Glucose deprivation increases hydrogen peroxide level in immunostimulated rat primary astrocytes

Activated astrocytes produce a large amount of bioactive molecules, including reactive oxygen and nitrogen species. Astrocytes are in general resistant to those reactive species. However, we previously reported that immunostimulated astrocytes became highly vulnerable to metabolic insults, such as glucose deprivation. In this study, we investigated whether H(2)O(2) production was associated with the increased vulnerability. Glucose deprivation for up to 8 hr did not change the intracellular level of H(2)O(2) in astrocytes. Treatment with lipopolysaccharide plus interferon-gamma for 48 hr evoked astroglial H(2)O(2) production; however, no apparent death or injury was observed in immunostimulated astrocytes. Glucose deprivation after 48 hr of immunostimulation markedly increased H(2)O(2) level, depleted adenosine triphosphate (ATP), and enhanced lactate dehydrogenase (LDH) release. The ATP depletion and LDH release were in part prevented by catalase, mannitol, and N-acetyl-L-cysteine. The enhanced level of H(2)O(2) in glucose-deprived immunostimulated astrocytes appeared to be secondary to the depletion of reduced glutathione. 4-(2-Aminoethyl)bebzenesulfonyl fluoride (AEBSF), an inhibitor of NADPH oxidase, reduced H(2)O(2) level and LDH release in glucose-deprived immunostimulated astrocytes. H(2)O(2), either endogenously produced or exogenously added, depolarized mitochondrial transmembrane potential in glucose-deprived astrocytes, leading to their ATP depletion and death. The present results strongly indicate that glucose deprivation causes deterioration of immunostimulated astrocytes by increasing the intracellular concentration of H(2)O(2).     

Adenosine and purine nucleosides protect rat primary astrocytes from peroxynitrite-potentiated,glucose deprivation-induced death: preservation of intracellular ATP level

Previously we have reported that immunostimulated astrocytes became highly vulnerable to glucose deprivation. In the present study we examined the effect of various kinds of nucleosides on the augmented death of glucose-deprived immunostimulated astrocytes. Preincubation with interferon-gamma (100 U/ml) and lipopolysaccharide (1 microg/ml) for 48 h and continuous exposure to glucose deprivation (4 h) significantly induced the lactate dehydrogenase (LDH) release, as a marker of cell injury or death, from astrocytes. The glucose deprivation-induced augmented cell death in immunostimulated astrocytes was mimicked by exogenous peroxynitrite generator 3-morpholinosydnonimine (SIN-1). The increased death in immunostimulated or SIN-1-treated astrocytes deprived of glucose was blocked by adenosine and ATP. Other purine nucleos(t)ides, not pyrimidine nucleotides, also showed similar protective effects. Adenosine receptor agonist R(-)-N-(2-phenylisopropyl)-adenosine or N-cyclohexyladenosine did not alter the augmented cell death. Adenosine receptor antagonists 8-cyclopentyl-1,3-dipropylxanthine, xanthine amine congener or 3,7-dimethyl-1-propargylxanthine also did not reverse the protective effect of adenosine. Intracellular ATP levels rapidly decreased prior to the LDH release in glucose-deprived immunostimulated astrocytes. The loss of intracellular ATP was prevented by adenosine and other purine nucleotides. The present results suggest that adenosine and their metabolites may protect astrocytes from peroxynitrite-potentiated, glucose deprivation-induced death by serving as substrates for intracellular ATP generation.     

