Rat Astroglial Cells Research Articles

Knocking Down lncRNA HOXA-AS2 Mitigates the Progression of Epilepsy via Regulation of the miR-372-3p/STAT3 Axis.

To explore the potential activity of HOXA cluster antisense RNA 2 (HOXA-AS2), a long non-coding RNA (lncRNA), in epilepsy progression, as well as the mechanisms behind its activity. Kainic acid (KA) was used to treat rat astroglial CTX-TNA2 cells to establish a cellular model of epilepsy. Reverse transcription-quantitative PCR was conducted to examine the expression levels of HOXA-AS2, microRNA (miR)-372-3p and STAT3. Cell Counting Kit-8, flow cytometry and western blot assays were performed to analyze cell viability and apoptosis. The secretion levels of various inflammatory factors (IL-6, IL-1? and TNF-?) were identified by ELISA. To validate the functional interaction between HOXA-AS2/STAT3 and miR?372-3p, dual-luciferase reporter assay was performed. The HOXA-AS2 and STAT3 expression levels were notably upregulated, whereas miR?372-3p was downregulated in KAtreated CTX-TNA2 cells. Silencing HOXA-AS2 or overexpressing miR-372-3p inhibited the secretion of inflammatory factors and apoptosis in KA-treated CTX-TNA2 cells. HOXA-AS2 negatively regulated miR?372-3p, and miR?372-3p targeted STAT3 mRNA. Suppression of miR-372-3p or overexpression of STAT3 abrogated the rescue effect of small interfering HOXA-AS2 in KA-treated CTX-TNA2 cells. The current study suggested that targeting HOXA-AS2 could alleviate cellular damages in the epileptic model by regulating the miR-372-3p/STAT3 axis. Therefore, HOXA-AS2 may serve as a potential anti-epilepsy therapeutic target.

Metformin mediates neuroprotection and attenuates hearing loss in experimental pneumococcal meningitis

BackgroundPneumococcal meningitis is associated with high risk of neurological sequelae such as cognitive impairment and hearing loss. These sequelae are due to parenchymal brain and inner ear damage primarily induced by the excessive inflammatory reaction in response to bacterial brain invasion. Metformin—a biguanide drug to treat diabetes mellitus type 2—was recently found to suppress neuroinflammation and induce neuroregeneration. This study evaluated the effect of metformin adjunctive to antibiotics on neuroinflammation, brain and inner ear damage, and neurofunctional outcome in experimental pediatric pneumococcal meningitis.MethodsEleven-day-old Wistar rats were infected intracisternally with 5.22 ± 1.27 × 103 CFU Streptococcus pneumoniae and randomized for treatment with metformin (50 mg/kg, i.p., once daily for 3 weeks) plus ceftriaxone (100 mg/kg, i.p., bid, n = 61) or ceftriaxone monotherapy (n = 79). Cortical damage and hippocampal apoptosis were evaluated histomorphometrically 42 h post infection. Cerebrospinal fluid cytokine levels were analyzed during acute infection. Five weeks post infection, auditory brainstem responses were measured to determine hearing thresholds. Spiral ganglion neuron density and abundance of recently proliferated and integrated hippocampal granule neurons were assessed histologically. Additionally, the anti-inflammatory effect of metformin was studied in primary rat astroglial cells in vitro.ResultsUpon pneumococcal infection, metformin treatment significantly reduced levels of inflammatory cytokines and nitric oxide production in cerebrospinal fluid and in astroglial cell cultures in vitro (p < 0.05). Compared to animals receiving ceftriaxone monotherapy, adjunctive metformin significantly reduced cortical necrosis (p < 0.02) during acute infection and improved median click-induced hearing thresholds (60 dB vs. 100 dB, p < 0.002) 5 weeks after infection. Adjuvant metformin significantly improved pure tone hearing thresholds at all assessed frequencies compared to ceftriaxone monotherapy (p < 0.05) and protected from PM-induced spiral ganglion neuron loss in the inner ear (p < 0.05).ConclusionAdjuvant metformin reduces brain injury during pneumococcal meningitis by decreasing the excessive neuroinflammatory response. Furthermore, it protects spiral ganglion neurons in the inner ear and improves hearing impairments after experimental pneumococcal meningitis. These results identify adjuvant metformin as a promising therapeutic option to improve the outcome after pediatric pneumococcal meningitis.

