Inhibition of nitric oxide synthase by antisense techniques: investigations of the roles of NO produced by murine macrophages

1. An antisense approach to block nitric oxide (NO) synthesis was developed, complementing the widely used chemical inhibitors and overcoming problems associated with their use in studying the roles of NO.
2. Murine macrophage cell lines (J774.2) were generated expressing a 500 bp sequence from inducible NO synthase (iNOS) in either the antisense or sense orientation, driven by the SV40 promoter/enhancer region.
3. Messenger RNA derived from the transfected sequences was detected by a specific cDNA probe. Cells expressing sense and antisense iNOS RNA were characterized further.
4. The antisense lines produced 22–97% less NO than the sense lines on stimulation with lipopolysaccharide (LPS) in the range 1 ng ml⁻¹–10 μg ml⁻¹, as determined by nitrite production. One antisense line in particular, A10, expressed substantially less iNOS protein on LPS stimulation as determined by western blot analysis.
5. Adhesion of the antisense line, A10, to cytokine-stimulated murine endothelial cells (sEnd.1 line) was significantly higher than adhesion of the sense lines. There was a negative correlation between the amount of NO produced, as determined by nitrite accumulation, and the level of adhesion of the transfected lines. This indicates an anti-adhesive role of NO, produced by macrophages during the 15 min of the assay, in adhesion to endothelial cells.
6. This novel approach allowed the roles of NO in adhesion to be investigated with the substantial advantage that the contribution of NO produced rapidly by activated macrophages could be studied separately from that produced in a continuous manner by endothelial cells.
7. These lines, and the extension of this approach, will be of great use in dissecting the contributions of NO produced by different cell types to its many potential functions.