Peroxynitrite mediated damage and lowered superoxide tolerance in primary cortical glial cultures after induction of the inducible isoform of NOS.
The effect of the induction of i-NOS in primary glial cultures was studied with respect to the protein levels of reactive oxygen species (ROS) scavenging enzymes and the cytotoxicity of nitric oxide (.NO) formation at different levels of artificially generated superoxide. Stimulation of the cultures by bacterial lipopolysaccharides and gamma-interferon resulted in an induction of i-NOS exclusively in microglial cells. Among the ROS scavenging enzymes superoxide dismutase (Cu/Zn- and Mn-isoform), glutathione peroxidase and catalase only mitochondrial Mn-SOD was found to be upregulated in the course of i-NOS induction (Western blots). Although .NO formation did not affect cell viability at physiological levels of superoxide over a time period of 4 days, it caused an oxidative load particularly in microglial cells as observed by monitoring the oxidation of dichloro-dihydrofluorescein, an indicator for the formation of peroxynitrite and ROS. Elevated levels of superoxide, generated either intracellularly by paraquat or extracellularly via xanthine oxidase and hypoxanthine, resulted dose-dependently in a larger decline of cell viability in the .NO forming cultures compared to controls (release of lactate dehydrogenase, citrate synthase, stainability by propidium iodide, and tetramethylrhodamine). NOS-inhibitors reduced the degree of cell damage to that seen for control cultures, indicating an ONOO--/.NO mediated mechanism of cell damage. Our data support the concept that i-NOS catalyzed .NO-formation leads to an ONOO--mediated increased oxidative load. At physiological levels of superoxide and within a wide range of higher superoxide levels this nitrosative stress is well balanced in cultured glial cells by protective mechanisms.
Noack
H
H
Institut für Medizinische Neurobiologie, Otto-von-Guericke-Universität, Magdeburg, Germany. Heiko.Noack@Medizin.Uni-Halle.de
Possel
H
H
Rethfeldt
C
C
Keilhoff
G
G
Wolf
G
G
D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D002950:Citrate (si)-Synthase / Q000378:metabolism; D004790:Enzyme Induction / Q000187:drug effects; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000096:biosynthesis; D009457:Neuroglia / Q000187:drug effects / Q000201:enzymology*; D009566:Nitrates / Q000633:toxicity*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II; D009573:Nitrites / Q000378:metabolism; D016877:Oxidants / Q000633:toxicity*; D010269:Paraquat / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013481:Superoxides / Q000378:metabolism / Q000633:toxicity*; D014969:Xanthine Oxidase / Q000494:pharmacology
D009419:Nerve Tissue Proteins; D009566:Nitrates; D009573:Nitrites; D016877:Oxidants; D013481:Superoxides; C039642:peroxynitric acid; D007770:L-Lactate Dehydrogenase; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D014969:Xanthine Oxidase; D002950:Citrate (si)-Synthase; D010269:Paraquat