Computational assessment of airway wall stiffness in vivo in allergically inflamed mouse models of asthma.

Allergic inflammation is known to cause airway hyperresponsiveness in mice. However, it is not known whether inflammation affects the stiffness of the airway wall, which would alter the load against which the circumscribing smooth muscle shortens when activated. Accordingly, we measured the time course of airway resistance immediately following intravenous methacholine injection in acutely and chronically allergically inflamed mice. We estimated the effective stiffness of the airway wall in these animals by fitting to the airway resistance profiles a computational model of a dynamically narrowing airway embedded in elastic parenchyma. Effective airway wall stiffness was estimated from the model fit and was found not to change from control in either the acute or chronic inflammatory groups. However, the acutely inflamed mice were hyperresponsive compared with controls, which we interpret as reflecting increased delivery of methacholine to the airway smooth muscle through a leaky pulmonary endothelium. These results support the notion that acutely inflamed BALB/c mice represent an animal model of functionally normal airway smooth muscle in a transiently abnormal lung.     

Parenchymal tethering, airway wall stiffness, and the dynamics of bronchoconstriction.

We do not yet have a good quantitative understanding of how the force-velocity properties of airway smooth muscle interact with the opposing loads of parenchymal tethering and airway wall stiffness to produce the dynamics of bronchoconstriction. We therefore developed a two-dimensional computational model of a dynamically narrowing airway embedded in uniformly elastic lung parenchyma and compared the predictions of the model to published measurements of airway resistance made in rats and rabbits during the development of bronchoconstriction following a bolus injection of methacholine. The model accurately reproduced the experimental time-courses of airway resistance as a function of both lung inflation pressure and tidal volume. The model also showed that the stiffness of the airway wall is similar in rats and rabbits, and significantly greater than that of the lung parenchyma. Our results indicate that the main features of the dynamical nature of bronchoconstriction in vivo can be understood in terms of the classic Hill force-velocity relationship operating against elastic loads provided by the surrounding lung parenchyma and an airway wall that is stiffer than the parenchyma.     

Mechanical response to methacholine and deep inspiration in supine men.

The effects of supine posture on airway responses to inhaled methacholine and deep inspiration (DI) were studied in seven male volunteers. On a control day, subjects were in a seated position during both methacholine inhalation and lung function measurements. On a second occasion, the whole procedure was repeated with the subjects lying supine for the entire duration of the study. On a third occasion, methacholine was inhaled from the seated position and measurements were taken in a supine position. Finally, on a fourth occasion, methacholine was inhaled from the supine position and measurements were taken in the seated position. Going from sitting to supine position, the functional residual capacity decreased by approximately 1 liter in all subjects. When lung function measurements (pulmonary resistance, dynamic elastance, residual volume, and maximal flows) were taken in supine position, the response to methacholine was greater than at control, and this was associated with a greater dyspnea and a faster recovery of dynamic elastance after DI. By contrast, when methacholine was inhaled in supine position but measurements were taken in sitting position, the response to methacholine was similar to control day. These findings document the potential of the decrease in the operational lung volumes in eliciting or sustaining airflow obstruction in nocturnal asthma. It is speculated that the exaggerated response to methacholine in the supine posture may variably contribute to airway smooth muscle adaptation to short length, airway wall edema, and faster airway renarrowing after a large inflation.     

Airway response to deep inspiration: role of inflation pressure.

Deep inspirations (DIs) have been shown to have both bronchoprotective and bronchodilator effects in healthy subjects; however, the bronchodilator effects of a DI appear to be impaired in asthmatic compared with healthy subjects. Because the ability to generate high transpulmonary pressures at total lung capacity depends on both the lung properties and voluntary effort, we wondered how the response of airways to DI might be altered if the maneuver were done with less than maximal inflation. The present work was undertaken to examine the effects of varying the magnitude of lung inflation during the DI maneuver on subsequent airway caliber. In five anesthetized and ventilated dogs during methacholine infusion, changes in airway size after DIs of increasing magnitude were measured over the subsequent 5-min period using high-resolution computed tomography. Results show that the magnitude of lung inflation is extremely important, leading to a qualitative change in the airway response. A large DI (45 cmH(2)O airway pressure) caused subsequent airway dilation, whereas smaller DIs (< or =35 cmH(2)O) caused bronchoconstriction. The precise mechanism underlying these observations is uncertain, but it seems to be related to an interaction between intrinsic properties of the contracted airway smooth muscle and the response to mild stretch.     

