RESUMEN
Many interesting applications of magnetic iron oxide nanoparticles (IONPs) have recently been developed based on their magnetic properties and promising catalytic activity. Depending on their intended use, such nanoparticles (NPs) are frequently functionalized with proteins, polymers, or other organic molecules such as meso-2,3-dimercaptosuccinic acid (DMSA) to improve their colloidal stability or biocompatibility. Although the coating strongly affects the colloidal properties and environmental behaviour of NPs, quantitative analysis of the coating is often neglected. To address this issue, we established an ion chromatographic method for the quantitative analysis of surface-bound sulfur-containing molecules such as DMSA. The method determines the amount of sulfate generated by complete oxidation of sulfur present in the molecule. Quantification of the DMSA content of DMSA-coated IONPs showed that reproducibly approximately 38% of the DMSA used in the synthesis was adsorbed on the IONPs. Tests for the biodegradability of free and NP-bound DMSA using a microbial community from a wastewater treatment plant showed that both free and NP-bound DMSA was degraded to negligible extent, suggesting long-term environmental stability of DMSA-coated IONPs.
RESUMEN
The specific activity of brain glutamine synthetase (GS) is lowered in several neurodegenerative diseases that involve iron-mediated oxidative stress. The present study has investigated whether H2O2 directly inactivates GS or whether GS is primarily inactivated by hydroxyl radicals that are produced by the Fenton reaction when H2O2 reacts with ferrous iron. Exposure of purified sheep brain GS to supraphysiological concentrations of H2O2 (1 mM for 30 min) reduced its specific activity by only 41%, indicating that the enzyme is fairly resistant to oxidation by peroxide. However, the enzyme was completely inactivated when co-incubated with H2O2, iron and ascorbate, indicating a vulnerability to oxidation by conditions that favour the production of hydroxyl radicals. Similarly, specific GS activity in cultured mouse astrocytes was resistant to supraphysiological concentrations of H2O2, with approximately 37% of activity remaining 3h after incubation with 1mM H2O2. This inactivation was prevented by the iron chelators 2,2'-dipyridyl or 1,10-phenanthroline, but not by their non-chelating analogues. These data suggest that inactivation of astrocytic GS is caused by H2O2 indirectly via the Fenton reaction as it required the presence of chelatable intracellular iron.
Asunto(s)
Astrocitos/efectos de los fármacos , Glutamato-Amoníaco Ligasa/metabolismo , Peróxido de Hidrógeno/farmacología , Hierro/metabolismo , Oxidantes/farmacología , 2,2'-Dipiridil/farmacología , Análisis de Varianza , Animales , Encéfalo/citología , Células Cultivadas , Quelantes/farmacología , Relación Dosis-Respuesta a Droga , L-Lactato Deshidrogenasa/metabolismo , Ratones , Ratones Endogámicos C57BL , Fenantrolinas/farmacología , Factores de TiempoRESUMEN
Glutamine synthetase (GS) is an astrocytic enzyme that is essential for the glutamate-glutamine cycle between neurons and astrocytes. To measure the effects of oxidative stress on the activity of GS in astrocytes, astrocyte-rich primary cultures from the brains of wild-type and glutathione peroxidase-1 deficient mice (GPx1(-/-)) were exposed to a chronic hydrogen peroxide-generating system consisting of xanthine oxidase, hypoxanthine and superoxide dismutase. The specific activity of GS was strongly diminished by chronic exposure to hydrogen peroxide in astrocytes cultured from both mouse lines. After 60 min of oxidative stress in the presence of 5 mU/mL, 10 mU/mL and 20 mU/mL of xanthine oxidase, the specific GS activity of wild-type astrocytes was reduced to 47%, 22% and 13% of the initial activity, respectively. For all activities of xanthine oxidase applied, astrocytes from GPx1(-/-) mice experienced a significantly greater rate of GS inactivation compared to their wild-type counterparts. These results confirm that GS is sensitive to inactivation by chronic peroxide stress in viable astrocytes and show that glutathione peroxidase-1 helps to protect GS from inactivation by oxidative stress.