Antidepressant amitriptyline-induced matrix metalloproteinase-9 activation is mediated by Src family tyrosine kinase, which leads to glial cell line-derived neurotrophic factor mRNA expression in rat astroglial cells.

BackgroundAstrocytes have been implicated in the pathophysiology of mood disorders and in the mechanism of the pharmacological effects of antidepressant drugs by the production of neurotrophic/growth factors. Previous studies have identified astrocyte‐expressed Gαi/o‐coupled lysophosphatidic acid receptor 1 (LPAR1), as being involved in antidepressant‐induced production of glial cell line‐derived neurotrophic factor (GDNF) and matrix metalloproteinase‐9 (MMP‐9) activation, an important step in the production of GNDF. However, the precise mechanism of MMP‐9 activation by antidepressants has yet to be identified, in particular the intracellular signaling pathway between LPAR1/Gαi/o and MMP‐9.Methods and ResultsTreatment of rat C6 astroglial cells (C6 cells) with amitriptyline increased Src family tyrosine kinase phosphorylation in a time and concentration‐dependent manner. Amitriptyline‐induced GDNF mRNA expression was blocked by Src family tyrosine kinase inhibitors. In addition, inhibiting Src family tyrosine kinase blocked amitriptyline‐induced zymographic MMP‐9 activation in C6 cells. The amitriptyline‐induced zymographic MMP‐9 activity was completely blocked by selective inhibition of Gαi/o protein and LPAR1. Furthermore, the amitriptyline‐induced Src family tyrosine kinase phosphorylation was blocked by LPAR1, but not MMP‐9 inhibition, indicating that Src family tyrosine kinase involvement is downstream of LPAR1.ConclusionsThe current findings suggest that the pharmacological effect of antidepressant such as amitriptyline is mediated through an intracellular signaling pathway via the LPAR1/Gαi/o/Src family tyrosine kinase, which leads to MMP‐9 activation and GDNF production.

Antioxidant Activities of Solanum nigrum L. Leaf Extracts Determined in In Vitro Cellular Models

Several medicinal foods abound in traditional medicine with antioxidant potentials that could be of importance for the management of several diseases but with little or no scientific justification to substantiate their use. Thus, the objective of this study was the assessment of the antioxidant effect of two leave extracts of Solanum nigrum L. (SN), which is a medicinal plant member of the Solanaceae family, mainly used for soup preparation in different parts of the world. Then methanolic/water (80:20) (SN1) and water (SN2) leaves extracts were prepared. The total polyphenolic content and the concentration of phenolic acids and flavones compounds were determined. In order to verify whether examined extracts were able to restore the oxidative status, modified by glutamate in primary cultures of astrocytes, the study evaluated the glutathione levels, the intracellular oxidative stress, and the cytotoxicity of SN1 and SN2 extracts. Both extracts were able to quench the radical in an in vitro free cellular system and restore the oxidative status in in vitro primary cultures of rat astroglial cells exposed to glutamate. These extracts prevented the increase in glutamate uptake and inhibited glutamate excitotoxicity, which leads to cell damage and shows a notable antioxidant property.

