title: Induction of nitric oxide synthase inhibits gap junction permeability in cultured rat astrocytes.
abstract: Nitric oxide (.NO) synthase (NOS) was induced in cultured rat astrocytes by incubation with lipopolysaccharide (LPS) for 18 h and gap junction permeability was assessed by the scrape-loading/Lucifer yellow transfer technique. Induction of NOS was confirmed by determining either the NG-methyl-L-arginine (NMMA)-inhibitable production of nitrites and nitrates or the conversion of L-[3H]arginine to L-[3H]citrulline. Incubation with LPS dose-dependently inhibited gap junction permeability to 63.3% at 0.05 microgram/ml LPS and no further inhibition was observed on increasing the LPS concentration up to 0.5 microgram/ml. LPS-mediated gap junction inhibition was irreversible but was prevented by incubation with the NOS inhibitor NMMA and with the superoxide anion (O2.-) scavenger superoxide dismutase. Incubation of the cells with both the .NO donor S-nitroso-N-acetylpenicillamine and the O2.(-)-generating system xanthine/xanthine oxidase inhibited gap junction permeability. These results suggest that the in situ reaction between .NO and O2.-, to form the peroxynitrite anion (ONOO-), may be responsible for the inhibition of gap junction permeability. Scavenging the ONOO- derivative hydroxyl radical (.OH) with either dimethyl sulfoxide or mannitol prevented the LPS-mediated inhibition of gap junction permeability. Finally, exposure of astrocytes to authentic ONOO- caused a dose-dependent inhibition of gap junction permeability (65.7% of inhibition at 0.5 mM ONOO-). The pathophysiological relevance of ONOO(-)-mediated inhibition of gap junctional communication in astrocytes after NOS induction by LPS is discussed, stressing the possible role played by this mechanism in some neurodegenerative diseases.
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Salamanca, Spain.
authors.forename: J P
authors.initials: JP
authors.lastname: Bolaños
authors.forename: J M
authors.initials: JM
authors.lastname: Medina
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D004790:Enzyme Induction; D005456:Fluorescent Dyes; D017629:Gap Junctions / Q000378:metabolism*; D007546:Isoquinolines; D008070:Lipopolysaccharides / Q000494:pharmacology; D009566:Nitrates / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010396:Penicillamine / Q000031:analogs & derivatives / Q000494:pharmacology; D010539:Permeability; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000502:physiology; D026423:S-Nitroso-N-Acetylpenicillamine; D013482:Superoxide Dismutase / Q000494:pharmacology; D019323:omega-N-Methylarginine / Q000494:pharmacology
chemical_list: D005456:Fluorescent Dyes; D007546:Isoquinolines; D008070:Lipopolysaccharides; D009566:Nitrates; D017382:Reactive Oxygen Species; C039642:peroxynitric acid; D019323:omega-N-Methylarginine; D026423:S-Nitroso-N-Acetylpenicillamine; C017475:lucifer yellow; D019001:Nitric Oxide Synthase; D013482:Superoxide Dismutase; D010396:Penicillamine