INTRODUCTION

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Nonspecific bronchial hyperresponsiveness (NSBH) is the exaggerated airway narrowing that develops during airway smooth muscle (ASM) contraction in response to pharmacologic smooth muscle agonists and irritants. It is most characteristic of asthma where its presence is required in some definitions (1). NSBH is characterized both by an increase in airway sensitivity and maximal airway narrowing produced by smooth muscle stimulants. In tests in which a deep inspiration (DI) is incorporated into the protocol (FEV₁, PEFR) many normal subjects develop a plateau on the dose-response curve at relatively minor degrees of airway narrowing (2). Fish and colleagues (5) were the first to suggest that the distinguishing feature of asthma was a failure of a deep inspiration to reverse airway narrowing rather than an exaggerated capacity of the airways to narrow. Burns and associates (6) have proposed that the degree of bronchodilation following DI is related to the relative magnitude of airway and parenchymal hysteresis. They have shown that the degree of reversal of airway narrowing is variable in asthmatics; during spontaneous bronchoconstriction, DI may actually cause an exaggeration of airway narrowing in some asthmatics. During induced bronchoconstriction of the same magnitude, the dilating effect of DI is less effective in asthmatics than in normal subjects. Recently Skloot and coworkers (7) challenged normal subjects and patients with asthma with methacholine using a protocol in which DI was prohibited. They showed that the dose- response curve was similar in asthmatics and normal subjects over the range of doses that they both received and that the degree of bronchoconstriction in normal subjects was markedly increased compared with that after a standard inhalation challenge test. They suggested that prohibition of DI in normal subjects makes them resemble asthmatics in that bronchodilation after DI is reduced. Prolonged prohibition of DI may allow airway smooth muscle to become stiff and noncompliant and/or lead to a failure of transmission of the stretch provided to the muscle by deep inspiration.

In the present study, we tested whether the fall in FEV₁ during inhibition of DIs was actually due to increased airway narrowing or due to a failure to inspire fully, and also whether repeated forced exhalations to residual volume (RV) were necessary for the effect.

	METHODS

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Ten nonasthmatic, nonsmoking subjects, five male and five female, volunteered for the study. Three of the subjects had a history of seasonal rhinitis.

Pulmonary function tests and a methacholine challenge were performed on three separate days, at the same time each day. Partial and complete flow-volume curves were performed, and FVC and FEV₁ were measured from these curves. FRC was measured using Boyle's law technique. Total lung capacity (TLC) was calculated by adding inspiratory capacity (IC) to FRC, and RV was calculated by subtracting FVC from TLC. All pulmonary function values were recorded in liters and expressed as percent predicted based on the equations of Crapo for spirometry (8) and Goldman and Becklake for lung volumes (9). Equipment specifications have been documented previously (4).

Methacholine challenge was carried out according to the protocol of Juniper and coworkers (10). Subjects inhaled an aerosol of methacholine in doubling concentrations from 1 mg/ml to a maximum of 64 mg/ml.

Subjects first performed an FRC maneuver, then a partial, and a complete forced expiratory maneuver, in triplicate, to establish baseline values on each day. On the first study day (Day 1) after each dose, partial and complete flow-volume maneuvers were repeated. The challenge was stopped if FEV₁ dropped 20% from baseline, if the subject requested to stop, or when the final dose was reached. Sequential measurements of FRC and partial and complete forced expiratory maneuvers were performed after the final dose, at least three times or until baseline values were reestablished.

On the second visit (Day 2), subjects were not allowed to take a DI during the challenge. They performed the same baseline pulmonary function tests, but throughout the challenge only partial curves were performed. FEV₁ and FVC from the partial curves were recorded, and the challenge was stopped if the partial FEV₁/FVC ratio dropped below 0.55, at the patient's request, or at the highest dose reached on Day 1. When the challenge was stopped, the subject was asked to perform an FRC maneuver, then a partial and complete flow-volume curve. This was repeated three times. The partial FEV₁/FVC ratio was used to monitor the bronchoconstriction on the second day in keeping with the protocol described by Skloot and colleagues (7).

