Cyclic AMP Analogue as a Triggering Signal for the Induction of Nitric Oxide Synthesis in Murine Peritoneal Macrophages

To understand the role of cAMP during macrophage activation, we investigated the effects of various cAMP analogues in the induction of nitric oxide (NO) synthesis in murine peritoneal macrophages. Intracellular cAMP modulators such asN6,O2′-dibutyryl cyclic AMP (DB-cAMP), 8-bromo-cAMP, or 8-chloro-cAMP had no effect on NO synthesis by themselves, whereas cAMP analogues in combination with interferon-γ (IFN-γ) had a marked cooperative induction of NO synthesis in a dose-dependent manner. This increase in NO synthesis was reflected as an increased amount of inducible NO synthase mRNA, as determined by Northern blotting. To find the point in the signaling pathways of macrophage activation at which cAMP is involved, we carried out several of the following experiments. Although DB-cAMP showed synergistic action with rIFN-γ to induce NO synthesis when the cells were treated with DB-cAMP after or with simultaneous treatment with rIFN-γ, there is no synergistic induction of NO synthesis when the cells were treated with DB-cAMP 6 hr before treatment with rIFN-γ. In addition, when phorbol 12-myristate 13-acetate (PMA), which is known to provide a triggering signal in the induction of NO synthesis in murine macrophages, was added to the cells 6 hr after the treatment with DB-cAMP, PMA showed no synergistic cooperation with DB-cAMP. On the other hand, DB-cAMP alone induced the release of NO to the incubation medium from bacillus Calmette-Guerin-infected peritoneal macrophages just as lipopolysaccharide (LPS) did. However, DB-cAMP, unlike LPS, decreased the secretion of tumor necrosis factor-α from IFN-γ-treated macrophages. Based on the results obtained in this study, we suggest that cAMP analogue could give a “triggering” signal which might be different from one given by LPS in the production of NO by primed macrophages.