Dehydroepiandrosterone inhibits the death of immunostimulated rat C6 glioma cells deprived of glucose

Pretreatment of interferon-gamma and lipopolysaccharides made C6 glioma cells highly vulnerable to glucose deprivation. Neither 12 h of glucose deprivation nor 2-day treatment with interferon-gamma (100 U/ml) and lipopolysaccharides (1 microg/ml) altered the viability of C6 glioma cells. However, significant death of immunostimulated C6 glioma cells was observed after 5 h of glucose deprivation. The augmented death was prevented by dehydroepiandrosterone (DHEA) treatment during immunostimulation, but not by DHEA treatment during glucose deprivation. DHEA reduced the rise in nitrotyrosine immunoreactivity, a marker of peroxynitrite, and superoxide production in glucose-deprived immunostimulated C6 glioma cells. DHEA, however, did not protect glucose-deprived C6 glioma cells from the exogenously produced peroxynitrite by 3-morpholinosydnonimine. Further, DHEA did not alter the production of total reactive oxygen species and nitric oxide in immunostimulated C6 glioma cells. Superoxide dismutase (SOD) and the synthetic SOD mimetic Mn(III)tetrakis (4-benzoic acid) porphyrin inhibited the death of glucose-deprived immunostimulated C6 glioma cells. In addition, a superoxide anion generator paraquat reversed the protective effect of DHEA on the augmented death. The data indicate that DHEA prevents the glucose deprivation-evoked augmented death by inhibiting the production of superoxide anion in immunostimulated C6 glioma cells.     

Glucocorticoids exacerbate peroxynitrite mediated potentiation of glucose deprivation-induced death of rat primary astrocytes

Glucocorticoids have been implicated in the exacerbation of several types of neurotoxicity in various neuropathological situations. In this study, we investigated the effect of a glucocorticoid dexamethasone on glucose deprivation induced cell death of immunostimulated rat primary astrocytes, which is dependent on the production of peroxynitrite from the immunostimulated cells [Choi et al. Glia, 31(2001) 155-164; J. Neuroimmunol. 112 (2001) 55-62]. Glucose deprivation in immunostimulated rat primary astrocytes results in the release of lactate dehydrogenase (LDH) after 5 h and co-treatment with dexamethasone (1-1000 nM) dose-dependently increased LDH release. Treatment of the exogenous peroxynitrite generator SIN-1 (20 microM), plus glucose deprivation, also increased LDH release after 6 h and co-treatment with dexamethasone dose-dependently increased LDH release. A glucocorticoid receptor antagonist, RU-486, reversed the potentiation of cell death by dexamethasone. Glucose deprivation in immunostimulated cells decreased the intracellular ATP levels, which preceded LDH release from the cell, and co-treatment with dexamethasone dose-dependently potentiated the depletion of intracellular ATP levels. In addition, dexamethasone further deteriorated SIN-1 plus glucose deprivation-induced decrease in mitochondrial transmembrane potential in rat primary astrocytes, which was reversed by RU-486. The results from the present study suggest that glucocorticoids may be detrimental to astrocytes in situations where activation of glial cells are observed, including ischemia and Alzheimer's disease, by mechanisms involving depletion of intracellular ATP levels and deterioration of mitochondrial transmembrane potentials.     

Potentiated glucose deprivation-induced death of astrocytes after induction of iNOS

Astrocytes play an essential role in the maintenance of normal neuronal function. Here we report that pretreatment of interferon-γ (IFN-γ) and lipopolysaccharides (LPS) made murine astrocytes highly vulnerable to glucose deprivation-induced death. Neither 12-hr glucose deprivation nor 2-day treatment with IFN-γ (100 U/ml) and LPS (1 μg/ml) altered the viability of astrocytes. However, significant death of IFN-γ/LPS-treated astrocytes was observed after 4-hr glucose deprivation. This augmented death was mimicked by the nitric oxide releasing reagent 3-morpholinosydnonimine and was in part prevented by the nitric oxide synthase inhibitor N^G-nitroarginine. The data indicate that immunostimulated astrocytes can undergo suicidal death during glucose deprivation through the expression of inducible nitric oxide synthase. J. Neurosci. Res. 54:870–875, 1998. © 1998 Wiley-Liss, Inc.     

