ANTISENSE OLIGODEOXYNUCLEOTIDE TO INDUCIBLE NITRIC-OXIDE SYNTHASE INHIBITS NITRIC-OXIDE SYNTHESIS IN RAT PULMONARY-ARTERY SMOOTH-MUSCLE CELLS IN CULTURE

Background. We recently demonstrated the induced expression of the ind ucible nitric oxide synthase (iNOS) gene in cultured rat pulmonary art ery smooth muscle (RPASM) in response to lipopolysaccharide and cytoki nes, using a complementary DNA probe to murine macrophage iNOS. Becaus e nitric oxide (NO) can be cytotoxic, iNOS in the pulmonary vasculatur e may contribute to lung injury in sepsis. We designed an antisense ol igodeoxynucleotide complementary to the iNOS messenger RNA (mRNA) sequ ence to determine whether the probe prevented iNOS translation. Method s. RPASM, preincubated in the presence of antisense and sense oligodeo xynucleotide to the first 18 bases after the initiation codon of iNOS mRNA, was exposed to interferon-gamma and tumor necrosis factor-alpha to induce NO production (as measured by NO2-, the stable end product o f NO formation). Results. Interferon-gamma and tumor necrosis factor-a lpha induced NO production in RPASM. The antisense probe caused up to a 36% decrease in cytokine-induced NO2 production in a concentration-d ependent manner (1 to 10 mumol/L). The sense probe had no effect. Conc lusions. Increased transcription of iNOS mRNA is an essential step in the induced production of NO by RPASM. Antisense probes partially inhi bit iNOS expression in vitro, suggesting its use to inhibit iNOS expre ssion under pathologic conditions.