Increase In Glutamine Synthetase Activity Research Articles

Ammonium, but not nitrate, stimulates an increase in glutamine concentration in the haemolymph of Tridacna gigas

The concentration of glutamine in Tridacna gigas haemolymph increased >35-fold following exposure to sea water supplemented with ammonium (20 μM), but no increase was observed with nitrate (20 μM). Lack of a diel cycle, no decrease in haemolymph glucose levels, the expression patterns of glutamine synthetase in zooxanthellae and host, and the lack of glutamine release in response to nitrate supplementation all support the proposition that the increase in haemolymph glutamine is a product of the host and not the zooxanthellae. Unlike ammonium, nitrate accumulates rapidly in the haemolymph. It has no effect on the concentration of glutamine in the haemolymph, but there is an increase in arginine, histidine and lysine in the haemolymph, suggesting the release of these essential amino acids from zooxanthellae. Glutamine synthetase (GS) activity decreased markedly in the gill and less so in the mantle over a period of 6 d exposure to elevated ammonium (20 μM). In contrast, GS activity in zooxan- thellae doubled. The response of zooxanthellae in situ was confirmed by incubating freshly isolated zooxanthellae for 4 d in ammonium, which resulted in a ten-fold increase in GS activity. Comparison of the in situ response of zooxanthellae with that obtained in vitro indicates that the symbionts are likely to be exposed to ammonium concentrations lower than that found in the haemolymph.

Glutamine synthetase expression and activity are regulated by 3,5,3 -TRIODO-l-THYRONINE and hydrocortisone in rat oligodendrocyte cultures

Glutamine synthetase plays a central role in the detoxification of brain ammonia. Previously, we demonstrated that in vitro glutamine synthetase is expressed by all macroglial cell types and is developmentally regulated in oligodendrocyte lineage. Furthermore, glutamine synthetase is increased in secondary cultures of oligodendrocytes following a 72 h treatment with 30 nM 3,5,3′-triodo-l-thyronine [Baas, D., Bourbeau, D., Sarliève, L. L., Ittel, M. E., Dussault, J. H. and Puymirat, J., Oligodendrocyte maturation and progenitor cell proliferation are independently regulated by thyroid hormone. Glia, 1997, 19, 324–332]. Hydrocortisone also increases glutamine synthetase activity after 72 h [Fressinaud, C., Weinrauder, H., Delaunoy, J. P., Tholey, G., Labourdette, G. and Sarliève, L. L., Glutamine synthetase expression in rat oligodendrocytes in culture: regulation by hormones and growth factors. J. Cell. Physiol., 1991, 149, 459–468]; however, it is still unknown whether these increases in glutamine synthetase expression in oligodendrocytes after 3,5,3′-triodo-l-thyronine and hydrocortisone application are dose- and time-dependent. To further investigate this issue, we measured glutamine synthetase levels by Northern analysis, immunostaining and determination of glutamine synthetase activity after 3,5,3′-triodo-l-thyronine or hydrocortisone stimulation. We find that in rat oligodendrocyte secondary cultures, 3,5,3′-triodo-l-thyronine and hydrocortisone cause a dose- and time-dependent increase in glutamine synthetase mRNA, protein and activity. However, these hormones do not exert an additive or synergistic effect. Because purines, pyrimidines, and certain amino acids necessary for the synthesis of myelin components, are, in part, provided by the glutamine synthetase pathway, 3,5,3′-triodo-l-thyronine effect on myelination development and maturation could be mediated in part, through the glutamine synthetase gene regulation.

Transcriptional regulation of the rat glutamine synthetase gene by tumor necrosis factor-alpha.

The expression of glutamine synthetase (GS) is induced in rat skeletal muscle cells (L-6) in response to treatment with the inflammatory cytokine tumor necrosis factor alpha (TNF-alpha). This paper reports the regulation of GS expression in rat skeletal muscle which expresses high levels of GS. TNF-alpha treatment leads to a 3-4-fold increase in GS activity in a dose-dependent and time-dependent manner. Northern-blot analysis of GS mRNA revealed an increased mRNA concentration, reaching a peak at 12 h in response to TNF-alpha treatment. To monitor transcriptional activation of GS by TNF-alpha, and to identify a TNF-alpha-responsive element in the GS promoter, L6 cells were treated with TNF-alpha following transfection of GS-chloramphenicol-acetyltransferase (CAT) constructs. The first 251-bp fragment at the GS upstream sequence showed basal promoter activity, but failed to show any TNF-alpha-inducible activity. However, a 2.5-3-fold induction was noted in constructs extending up to 1.1 kb. This data demonstrates that the rat GS gene is transcriptionally regulated by TNF-alpha and identifies a TNF-alpha-responsive region at the 5' flanking sequence of the GS gene.

