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Discipline of Physiology, School of Biomedical and Chemical Science, University of Western Australia, Perth 6009, Australia
Submitted 10 March 2004 ; accepted in final form 12 May 2004
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ABSTRACT |
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bronchoconstriction; airway smooth muscle; deep inspiration
The causes of bronchodilation and bronchoprotection are not fully understood but may involve the effects of cyclical force on ASM (6, 34, 36). With normal tidal breaths and DI, airways expand in diameter and also elongate (1, 12, 19). The effects of cyclical expansion on airway function has been modeled in a number of studies. Isolated airway and ASM preparations exhibit bronchodilation when they are cycled in the presence of ASM tone (7, 9, 11). Evoked ASM force is also depressed after an earlier period of stretch, implicating ASM in bronchoprotection (37). The effect of dynamic stress on ASM appears to involve the reorganization of cytoskeletal structures (10, 26, 34). However, a recent study (23) using whole airway segments failed to demonstrate bronchoprotection despite a reduction in force generation by isolated ASM, suggesting that the airway wall modifies the response of ASM to dynamic load. Force-generating and load-bearing parts of the airway wall are also subject to dynamic stress when the airway lengthens. For example, the length of ASM cells may be altered as an airway lengthens and the cells broaden transversely, depending on the geometric arrangement of the muscle in the airway wall (29). It is not known whether cyclical elongation or stretching of an airway regulates the physiological response of the airway to contractile stimulation and whether elongation contributes to the effects of DI.
Broadly, this study sought to determine whether cyclical elongation of an isolated airway altered bronchoconstrictor responses. Two types of protocols were carried out. In the first, relaxed bronchi were cycled over a range of lengths and frequencies. The effect, if any, of cycling on subsequent bronchoconstrictor responses to electrical field stimulation (EFS) was determined. In the second, airways were pretoned with carbachol and then cycled in length to determine the airway response.
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METHODS |
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2-mm inner diameter) was mounted horizontally in a 40-ml organ bath containing Krebs solution [in mM: 121 NaCl, 5.4 KCl, 1.2 MgSO4, 25 NaHCO3, 5.0 Na morpholinopropane sulfonic acid (pH 7.3), 11.5 glucose, and 2.5 CaCl2] and gassed with 95% O2-5% CO2. At one end of the airway, the lumen was mounted on a cannula fixed to one side of the organ chamber. The other end of the airway was mounted on an adjustable cannula that could slide in or out of the organ bath, allowing the airway length to be freely changed. The cannulas and the airway lumen were filled with Krebs solution via a reservoir, the height of which set the lumen (transmural) pressure. The connectors at each end of the airway segment also incorporated three-way taps, which could be opened to flush the airway with Krebs solution or closed to make the airway lumen isovolumic. The pressure in the airway lumen was recorded with a pressure transducer attached to one arm of a three-way tap. The segment was initially mounted at its resting length, defined as the length of the segment when dissected free from the parenchyma at zero transmural pressure (Li). The length of the airway segment could be changed by moving the adjustable connector described above, using custom-designed software to control a stepping motor. In this manner, the segment could be lengthened by known increments or it could be oscillated at preset frequencies and amplitudes of displacement.
Protocols. At each of the airway lengths studied, a pressure test was conducted to ensure there were no leaks. Segments were then rested for 1 h, during which the lumen and adventitia were regularly flushed with fresh Krebs solution. Evoked contractions were recorded from the pressure transducer connected to the airway lumen under isovolumic conditions. Airways were first stimulated with a 10–4 M dose of acetylcholine to establish viability. After washout and recovery, contractile responses were obtained to EFS via platinum wire electrodes and a Grass stimulator (60 V, 3 ms, 30 Hz) or by adding carbachol to the bathing solution. Airways were subjected to trains of electrical stimulation until maximum contraction was reached (typically 20 s). EFS-induced contractions were evoked at 5-min intervals with the airway at Li (unless specified) and at 5 cmH2O passive lumen pressure, i.e., the pressure that provides optimal airway responses (8, 32).
