Cultured Rat Astroglial Cells Research Articles

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.

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.

Antioxidant properties of Berberis aetnensis C. Presl (Berberidaceae) roots extract and protective effects on astroglial cell cultures.

Berberis aetnensis C. Presl (Berberidaceae) is a bushy-spiny shrub common on Mount Etna (Sicily). We demonstrated that the alkaloid extract of roots of B. aetnensis C. Presl contains prevalently berberine and berbamine, possesses antimicrobial properties, and was able to counteract the upregulation evoked by glutamate of tissue transglutaminase in primary rat astroglial cell cultures. Until now, there are no reports regarding antioxidant properties of B. aetnensis C. Presl collected in Sicily. Air-dried, powdered roots of B. aetnensis C. Presl were extracted, identified, and quantified by HPLC. We assessed in cellular free system its effect on superoxide anion, radicals scavenging activity of antioxidants against free radicals like the 1,1-diphenyl-2-picrylhydrazyl radical, and the inhibition of xanthine oxidase activity. In primary rat astroglial cell cultures, exposed to glutamate, we evaluated the effect of the extract on glutathione levels and on intracellular production of reactive oxygen species generated by glutamate. The alkaloid extract of B. aetnensis C. Presl inhibited superoxide anion, restored to control values, the decrease of GSH levels, and the production of reactive oxygen species. Potent antioxidant activities of the alkaloid extract of roots of B. aetnensis C. Presl may be one of the mechanisms by which the extract is effective against health disorders associated to oxidative stress.

Parp and Cell Death or Protection in Rat Primary Astroglial Cell Cultures Under LPS/IFNγ Induced Proinflammatory Conditions

The enzyme poly(ADP-ribose)polymerase (PARP) has a leader role in the DNA damage survey mechanisms by its nick-sensor function, but it is also involved in the early events of the programmed cell death, particularly during inflammatory injury, as a coactivator of NF-kB. In the present study, we evaluated the PARP involvement in the mechanisms of protection and/or cell death in rat astroglial cell cultures during the early phase of proinflammatory commitment after lipopolysaccharide and interferon gamma treatment. According with the recent findings that PARP-1 phosphorylation by MAPK/ERK-2 pathway seems to modulate PARP activation, in time course experiments we demonstrated that a very early PARP activation and expression is able to trigger a cell death pathway, DNA damage independent, during strong proinflammatory insults, maintaining its role of guardian of the genome stability only during the normal cell cycling.

Activated microglia modulate astroglial enzymes involved in oxidative and inflammatory stress and increase the resistance of astrocytes to oxidative stress in Vitro

Neuropathological processes in the central nervous system are commonly accompanied by an activation of microglia and astrocytes. The involvement of both cell populations in the onset and progress of neurological disorders has been widely documented, implicating both beneficial and detrimental influences on the neural tissue. Nevertheless, little is known about the interplay of these glial cell populations, especially under diseased conditions. To examine the effects of activated microglia on astrocytes purified rat astroglial cell cultures were treated with medium conditioned by purified quiescent (MCM[-]) or lipopolysaccharide (LPS)-activated rat microglia (MCM[+]) and subjected to a comparative proteome analysis based on two-dimensional gel electrophoresis. No significant down regulation of proteins was observed. The majority of the 19 proteins identified by means of nano HPLC/ESI-MS/MS in the 12 most prominent protein spots significantly overexpressed (> or =2-fold) in MCM[+] treated astrocytes are involved in inflammatory processes and oxidative stress response: superoxide dismutases (Sod), peroxiredoxins, glutathione S-transferases (Gst), nucleoside diphosphate kinase B, argininosuccinate synthase (Ass), and cellular retinol-binding protein I (Rbp1). Sod2, Rbp1, Gstp1, and Ass were also significantly increased on the mRNA level determined by quantitative RT-PCR. The upregulation of antioxidative enzymes in astrocytes was accompanied by a higher resistance to oxidative stress induced by H2O2. These results show that activated microglia change the expression of antioxidative proteins in astrocytes and protect them against oxidative stress, which might be an effective way to increase the neuroprotective potential of astrocytes under pathological conditions associated with oxidative stress and inflammation.

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.

