Airway smooth muscle tone increases actin filamentogenesis and contractile capacity.

Force adaptation of airway smooth muscle (ASM) is a process whereby the presence of tone (i.e., a sustained contraction) increases the contractile capacity. For example, tone has been shown to increase airway responsiveness in both healthy mice and humans. The goal of the present study is to elucidate the underlying molecular mechanisms. The maximal force generated by mouse tracheas was measured in response to 10-4 M of methacholine following a 30-min period with or without tone elicited by the EC30 of methacholine. To confirm the occurrence of force adaptation at the cellular level, traction force generated by cultured human ASM cells was also measured following a similar protocol. Different pharmacological inhibitors were used to investigate the role of Rho-associated coiled-coil containing protein kinase (ROCK), protein kinase C (PKC), myosin light chain kinase (MLCK), and actin polymerization in force adaptation. The phosphorylation level of the regulatory light chain (RLC) of myosin, the amount of actin filaments, and the activation level of the actin-severing protein cofilin were also quantified. Although ROCK, PKC, MLCK, and RLC phosphorylation was not implicated, force adaptation was prevented by inhibiting actin polymerization. Interestingly, the presence of tone blocked the activation of cofilin in addition to increasing the amount of actin filaments to a maximal level. We conclude that actin filamentogenesis induced by tone, resulting from both actin polymerization and the prevention of cofilin-mediated actin cleavage, is the main molecular mechanism underlying force adaptation.

Assessment of Respiratory Function in Conscious Mice by Double-chamber Plethysmography.

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to non-invasively assess some features of the respiratory function in humans as well as in laboratory animals. The present article focuses on the application of the double-chamber plethysmography (DCP) in small animals. It provides background information on the methodology as well as a detailed step-by-step procedure to successfully assess respiratory function in conscious, spontaneously breathing animals in a non-invasive manner. The DCP can be used to monitor the respiratory function of multiple animals in parallel, as well as to identify changes induced by aerosolized substances over a chosen time period and in a repeated manner. Experiments on control and allergic mice are used herein to demonstrate the utility of the technique, explain the associated outcome parameters, as well as to discuss the related advantages and shortcomings. Overall, the DCP provides valid and theoretically sound readouts that can be trusted to evaluate the respiratory function of conscious small animals both at baseline and after challenges with aerosolized substances.

Repeated airway constrictions in mice do not alter respiratory function.

It is suggested that the frequent strain the airways undergo in asthma because of repeated airway smooth muscle (ASM)-mediated constrictions contributes to airway wall remodeling. However, the effects of repeated constrictions on airway remodeling, as well as the ensuing impact of this presumptive remodeling on respiratory mechanics, have never been investigated in subjects without asthma. In this study, we set out to determine whether repeated constrictions lead to features that are reminiscent of asthma in mice without asthma. BALB/c mice were subjected to a 30-min constriction elicited by aerosolized methacholine every other day over 6 wk. Forty-eight hours after the last constriction, the mechanics of the respiratory system was evaluated at baseline and in response to incremental doses of nebulized methacholine with the flexiVent. The whole-lung lavages, the tracheas, and the lungs were also collected to evaluate inflammation, the contractile capacity of ASM, and the structural components of the airway wall, respectively. The resistance and the compliance of the respiratory system, as well as the Newtonian resistance and the resistive and elastic properties of the lung tissue, were not affected by repeated constrictions, both at baseline and in response to methacholine. All the other examined features also remained unaltered, except the number of goblet cells in the epithelium and the number of macrophages in the whole-lung lavages, which both increased with repeated constrictions. This study demonstrates that, despite causing goblet cell hyperplasia and a mild macrophagic inflammation, repeated constrictions with methacholine do not lead to structural changes that adversely impact the physiology. NEW & NOTEWORTHY Repeated airway constrictions led to signs of remodeling that are typically observed in asthma, which neither altered respiratory mechanics nor the contractile capacity of airway smooth muscle. These findings shed light on a debate between those claiming that constrictions induce remodeling and those convinced that methacholine challenges are harmless. Insofar as our results with mice relate to humans, the findings indicate that repeated challenges with methacholine can be performed safely.

