Adaptation of Airway Smooth Muscle to Basal Tone Relevance to Airway Hyperresponsiveness Ynuk Bosse´1, Leslie Y. M. Chin1,3, Peter D. Pare´1,2, and Chun Y. Seow1,3 1 The James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, Providence Health Care/St. Paul’s Hospital; 2Department of Medicine, Respiratory Division; and 3Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada
Lung inflammation and airway hyperresponsiveness (AHR) are hallmarks of asthma, but their interrelationship is unclear. Excessive shortening of airway smooth muscle (ASM) in response to bronchoconstrictors is likely an important determinant of AHR. Hypercontractility of ASM could stem from a change in the intrinsic properties of the muscle, or it could be due to extrinsic factors such as chronic exposure of the muscle to inflammatory mediators in the airways. The latter could be the link between lung inflammation and AHR. The present study was designed to examine the influence of chronic exposure to a contractile agonist on the force-generating capacity of ASM. Force generation in response to electric field stimulation (EFS) was measured in ovine trachealis with or without a basal tone induced by acetylcholine (ACh). While the tone was maintained, the EFS-induced force decreased transiently but increased over time to reach a plateau in approximately 50 minutes. The total force (ACh tone 1 EFS force) increased monotonically and in proportion to ACh concentration. The results indicate that the muscle adapted to the basal tone and regained its contractile ability in response to a second stimulus (EFS) over time. Analysis suggests that this is due to a cytoskeletal transformation that allows the cytoskeleton to bear force, thus freeing up actomyosin crossbridges to generate more force. Force adaptation in ASM as a consequence of prolonged exposure to the many spasmogens found in asthmatic airways could be a mechanism contributing to AHR seen in asthma. Keywords: muscle contraction; spasmogen; acetylcholine; muscle tone; asthma
One of the hallmarks of asthma is airway hyperresponsiveness (AHR). Although the underlying mechanism for AHR is not clear, hypercontractility of airway smooth muscle (ASM) could be involved (1). It is clear that the amount of ASM shortening is an important determinant of airway narrowing and airway obstruction. Because ASM shortens as long as the muscle is capable of generating more force than the load against which it shortens, it is believed that the extent of airway narrowing depends on the force produced by the ASM. This presumption has led many investigators to compare the force generated by ASM derived from both individuals with asthma and those without asthma (see review in Ref. 2). Since there are conflicting results as to whether asthmatic ASM generates more force, this remains an unsettled issue. Previous studies from our laboratory (3) suggest that even if asthmatic ASM generates a normal amount of force, the asthmatic airway environment could favor (Received in original form April 17, 2008 and in final form June 5, 2008) This work was supported by operating grants from Canadian Institutes of Health Research (CIHR) (MOP-13271, MOP-4725). Y.B. is supported by a fellowship from Fonds de la Recherche en Sante´ Que´bec (FRSQ) and a CIHR Strategic Training Initiative in Health Research-IMPACT fellowship. L.Y.M.C. is supported by a fellowship from Michael Smith Foundation for Health Research (MSFHR). Correspondence and requests for reprints should be addressed to Ynuk Bosse´, Ph.D., James Hogg iCAPTURE Centre/St. Paul’s Hospital, Room 166–1081, Burrard Street, Vancouver, BC, V6Z 1Y6 Canada. E-mail:
[email protected] Am J Respir Cell Mol Biol Vol 40. pp 13–18, 2009 Originally Published in Press as DOI: 10.1165/rcmb.2008-0150OC on July 10, 2008 Internet address: www.atsjournals.org
CLINICAL RELEVANCE A basal tone increased the force-generating capacity of airway smooth muscle over time. A tone in asthmatic airways, such as the one induced by inflammatory spasmogens, may foster this force adaptation process and underlie airway hyperresponsiveness.
adaptation of ASM to abnormally short lengths and lead to excessive narrowing of the airways. The results suggest that asthmatic ASM per se can be normal, but its ability to shorten can be enhanced by an abnormal airway environment acting through the mechanism of length adaptation (3). Another scenario that has not been explored (that does not involve adaptation of ASM to pathologically short lengths) is enhancement of ASM force-generating capacity in an asthmatic airway environment, where inflammatory mediators are chronically present. Whereas ASM is the effector tissue for excessive airway narrowing, inflammation contributes unequivocally to several facets of asthma, including airway remodeling. Many inflammation-derived spasmogens, such as histamine (4), leukotrienes (5), endothelin-1 (6), prostaglandin D2 (7), thromboxane A2 (8), adenosine (9), bradykinin (10), anaphylatoxin C3a and C5a (11) and substance P (12), have been shown to be up-regulated in asthmatic airways. Recently, the influence of acetylcholine (ACh) on ASM behavior was also proposed to be increased in asthma (13). These spasmogens are undoubtedly involved in the acute and chronic phases of asthma. An important question therefore is: how does chronic exposure to spasmogens (which increase ASM tone) influence the contractile properties of ASM? The present study seeks to answer this question.