Asunto(s)
Astrocitos/fisiología , Glutamato-Amoníaco Ligasa/metabolismo , Glutatión Peroxidasa/fisiología , Estrés Oxidativo/fisiología , Animales , Astrocitos/enzimología , Astrocitos/patología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Glutatión Peroxidasa/genética , Peróxido de Hidrógeno/toxicidad , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados/fisiología , Proteínas del Tejido Nervioso/metabolismo , Oxidantes/toxicidad , Glutatión Peroxidasa GPX1RESUMEN
Astroglia-rich primary cultures from rat brain were used to investigate the presence in glial cells of Nepsilon-(carboxymethyl)lysine (CML), an advanced glycation endproduct. Westernblot analysis of homogenates of rat brain as well as of astroglia-rich cultures demonstrated the presence of CML-modified proteins in these samples. Immunocytochemical staining of astroglia-rich cultures revealed that only a minority of the cells in these cultures were intensively stained for CML. The staining intensity of CML-positive cells was strongly reduced, if the cells were not permeabilized, indicating that intracellular proteins were CML-modified. The CML-positive cells were identified as astrocytes and oligodendrocytes by double-labelling immunocytochemical staining for CML and the cellular markers galactocerebroside, myelin basic protein and glial fibrillary acidic protein. In contrast to other glial cells, microglial cells in astroglia-rich cultures were CML-negative. The finding that only a minority of cells in astroglia-rich cultures contains high amounts of intracellular CML-modified proteins indicates that specific properties of these CML-positive cells are responsible for the CML-formation in these cells.
Asunto(s)
Astrocitos/química , Productos Finales de Glicación Avanzada/análisis , Lisina/análogos & derivados , Lisina/análisis , Animales , Astrocitos/metabolismo , Western Blotting , Química Encefálica , Técnicas de Cultivo de Célula , Electroforesis en Gel de Poliacrilamida , Productos Finales de Glicación Avanzada/metabolismo , Inmunohistoquímica , Lisina/metabolismo , Ratas , Ratas WistarRESUMEN
The serotonin transporter (5HTT) is the molecule responsible for the high-affinity reuptake of 5HT from the synaptic cleft. Mice lacking the 5HTT exhibit highly elevated extracellular concentrations of 5HT. We assessed whether the glutathione detoxification system is altered in 5HTT-deficient mice. While levels of reduced and oxidized glutathione were unchanged, glutathione metabolising enzymes showed a differential pattern of modulation. Glutathione peroxidase was reduced in frontal cortex, brainstem, and cerebellum of 5HTT-deficient mice, though not to a statistically significant extent, while a putative isoform of the detoxifying enzyme glutathione-S-transferase pi was decreased in a number of brain regions, especially in brainstem. At the level of the DNA, we found an increase of oxidative DNA adducts in the hippocampus of 5HTT-deficient mice. Given the importance of the hippocampus in learning and memory, this may be the most important neurochemical consequence of the absence of the 5HTT.
Asunto(s)
Proteínas Portadoras/fisiología , ADN/metabolismo , Hipocampo/metabolismo , Glicoproteínas de Membrana/fisiología , Proteínas de Transporte de Membrana , Proteínas del Tejido Nervioso , Animales , Encéfalo/enzimología , Proteínas Portadoras/genética , Aductos de ADN/metabolismo , Glutatión/metabolismo , Disulfuro de Glutatión/metabolismo , Glutatión Peroxidasa/metabolismo , Gutatión-S-Transferasa pi , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados/genética , Oxidación-Reducción , Estrés Oxidativo/fisiología , ARN Mensajero/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática , Distribución TisularRESUMEN
Neurons in culture rely on the supply of exogenous cysteine for their glutathione synthesis. After application of cysteine to neuron-rich primary cultures, the glutathione content was doubled after a 4-hr incubation. The dipeptide cysteinylglycine (CysGly) was able to substitute for cysteine as exogenous glutathione precursor. In kidneys, the ectopeptidase aminopeptidase N (ApN) has been reported to hydrolyze CysGly. Expression of mRNA of ApN in rat brain and cultured rat neurons was demonstrated by reverse transcriptase polymerase chain reaction and sequencing of the cDNA fragment obtained. In addition, the presence of ApN protein in cultured neurons was demonstrated by its immunocytochemical localization. In the presence of an activity-inhibiting antiserum against ApN the utilization of CysGly as neuronal glutathione precursor was completely prevented, whereas that of cysteine plus glycine was not affected. The data presented demonstrates that cultured rat neurons express ApN and that this ectopeptidase participates in the utilization of CysGly as precursor for neuronal glutathione.
