Effects of Y-27632 on acetylcholine-induced contraction of intact and permeabilized intrapulmonary bronchial smooth muscles in rats

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Abstract

In the present study, the effects of a selective Rho-associated coiled-coil forming protein kinase (ROCK) inhibitor, Y-27632 [(+)-(R)-trans-4-(1-aminoethyl)-(4-pyridyl)cyclohexanecarboxamide dihydrochloride] on acetylcholine-induced contraction and Ca2+ sensitization of rat bronchial smooth muscle were examined. Intact and β-escin-permeabilized muscles of the third branch of intrapulmonary bronchi were used. In intact muscles, Y-27632 (10−6–10−4 M) concentration-dependently inhibited acetylcholine-induced contractile responses. In acetylcholine (10−3 M)-precontracted intact muscles, the maximal relaxation (about 50% inhibition of contraction) was obtained by a concentration of 10−4 M Y-27632, which had no effect on the resting tone. In β-escin-permeabilized muscles, addition of acetylcholine (10−5–10−3 M) plus GTP (100 μM) induced a further contraction, i.e., Ca2+ sensitization at a constant Ca2+ concentration of pCa=6.0. The acetylcholine-induced Ca2+ sensitization was completely blocked in the presence of 10−4 M Y-27632, whereas the Ca2+-induced contraction itself was not affected by Y-27632. Immunoblot study revealed the expression of ROCK-I and ROCK-II proteins in the intrapulmonary bronchi of rats. These findings suggest that Y-27632 dilates acetylcholine-mediated contraction of rat bronchial smooth muscle by inhibiting RhoA/ROCK-mediated Ca2+ sensitization.

Introduction

In general, smooth muscle contraction has been thought to be induced by an increase in cytosolic Ca2+ via the activation of plasma membrane Ca2+ channels and/or Ca2+ release from sarcoplasmic reticulum. However, additional mechanisms have been suggested in agonist-induced smooth muscle contraction by studies which used the simultaneous measurements of force development and intracellular Ca2+ concentration (Sato et al., 1988), and chemically permeabilized preparations (Fujita et al., 1995) in various types of smooth muscles including airways Ozaki et al., 1990, Chiba et al., 1999b. It has been demonstrated that agonist stimulation increases myofilament Ca2+ sensitivity in β-escin-permeabilized smooth muscles of the rat coronary artery (Satoh et al., 1994), guinea pig vas deferens (Fujita et al., 1995), canine trachea (Bremerich et al., 1997) and rat bronchus (Chiba et al., 1999b). Although the detailed mechanism is not fully understood, a participation of Rho protein, a monomeric GTP binding protein, in the agonist-induced Ca2+ sensitization has been suggested by many investigators (e.g., Fujita et al., 1995, Otto et al., 1996, Gong et al., 1997, Chiba et al., 1999b).

Recently, several target proteins have been identified as effectors of RhoA, including Rho-associated coiled-coil forming protein kinase-I (ROCK-I) (Narumiya et al., 1997) and ROCK-II (Nakagawa et al., 1996). The importance of ROCKs in the induction of smooth muscle Ca2+ sensitization has been demonstrated in various types of smooth muscle Fu et al., 1998, Iizuka et al., 1999, Yoshii et al., 1999, Swärd et al., 2000, Yamagata et al., 2000 by using a selective ROCK inhibitor, (+)-(R)-trans-4-(1-aminoethyl)-(4-pyridyl)cyclohexanecarboxamide dihydrochloride (Y-27632; Uehata et al., 1997). The RhoA/ROCK-mediated Ca2+ sensitization has also been reported in airway smooth muscle Iizuka et al., 1999, Yoshii et al., 1999, Yamagata et al., 2000. In permeabilized rabbit tracheal smooth muscle, carbachol (in the presence of GTP)- and guanosine-5′-O-(3′-thiotriphosphate) (GTPγS)-induced contractions at a constant Ca2+ concentration, which are mediated by Ca2+ sensitization, were completely inhibited by Y-27632 Iizuka et al., 1999, Yoshii et al., 1999. Furthermore, in intact (nonpermeabilized) rabbit tracheal smooth muscle, Y-27632 completely reversed carbachol-induced contraction (Yoshii et al., 1999). Similar results have also been reported using human bronchial smooth muscle Yoshii et al., 1999, Yamagata et al., 2000. Thus, it is suggestive that inhibition of RhoA/ROCK pathway may become a new strategy to treatment of bronchial asthma.

We recently revealed the existence of acetylcholine-induced Ca2+ sensitization in rat bronchial smooth muscle contraction by using β-escin-permeabilized muscle strips (Chiba et al., 1999b). The acetylcholine-induced Ca2+ sensitization was completely blocked by treatment with C3 exoenzyme, indicating an involvement of RhoA in the acetylcholine-induced Ca2+ sensitization of rat bronchial smooth muscle. However, there is no information about the downstream pathway of RhoA in bronchial smooth muscle of rats. Moreover, it is of interest whether the RhoA/ROCK system is inherent in smooth muscle over species. Therefore, in the present study, the effects of Y-27632 on acetylcholine-induced contractile response of rat intrapulmonary small bronchi were examined to elucidate the downstream pathway of acetylcholine-mediated Ca2+ sensitization of bronchial smooth muscle contraction in rats.

Section snippets

Tissue preparation

Male Wistar rats (170–190 g, specific pathogen-free) were used. Animals were killed by exsanguination from the abdominal aorta under anesthetization by chloral hydrate (400 mg/kg, i.p.). The third branch of the intrapulmonary bronchus was isolated by the method described previously (Chiba et al., 1999b). In brief, the tissue was carefully cleaned of lung parenchyma and adhering connective tissue, and then cut into ring strips (about 200 μm width, 500 μm diameter). The epithelium was removed by

Results

In intact (nonpermeabilized) intrapulmonary bronchial smooth muscle of rat that was precontracted by 10−3 M acetylcholine (which induced the maximal contraction), cumulatively applied Y-27632 (10−6–10−4 M) induced a concentration-dependent relaxation (Fig. 1). The plateau responses induced by respective concentrations of Y-27632 were reached 15–20 min after the application. The effective concentration of Y-27632 was 10−5–10−4 M and no further relaxation was observed at the concentration of 10−3

Discussion

We previously reported the existence of acetylcholine-induced Ca2+ sensitization in rat intrapulmonary bronchial smooth muscle contraction, which was sensitive to C3 exoenzyme (Chiba et al., 1999b). In the present study, the acetylcholine-induced Ca2+ sensitization of β-escin-permeabilized muscle was also inhibited by Y-27632, a selective inhibitor of ROCKs (Uehata et al., 1997). It is, therefore, suggested that like other airway smooth muscles Iizuka et al., 1999, Yoshii et al., 1999, Yamagata

Acknowledgements

We thank Tetsuro Uchida and Shintaro Sakurai for their help in technical assistance. We also thank WelFide (Yoshitomi Pharmaceutical Industries) for generous gift of Y-27632.

This work was supported by a Grant-in-Aid for Encouragement of Young Scientists from the Ministry of Education, Science, Sports and Culture of Japan and by a grant from the Pharmacological Research Foundation, Tokyo.

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