Messenger RNA coding for glutamine synthetase in cerebral hemispheres and astroglial cultures from mouse brain: A developmental study

Messenger RNAs from mouse brain hemispheres and from an enriched astroglial population of the same area were isolated, characterized and used to study Glutamine Synthetase (GS) processing during postnatal development using a m-RNA stimulated reticulocyte lysate system.The RNA preparations yielded distinct polypeptide products upon translation including high molecular weight species. Polypeptides in the range of 43–55 kDa appeared developmentally regulated in brain hemispheres but not in astroglia. After immunoprecipitation of the translation products with a GS antibody a major monomeric polypeptide was isolated on SDS/PAGE which migrated at the same position as the purified brain GS antigen (43 kDa).The translatable mRNA were optimal in the perinatal period and decreased until 300 days while GS-mRNA increased during the same period of time and closely paralleled the previously described GS activity profile in this brain area reaching an optimum at 15 days. Astroglial mRNAs were optimal at 18 days in vitro and decreased thereafter. The GS-mRNA was much lower in control astroglial cultures than in brain tissue, but in the presence of 10⁻⁶M hydrocortisone increased all over the growth period. The highest stimulation of GS-mRNA was observed at 18 days whereas the global mRNA decreased in the presence of the hormone.The GS-mRNAs from either 15-day-old brain hemispheres or 18-day in vitro hydrocortisone stimulated cultures were partially purified on a 5–30% linear sucrose gradient. Two GS-mRNAs, which sedimented respectively at 17S and 23S, were characterized. In addition, based on the profile of total proteins translated in vitro, we estimated that GS-mRNA constituted 0.01% of the brain hemisphere fraction and 0.3% in the astroglial hormone stimulated cells.     

Expression and modulation of the intermediate- conductance Ca2+-activated K+ channel in glioblastoma GL-15 cells.

We report here the expression and properties of the intermediate-conductance Ca(2+)-activated K(+) (IK(Ca)) channel in the GL-15 human glioblastoma cell line. Macroscopic IK(Ca) currents on GL-15 cells displayed a mean amplitude of 7.2+/-0.8 pA/pF at 0 mV, at day 1 after plating. The current was inhibited by clotrimazole (CTL, IC(50)=257 nM), TRAM-34 (IC(50)=55 nM), and charybdotoxin (CTX, IC(50)=10.3 nM). RT-PCR analysis demonstrated the expression of mRNA encoding the IK(Ca) channel in GL-15 cells. Unitary currents recorded using the inside-out configuration had a conductance of 25 pS, a K(D) for Ca(2+) of 188 nM at -100 mV, and no voltage dependence. We tested whether the IKCa channel expression in GL-15 cells could be the result of an increased ERK activity. Inhibition of the ERK pathway with the MEK antagonist PD98059 (25 muM, for 5 days) virtually suppressed the IK(Ca) current in GL-15 cells. PD98059 treatment also increased the length of cellular processes and up-regulated the astrocytic differentiative marker GFAP. A significant reduction of the IKCa current amplitude was also observed with time in culture, with mean currents of 7.17+/-0.75 pA/pF at 1-2 days, and 3.11+/-1.35 pA/pF at 5-6 days after plating. This time-dependent downregulation of the IK(Ca) current was not accompanied by changes in the ERK activity, as assessed by immunoblot analysis. Semiquantitative RT-PCR analysis demonstrated a ~35% reduction of the IK(Ca) channel mRNA resulting from ERK inhibition and a approximately 50% reduction with time in culture.     

A Time-Dependent Increase in Glial Fibrillary Acidic Protein Expression and Glutamine Synthetase Activity in Long-Term Subculture of the GL15 Glioma Cell Line