Carnosine interaction with nitric oxide and astroglial cell protection

The neuropeptide carnosine (beta-amyloid peptide aggregation has been demonstrated. Carnosine protection against peroxynitrite damage is particularly relevant, but until now there has been no evidence of any direct interaction with nitric oxide. In this study we examined the protection that carnosine provides against nitric oxide (NO)-induced cell death in primary rat astroglial cell cultures treated with lipopolysaccharide (LPS) and interferon gamma (INFgamma), a well-known neurotoxic proinflammatory condition. A correlation was found between cell protection and NO free-radical scavenging activity of carnosine. Moreover, by competitive spectrophotometric measurement and electrospray mass spectrometry analysis in cell-free experiments, we demonstrated a direct interaction of the dipeptide with NO. A comparison of carnosine with its homologues or derivatives (homocarnosine and carcinine) as well as with its amino acid constituents (L-histidine and beta-alanine) highlighted that only histidine showed significant scavenging activity. Therefore, carnosine shows direct NO-trapping ability and may be a valuable multifunctional molecule in the treatment of neurodegenerative disorders.

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.

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.

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.

Changes in antioxidant enzyme expression in response to hydrogen peroxide in rat astroglial cells.

Oxidative stress has been causally linked to a variety of neurodegenerative diseases. To clarify the role of the antioxidant enzyme (AOE) system in oxidative brain damage primary cultures of rat astroglial cells were exposed to hydrogen peroxide (H2O2). Expression of AOEs and several parameters for cell viability and functionality were measured. In our experiments astrocytes responded to low concentrations of H2O2 exposure with a pronounced generation of ROS which ran parallel with induction of lipid peroxidation. This distinct oxidative stress was not reflected in cell viability or functionality parameters measured. Cytotoxicity, a decrease in glutathione content of astrocytes, and impairment of mitochondrial functions became obvious only for higher concentrations of H2O2. After H2O2 exposure catalase, manganese superoxide dismutase, and glutathione peroxidase expression levels were found to be increased, whereas copper/zinc superoxide dismutase mRNA expression was not affected. These data indicate that the AOE system of astrocytes can be directly regulated by oxidative stress and may thus contribute to protection of cells against oxidative insults.

The influence of oxidative stress on catalase and MnSOD gene transcription in astrocytes

The brain is particularly vulnerable to oxygen free radicals, which have been implicated in the pathology of several neurological disorders. The antioxidant enzyme (AOE) system of the brain may play an important role in the protection against such oxidative stress. We investigated the influence of oxidative stress on the transcription of catalase and MnSOD mRNA. Primary rat astroglial cell cultures were treated either with hydrogen peroxide (H 2O 2), as a direct mediator of oxidative stress, or with the redox cycling compound paraquat. Both substances led to an increase of catalase and MnSOD mRNA levels. To further elucidate the mechanisms residing behind this increase, transfection experiments were performed. Transient transfection of primary astroglial cells with a reporter plasmid containing the upstream region of the catalase gene showed a decrease in reporter gene activity after exposure of transfected cells to either H 2O 2 or paraquat. In contrast, transfection experiments done with reporter plasmids for the MnSOD upstream region resulted in an increase of reporter gene activity after H 2O 2 as well as after paraquat treatment of transfected cells. These results indicate transcriptional regulation of MnSOD and post-transcriptional regulation of catalase gene expression after oxidative stress in primary rat astrocytes.

Neosynthesis and Activation of Rho by Escherichia coli Cytotoxic Necrotizing Factor (CNF1) Reverse Cytopathic Effects of ADP-ribosylated Rho

Clostridium botulinum exoenzyme C3 inactivates the small GTPase Rho by ADP-ribosylation. We used a C3 fusion toxin (C2IN-C3) with high cell accessibility to study the kinetics of Rho inactivation by ADP-ribosylation. In primary cultures of rat astroglial cells and Chinese hamster ovary cells, C2IN-C3 induced the complete ADP-ribosylation of RhoA and concomitantly the disassembly of stress fibers within 3 h. Removal of C2IN-C3 from the medium caused the recovery of stress fibers and normal cell morphology within 4 h. The regeneration was preceded by the appearance of non-ADP-ribosylated RhoA. Recovery of cell morphology was blocked by the proteasome inhibitor lactacystin and by the translation inhibitors cycloheximide and puromycin, indicating that intracellular degradation of the C3 fusion toxin and the neosynthesis of Rho were required for reversal of cell morphology. Escherichia coli cytotoxic necrotizing factor CNF1, which activates Rho by deamidation of Gln(63), caused reconstitution of stress fibers and cell morphology in C2IN-C3-treated cells within 30-60 min. The effect of CNF1 was independent of RhoA neosynthesis and occurred in the presence of completely ADP-ribosylated RhoA. The data show three novel findings; 1) the cytopathic effects of ADP-ribosylation of Rho are rapidly reversed by neosynthesis of Rho, 2) CNF1-induced deamidation activates ADP-ribosylated Rho, and 3) inhibition of Rho activation but not inhibition of Rho-effector interaction is a major mechanism underlying inhibition of cellular functions of Rho by ADP-ribosylation.

