Induction of Nitric Oxide Synthase Inhibits Gap Junction Permeability in Cultured Rat Astrocytes

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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 µg/ml LPS and no further inhibition was observed on increasing the LPS concentration up to 0.5 µg/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.

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