Interferon-gamma alone triggers the production of nitric oxide from serum-starved BNL CL.2, murine embryonic liver cells

A previous study has demonstrated that both interferon-gamma (IFN-gamma) an d lipopolysaccharide (LPS) were needed to induce the production of nitric o xide (NO) in BNL CL.2 cells, murine embryonic liver cells. We here demonstr ate that when BNL CL.2 cells were cultured with serum-free medium, they wer e induced to produce NO by the stimulation of IFN-gamma alone. BNL CL.2 cel ls were cultured with serum-free or serum-containing medium for 1-3 days an d then stimulated to synthesize NO by IFN-gamma. Surprisingly, only serum-s tarved cells showed significant amount of nitrite accumulation and iNOS pro tein expression in response to IFN-gamma in dose- and time-dependent manner s, but serum-supplied cells did not. When the cells were stimulated with IF N-gamma, tumor necrosis factor-alpha (TNF-alpha), or LPS in combinations, o nly the combination of IFN-gamma and LPS produced more NO than that produce d by IFN-gamma alone, The production of NO by the cells stimulated with IFN -gamma or IFN-gamma plus LPS was blocked by the addition of N-G-monomethyl- L-arginine (N(G)MMA), a NO synthesis inhibitor. To address the intracellula r signal pathway responsible for the production of NO by the cells stimulat ed with IFN-gamma alone or IFN-gamma plus LPS, we examined the effects of s everal protein kinase inhibitors on the production of NO from the cells. Th e production of NO was significantly inhibited by protein tyrosine kinase ( PTK) inhibitors, genistein and herbimycin A, but not by protein kinase A or C inhibitors. These results suggest that the deprivation of serum from BNL CL.2 cell culture medium might prime the cells to induce NO synthesis when the cells are triggered by IFN-gamma and the involvement of PTK signal tra nsduction pathway in the expression of inducible NO synthase gene in murine hepatoma cells.