A Putative Tetrapeptide Antagonist Prevents β-Amyloid-Induced Long-Term Elevation of [Ca2+]iin Rat Astrocytes

Comparative fluorimetric studies on the long-term (8-hour) action of β[1-42]amyloid and its shorter fragments β[1-40], β[25-35] and β[31-35] on the steady-state intracellular Ca2+concentration in primary cultures of rat astroglial cells using the Ca2+-sensitive fluorescent probe Fura-2 AM revealed higher 340/380 fluorescence excitation ratios in the treated cells as compared to the untreated controls. All the peptides were found to induce similar cellular effects, suggesting the [31-35] region as the putative active centre of the molecule. No significant alteration was detectable in Fura-2 fluorescence using the Ca2+-insensitive excitation wavelength of 367 nm, indicating that the observed changes reflect a real alteration in the Ca2+concentration of the cells. Moreover, no considerable difference was observed in the total protein content of treated and untreated cells. Co-treatment of the cells with Pr-Ile-Ile-Gly-Leu-NH2(Pr-IIGL) peptide, an analogue of the [31-34] region of β[1-42]-amyloid, was found to effectively antagonize the β[1-42]-amyloid-induced elevation of the fluorescence excitation ratio, leaving the 367-nm fluorescence unaffected. To the best of the authors’ knowledge, this is the first report on an analogue of β-amyloid peptide capable of blocking one of its physiological effects, thereby raising the possibility that this sequence could prove to be a lead compound for designing effective β-amyloid antagonists.

Insulin promotes the hydrolysis of a glycosyl phosphatidylinositol in cultured rat astroglial cells.

Glycosyl phosphatidylinositols have been implicated in insulin signaling through their action as precursors of second messenger molecules in peripheral tissues. In the present study, cultured rat astrocytes were used to investigate whether glycosyl phosphatidylinositol might be involved in the mechanism of insulin signal transduction in neural cells. A glycosyl phosphatidylinositol sensitive to hydrolysis by both phosphatidylinositol-specific phospholipase C and glycosyl phosphatidylinositol-specific phospholipase D and to nitrous acid deamination was purified. When astrocytes were exposed to 10 nM insulin, a rapid and significant reduction in the content of glycosyl phosphatidylinositol was observed within 1-2 min. In addition, an inverse concentration-dependent relationship between glycosyl phosphatidylinositol and diacylglycerol levels was found, suggesting a phospholipase C-mediated hydrolysis of glycosyl phosphatidylinositol in response to insulin. The effects of insulin were mediated through its own receptors and not through insulin-like growth factor (IGF)-I and/or IGF-II receptors, as demonstrated by affinity cross-linking studies. Also, the effects of 5 nM IGF-1 or 5 nM IGF-II on glycosyl phosphatidylinositol and diacylglycerol levels were different from those caused by insulin and were not essentially modified by pretreatment of the cells with either platelet-derived growth factor (PDGF) or epidermal growth factor (EGF). When cells were sequentially incubated with PDGF and EGF, a reduction in both glycosyl phosphatidylinositol and diacylglycerol contents was observed; the diacylglycerol but not the glycosyl phosphatidyl content was reversed after incubation with IGF-I, and especially with IGF-II, for 10 min. Despite the remarkable homology among insulin, IGF-I, and IGF-II, our results indicate that in astrocytes these compounds probably use different signal transduction pathways.

Modulation of oxygen-radical-scavenging enzymes by oxidative stress in primary cultures of rat astroglial cells.

Cytosolic and mitochondrial alterations induced by exposure of rat astroglial primary cultures to reactive oxygen species (ROS) generated by a xanthine/xanthine oxidase (X/XO) mixture or by lipopolysaccharide (LPS) have been investigated biochemically and immunochemically. In the presence of ROS generated by X/XO, a significant decrease in Cu,Zn superoxide dismutase (Cu,Zn-SOD) and in glutamine synthetase (GS) activity was observed whereas mitochondrial Mn-SOD activity and enzyme protein levels were significantly enhanced. Similar effects on GS, Cu,Zn- and Mn-SOD activities were observed by glucose/glucose oxidase treatment of the cells. Addition of LPS to the cell growth medium also specifically induces Mn-SOD synthesis but was without effect on Cu,Zn-SOD. It is suggested that in all these tested situations, hydrogen peroxide could represent a specific inducer of the observed phenomenon and it may therefore be considered as an intracellular messenger involved in the regulation of some aspects of astroglial oxidative metabolism, particularly the defence against ROS.

