Figure 5.

Role of intracellular Ca²⁺ in mediating ET-1-induced activation of ERK1/2 in HASMCs. Serum-starved cells were treated with 10 nM of ET-1 for 10 min after different treatments. A, L-type Ca²⁺ channel inhibitor nifedipine was treated for 30 min before addition of ET-1, bar graph shows effects of nifedipine at 2 μM and 10 μM on phosphorylated ERK1/2 induced by ET-1. Phosphorylated ERK1/2 was determined by immunofluorescence with an anti-phospho-ERK1/2 antibody. B, 10 μM of KN-62 was given for 30 min before addition of ET-1, bar graph shows effect of KN-62 on phosphorylated ERK1/2 induced by ET-1. C, 5 mM of the Ca²⁺ chelator EGTA was administered 15 min before addition of ET-1, bar graph shows effect of EGTA on phosphorylated ERK1/2 induced by ET-1. D, the cells were treated with 1 μM of thapsigargin with 5 mM of EGTA for 15 min before addition of ET-1, bar graph shows effect of thapsigargin on phosphorylated ERK1/2 induced by ET-1 in the presence of EGTA. E, the treatment of cells with 10 μM of nifedipine, 5 mM of EGTA, or 1 μM of thapsigargin with 5 mM of EGTA before addition of ET-1, bar graph shows effects of the different treatments on phosphorylated ERK1/2 activity induced by ET-1 as determined by the phosphoELISA assay. The upper panels of A, B, C and D indicate representative images of immunofluorescence illustrating the phosphorylated ERK1/2 from samples given the different treatments prior to addition of ET-1. The scale bar in each image represents 20 μm. Data represent mean ± S.E.M. ns = non-significant. p = phosphorylation.