produced by both dbcAMP and increasing culture passage and expressed on a DNA basis was 5.6-fold. The increase in glutamine synthetase activity was generally linear during the first 20 h of drug treatment, after which enzyme activity remained nearly constant up to 72 h. Ninety percent or more of the dbcAMP remained in the medium at the end of 48-h exposure of cells to dbcAMP. 8-br-Cyclic AMP also increased glutamine synthetase activity of C-6-cels, but n-butyrate did not. Isoproterenol, which increases cyclic AMP in C-6-cells, increased glutamine synthetase activity. The effect of isoproterenol on glutamine synthetase was inhibited by the beta-adrenergic blocking agent sotalol. Cycloheximide (10 micrograms/ml) inhibited the dbcAMP effect on glutamine synthetase activity and also decreased the control enzyme activity by 60%.     

Effects of insulin secretagogues and inhibitors on phospholipid metabolism in Langerhans' islets of rat pancreas

Glucose stimulation of [32P]-prelabelled pancreatic islets induced an increased incorporation of radioactivity into phosphatidylinositol. Glucagon and agents that increases cAMP in the islet did not affect phosphatidylinositol turnover, in spite of increased release of insulin. Furthermore, the potent inhibitor of insulin release, somatostatin did not alter phospholipid metabolism. Colchicine inhibited glucose-stimulated turnover of phosphatidylinositol as well as insulin release. These results may suggest that: (1) cAMP and phosphatidylinositol turnover are involved in different transmembrane control system for regulating insulin release; and (2) the function of microtubules modulate phospholipid metabolism.     

Effect of dibutyryl cAMP on cell cycle progression of rat brain tumor cells in vitro

Summary Autoradiographic and flow microfluorometry analyses have been applied to a study of perturbed cell kinetics in 9L rat brain tumor cells treated with dibutyryl cyclic AMP and theophylline alone and in combination in vitro. At a concentration of 1 mM each, cell growth ceased shortly after the administration of these drugs. The results indicate that cells in S and G₂ phase at the time of drug administration can undergo mitosis even though a considerable prolongation of G₂ phase was apparent. However, cells in G₁ at the time of drug administration were arrested in that phase whereas those cells in S or G₂ were able to complete one mitosis before becoming arrested in the G₁ phase. This blocking effect was reversible, and cells resumed proliferation at a normal rate shortly after the removal of these drugs. This work was supported in part by NIH Cancer Research Center Grant CA-13525 and CA-19992 from NCI, and by the Association for Brain Tumor Research. Presented at the 6th International Cell Cycle Conference, March, 1976, New Orleans, Louisiana. The tumor used in this study was provided by William H. Sweet, Paul T. Kornblith, Janette L. Messer and Beverly O. Whitman of the Massachusetts General Hospital, Boston, Massachusetts.