Intrinsic and antigen-induced airway hyperresponsiveness are the result of diverse physiological mechanisms.

Airway hyperresponsiveness (AHR) is a defining feature of asthma. We have previously shown, in mice sensitized and challenged with antigen, that AHR is attributable to normal airway smooth muscle contraction with exaggerated airway closure. In the present study we sought to determine if the same was true for mice known to have intrinsic AHR, the genetic strain of mice, A/J. We found that A/J mice have AHR characterized by minimal increase in elastance following aerosolized methacholine challenge compared with mice (BALB/c) that have been antigen sensitized and challenged [concentration that evokes 50% change in elastance (PC(50)): 22.9 +/- 5.7 mg/ml for A/J vs. 3.3 +/- 0.4 mg/ml for antigen-challenged and -sensitized mice; P < 0.004]. Similar results were found when intravenous methacholine was used (PC(30) 0.22 +/- 0.08 mg/ml for A/J vs. 0.03 +/- 0.004 mg/ml for antigen-challenged and -sensitized mice). Computational model analysis revealed that the AHR in A/J mice is dominated by exaggerated airway smooth muscle contraction and that when the route of methacholine administration was changed to intravenous, central airway constriction dominates. Absorption atelectasis was used to provide evidence of the lack of airway closure in A/J mice. Bronchoconstriction during ventilation with 100% oxygen resulted in a mean 9.8% loss of visible lung area in A/J mice compared with 28% in antigen-sensitized and -challenged mice (P < 0.02). We conclude that the physiology of AHR depends on the mouse model used and the route of bronchial agonist administration.     

Kinetics of respiratory system elastance after airway challenge in dogs.

We compared the time courses of lung mechanical changes with intravenous (iv) injection vs. aerosol administration of histamine, methacholine, and ACh in dogs. We interpret these results in terms of a spring-and-dashpot model of airway smooth muscle receiving activation via a tissue compartment when agonist is delivered by the iv route and through an additional airway wall compartment when it is delivered by the aerosol route. The model accurately accounts for the principal features of the respiratory system elastance response curves. It also accounts for the differences between iv and aerosol responses, supporting the notion that agonist delivered by aerosol has to traverse a longer pathway to the airway smooth muscle than does agonist delivered iv. The time constants representing diffusive exchange of agonist between compartments were not significantly different for the three agonists, suggesting that the three agonists shared a common principal means of clearance, which was presumably blood flow.     

Bronchoprotective and bronchodilatory effects of deep inspiration in rabbits subjected to bronchial challenge.

The effect of deep inspiration (DI) on airway responsiveness differs in asthmatic and normal human subjects. The mechanism for the effects of DI on airway responsiveness in vivo has not been identified. To elucidate potential mechanisms, we compared the effects of DI imposed before or during induced bronchoconstriction on the airway response to methacholine (MCh) in rabbits. The changes in airway resistance in response to intravenous MCh were continuously monitored. DI depressed the maximum response to MCh when imposed before or during the MCh challenge; however, the inhibitory effect of DI was greater when imposed during bronchoconstriction. Because immature rabbits have greater airway reactivity than mature rabbits, we compared the effects of DI on their airway responses. No differences were observed. Our results suggest that the mechanisms by which DI inhibits airway responsiveness do not depend on prior activation of airway smooth muscle (ASM). These results are consistent with the possibility that reorganization of the contractile apparatus caused by stretch of ASM during DI contributes to depression of the airway response.     

Role of Dystrophin in Airway Smooth Muscle Phenotype,Contraction and Lung Function

Dystrophin links the transmembrane dystrophin-glycoprotein complex to the actin cytoskeleton. We have shown that dystrophin-glycoprotein complex subunits are markers for airway smooth muscle phenotype maturation and together with caveolin-1, play an important role in calcium homeostasis. We tested if dystrophin affects phenotype maturation, tracheal contraction and lung physiology. We used dystrophin deficient Golden Retriever dogs (GRMD) and mdx mice vs healthy control animals in our approach. We found significant reduction of contractile protein markers: smooth muscle myosin heavy chain (smMHC) and calponin and reduced Ca²⁺ response to contractile agonist in dystrophin deficient cells. Immunocytochemistry revealed reduced stress fibers and number of smMHC positive cells in dystrophin-deficient cells, when compared to control. Immunoblot analysis of Akt1, GSK3β and mTOR phosphorylation further revealed that downstream PI3K signaling, which is essential for phenotype maturation, was suppressed in dystrophin deficient cell cultures. Tracheal rings from mdx mice showed significant reduction in the isometric contraction to methacholine (MCh) when compared to genetic control BL10ScSnJ mice (wild-type). In vivo lung function studies using a small animal ventilator revealed a significant reduction in peak airway resistance induced by maximum concentrations of inhaled MCh in mdx mice, while there was no change in other lung function parameters. These data show that the lack of dystrophin is associated with a concomitant suppression of ASM cell phenotype maturation in vitro, ASM contraction ex vivo and lung function in vivo, indicating that a linkage between the DGC and the actin cytoskeleton via dystrophin is a determinant of the phenotype and functional properties of ASM.     

