Stimulated Murine Macrophages as a Bioassay for H. pylori-related Inhibition of Nitric Oxide Production

Abstract

Background: Interference with the L-arginine/nitric oxide pathway may be a virulence strategy for the gastric pathogen Helicobacter pylori. This study evaluates a bioassay for such inhibitory actions on nitric oxide synthase. Methods: Cultured murine macrophages were stimulated by lipopolysaccharide and interferon-gamma. Nitric oxide synthesis and the expression of inducible nitric oxide synthase (iNOS) at increasing concentrations of L-arginine were analysed using chemiluminescence and Western blotting, respectively. Results: The bioassay was evaluated against nitrite accumulation and two established NOS inhibitors. Bacterial extracts or whole cells of one H. pylori strain inhibited nitric oxide production at low L-arginine concentrations (2–20 μM). A higher concentration of L-arginine (200 μM) was not associated with such inhibition. The iNOS expression was not affected by any of the additives compared to stimulated controls. Conclusions: This bioassay is a reliable and simple method for analysing iNOS inhibition, resolving effects on enzyme activity or enzyme expression. H. pylori water extract and whole cells exert an L-arginine-dependent NOS inhibition, not influencing iNOS expression.

Key words:

• Bioassay
• H. pylori
• iNOS
• L-arginine
• macrophage
• nitric oxide