Synergistic cooperation between cAMP and IFN-γ for induction of nitric oxide synthesis in ROS 17/2.8 osteoblasts

The gas radical, nitric oxide (NO), is a major autacoid regulating cell behavior in the cardiovascular, immune and central nervous systems. Recently, it was shown that it is produced by both the osteoblast and osteoclast and that NO appears to be an important regulatory molecule in the processes of bone remodeling. By measurements of nitrite production, Northern blot analysis, and Western blot analysis, we studied the effects of a membrane- permeable cAMP derivative, DBcAMP and 8-bromo-cAMP, on the expression of inducible nitric oxide synthase (iNOS) mRNA and protein and the synthesis of nitrite in ROS 17/2.8 cells. DBcAMP and 8-bromo-cAMP stimulated NO production and iNOS mRNA and protein expression in IFN-γ-treated ROS 17/2.8 cells in a dose-dependent manner. Compounds that increase intracellular cAMP levels (forskolin, theophylline, PGE₂), all stimulated NO production and iNOS mRNA and protein expression in IFN-γ-treated ROS 17/2.8 cells. Sp-cAMPS (a selective activator of cAMP-dependent protein kinase I and II) also increased NO production and iNOS mRNA and protein expression in IFN-γ-treated ROS 17/2.8 cells. N(G)-monomethyl-L-arginine, a NOS inhibitor, completely blocked the cAMP-elevating agents-induced NO production in IFNγ-treated ROS 17/2.8 cells. These observations indicate that cAMP is important in considering experiments involving the production of nitric oxide with IFN-γ.