Deficiency of RAMP1 Attenuates Antigen-Induced Airway Hyperresponsiveness in Mice

Asthma is a chronic inflammatory disease affecting the lung, characterized by breathing difficulty during an attack following exposure to an environmental trigger. Calcitonin gene-related peptide (CGRP) is a neuropeptide that may have a pathological role in asthma. The CGRP receptor is comprised of two components, which include the G-protein coupled receptor, calcitonin receptor-like receptor (CLR), and receptor activity-modifying protein 1 (RAMP1). RAMPs, including RAMP1, mediate ligand specificity in addition to aiding in the localization of receptors to the cell surface. Since there has been some controversy regarding the effect of CGRP on asthma, we sought to determine the effect of CGRP signaling ablation in an animal model of asthma. Using gene-targeting techniques, we generated mice deficient for RAMP1 by excising exon 3. After determining that these mice are viable and overtly normal, we sensitized the animals to ovalbumin prior to assessing airway resistance and inflammation after methacholine challenge. We found that mice lacking RAMP1 had reduced airway resistance and inflammation compared to wildtype animals. Additionally, we found that a 50% reduction of CLR, the G-protein receptor component of the CGRP receptor, also ameliorated airway resistance and inflammation in this model of allergic asthma. Interestingly, the loss of CLR from the smooth muscle cells did not alter the airway resistance, indicating that CGRP does not act directly on the smooth muscle cells to drive airway hyperresponsiveness. Together, these data indicate that signaling through RAMP1 and CLR plays a role in mediating asthma pathology. Since RAMP1 and CLR interact to form a receptor for CGRP, our data indicate that aberrant CGRP signaling, perhaps on lung endothelial and inflammatory cells, contributes to asthma pathophysiology. Finally, since RAMP-receptor interfaces are pharmacologically tractable, it may be possible to develop compounds targeting the RAMP1/CLR interface to assist in the treatment of asthma.     

CD38-deficient mice have reduced airway hyperresponsiveness following IL-13 challenge

The transmembrane glycoprotein CD38 in airway smooth muscle is the source of cyclic-ADP ribose, an intracellular calcium-releasing molecule, and is subject to regulatory effects of cytokines such as interleukin (IL)-13, a cytokine implicated in asthma. We investigated the role of CD38 in airway hyperresponsiveness using a mouse model of IL-13-induced airway disease. Wild-type (WT) and CD38-deficient (CD38KO) mice were intranasally challenged with 5 microg of IL-13 three times on alternate days under isoflurane anesthesia. Lung resistance (R(L)) in response to inhaled methacholine was measured 24 h after the last challenge in pentobarbital-anesthetized, tracheostomized, and mechanically ventilated mice. Bronchoalveolar cytokines, bronchoalveolar and parenchymal inflammation, and smooth muscle contractility and relaxation using tracheal segments were also evaluated. Changes in methacholine-induced R(L) were significantly greater in the WT than in the CD38KO mice following intranasal IL-13 challenges. Airway reactivity after IL-13 exposure, as measured by the slope of the methacholine dose-response curve, was significantly higher in the WT than in the CD38KO mice. The rate of isometric force generation in tracheal segments (e.g., smooth muscle reactivity) was greater in the WT than in the CD38KO mice following incubation with IL-13. IL-13 treatment reduced isoproterenol-induced relaxations to similar magnitudes in tracheal segments obtained from WT and CD38KO mice. Both WT and CD38KO mice developed significant bronchoalveolar and parenchymal inflammation after IL-13 challenges compared with na?ve controls. The results indicate that CD38 contributes to airway hyperresponsiveness in lungs exposed to IL-13 at least partly by increasing airway smooth muscle reactivity to contractile agonists.     