Acetaldehyde-induced growth inhibition in cultured rat astroglial cells

Due to the important role of glial cells in brain maturation and reports on delayed astroglial proliferation following ethanol exposition, it was of great interest to examine the effects of the primary metabolite of ethanol-acetaldehyde-on astroglial cell growth. This was carried out by examining biochemical parameters of astroglial cells cocultured with Chinese hamster ovary cell line (CHO) transfected with alcohol dehydrogenase (ADH), able to generate acetaldehyde from ethanol. Acetaldehyde generated from ethanol by ADH-transfected CHO cells had an inhibitory effect on the growth of astroglial cells as assessed by measuring marker enzyme activities and culture protein levels. Moreover, both acetaldehyde and ethanol altered cell cycle and increased astroglial superoxide dismutase activity. Additionally, acetaldehyde, but not ethanol, increased malondialdehyde levels in cultured astroglia. These results clearly show that acetaldehyde may participate in the development of the Fetal Alcohol Syndrome.

Pneumococcal cell wall components induce nitric oxide synthase and TNF-alpha in astroglial-enriched cultures.

Astroglia and microglia, the most numerous cells in the central nervous system (CNS), have been shown to produce the inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-alpha) upon stimulation with the cytokines IFN-gamma, IL-1-beta, or bacterial lipopolysaccharides (LPS). However, it is not known whether gram-positive bacteria like Streptococcus pneumoniae cause astroglial cells to release nitric oxide (NO) and TNF-alpha. S. pneumoniae meningitis still has a high incidence and mortality in spite of antibiotic therapy. Cell wall components from S. pneumoniae (pneumococcal cell-wall components, PCW) and TNF-alpha have been shown to cause meningeal inflammation and cerebrovascular changes in experimental meningitis. Addition of PCW to cultured rat astroglial cells increased nitrite in the supernatant significantly after 24 h, from 17 +/- 21 to 133 +/- 62 nM/micrograms protein. Nitrite release was dose-dependent in a range shown to cause meningeal inflammation in vivo and was inhibited by the competitive NO synthase inhibitor NW-nitro-L-arginine (L-NA 10(-4 M)) and dexamethasone (10(-6 M)), with transcriptional and translational inhibition by actinomycin D and cycloheximide, respectively. PCW caused a significant increase in the release of TNF-alpha from astroglial cells after 4 h, from 2 +/- 3.5 pg/ml to 102 +/- 13.5 pg/ml, which was inhibited by dexamethasone (10(-6 M)). Our results suggest a role for astroglial-derived NO and TNF-alpha as mediators of vascular and inflammatory response in the early phase of experimental pneumococcal meningitis.

Identification by photoaffinity labelling of a pyridine nucleotide-dependent tri-iodothyronine-binding protein in the cytosol of cultured astroglial cells

High-affinity 3,3',5-tri-iodo-L-thyronine (T3) binding (Kd approximately 0.3 nM) to the cytosol of cultured rat astroglial cells was strongly activated in the presence of pyridine nucleotides. A 35 kDa pyridine nucleotide-dependent T3-binding polypeptide (35K-TBP) was photoaffinity labelled using underivatized [125I]T3 in the presence of pyridine nucleotides and the free-radical scavenger dithiothreitol. Maximum activations of T3 binding and 35K-TBP photolabelling were obtained at approx. 1 x 10(-7) M NADP+ or NADPH, or 1 x 10(-4) M NADH. NAD+ and other nucleotides were without effect. NADPH is the form which activates T3 binding and 35K-TBP photolabelling, since cytosol contains NADP(+)-reducing activity, and the activation of both processes in the presence of NADPH and NADP+ was prevented by an exogenous NADPH oxidation system. NADPH behaved as an allosteric activator of T3 binding. The NADPH oxidation system promoted the release of bound T3 in the absence of any change in the total concentration of the hormone. The 35K-TBP photolabelling and [125I]T3 binding were similarly inhibited by non-radioactive T3 (half-maximum effect at 0.5-1.0 nM T3). The concentrations of iodothyronine analogues that inhibited both processes were correlated (3,3',5-tri-iodo-D-thyronine > or = T3 > L-thyroxine > tri-iodothyroacetic acid > 3,3'5'-tri-iodo-L-thyronine). Molecular sieving and density-gradient centrifugation of cytosol identified a 65 kDa T3-binding entity, which included the 35K-TBP. These results indicate that 35K-TBP is the cytosolic entity involved in the pyridine nucleotide-dependent T3 binding, and suggest that the sequestration and release of intracellular thyroid hormones are regulated by the redox state of astroglial cell compartment(s).