1. Astrocytes are the most numerous cellular elements in the central nervous tissue, where they play a critical role in physiological and pathological events. The biological signals regulating astrocyte growth and differentiation are relevant for both physiology and pathology, but they are still little understood.2. Using a poorly differentiated glioma cell line, GL15, we investigated whether, in long-term subculture, this could upregulate the expression of glial fibrillary acidic protein (GFAP), as described in some rodent astrocyte cell lines. Under the same culture conditions, we investigated glutamine synthetase (GS) activity, growth-associated protein (GAP)-43 expression, and expression of several neutrotrophic factors.3. A dramatic increase in GFAP expression was evidenced by Western blotting during progressive in vitro growth of GL15 cells. GS specific activity was also upregulated in long-term culture. The time spent in vitro by GL15 cells did not affect GAP-43 and neutrophic factor BDNF and NT3 expression as revealed by RT-PCR analysis.4. Our results suggest that, in GL15, GFAP and GS genes may have common or integrated regulatory mechanisms elicited at the cell confluency which could be relevant for both astrocyte physiology and astrocyte pathology. These mechanisms are not involved in GAP-43 and neutrophic factor BDNF and NT3 expression.     

Influence of Different Concentrations of NO-3 and NH+4 on the Activity of Glutamine Synthetase and Other Relevant Enzymes of Nitrogen Metabolism in Wheat Roots

Influence of different concentrations of NO3 and NH+ on the activity of glutamine synthetase (GS), asparagine synthetase (AS), glutamate dehydrogenase (GDH), nitrate reductase (NR) and the changes of GS-mRNA in wheat roots have been studied with enzymes activity assay and Northern blot. The results showed that the higher GS activity was found in roots of wheat when NH+4-N was the sole nitrogen source than when NO3-N was the sole nitrogen source. GS-mRNA of Northern blot was simillar to GS activity. 3 mmol/L NO3- promoted the activity of AS. The change of AS was independent of the change of GS. GDH activity was not been detected, and change in regulation of NR activity was not found.     

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.     

Phase-Dependent Astroglial Alterations in Li–Pilocarpine-Induced <Emphasis Type="Italic">Status Epilepticus</Emphasis> in Young Rats

Epilepsy prevalence is high in infancy and in the elderly population. Lithium–pilocarpine is widely used to induce experimental animal models of epilepsy, leading to similar neurochemical and morphological alterations to those observed in temporal lobe epilepsy. As astrocytes have been implicated in epileptic disorders, we hypothesized that specific astroglial changes accompany and contribute to epileptogenesis. Herein, we evaluated time-dependent astroglial alterations in the hippocampus of young (27-day-old) rats at 1, 14 and 56 days after Li–pilocarpine-induced status epilepticus (SE), corresponding to different phases in this model of epilepsy. We determined specific markers of astroglial activation: GFAP, S100B, glutamine synthetase (GS), glutathione (GSH) content, aquaporin-4 (AQP-4) and potassium channel Kir 4.1; as well as epileptic behavioral, inflammatory and neurodegenerative changes. Phase-dependent signs of hippocampal astrogliosis were observed, as demonstrated by increments in GFAP, S100B and GS. Astrocyte dysfunction in the hippocampus was characterized, based on the decrease in GSH content, AQP-4 and Kir 4.1 channels. Degenerating neurons were identified by Fluoro-Jade C staining. We found a clear, early (at SE1) and persistent (at SE56) increase in cerebrospinal fluid (CSF) S100B levels. Additionally, serum S100B was found to decrease soon after SE induction, implicating a rapid-onset increase in the CSF/serum S100B ratio. However, serum S100B increased at SE14, possibly reflecting astroglial activation and/or long-term increase in cerebrovascular permeability. Moreover, we suggest that peripheral S100B levels may represent a useful marker for SE in young rats and for follow up during the chronic phases of this model of epilepsy. Together, results reinforce and extend the idea of astroglial involvement in epileptic disorders.     

