Relaxation of contracted rabbit tracheal and human bronchial smooth muscle by Y-27632 through inhibition of Ca2+ sensitization

The mechanism of Ca2+ sensitization of contraction has not been elucidated in airway smooth muscle (SM), To determine the role of a small G protein, rhoA p21, and its target protein, rho-associated coiled coil-forming protein kinase (ROCK), in receptor-coupled Ca2+ sensitization of airway SM, we studied the effect of(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexane carboxamide dihydrochloride, monohydrate (Y-27632), a ROCK inhibitor, on isometric contractions in rabbit tracheal and human bronchial SM. Y-27632 completely reversed 1 mu M carbachol (CCh)-induced contraction of intact trachea with a concentration producing half-maximum inhibition of effect (IC50) of 1.29 +/- 0.2 mu M (n = 5). Although 4 beta-phorbol 12,13-dibutyrate (1 mu M)-induced Ca2+ sensitization was relatively resistant to Y-27632 in alpha-toxin-permeabilized trachea, CCh (100 mu M) plus guanosine triphosphate (GTP) (3 mu M)- and guanosine 5'-O-(3'-thiotriphosphate) (10 mu M)-induced contractions were relaxed completely by Y-27632 with IC50 of 1.44 +/- 0.3 (n = 6) and 1.15 +/- 0.3 mu M (n = 6). Endothelin-l (1 mu M) plus GTP (3 mu M)developed force was also reversed by Y-37632 with IC50 of 4.10 +/- 1.1 mu M (n = 6) in the alpha-toxin-permeabilized bronchus. Both the rabbit and human SM expressed rhoA p21, ROCK I, and its isoform ROCK II. Collectively, rho/ROCK-mediated Ca2+ sensitization plays a central role in the sustained phase of airway SM contraction, and selective inhibition of this pathway may become a new strategy to resolve airflow limitation in asthma.