Bronchial smooth muscle mechanics of a canine model of allergic airway hyperresponsiveness HE JIANG, KANG RAO, ANDREW J. HALAYKO, WAYNE KEPRON, AND NEWMAN L. STEPHENS Departments of Physiology and Immunology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba R3E OW3, Canada JIANG, HE, KANG RAO, ANDREW J. HALAYKO, WAYNE KEPRON,ANDNEWMAN L. STEPHENS.~~~OIIC~~~~S~OO~~~~~~~ mechanics of a canine model of allergic airway hyperresponsiueness. J. Appl. Physiol. 72(l): 39-45, 1992.-Although we have reported that tracheal smooth muscle from sensitized dogs shows altered mechanical properties, we did not know, because of technical difficulties with the preparation, whether similar changes occur in the properties of sensitized central bronchial smooth muscle (NM), the site at which the acute asthmatic response is believed to develop. We have now succeeded in developing a cartilage-free BSM preparation that retains optimal mechanical properties. Such strips were obtained from mongrel dogs that had been sensitized to ragweed pollen. Controls were littermates injected with adjuvant alone. Length-tension relationships were obtained for both control and sensitized BSM strips (CBSM and SBSM, respectively). The maximal active stresses were the same (P > 0.05) when normalized to muscle fraction in total tissue cross-sectional area [6.2 t 0.6 X lo4 and - 0.6 X lo4 (SE) for SBSM and CBSM, respectively]. This 5.9 + suggests that optimal tension is an insensitive indicator of bronchial hyperresponsiveness and that isotonic studies might be more revealing. The maximal shortening velocity (V,) for SBSM at 2 s [0.35 ,t 0.017 (SE) I,ls, where I, signifies optimal muscle length], in the course of a 10-s contraction, was significantly greater (P < 0.05) than V, measured for CBSM (0.27 t 0.015 I,ls). However, V, did not differ at the 8-s point of contraction. The sensitized group demonstrated a statistically significantly greater maximal shortening capacity (0.67 t 0.04 E,) than the control group (0.51 t 0.04 I,). At 2 s of contraction, 80% of maximal SBSM shortening had been completed and was significantly greater than for CBSM. These data reflect alterations in the early dynamic mechanical properties of SBSM, which indicate that normally cycling cross-bridges are likely involved mechanistically in the changes observed. The increased shortening velocity of SBSM could be due to increased myosin light chain phosphorylation, and the increased shortening capacity could be the result of increased compliance of the muscle’s so-called internal resistor.
bronchial smooth muscle hyperresponsiveness; ragweed-sensitized canine asthma model; mechanism for airway hyperresponsiveness ALTHOUGHTHEPATHOPHYSIOLOGICAL changesleading to asthma are not yet well known, increased airway responsiveness to a number of allergic and nonallergic stim-
uli is an important aspect of the disease (7). Sensitized canine tracheal smooth muscle has been shown to undergo nonspecific alterations in mechanical properties (1, 2, 4, 28), which, along with the observation of Cock0161-7567/92
$2.00 Copyright
croft et al. (7), suggests that the essential
defect in aller-
gic bronchial hyperresponsiveness might be at muscle cell level. The role of airway smooth muscle as the effector in allergic bronchial hyperresponsiveness has been considerably investigated (27). Studies in our laboratory have focused on the mechanical properties of tracheal smooth muscle, which we believe to be a mechanical model for airways down to those generations of bronchi where allergic bronchial hyperresponsiveness occurs. However, the tracheal smooth muscle is not a perfect model for bronchial smooth muscle, because we have shown that although the bronchial smooth muscle possesses the same qualitative
mechanical
properties
as the
trachealis, there are quantitative differences, as well as differences in pharmacology (25). Therefore it was necessary to employ the bronchial smooth muscle preparation (15) itself for mechanical study if elucidation of the pathogenesis of asthma was to be successful. This had not been attempted in the past because of the belief that attempts to remove cartilaginous plaques, a sine qua non if isotonic studies were to be undertaken, would seriously damage the attached smooth muscle. However, in an examination of histological slides of the bronchi, we noted that the muscle was not attached directly to cartilage at any point. Attachment was via connective tissue. We demonstrated that removal of the cartilage and provision of an intact muscle was feasible (15). The present study was undertaken to determine whether the mechanical properties of bronchial smooth muscle from dogs are altered after ragweed pollen sensitization. METHODS