Ammonium and soluble amide-bound nitrogen in leaves of Brassica napus as related to glutamine synthetase activity and external N supply

Short and long term effects of variations in external N supply on the content of soluble N compounds and glutamine synthetase (GS) activity were investigated in leaves of vegetatively growing oilseed rape ( Brassica napus L., cv. Global). Only the chloroplastic isoform of GS had a detectable activity. Plants growing continuously for 5 weeks at elevated nitrogen levels had 3-fold higher levels of NH 4 + and soluble amides compared with plants growing at low levels of N supply. In contrast, GS activities were similar at the different levels of N supply. Addition of a pulse of nitrogen to plants of medium N status caused a rapid and large increase in NH 4 + and soluble amides within the leaves. Pulse addition of nitrogen also triggered a temporary increase in GS activity, thereby maintaining the concentration of NH 4 + below 14 μmol NH 4 +·g −1 leaf fresh weight (corresponding to 21 mM NH 4 + ammonium on tissue water basis). This level of NH 4 + was equal to the maximum level observed in leaves of plants growing permanently at elevated N supplies in spite of the fact that the latter plants received a higher total amount of nitrogen. It is concluded that tissue concentrations of NH 4 + and soluble amides in B. napus may vary considerably with external N supply, although accumulation of exceedingly high NH 4 + concentrations can be avoided by stimulation of GS activity.

Cloning of an EF-P homologue from Bacteroides fragilis that increases B. fragilis glutamine synthetase activity in Escherichia coli.

Investigations of possible regulators of Bacteroides fragilis glutamine synthetase (GS) activity were done in Escherichia coli using a compatible dual-plasmid system. The B. fragilis glnA gene, together with upstream and downstream flanking regions, was cloned onto the low copy number plasmid pACYC184 and expressed in the E. coli glnA ntrB ntrC deletion strain, YMC11. GS activity was monitored following co-transformation with a B. fragilis genomic library carried on the compatible plasmid pEcoR251. A gene was cloned that caused a twofold increase in B. fragilis GS activity but did not affect the activity of the E. coli GS enzyme or the B. fragilis sucrase (ScrL). Deletion of the B. fragilis glnA downstream region decreased basal levels of GS activity, but did not affect the ability of the cloned gene to increase the B. fragilis GS activity. Reporter gene analysis, using the B. fragilis glnA promoter region fused to the promoterless Clostridium acetobutylicum endoglucanase gene, showed no increase in reporter gene activity. This demonstrated that the increase in GS activity was not regulated at the transcriptional level, and that the cloned gene product was not affecting the copy number of the plasmid in trans. Sequence data indicated that the cloned gene had good amino acid identity to a range of elongation factor P (EF-P) proteins, the highest being to that of a Synechocystis sp (48%), and the least to Mycobacterium genitalium (27%). Amino acid identity to the E. coli EF-P was intermediate (37%). A possible role for EF-P in enhancing translation of the B. fragilis glnA mRNA is proposed.

Programmed cell death in cortical chick embryo astrocytes is associated with activation of protein kinase PK60 and ceramide formation.

Embryonic astrocytes respond readily to serine/threonine kinase regulation in terms of cytoskeleton assembly, mitotic activity, and cell fate. We now present evidence that these responses include apoptosis. Staurosporine induced apoptosis in astrocyte cultures derived from chick embryo cerebral hemispheres, as assayed both by immunocytochemical detection of new 3-hydroxy DNA ends and production of 200-bp DNA fragment laddering. Staurosporine treatment also resulted in the prolonged (>24 h) activation of a 60-kDa serine/threonine protein kinase (PK60), increased ceramide formation (fourfold after 24 h), increased glutamine synthetase activity, and significant apoptosis (40%) after 24 h. PK60 was shown to be cytoskeleton associated and its activity, as measured by phosphorylation of myelin basic protein, was rapid, increased for up to 3 h, and was stable for at least 24 h. Other protein kinase C inhibitors, H8, sphingosine, calphostin C, or the protein kinase A inhibitor KT5720 did not induce either PK60 activation or apoptosis. The dose-dependent increase in [3H]palmitate labeling of ceramide and a specific decrease in labeling of its precursor sphingomyelin were not blocked by the biosynthetic inhibitor fumonisin beta1 but were increased (in a dose-dependent manner) by the coaddition of the ceramidase inhibitor oleoylethanolamine. Exogenous addition of C2-ceramide induced apoptosis but did not activate PK60. These results suggest that apoptosis in embryonic astrocytes involves pathways similar to those described in other cell types and that the activation of PK60 and formation of ceramide are early events in the pathway.

