Fig. 1.
Cell-permeant exoenzyme C3 transferase inhibits ACh-induced RhoA activation and tension development in tracheal smooth muscle tissues. Tracheal muscle tissues were incubated in C3 transferase, and the effects on RhoA activity and contractile tension in response to ACh were evaluated. RhoA activation was determined by a pull-down assay using glutathione S-transferase (GST)-tagged Rho-binding domain (RBD) region of Rhotekin to precipitate activated RhoA from extracts of unstimulated (US) and 10−5 M ACh-stimulated (ACh) muscle strips. A: immunoblot of precipitates from extracts of 1 US and 4 ACh-stimulated muscle tissues treated with 0–20 μg/ml C3 and probed with RhoA antibody to quantitate activated RhoA. The amount of activated RhoA was greater in extracts of tracheal muscle strips stimulated with 10−5 M ACh and was reduced in extracts of muscle tissues treated with 2.5–20 μg/ml C3 exoenzyme. B: isometric force in response to 10−5 M ACh in 2 tissues before and after treatment with 10 μg/ml exoenzyme C3 transferase or with no treatment. C: treatment of muscle tissues with 2.5–20 μg/ml C3 significantly depressed ACh-stimulated force and RhoA activation. Values for activated RhoA and force in muscles treated with C3 are normalized to values with no C3 (0 C3). *Significantly different compared with ACh-stimulated tissues not treated with C3 (P < 0.05, n = 3–8). Values are means ± SE.