Antisense oligodeoxynucleotides to inducible NO synthase rescue epithelial cells from oxidative stress injury

Abstract

Until recently, the lack of specific inhibitors of various forms of nitric oxide synthase (NOS) hampered a stringent evaluation of the role played by inducible NOS (iNOS) in cell damage. Phosphorothioate derivatives of iNOS antisense and control sense or scrambled oligodeoxynucleotides (S-ODNs) were synthesized, and their effect on epithelial cell viability was examined under oxidant stress. Exposure of BSC-1 kidney tubular epithelial cells to H2O2 resulted in elevation of NO release, accompanied by a significant decrease in the population of viable cells (from 97.4 +/- 1.7% to 72.4 +/- 2.4% population). Nitrite production by BSC-1 cells exposed to H2O2 increased almost 10-fold compared with control. Pretreatment of the cells with 10 microM antisense ODNs significantly blunted this response, whereas sense or scrambled ODNs did not modify it. Pretreatment of BSC-1 cells with 10 microM antisense ODNs virtually prevented lethal cell damage in response to H2O2, whereas sense ODNs were ineffective. Lipopolysaccharide induction of iNOS, also preventable by the antisense construct, resulted in a lesser compromise to cell viability. Immunocytochemistry of iNOS in cells pretreated with antisense ODNs showed minimal cytoplasmic staining, as opposed to the untreated or sense ODN-treated positively stained cells. Staining with antibodies to nitrotyrosine was conspicuous in stressed cells but undetectable in antisense ODN-treated cells. In conclusion, oxidant stress is accompanied by the induction of iNOS, increased production of NO, and impaired cell viability; selective inhibition of iNOS using the designed antisense ODNs dramatically improved BSC-1 cell viability after oxidant stress.