Endothelial cell conditioned media mediated regulation of glutamine synthetase activity in glial cells

The responsiveness of late passage C-6 glial cells to human retinal endothelial cell-conditioned medium (HREC-CM) was examined using glutamine synthetase (GS) activity as test parameter. Treatment with 50% or 100% HREC-CM for 4–5 days slightly affected the morphology but significantly increased GS activity. Increased glial GS activity induced by vascular endothelial cells is of relevance in preventing extracellular glutamate toxicity and regulating the brain/retinal blood barrier.

Contribution of Glial Metabolism to Neuronal Damage Caused by Partial Inhibition of Energy Metabolism in Retina

Glial cells are relatively resistant to energy impairment, although little is known of the extent to which glial metabolism is affected during partial energy impairment and how this influences neurons. Fluorocitrate has been shown to be a glial specific metabolic inhibitor. Its selective effect on chick retinal Müller cells was verified by measuring incorporation of radiolabel from3H-acetate and U-14C-glucose into glutamate and glutamine following exposure of isolated embryonic day 15–18 chick retina to 20 μmfluorocitrate. Fluorocitrate significantly reduced the incorporation of radiolabel from acetate and glucose into glutamine, with less effect on incorporation of label from acetate into glutamate and no reduction of label from glucose into glutamate. The relative specific activity (RSA; ratio of glutamine to glutamate) increased between embryonic day 15 and 18 consistent with the increase in glutamine synthetase activity that occurs in Müller cells at this time in chick retinal development. As with previous findings, mild energy stress produced by inhibiting glycolysis with the general inhibitor iodoacetate (IOA) for up to 45 min, caused acute neuronal damage that was predominately NMDA receptor mediated and occurred in the absence of a net efflux of excitatory amino acids. Acute NMDA-mediated toxicity in this preparation is characterized by the selective damage to amacrine and ganglion cells and quantitatively, by GABA release into the medium. When IOA was combined with fluorocitrate, acute toxicity was potentiated and temporally accelerated. Acute damage was first noted at 15 min, occurred throughout all retinal layers and was accompanied by an overflow of excitatory amino acids at 30 and 45 min. Blocking NMDA receptors with MK-801 during IOA plus fluorocitrate exposure attenuated the rise in excitatory amino acids and prevented the swelling in neuronal, but not Müller cells. Following incorporation of radiolabel from acetate and glucose into glutamate and glutamine after different times of exposure to IOA showed that while the effects of incorporation of label from glucose were immediate, glutamine synthesis from acetate was preserved for a longer period of time. These findings suggest that during a partial energy impairment, neuronal metabolism is affected to a greater extent than is glial metabolism. Glial cells may play a protective role in this situation, and can delay the onset of acute neuronal damage.

Microtubule dynamics regulates the level of endothelin-B receptor in rat cultured astrocytes.

We investigated the effect of cytoskeleton modulators on endothelin-B (ET(B)) receptor expression in rat primary cultured astrocytes. Northern blot analysis and a binding study revealed that colchicine and nocodazole, microtubule-disrupting agents, decreased the levels of both ET(B) receptor mRNA and the number of ET-1 binding sites in quiescent astrocytes. Down-regulation of both ET(B) receptor mRNA and the number of binding sites for ET-1 was also observed in quiescent astrocytes treated with taxol, a microtubule-stabilizing agent. In contrast, neither beta-lumicolchicine, an inactive isomer of colchicine, nor cytochalasin D, a microfilament-disrupting agent, influenced ET(B) receptor expression. The level of ET(B) receptors in astrocytes was affected by the cell state, namely, proliferative, quiescent, or differentiated state. The order of ET(B) receptor expression according to the cell state was proliferative state < quiescent state << differentiated state induced by dibutyryl cyclic AMP. Also, in proliferative astrocytes and differentiated astrocytes, colchicine significantly down-regulated both ET(B) receptor mRNA and the number of binding sites for ET-1. However, thymidine assay revealed that colchicine did not change quiescent astrocytes and differentiated astrocytes to a proliferative state. Furthermore, the increase in glutamine synthetase activity in differentiated astrocytes was not affected by colchicine. These results suggest that microtubule dynamics possibly regulates ET(B) receptor expression in astrocytes without affecting the cell state.

