HTRF cGMP Assays

The cGMP accumulation assays in HPASMCs followed standard protocols. Homogeneous time-resolved fluorescence (HTRF) cGMP assay was performed according to the cGMP kits (Cisbio). HPASMCs were resuspended in PBS containing 1 mM PDE-5 inhibitor 3-isobutyl-1-methylxanthine (IBMX) and 0.2% BSA at 1 × 10⁵ cells/ml with or without 10 μM sGC inhibitor 1H-[1,2,4]oxadiazolo [4,3-a] quinoxalin-1-one (ODQ), and allocated into 384-well plates (HTRF^®) at 5 μl/well. Test compounds were solubilized to 100 mM in DMSO and serially diluted by the diluent of the cGMP kits to achieve a 2 × stock, which was diluted using 10-fold dilutions to produce a 6-point dose-response curve with a top concentration of 200 μM. Diluted compounds were then transferred to a triplicate set of assay plates (5 μl/well). After 1 hour incubation, 5 μl cGMP-d2 reagent diluted in lysis buffer was added to each well followed by 5 μl europium cryptate reagent. Next, the plates were mounted and incubated for 1 hour before reading on an HTRF^® compatible reader (Bio Tek, United States).