Role of thyroid transcription factor-1 in transcriptional regulation of heme oxygenase-1

Here, we report thyroid transcription factor 1 (TTF-1) as an important transcription factor for the expression of heme oxygenase-1 (HO-1). HO-1 is a well-known cytoprotective enzyme against inflammation. We observed that HO-1 co-expressed with TTF-1 in mouse hypothalamic cells. Results from luciferase and chromatin immunoprecipitation assays revealed that TTF-1 directly activated HO-1 transcription by binding to binding domains in the 5′-flanking region of the HO-1 gene. A proinflammatory cytokine, tumor necrosis factor-alpha (TNF-α), induced nuclear translocation of TTF-1 and increased binding affinity of TTF-1 to its binding sites on the HO-1 gene. HO-1 mRNA increased with TTF-1 overexpression but decreased with RNA interference of TTF-1 expression in rat astroglial C6 cells. Together with results showing involvement of TTF-1 in the TNF-α-induced increase in interleukin 1 beta and monocyte chemotactic protein 1 production, this study suggests that TTF-1 plays an important role in the mouse hypothalamus TNF-α-induced inflammatory response for regulating HO-1 gene expression.

The expression of glial cell line-derived neurotrophic factor mRNA by antidepressants involves matrix metalloproteinase-9 activation in rat astroglial cells

Neurotrophic/growth factors derived from glial cells, especially astrocytes, have been implicated in mood disorders and the pharmacological effects of antidepressant drugs. Previous studies demonstrated that the release of glial cell line-derived neurotrophic factor (GDNF) induced by the tricyclic antidepressant amitriptyline was significantly inhibited by a broad-spectrum matrix metalloproteinase (MMP) inhibitor in rat C6 astroglial cells (C6 cells). However, it is unknown whether amitriptyline affects MMP enzymatic activity or expression, and the MMP subtype has yet to be identified. The current study measured the effect of antidepressants on MMP activity with gelatin zymography, an in vitro assay for enzymatic activity, in C6 cells and primary cultured rat astrocytes (primary astrocytes). Treatment with amitriptyline increased zymographic MMP-9 activity without changing MMP-9 mRNA expression in C6 cells. Several different classes of antidepressants significantly increased zymographic MMP-9 activity in C6 cells and primary astrocytes, whereas antipsychotic drugs without antidepressant pharmacological activity did not. The amitriptyline-induced expression of GDNF mRNA was completely blocked by selective inhibition of MMP-9 in C6 cells. Treatment of C6 cells and primary astrocytes with exogenous recombinant MMP-9 increased GDNF mRNA expression, similar to that observed with amitriptyline. Inhibiting MMP-3 blocked amitriptyline-induced zymographic MMP-9 activation in C6 cells and primary astrocytes, indicating that MMP-3 is necessary for MMP-9 activity. The current study suggests that MMP-9 activation is indispensable in the amitriptyline-induced expression of GDNF mRNA in astrocytes and further supports a role of astrocytic neurotrophic/growth factors in the pharmacological effect of antidepressants.

Ammonia Affects Astroglial Proliferation in Culture

Primary cultures of rat astroglial cells were exposed to 1, 3 and 5 mM NH4Cl for up to 10 days. Dose- and time-dependent reductions in cell numbers were seen, plus an increase in the proportion of cells in the S phase. The DNA content was reduced in the treated cells, and BrdU incorporation diminished. However, neither ammonia nor ammonia plus glutamine had any effect on DNA polymerase activity. iTRAQ analysis showed that exposure to ammonia induced a significant reduction in histone and heterochromatin protein 1 expression. A reduction in cell viability was also noted. The ammonia-induced reduction of proliferative activity in these cultured astroglial cells seems to be due to a delay in the completion of the S phase provoked by the inhibition of chromatin protein synthesis.

Age-dependent astroglial vulnerability to hypoxia and glutamate: the role for erythropoietin.

