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 microM carbachol (CCh)-induced contraction of intact trachea with a concentration producing half-maximum inhibition of effect (IC50) of 1.29 +/- 0.2 microM (n = 5). Although 4beta-phorbol 12,13-dibutyrate (1 microM)-induced Ca2+ sensitization was relatively resistant to Y-27632 in alpha-toxin-permeabilized trachea, CCh (100 microM) plus guanosine triphosphate (GTP) (3 microM)- and guanosine 5'-O-(3'-thiotriphosphate) (10 microM)-induced contractions were relaxed completely by Y-27632 with IC50 of 1.44 +/- 0.3 (n = 6) and 1.15 +/- 0.3 microM (n = 6). Endothelin-1 (1 microM) plus GTP (3 microM)- developed force was also reversed by Y-27632 with IC50 of 4. 10 +/- 1.1 microM (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.
G protein‐mediated Ca2+ sensitization of airway smooth muscle contraction was investigated with respect to the relative importance of Rho‐associated coiled coil forming protein kinase (ROCK) and protein kinase C (PKC). We examined the effects of Y‐27632, a ROCK inhibitor, and GF 109203X, a PKC inhibitor, on guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS)‐induced contraction in α‐toxin‐ or β‐escin‐permeabilized rabbit trachea.
Although pre‐treatment with Y‐27632 dose‐dependently inhibited GTPγS (10 μM)‐induced Ca2+ sensitization of α‐toxin‐permeabilized trachea, a Y‐27632‐insensitive component (approximately 16% of the maximum contraction) was retained during the early phase of the GTPγS response in the presence of Y‐27632 (100 μM).
GF 109203X (5 μM) abolished 1 μM 4β‐phorbol 12, 13‐dibutyrate (PDBu)‐induced, but only partially inhibited the GTPγS‐induced Ca2+ sensitization. A combination of Y‐27632 (100 μM) and GF 109203X (5 μM) totally abolished the GTPγS response.
GTPγS caused only a small contraction in the absence of Ca2+. Wortmannin (30 μM), a myosin light chain kinase (MLCK) inhibitor, completely inhibited Ca2+‐induced contraction. ATP‐triggered contraction of the strip which had been treated with calyculin A (1 μM), a phosphatase inhibitor, in rigor solutions was markedly slowed by worthmannin (30 μM), but not by Y‐27632 (100 μM), in the presence of GTPγS and Ca2+.
GTPγS, but not PDBu, contracted the β‐escin‐permeabilized trachea in the absence of Ca2+, but the presence of Ca2+‐independent MLCK.
We conclude that ROCK plays a primary role in G‐protein‐mediated Ca2+ sensitization, which requires MLCK activity, with minor contribution of PKC to the early phase of contraction, and PDBu utilizes conventional PKC(s) in airway smooth muscle.
British Journal of Pharmacology (1999) 128, 925–933; doi:
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