MATERIALS AND METHODS Tissue Preparation and Determination of In Situ Muscle Length Ovine tracheas were obtained from a local abattoir. Immediately after the sheep were killed, the tracheas were removed and immediately placed in room-temperature physiologic Krebs’ solution (pH 7.4; 118 mM NaCl, 4 mM KCl, 1.2 mM NaH2PO4, 22.5 mM NaHCO3, 2 mM MgSO4, 2 mM CaCl2, and 2 g/L dextrose). After transportation to the laboratory, tracheas were stored at 48C until use in experiments. A tracheal segment was removed from the trachea, and the in situ length of a relaxed tracheal smooth muscle bundle connecting the C-shaped cartilage ring was measured. The tracheal rings were then cut open. Adventitial connective tissue and the epithelium were dissected away from the tracheal smooth muscle layer, and muscle strips (z7 mm long, 1 mm wide, and 0.3 mm thick) were isolated. The muscle strips were attached on both ends with aluminum foil clips and mounted vertically on two hooks in a warm-water (378C) jacketed bath filled with Krebs’ solution. One of the hooks was stationary and the other was connected to the lever arm of a servo-controlled force-length transducer via a piece of surgical thread (Size 6-0), allowing measurements of the
14
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 40 2009
tension generated by the ASM strip in response to electrical field stimulation (EFS) and/or acetylcholine (ACh) stimulation. The muscle strips were stretched to their in situ length (i.e., the length of the relaxed muscle before it was cut from the cartilage ‘‘C’’ ring) before the equilibration period (see below). The in situ length was used throughout the experiments as a reference length (Lref) for normalization of data.
Equilibration of Muscle Preparation Muscle ‘‘equilibration’’ was done before beginning any experiment to allow the muscle to recover from mechanical and metabolic perturbations caused by dissection, lack of perfusion, and low storage temperature (48C). During the equilibration period, muscle strips were subjected to a repeated cycle of 9-second EFS that produced tetani at 5-minute intervals. Equilibration was considered complete when stimulations produced a stable maximal isometric force (Fmax) with low resting tension (around 1 to 2 mN, or z2% Fmax). The process took approximately 1.5 h. During the equilibration period, prewarmed (378C) Krebs’ solution was changed regularly and the pH was maintained at 7.4 at all times by bubbling the solution with carbogen gas (mixture of 95% O2 and 5% CO2).
Experimental Protocol for Force Measurement After the equilibration period, active isometric force produced by EFS (Fmax) before administration of ACh was recorded (active force 5 total force 2 baseline force). The baseline force at in situ length was usually insignificant, less than 2% of Fmax. ACh was then added to the Krebs’ solution (1027 M unless otherwise indicated) to produce basal tone. The active force produced by EFS after ACh administration was normalized to the respective Fmax for each ASM preparation tested before grouping the data together to obtain means and standard errors. A 5-minute cycle of EFS was maintained throughout the experiment and fresh ACh-containing Krebs was added to the bath after every EFS to maintain constant tone. Force traces with or without ACh are schematically illustrated in Figure 1A. The continued presence of ACh established a basal tone, which was proportional to ACh concentration.
Figure 1. Schematic illustration of electrical field stimulation (EFS) with or without an ACh-induced tone. (A) After establishing a maximal and stable EFS-induced maximal force (Fmax) during the equilibration period, a predetermined concentration of ACh was added to the muscle bath to induce a muscle tone. The muscle was stimulated briefly (9 s) by EFS once every 5 minutes throughout the experiment, and the basal tone, EFS-induced force, and total force (ACh tone plus EFS force) were recorded. (B) Same as in A, except that a quick isotonic release was applied to the muscle 180 seconds after each EFS. The load to which the muscle was released to was set at 5% Fmax. The shortening velocity was determined as the slope of the length trace 200 milliseconds after the onset of the quick release.
