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

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 μM antisense ODNs significantly blunted this response, whereas sense or scrambled ODNs did not modify it. Pretreatment of BSC-1 cells with 10 μM 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.