Extracellular accumulation of toxic concentrations of glutamate (Glu) is a hallmark of many neurodegenerative diseases, often accompanied by hypoxia and impaired metabolism of this neuromediator. To address the question whether the multifunctional neuroprotective action of erythropoietin (EPO) extends to the regulation of extracellular Glu-level and is age-related, young and culture-aged rat astroglial primary cells (APC) were simultaneously treated with 1mM Glu and/or human recombinant EPO under normoxic and hypoxic conditions (NC and HC). EPO increased the Glu uptake by astrocytes under both NC and especially upon HC in culture-aged APC (by 60%). Moreover, treatment with EPO up-regulated the activity of glutamine synthetase (GS), the expression of glutamate-aspartate transporter (GLAST) and the level of EPO mRNA. EPO alleviated the Glu- and hypoxia-induced LDH release from astrocytes. These protective EPO effects were concentration-dependent and they were strongly intensified with age in culture. More than a 4-fold increase in apoptosis and a 2-fold decrease in GS enzyme activity was observed in APC transfected with EPO receptor (EPOR)-siRNA. Our in vivo data show decreased expression of EPO and a strong increase of EPOR in brain homogenates of APP/PS1 mice and their wild type controls during aging. Comparison of APP/PS1 and age-matched WT control mice revealed a stronger expression of EPOR but a weaker one of EPO in the Alzheimer’s disease (AD) model mice. Here we show for the first time the direct correlation between the extent of differentiation (age) of astrocytes and the efficacy of EPO in balancing extracellular glutamate clearance and metabolism in an in-vitro model of hypoxia and Glu-induced astroglial injury. The clinical relevance of EPO and EPOR as markers of brain cells vulnerability during aging and neurodegeneration is evidenced by remarkable changes in their expression levels in a transgenic model of AD and their WT controls.

Octopamine, unlike other trace amines, inhibits responses of astroglia-enriched cultures to lipopolysaccharide via a β-adrenoreceptor-mediated mechanism

Trace amines (TAs), i.e. β-phenylethylamine, tyramine and octopamine, are generally regarded as sympathomimetic compounds with structural and functional analogy with catecholamines. Previous reports have shown particularly high levels of circulating TAs in migraine and cluster headache patients. However, no clues are yet available as to the pathophysiological significance of these alterations. The effect of different TAs on the release of nitric oxide was investigated in rat astroglial cells stimulated with lipopolysaccharide (LPS). Octopamine substantially inhibited the release of NO evoked by LPS. Tyramine and β-PEA were ineffective. The inhibitory effect of octopamine was fully reverted by two selective antagonists of β-adrenergic receptors, while α-adrenergic blockade was ineffective. These data, consistent with a role of octopamine as a modulator of NO release, uncover an interaction between octopamine and β-adrenergic receptors in astroglial cells. These results may have an impact in understanding the mechanisms underlying migraine pathophysiology.

The effect of angiotensin II and IV on ERK1/2 and CREB signalling in cultured rat astroglial cells

Angiotensin peptides produced by the brain renin-angiotensin system have established roles in cognition, but there is no mechanistic basis of angiotensin effects on memory. Astroglial cells present throughout the whole brain, synthesize all the components of the renin-angiotensin system and express angiotensin receptors; therefore our aim was to assess changes in intracellular signalling pathways related to memory formation, particularly the activation of CREB and ERK1/2 in astroglial cells grown in the presence of angiotensin peptides. Cultured rat astroglial cells were treated for 24 h with 10 microM angiotensin II and/or 10 microM angiotensin IV in the presence or absence of 100 microM losartan (AT1-receptor antagonist) or 100 microM PD123319 (AT2-receptor antagonist). Both angiotensin peptides alone were without effect on culture protein levels and cell viability and did not induce oxidative stress, but both peptides together slightly elevated cell growth rates and increased damaged, apoptotic cell numbers. This effect was most probably mediated by the AT1 receptor. Angiotensin II but not angiotensin IV increased intracellular calcium via activation of AT1 receptor. Angiotensin IV but not angiotensin II increased extracellular-regulated protein kinases 1 and 2 (ERK1/2) by 65% and T202, T204 phosphorylated ERK1/2 levels by 36%; this effect was blocked in part by both losartan and PD123319. Angiotensin II but not angiotensin IV increased cyclic AMP-responsive element binding protein (CREB) expression by almost 100% and elevated Ser 133-phosphorylated CREB levels by 56%. These effects were also inhibited in part by both losartan and PD123319. Our results indicate that CREB activation in cultured rat glial cells is mediated mostly by angiotensin II. Angiotensin IV appears to affect the ERK1/2 pathway.

