Fig. 2.
Expression of RhoA T19N inhibits ACh-induced RhoA activation and tension development in tracheal smooth muscle tissues. Hemagglutinin (HA)-RhoA T19N was expressed in muscle tissues, and the effects on ACh-induced contraction and RhoA activity were evaluated. A: immunoblots against RhoA from extracts from untreated and HA-RhoA T19N-transfected tissues. B: RhoA activation was determined by a pull-down assay using GST-tagged RBD region of Rhotekin to precipitate activated RhoA from extracts of unstimulated and ACh-stimulated muscle strips. Immunoblot against RhoA in activated RhoA precipitates from extracts of tissue expressing RhoA T19N or sham-treated tissues is shown. C: RhoA activity increased significantly in response to 10−5 M ACh stimulation in sham-treated tissues and was significantly inhibited in tissues expressing RhoA T19N. Values are means ± SE (n = 8, P < 0.05). Values for activated RhoA are normalized to values for ACh-stimulated sham-treated muscles. *Significant difference between ACh-stimulated tissues treated with RhoA T19N and sham-treated ACh-stimulated tissues. There were no significant differences in values for unstimulated tissues. D: isometric force in response to 10−5 M ACh in 2 tissues before and after treatment with RhoA T19N or sham treatment. E: RhoA T19N significantly inhibited contractile force in response to 5-min stimulation with 10−5 M ACh relative to sham-treated smooth muscle. Values for force are normalized to values for sham-treated muscles. *Significant difference between tissues treated with RhoA T19N and sham-treated tissues with ACh stimulation (n = 8, P < 0.05). Values are means ± SE.