Fig. 6.

Role of Epac1-Rap1 signaling in mediating effects of PDE1 inhibition. a Collagen synthesis measured via [³H]-proline incorporation in cardiac fibroblasts transfected with siRNA against non-targeting control, Rap1, or Epac1 followed by treatment with vehicle or the PDE1 inhibitor IC86340 (30 μmol/L) and Ang II (100 nmol/L) stimulation for 48 h, and normalized to total protein levels. b Collagen synthesis in cardiac fibroblasts transduced with Ad-Lacz shRNA or PDE1A shRNA for 48 h followed by Ang II stimulation, and normalized to total protein levels. Values represent mean ± SD of triplicates from n = 3 independent experiments. *P < 0.05 versus Ang II or LacZ shRNA + Ang II. c Rap1 activity determined using GST-RalGDS-RBD to pulldown Rap1-GTP in cardiac fibroblasts stimulated with Ang II (100 nmol/L) for 24 h prior to treatment with PDE1 inhibitor IC86340 (30 μmol/L) for the indicated times. Rap1-GTP levels shown below each blot were quantitated by densitometry in a linear range and normalized to total Rap1 levels. Values (normalized to serum-free control) are averaged from two independent experiments