Regulation of airway smooth muscle RhoA/ROCK activities by cholinergic and bronchodilator stimuli

The current study set out to compare the temporal relationships of Rho activity, Rho kinase (ROCK) activity and tone following cholinergic stimulation in the presence and absence of three different bronchodilators. Bovine trachea challenged with a half-maximally effective concentration of carbachol (CCh) was flash-frozen at different times, then assayed for Rho (rhotekin pull-down assay) and ROCK (Western blot; radiometric assay) activities. Rho was activated within 30 s, followed by ROCK (peak at 2 min); both returned to baseline by 20 min, although tone continued to rise over that period. Increasing the concentration of CCh greatly increased the magnitudes and rates of stimulation of Rho, ROCK and tone. These CCh-induced changes were next compared in tissues pre-treated with isoproterenol, salmeterol or the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP). Neither the time course nor the magnitude of Rho-activation were reduced by the beta-agonists; SNAP slowed Rho activation but it did not alter the peak magnitude. These observations were mirrored in ROCK activation and contraction. When tissues were pre-constricted with CCh and then challenged with the bronchodilators, however, all three agonists reversed cholinergically stimulated Rho, ROCK and myosin light chain kinase activities as well as tone. In conclusion, bronchodilators can suppress RhoA and Rho kinase activities, although their major effect appears to be on myosin light chain kinase activity.