INTRODUCTION

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The ability of airways to narrow excessively during airway smooth-muscle (ASM) contraction is characteristic of asthmatic airways. A number of possible mechanisms could cause excessive airway narrowing. A primary abnormality of ASM, such as increased contractility and/or mass, could cause excessive narrowing by increasing the force that is generated during activation. Alternatively, a decrease in the load on ASM could allow excessive shortening and airway narrowing.

Recently, it has been suggested that a fundamental difference in the response of ASM to the dynamic loading caused by tidal stretch and/or deep inspiration (DI) may be the basis of the excessive airway narrowing in asthma. Skloot and colleagues (1) and Moore and coworkers (2) showed that in normal subjects, prohibition of DI during the course of generation of a methacholine dose-response curve caused excessive airway narrowing and an ineffectual reversal of bronchoconstriction when DIs were ultimately allowed. These results mimic altered airway function in asthmatic individuals, in whom impaired bronchodilation in response to DI accompanies excessive airway narrowing (3).

The dynamic response of ASM to externally applied stress could be impaired for a number of reasons. A reduced load against which ASM acts may alter the DI response in asthmatic patients. Unlinking of airway-parenchymal interdependence would reduce the amplitude of peribronchial pressure swings and/or might make peribronchial pressure less negative (6). Reduced airway wall compliance would also have a similar effect. Impaired pulmonary surfactant function could increase surface tension on the airway mucosal surface and add to the forces that favor airway narrowing. A reduced load on ASM, a noncompliant airway wall, or increased mucosal surface tension could all reduce the cyclical stress applied to ASM during tidal breathing, which could alter the contractile properties of ASM (7).

It has been shown that the mechanics and biochemistry of ASM contraction change during the course of stimulation. Early in ASM stimulation, there is rapid cycling of crossbridges and a rapid velocity of shortening, but the muscle is relatively compliant. Later in contraction there is less rapid cycling of crossbridges but the muscle is stiffer, presumably owing to a greater percentage of attached crossbridges. Facilitation of this process, which has been termed the "latch bridge state," has been suggested as a mechanism by which excessive airway narrowing and deficient response to DI could develop in asthmatic subjects (7). Recently, a number of investigators have suggested alternate or complementary mechanisms by which the length- and time-dependent behavior of ASM could change. Gunst and associates showed that ASM was stiffer when contracted at shorter lengths (8), and Ford and colleagues have shown that ASM exhibits plasticity, adapting by generating greater force at shorter lengths after repeated stimulation.

To characterize the time course of the adaptation of ASM to prohibition of DI, we repeatedly challenged normal subjects, using a constant dose of methacholine and allowing DI after from one to five doses.

	METHODS

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Subjects

Ten subjects (five males and five females) were recruited from the staff of St. Paul's Hospital and the University of British Columbia, and written informed consent was obtained from them. The study was approved by the Ethical Review Committees of St. Paul's Hospital and the University of British Columbia. All subjects denied a history of chronic respiratory disease, regular medication use, and acute or chronic respiratory symptoms.

Protocol and Lung Function

Pulmonary function tests and methacholine challenge were performed on five separate days, at the same time each day. On the first day, complete flow-volume curves were generated and FEV₁ and FVC were measured. Measurements were accepted if two or more values of FEV₁ and FVC were within 5% of each other, and the mean of these values was used as the baseline value. All values were recorded in liters at body temperature-atmospheric pressure-saturated (BTPS), and were expressed as percents of predicted values based on the equations of Crapo and colleagues (9). Five 16-mg/ml doses of methacholine were given every 5 min by nebulizer, for 2 min, and spirometry was conducted 1 min after each dose. On the four subsequent days, two, three, four, or five doses of methacholine were administered; the number of doses on a given day was chosen randomly. The administration of methacholine was done in an identical manner to that on the first day. However, flow-volume curves were generated only at baseline and after the last dose of methacholine, when at least three curves were recorded until two FEV₁ and FVC values were within 5% of each other. During all challenges subsequent to the Day 1 challenge, the subjects were asked not to take any DIs except during the maximal FVC maneuvers at the start and end of the challenge. When required, salbutamol at 200 µg was administered at the end of the protocol by metered dose inhaler, using a spacer device, to relieve bronchoconstriction.

Data Analyses

Baseline predicted FEV₁ and FVC were calculated for each subject as the mean of all baseline values from each challenge. The decrease in FEV₁ and FVC on Day 1 was compared with the decrease on subsequent days after the same number of doses of methacholine. Any differences in the magnitude of decrease could then be attributable to the absence of DI. Paired t tests were used to compare the decrease in FEV₁ and FVC on Day 1 with the decrease on the subsequent days after the same dose of methacholine. The results were considered significant if p < 0.05. Data are presented as the mean ± SEM unless otherwise specified.

