Fig. 5.

MR-L2 treatment-mediated MDCK cyst suppression is independent of agonist drive and suppresses PGE2-mediated CFTR activation and membrane depolarization. (A) Forskolin treatment drives cyst formation and expansion in a concentration-dependent manner. Activator compound MR-L2 (3 µM) suppressed forskolin-induced cyst formation. Data are displayed as mean ± SEM of all cysts measured in each experimental condition. (B) PGE2-stimulated MDCK cyst formation was also suppressed by PDE4 long-form activation with 3 µM MR-L2. Data are displayed as mean ± SEM of all cysts measured in each experimental condition. (C) MDCK cells exhibit a concentration-dependent membrane depolarization in response to PGE2-mediated CFTR activation using a fluorescent dye-based assay for membrane potential. Individual fluorescence traces over time are shown for each PGE2 concentration. T1 highlights the time point at which PGE2 was added. (D) Cells were preincubated (40 min) with concentrations of the PDE4 long-form activator compound MR-L2, and then challenged with 100 nM PGE2. Increasing concentrations of MR-L2 suppress the PGE2-stimulated increase in fluorescence, indicating that activation of PDE4 is sufficient to dampen the cAMP-driven activation of the CFTR. (E and F) Cyst assays were conducted in parallel with CFTR assessment. When MDCK cells are stimulated to form cysts with either 300 nM PGE2 (E) or 100 nM PGE2 (F), the resulting concentration-dependent suppression of cystic expansion by MR-L2 (black bars, shown as percentage of PGE2 alone; error bars are SEM of all cysts measured in each treatment) closely follows the down-regulation of CFTR-mediated membrane depolarization (gray bars, fold change in fluorescence).