Amino acid metabolism in the kidneys of genetic and nutritionally obese rats.

The ability of the kidney to take up and/or release amino acids has been determined in two models of obesity in Zucker rats, one genetic and the other nutritional (diet-obese). There was a noticeable increase in gluconeogenic amino acids in the arterial blood of diet-obese animals whereas the genetically obese rats showed small variations in the levels of these amino acids. There were significant decreases in renal Gly and Ser, only in the genetically obese rats. Genetically obese animals showed an increase in Glutamine synthetase activity. The uptake and/or release of amino acids showed important variations between the groups. The diet-obese group exhibited greater variation, since this group took up Glu, Ala, Gy, Phe and Citrulline and released Gln, Ser, Arg and Tyr. Genetically obese rats took up Gln, His and Taurine and released Ser. These different patterns may be related to variations in the whole body metabolic rate, since the diet-obese group was more active than the genetically obese group.

GLUTAMINE DEPRIVATION IN RAT INTESTINAL CELLS. 476

Our objective is to establish the importance of both endogenous and exogenous sources of glutamine (GLN) for proliferation and differentiation of small intestinal epithelium. We hypothesize that exposure to sub-physiologic concentrations of GLN (<0.4 mM) in cell culture media (exogenous GLN source) results in upregulation of glutamine synthetase (GS) activity(endogenous GLN source). Increased GS activity should compensate for the lack of exogenous GLN and result in no differences in variables indicative of cell growth. IEC-6 cells were grown in GLN-free DMEM containing 10% FBS. Confluent stock cultures were trypsinized and seeded (1 × 105 cells/well) onto plates grown in media containing 1 of 6 different concentrations of supplemental GLN (4, 2, 0.4, 0.2, 0.02, and 0 mM). The approximate percentage of area covered by cells, an indicator of cell growth, was monitored each day and time to confluency was noted. Cells were trypsinized and processed for determination of protein concentration, GS activity (μ M•mg-1protein•h-1), and 3H-thymidine incorporation (dp m•mg protein-1). Results (means± SD, *p<0.05): All variables decreased in response to decreased [GLN] in a dose-dependent manner. Table

Oligodendrocyte maturation and progenitor cell proliferation are independently regulated by thyroid hormone.

The development of oligodendrocyte progenitor cells is regulated by epigenetic factors which control their proliferation and differentiation. When oligodendrocyte progenitor cells, purified on a Percoll centrifugation gradient from neonate rat brain, are cultured in serum-free medium in the presence of platelet-derived-growth factor (PDGF), they divide and their differentiation is delayed. Triiodothyronine (T3) treatment of progenitor cells blocks their proliferation and induces their differentiation into oligodendrocytes. T3 also induces morphological differentiation of oligodendrocytes as indicated by the marked increase in the length of oligodendrocyte processes. To determine whether the effects of T3 on progenitor cell proliferation and oligodendrocyte maturation are causally related, or instead, are independent, we examined the influence of T3 on secondary cultures of postmitotic oligodendrocytes. We show that T3 increases morphological and functional maturation of postmitotic oligodendrocytes as indicated by a well developed network of branched processes and by the expression of myelin/oligodendrocyte glycoprotein (MOG) and glutamine synthetase (GS). T3 increases glutamine synthetase activity and its message level after a lag period of 24-48 h, and these levels increase through a posttranscriptional event. In contrast, no effect of T3 was observed on myelin basic protein (MBP) gene expression as determined by Northern blot analysis. Our results indicate that thyroid hormones participate in the control of the progenitor cell proliferation and differentiation as well as in oligodendrocyte maturation and that these two T3-regulated events are independent.

Induction of glutamine synthetase by l- α-aminoadipate in developmental stages of cultured astrocytes

Effect of l- α-aminoadipate ( l αAA), a gliotoxic l-glutamate analogue, on glutamine synthetase (GS) activity of rat cultured astrocytes was examined. l αAA at sub-toxic concentrations (less than 0.5 mM, for 48 h) increased GS activity of cultured astrocytes. This increase was prevented by 10 μM cycloheximide, an inhibitor of protein synthesis. l αAA increased GS activities of astrocytes cultured in three different conditions, i.e. 12-day-old, 5-week-old and dibutyryl cAMP(DBcAMP)-induced differentiated cultures. Insulin (10 μg/ml) and hydrocortisone (10 μM) increased GS activity of 12-day-old cultured cells, but not that of 5-weeks-old and DBcAMP-treated cells. The increase in GS activity was observed after a transient treatment with l αAA for 4 h. These results show that the induction of GS by l αAA is not related to developmental stages of astrocytes in culture.