	RESULTS

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Baseline spirometric parameters were normal in all subjects, as shown in Table 1. Baseline values also did not differ between study days. All subjects were able to receive five doses of methacholine during the Day 1 challenge. Subjects 4 and 5 were only able to receive three and four doses of methacholine, respectively, on subsequent day challenges, owing to discomfort associated with large decreases in FEV₁ (of 68% and 65% of baseline, respectively) which occurred after these doses. The other subjects completed all subsequent challenges.

Figure 1 shows the decreases in FEV₁ and FVC during Day 1 and subsequent challenge days. On Day 1 the maximal decreases in FEV₁ and FVC were 10 ± 1.5% and 3 ± 1.7%, respectively. On the subsequent study day challenges, the maximal decreases in FEV₁ and FVC were 28 ± 6.0% and 18 ± 6.7%, respectively. The decreases in FEV₁ after three or more doses of methacholine were significantly greater when DIs were prohibited (p < 0.01), and also plateaued after three doses. The decrease in FVC was also greater during the challenges without DIs (p  0.05 for Doses 3, 4, and 5).

View larger version (15K): [in this window] [in a new window]   Figure 1.   Mean (± SEM) percent decrease in FEV1 and FVC after each dose of methacholine. Comparison between challenges with DIs (open circles) and with inhibition of DIs (closed circles) (*p < 0.05).

Figure 2 shows the magnitude of reversal of airway narrowing by three DIs after each dose of methacholine when DIs were taken as compared with when they were not taken. After two and three doses of methacholine, the mean FEV₁ after three DIs was not significantly different for the two types of challenge. After methacholine Doses 4 and 5 without DIs, there was incomplete reversal of airway narrowing; the mean FEV₁ after the three DIs remained less, throughout the challenge, than the mean FEV₁ after the same number of doses when DIs had been taken (p < 0.05). There was a similar pattern of reversal for FVC. The mean FVC after three DIs was significantly smaller after Dose 4 (p < 0.05), although not after Dose 5. The difference in the degree of reversal of narrowing due to absent DIs after Dose 5 may not have been significant because FVC was recorded from only seven subjects after this dose (one subjected terminated the FVC maneuver early, one subject completed only Doses 1 through 3, and another subject completed only Doses 1 through 4).

View larger version (16K): [in this window] [in a new window]   Figure 2.   Mean (± SEM) degree of reversal of airway narrowing with successive DIs after each dose of methacholine. Comparison between challenges with DIs (open circles) and with inhibition of DIs (closed circles) (*p < 0.05).

	DISCUSSION

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The results of this study show that the increased airway narrowing resulting from inhibition of DI during ASM contraction occurs after approximately 10 min, but that the degree of airway narrowing subsequently remains unchanged. The reversal of airway narrowing by DIs, after a period of their absence, becomes incomplete after 15 min, and there is a trend suggesting that the reversal becomes more impaired with increasing duration of inhibition of DI. The FEV₁/FVC ratio decreases when DIs are inhibited for 10 min or more, in comparison with when DIs are taken.

A number of investigators have previously suggested that the primary defect in asthma could be an impairment of the bronchodilator response to DI, rather than enhanced end-organ responsiveness (1, 3, 10). Several possible mechanisms could explain this phenomenon, including changes in ASM contractile function, changes in non-ASM constituents of the airway wall (such as increased thickness and unlinking of parenchymal interdependence), and/or changes in the fluid lining the airway lumen. The possibilities are considered in the following discussion.

ASM plasticity, defined as the ability of ASM to maximize force generation according to existing muscle length, could lead to excessive airway narrowing during agonist challenges in which DIs are prohibited. If ASM were activated and not intermittently lengthened, force generation would increase over time, leading to progressively greater shortening. The reversal of airway narrowing produced by DI after methacholine challenge is likely to be due to force reduction in the ASM. Stretching of the muscle during DI could disrupt the arrangement of the contractile filaments (13) and/or their attachment to the cytoskeleton (8). Thus, one explanation for the greater decrease in FEV₁ during prohibition of DI in our study (Figure 1) is that the contracted ASM adapted to a shorter muscle length at the end of the study protocol. The time course of the decrease in FEV₁ is comparable to that of canine trachealis adaptation after a change in length (13). The time course of FEV₁ recovery with DI (Figure 2) can be regarded as the time course of "unadaptation" of the ASM if the decrease in FEV₁ is attributed to ASM adaptation to shorter muscle lengths. On the basis of experiments by Shen and coworkers (14), it seems that the time course of unadaptation is a function of both the amplitude and frequency of the oscillation in length. The speed and completeness of recovery are time dependent (Figure 2), such that the longer the ASM has been adapted at a certain length, the more difficult it is for the muscle to unadapt to that length. After the fourth dose of methacholine in our study, it seems that the muscle was no longer able to relax completely after three DIs. This raises the interesting question of whether long-term adaptation of ASM at short lengths could cause an irreversible decrease in FEV₁, such as observed in some asthmatic subjects.