Mimosine prevents the death of glucose-deprived immunostimulated astrocytes by scavenging peroxynitrite

Immunostimulated astrocytes become highly vulnerable to glucose deprivation (Choi and Kim: J Neurosci Res 54:870-875, 1998a). The increased vulnerability is caused by the enhanced level of peroxynitrite endogenously produced in glucose-deprived immunostimulated astrocytes. In the present study, we report that the plant amino acid mimosine can attenuate the increased death by scavenging peroxynitrite. Treatment with mimosine blocked the increase of nitrotyrosine immunoreactivity, a marker of peroxynitrite, in glucose-deprived immunostimulated astrocytes. Furthermore, mimosine directly inhibited the nitration of tyrosine residues of bovine serum albumin and the oxidation of dihydrorhodamine-123 to rhodamine-123 by peroxynitrite. Mimosine has been used experimentally as a cell cycle G1/S phase transition blocker (Lalande: Exp Cell Res 186:332-339, 1990; Hoffman et al.: Cytometry 12:26-32, 1991). Flow cytometry analysis, however, showed that the cytoprotective effect of mimosine was not attributed to its inhibition of cell cycle progression. Furthermore, under our experimental conditions, mimosine did not alter the levels of cell cycle regulatory proteins, including p21(WAF1/CIP1), cyclins D1 and E, and proliferating cell nuclear antigen. In addition, cyclin-dependent kinase inhibitors olomoucine and roscovitine did not block the increased death. These results indicate that mimosine inhibits the augmented death of glucose-deprived immunostimulated astrocytes by scavenging peroxynitrite rather than suppressing the cell cycle progression.     

Selective upregulation of inducible nitric oxide synthase (iNOS) by lipopolysaccharide (LPS) and cytokines in microglia: in vitro and in vivo studies

A role for free radicals has been proposed in infectious brain disease, where resident microglia cells upregulate the inducible nitric oxide synthase isoform (iNOS), and thus are capable of producing nitric oxide at enhanced rates. Using the constitutively expressed NADPH oxidase, microglial cells can generate superoxide, which reacts with nitric oxide to form the powerful oxidant peroxynitrite. In a mixed cell culture system of astrocytes and microglial cells, nitrite levels, used as an indicator of nitric oxide production, were elevated after the addition of lipopolysaccharide (LPS) and cytokines. Immunohistochemistry and the NADPH diaphorase technique demonstrated selective localization of the iNOS protein in microglial cells, whereas no iNOS protein or NADPH diaphorase activity was detected in astrocytes. A similar cellular distribution was observed in vivo following injection of LPS and cytokines into the rat striatum. By contrast, LPS and interferon-gamma led to translocation of NF-kappaB in microglia and in astrocytes, demonstrating that both cell types are responsive to the stimulus. Therefore, downstream control in iNOS expression is cell type-specific.     

Catalpol protects primary cultured astrocytes from in vitro ischemia-induced damage

Catalpol, an iridoid glycoside abundant in the roots of Rehmannia glutinosa, has been previously found to prevent the loss of CA1 hippocampal neurons and to reduce working errors in gerbils after ischemia-reperfusion injury. In the present study, we investigated the effects of catalpol on astrocytes in an ischemic model to further characterize its neuroprotective mechanisms. Primary cultured astrocytes exposed to oxygen-glucose deprivation (OGD) followed by reperfusion (adding back oxygen and glucose, OGD-R), were used as an in vitro ischemic model. Treatment of the astrocytes with catalpol during ischemia-reperfusion increased astrocyte survival significantly in a concentration-dependent manner, as demonstrated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) release and morphological observation. In addition, catalpol prevented the decrease in mitochondrial membrane potential, inhibited the formation of reactive oxygen species (ROS) and the production of nitric oxide (NO), decreased the level of lipid peroxide and the activity of inducible nitric oxide synthase (iNOS), and elevated the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and the content of glutathione (GSH). Our results suggest that catalpol exerts the most significant cytoprotective effect on astrocytes by suppressing the production of free radicals and elevating antioxidant capacity.     

