Figure 3.

ET-1 stimulates OPC migration. Agarose drop assays were used to measure ET-1 (200 nm) effects on OPC migration. A, ET-1 stimulates OPC migration in the presence of PDGF, compared with PDGF alone (*significantly different from control at corresponding time points; Student's t test; **p < 0.01). ET-R pan-antagonist bosentan (2 μm) completely prevented ET-1 effects on OPC migration (PDGF/PDGF + ET-1 + Bos vs PDGF/PDGF + ET-1, significantly different at days 4, 5, and 6; p < 0.01). B, Cell density at different distances from the edge of the drop (expressed as number of cells per unit area) in assays performed for 6 d in the absence (Sato medium) or presence of PDGF (10 ng/ml) and/or ET-1 (200 nm). At 6 d in the presence of PDGF, ET-1 significantly increased the number of cells migrating out of the agarose drop (Student's t test, p < 0.05). Data are means ± SEM of two independent experiments performed in triplicate. C, D, Staining of the agarose drop assay after 48 h with A2B5 (green) and anti-Olig2 antibodies (red) and counterstained with DAPI (blue). Most cells were A2B5⁺ and Olig2⁺, and remaining cells were O4⁺ (data not shown). ET-1 stimulated cell migration out of the drop without changing relative proportion of A2B5⁺ or Olig2⁺ cells (>95% A2B5⁺/Olig2⁺ cells under both conditions). E, Effects of ET_A-R antagonist JKC-301 (1 μm) and ET_B-R antagonist IRL-1038 (1 μm) on OPC migration were determined in the presence of ET-1 (200 nm) and PDGF (10 ng/ml). ET-1 stimulated OPC migration in the presence of PDGF, compared with control (PDGF alone). At 6 d, ET_A-R and ET_B-R antagonists JKC-301 and IRL-1038 (1 μm) significantly inhibited ET-1-stimulated migration (34 ± 5 and 75 ± 4%, respectively; Student's t test, *p < 0.05, **p < 0.01). F, ET_A-R and ET_B-R antagonists JKC-301 and IRL-1038 (both 1 μm) inhibited effects of ET-1 on P-ERK phosphorylation. Student's t test, **p < 0.01. Equal loading in Western blot was demonstrated by detection of total ERK.