Seven of the subjects were tested a third time (Day 3) in which baseline values were obtained as in the first two protocols, but no measurements were made until the end of the challenge. This was to test if exhalation to RV was the cause of the increased bronchoconstriction when DI was prohibited. The challenge was continued until the final dose administered on Day 2 was reached or until the patient asked to stop. Triplicate measurements of FRC and partial and complete flow-volume curves were made after the last dose.

To eliminate any differences in response due to variable timing, the time between doses was kept consistent at 5 min on all 3 d.

The response to the different challenge protocols was assessed by changes in spirometry and subdivisions of lung volume following the challenge. The baseline values were defined as the mean of the three measurements obtained prior to challenge. The percent change from baseline values of FEV₁, FVC, FEV₁/FVC, FRC, TLC, RV, and IC were calculated using the first set of values postchallenge.

A two-tailed t test was used to test for differences in pulmonary function values from baseline, on Day 1. Repeated measures analysis of variance (ANOVA) was used to test for differences between days. The results were considered significant if p < 0.05.

	RESULTS

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Table 1 shows that baseline pulmonary function was normal in all subjects. Baseline values were not different between days. Nine of the 10 subjects went to the final dose of 64 mg/ml during the routine challenge and one stopped at 32 mg/ml. Four subjects (including the one who stopped early Day 1) were stopped before the highest dose on the modified challenge (Day 2) because partial FEV₁/FVC dropped to 0.55 or lower, or at their request. The subjects who were studied a third time received the same doses as on Day 2. Although some subjects did not receive the maximal dose on all 3 d, the geometric mean of the maximal dose achieved on all 3 d was not significantly different.

Figure 1 shows the changes in lung function after the final dose on each of the 3 d. On Day 1, following a conventional challenge, there were significant decreases in FEV₁, FVC, and FEV₁/FVC, and significant increases in FRC and RV. The change in FEV₁, FVC, and FEV₁/FVC were all significantly greater on Days 2 and 3 than on Day 1. There were no significant differences in the change in FRC or TLC between the days, although hyperinflation (increased FRC) was seen on all days. The change in RV was significantly greater on Day 3 than Day 1. There were no significant differences in any lung function variable between Day 2 and Day 3. 

View larger version (18K): [in this window] [in a new window]   Figure 1.   Mean (± SE) changes in pulmonary function between study days. There was a significantly greater decrease in FEV1, FVC, and FEV1/FVC on Days 2 and 3 compared with Day 1.

Figure 2 shows recovery data for FEV₁ and FEV₁/FVC on all 3 d. FEV₁ recovered significantly after three blows (forced expiratory maneuver) on all 3 d but on none of the 3 d was recovery complete (FEV₁ did not reach baseline values after 3 maximal flow-volume curves). There was an improvement in FEV₁ on blow 3 compared with blow 2 on Days 2 and 3. There was a larger recovery on Days 2 and 3 than on Day 1, and on Day 3 compared with Day 2. Interestingly, there was no significant recovery of the ratio of FEV₁ to FVC even after three blows.

View larger version (13K): [in this window] [in a new window]   Figure 2.   Recovery data for FEV1 and FEV1/FVC on all 3 d (mean ± SE). There was significant improvement in FEV1 with each successive blow on each day.

	DISCUSSION

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The results of this study confirm and extend those of Skloot and colleagues (7). Prohibition of DI for the duration of a methacholine challenge produces more bronchoconstriction than a similar inhalation challenge during which a deep inspiration is permitted after each dose. Skloot and colleagues could not be sure that the DI after the modified challenge was to the same absolute lung volume as at baseline since they did not measure absolute lung volume. If prolonged prohibition of DI combined with methacholine challenge had led to a decreased inspiratory effort, the postchallenge FEV₁ would have been lowered due to the failure to reach prechallenge TLC. Our finding that postchallenge TLC did not differ between days is not consistent with this hypothesis and supports greater airway narrowing and/or closure as the cause of the exaggerated response.