Three lengthening protocols were used. First, baseline response to EFS were established under isovolumic conditions with airway length at Li. The airway was then stretched stepwise by 1-mm increments while a constant lumen pressure was maintained. At each successive length, EFS responses were recorded. Second, after baseline EFS responses were established, airways were subjected to 5 min of length oscillation (see below) while a constant lumen passive pressure was maintained. After oscillation, an EFS-induced response was recorded and repeated at 5-min intervals. Third, the airway was contracted with a submaximal dose (10–6 M) of carbachol under isovolumic conditions. When a plateau in response was obtained, the length of the segment was oscillated isovolumically for 5 min. A schematic of protocol 3 is shown in Fig. 1.
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Histology and morphometry. Airways were fixed in 5% (bath concentration) formaldehyde at a transmural pressure of 5 cmH2O at lengths of Li, or 105, 115, or 130% Li. A midlength portion of the bronchus was then processed into wax blocks, cut into 6-µm sections, and stained with hematoxylin and eosin.
The following morphological measurements were made with Optimas image analysis software: the perimeter of the airway epithelium, the outer perimeter of the cartilage plates, and lumen area (the area enclosed by the perimeter of the airway epithelium). Airway cross-sectional area was the area enclosed by the outer perimeter of the cartilage plates. Total wall area was calculated by subtracting lumen area from cross-sectional area. Measurements from four to six sections per airway were averaged.
Statistics. Oscillation amplitudes reported refer to the peak-to-trough amplitude as a percentage of Li. In precontracted airways, oscillatory airway pressure during length cycling was recorded at the origin (midpoint) of the pressure cycle. Unless specified differently, changes in pressure of pretoned airways refer to the change from the origin of the first complete cycle at the start of oscillation to the origin of the cycle of interest (Fig. 1). Mean values were compared by ANOVA, using GraphPad Prism software. Relationships between airway length and morphology were analyzed by linear regression. Values reported are means ± SE. P < 0.05 was taken as significant.
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RESULTS |
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115% Li), EFS-induced responses increased slightly but then fell away more steeply with greater stretch (Fig. 2). Airways fixed at different lengths (but constant lumen pressure, 5 cmH2O) showed that the total wall area was inversely related to the degree of airway stretch (Fig. 3) (P < 0.01, R2 = 0.9662). The lumen area tended to be reduced with stretching but not significantly (R2 = 0.7532).
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Cycling on precontracted isovolumic airways. The response of pretoned airways (carbachol, 10–6 M) to cyclical stretch was complex and included both an immediate passive and a slower active (see below) change in pressure. The immediate response on stretching airways was an abrupt drop in lumen pressure (Fig. 5). When airway length was cycled, lumen pressure showed oscillatory variation corresponding to the parameters of length cycling (Fig. 5). The amplitude of the pressure oscillations increased with amplitude of length cycling but was reduced by the frequency at which the airway was cycled. At the origin of the cycle, however, pressure was frequency independent. Similar responses were observed in passively pressurized airways and in segments of rubber tubing oscillated under the same conditions (data not shown), suggesting that these immediate responses reflect passive rather than active phenomena.
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10 s from the start of oscillation. For example, cycling at 30% amplitude at 2 Hz reduced pressure by 22.2 ± 1.5% (Fig. 6B). This transient fall in pressure was followed by a gradual increase in pressure reaching steady state within 2–3 min of cycling that was sustained until cycling stopped (Figs. 1 and 6, A and C). At lower oscillation parameters, only the excitatory response to length cycling was present. The increase in pressure from the previous low point was significant (P < 0.001–0.05, point B to C in Fig. 1) at all frequencies and amplitudes. Under most conditions of cycling, the pressure increased above the initial pressure recorded on the first cycle. For example, at 0.5 Hz and 30% amplitude, the pressure increased by 20.5 ± 1.2% above the initial pressure (P < 0.001) (Figs. 1 and 6C). Neither the sustained rise in pressure nor the transient fall was present in rubber tubes, suggesting that they were an active physiological response.
