Induction of nitric oxide synthase inhibits gap junction permeability in cultured rat astrocytes.
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.
Bolaños
J P
JP
Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Salamanca, Spain.
Medina
J M
JM
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
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