Inhibition of Rho-kinase normalizes nonspecific hyperresponsiveness in passively sensitized airway smooth muscle preparations

https://doi.org/10.1016/j.ejphar.2005.12.043Get rights and content

Abstract

Currently, little is known about mechanisms underlying passive sensitization-induced nonspecific airway hyperresponsiveness. We sought to determine whether the nonspecific airway hyperresponsiveness observed after passive sensitization involves an increased role of Rho-kinase in airway smooth muscle contraction. In addition, the contribution of Rho-kinase to specific allergen-induced airway smooth muscle contraction was studied. Guinea pig tracheal smooth muscle preparations were incubated for 16 h, in the presence of serum obtained from nonsensitized guinea pigs or atopic serum obtained from actively ovalbumin-sensitized guinea pigs. After incubation, the contribution of Rho-kinase to histamine-, methacholine- or ovalbumin-induced isometric contractions was determined, using the specific Rho-kinase inhibitor Y-27632.

Maximal contractions induced by histamine and methacholine were significantly increased in passively sensitized preparations, without a change in potency (− logEC50). In control preparations, Y-27632 reduced the potency of both agonists, without affecting maximal contraction. Remarkably, the increased agonist responsiveness induced by passive sensitization was fully normalized by Y-27632. Treatment with Y-27632 also reduced ovalbumin-induced contraction in these preparations.

This study shows that the nonspecific airway smooth muscle hyperresponsiveness as well as the specific allergen responsiveness induced by passive sensitization are dependent on Rho-kinase. The complete inhibition by Y-27632 of the passive sensitization-induced increased responsiveness toward histamine and methacholine indicates a pivotal role of Rho-kinase in this process.

Introduction

Passive sensitization of airway smooth muscle preparations has successfully been used as a model system to investigate mechanisms of airway hyperresponsiveness (Schmidt and Rabe, 2000). Thus, in nonsensitized human airway smooth muscle preparations it has been demonstrated that pre-incubation with atopic serum not only induced a ‘specific’ contractile response to the allergen, but also enhanced the responsiveness to various ‘nonspecific' stimuli, including histamine, leukotriene C4, contractile neuropeptides and KCl (Black et al., 1989, Marthan et al., 1992, Schmidt et al., 1999, Ben-Jebria et al., 1993). In addition, passive sensitization of human bronchi has been shown to increase maximal shortening velocity and maximal shortening capacity of the smooth muscle in response to electrical field stimulation (Mitchell et al., 1994).

The allergen-induced contractile response is fully dependent on immunoglobulin E (IgE), as no response was observed after incubation with IgE-depleted serum or serum containing anti-IgE-antibodies (Watson et al., 1998, Rabe et al., 1998). In contrast, neither the removal of IgE (Watson et al., 1998), nor the presence of anti-IgE-antibodies (Rabe et al., 1998) affected the nonspecific hyperresponsiveness toward histamine, indicating that other factors present in the serum of allergic asthmatics, possibly cytokines (Schmidt and Rabe, 2000), are likely to be major determinants of the increased airway smooth muscle responsiveness.

Remarkably, little is known about sensitization-induced alterations in signaling mechanisms of contractile agonists. Both in canine (Jiang et al., 1992) and in human airway smooth muscle (Ammit et al., 2000), it has been demonstrated that active allergic sensitization is capable of increasing myosin light chain kinase (MLCK)-content. This increase might be responsible for the enhanced reactivity of sensitized tissue, since agonist-induced airway smooth muscle contraction involves myosin light chain (MLC) phosphorylation induced by Ca2+-calmodulin-mediated activation of MLCK. Increased MLC phosphorylation can also occur as a consequence of Ca2+-sensitization (Fukata et al., 2001, Somlyo and Somlyo, 2003), which involves inhibition of myosin light chain phosphatase (MLCP) by the Rho/Rho-kinase pathway (Yoshii et al., 1999, Ito et al., 2001, Somlyo and Somlyo, 2003, Amrani et al., 2004).

Agonist-induced Rho-mediated Ca2+-sensitization of airway smooth muscle has been found to be increased in repeatedly allergen-challenged rats with airway hyperresponsiveness (Chiba et al., 1999). In addition, the specific Rho-kinase inhibitor Y-27632 suppressed allergen-induced airway hyperresponsiveness in mice repeatedly challenged with ovalbumin after active sensitization in the absence and presence of respiratory syncytial virus infection (Hashimoto et al., 2002). We have recently demonstrated that the process of active allergic sensitization by itself, without subsequent allergen exposure, is sufficient to induce an enhanced role of Rho-kinase in guinea pig airway smooth muscle contraction ex vivo and airway responsiveness in vivo (Schaafsma et al., 2004).

Based on these observations, we hypothesize that the contribution of Rho-kinase to airway smooth muscle contraction after passive sensitization is increased, which could underlie the nonspecific hyperresponsiveness in this model. Therefore, the present study focused on the role of Rho-kinase in the increased responsiveness of passively sensitized guinea pig tracheal smooth muscle preparations to histamine and methacholine. Moreover, the Rho-kinase dependency of ovalbumin-induced contraction in these preparations was investigated as well.

Section snippets

Animals

Outbred specified pathogen-free male Dunkin Hartley guinea pigs (Harlan, Heathfield, U.K.) were used. All protocols described in this study were approved by the University of Groningen Committee for Animal Experimentation.

Active sensitization procedure

Guinea pigs were actively IgE-sensitized to ovalbumin as described previously (Van Amsterdam et al., 1989). In short, 0.5 ml of an allergen solution containing 100 μg/ml ovalbumin and 100 mg/ml Al(OH)3 in saline was injected intraperitoneally, while another 0.5 ml was divided

Effects of Rho-kinase inhibition on passive sensitization-induced airway smooth muscle hyperresponsiveness

No effect of passive sensitization was observed on basal myogenic tone (i.e. the resting tension compared to the maximal relaxation established with isoproterenol) (Fig. 1). In contrast, maximal contraction (Emax) induced by histamine (Fig. 1, A) and methacholine (Fig. 1, B) was significantly increased after passive sensitization, whereas no effect on the potency (− logEC50) of these agonists was observed (Table 1). In control serum-treated preparations a significant decrease in potency of both

Discussion

In the present study, we demonstrated that passive sensitization-induced nonspecific hyperresponsiveness of guinea pig tracheal preparations toward histamine and methacholine, is fully normalized in the presence of Y-27632, indicating a crucial role for Rho-kinase in this process. This finding corresponds well with our previous study, which demonstrated that the contribution of Rho-kinase to contractile airway responsiveness, both in vivo and ex vivo, is augmented after active allergic

Acknowledgements

This work was financially supported by the Netherlands Asthma Foundation, grant NAF 01.83 (D.S.).

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