Role of tyrosine kinase activity in 2,2',2''-tripyridine-induced nitricoxide generation in macrophages.

In this paper, we demonstrated that 2,2',2"-tripyridine (TP, 1-20 microM) is a potent inducer of nitric oxide (NO) synthase in the cultured murine macrophage RAW 264.7 cell line. TP increased not only nitrite but also inducible NO synthase (iNOS) protein and mRNA production. Co-treatment with either NOS inhibitors (N(G)-monomethyl-L-arginine and aminoguanidine) or cycloheximide and actinomycin D all inhibited TP-induced nitrite production, indicating the requirement of protein and mRNA synthesis. The signaling pathway of TP-induced iNOS expression was explored, and the results obtained suggested that increased tyrosine kinase activity followed by inhibitor of nuclear factor for immunoglobulin kappa chain in B cells (IkappaB) degradation and then nuclear factor kappaB (NFkappaB) activation was involved in TP-induced iNOS expression. Tyrosine kinase inhibitors (e.g. genistein and tyrphostin AG126) inhibited both TP-induced nitrite and iNOS protein production. Whether the metalochelating property of TP was involved in these effects was explored by saturating TP with FeCl3. Although the ferrated TP became inactive, the specific iron chelator desferrioxamine, at a very high concentration of 400 microM, induced only a weak enhancement of nitrite production in this RAW cell line. It was thereby concluded that TP induces NO production through an increase in iNOS expression, which is initiated by a signaling pathway via tyrosine kinases leading to an activation of NFkappaB. Since TP is much more potent than desferrioxamine in increasing nitrite production, it is suspected that the primary event induced by TP was possibly mediated by TP's interacting with certain macromolecules in addition to its metal-chelating property.