Glutamine synthetase in brain: effect of ammonia

Glutamine synthetase (GS) in brain is located mainly in astrocytes. One of the primary roles of astrocytes is to protect neurons against excitotoxicity by taking up excess ammonia and glutamate and converting it into glutamine via the enzyme GS. Changes in GS expression may reflect changes in astroglial function, which can affect neuronal functions.Hyperammonemia is an important factor responsible of hepatic encephalopathy (HE) and causes astroglial swelling. Hyperammonemia can be experimentally induced and an adaptive astroglial response to high levels of ammonia and glutamate seems to occur in long-term studies. In hyperammonemic states, astroglial cells can experience morphological changes that may alter different astrocyte functions, such as protein synthesis or neurotransmitters uptake. One of the observed changes is the increase in the GS expression in astrocytes located in glutamatergic areas. The induction of GS expression in these specific areas would balance the increased ammonia and glutamate uptake and protect against neuronal degeneration, whereas, decrease of GS expression in non-glutamatergic areas could disrupt the neuron-glial metabolic interactions as a consequence of hyperammonemia.Induction of GS has been described in astrocytes in response to the action of glutamate on active glutamate receptors. The over-stimulation of glutamate receptors may also favour nitric oxide (NO) formation by activation of NO synthase (NOS), and NO has been implicated in the pathogenesis of several CNS diseases. Hyperammonemia could induce the formation of inducible NOS in astroglial cells, with the consequent NO formation, deactivation of GS and dawn-regulation of glutamate uptake. However, in glutamatergic areas, the distribution of both glial glutamate receptors and glial glutamate transporters parallels the GS location, suggesting a functional coupling between glutamate uptake and degradation by glutamate transporters and GS to attenuate brain injury in these areas.In hyperammonemia, the astroglial cells located in proximity to blood-vessels in glutamatergic areas show increased GS protein content in their perivascular processes. Since ammonia freely crosses the blood-brain barrier (BBB) and astrocytes are responsible for maintaining the BBB, the presence of GS in the perivascular processes could produce a rapid glutamine synthesis to be released into blood. It could, therefore, prevent the entry of high amounts of ammonia from circulation to attenuate neurotoxicity. The changes in the distribution of this critical enzyme suggests that the glutamate-glutamine cycle may be differentially impaired in hyperammonemic states.     

Glial fibrillary acidic protein in rat brain structures under conditions of training for a passive defensive habit

Under conditions of training for a passive defensive habit in rats, changes were observed in the content of an intermediate filament marker of the astroglial cellular cytoskeleton, glial fibrillary acidic protein (GFAP), in different brain structures (frontal cortex, medial thalamus, striatum, hippocampus, and pons). It is hypothesized that GFAP is involved in the control of synaptic plasticity underlying memory formation.     

Further similarities between astrocytes and perisinusoidal stellate cells of liver (Ito cells): Colocalization of desmin and glial fibrillary acidic protein in astroglial primary cultures

The colocalization of desmin and glial fibrillary acidic protein (GFAP) in astrocytes was inferred from previous studies demonstrating a unique antigenic composition comprising GFAP, desmin and vimentin in perisinusoidal stellate cells (PSC) of liver which share several features with astrocytes. In the present study the colocalization of GFAP and desmin was investigated by double-immunolabeling experiments in 12-day-old rat astroglial primary cultures with antiserum against GFAP and two commercial monoclonal antibodies against desmin, antibodies of clone DEU-10 and clone DEB-5. These antibodies selectively decorated the perisinusoidal stellate cells (PSC) of liver for which desmin is known to be a marker. The results obtained with astroglial cells demonstrate that both GFAP and desmin are coexpressed in morphologically different types, process-bearing and process-lacking astrocytes. The expression of desmin was apparently more pronounced in process-lacking astrocytes and was considerably lower in process-bearing ones. In process-lacking astrocytes, in contrast to filamentous cytoplasmic staining for GFAP, the immunoreactivity for desmin was non-filamentous and was irregularly spread in the perinuclear cytoplasm of the cells, while in process-bearing astrocytes the pattern of staining for desmin was similar to that of GFAP. The variability in the intensity and pattern of staining for desmin in astrocytes might be due to transitional stages of differentiation for part of the cells. This interpretation was supported by the presence of GFAP in the cells weakly expressing smooth muscle alpha-actin and the absence of GFAP in the cells enriched with microfilaments.     

Specific Insulin-Mediated Regulation of Glutamine Synthetase in Cultured Chick Astroglial Cells

The expression of glutamine synthetase (GS; L-glutamate ammonia ligase; EC 6.3.1.2) in primary cultures of chick astroglial cells and neurons grown in a chemically defined medium, with and without insulin added, was investigated. An inhibitory effect of insulin toward GS activity, and specific to chick astroglial cells, was observed. Neurons in culture were not sensitive to the hormone effect. Modulation of the activating effect of hydrocortisone on glial GS by insulin was also observed. The data suggest that insulin contributes to the regulation of the metabolism of amino acid neurotransmitters via its effect on GS.