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 inducible nitric oxide synthase (iNOS) gene in cultured rat pulmonary artery smooth muscle (RPASM) in response to lipopolysaccharide and cytokines, using a complementary DNA probe to murine macrophage iNOS. Because nitric oxide (NO) can be cytotoxic, iNOS in the pulmonary vasculature may contribute to lung injury in sepsis. We designed an antisense oligodeoxynucleotide complementary to the iNOS messenger RNA (mRNA) sequence to determine whether the probe prevented iNOS translation.

METHODS

RPASM, preincubated in the presence of antisense and sense oligodeoxynucleotide 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 of NO formation).

RESULTS

Interferon-gamma and tumor necrosis factor-alpha induced NO production in RPASM: The antisense probe caused up to a 36% decrease in cytokine-induced NO2- production in a concentration-dependent manner (1 to 10 mumol/L). The sense probe had no effect.

CONCLUSIONS

Increased transcription of iNOS mRNA is an essential step in the induced production of NO by RPASM: Antisense probes partially inhibit iNOS expression in vitro, suggesting its use to inhibit iNOS expression under pathologic conditions.