Skloot and colleagues (7) speculated that it could be the repeated exhalation to low lung volumes (RV) coupled with bronchoconstriction, which could amplify the effects of methacholine, perhaps by unloading the smooth muscle. Ding and coworkers (11) showed enhanced airway narrowing when subjects breathed at FRC 500 ml and attenuated airway narrowing at volumes above FRC. Gibbons and associates (12) found that challenge in the supine position, which was associated with a small decrease in FRC, enhanced airway narrowing, and abolished the plateau in some normal subjects. Similar degrees of narrowing on Day 2 (repeated exhalation to RV) and Day 3 (no exhalation to RV) in our study suggest that prohibition of deep inspiration, rather than the forced expiration to RV, is responsible for the excessive narrowing.

A decrease in FVC during challenge suggests airway closure, whereas a decrease in FEV₁/FVC suggests airway narrowing (13). Our findings of a larger decrease in FVC and FEV₁/FVC following the modified protocols suggest both greater airway closure and greater narrowing. Decreased expiratory flow at a given lung volume could be due to a decrease in lung recoil at that volume or to more airway narrowing. We did not measure the pressure-volume relationship of the lung after the modified challenge, but others have shown that challenge with cholinergic agonists does not modify the static pressure- volume relationship of the lung (14); a possibility that we cannot address is a change of the dynamic pressure-volume relationship of the lung.

Deep inspiration dilates the airways by exerting an increase in the airway transmural pressure. The magnitude of the pressure change following a DI is related to the size of the inspiration, the elastic recoil properties of the lung, the pressure/area relationship of the airways, and the coupling between airway and parenchyma (interdependence). There are at least three possible explanations for the exaggerated airway narrowing during the modified protocol. First, the stress applied to the airway smooth muscle by a deep inspiration could be attenuated after a prolonged period of prohibition of deep inspiration if there was an accumulation of fluid in the space between the parenchyma and airway smooth muscle, thereby decreasing interdependence. Although this is theoretically possible, there is no evidence that methacholine challenge causes peribronchial accumulation of fluid; in fact, Okazawa and coworkers (15) recently showed that the airway area decreases following methacholine challenge in normal subjects.

Second, prolonged failure to take a deep breath and stretch airway smooth muscle could alter the contractile properties of the smooth muscle. Gunst and Wu (16) recently reported that airway smooth muscle becomes stiffer when it is not stretched for prolonged periods; a similar effect could occur in vivo. There is evidence that there are two states of contractile apparatus activity in smooth muscle. During the initial activation, there is a period of rapid cross-bridge cycling and rapid velocity of shortening. With prolonged stimulation, there is slower cycling of cross-bridges. The change, which has been called the "latch phenomenon," is controversial, but Fredberg and colleagues (17) have postulated that it is the uninhibited formation of latch bridges in asthmatic airways that causes their diminished response to DI. Airway smooth muscle also shows plasticity of its length tension relationship. Pratusevich and coworkers (18) have hypothesized that contractile elements in series can be moved to a mechanically parallel arrangement at shorter lengths. This hypothesis suggests that repeated stimulation at short lengths will result in a subtraction of contractile units in series with maintenance or augmentation of units in parallel so that the force-generating ability of the muscle is preserved or increased despite decreased length. Similar mechanical alterations in smooth muscle behavior have been suggested by Harris and Warshaw (19). Gunst and colleagues (20) have suggested alterations in the arrangement of the cytoskeleton to explain the plastic behavior.

A third possible explanation is related to surface phenomena. Pulmonary surfactant is thought to be important in peripheral airway stability. Preventing stretch of the surfactant lining (by prolonged prohibition of DI) could lead to increased surface tension in small airways, which could enhance narrowing and promote closure. With repeated deep inspiration at the termination of the study, there was progressive increase in FEV₁ (Figure 2) and FVC but not a significant increase in the FEV₁/FVC ratio (Figure 2). This suggests that airway closure may contribute to the exaggerated response.

The results of this study confirm that prohibition of deep inspiration during methacholine challenge produces more bronchoconstriction than a conventional challenge, which mandates deep breaths in order to perform FEV₁ measurement. The novel results of this study are that the amplified effect is not due to failure to fully inspire to TLC or to repeated exhalation to RV. A more complete understanding of the phenomena of exaggerated airway narrowing in normal subjects after prohibition of deep inspiration may yield important information regarding the basic mechanisms of exaggerated airway narrowing in asthma.