Experimental Protocol for Velocity Measurement To assess the change in the cycling rate of the muscle cross-bridges during the prolonged exposure to ACh-induced tone, a different set of experiments was carried out to measure the shortening velocity of muscle preparations activated by ACh during the time course of exposure. In these experiments, as illustrated in Figure 1B, both isometric force induced by EFS (measured at the peak of contraction) and the shortening velocity of ASM against a constant load were measured (180 s after the EFS-induced force had returned to its baseline tone). The constant (isotonic) load was set to 5% of Fmax and the velocity was measured 200 milliseconds after the transition from an isometric to an isotonic contraction (the quick release). The velocity was taken as the slope of the length trace 200 milliseconds after the onset of quick release; the short delay allowed the passive recoil of the muscle to settle after the quick release. ACh-containing Krebs was added every EFS to ensure that the tone induced by ACh was constant throughout the experiment.
Statistical Analyses In all experiments, force data from each animal were normalized to Fmax and velocity data from each animal were normalized to Lref, before averaging the means from different animals. Data shown are means 6 SE. Repeated measures ANOVA followed by Tukey’s a posteriori test for comparison between all pairs of contractions were performed to compare Fmax or shortening velocity before and throughout the experiment after ACh administration. The same tests were used for paired comparisons between control (without ACh) and every concentrations of ACh tested for both adapted and nonadapted ASM. Two-way ANOVA were used to determine the effect of adaptation, the effect of ACh concentrations and the interaction between them on total force or velocity. All statistical analyses were performed using Prism 4 (GraphPad Software, San Diego CA) and a P less than or equal to 0.05 was arbitrarily considered to be sufficient to reject the null hypothesis.
RESULTS The EFS-induced force decreased initially after the administration of ACh (1027 M). However, the force increased with a rate constant of 0.068 min21 and leveled off over a time period of approximately 50 minutes (Figure 2). During this period, the muscle tone induced by ACh was maintained between 35 and 40% of Fmax. As shown in Figure 2, the time course of force increase could be described quite accurately with a single exponential equation. The curve fit indicated that the force plateau was at about 91% Fmax; the force ‘‘recovery’’ was therefore incomplete (i.e., it did not reach Fmax), despite the significant (one-way ANOVA, P , 0.05) and substantial increase in force. The magnitude of the initial decrease in EFS-induced force and the extent of its subsequent recovery were different depending on the level of ACh-induced tone, as shown in Figure 3. At the low dose of ACh (3 3 1028 M), the initial decrease in EFS force was absent, and after the recovery period, the EFS force exceeded Fmax. At higher concentrations of ACh (> 1027 M) the EFS force decreased significantly initially and remained less than Fmax even after the recovery period. The higher the ACh tone, the more substantial was the decrease in both the initial and final EFS forces. Two-way ANOVA indicates that the differences between initial and final EFS force over the entire range of ACh-induced tone were significant (P 5 0.01) (Figure 3, solid circles). An interesting observation was that the total force (ACh tone plus EFS force) increased monotonically with ACh concentration, above and beyond Fmax. The difference between the initial and final total
Bosse´, Chin, Pare´, et al.: Force Adaptation in Airway Smooth Muscle
Figure 2. Time course of change in EFS-induced force before and after administration of ACh (1027 M). The data points represent peak EFS force (without ACh tone) recorded during the time course illustrated in Figure 1A. The data points after time zero were fitted with an exponential equation of the form: y 5 yo 1 a(12e2bx). yo 5 0.720 (Fmax) 6 0.012 (SE), a 5 0.192 (Fmax) 6 0.012, b 5 0.068 (min21) 6 0.013. The increase in force after time zero is statistically significant (one-way ANOVA, P , 0.05) (n 5 14).
force over the entire range of ACh tone was significant (twoway ANOVA, P 5 0.03; Figure 3, open squares). There was, however, no significant difference between the initial and final tone (Figure 3, open circles). There was a large variation in the muscle tone induced by a given concentration of ACh in different muscle preparations (for example, the range of response to 1027 M of ACh was 0.11– 0.79 Fmax). Therefore, in addition to examining the force changes as a function of [ACh], we also examined the initial and final EFS forces as functions of the level of ACh-induced tone. In Figure 4, data were plotted according to the amplitude of the ACh-induced tone versus EFS-induced force, both at the first contraction after ACh administration (open circles) and
Figure 3. Changes in muscle tone, EFS force, and total force as functions of [ACh]. The initial tone or force was measured 1 minute after exposure of the muscle to ACh; the final tone or force was measured at the end of a recovery period (as depicted in Figure 2). Two-way ANOVA indicates that the initial and final EFS forces are different (P 5 0.01), and that the initial and final total forces are different (P 5 0.03), whereas there is no significant difference between the initial and final tones (n 5 4).