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A separate group of pretoned airways were cycled in the presence of indomethacin (10–6 M) to assess a possible contribution from prostaglandins to the airway response. The reduction in lumen pressure normally present 10 s after the beginning of cycling was also present in indomethacin-treated airways (Fig. 7). However, the degree of relaxation was significantly greater (P < 0.01) than that observed without indomethacin. As in nontreated airways, indomethacin-exposed airways also showed a secondary rise in pressure after bronchodilation (point B to C in Fig. 1; P < 0.001–0.01). This size of the contractile effect was not different from nontreated airways. However, because of the strong early bronchodilator response preceding the excitatory response, the lumen pressure did not rise above the pressure at the start of cycling (point A to C in Fig. 1) as previously observed in experiments without indomethacin.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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We first showed that EFS-induced contractions were length dependent when responses were recorded under static conditions. Figure 2 shows a blunt length-response curve in which peak contractions were present in airways stretched 15% above Li (i.e., 115% Li). These findings extend the range of airway lengths studied in a previous report from this laboratory (35). In a study of human airways, the EC50 of acetylcholine was not changed by lengthening the airway (20), which along with our own findings suggests that stretching an airway alters the maximum response rather than airway sensitivity. The cause of the static length dependency of airway responses to EFS is uncertain but may involve changes to ASM length accompanying airway elongation. Studies have shown a spiraled orientation of ASM in the bronchi (4, 29). The angle of pitch varies between species (10–30%) and varies greatly at different locations (e.g., at branching points) within species (29). The effect of airway stretch on ASM length depends on the angle of ASM pitch and on the change in airway circumference on stretching. For an ideal compliant tube with a constant wall volume, when the tube is lengthened, the wall area of the tube will fall, producing changes in thickness and circumference. Histology on airways fixed under isobaric conditions indicated that the airway wall area fell on stretching, a result broadly in agreement with studies in other species (13). In the ideal case of an isotropic tube where the wall is thin compared with tube caliber, the circumference will fall on stretching the tube. If ASM is aligned circumferentially around the airway, the ASM length will fall with circumference as the airway stretches. In a contrasting case where the ASM runs axially, ASM length increases in proportion to airway length. The effect of airway stretch on ASM length therefore depends on the pitch angle of the ASM, which itself changes with airway length.
Indirect studies suggest that the angle of pitch of ASM in pig airways is smaller than in some other species (21); i.e., the ASM is close to perpendicular. A fall in ASM length is therefore predicted when the airway is stretched. A physiological effect of increasing or decreasing airway length would depend partly on the position of ASM on its length-tension curve. If the static length dependency of airway responsiveness reported in the present study (Fig. 2) is related to changes in ASM length, the increased size of EFS-induced contractions as airways were stretched from Li to 115% Li could only occur if ASM was initially beyond its optimal length at Li and 5 cmH2O lumen pressure, as used. In previous studies, our laboratory found that optimal length for isolated pig bronchi occurs at transmural pressures between 5 and 10 cmH2O (8, 32); however, in those studies, the airway was stretched beyond Li. We suggest that ASM operating length is dependent on both the diameter of the airway and its length. An implication is that evoked ASM force and the resulting bronchoconstriction could vary with lung volume through changes to airway length as well as directly by airway circumferential expansion. In addition to a potential effect of airway length on ASM length, stretching airways may also stretch ASM cells transversely to their axis of contraction. A possible effect of transverse stretch on ASM responsiveness is not known. Cultured ASM align themselves perpendicular to applied stress (30) and show increased force and shortening (31), suggesting that transverse stretch might have an effect on ASM contraction.