Augmented death in immunostimulated astrocytes deprived of glucose: inhibition by an iron porphyrin FeTMPyP

The present study shows that under glucose-deprived conditions immunostimulated astrocytes rapidly undergo death due to their increased susceptibility to endogenously produced peroxynitrite. Fe(III)tetrakis(N-methyl-4'-pyridyl)porphyrin (FeTMPyP), but not the structurally related compounds ZnTMPyP and H(2)TMPyP, prevented the death in glucose-deprived immunostimulated astrocytes. Consistently, FeTMPyP, not ZnTMPyP and H(2)TMPyP, completely blocked the elevation of nitrotyrosine immunoreactivity (a marker of peroxynitrite) and the depolarization of the mitochondrial transmembrane potential in glucose-deprived immunostimulated astrocytes. The present data suggest that peroxynitrite may be associated with glial cell death during metabolic deterioration in the cerebral ischemic penumbra.     

Gradual inhibition of inducible nitric oxide synthase but not of interleukin-1β production in rat microglial cells of endotoxin-treated mixed glial cell cultures

In cultures of purified microglial cells and astrocytes from newborn rats, the immunocytochemical localization of interleukin-1β (IL-1β) and inducible nitric oxide synthase (iNOS) using recently developed antibodies, as well as the release of IL-1β and nitric oxide (NO), was studied following exposure of the cells to endotoxin [lipopolysaccharide (LPS)]. In the absence of LPS, IL-1β- and iNOS-immunoreactive microglial cells and IL-1β or NO release were not observed, whereas in the presence of the endotoxin, the production of NO and IL-1β by microglial cells dramatically exceeded their synthesis and release by astrocytes. Interestingly, microglial cells cultured for 4–8 days in the presence of astrocytes appeared to lose their ability to produce iNOS, whereas the release of IL-1β remained unaltered. Moreover, endotoxin-stimulated microglial cells appeared to regain their ability to synthesize iNOS following their separation from astrocytes. These data show that microglia are primarily responsible for NO and IL-1β production in mixed glial cell cultures upon endotoxin stimulation. Moreover, in the presence of astrocytes the induction of iNOS, but not that of IL-1β in microglial cells is gradually inhibited. © 1996 Wiley-Liss, Inc.     

Neuronal expression of inducible nitric oxide synthase after oxygen and glucose deprivation in rat forebrain slices

Nitric oxide (NO) overproduction has been postulated to contribute significantly to ischaemia-reperfusion neurotoxicity. Inducible or type II NO synthase (iNOS) synthesizes NO in large quantities for long periods of time. Therefore we investigated the expression and localization of iNOS after oxygen and glucose deprivation in rat forebrain slices. In this experimental model, calcium-independent NOS activity reached a maximum 180 min after the end of a 20 min oxygen–glucose deprivation period. During the same period of time, the calcium-independent activity was absent in control forebrain slices. To test whether this calcium-independent NOS activity was due to the expression of iNOS, the effects of the addition of dexamethasone, cycloheximide and pyrrolidine dithiocarbamate were determined. All of them inhibited the induction of the calcium-independent NOS activity measured in the rat forebrain slices after oxygen and glucose deprivation. Furthermore, oxygen and glucose deprivation caused the expression of the gene encoding iNOS in rat forebrain slices, as assessed by the detection of iNOS message and protein in these samples. A sixfold increase in the iNOS mRNA levels was observed at 180 min and the time-course of the expression of iNOS mRNA was in agreement with the temporal profile of iNOS enzymatic activity. Immunohistochemistry analysis revealed that iNOS was highly expressed in neurones, astrocytes and microglial cells. These results demonstrate for the first time that iNOS is expressed in neurones after oxygen and glucose deprivation, and that this expression occurs in short periods of time. These findings suggest that NO can play an important pathogenic role in the tissue damage that occurs after cerebral ischaemia.     

