Figure 5
Sildenafil (SIL) inhibits the effects of ET-1 on calcineurin/NFATc4 activation and TRPC6 expression in PASMCs. (a) PASMCs were stimulated with ET-1 (100 nM) for 30 min with or without pre-treatment of cells with sildenafil (1 μM for 30 min) or sildenafil plus Rp-8Br-cGMPs (25 μM), calcineurin activity was determined using a calcineurin assay kit (n = 4 each group). (b) PASMCs were stimulated with ET-1 (100 nM) for 3 h with or without pre-treatment of cells with sildenafil (1 μM for 30 min) or sildenafil plus Rp-8Br-cGMPs (25 μM), phosphorylation of NFATc4 was measured by immunoblotting, β-actin served as a loading control (n = 4 each group). (c) PASMCs were stimulated with ET-1 (100 nM) for 3 h with or without pre-treatment of cells with sildenafil (1 μM for 30 min) or sildenafil plus Rp-8Br-cGMPs (25 μM), subcellular localization of NFATc4 was determined using immunofluorescence staining. Cells were stained with a primary antibody for NFATc4 and an Alexa Fluor 488-conjugated secondary antibody. Cell nuclei were stained with DAPI. (d) PASMCs were stimulated with ET-1 (100 nM) for 3 h with or without pre-treatment of cells with sildenafil (1 μM for 30 min) or sildenafil plus Rp-8Br-cGMPs (25 μM), protein level of NFATc4 in the cytoplasmic and nuclear fractions were determined with immunoblotting. Lamin B and β-actin served as loading controls for the nuclear and cytoplasmic fractions, respectively (n = 4 each group). (e) PASMCs were stimulated with ET-1 (100 nM) for 24 h with or without pre-treatment of cells with sildenafil (1 μM for 30 min) or sildenafil plus Rp-8Br-cGMPs (25 μM), TRPC6 mRNA level was determined using qRT-PCR, β-actin served as an internal control (n = 4 each group). (f) PASMCs were stimulated with ET-1 (100 nM) for 48 h with or without pre-treatment of cells with sildenafil (1 μM for 30 min) or sildenafil plus Rp-8Br-cGMPs (25 μM), TRPC6 protein level was determined using immunoblotting, β-actin served as a loading control (n = 4 each group). *P < 0.05 versus control, #P < 0.05 versus ET-1-treated cells, ‡P < 0.05 versus sildenafil and ET-1 co-treated cells.
