Nicotinamide inhibits nitric oxide synthase mRNA induction in activated macrophages

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Abstract
Nitric oxide (NO) is a potent mediator involved in many biological functions including inflammation and non-specific immunity. Murine macrophages possess the prototype of high-output NO synthase which is not constitutively expressed but induced within a few hours by immunological stimuli. In this study, we explored the possibility of controlling the activity of the inducible NO synthase by interfering with the transduction signal which triggers its induction, in the RAW 264.7 macrophage cell line. We found that nicotinamide, an inhibitor of ADP-ribosylation, prevented NO synthase induction in RAW 264.7 cells after stimulation with interferon gamma (IFN-gamma) and lipopolysaccharide (LPS). Furthermore, the level of NO synthase mRNA was measured by Northern-blot analysis and we found that nicotinamide prevents expression of NO synthase mRNA in IFN-gamma- and LPS-stimulated cells. Nicotinamide was also found to inhibit other macrophage functions expressed in response to IFN-gamma, i.e. tumour necrosis factor secretion and the expression of the Ia antigen of the major histocompatibility complex. Analysis of the pattern of ADP-ribosylated proteins revealed that nicotinamide as well as cholera toxin prevented the ADP-ribosylation of a 107-117 kDa protein found constitutively ADP-ribosylated in stimulated and non-stimulated macrophage extracts. Together, our results indicate ADP-ribosylation as a crucial point of the signalling pathway which leads to NO synthase mRNA induction.