INTRODUCTION

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Airway hyperresponsiveness (AHR) is a characteristic feature of asthma; airways narrow excessively in response to inhaled stimuli and do so at lower doses of stimuli. The bronchodilating effect of deep inspiration (DI) is diminished or absent in asthmatics compared with nonasthmatic subjects. This has led to the suggestion that excessive narrowing could be the result of failure of DI to dilate the airways (1).

Skloot and coworkers (2) studied normal subjects who had a decrease in FEV₁ less than 20% during a standard methacholine challenge. When the challenge was repeated and DIs were inhibited throughout the test, the decrease in FEV₁ was greater and was more than 20% in six out of 10 subjects. In previous studies, we have confirmed the studies of Skloot and coworkers and showed that after approximately 10 min of activation of airway smooth muscle (ASM) during which DIs were inhibited, airway narrowing was increased and the bronchodilating effect of DI was impaired in normals (3). We hypothesized that if the bronchodilating effect of DI were impaired in asthmatics, then inhibition of DIs during ASM activation would not be associated with an increase in airway narrowing.

	METHODS

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Subjects

Eight subjects were recruited from among patients attending the Asthma Education Program, St. Paul's Hospital and from recruitment notices. Written informed consent was obtained and the ethics committee of St. Paul's Hospital approved this study. All patients were considered to have a diagnosis of asthma based on World Health Organization/National Heart, Lung, and Blood Institute (WHO/NHLBI) guidelines (4).

Study Protocol and Lung Function

This study was conducted on six separate days but at the same time of day. On the first day, a clinical history was obtained, and routine methacholine challenge was performed (5). Methacholine was administered in doubling doses using a Hudson-Bennett Twin Jet nebulizer every 5 min, for 2 min, and spirometry was measured 1 min after each dose. On the second day, the concentration of methacholine causing a 15% reduction in FEV₁ (PC₁₅) was given every 5 min for 5 doses. Spirometry was measured in triplicate after each dose. On the four subsequent study days, the baseline spirometry was measured, and either 2, 3, 4, or 5 PC₁₅ doses were given, but DIs were prohibited and FEV₁ were not measured until after the final dose had been administered. The order of the number of doses given was randomized. Baseline spirometry required reproducibility to within ± 5%, and the largest FEV₁ was used. All values were recorded in liters BTPS and were expressed as percentage of predicted based on the equations of Crapo and colleagues (6).

Data Analyses

Data are presented as mean ± SEM. We compared the changes in FEV₁ that occurred during inhibition of DIs with the corresponding changes in FEV₁ that occurred when DIs were allowed, using paired t tests, (i.e., we compared the first spirometry in the no DI challenge with the first spirometry of the DI challenge, etc.). The effects of DI on the changes in FEV₁ over all the whole challenge were examined using a repeated measures analysis of variance (ANOVA). Changes were considered significant at p < 0.05.

	RESULTS

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Table 1 shows the subject details. All subjects were nonsmokers except Subject DF who was a smoker at the time of the study. The geometric mean PC₁₅ (95% confidence interval) was 1.6 (0.7 to 3.8) mg/ml. All subjects completed five inhalations of their PC₁₅ dose of methacholine on both the day when DIs were allowed after each dose and also on all days when DIs were prohibited.

Figures 1A and 1B show the decreases in FEV₁ and FVC after each dose of methacholine when DIs were allowed and when DIs were inhibited. There were no differences in baseline FEV₁ between days (p > 0.05, ANOVA). During methacholine administration when DIs were allowed, there was a plateau in the decrease in FEV₁ over the last 3 doses. When DIs were inhibited, there was a larger decrease in FEV₁ compared with when DIs were allowed (p < 0.05, repeated measures ANOVA). For individual doses, the decreases in FEV₁ were significantly different after the fourth and fifth doses (p < 0.05), which were approximately 15 and 20 min after the start of the methacholine challenge respectively. The decrease in FEV₁ when DIs were inhibited did not appear to plateau, suggesting that its effects were cumulative. After Doses 4 and 5, the first two DIs did not completely reverse the decreases in FEV₁ compared with when DIs were allowed after methacholine inhalations. The changes in FVC (Figure 1B) were similar to FEV₁ except the decreases were different only after the fifth dose.

View larger version (13K): [in this window] [in a new window]   Figure 1.   Changes in FEV1 (A) and FVC (B) after methacholine inhalation when DIs were allowed (open circles) compared with when DIs were inhibited (closed circles). Three successive FEV1 measurements are shown after each methacholine dose. Data are from eight subjects; error bars are ± SEM.

These results are similar to those found in an identical study of normal subjects (3), except that the magnitude of decrease in FEV₁ was greater in asthmatic subjects both with and without DIs.

	DISCUSSION

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In this study, we have examined the effect of inhibition of DI during methacholine inhalation in asthmatic subjects. Inhibition of DI was found to increase airway narrowing after 15 to 20 min in asthmatic subjects. We therefore suggest that stretch applied to the airway by DIs is an important mechanism for limiting acutely induced airway narrowing in asthmatic subjects.

