Figure 2

Roles of endothelin receptor subtypes in mediating ET-1-induced activation of ERK1/2 in HASMCs. Serum-starved cells were stimulated with S6c for 5, 10 or 15 min or ET-1 for 10 min. 5 μM of BQ123, 5 μM of BQ788, 5 μM or 10 μM of bosentan were given for 30 min before addition of ET-1. A, representative autoradiograph of western blot showing the phosphorylated ERK1/2 and total ERK1/2 from samples treated with 100 nM of S6c at different time points. B, bar graph shows time-dependent activation of ERK1/2 by 1 μM of S6c. Phosphorylated ERK1/2 was determined by immunofluorescence with an anti-phospho-ERK1/2 antibody. C, bar graph shows inhibitory effects of ET receptor inhibitors on phosphorylated ERK1/2 induced by 10 nM of ET-1. Phosphorylated ERK1/2 was determined by immunofluorescence with an anti-phospho-ERK1/2 antibody. D, inhibitory effects of ET receptor inhibitors on phosphorylated ERK1/2 activity induced by 10 nM of ET-1. Phosphorylated ERK1/2 activity was determined by phosphoELISA assay as described in Methods. The upper panels of B and C indicate representative images of immunofluorescence showing the phosphorylated ERK1/2 from samples treated with S6c at different time points and treated with ET receptor inhibitors prior to addition of ET-1, respectively. The scale bar in each image represents 20 μm. Data represent mean ± S.E.M. *** p < 0.001 compared with the vehicle value (B). ** p < 0.01, *** p < 0.001 compared with the ET-1-stimulated states after DMSO treatment (C,D). # p < 0.05, ## p < 0.01, ### p < 0.001; p = phosphorylated; t = total, ns = non-significant.