ANTISENSE OLIGODEOXYNUCLEOTIDES TO INDUCIBLE NO SYNTHASE RESCUE EPITHELIAL-CELLS FROM OXIDATIVE STRESS INJURY

Until recently, the lack of specific inhibitors of various forms of ni tric oxide synthase (NOS) hampered a stringent evaluation of the role played by inducible NOS (iNOS) in cell damage. Phosphorothioate deriva tives of iNOS antisense and control sense or scrambled oligodeoxynucle otides (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 cell s (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 mu M antisense ODNs signif icantly blunted this response, whereas sense or scrambled ODNs did not modify it. Pretreatment of BSC-1 cells with 10 mu M antisense ODNs vi rtually prevented lethal cell damage in response to H2O2, whereas sens e ODNs were ineffective. Lipopolysaccharide induction of iNOS, also pr eventable by the antisense construct, resulted in a lesser compromise to cell viability. Immunocytochemistry of iNOS in cells pretreated wit h antisense ODNs showed minimal cytoplasmic staining, as opposed to th e untreated or sense ODN-treated positively stained cells. Staining wi th antibodies to nitrotyrosine was conspicuous in stressed cells but u ndetectable in antisense ODN-treated cells. In conclusion, oxidant str ess is accompanied by the induction of iNOS, increased production of N O, and impaired cell viability; selective inhibition of iNOS using the designed antisense ODNs dramatically improved BSC-1 cell viability af ter oxidant stress.