Darusentan (Lu-135252) is a potent inhibitor of endothelin signaling and function in both large and small arteries

Briefly, isolated rat aortic smooth muscle cells were incubated overnight in complete media in a 96-well microplate at a density of 50 000 cells/well. The next day, cells were starved for 24 h in 0% serum conditions. Following starvation, medium was removed and cells were dosed with 20 μL of increasing concentrations of darusentan (0, 0.001, 0.01, 0.1, 1, and 10 μmol/L) made in stimulation buffer containing 50 mmol/L LiCl. Cells were incubated at 37 °C, 5% CO2 for 30 min before the addition of 20 μL of ET-1 in dose-response fashion. Cells were incubated in the presence or absence of darusentan and ET-1 at 37 °C, 5% CO2 for 60 min. Following this incubation, cells were lysed using 20 μL of 2.5% lysis solution for 30 min and lysates were transferred to a 96-well microplate pre-coated with goat anti-mouse IgG. After transfer into the ELISA plate, kit-supplied IP1-horseradish peroxidase (HRP) conjugate and anti-IP1 mAb were added to the lysates. Assays were incubated for 3 h at room temperature on a platform shaker. Assay plates were then washed 6 times with 1× ELISA wash solution (250 μL/well) before the addition of HRP substrate TMB (3, 3', 5, 5'-tetramethylbenzidine). After 20 min, the reaction was stopped and the optical density (OD) was measured at 450 nm with a correction of 620 nm. Measurements were performed in triplicate.