An alternative or complementary mechanism for the exaggerated airway narrowing seen during prohibition of DI has been suggested. Slowing of crossbridge cycling rates during ASM contraction in the absence of high-force stretches could increase the stiffness of the muscle, making it more resistant to lengthening during tidal breathing and also to high-force stretches when they are finally applied. Fredberg and associates have postulated that when slowly cycling latch bridges are present, ASM is less responsive to the stretch that occurs during tidal breathing, in that the muscle will lengthen less during tidal stretching (7). This then encourages the further formation of slowly cycling latch bridges, and so on. Results of experiments on ASM in vitro demonstrate that constant force oscillations reduce ASM stiffness and increase the hysteresivity of activated ASM over a period of approximately 15 min (24). Thus, the time course of unadaptation of ASM preparations appears to be similar to that of airway narrowing in vivo.

Non-smooth-muscle components of the airway could also be affected by continued ASM contraction without large-amplitude stretches. During ASM contraction, a decrease in peribronchial interstitial pressure could increase the transudation of fluid into the airway wall; given sufficient time, the accumulated fluid could affect the mechanical function of the airway. Increased airway wall fluid could affect airway function by increasing airway wall thickness, and could thereby reduce the effectiveness of parenchymal interdependence (6, 15). Support for this mechanism of exaggerated airway narrowing has been provided by experimental data from sheep that were challenged with an intravenous infusion of methacholine with or without bradykinin (16). Bradykinin caused an increase in airway wall thickness and was associated with slower reversal of the methacholine-induced airway narrowing.

The load against which ASM contracts has been shown to be an important determinant of airway narrowing since reducing the load by breathing at a slightly lower lung volume abolishes the maximal dose-response plateau in normal subjects (17). The results of the present study suggest that the effect of DIs during ASM activation is also an important determinant of airway narrowing and that with respect to time, rather than dose, there is a plateau. The effect of inhibiting DIs on the dose- response plateau, however, cannot be inferred from these results.

Changes in the distribution and properties of surfactant in the pulmonary surface fluid may also be affected by an absence of DIs. The peripheral airways are more prone to instability and collapse during ASM activation and/or low transpulmonary pressure because of their relatively greater proportion of ASM (18) and their small radius of curvature. Pulmonary surfactant stabilizes alveoli but is also present in peripheral airways, where it reduces surface tension and helps maintain airway caliber (19, 20). Surfactant attenuates airway narrowing at lung volumes above FRC but is relatively ineffective at lower volumes, where ASM is able to shorten enough to cause airway closure (21). In the present study, ASM activation accompanied by prohibition of DI caused a greater decrease in FVC than occurred when DIs were allowed, suggesting greater airway closure under the former condition. During peripheral airway closure, the mucosal fluid coalesces and forms a meniscus (liquid bridge formation) (22) that could require several DIs to overcome; the longer the absence of DIs the more disruption to the surfactant and the less effective DIs would be in restoring airway caliber.

Malmberg and colleagues also examined the time course of the effect of DI on the magnitude of airway narrowing after methacholine inhalation in normal subjects (23). They studied the effect of varying the time before an FEV₁ maneuver after a single dose of methacholine, which was or was not preceded by a DI. They found that performing DIs prior to methacholine inhalation reduced the subsequent amount of airway narrowing for up to 10 min; the shorter the time interval between the methacholine and the FEV₁ maneuver, the greater the attenuation of the airway narrowing.

In our study, the effectiveness of DI in reversing airway narrowing decreased with an increasing duration of prohibition of DI, and this trend suggests that the failure to reverse bronchoconstriction would have continued to worsen if the study had been prolonged (Figure 2). The maximal decrease in FEV₁, however, appeared to plateau at 10 min. The difference in time course of these two effects suggests that there may be differences in the factors that determine maximal airway narrowing and the effectiveness of reversal of narrowing.

In summary, the results of this study confirm that there is an increase in airway narrowing associated with prohibition of DIs during ASM activation, and show that the increased response plateaus after 10 min. The ability of DIs to reverse the airway narrowing becomes impaired at 15 min and may continue to worsen after 20 min. Studies designed to elucidate the mechanisms responsible for this altered airway behaviour in normal subjects may provide insights into the pathophysiologic mechanisms of asthma.