DI can affect acute airway narrowing by two mechanisms: "bronchoprotection" and "bronchodilation." Scichilone and coworkers (7) studied bronchoprotection and bronchodilation in normal subjects and Wang and coworkers (8) studied these mechanisms in an in vivo preparation of porcine ASM. Bronchoprotection was defined in these studies as "the reduction in bronchoconstriction resulting from the act of DI before the ASM has been stimulated to contract" (8) whereas bronchodilation was the reduction in bronchoconstriction after ASM activation by a spasmogen. Scichilone and coworkers reported that the bronchoprotective effects of DI were stronger than the bronchodilatory effects and also that bronchoprotection was absent in asthmatics (7, 9). The effects of stretch on ASM preparations were found to be consistent with the changes observed in in vivo studies (8), which suggests that the effects of DI on airway narrowing are indeed mediated by its action on ASM.

The protocol that we used in the present study involved prohibiting DIs after successive doses of methacholine. The progressive increase in airway narrowing that we observed with each subsequent dose of methacholine when DIs were prohibited, compared with when DIs were allowed, could be due both to the increasing duration of time since the last DI and the cumulative effect of the additional methacholine doses. The changes in FEV₁ observed when DIs were taken after each dose (Figure 1) show that there is a cumulative effect of dose that occurs up to the third dose. Interestingly, this was also observed in our study of normal subjects (3). However, the airway narrowing continued to be cumulative, even after the third dose when DIs were inhibited and this was also observed in normals. This result suggests that both time since last DI and repetitive doses contribute to the cumulative increase in narrowing. The most likely physiologic explanation for these observations is an effect on ASM. Increasing force generation and stiffness with increasing time since last DI and repeated methacholine doses, could occur if there were changes in the mechanical properties of ASM owing to plasticity (10), cytoskeletal rearrangement (11), or latch bridge formation (12).

It has been shown that in asthmatics there is impaired dilatation of the airways in response to stretch (2, 13). This is based on the differences in the response of asthmatic subjects to DI compared with normals. Fish and associates (13) measured changes in airway conductance and partial flow-volume curves in response to methacholine inhalation in asthmatics and patients with allergic rhinitis before and after a DI. Although asthmatics showed greater airway narrowing as assessed by either measure, the difference between the asthmatics and rhinitics was much less prior to, than after DI. They suggested that failure of bronchodilatation with DI was an important cause of AHR in asthma.

In asthmatics, the magnitude of the dilatation caused by DI varies with the degree of spontaneous airway narrowing; less dilatation is associated with a lower baseline FEV₁ (14). The response to DI may also vary depending on the cause of the airway narrowing. During a spontaneous exacerbation of asthma, the bronchodilating effect of DI is impaired and may even lead to further narrowing. After recovery when a similar degree of airway narrowing is reproduced by bronchial challenge, DI causes more dilatation than was observed during the asthma exacerbation (15).

More recently, Skloot and coworkers performed standard and modified methacholine challenge tests in normals and asthmatics. During the standard challenge, spirometry and hence DIs, were taken after each dose and during the modified challenge, DIs were inhibited and only partial flow-volume curves were performed (2). Using the time constant derived from the partial flow-volume curve as a measure of airway narrowing, they found that inhibition of DIs during a methacholine challenge test in normals was associated with a degree of airway narrowing similar to that in asthmatics. However, inhibition of DIs did not affect the dose-response curves in asthmatics. Furthermore, the findings were similar for FEV₁ measured at the end of the challenge tests, i.e., prohibition of DI augmented the decreases in FEV₁ in normals (approximately 10% versus 36%) but not in asthmatics (approximately 30% versus 32%). In another study in which isovolume flow on partial flow-volume curves at 25% of vital capacity (VC) was used as the measure of airway narrowing, the dose- response curves were also unchanged in asthmatics by DI inhibition but were shifted to the left in normals (16). In a previous study, we confirmed that inhibition of DI accentuates bronchoconstriction in normals with a time course of approximately 10 min (3).

Our findings of increases in airway narrowing associated with inhibition of DI in asthmatic subjects therefore differ from findings in the aforementioned studies. This difference may be a result of differences in the study protocols. Whereas Skloot and coworkers (2) and Brusasco and coworkers (16) used doubling doses of methacholine, we gave the PC₁₅ concentration repeatedly. This would mean that during our modified challenge protocol, airways would be narrowed earlier. This could be important because the bronchodilating effect of DI appears to be related to the magnitude of airway narrowing (7). At lower doses of agonist and when airway narrowing was less, inhibition of DI may have little effect in asthmatics. As airway narrowing increases, there is more potential for bronchodilatation with DI so that inhibition of DIs could have more effect on airway narrowing. Thus, it is possible that if we had matched the decrease in FEV₁ in asthmatics with that seen in the normal subjects in our previous study, we may have found less of an effect of inhibition of DI, as had been found in previous studies. Another possible explanation is that there may be an inherent difference in response to DI between our asthmatic subjects and those studied by the other groups, although comparison of baseline physiologic data and medication use shows no obvious differences.

Finally, our use of FEV₁ as the measure of airway narrowing could also explain the apparently disparate results. Skloot and coworkers (2) and Brusasco and coworkers (16) measured airway caliber using tests that did not require a DI, viz. partial flow-volume curves and airway resistance, respectively. These measurements are made without a DI and therefore without distending the airway. We used the first FEV₁ after methacholine inhalation and inhibition of DIs, which necessarily requires a DI. It is possible that there may be differences in the distensibility of asthmatic and normal airways, which are relevant to our use of FEV₁, which might explain differences between our findings and those of others.

In conclusion, the similarity in response of asthmatics and normals to inhibition of DI during our modified challenges suggests that DI is important in limiting ASM shortening in both groups, when there is ASM activation and shortening for 15 min or more.