Protection by a manganese porphyrin of endogenous peroxynitrite-induced death of glial cells via inhibition of mitochondrial transmembrane potential decrease

In the cerebral ischemic penumbra, progressive metabolic deterioration eventually leads to death of glial cells. The exact mechanism for the death of glial cells is unclear. Here we report that under glucose-deprived conditions immunostimulated glial cells rapidly underwent death via production of large amounts of peroxynitrite. The cell-permeable Mn(III)tetrakis(N-methyl-4'-pyridyl)porphyrin (MnTMPyP) caused a concentration-dependent attenuation of the increased death in glucose-deprived immunostimulated glial cells. The structurally related compound H(2)TMPyP, which lacks metals, did not attenuate this augmented cell death. MnTMPyP prevented the elevation in nitrotyrosine immunoreactivity (a marker of ONOO(-)) in glucose-deprived immunostimulated glial cells. In glucose-deprived glial cells, MnTMPyP also completely blocked the augmented death and nitrotyrosine immunoreactivity induced by the ONOO(-)-producing reagent 3-morpholinosydnonimine (SIN-1). The mitochondrial transmembrane potential (MTP), as measured using the dye JC-1, was rapidly decreased in immunostimulated or SIN-1-treated glial cells deprived of glucose. MnTMPyP, but not H(2)TMPyP, blocked the depolarization of MTP in those glial cells. The present data, at least in part, provide evidence for how glial cells die in the postischemic and/or recurrent ischemic brain.     

Lipopolysaccharide and proinflammatory cytokines require different astrocyte states to induce nitric oxide production

Nitric oxide (NO) production by astrocytes is a significant factor affecting brain physiology and pathology, but the mechanism by which it is regulated is not known. Previous studies using different specimens and stimuli might have described different aspects of a complex system. We investigated the effect of culture and stimulus conditions on NO production by cultured astrocytes and identified two combinations of these allowing NO production. Lipopolysaccharide (LPS)-induced NO production required a high seeding cell density and was independent of the serum concentration, whereas that induced by proinflammatory cytokines required simultaneous treatment with interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma and low-serum conditions but was less affected by the seeding density. These two pathways showed differential sensitivity to protein kinase inhibitors. Both LPS and cytokines induced expression of inducible nitric oxide synthase (iNOS). Although LPS-induced iNOS expression required a high seeding cell density, cytokine-induced iNOS expression, in contrast to NO production, was not affected by the serum concentration. These results suggest that astrocytes interact with the environment and alter their responsiveness to NO production-inducing stimuli by regulating iNOS expression and activity. This is the first evidence for the selective use of two different regulatory pathways in any cell type.     

Immunohistochemical and ultrastructural evidence for the pathogenesis of white matter degeneration in patients with panencephalopathic-type Creutzfeldt–Jakob disease: Inducible nitric oxide synthase overexpression in bizarre astrocytes

Excessive production of nitric oxide (NO) due to the overinduction of inducible nitric oxide synthase (iNOS) has a severe cytotoxic effect, which may relate to the pathogenesis of neurodegenerative disorders. In this study, we report the novel finding that iNOS is overinduced in a large number of bizarre astrocytes in the white matter of patients with panencephalopathic (PE)-type Creutzfeldt–Jakob disease (CJD). This study was carried out on brain tissue from seven patients with PE-type CJD. As controls, 12 normal individuals and nine patients with cerebral infarction were examined. We identified a large number of bizarre astrocytes in the degenerative cerebral white matter in PE-type CJD. Using immunohistochemistry, only bizarre astrocytes in PE-type CJD showed strong immunoreactivity for both iNOS and superoxide dismutase 1 (SOD1). Ultrastructural examination demonstrated that these bizarre astrocytes contained many free polyribosome-like granules. No significant iNOS immunoreactivity was observed in either the astrocytes of patients with cerebral infarcts or in the normal controls. This study suggests that the iNOS-overexpressing astrocytes, especially iNOS-overexpressing bizarre astrocytes, could play an important role in the development of white matter lesions in PE-type CJD. Our data also suggest that the bizarre astrocytes could be protecting themselves from the cytotoxicity of NO by producing SOD1. These immunohistochemical findings are supported by the ultrastructural observation of numerous polyribosome granules restricted to the cytoplasm of these bizarre astrocytes.     