Effect of resting smooth muscle length on contractile response in resistance airways

We studied the effect of resting smooth muscle length on the contractile response of the major resistance airways (generations 0-5) in 18 mongrel dogs in vivo using tantalum bronchography. Dose-response curves to 10(-10) to 10(-7) mol/kg methacholine (MCh) were generated [at functional residual capacity (FRC)] by repeated intravenous bolus administration using tantalum bronchography after each dose. Airway constriction varied substantially with dose-equivalent stimulation and varied sequentially from trachea (8.8 +/- 2.2% change in airway diam) to fifth-generation bronchus (49.8 +/- 3.0%; P less than 0.001). Length-tension curves were generated for each airway to determine the airway diameter (i.e., resting in situ smooth muscle length) at which maximal constriction was elicited using bolus intravenous injection of 10(-8) mol/kg MCh. A Frank-Starling relationship was obtained for each airway; the transpulmonary pressure at which maximal constriction was elicited increased progressively from 2.50 +/- 1.12 cmH2O for trachea (approximately FRC) to 18.3 +/- 1.05 cmH2O for fifth-generation airways (approximately 50% TLC) (P less than 0.001). A similar relationship was obtained when change in airway diameter was plotted as a function of airway radius. We demonstrate substantial heterogeneity in the lung volumes at which maximal constriction is elicited and in distribution of parasympathomimetic constriction within the first few generations of resistance bronchi. Our data also suggest that lung hyperinflation may lead to augmented airway contractile responses by shifting resting smooth muscle length toward optimum resting smooth muscle length.     

Basal lung mechanics and airway and pulmonary vascular responsiveness in different inbred mouse strains.

Little is known about interstrain variations in baseline lung functions or smooth muscle contractility in murine lungs. We therefore examined basal lung mechanics and airway, as well as vascular reactivity to methacholine, thromboxane (using U-46619), and endothelin-1 (ET-1), A/J, AKR, BALB/c, C3H/HeN, C57BL/6, and SCID mice. All experiments were performed with isolated perfused mouse lungs. Except AKR mice (which were excluded from further analysis), all other strains showed stable pulmonary compliance, pulmonary resistance, and pulmonary arterial pressure within a control period of 45 min. Among these strains, C3H/HeN mice exhibited higher dynamic pulmonary compliance and lower pulmonary resistance, whereas SCID mice had higher baseline pulmonary resistance than the other strains. Concentration-response experiments with methacholine showed a lower airway reactivity for C57BL/6 mice compared with the other strains. Perfusion with 1 microM U-46619 or 100 nM ET-1 revealed a similar pattern: the agonist-inducible broncho- and vasoconstriction was lower in C57BL/6 mice than in all other strains, whereas it tended to be higher in SCID mice. The present study demonstrates a correlation between airway and vascular responsiveness in all tested strains. SCID mice are hyperreactive, whereas C57BL/6 mice are hyporeactive, to smooth muscle constrictors. Lung mechanics, as well as airway and vascular responsiveness, appear to be genetically controlled.     

Desmin modulates lung elastic recoil and airway responsiveness

Desmin is a structural protein that is expressed in smooth muscle cells of both airways and alveolar ducts. Therefore, desmin could be well situated to participate in passive and contractile force transmission in the lung. We hypothesized that desmin modulates lung compliance, lung recoil pressure, and airway contractile response. To test this hypothesis, respiratory system complex impedance (Zin,rs) at different positive end-expiratory pressure (PEEP) levels and quasi-static pressure-volume data were obtained in desmin-null and wild-type mice at baseline and during methacholine administration. Airways and lung tissue properties were partitioned by fitting Zin,rs to a constant-phase model. Relative to controls, desmin-null mice showed 1) lower values for lung stiffness and recoil pressure at baseline and induced airway constriction, 2) greater negative PEEP dependence of H and airway resistance under baseline conditions and cholinergic stimulation, and 3) airway hyporesponsiveness. These results demonstrate that desmin is a load-bearing protein that stiffens the airways and consequently the lung and modulates airway contractile response.     

