Am. J. Respir. Cell Mol. Biol., Volume 20, Number 6, June, 1999 1190-1200

Relaxation of Contracted Rabbit Tracheal and Human Bronchial Smooth Muscle by Y-27632 through Inhibition of Ca²⁺ Sensitization

Akihiro Yoshii, Kunihiko Iizuka, Kunio Dobashi, Takeo Horie, Takashi Harada, Tsugio Nakazawa, and Masatomo Mori

First Department of Internal Medicine, Faculty of Medicine, School of Medicine; and Faculty of Health Sciences, Gunma University, Maebashi, Gunma, Japan

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 µM carbachol (CCh)-induced contraction of intact trachea with a concentration producing half-maximum inhibition of effect (IC₅₀) of 1.29 ± 0.2 µM (n = 5). Although 4-phorbol 12,13-dibutyrate (1 µM)-induced Ca²⁺ sensitization was relatively resistant to Y-27632 in -toxin-permeabilized trachea, CCh (100 µM) plus guanosine triphosphate (GTP) (3 µM)- and guanosine 5'-O-(3'-thiotriphosphate) (10 µM)-induced contractions were relaxed completely by Y-27632 with IC₅₀ of 1.44 ± 0.3 (n = 6) and 1.15 ± 0.3 µM (n = 6). Endothelin-1 (1 µM) plus GTP (3 µM)- developed force was also reversed by Y-27632 with IC₅₀ of 4.10 ± 1.1 µM (n = 6) in the -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.

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