Sodium L‐lactate differently affects brain‐derived neurothrophic factor,inducible nitric oxide synthase,and heat shock protein 70 kDa production in human astrocytes and SH‐SY5Y cultures

The present study analyzed the in vitro effects induced by sodium L‐lactate on human astrocytes and the SH‐SY5Y cell line, when added at concentrations of 5, 10, and 25 mmol/liter. Expression of brain‐derived neurotrophic factor (BDNF), inducible nitric oxide synthase (iNOS), and heat shock protein 70 kDa (HSP70) was evaluated by Western blot analysis. Cell viability with MTT, release of nitric oxide (NO) through the Griess reaction, and production of BDNF by enzyme‐linked immunoassay was determined. Data indicate that, in SH‐SY5Y as well as in cortical astrocytes, after 4 hr sodium L‐lactate increases the expression and release of BDNF, iNOS, and NO; after 24 hr, it turns is ineffective for the production of the neurotrophin in SH‐SY5Y and not in astrocytes, but the expression of iNOS and release of NO appear to be further increased compared with those after 4 hr. Sodium L‐lactate influences differently the expression of HSP70 in SH‐SY5Y compared with astrocytes. We propose, based on these findings, that sodium L‐lactate affects the expression of BDNF in SH‐SY5Y and astrocytes in a different manner: high levels of iNOS and NO expressed in SH‐SY5Y have a profound inhibitory effect on the release of BDNF related to a more limited production of HSP70 by SH‐SY5Y. In conclusion, the results demonstrate differences in the responses of SH‐SY5Y and astrocytes to stimulation by high levels of sodium L‐lactate. Sodium L‐lactate differently and dose and time dependently influences the expression and release of BDNF, iNOS, NO, and HSP70 depending on the cell type. © 2012 Wiley Periodicals, Inc.     

西洛他唑对大鼠皮层细胞糖氧剥离损伤的保护作用

目的探讨磷酸二酯酶抑制剂西洛他唑对糖氧剥离后大鼠皮层细胞培养的影响及作用机制。方法原代混合培养大鼠皮层细胞,建立糖氧剥离的细胞损伤模型模拟细胞"缺血损伤",然后进行干预。测定细胞培养上清中乳酸脱氢酶(LDH)、丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)、神经元型一氧化氮合酶(nNOS)及诱导型一氧化氮合酶(i NOS)的含量;测定一氧化氮(NO)的分泌水平;测定细胞内环磷酸腺苷(cAMP)水平及四唑盐(MTT)比色试验测定细胞活力。结果西洛他唑组及依达拉奉组与糖氧剥离模型组比较,LDH、MDA漏出量显著减少(P均0.05),GSH-Px释放量明显升高(P均0.05),nNOS、i NOS的水平及NO的分泌量显著下降(P均0.05),细胞内cAMP水平明显升高(P均0.05);细胞存活率显著提高(P均0.05);西洛他唑与依达拉奉组比较,LDH、MDA漏出量及GSH-Px的释放量无差别,nNOS、i NOS和NO的水平明显降低(P均0.05),细胞内cAMP水平显著升高(P0.05);细胞存活率明显提高(P0.05)。结论西洛他唑对培养大鼠皮层细胞在糖氧剥离损伤中具有保护作用,其作用机制可能通过抗氧化、降低nNOS及i NOS的水平从而降低NO的分泌、升高细胞内cAMP水平来实现的。