Glutamine synthetase induction by glucocorticoids is preserved in skeletal muscle of aged rats.

Glutamine synthetase (GS) is a glucocorticoid-inducible enzyme that has a key role for glutamine synthesis in muscle. We hypothesized that the glucocorticoid induction of GS could be altered in aged rats, because alterations in the responsiveness of some genes to glucocorticoids were reported in aging. We compared the glucocorticoid-induced GS in fast-twitch and slow-twitch skeletal muscles (tibialis anterior and soleus, respectively) and heart from adult (age 6-8 mo) and aged (age 22 mo) female rats. All animals received dexamethasone (Dex) in their drinking water (0.77 +/- 0.10 and 0.80 +/- 0.08 mg/day per adult and aged rat, respectively) for 5 days. Dex caused an increase in both GS activity and GS mRNA in fast-twitch and slow-twitch skeletal muscles from adult and aged rats. In contrast, Dex increased GS activity in heart of adult rats, without any concomitant change in GS mRNA levels. Furthermore, Dex did not affect GS activity in aged heart. Thus the responsiveness of GS to an excess of glucocorticoids is preserved in skeletal muscle but not in heart from aged animals.

Effect of growth factors on the in vitro growth and differentiation of early and late passage C6 glioma cells

The effect of different hormones and growth factors was assayed on the in vitro growth and enzymatic activities of 2′,3′-cyclic nucleotide 3′phosphohydrolase (CNP) and glutamine synthetase (GS) of rat glioma C6 cells at two different passages in culture. Young cultures (passage 26), mainly oligodendrocytic, and older cultures (passage 134), predominantly astrocytic, were treated with 10 μM dexamethasone, 20 ng/ml transforming growth factor alpha (TGFα), 10 ng/ml insulin, 20 ng/ml platelet-derived growth factor (PDGF), and 20 ng/ml, epidermal growth factor (EGF) in serum-free chemically defined media. In vitro growth rate was measured in terms of DNA content, by a fluorometric method of diaminobenzoic acid, and rate of DNA synthesis by 3H-thymidine incorporation. CNP activity (marker for in vitro oligodendrocytes) and GS activity (marker for astrocytes) were determined spectrophotometrically. Dexamethasone reversibly and significantly inhibited growth of C6 glioma in early and late passages. PDGF and insulin promoted in vitro growth only in late passage but not in early passage cells, whereas EGF and TGFα did not significantly affect growth. An increase in CNP activity was observed in early passage cells under the effect of PDGF and insulin. The increase in GS activity induced by insulin and dexamethasone suggests a differentiating role for these factors in C6 glioma cells. These results further present the C6 glioma cell line as a useful model for studies on glial cell properties and responsiveness in culture and support its use in experimental aging in vitro.

Differential responsiveness of late passage C-6 glial cells and advanced passages of astrocytes derived from aged mouse cerebral hemispheres to cytokines and growth factors: glutamine synthetase activity.

In this study, we were interested to compare the responsiveness to growth factors, NGF, b-FGF and EGF and cytokines, IL1 beta, and TNF-alpha, in late passages (74-79) C6 glial cells committed astrocytes and astrocytes of advanced passages (26-28) in cultures derived from aged mouse cerebral hemispheres (MACH). Cultures were grown in either DMEM or chemically defined medium (CDM/TIPS) in order to test the effects of growth factors or cytokines. The activity of glutamine synthetase (GS), a marker for astrocytes, was used as a test parameter. We found that treatment with growth factors increased GS activity in both glial cell culture systems with the exception of EGF in C-6 glial cells. Treatment with cytokines markedly decreased GS activity in the late passage C6 glial cells whereas only TNF-alpha had a similar effect on MACH astrocytes. In view of the generally opposite effects of growth factors and cytokines on GS activity, we speculate that these molecules which are also endogenously present in glial cells may play a role in the maintenance of cellular homeostasis.