Effect of lead on ERK activity and the protective function of bFGF in rat primary culture astroglia

To observe the effects of lead on levels of phosphorylated extracellular signal regulated kinase (p-ERK) in the cytoplasm of primary cultures of rat astroglial cells and the possible protective effect of basic fibroblast growth factor (bFGF) on lead-induced effects. The primary astroglia cells from 1~6 d old Wistar rats were cultured. The cells pretreated with the MEK1 (mitogen-activated protein kinase kinase 1) inhibitor PD98059 and bFGF, respectively, were exposed to Pb acetate of different concentrations for different times. Western blotting and reverse transcription polymerase chain reaction (RT-PCR) methods were used to detect the protein and mRNA expressions of ERK. mRNA expression for ERK peaked 15 min after initiation of lead exposure (P<0.05) and protein expression of p-ERK peaked at 30 min (P<0.05). ERK mRNA levels and p-ERK protein levels returned to baseline after 60 and 120 min of lead exposure, respectively (P>0.05). The increase in p-ERK levels in lead-treated cells could be inhibited by PD098059. Activation of ERK in the cells by lead was prevented by pretreatment with bFGF. Total ERK protein levels did not change under the same experimental conditions (P>0.05). Low-level lead exposure resulted in transient activation of ERK through the MEK pathway, which then returned to basal levels in the continued presence of lead. Exogenous bFGF protected ERK signaling components in astroglia from lead poisoning.

Effect of ammonia on ciliary neurotrophic factor mRNA and protein expression and its upstream signalling pathway in cultured rat astroglial cells: possible implication of c‐fos, Sp1 and p38MAPK

Ciliary neurotrophic factor (CNTF) may be implicated in the pathogenetic mechanisms of hepatic encephalopathy. We tested this hypothesis by treating confluent primary cultures of rat astroglial cells with ammonium chloride for various periods and analysing the effect of ammonia on the signalling pathway that regulates CNTF mRNA and protein expression. Ammonia treatment induced a dose- and time-dependent reduction in CNTF mRNA and protein expression. Surface-enhanced laser desorption/ionization-time-of-flight mass spectrometry analysis of CNTF in the culture medium demonstrated that ammonia also induced a significant decrease in CNTF release. In addition, ammonia affected Sp1 and c-fos, transcription factors that regulate CNTF mRNA and protein expression, which showed partial dephosphorylation and significantly lower mRNA and protein levels. Total content of p38MAPK (for which Sp1 and c-fos are substrates) was unaffected by ammonia, although the diphosphorylated (active) form was significantly reduced after ammonia exposure.

Substance P induces expression of the corticotropin-releasing factor receptor 1 by activation of the neurokinin-1 receptor

The neuropeptide substance P (SP) has been found to be possibly involved in the etiology of affective and anxiety disorders. However, the molecular mechanisms underlying this involvement are still poorly understood. In this study, we used macroarrays to investigate the differential gene expression profile induced by SP, particularly of genes which have been shown to be involved in the pathophysiology of affective disorders. As a model system, we used the human astrocytoma cell line U373 MG as well as primary rat astroglial cells, which both are known to express functional neurokinin-1 receptors (NK-1-R) and to secret various cytokines upon stimulation with SP. Among several regulated genes, we found that SP (100 and 1000 nM) induced the expression of the corticotropin-releasing factor receptor 1 (CRF1 receptor). Further analyses revealed that this induction was mediated (a) via NK-1-R, as the selective NK-1-R-antagonist L-733,060 (1 μM) strongly inhibited SP-induced CRF1 receptor expression, and (b) intracellularly, by protein kinase C, p42/44 and p38 mitogen-activated protein kinases (MAPK), as shown by using specific inhibitors of signal transduction pathways. In conclusion, this study demonstrates that SP induces CRF1 receptor expression in cells of the CNS, which may be of potential interest for a better understanding of the interplay between SP and the stress hormone axis and, thus, diseases like affective or anxiety disorders. Further studies are needed to substantiate this link in vivo.