Airways in smooth muscle α-actin null mice experience a compensatory mechanism that modulates their contractile response

We hypothesized that ablation of smooth muscle α-actin (SM α-A), a contractile-cytoskeletal protein expressed in airway smooth muscle (ASM) cells, abolishes ASM shortening capacity and decreases lung stiffness. In both SM α-A knockout and wild-type (WT) mice, airway resistance (Raw) determined by the forced oscillation technique rose in response to intravenous methacholine (Mch). However, the slope of Raw (cmH(2)O·ml(-1)·s) vs. log(2) Mch dose (μg·kg(-1)·min(-1)) was lower (P = 0.007) in mutant (0.54 ± 0.14) than in WT mice (1.23 ± 0.19). RT-PCR analysis performed on lung tissues confirmed that mutant mice lacked SM α-A mRNA and showed that these mice had robust expressions of both SM γ-A mRNA and skeletal muscle (SKM) α-A mRNA, which were not expressed in WT mice, and an enhanced SM22 mRNA expression relative to that in WT mice. Compared with corresponding spontaneously breathing mice, mechanical ventilation-induced lung mechanical strain increased the expression of SM α-A mRNA in WT lungs; in mutant mice, it augmented the expressions of SM γ-A mRNA and SM22 mRNA and did not alter that of SKM α-A mRNA. In mutant mice, the expression of SM γ-A mRNA in the lung during spontaneous breathing and its enhanced expression following mechanical ventilation are consistent with the likely possibility that in the absence of SM α-A, SM γ-A underwent polymerization and interacted with smooth muscle myosin to produce ASM shortening during cholinergic stimulation. Thus our data are consistent with ASM in mutant mice experiencing compensatory mechanisms that modulated its contractile muscle capacity.     

Association between reduced bronchodilatory effect of deep inspiration and loss of alveolar attachments

Background

We have previously shown that the bronchodilatory effect of deep inspiration is attenuated in individuals with COPD. This study was designed to investigate whether the impairment in this effect is associated with loss of alveolar attachments.

Methods

We measured deep inspiration (DI)-induced bronchodilation in 15 individuals with and without COPD (67 ± 2.2 yrs of age, mean ± SEM) undergoing lobar resection for peripheral pulmonary nodule. Prior to surgery, we measured TLCO and determined the bronchodilatory effect of deep inspiration after constricting the airways with methacholine. The number of destroyed alveolar attachments, as well as airway wall area and airway smooth muscle area, were determined in tumor-free, peripheral lung tissue.

Results

The bronchodilatory effect of deep inspiration correlated inversely with the % destroyed attachments (r = -0.51, p = 0.05) and directly with the airway smooth muscle area (r = 0.59, p = 0.03), but not with the total wall area (r = 0.39, p = 0.15).

Conclusion

We postulate that attenuation of airway stretch due to loss of alveolar attachments contributes to the loss of the bronchodilatory effect of lung inflation in COPD.     

Effect of lung inflation in vivo on airways with smooth muscle tone or edema

Brown, Robert H., Wayne Mitzner, Yonca Bulut, and ElizabethM. Wagner. Effect of lung inflation in vivo on airways with smoothmuscle tone or edema. J. Appl.Physiol. 82(2): 491-499, 1997.Fibrousattachments to the airway wall and a subpleural surrounding pressurecan create an external load against which airway smooth muscle mustcontract. A decrease in this load has been proposed as a possible causeof increased airway narrowing in asthmatic individuals. To study theinteraction between the airways and the surrounding lung parenchyma, weinvestigated the effect of lung inflation on relaxed airways, airwayscontracted with methacholine, and airways made edematous by infusion ofbradykinin into the bronchial artery. Measurements were made inanesthetized sheep by using high-resolution computed tomography tovisualize changes in individual airways. During methacholine infusion,airway area was decreased but increased minimally with increases intranspulmonary pressure. Bradykinin infusion caused a 50% increase inairway wall area and a small decrease in airway luminal area. Incontrast to airways contracted with methacholine, the luminal areaafter bradykinin increased substantially with increases intranspulmonary pressure, reaching 99% of the relaxed area at totallung capacity. Thus airway edema by itself did not prevent fulldistension of the airway at lung volumes approaching total lungcapacity. Therefore, we speculate that if a deep inspiration fails torelieve airway narrowing in vivo, this must be a manifestation ofairway smooth muscle contraction and not airway wall edema.

     

Impedance, gas mixing, and bimodal ventilation in constricted lungs.