Asunto(s)
Antígenos CD13/genética , Sistema Nervioso Central/enzimología , Cisteína/metabolismo , Dipéptidos/metabolismo , Glutatión/biosíntesis , Glicina/metabolismo , Neuronas/enzimología , Animales , Antígenos CD13/antagonistas & inhibidores , Antígenos CD13/metabolismo , Células Cultivadas , Cisteína/farmacología , ADN Complementario/metabolismo , Dipéptidos/farmacología , Feto , Glicina/farmacología , Inmunohistoquímica , Enfermedades Neurodegenerativas/enzimología , Enfermedades Neurodegenerativas/fisiopatología , Estrés Oxidativo/fisiología , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Astrocytes are the most numerous cell type within the central nervous system. Early during development they act as guiding structures for migratory neurons; later they are not only the main source for nutrients and growth factors in the brain, but they are also communication partners of neighboring neurons. For this purpose astrocytes are equipped with several types of transmitter receptors and the capacity to release neuroactive substances. In addition, they form an extended syncytium via gap junction channels which allows fast intercellular signaling pathways. The pivotal involvement of astrocytes in brain function during disease situations is the topic of many studies. Here, we will review the role of astrocytic gap junctions, astroglial metabolism and neuron-astrocyte signaling. Identification of the molecular mechanisms of these three functions will improve our understanding of neuroprotection.
Asunto(s)
Astrocitos/metabolismo , Encéfalo/metabolismo , Uniones Comunicantes/metabolismo , Neuronas/metabolismo , Animales , Barrera Hematoencefálica , Comunicación Celular , Supervivencia Celular , Conexinas/metabolismo , Regulación hacia Abajo , Humanos , Red Nerviosa , Neurotransmisores/biosíntesis , Transducción de SeñalRESUMEN
The reaction of proteins with reducing sugars leads to the formation of 'advanced glycation endproducts' (AGEs). They accumulate in Alzheimer's disease brain in the vicinity of beta-amyloid plaques. AGEs are cytotoxic by a mechanism involving reactive oxygen species, which implies that they could compromise glutathione redox status. In this study, we show that AGEs (BSA-AGE and beta-amyloid-AGE) persistently increase the ratio of oxidized to reduced glutathione in a dose- and time-dependent manner in SH-SY5Y neuroblastoma cells. The level of oxidized glutathione accounted to 10-14% and persisted for up to 24 h in the presence of added AGEs. In contrast, the unmodified beta-amyloid peptides A beta (1-40) and A beta (25-35) had no significant effect on glutathione redox status. The AGE-induced increase in oxidized glutathione could be prevented by the radical scavengers N-acetylcysteine, alpha-lipoic acid and 17beta-estradiol or by application of catalase, indicating that superoxide and hydrogen peroxide production precedes the AGE-mediated depletion of reduced glutathione.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Encéfalo/metabolismo , Glutatión/metabolismo , Productos Finales de Glicación Avanzada/metabolismo , Peróxido de Hidrógeno/metabolismo , Neuronas/metabolismo , Enfermedad de Alzheimer/fisiopatología , Péptidos beta-Amiloides/farmacología , Antioxidantes/farmacología , Encéfalo/fisiopatología , Glutatión/efectos de los fármacos , Productos Finales de Glicación Avanzada/farmacología , Humanos , Fármacos Neuroprotectores/farmacología , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Fragmentos de Péptidos/farmacología , Albúmina Sérica Bovina/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismoRESUMEN
The release of glutathione disulfide has been considered an important process for the maintenance of a reduced thiol redox potential in cells during oxidative stress. In cultured rat astrocytes, permanent hydrogen peroxide-induced oxidative stress caused a rapid increase in intracellular glutathione disulfide, which was followed by the appearance of glutathione disulfide in the medium. Under these conditions, the viability of the cells was not compromised. In the presence of cyclosporin A and the quinoline-derivative MK571, inhibitors of multidrug resistance proteins (MRP1 and MRP2), glutathione disulfide accumulated in cells and the release of glutathione disulfide from astrocytes during H2O2 stress was potently inhibited, suggesting a contribution of MRP1 or MRP2 in the release of glutathione disulfide from astrocytes. Using RT-PCR we amplified a cDNA from astroglial RNA with a high degree of homology to MRP1 from humans and mouse. In contrast, no fragment was amplified by using primers specific for rat MRP2. In addition, the presence of MRP1 protein in astrocytes was demonstrated by its immunolocalization in cells expressing the astroglial marker protein glial fibrillary acidic protein. Our data identify rat astrocytes as a MRP1-expressin, brain cell type and demonstrate that this transporter participates in the release of glutathione disulfide from astrocytes during oxidative stress.