Figure 9.

ET-1-induced DMR in MRC-5 human lung fibroblasts in absence and presence of bosentan (Bos) or the ET-A (BQ123) or ET-B (BQ788) receptor selective antagonist. Cells were seeded in a density of 15 000 cells per well in 384-well Epic fibronectin-coated microplates with 40 μL of growth medium cultured at 37°C for 20 h to achieve confluent cell layers. Thereafter, cell culture medium was replaced by 30 μL assay buffer per well (HBSS with 20 mM HEPES, pH 7.0), and cells were allowed to rest in assay buffer in absence or presence of antagonists for 2 h in the Epic® reader at 28°C. After addition of 10 μL of ET-1 (final concentrations 0.01–1 μM), DMR responses were monitored for 1 h. Indicated are real-time recordings of ET-1-induced DMR-signals from representative experiments (A, C and E, means ± SEM of three to four measurements run in parallel). ET-1-induced concentration–effect curve (B) resulted from the maximal DMR signals within 1800 s: log IC₅₀ is 7.25 ± 0.11 (ET-1 1 μM, bottom and slope factor are fixed to 100 %, 0% and 1, respectively; n = 5). Height of columns (D and F): maximal DMR signal evoked by 0.3 μM ET-1 in presence of antagonists at the concentrations given, expressed as % of the ET-1 response in absence of antagonists, means ± SEM, n = 3–5. Significance of differences: *P < 0.05; **P < 0.01 versus ET-1 response in absence of antagonists.