Peroxynitrite mediated damage and lowered superoxide tolerance in primary cortical glial cultures after induction of the inducible isoform of NOS

✍ Scribed by Heiko Noack; Heiko Possel; Christoph Rethfeldt; Gerburg Keilhoff; Gerald Wolf

• Publisher: John Wiley and Sons
• Year: 1999
• Tongue: English
• Weight: 603 KB
• Volume: 28
• Category: Article
• ISSN: 0894-1491
• DOI: 10.1002/(sici)1098-1136(199910)28:1<13::aid-glia2>3.0.co;2-l

✦ Synopsis

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 ( и NO) formation at different levels of artificially generated superoxide. Stimulation of the cultures by bacterial lipopolysaccharides and ␥-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 и 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 и 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 Ϫ -/ и NO mediated mechanism of cell damage. Our data support the concept that i-NOS catalyzed и NO-formation leads to an ONOO Ϫ -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.