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP , Astrocitos/metabolismo , Disulfuro de Glutatión/metabolismo , Proteínas de la Membrana , Estrés Oxidativo/fisiología , Receptores de Leucotrienos , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Alopurinol/farmacología , Animales , Antioxidantes/farmacología , Astrocitos/citología , Astrocitos/efectos de los fármacos , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Catalasa/farmacología , Células Cultivadas , Ciclosporina/farmacología , Inhibidores Enzimáticos/farmacología , Peróxido de Hidrógeno/farmacología , Antagonistas de Leucotrieno , Datos de Secuencia Molecular , Oxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , Propionatos/farmacología , Quinolinas/farmacología , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de AminoácidoRESUMEN
The expression of catalase in cells of astroglia-rich primary cultures derived from the brains of newborn rats was investigated by double-labelling immunocytochemical staining. Strong catalase immunoreactivity was found in cells positive for glial fibrillary acidic protein and galactocerebroside, cellular markers for astroglial and oligodendroglial cells, respectively. The cells of these cultures dispose of exogenously applied hydrogen peroxide (initial concentration 200 microM) quickly with first order kinetics. In contrast, after inhibition of glutathione peroxidases by mercaptosuccinate the rate of the catalase-dependent disposal of H(2)O(2) declined with time and after about 10 min the extracellular concentration of H(2)O(2) remained almost constant at a concentration of about 100 microM. Catalase activity after 10 min of incubation under these conditions was no longer detectable. In contrast, in the absence of mercaptosuccinate catalase activity was maintained during H(2)O(2) disposal. These results demonstrate that in astroglia-rich cultures catalase is strongly expressed in the predominant astroglial cells and in the minor population of oligodendroglial cells and that the enzyme is rapidly inactivated during the disposal of H(2)O(2), if the glutathione system of the cells is compromised.
Asunto(s)
Astrocitos/enzimología , Catalasa/análisis , Peróxido de Hidrógeno/metabolismo , Oxidantes/metabolismo , Animales , Anticuerpos , Astrocitos/citología , Encéfalo/citología , Encéfalo/enzimología , Catalasa/inmunología , Células Cultivadas , Activación Enzimática/fisiología , Glutatión/metabolismo , Peróxido de Hidrógeno/farmacología , Inmunohistoquímica , Oxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Ratas , Ratas WistarRESUMEN
To obtain information on the glutathione metabolism of microglial cells, the content of glutathione and activities of enzymes involved in the defense against peroxides were determined for microglia-rich cultures from rat brain. These cultures contain approximately 90% microglia cells as determined by immunocytochemical staining for glial markers, by the phagocytosis activity of the cells and by the production of superoxide after stimulation of the cells with phorbolester. For these cultures, a glutathione content of 41.2 +/- 11.2 nmol/mg protein and a specific activity of glutathione reductase of 15.2 +/- 3.2 nmol/(min x mg protein) were determined. These values are significantly higher than those found for astroglial or neuronal cultures. In addition, with 68.7 +/- 23.5 nmol/(min x mg protein), the specific activity of glutathione peroxidase in microglial cultures was 78% higher than in cultured neurons. The specific catalase activity of microglial cultures was less than 40% that of astroglial or neuronal cultures. Microglial cultures contain only marginal amounts of oxidized glutathione. However, on application of oxidative stress by incubation of microglial cultures with hydrogen peroxide or with the superoxide-producing hypoxanthine/xanthine oxidase system, cellular glutathione was rapidly oxidized. These results demonstrate that microglial cells have a prominent glutathione system, which is likely to reflect the necessity for self-protection against reactive oxygen species when produced by these or surrounding brain cells.