Possible implication of ciliary neurotrophic factor (CNTF) and β-synuclein in the ammonia effect on cultured rat astroglial cells: A study using DNA and protein microarrays

Astrocytes are considered the key cell in hepatic encephalopathy; although their precise role in the disease has not yet been determined, exposure to ammonia appears to have an important pathogenic effect. We exposed confluent cultures of rat astroglial cells to ammonia (5 mM NH 4Cl) for 1, 3, 5 and 7 days, and determined astroglial levels of actin, glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), GLAST glutamate transporter, 25 kDa heat-shock protein (HSP25), HSP60 and HSP70 by Western blot; the glutamine content in culture medium was measured by mass spectrometry. Significant increases were observed for GS, HSP60 and glutamine, and significant reductions for actin and GFAP. Astrocytes exposed to ammonia for 4 days were used to analyze the effect of ammonia in protein and DNA microarrays. After protein microarray data filtration by signal intensity, x-fold change and z-score, 11 proteins were selected, among which the significant increase in β-synuclein was confirmed by Western blot. DNA microarray data filtration by intensity signal, x-fold change and p-value selected almost 600 genes. The significant increase in α-synuclein mRNA was confirmed by quantitative RT-PCR, but no change was observed in α-synuclein protein levels. A notable decrease in ciliary neurotrophic factor (CNTF) was demonstrated by Western blot after ammonia treatment, concurring with the reduction in CNTF mRNA observed in DNA microarrays. We discuss the possibility of a pathogenic role for CNTF and a protective role for β-synuclein in experimental hyperammonemia. This study demonstrates the use of microarrays as tools to ascertain the possible implication of previously unidentified proteins in the pathogenesis of hepatic encephalopathy.

Similar protective effects of BQ-123 and erythropoietin on survival of neural cells and generation of neurons upon hypoxic injury

Our recent study [Danielyan et al., 2005. Eur. J. Cell Biol. 84, 567–579] showed an additive protective effect of endothelin (ET) receptor A (ETA-R) blockade and erythropoietin (EPO) on the survival and rejuvenation of rat astroglial cells exposed to hypoxia. Whether the effects observed with rodent astroglial cells can be reproduced in human astrocytes and whether these effects of ETA-R blockade and EPO on astrocytes are associated with neuronal survival remained open. Therefore, in the present study, the effects of the ETA-R antagonist BQ-123 and EPO on the maintenance of the neuronal population and survival of the human fetal astroglial cell line (SV-FHAS) under normoxic and hypoxic conditions (NC and HC, respectively) were investigated. Rat brain primary cultures exposed to BQ-123 and/or EPO revealed an increase in the number of β-III tubulin-positive neurons under NC. The hypoxia-caused loss of neurons was abolished by administration of BQ-123 or EPO. Simultaneous application of EPO and BQ-123 led to an additive protective effect on the generation of neurons under NC only. By contrast, BQ-788, the selective ETB-R antagonist, diminished the neuronal population both in NC and HC. Both under NC and HC the number of non-differentiated nestin +/GFAP − neural cells increased upon application of EPO or BQ-123. SV-FHAS responded to BQ-123 or EPO by a decrease in LDH activity in the culture medium under NC (35%) and HC (26% LDH decrease). Concomitant effects of EPO and BQ-123 were illustrated in an additional increase in the survival of human astrocytes (33% under NC and 17% under HC). These data hint at a neuroprotective therapeutic potency of ETA-R blockade, which either alone or in combination with EPO may improve the survival of astroglial and neuronal cells upon hypoxic injury.