15
Figure 4. Initial and final EFS forces as functions of ACh tone. Each group was fitted with a linear function of the form: y 5 yo 1ax. For the initial force group (open circles), yo 5 1.029 (Fmax) 6 0.019 (SE), a 5 20.686 6 0.040. For the final force group (solid circles), yo 5 1.110 (Fmax) 6 0.015, a 5 20.502 6 0.026. The two groups of data were significantly different from each other according to two-way ANOVA (P 5 0.001). The slopes (a values), however, are not significantly different (P . 0.05). The final forces obtained at ACh tone less than 20% Fmax (solid circles above the dotted line) are significantly higher than Fmax (P 5 0.004).
after force adaptation (vertically aligned solid circles). Two-way ANOVA indicates a significant difference (P 5 0.001) between the initial (before adaptation) and final (after adaptation) EFSinduced forces. Linear relationships were obtained for both forces as functions of ACh-induced tone, suggesting that the initial decrease in EFS-induced force after the administration of ACh was directly proportional to the level of ACh tone (Figure 4, open circles). A similar relationship was found for the final EFS-induced force (Figure 4, solid circles). The slopes for both the linear fits were not significantly different from each other (P . 0.05). An interesting observation was made for the EFSinduced forces obtained at levels of ACh-induced tone less than 20% of Fmax. While there was no significant decrease in the initial force from Fmax (Figure 4, open circles), the final force was significantly higher (P , 0.05) than Fmax, indicating potentiation of EFS force at low levels of tone. To probe the mechanism underlying the tone, we examined the shortening velocity of the muscle at different time points during a period in which muscle tone was maintained at a constant level. If the tone was ‘‘active,’’ (that is, maintained by active actomyosin interaction), we should see significant rate of cycling of the cross-bridges, and therefore a significant shortening velocity. If, on the other hand, the tone was ‘‘passive,’’ (that is, it was not maintained by cross-bridges interacting with actin filaments, but instead by other mechanisms such as cross-linking of cytoskeletal filaments), then the muscle might not be capable of shortening. Figure 5 shows the time course of the decrease in velocity during a period of maintained constant ACh tone. At low Ach-induced tone (, 20% Fmax), the shortening velocity declined with a rate constant of 0.0504 min21 and approached zero velocity in about 30 minutes (Figure 5A). At higher Ach-induced tone (> 20% Fmax), the velocity declined at a somewhat higher rate (0.181 min21) but did not approach zero velocity (Figure 5B). To exclude the possibility that the decline in shortening velocity was due to periodic EFS or quick releases, control experiments were performed in the presence of ACh tone but without periodic EFS (Figure 6, open circles), and without intervening quick releases (Figure 6, solid circles). Velocity of the toneinduced shortening decreased over time in the same fashion as
16
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 40 2009
Figure 6. Decline of velocity during the time course of a maintained muscle tone without periodic EFS (open circles). As in Figure 5, the data were normalized to the initial velocity (Vi, first measurement after ACh administration) and fitted with the same exponential equation. The ACh tone was 20 to 30% Fmax. The shortening velocity decreased exponentially with a rate constant (b) of 0.132 (min21) 6 0.019. The velocity at time infinity (yo) was 0.50 (Vi) 6 0.019. Vi 5 5.76 (%Lref/s) 6 0.74. The solid circles were obtained without periodic EFS and without the intervening quick releases for 26 minutes after the administration of ACh (n 5 3 for all data points).
Figure 5. Decline of velocity during the time course of a maintained muscle tone. The data were normalized to the initial velocity (Vi, first measurement after ACh administration) and fitted with an exponential equation of the form: y 5 yo 1 ae2bx. (A) At low ACh tone (,20% Fmax), the shortening velocity decreased exponentially with a rate constant (b) of 0.0504 (min21) 6 0.0354 (SE). The velocity at time infinity (yo) was 20.148 (Vi) 6 0.402, not different from zero (P . 0.05). Vi 5 2.27 (%Lref/s) 6 0.58. (B) At higher ACh tone (> 20% Fmax), the shortening velocity decreased exponentially with a rate constant of 0.181 (min21) 6 0.044. The velocity at time infinity (yo 5 0.584 (Vi) 6 0.025) was significantly different from zero (P , 0.05). Vi 5 7.47 (%Lref/s) 6 1.21. Numbers besides the symbols indicate the number of experiments (n).
that in the presence of periodic EFS (Figure 5B). The rate constants obtained from fitting the data in Figures 5B and 6 were not different (P . 0.05). Velocities obtained without periodic EFS and quick releases (Figure 6, solid circles) were not different from those obtained with prior history of periodic quick releases (open circles at the same time points in Figure 6) (P . 0.05). The control observation indicated that it was the constant presence of ACh but not the periodic EFS or quick releases that was responsible for the observed decrease of shortening velocity over time.