To evaluate the effect of increasing smooth muscle activation on the distribution of ventilation, lung impedance and expired gas concentrations were measured during a 16-breath He-washin maneuver in five nonasthmatic subjects at baseline and after each of three doses of aerosolized methacholine. Values of dynamic lung elastance (El,dyn), the curvature of washin plots, and the normalized slope of phase III (S(N)) were obtained. At the highest dose, El,dyn was 2.6 times the control value and S(N) for the 16th breath was 0.65 liter(-1). A previously described model of a constricted terminal airway was extended to include variable muscle activation, and the extended model was tested against these data. The model predicts that the constricted airway has two stable states. The impedances of the two stable states are independent of smooth muscle activation, but driving pressure and the number of airways in the high-resistance state increase with increasing muscle activation. Model predictions and experimental data agree well. We conclude that, as a result of the bistability of the terminal airways, the ventilation distribution in the constricted lung is bimodal.     

Effects of chest wall strapping on mechanical response to methacholine in humans.

We examined the effects of chest wall strapping (CWS) on the response to inhaled methacholine (MCh) and the effects of deep inspiration (DI). Eight subjects were studied on 1 day with MCh inhaled without CWS (CTRL), 1 day with MCh inhaled during CWS (CWSon/on), and 1 day with MCh inhaled during temporary removal of CWS (CWSoff/on). On the CWSon/on day, MCh caused greater increases in pulmonary resistance, upstream resistance, dynamic elastance, residual volume, and greater decreases in maximal expiratory flow than on the CTRL day. On the CWSoff/on day, the changes in these parameters with MCh were not different from the CTRL day. Six of the subjects were again studied using the same protocol on CTRL and CWSon/on days, except that, on a third day, MCh was given after applying the CWS, but the measurements before and after the inhalation were made without CWS (CWSon/off). The latter sequence was associated with more severe airflow obstruction than during CTRL, but less than with CWSon/on. The bronchodilator effects of a DI were blunted when CWS was applied during measurements (CWSon/on and CWSoff/on) but not after it was removed (CWSon/off). We conclude that CWS is capable of increasing airway responsiveness only when it is applied during the inhalation of the constrictor agent. We speculate that breathing at low lung volumes induced by CWS enhances airway narrowing because the airway smooth muscle is adapted at a length at which the contractile apparatus is able to generate a force greater than normal.     

Exaggerated airway narrowing in mice treated with intratracheal cationic protein.

Airway hyperresponsiveness in mice with allergic airway inflammation can be attributed entirely to exaggerated closure of peripheral airways (Wagers S, Lundblad LK, Ekman M, Irvin CG, and Bates JHT. J Appl Physiol 96: 2019-2027, 2004). However, clinical asthma can be characterized by hyperresponsiveness of the central airways as well as the lung periphery. We, therefore, sought to establish a complementary model of hyperresponsiveness in the mouse due to excessive narrowing of the airways. We treated mice with a tracheal instillation of the cationic protein poly-l-lysine (PLL), hypothesizing that this would reduce the barrier function of the epithelium and thereby render the underlying airway smooth muscle more accessible to aerosolized methacholine. The PLL-treated animals were hypersensitive to methacholine: they exhibited an exaggerated response to submaximal doses but had a maximal response that was similar to controls. With the aid of a computational model of the mouse lung, we conclude that the methacholine responsiveness of PLL-treated mice is fundamentally different in nature to the hyperresponsiveness that we found previously in mice with allergically inflamed lungs.     

Selected contribution: airway caliber in healthy and asthmatic subjects: effects of bronchial challenge and deep inspirations.

In 9 healthy and 14 asthmatic subjects before and after a standard bronchial challenge and a modified [deep inspiration (DI), inhibited] bronchial challenge and after albuterol, we tracked airway caliber by synthesizing a method to measure airway resistance (Raw; i.e., lung resistance at 8 Hz) in real time. We determined the minimum Raw achievable during a DI to total lung capacity and the subsequent dynamics of Raw after exhalation and resumption of tidal breathing. Results showed that even after a bronchial challenge healthy subjects can dilate airways maximally, and the dilation caused by a single DI takes several breaths to return to baseline. In contrast, at baseline, asthmatic subjects cannot maximally dilate their airways, and this worsens considerably postconstriction. Moreover, after a DI, the dilation that does occur in airway caliber in asthmatic subjects constricts back to baseline much faster (often after a single breath). After albuterol, asthmatic subjects could dilate airways much closer to levels of those of healthy subjects. These data suggest that the asthmatic smooth muscle resides in a stiffer biological state compared with the stimulated healthy smooth muscle, and inhibiting a DI in healthy subjects cannot mimic this.