PDE2A activity assay.

SG from WKY rats and SHRs or from humans were dissected and rapidly frozen in liquid nitrogen. PDE activity was measured using the PDE Activity Assay Kit (Colorimetric) (Abcam, ab139460) according to the manufacturer’s instructions. Briefly, tissues were homogenized in lysis buffer and protease inhibitor cocktail (Sigma-Aldrich), centrifuged at 9,600 g (4°C, 10 minutes) in a microfuge. Protein concentration was quantified by Bradford assay; 7–15 μg of protein was used per sample. Samples were desalted using 0.5-ml Zeba Spin Desalting Columns (Thermo Fisher Scientific, 89882) to remove endogenous free phosphate. Samples were assayed in a reaction mixture (total volume, 50 μl/well) containing 200 μmol/l cGMP substrate, 5′-nucleotidase (50 kU/well), and PDE enzyme (20 mU/well) for 17 minutes at 30°C. The reaction was terminated by addition of Green Assay Reagent (100 μl/well). After incubation at room temperature for 25 minutes, the phosphate-dependent color reaction was measured by reading OD_620nm in a microplate-reading spectrophotometer. Individual PDE activities were measured in the absence of the inhibitor as well as in the presence of 1 μmol/l Bay 60-7550, a highly specific PDE2A inhibitor. PDE2A activity is shown as pmol 5′-GMP produced per milligram of protein per minute inhibited by Bay 60-7550.

To judge the contribution of PDE2A to cAMP hydrolysis in SG tissues of 4-week-old WKY rats and SHRs, the same protocol as above was performed except 4–7 μg of protein per sample was used, and assayed in 200 μmol/l cAMP (instead of cGMP) substrate plus 1 μM cGMP as an allosteric activator, and incubated for 10 minutes at 30°C.