DISCUSSION The most important finding of this study is that the ability of ASM to generate force can be enhanced by chronic exposure of the muscle to submaximal concentrations of a contractile agonist—in this case ACh. This suggests that prolonged exposure of ASM in vivo to spasmogens (a likely scenario in asthmatic airways) could lead to increased force production in the muscle and airway hyperresponsiveness. There are four interesting aspects of our main finding. First, the total force (ACh-tone plus EFS-force) is always greater than
the maximal EFS force (Fmax) without ACh-tone (Figure 3). This shows that a combination of two stimuli (EFS and ACh) boosts the force-generating capacity of ASM, either by recruiting more contractile units or through other mechanisms (as discussed in more details later). Second, the total force can be further increased through a process of repeated EFS in the presence of a constant tone. This force increase over time is solely due to the increase in the EFS-induced force over time (Figure 2). Third, the greater the ACh-induced tone, the greater is the increase in total force (Figure 3). Finally, at low levels of muscle tone, EFS force is augmented by a synergistic mechanism so that it exceeds the Fmax obtained in the absence of the tone. The phenomenon of ASM adapting to elevated muscle tone and generating more total force over time has never been described. This muscle behavior shares many similarities to the phenomenon of length adaptation found in the same muscle (14). Length adaptation is defined as the process of force recovery after a change in muscle length. After a length perturbation, force increases during the recovery period after a single exponential time course to reach a plateau in approximately 30 minutes, provided the muscle goes through several cycles of activation and relaxation (14). Here we define the process of force recovery after exposure of the muscle to an elevated tone as force adaptation; the increase in active force also follows a single exponential time course, although it has a slower rate constant, reaching a plateau in approximately 50 minutes (Figure 2). While several mechanisms underlying length adaptation have been proposed (14–20), nothing is known about the mechanisms driving force adaptation. The four aspects of force adaptation described above may or may not share the same underlying mechanism(s). To facilitate discussion, we group the results in different categories. Relationship between Initial Force and Tone: The Unadapted State
Because EFS activates ASM mainly through pre-junctional release of ACh (21, 22), one would expect that once the postjunctional ACh receptors are saturated with the agonists,
Bosse´, Chin, Pare´, et al.: Force Adaptation in Airway Smooth Muscle
a constant maximal response (manifested as the total force produced by the muscle) would be obtained. This implies that the muscle tone induced by exogenous ACh plus that induced by EFS would add up to be a constant, and that ACh tone and EFS-induced force will be reciprocally related. Although we observed the reciprocal relationship between EFS-induced force and ACh-induced tone, the two do not add up to a constant (Figure 3). The results suggest that the exogenous ACh together with ACh released by EFS have activated a pool of post-junctional ACh receptors that were not activated by EFS alone. In other words, ACh released by EFS in our preparation did not saturate the post-junctional ACh receptors. This explanation of course relies on the assumption that the receptor occupancy is directly translated into activation of cross-bridges and force generation. If this is the case, a low level of tone induced by a low dose of ACh (3 3 1028 M, Figure 3) appears to be associated with a population of cross-bridges separated from that activated by EFS, because the EFS force was not reduced (from Fmax) at that level of tone. At higher concentrations of ACh, there appears to be an overlap between the pools of cross-bridges activated by exogenous ACh (added to the muscle bath) and endogenous ACh (from EFS). It is possible too that the increase in the initial total force (Figure 3) (before force adaptation) is due to calcium sensitization. For this explanation to be valid, we have to assume that calcium sensitization is induced only by chronic presence of exogenous ACh, and not by ACh release from the relatively brief EFS. Relationship between Final Force and Tone: The Adapted State
The increase in final total force (Figure 3) is mostly driven by the increase in EFS force over time (Figure 2), with a possible minor contribution from the slight decrease in ACh-induced tone (Figure 3). This increase in the final adapted total force occurs at all levels of tones examined in this study (Figure 3). An interesting observation from Figure 3 is that the increment in total force which occurs after force adaptation (i.e., the difference between initial and final total force) is independent of ACh concentration. That is, within the [ACh] range of 3 3 1028 to 1026 M, the final total force is shifted upward by a constant amount from the initial total force. To explain this additional gain in total force after adaptation, we hypothesize that during the adaptation process, force generated and borne by the cross-bridges is gradually transferred to a passive (non-cross-bridge related) structure of the cytoskeleton which functions in parallel with the contractile filaments in bearing the total force. Such a structure could be formed by cross-linking of cytoskeletal filaments, or by other mechanisms that transform a non–force-bearing cytoskeletal structure into one that is capable of bearing tension. Recent findings from Fredberg’s laboratory (18–20) have provided strong evidence for the existence of such a dynamic structure in smooth muscle cells. If prolonged exposure to agonist stimulation led to the formation of a passive force-bearing structure in the cytoskeleton of smooth muscle cells, one would expect that such a structure would not generate active shortening if the muscle is quickly released to a load lower than the ‘‘tone’’ (i.e., the force borne by the structure before the quick release). When we examined shortening velocities of the muscle after quick releases, we found that for low levels of muscle tone (, 20% Fmax), velocity declined exponentially toward zero during the time course of force adaptation (Figure 5A). At the end of a force-adaptation protocol, when the EFS-induced force reached a plateau (Figure 2), the baseline tone induced by ACh was no longer capable of generating active shortening in the
