Skip to main content
. 2011 Jan 27;300(5):G749–G760. doi: 10.1152/ajpgi.00306.2010

Fig. 5.

Fig. 5.

Actions of ACh on expression and activation of RhoA and ROCK. A: in H508 colon cancer cells, incubation with 100 μM ACh for up to 8 h did not alter expression of RhoA. RhoA expression was determined by immunoblotting proteins from H508 cell lysates after incubation for the times indicated. For each time point, the ratio of signal intensity for RhoA compared with that for β-actin (loading control) was calculated. Immunoblots shown are representative of 3 others. B: chemical inhibitors of RhoA [exoenzyme C3 (ExoC3), 5 μg/ml] and ROCK (Y27632, 75 μM) abolished ACh-induced H508 cell migration. After preincubation with inhibitors, cells were plated at confluence before a linear wound was made. Cells were incubated with or without ACh (100 μM) for an additional 8 h. As described in materials and methods, photomicrographs were taken immediately after a linear wound was made and again 8 h after addition of ACh. **P < 0.01 compared with ACh alone. Cell migration with ACh plus ExoC3 and Y27632 was not significantly different than control migration with vehicle alone. C: time course for ACh-induced activation of RhoA. In H508 cells, RhoA activation was measured using an ELISA-based assay (materials and methods) before (time = 0 min) and at the indicated times after adding ACh (solid bars). Results are expressed as a percentage of the maximal response obtained with 100 μM ACh (5 min). *P < 0.05 compared with basal RhoA activation (0 min). D: ACh-induced RhoA activation was blocked by inhibitors of muscarinic receptor, ERBB1, ERK, and PI3K signaling. After 5 min incubation with test agents, RhoA activation was measured as described in materials and methods. Bar graph shows effects of adding water-soluble (atropine, ExoC3) and methanol-soluble (PD168393, LY294002, U0126) inhibitors to 100 μM ACh. Vehicle (water and 0.5% methanol), atropine (5 μM), and ExoC3 (5 μg/ml) were used as controls. Basal RhoA activation was not altered by PD168393, LY294002, and U0126 (all 10 μM; data not shown) but these inhibitors all attenuated ACh-induced RhoA activation. ***P < 0.001 for 100 μM ACh compared with vehicle alone (either water or 0.5% methanol). Values shown in bar graphs (B–D) represent means ± SE from at least 3 separate experiments.