CD34 Differentially Regulates Contractile and Noncontractile Elements of Airway Reactivity.

Airway hyperresponsiveness (AHR), a major hallmark of asthma, results from alterations of contractile and noncontractile elements of airway reactivity. CD34 is a sialomucin that is expressed on various cells involved in asthma, such as eosinophils and airway smooth muscle precursors, highlighting its potential influence in AHR. To study the role of CD34 in regulating the contractile and noncontractile elements of AHR, AHR was induced by chronic exposure to house dust mite (HDM) antigen. To assess the role of CD34 on the contractile elements of AHR, airway reactivity and airway smooth muscle contractility in response to methacholine were measured. To assess CD34's role in regulating the noncontractile elements of AHR, a chimeric mouse model was used to determine the impact of CD34 expression on inflammatory versus microenvironmental cells in AHR development. Extracellular matrix production, mucus production, and mast cell degranulation were also measured. Whereas wild-type mice developed AHR in response to HDM, a loss of airway reactivity was observed in Cd34-/- mice 24 hours after the last exposure to HDM compared with naive controls. This was reversed when airway reactivity was measured 1 week after the last HDM exposure. Additionally, mast cell degranulation and mucus production were altered in the absence of CD34 expression. Importantly, simultaneous expression of CD34 on cells originating from the hematopoietic compartment and the microenvironment was needed for expression of this phenotype. These results provide evidence that CD34 is required for AHR and airway reactivity maintenance in the early days after an inflammatory episode in asthma.

An in vitro study examining the duration between deep inspirations on the rate of renarrowing.

The factors altering the bronchodilatory response to a deep inspiration (DI) in asthma are important to decipher. In this in vitro study, we investigated the effect of changing the duration between DIs on the rate of force recovery post-DI in guinea pig bronchi. The airway smooth muscle (ASM) within the main bronchi were submitted to length oscillation that simulated tidal breathing in different contractile states during 2, 5, 10 or 30min prior to a larger length excursion that simulated a DI. The contractile states of ASM were determined by adding either methacholine or isoproterenol. Irrespective of the contractile state, the duration between DIs neither affected the measured force during length oscillation nor the bronchodilator effect of DI. Contrastingly, the rate of force recovery post-DI in contracted state increased as the duration between DIs decreased. Similar results were obtained with contracted parenchymal strips. These findings suggest that changing the duration between DIs may alter the rate of ASM force recovery post-DI and thereby affect the rate of renarrowing and the duration of the respiratory relief afforded by DI.

Airway smooth muscle tone increases airway responsiveness in healthy young adults.

Force adaptation, a process whereby sustained spasmogenic activation (viz., tone) of airway smooth muscle (ASM) increases its contractile capacity, has been reported in isolated ASM tissues in vitro, as well as in mice in vivo. The objective of the present study was to assess the effect of tone on airway responsiveness in humans. Ten healthy volunteers underwent methacholine challenge on two occasions. One challenge consisted of six serial doses of saline followed by a single high dose of methacholine. The other consisted of six low doses of methacholine 5 min apart followed by a higher dose. The cumulative dose was identical for both challenges. After both methacholine challenges, subjects took a deep inspiration (DI) to total lung capacity as another way to probe ASM mechanics. Responses to methacholine and the DI were measured using a multifrequency forced oscillation technique. Compared with a single high dose, the challenge preceded by tone led to an elevated response measured by respiratory system resistance (Rrs) and reactance at 5 Hz. However, there was no difference in the increase in Rrs at 19 Hz, suggesting a predominant effect on smaller airways. Increased tone also reduced the efficacy of DI, measured by an attenuated maximal dilation during the DI and an increased renarrowing post-DI. We conclude that ASM tone increases small airway responsiveness to inhaled methacholine and reduces the effectiveness of DI in healthy humans. This suggests that force adaptation may contribute to airway hyperresponsiveness and the reduced bronchodilatory effect of DI in asthma.