17
muscle (Figure 5A), suggesting that the tone has been transformed from one that is maintained by actomyosin cross-bridges to one that is not cross-bridge related. The time courses of velocity decline (Figure 5A) and EFS force recovery (Figure 2) may be manifestations of this transformation. At high levels of ACh tone (. 20% Fmax), quick releases did not produce zero shortening velocity at the end of our adaptation protocol (Figure 5B). It could be that the putative passive cytoskeletal structure can only bear a limited amount of force (e.g., , 20% Fmax), and that the higher tone contains an active force component (borne by cross-bridges), so that when the muscle is quickly released to a low load, the cross-bridges are able to overcome the impedance provided by the passive structure and cause active shortening. This could explain why active shortening persists in muscles released from a higher tone, even when it is at the end of the adaptation protocol (Figure 5B). The notion that the passive structure bears a constant and relatively small amount of tension is consistent with the observation that the gain in total force after force adaptation (Figure 3, square symbols) is constant at all tested levels of ACh tone and the difference between the final and initial total forces is , 20% Fmax. One could argue that the decline in velocity is due the formation of a population of slowly cycling bridges as described by Murphy’s group (23, 24). To adopt this explanation, one has to assume that true latch bridges exist (to explain the zero velocity found in this study; Figure 5A). The model of Hai and Murphy (24), however, assumes a nonzero velocity for their slowly cycling bridges. The model of slowly cycling bridges cannot explain easily the increase in EFS-induced force or total force after force adaptation (Figure 3), unless it is assumed that there is a population of truly latched bridges (incapable of cycling when the muscle is released) and that these bridges are capable of maintaining more force per bridge than the normal bridges. Another possible explanation is provided by Siegman and colleagues (25). They have described a state of weak crossbridge attachment to thin filaments at basal levels of calcium in resting state that is capable of resisting stretch but incapable of causing the muscle to shorten or develop tone. The behavior of these weakly attached cross-bridges in resting state fits our description of the force-bearing ‘‘passive’’ structure, but, similar to the slowly-cycling-bridge model (24), it does not offer a simple explanation for the phenomenon of force adaptation, in which the total force generated by the muscle increases over time. Because both of these models assume that the forcebearing structures are cross-bridges themselves, to explain force adaptation one has to invoke a mechanism that involves recruitment of new cross-bridges, or a mechanism that increases force generated per cross-bridge. We are not aware of any evidence supporting either of the mechanisms. Yet another possible explanation for force adaptation is the mechanism of calcium sensitization (26). It is possible that prolonged exposure to exogenous ACh sensitizes the muscle so that for the same amount of Ca21 released by each EFS more force is generated over time. Presumably this happens when myosin light chain phosphatase (MLCP) is inhibited. For this mechanism to work, we have to assume that EFS alone does not lead to inhibition of MLCP in ASM. Calcium sensitization, however, does not explain the abolishment of shortening velocity observed in this study (Figure 5A). Relevance to Airway Hyperresponsiveness
Regardless of the mechanism(s) underlying the phenomenon of force adaptation, the fact that prolonged exposure to contractile agonists can lead to enhancement of force-generating ability of a second stimulus should be of interest to investigators looking
18
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 40 2009
for mechanisms underlying airway hyperresponsiveness, such as that seen in asthma. It has been shown that inflammatory mediators are up-regulated in asthmatic airways (4–12), and endogenous ACh may promote changes in ASM phenotype triggered by the presence of these mediators (27, 28). Results from the present study suggest that inflammatory mediators may not only regulate phenotypic changes in ASM but when coupled with elevated [ACh] could also alter the mechanical function of ASM. The most important consequence of force adaptation in ASM is the increase in total force that the muscle can generate. This could lead to excessive shortening of the muscle and exaggerated airway narrowing. Even if the airways are not totally occluded, the enhancement in ASM force through force adaptation could make the partially narrowed airways more resistant to stretch. This could explain the refractoriness of asthmatic airways to the bronchodilating effect of deep inspiration (29) that normally stretches the airways and relaxes the ASM (29, 30).