Asunto(s)
Disulfuro de Glutatión/biosíntesis , Glutatión/biosíntesis , Microglía/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Antígenos de Diferenciación/biosíntesis , Astrocitos/citología , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Catalasa/metabolismo , Células Cultivadas , Colorantes Fluorescentes , Glutatión Peroxidasa/metabolismo , Glutatión Reductasa/metabolismo , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacología , Hipoxantina/metabolismo , Hipoxantina/farmacología , Microglía/citología , Microglía/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Ésteres del Forbol/farmacología , Ratas , Ratas Wistar , Superóxidos/metabolismo , Xantina Oxidasa/metabolismoRESUMEN
The cells of the adult human brain consume approximately 20% of the oxygen utilized by the body although the brain comprises only 2% of the body weight. Reactive oxygen species, which are produced continuously during oxidative metabolism, are generated at high rates within the brain. Therefore, the defense against the toxic effects of reactive oxygen species is an essential task within the brain. An important component of the cellular detoxification of reactive oxygen species is the antioxidant glutathione. The main focus of this short review is recent results on glutathione metabolism of brain astrocytes and neurons in culture. These two types of cell prefer different extracellular precursors for glutathione. Glutathione is involved in the disposal of exogenous peroxides by astrocytes and neurons. In coculture astrocytes protect neurons against the toxicity of reactive oxygen species. One mechanism of this interaction is the supply by astrocytes of glutathione precursors to neurons.
Asunto(s)
Astrocitos/metabolismo , Encéfalo/metabolismo , Glutatión/metabolismo , Neuronas/metabolismo , Especies Reactivas de Oxígeno , Adulto , Animales , Comunicación Celular , Humanos , Consumo de OxígenoRESUMEN
Regeneration of reduced nicotinamide adenine dinucleotide phosphate (NADPH) is essential for the activity of glutathione redox cycling during cellular peroxide detoxification. In order to test for a function of astroglial glycogen to serve as endogenous precursor for glucose-6-phosphate, the substrate for the regeneration of NADPH by the pentose phosphate pathway, the content of glycogen in astroglia-rich primary cultures derived from the brains of newborn rats was determined after application of peroxides. In the presence of hydrogen peroxide or cumene hydroperoxide in concentrations of 200 microM glycogen was mobilized with a half-life of 16 min in incubation medium containing 20 mM glucose, whereas in the absence of peroxides the glycogen content decreased more slowly with a half-life of 42 min. After 30 min of incubation with or without peroxides 30 and 73%, respectively, of the initial glycogen content was found. The degree of glycogen mobilization was reduced by lowering the initial concentration of the peroxides. These results demonstrate that in astroglial cells (i) glucosyl residues of glycogen are mobilized after application of peroxides despite the presence of exogenous glucose, and (ii) that the demand for glucose-6-phosphate as substrate for NADPH regeneration via the pentose phosphate pathway can, at least partially, be met by mobilization of glycogen.
Asunto(s)
Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Metabolismo Energético/fisiología , Glucógeno/metabolismo , NADP/metabolismo , Peróxidos/metabolismo , Peróxidos/farmacología , Animales , Animales Recién Nacidos , Astrocitos/citología , Células Cultivadas , Ratas , Ratas WistarRESUMEN
The tripeptide glutathione is the thiol compound present in the highest concentration in cells of all organs. Glutathione has many physiological functions including its involvement in the defense against reactive oxygen species. The cells of the human brain consume about 20% of the oxygen utilized by the body but constitute only 2% of the body weight. Consequently, reactive oxygen species which are continuously generated during oxidative metabolism will be generated in high rates within the brain. Therefore, the detoxification of reactive oxygen species is an essential task within the brain and the involvement of the antioxidant glutathione in such processes is very important. The main focus of this review article will be recent results on glutathione metabolism of different brain cell types in culture. The glutathione content of brain cells depends strongly on the availability of precursors for glutathione. Different types of brain cells prefer different extracellular glutathione precursors. Glutathione is involved in the disposal of peroxides by brain cells and in the protection against reactive oxygen species. In coculture astroglial cells protect other neural cell types against the toxicity of various compounds. One mechanism for this interaction is the supply by astroglial cells of glutathione precursors to neighboring cells. Recent results confirm the prominent role of astrocytes in glutathione metabolism and the defense against reactive oxygen species in brain. These results also suggest an involvement of a compromised astroglial glutathione system in the oxidative stress reported for neurological disorders.