The blockade of endothelin A receptor protects astrocytes against hypoxic injury: Common effects of BQ-123 and erythropoietin on the rejuvenation of the astrocyte population

In the present study the role of endothelin (ET) and its receptors (ETA-R and ETB-R) in cellular mechanisms underlying the resistance of astroglial cells to low oxygen level and development of hypoxia has been investigated. To define the influences of ET and its receptors on survival and on antigenic as well as morphologic differentiation of rat astroglial cells in normoxic (NC) and hypoxic culture (HC) the selective antagonists of ETA-R (BQ-123) and ETB-R (BQ-788) were used. Treatment of HC with BQ-123 caused an increase in cell number and inhibited the hypoxia-induced apoptosis by 37%. BQ-123 decreased the hypoxia-induced cytotoxicity in HC. These effects of BQ-123 were abolished in cultures simultaneously treated with BQ-123 and BQ-788. Administration of BQ-788 alone decreased the number of living cells in NC, but not in HC. The activity of caspase-3/-7 was not changed by exposure of NC and HC to BQ-788. The protection provided by BQ-123 to astroglial cells against cytotoxicity in NC and HC was similar to that of erythropoietin (EPO), a cytokine with established neuroprotective effects. The functional improvement of astroglial cells and slowing down of their differentiation under exposure to BQ-123, or EPO, or BQ-123+EPO has been evidenced by an increased number of nestin +/glial fibrillary acidic protein-positive (GFAP +) astrocytes accompanied by decrease of nestin −/GFAP + cells. The simultaneous treatment with BQ-123 and EPO additionally decreased the activities of caspase-3/-7 (64%) and release of LDH into the medium (94%). The benefits in the functional states of astrocytes obtained by combined treatment of HC with BQ-123 and EPO suggest a new therapeutic strategy in treatment of hypoxic brain injury.

Lead induces increased water permeability in astrocytes expressing aquaporin 4

The water channel aquaporin 4 (AQP4) is abundantly expressed in astrocytes. There is now compelling evidence that AQP4 may contribute to an unfavorable course in brain edema. Acute lead intoxication is a condition that causes brain damage preceded by brain edema. Here we report that lead increases AQP4 water permeability (P f) in astrocytes. A rat astrocyte cell line that does not express aquaporin 4 was transiently transfected with aquaporin 4 tagged with green fluorescent protein (GFP). Using confocal laser scanning microscopy we measured water permeability in these cells and in AQP4-negative cells located on the same plate. AQP4-expressing astrocytes had a three-fold higher water permeability than astrocytes not expressing AQP4. Lead exposure induced a significant, 40%, increase in water permeability in astrocytes expressing AQP4, but had no effect on P f in astrocytes not expressing AQP4. The increase in water permeability persisted after lead washout, while treatment with a lead chelator, meso-2,3-dimercaptosuccinic acid, abolished the lead-induced increase in P f. The effect of lead was attenuated in the presence of a calcium (Ca 2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor, but not in the presence of a protein kinase C inhibitor. In cells expressing AQP4 where the consensus site for CaMKII phosphorylation was mutated, lead failed to increase water permeability. Lead exposure also increased P f in rat astroglial cells in primary culture, which express endogenous AQP4. Lead had no effect on P f in astrocytes transfected with aquaporin 3. In situ hybridization studies on rat brain after oral lead intake for three days showed no change in distribution of AQP4 mRNA. It is suggested that lead-triggered stimulation of water transport in AQP4-expressing astrocytes may contribute to the pathology of acute lead intoxication.

TNFα increases activity of γ‐glutamyl transpeptidase in cultured rat astroglial cells