CONCLUSIONS The present study describes a new phenomenon in ASM behavior: force adaptation. Although force and length adaptation are similar, it is likely that they stem from different underlying mechanisms and both could independently contribute to exaggerated airway narrowing in asthma. The increased total force that occurs during force adaptation in ASM appears to involve transformation of a cytoskeletal component from a non–force-bearing structure to one that is able to withstand tension. This transformation allows transfer of force generated by the actomyosin cross-bridges to ‘‘passive’’ structures and in turn frees up cross-bridges to generate ‘‘active’’ force when they are called upon to do so. The ‘‘passive’’ structures have no contractile function, because they do not cause the muscle to actively shorten when the tension on these structures is released. Conflict of Interest Statement: P.D.P. is the principal investigator of a project funded by GlaxoSmithKline to develop CT-based algorithms to quantify emphysema and airway disease in COPD. With collaborators he received approximately $300,000 to develop and validate these techniques. The funds he has applied solely to the research to support programmers and technicians. He is also principal investigator of a Merck Frosst–supported research program to investigate gene expression in the lungs of patients who have COPD. He and collaborators have received approximately $200,000 for this project. These funds have supported the technical personnel and expendables involved in the project. None of the other authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
References 1. An SS, Bai TR, Bates JH, Black JL, Brown RH, Brusasco V, Chitano P, Deng L, Dowell M, Eidelman DH, et al. Airway smooth muscle dynamics: a common pathway of airway obstruction in asthma. Eur Respir J 2007;29:834–860. 2. McParland BE, Macklem PT, Pare PD. Airway wall remodeling: friend or foe? J Appl Physiol 2003;95:426–434. 3. McParland BE, Tait RR, Pare PD, Seow CY. The role of airway smooth muscle during an attack of asthma simulated in vitro. Am J Respir Cell Mol Biol 2005;33:500–504. 4. White J, Eiser NM. The role of histamine and its receptors in the pathogenesis of asthma. Br J Dis Chest 1983;77:215–226. 5. Lam S, Chan H, LeRiche JC, Chan-Yeung M, Salari H. Release of leukotrienes in patients with bronchial asthma. J Allergy Clin Immunol 1988;81:711–717. 6. Mattoli S, Soloperto M, Marini M, Fasoli A. Levels of endothelin in the bronchoalveolar lavage fluid of patients with symptomatic asthma and reversible airflow obstruction. J Allergy Clin Immunol 1991;88:376–384.
