Fig. 6.

Epac-1 is not involved in mediating the inhibitory actions of NECA or forskolin on LPS-induced TNF-α release. A and B, treatments with NECA and forskolin (forsk) continue to inhibit LPS-induced TNF-α release from macrophages after siRNA-mediated knockdown of Epac-1. C, treatment with the Epac agonist 8′-CPT-cAMP-O-Me failed to inhibit LPS-induced TNF-α release. For the siRNA studies, macrophages subjected 72 h earlier to transfection with 400 nM anti-Epac-1 siRNA (siRNA) or mock transfection were pretreated with NECA (1 μM) or forskolin (50 μM) 30 min before stimulation with LPS (10 μg/ml). For studies with the Epac agonist, macrophages were pretreated for 30 min with increasing concentrations of 8′-CPT-cAMP-O-Me before simulation with LPS (10 μg/ml). In all studies, the concentration of TNF-α was measured in cell culture media 4 h after stimulation with LPS. The data are presented as a percentage of TNF-α released compared with that for the LPS-stimulated control group. *, p < 0.05 versus vehicle-treated group by Student's t test. n = 4 to 6.