Asunto(s)
Encéfalo/metabolismo , Glutatión/metabolismo , Animales , Astrocitos/citología , Astrocitos/metabolismo , Transporte Biológico/fisiología , Comunicación Celular/fisiología , Células Cultivadas , Espacio Extracelular/metabolismo , Glutatión/deficiencia , Humanos , Microglía/citología , Microglía/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Neuronas/citología , Neuronas/metabolismo , Oligodendroglía/citología , Oligodendroglía/metabolismo , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Metabolic pathways underlying the regeneration of reduced glutathione were investigated in acutely isolated metabolically active mitochondria from rat forebrain. The application of hydrogen peroxide to the organelles was accompanied by a transient increase in glutathione disulfide. The recovery of reduced glutathione was significantly improved in the presence of alternatively succinate, malate, citrate, isocitrate, or beta-hydroxybutyrate. Inhibition of succinate dehydrogenase by malonate abolished the beneficial effect of succinate on the reduction of glutathione disulfide but did not influence the effect of isocitrate. Fluorocitrate, an inhibitor of aconitase, blocked the effect exerted by citrate but did not inhibit the effects of malate or beta-hydroxybutyrate. Uncoupling of the respiratory chain by carbonyl cyanide m-chlorophenylhydrazone prevented the beneficial effect of beta-hydroxybutyrate but did not abolish the improved reduction of mitochondrial glutathione disulfide in the presence of malate and isocitrate. These results suggest that NADP+-dependent isocitrate dehydrogenase as well as malic enzyme and nicotinamide nucleotide transhydrogenase contribute to the regeneration of NADPH required for the reduction of glutathione disulfide in brain mitochondria.
Asunto(s)
Glutatión/metabolismo , Mitocondrias/metabolismo , NADP/metabolismo , Prosencéfalo/metabolismo , Animales , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacología , Isocitrato Deshidrogenasa/metabolismo , Malato Deshidrogenasa/metabolismo , Mitocondrias/efectos de los fármacos , NADP Transhidrogenasas/metabolismo , Oxidación-Reducción , Estrés Oxidativo , Ratas , Ratas WistarRESUMEN
Using a new polyclonal antibody against prostacyclin (PGI2)-synthase this enzyme was shown to be present in neuronal cells of bovine, rat and human brain, most abundantly in Purkinje cells of the cerebellum and cortical neurons, but not in glial cells. Western blots confirmed the specificity of the antibody and applied to enriched neuronal and astrocyte cultures supported these immunohistochemical data. It was further shown that staining with an anti-nitrotyrosine antibody was positive for PGI2-synthase containing cells. Possible physiological and/or pathophysiological functions of the enzyme in brain are discussed.
Asunto(s)
Corteza Cerebral/enzimología , Sistema Enzimático del Citocromo P-450/análisis , Oxidorreductasas Intramoleculares/análisis , Células de Purkinje/enzimología , Anciano , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Anticuerpos , Astrocitos/citología , Western Blotting , Bovinos , Células Cultivadas , Corteza Cerebral/citología , Sistema Enzimático del Citocromo P-450/inmunología , Técnica del Anticuerpo Fluorescente , Humanos , Oxidorreductasas Intramoleculares/inmunología , Persona de Mediana Edad , Datos de Secuencia Molecular , Células de Purkinje/citología , Ratas , Ratas Wistar , Tirosina/análogos & derivados , Tirosina/análisis , Tirosina/inmunologíaRESUMEN
Glutathione reductase (GR) is an essential enzyme for the glutathione-mediated detoxification of peroxides because it catalyzes the reduction of glutathione disulfide. GR was purified from bovine brain 5,000-fold with a specific activity of 145 U/mg of protein. The homogeneity of the enzyme was proven by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the gel. The purified GR from bovine brain is a dimer of two subunits that have an apparent molecular mass of 55 kDa. The purified GR was used to generate a rabbit antiserum with the intention to localize GR in brain cells. The antiserum was useful for the detection of GR by double-labeling immunocytochemical staining in astroglia-rich and neuron-rich primary cultures from rat brain. In homogenates of these cultures, no significant difference in the specific activities of GR was determined. However, not all cell types present in these cultures showed identical staining intensity for GR. In astroglia-rich primary cultures, strong GR immunoreactivity was found in cells positive for the cellular markers galactocerebroside and C3b (antibody Ox42), indicating that oligodendroglial and microglial cells, respectively, contain GR. In contrast, only weak immunoreactivity for GR was found in cells positive for glial fibrillary acidic protein. In neuron-rich primary cultures, GAP43-positive cells stained with the antiserum against GR. These data demonstrate that, in cultures of neural cells, neurons, oligodendroglial cells, and microglial cells express high levels of GR.