7. Murray JJ, Tonnel AB, Brash AR, Roberts LJ II, Gosset P, Workman R, Capron A, Oates JA. Release of prostaglandin D2 into human airways during acute antigen challenge. N Engl J Med 1986;315:800–804. 8. Wenzel SE, Westcott JY, Larsen GL. Bronchoalveolar lavage fluid mediator levels 5 minutes after allergen challenge in atopic subjects with asthma: relationship to the development of late asthmatic responses. J Allergy Clin Immunol 1991;87:540–548. 9. Driver AG, Kukoly CA, Ali S, Mustafa SJ. Adenosine in bronchoalveolar lavage fluid in asthma. Am Rev Respir Dis 1993;148:91–97. 10. Liu MC, Hubbard WC, Proud D, Stealey BA, Galli SJ, Kagey-Sobotka A, Bleecker ER, Lichtenstein LM. Immediate and late inflammatory responses to ragweed antigen challenge of the peripheral airways in allergic asthmatics: cellular, mediator, and permeability changes. Am Rev Respir Dis 1991;144:51–58. 11. Krug N, Tschernig T, Erpenbeck VJ, Hohlfeld JM, Kohl J. Complement factors C3a and C5a are increased in bronchoalveolar lavage fluid after segmental allergen provocation in subjects with asthma. Am J Respir Crit Care Med 2001;164:1841–1843. 12. Tomaki M, Ichinose M, Miura M, Hirayama Y, Yamauchi H, Nakajima N, Shirato K. Elevated substance P content in induced sputum from patients with asthma and patients with chronic bronchitis. Am J Respir Crit Care Med 1995;151:613–617. 13. Gosens R, Zaagsma J, Meurs H, Halayko AJ. Muscarinic receptor signaling in the pathophysiology of asthma and COPD. Respir Res 2006;7:73. 14. Pratusevich VR, Seow CY, Ford LE. Plasticity in canine airway smooth muscle. J Gen Physiol 1995;105:73–94. 15. Gunst SJ, Meiss RA, Wu MF, Rowe M. Mechanisms for the mechanical plasticity of tracheal smooth muscle. Am J Physiol 1995;268:C1267– C1276. 16. Kuo KH, Herrera AM, Wang L, Pare PD, Ford LE, Stephens NL, Seow CY. Structure-function correlation in airway smooth muscle adapted to different lengths. Am J Physiol Cell Physiol 2003;285:C384–C390. 17. Herrera AM, McParland BE, Bienkowska A, Tait R, Pare PD, Seow CY. ‘Sarcomeres’ of smooth muscle: functional characteristics and ultrastructural evidence. J Cell Sci 2005;118:2381–2392. 18. Bursac P, Lenormand G, Fabry B, Oliver M, Weitz DA, Viasnoff V, Butler JP, Fredberg JJ. Cytoskeletal remodelling and slow dynamics in the living cell. Nat Mater 2005;4:557–561. 19. Deng L, Trepat X, Butler JP, Millet E, Morgan KG, Weitz DA, Fredberg JJ. Fast and slow dynamics of the cytoskeleton. Nat Mater 2006;5:636–640. 20. Trepat X, Deng L, An SS, Navajas D, Tschumperlin DJ, Gerthoffer WT, Butler JP, Fredberg JJ. Universal physical responses to stretch in the living cell. Nature 2007;447:592–595. 21. Baker DG, Don HF, Brown JK. Direct measurement of acetylcholine release in guinea pig trachea. Am J Physiol 1992;263:L142–L147. 22. Wang Z, Robinson NE, Yu M. ACh release from horse airway cholinergic nerves: effects of stimulation intensity and muscle preload. Am J Physiol 1993;264:L269–L275. 23. Dillon PF, Aksoy MO, Driska SP, Murphy RA. Myosin phosphorylation and the cross-bridge cycle in arterial smooth muscle. Science 1981;211: 495–497. 24. Hai CM, Murphy RA. Regulation of shortening velocity by cross-bridge phosphorylation in smooth muscle. Am J Physiol 1988;255:C86– C94. 25. Siegman MJ, Butler TM, Mooers SU, Davies RE. Calcium-dependent resistance to stretch and stress relaxation in resting smooth muscles. Am J Physiol 1976;231:1501–1508. 26. Sanderson MJ, Delmotte P, Bai Y, Perez-Zogbhi JF. Regulation of airway smooth muscle cell contractility by Ca21 signaling and sensitivity. Proc Am Thorac Soc 2008;5:23–31. 27. Gosens R, Zaagsma J, Grootte Bromhaar M, Nelemans A, Meurs H. Acetylcholine: a novel regulator of airway smooth muscle remodelling? Eur J Pharmacol 2004;500:193–201. 28. Halayko AJ, Tran T, Ji SY, Yamasaki A, Gosens R. Airway smooth muscle phenotype and function: interactions with current asthma therapies. Curr Drug Targets 2006;7:525–540. 29. Fish JE, Peterman VI, Cugell DW. Effect of deep inspiration on airway conductance in subjects with allergic rhinitis and allergic asthma. J Allergy Clin Immunol 1977;60:41–46. 30. Nadel JA, Tierney DF. Effect of a previous deep inspiration on airway resistance in man. J Appl Physiol 1961;16:717–719.