In studying the effect of airway length under dynamic conditions, we chose stretch amplitudes and frequencies to span a physiological range. Estimates of lengthening in midsized bronchi between residual volume and total lung capacity are varied and range from 15 to 60% in different species (12, 19, 25). Our unpublished observations in pigs show that airways lengthen by 15% between 0 and 30 cmH2O transmural pressure. Cycling protocols were therefore chosen to approximate tidal breathing (cycling a peak-to-trough amplitude of 5% Li) and DI (15% amplitude) in this species. Greater lengths were also included (30% amplitude) in our analysis to increase the generality of our findings. The above amplitudes of stretch were imposed on airways set initially to Li (length at 0 cmH2O transmural pressure), which could be reliably standardized between experiments and which was close to the airway length expected at residual volume or functional residual capacity. Although resting lengths of 115% Li gave the strongest contractions to EFS, that starting length was not used routinely in oscillation experiments because it would be physiologically unrealistic to oscillate an airway at lengths above total lung capacity. However, for selective purposes, responses were also recorded in airways initially stretched out to 115% Li to determine whether active responses to cycling were dependent on resting length (see below).
To determine whether airway lengthening modified subsequent bronchoconstriction (i.e., whether it induced bronchoprotection), we cycled the length of relaxed airway segments and compared EFS-induced responses before and after cycling. The lumen pressure was held constant during cycling. Periods of cycling caused a small but significant increase in airway response to subsequent EFS. The effect was transient, lasting <5 min from the end of cycling. This effect of cycling required only small increments in airway length (e.g., 5% amplitude), and it showed no strong frequency or amplitude dependency over the ranges tested. EFS-induced contractions were always recorded on the return of the airway segment to the length initially set (i.e., Li) and 5 cmH2O lumen pressure (8, 32). It is possible that there was hysteresis in the relationship between airway length and wall tension, similar to the static length-response curve (Fig. 2) that might alter the size of contractions. To test this possibility, we also cycled airway length starting at 115% Li, which was the airway length giving maximum airway contractions. Under these conditions, there was still a significant increase in EFS-induced contraction subsequent to the period of cycling, suggesting that hysteresis was not the underlying cause of the increased contractions.
The effect of cyclical elongation on subsequent airway responses reported here is very similar to recently reported effects of cyclical expansion in pig airways (23). Circumferential expansion of bronchial segments produces a transient increase of EFS-induced contraction. The similarity in the response of intact airways to cyclical lengthening and cyclical expansion suggests that they may share some common mechanism(s). It is interesting that the effects of cycling on whole airways is in direct contrast to the behavior of isolated ASM, where cycling the length of the muscle reduces rather than increases subsequent force generation (23, 37). The cause of the difference in response of an intact airway and ASM to cyclical stretch has not been determined, but it suggests that properties of the airway wall somehow modify the response of ASM cells in situ. Along with our previous findings (23), our results also suggest that the phenomenon of bronchoprotection in vivo may involve the effects of breathing on other properties of the respiratory system apart from the airway or ASM alone.
In a second protocol, we studied pretoned airways. To maintain tone, these airway segments were cycled under isovolumic conditions. Before discussing the effects of a sustained period of cycling, a number of characteristic responses of pretoned airways to length change need consideration. A single-step increase to airway length produced an abrupt fall in lumen pressure, and when airway length was cycled, lumen pressure oscillated. The peak-to-trough amplitude in oscillatory pressure was frequency dependent with reduced amplitude at higher frequency of length cycling. The origin of the pressure oscillations by contrast held relatively constant across different frequencies of cycling. Similar step changes and oscillations in lumen pressure on cyclical lengthening were also seen in segments of rubber tubing and in passively pressurized airways, indicating a passive rather than active basis. Airway and tube segments were isovolumic and leak-free during these lengthening experiments, leading us to conclude that the passive changes in lumen pressure were produced by recoil forces in the wall of these tubular structures when they were stretched. Both dynamic and static properties of the airway wall (e.g., inertia and compliance) could contribute to the magnitude of the pressure oscillation. It is probable that breathing produces similar length-dependent physical phenomena in the airway in vivo, which may contribute to load on ASM with implications for evoked airway narrowing.