Asunto(s)
Astrocitos/enzimología , Encéfalo/enzimología , Glutatión Reductasa/análisis , Glutatión Reductasa/aislamiento & purificación , Neuroglía/enzimología , Neuronas/enzimología , Animales , Astrocitos/citología , Biomarcadores , Encéfalo/citología , Bovinos , Células Cultivadas , Cromatografía de Afinidad , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Glutatión Reductasa/metabolismo , Sueros Inmunes , Inmunohistoquímica , Cinética , Sustancias Macromoleculares , Microglía/citología , Microglía/enzimología , Peso Molecular , Neuroglía/citología , Neuronas/citología , Conejos , RatasRESUMEN
Hydrogen peroxide is often applied to cultured brain cells to investigate functions of these cells under oxidative stress. However, this peroxide might be quickly detoxified by cultured neural cells. At least cultured astroglial cells dispose of exogenous H(2)O(2) with a half-time in the minute range [R. Dringen, B. Hamprecht, Involvement of glutathione peroxidase and catalase in the disposal of exogenous hydrogen peroxide by cultured astroglial cells, Brain. Res. 759 (1997) pp. 67-75]. Therefore, application of hydrogen peroxide to astroglial cells leads only to a period of transient oxidative stress which depends on the ability of the cells to detoxify the peroxide. In order to apply a permanent H(2)O(2)-induced oxidative stress to astroglial cells the generation of H(2)O(2) by the coupled reactions of xanthine oxidase (XO) and superoxide dismutase (SOD) was studied and this system was applied to cultured astroglial cells. In the presence of astroglial cells, an almost constant steady state concentration of H(2)O(2) in the range up to 100 microM was generated, which depended on the activity of the XO applied. These steady state levels of H(2)O(2) were: (i) elevated by application of additional XO, (ii) slowly reduced by application of the XO inhibitor allopurinol, and (iii) immediately reduced to zero by application of catalase or a combination of allopurinol plus catalase. In conclusion, the method presented allows the application of an almost constant exogenous H(2)O(2) stress to cultured cells.
Asunto(s)
Astrocitos/efectos de los fármacos , Técnicas de Cultivo de Célula/métodos , Peróxido de Hidrógeno/toxicidad , Alopurinol/farmacología , Animales , Astrocitos/metabolismo , Encéfalo/citología , Catalasa/farmacología , Supervivencia Celular , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Peróxido de Hidrógeno/metabolismo , Estrés Oxidativo , Ratas , Ratas Wistar , Superóxido Dismutasa/metabolismo , Factores de Tiempo , Xantina Oxidasa/antagonistas & inhibidores , Xantina Oxidasa/metabolismoRESUMEN
The ability of astroglia-rich primary cultures derived from the brains of newborn rats to detoxify exogenously applied cumene hydroperoxide (CHP) was analyzed as a model to study glutathione-mediated peroxide detoxification by astrocytes. Under the conditions used, 200 microM CHP disappeared from the incubation buffer with a half-time of approximately 10 min. The half-time of CHP in the incubation buffer was found strongly elevated (a) in cultures depleted of glutathione by a preincubation with buthionine sulfoximine, an inhibitor of glutathione synthesis, (b) in the presence of mercaptosuccinate, an inhibitor of glutathione peroxidase, and (c) in the absence of glucose, a precursor for the regeneration of NADPH. The involvement of glutathione peroxidase in the clearance of CHP was confirmed by the rapid increase in the level of GSSG after application of CHP. The restoration of the initial high ratio of GSH to GSSG depended on the presence of glucose during the incubation. The high capacity of astroglial cells to clear CHP and to restore the initial ratio of GSH to GSSG was fully maintained when glucose was replaced by mannose. In addition, fructose and galactose at least partially substituted for glucose, whereas exogenous isocitrate and malate were at best marginally able to replace glucose during peroxide detoxification and regeneration of GSH. These results demonstrate that CHP is detoxified rapidly by astroglial cells via the glutathione system. This metabolic process strongly depends on the availability of glucose or mannose as hydride donors for the regeneration of the NADPH that is required for the reduction of GSSG by glutathione reductase.