Fig. 5— Reversal of cholinergically enhanced RhoA, Rho kinase (ROCK), myosin light chain kinase (MLCK) and mechanical activities by bronchodilators Tissues were pre-treated for 20 min with the M2-selective antagonist AFDX-116 (10-6 M), then pre-constricted with carbachol (CCh; 2×10-7 M) for another 20 min before addition of vehicle, isoproterentol (ISO; 10-7 M) or S-nitroso-N-acetylpenicillamine (SNAP; 10-5 M) for 20 min, or with salmeterol (SAL; 10-7 M) for 30 min. Tissues were then flash-frozen and assayed for Rho, ROCK or MLCK activities: these were expressed as a percentage of the activities measured in matched tissues challenged with CCh alone for 40 min. Tone existing immediately prior to flash-freezing was standardised as a percentage of that existing immediately prior to addition of the bronchodilators. Bars indicate the mean changes in enzyme activities or tone induced by ISO, SNAP or SAL. n>5 for all. ANOVA was used to determine whether the decrease from control levels were statistically significant. *: p<0.05; **: p<0.01.