ABSTRACT
We previously reported that in immunostimulated astrocytes, glucose deprivation induced cell death via the loss of ATP, reduced glutathione, and mitochondrial transmembrane potential. The cytotoxicity was due to reactive nitrogen and oxygen species and blocked by adenosine, a purine nucleoside, via the preservation of cellular ATP. Here, we investigated whether uridine, a pyrimidine nucleoside, could prevent the glucose deprivation-induced cytotoxicity in LPS+IFN-gamma-treated (immunostimulated) astrocytes. Glucose deprivation induced the death of immunostimulated cells, which was significantly reduced by uridine. Glucose deprivation rapidly decreased cellular ATP levels in immunostimulated astrocytes, which was also reversed by uridine. The inhibition of cellular uptake of uridine by S-(4-nitrobenzyl)-6-thioinosine attenuated the protective effect of uridine. mRNA and protein expression for uridine phosphorylase, an enzyme catalyzing reversible phosphorolysis of uridine, were observed in rat brain as well as primary astrocytes. 5-(Phenylthio)acyclouridine (PTAU), a specific inhibitor of uridine phosphorylase, inhibited the protective effect of uridine. Additionally, the loss of mitochondrial transmembrane potential and reduced glutathione by glucose deprivation in immunostimulated cells was attenuated by uridine, which was also reversed by PTAU. These results provide the first evidence that uridine protects immunostimulated astrocytes against the glucose deprivation-induced death by preserving intracellular ATP through the action of uridine phosphorylase.
Subject(s)
Astrocytes/physiology , Cell Death/physiology , Glucose/metabolism , Immunization , Uridine Phosphorylase/metabolism , Uridine/metabolism , Adenosine Triphosphate/metabolism , Animals , Astrocytes/cytology , Astrocytes/drug effects , Cells, Cultured , Glutathione/metabolism , Humans , Interferon-gamma/immunology , Interferon-gamma/pharmacology , Lipopolysaccharides/immunology , Lipopolysaccharides/pharmacology , Membrane Potentials , Neuroprotective Agents/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
Peroxynitrite is a potent neurotoxic molecule produced from a reaction between NO and superoxide and induces NO-mediated inflammation under neuropathological conditions. Previously, we reported that glucose deprivation induced ATP depletion and cell death in immunostimulated astrocytes, which was mainly due to peroxynitrite. In this study, the role of MAPKs (ERK1/2, p38MAPK, and JNK1SAPK) signal pathway in the SIN-1/glucose deprivation-induced death of astrocytes was examined. A combined treatment with glucose deprivation and 50 microM SIN-1, an endogenous peroxynitrite generator, rapidly and markedly increased the death in rat primary astrocytes. Also, SIN-1/glucose deprivation resulted in the activation of MAPKs, which was significantly blocked by the treatment with 20 microM MAPKs inhibitors (ERK1/2, PD98059; p38MAPK, SB203580; JNK/SAPK, SP600125). Interestingly, SIN-1/glucose deprivation caused the loss of intracellular ATP level, which was significantly reversed by MAPKs inhibitors. These results suggest that the activation of MAPKs plays an important role in SIN-1/glucose deprivation-induced cell death by regulating the intracellular ATP level.
Subject(s)
Astrocytes/metabolism , Astrocytes/pathology , Mitogen-Activated Protein Kinase 1/metabolism , Peroxynitrous Acid , p38 Mitogen-Activated Protein Kinases/metabolism , Adenosine Triphosphate/metabolism , Animals , Anthracenes/pharmacology , Astrocytes/drug effects , Brain Ischemia/metabolism , Brain Ischemia/prevention & control , Cell Culture Techniques , Cell Death/drug effects , Cells, Cultured , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Glucose/deficiency , Imidazoles/pharmacology , L-Lactate Dehydrogenase/analysis , L-Lactate Dehydrogenase/metabolism , MAP Kinase Signaling System/drug effects , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Molsidomine/analogs & derivatives , Molsidomine/pharmacology , Nitric Oxide Donors/pharmacology , Peroxynitrous Acid/biosynthesis , Pyridines/pharmacology , Rats , Rats, Sprague-Dawley , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitorsABSTRACT
Previously, we have shown that astrocytes deprived of glucose became highly vulnerable to peroxynitrite, and adenosine and its metabolites attenuated the gliotoxicity via the preservation of cellular ATP level. Here, we found that adenosine and related metabolites prevented the disruption of mitochondrial transmembrane potential (MTP) in glucose-deprived rat primary astrocytes exposed to 3-morpholinosydnonimine (SIN-1), a peroxynitrite releasing agent. Exposure to glucose deprivation and SIN-1 (2 h) significantly disrupted MTP in astrocytes, and adenosine prevented it in dose-dependent manner with an EC50 of 5.08 microM. Adenosine also partially prevented the cell death by myxothiazol, a well-known inhibitor of mitochondrial respiration. Blockade of adenosine deamination or intracellular transport with erythro-9-(-hydroxy-3-nonyl)adenosine (EHNA) or S-(4-nitrobenzyl)-6-thioinosine (NBTI), respectively, completely reversed the protective effect of adenosine. Other purine nucleos(t)ides including inosine, guanosine, ATP, ADP, AMP, ITP, and GTP also showed similar protective effects. This study indicates that adenosine and related purine nucleos(t)ides may protect astrocytes from peroxynitrite-induced mitochondrial dysfunction.
