Fig. 1.
Effect of the nonselective AR agonist NECA, the A2AAR-selctive agonist CGS 21680, and the direct adenylyl cyclase activator forskolin on cAMP accumulation and phosphorylation of CREB in murine macrophages. A, macrophages were stimulated with either 1 μM NECA, 1 μM CGS 21680, or 50 μM forskolin in the presence of the phosphodiesterase inhibitor Ro 20-1754 (20 μM) for 15 min, and cAMP accumulation in acid extracts was determined by radioimmunoassay. B and C, macrophages were stimulated with 1 μM NECA for the times indicated either in the presence or absence of vehicle or the PKA inhibitor H89 (10 μM) before preparation of detergent-soluble extracts. Samples were equalized for protein content and were subjected to SDS-PAGE electrophoresis for immunoblotting with anti-phospho-CREB antibodies, as described under Materials and Methods. B, representative immunoblots. C, consolidated data from four independent experiments. *, p < 0.05 versus the baseline or vehicle-treated groups by one-way ANOVA or Student's t test, as appropriate. n = 3 to 5.