After the immediate passive effects of length cycling discussed above, sustained cycling of carbachol-toned airways produced a bronchodilator effect, particularly at the higher amplitudes of cycling. Bronchodilation was not seen in rubber tubes, indicating that it was an active airway response to cycling. At 30% amplitude, the bronchodilation amounted to 20% relaxation. The inhibitory response peaked 10 s after the start of oscillation. Other studies have shown that cyclical dilation of toned airway preparations also produces a bronchodilator response (7, 11). The effect was not contingent on the starting length of the airway, because it was present whether the airway was cycled from Li or from 115% Li. Breaking cross bridges or cytoskeletal plasticity have been proposed as possible mechanisms accounting for the different effects of cycling in isolated ASM (10, 27, 34). In isolated muscle, only small length perturbations (1%) are needed to reduce force (27). Small changes to the length of precontracted ASM in the present study might also reduce muscle force by a similar process and lead to the bronchodilator effects observed in our study.
After the bronchodilator response, a prominent contractile response developed as cycling continued. The contractile response occurred with most oscillation parameters and was observed whether or not the airway had first shown bronchodilation as discussed above. The contractile response was without strong frequency or amplitude dependence and was not affected by the initial length of the airway.
The more general response of length cycling in relaxed and pretoned airways was thus excitatory. It appears that lengthening the airway stimulates ASM contraction. As discussed above, ASM cell length could be decreased and girth increased on stretching the airway. However, cyclical changes to ASM length are typically associated with reduced active force rather than an increase (7, 9, 11, 23, 37). No evidence was produced supporting indirect activation of ASM by excitatory arachidonic acid metabolites in pretoned airways, because indomethacin did not block excitatory responses. Other contractile mediators might, however, be released as a result of stretching airway tissue, or ASM might be activated directly by mechanical distortion. The increased ASM force produced during cycling is reminiscent of the myogenic response in other smooth muscles, such as blood vessels. An electrophysiological basis of myogenic activity has been widely studied (e.g., stretch-activated channels). Although myogenic contractile responses to stretch are not commonly observed in ASM, there is evidence for stretch-activated channels in airway epithelial cells and possibly ASM (2, 15). Furthermore, a phenomenon similar to myogenic contraction has been reported in ferret trachealis muscle (3) and also in canine ASM in the presence of potassium channel blockers (33). A possible involvement of myogenic activity in the progressive increase in ASM response to cycling in pretoned airways warrants consideration.
Although indomethacin had no effect on excitatory responses, it did appear to increase the size of the bronchodilator response to cycling, suggesting some involvement of prostaglandins. One explanation for this finding is that indomethacin inhibits a contractile prostaglandin that normally attenuates the primary bronchodilator mechanism. However, this explanation seems inconsistent with our inability to demonstrate a contribution from prostaglandins to the excitatory responses of the airway to cycling, discussed above. Complex intermediate steps or differential sensitivity to prostaglandins of ASM contractile and relaxant coupling mechanisms would have to be proposed to reconcile the different effects of indomethacin on the two components of the airway response to cycling reported in our study.
Considerable interest has focused on the potential effects of cyclical expansion of the airway on lung function. Some studies have provided evidence implicating ASM and the airway in the bronchodilator and bronchoprotective effects of DI. The possible contributions of airway length to the effects of DI in vivo appear not to have been considered. Results from our study indicate that static and cyclical changes to airway length influence airway contraction and could therefore influence the response of the whole lung. The paradoxical finding from this and a previous study (23) is that isolated airways fail to show bronchoprotection as seen in vivo and in isolated ASM. Further studies are needed in other species or under different conditions to determine the significance of these findings.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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