FIGURE 2.

cAMP/PKA axis is responsible for the potentiation by PGE₂ of LPS-induced NO production. A, AMs were pretreated with or without dibutyryl cAMP (dbcAMP, 1 μm) for 10 min followed by LPS for 24 h. B, AMs were pretreated for 10 min with PGE₂, PKA-specific cAMP analog 6-Bnz-cAMP (500 μm), Epac-specific cAMP analog, 8-pCPT-2-O-Me-cAMP (500 μm), or both analogs followed by 24 h incubation with LPS. C, AMs were pretreated (bottom) or not (top) with the PKA agonist 6-Bnz-cAMP for 10 min followed by incubation with LPS for the indicated time points. Lysates (30 μg of protein) were subjected to Western blot analysis for iNOS and GAPDH. Results from one experiment of three are shown. D, AMs were incubated with the PKA inhibitors KT5720 (1 μm) or PKI amide (10 μm) for 30 min before the addition of PGE₂ or 6-Bnz-cAMP for 30 min and subsequent 24 h incubation with LPS. E, AMs were incubated with KT5720 for 30 min or PGE₂ for 10 min before treatment with peptidoglycan (PGN) (1 μg/ml) for 24 h. F, cells were incubated for 10 min with site-selective cAMP analogs to activate PKA type I (10 μm 2-Cl-8-MA-cAMP) or PKA type II (10 μm 6-MBC-cAMP) and then incubated with LPS for 24 h. In A, B, D, E, and F, supernatants were harvested after 24 h, and nitrite was determined. The data are the means ± S.E. values of 3–7 separate experiments, each performed in triplicate. *, p < 0.05 versus LPS alone; #, p < 0.05 versus PGE₂ or PKA agonist alone.