Assay for PDE5 activity.

PDE5 activity was measured in immunoprecipitates of PDE5 by the method of Wyatt et al. (51), as previously described (33). PDE5 was immunoprecipitated from lysates of dispersed rabbit colonic muscle cells (3 × 10⁶ cells/ml) by using an anti-PDE5 antibody, and the immunoprecipitates were washed in a solution of 50 mM Tris·HCl (pH 7.5), 200 mM NaCl, and 5 mM EDTA. The immunoprecipitates were then incubated for 15 min at 30°C in a reaction mixture containing 100 mM Mes (pH 7.5), 10 mM EDTA, 0.1 M magnesium acetate, 0.9 mg/ml BSA, 20 µM cGMP, and [³H]cGMP. The samples were boiled for 3 min, chilled, and then incubated at 30°C for 10 min in 20 mM Tris·HCl (pH 7.5) containing 10 µl of Crotalus atrox snake venom (10 µg/µl). The samples were added to DEAE-Sephacel A-25 columns, and the radioactivity in the effluent was counted. The results were expressed as counts per minute per milligram of protein.