AbstractTo investigate the presence of γ‐glutamyl transpeptidase (γGT) in brain cells, cultures enriched for astroglial cells, neurons, oligodendroglial cells, and microglial cells were studied. Astroglial cultures contained a specific γGT activity of 2.3 ± 0.9 nmol/min/mg protein. A similar specific γGT activity was measured for oligodendroglial cultures, whereas microglial cells and neurons contained less than 30% of the specific γGT activity of astroglial cultures. The activity of γGT in astroglial cultures was elevated strongly by the presence of tumor necrosis factor‐α (TNFα) in a time‐ and concentration‐dependent manner. Maximal activity of γGT was observed after incubation of astroglial cultures for 3 days with 30 ng/mL TNFα. Under these conditions the specific γGT activity was increased by threefold compared to controls. Presence of the γGT‐inhibitor acivicin completely inhibited γGT activity both in TNFα‐treated and in control cells. In addition, the increase in astroglial γGT activity after application of TNFα was prevented completely by the presence of the protein synthesis inhibitor cycloheximide. γGT is involved in extracellular processing of glutathione (GSH) that is exported by astroglial cells. After TNFα‐treatment the concentration of GSH in the medium of astroglial cells was reduced significantly compared to control cells. In conclusion, the data presented demonstrate that TNFα stimulates γGT synthesis in astroglial cells and thereby improves the capacity to process GSH exported by these cells. © 2003 Wiley‐Liss, Inc.

Identification of a plasma membrane glutamine transporter from the rat hepatoma cell line H4-IIE-C3.

Glutamine is taken up into the rat hepatoma cell line H4-IIE-C3 by a Na+-dependent transport system which is specific for glutamine, alanine, serine, cysteine and asparagine and does not tolerate substitution of Na+ by Li+. Glutamine transport was relatively weakly inhibited by a 50-fold excess of leucine and was not inhibited by phenylalanine or N -methyl aminoisobutyrate. These general properties are characteristic of the recently identified ASCT/B0 family of transporters. Using a reverse transcriptase PCR-based homology cloning approach, we have characterized a cDNA for a novel member of this transporter family (H4-ASCT2) from H4-IIE-C3 cells. The cDNA encodes a 551-amino acid protein which exhibits similarities of between 75 and 85% with ASCT/B0 transporters previously cloned from other sources. When expressed in Xenopus oocytes, this transporter catalyses Na+-dependent glutamine uptake with characteristics very similar to those of glutamine uptake into the H4-IIE-C3 cells. This newly characterized transporter possesses a number of amino acid sequence differences from ASCT2 clones recently isolated from rat astroglial cells and from normal rat liver. In particular, the loop region between transmembrane helices 3 and 4 from H4-ASCT2 shares less than 60% sequence similarity with ASCT2 from rat liver; furthermore, there are some 25 single amino acid substitutions elsewhere in the H4-ASCT2 sequence compared with that from rat liver. Thus enhanced glutamine uptake in rat hepatoma cells is mediated by the expression of a novel ASCT/B0 transporter isoform rather than by increased expression of the ASCT2 mRNA found in normal rat liver.

Glutathione release from cultured brain cells: multidrug resistance protein 1 mediates the release of GSH from rat astroglial cells.

To investigate the release of glutathione (GSH) from brain cells, cultures enriched for astroglial cells, neurons, oligodendroglial cells, and microglial cells derived from rat brain were studied. During incubation of astroglial cultures, GSH accumulated in the medium with a rate of 3.1 +/- 0.6 nmol x h(-1) x mg protein(-1). In contrast, only marginal amounts of extracellular GSH were detectable in the media of the other brain cell cultures investigated. The mechanism of GSH release from astroglial cells, as yet, has not been reported. Multidrug resistance protein 1 (Mrp1), a transport protein known to mediate cellular export of glutathione disulfide and glutathione conjugates, is expressed in astroglial cultures. Inhibitors of Mrp1 were used to test for a function of this transporter in mediating GSH release from astroglial cells. The presence of the competitive Mrp1 inhibitor MK571 at a concentration of 50 microM inhibited the rate of GSH release by 63%. In contrast, the low concentration of 1 microM of MK571 increased the rate of GSH release by 83%. This bimodal concentration-dependent effect of MK571 is in accord with literature data for the effects of Mrp1 substrates on GSH release from cells. In addition, the presence of cyclosporin A (10 microM) reduced the GSH release rate significantly and completely blocked the stimulating effect of 1 microM MK571 on the release of GSH from astroglial cells. In conclusion, the data presented are a strong indication that Mrp1 participates in the release of GSH from astroglial cells.