Plasticity of astrocytes derived from aged mouse cerebral hemispheres: Changes with cell passage and immortalization

This study was targeted at the beginning to understand the functional status of glial cells derived from aged brain. We have previously characterized passaged cell cultures derived from aged mouse cerebral hemispheres (MACH) and found them to contain large populations of astrocytes, type 1, as well as limited numbers of astrocytes, type 2, oligodendrocytes, and progenitor cells. Using the activity of the astrocyte marker, glutamine synthetase (GS), as an index, we found that MACH astrocytes continue to respond to several microenvironmental signals, including the cAMP-enhancing agents dibutyrl cAMP and RO20-1724 (an inhibitor of phosphodiesterase). In addition, whereas the basal activity of GS increased with cell passage, their response to these agents was cell-passage dependent, increasing at early (21–22) passages and decreasing at later (46–51) passages. Because neurotrophins (i.e., NGF and EGF) also provide microenvironmental signals essential to normal glial function, MACH cultures were assessed for their response to these factors, MACH cultures at passage 35 responded to treatment with NGF and EGF with a dose-dependent increase in GS activity by both neurotrophins. With the intention of arresting these cultures at a specific stage of differentiation, these cells were immortalized at passage 19 by transfection with the gene encoding SV40 Large T antigen. These immortalized MACH responded to exposure to dBcAMP and RO20-1724 with a marked decrease in GS activity, mimicking the response of normal MACH glia at late passage. Finally, because it has been shown that glia from both immature and adult brain contain neurotrophins and respond to neurotrophins via a receptor-mediated pathway, we examined expression of NGF protein as well as NGF (p 75) and EGF receptor protein in various passages and colonies of normal and immortalized MACH cultures. We found a consistent expression of all three proteins in the various cell populations. Results of this study suggest that astrocytes from aging brain continue to function normally with respect to several parameters (i.e., response to neurotrophins and differentiating agents). Thus, they retain their plasticity to a great degree through early cell passages. However, with advancing cell passage this plasticity declines and cell homeostasis is impaired. We propose, therefore, that astrocytes undergo several critical periods in their functional lifespan, one of which is represented by the functional transition demonstrated in this study.

Interactions of Cortisol and Nitrogen Metabolism in the Ureogenic Gulf Toadfish Opsanus Beta

We examined the relationship between plasma cortisol levels, hepatic glutamine synthetase (GNS) activity and the form of nitrogen excreted (e.g. urea, ammonia) in undisturbed versus confined/crowded (acutely stressed) gulf toadfish. Specifically, we tested the hypothesis that acute increases in plasma cortisol levels are required to trigger the increase in GNS activity induced by the confinement/crowding stress. Toadfish responded to the stress of confinement/crowding with an initial cortisol surge (approximately 37 ng ml-1 at 2 h), which was rapidly cleared and was indistinguishable from resting levels (approximately 10 ng ml-1 by 24 h). Treatment of fish with metyrapone (which inhibits cortisol synthesis) successfully blocked the acute 2 h confinement/crowding-induced surge in plasma cortisol levels. Additionally, GNS activity in confined fish 24 h after metyrapone injection also did not differ from that of control fish, indicating that acute GNS activation probably requires the earlier (2 h) cortisol peak. In post-absorptive fish, a strong relationship between total nitrogen excretion rate and plasma cortisol levels was evident. The percentage of nitrogen excreted as ammonia was inversely related to liver GNS activity. However, GNS activity explained only part (at most 57%) of the variability in the percentage of nitrogen excreted as ammonia/urea, suggesting that this is not the sole factor setting the degree of ureogenesis. When toadfish are fed, the relationships between total nitrogen excretion rate and cortisol levels, and between percentage nitrogen excreted as ammonia and GNS activity, are virtually absent. Taken together, our results indicate that a stress response may be only one of several mechanisms by which ureogenesis is activated in gulf toadfish.

Astrocyte differentiation is enhanced in chick embryos treated with ethanol during early neuroembryogenesis.

In this study, we examined the effects of ethanol administered to chick embryos, on the maturation of astrocytes, using glutamine synthetase (GS) activity as an astrocyte marker. Ethanol (50 mM) was administered in ovo via the air sac, embryos were sacrificed at various days of embryonic development and GS activity was determined in cerebral hemispheres and cerebellum. We found that in both cerebral hemispheres and cerebellum, GS activity was higher in the ethanol-treated embryos, as compared to controls, during the embryonic periods, E6 to E10 in the cerebral hemispheres and E10 to E14 in the cerebellum. These periods are characterized by increased neuronal differentiation in these CNS areas. The increase in GS activity in the ethanol-treated embryos is speculated to reflect either a transient reactive gliosis and/or an enhancement in the differentiation of radial glia, immature glia, to more mature astrocytes.

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.