Stimulated murine macrophages as a bioassay for H. pylori-related inhibition of nitric oxide production.
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 microM). A higher concentration of L-arginine (200 microM) 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.
von Bothmer
C
C
Dept. of Gastrosurgical Research, Sahlgrenska Academy, Göteborg University, Gothenburg, Sweden. charlotte.bothmer@gastro.gu.se
Bölin
I
I
Pettersson
A
A
Fändriks
L
L
D000818:Animals; D001120:Arginine / Q000378:metabolism; D001681:Biological Assay; D016481:Helicobacter Infections / Q000201:enzymology*; D016480:Helicobacter pylori; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors*
D009569:Nitric Oxide; D001120:Arginine; D019001:Nitric Oxide Synthase