In Vivo Sensitization Dogs were sensitized according to the method of Kepron et al. (16). Briefly, newborn mongrel dogs received intraperitoneal injections of 500 pg of ragweed pollen extract in 30 mg of an aluminum hydroxide adjuvant within 24 h of birth. The booster injections were repeated weekly for 8 wk and at biweekly intervals thereafter. This method of immunization (16, 22) has been shown to induce prolonged immunoglobulin E antibody production of high titers against the ragweed pollen extract. Randomly selected littermates of these sensitized dogs received, at the same time, injections of the adjuvant alone and were used as controls. Sensitization to ragweed was determined by the homologous passive cutaneous ana-
0 1992 the American
Physiological
Society
39
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40
SENSITIZED
BRONCHIAL
phylaxis test. A sensitized dog demonstrating passive cutaneous anaphylaxis titers > 1:256 and its nonsensitized littermate control were selected randomly on a given day for the in vitro studies. In previous in vivo studies (16), all sensitized littermates with passive cutaneous anaphylaxis titers A:64 developed marked increases in specific airway resistance on bronchoprovocation with ragweed extract aerosol compared with controls similarly challenged. In addition, sensitized dogs were fourfold more responsive to acetylcholine than their littermate controls (3). In Vitro Mechanical
Studies
BronchiaL smooth muscle preparation. After induction of anesthesia with pentobarbital sodium (30 mg/kg body wt iv), 13 sensitized and 13 control dogs of both sexes were killed with intravenous injections of saturated KCl. The lungs were promptly removed and placed into icecold Krebs-Henseleit solution aerated with 95% O,-5% CO,. The branches of the bronchi were dissected in gassed Krebs-Henseleit solution, and segments of nonbranching fifth-generation bronchi (0.8-1.2 mm diam) were isolated, because the orientation of the muscle fibres in such regions is parallel (10,15). The rings were cut open into rectangular strips, and the connective tissues and cartilage plates were removed under the dissecting microscope, as described previously (15). The lower end of the muscle strip was anchored by a clamp at the bottom of the organ bath (50 ml), and the upper end was connected to an electromagnetic lever with 7-O braided surgical thread. The muscle strips were equilibrated for M-2 h in the muscle bath, which was maintained at a PO, of 600 Torr, a PCO, of 40 Torr, and pH of 7.40, at a temperature of 37OC. The Krebs-Henseleit solution in the bath was replaced every 30 min. The tissue baths in which the muscle strips were immersed contained a fixed volume (30 ml) of Krebs-Henseleit solution of the following composition (in mM): 115 NaCl, 25 NaHCO,, 1.38 NaH,PO,, 2.51 KCl, 2.46 MgSO, +7H,O, 1.91 CaC1,, and 5.56 dextrose. Mechanical data acquisition system. The muscle strips were stimulated every 6 min with an electrical stimulator that generated a 60-Hz 300-mA/cm2 current. The duration of the stimulus was 10 s. The supramaximal voltage (110% of the voltage that resulted in maximal response, 18 V) was used to elicit maximal activation of the contractile elements. The contractions of the muscle strips were obtained by the lever system that was originally
developed
by Brut-
SMOOTH
MUSCLE
spective lengths to obtain the length-tension relationships for sensitized and control bronchial smooth muscle. Tension was expressed in newtons per square meter of smooth muscle tissue in the whole cross-sectional area of the strips (14,15). The cross-sectional areas of muscle strips were computed from images captured by a video camera system described in Morphological Measurements. The fraction of the muscle tissue cross-sectional area in the whole strip cross-sectional area was determined by planimetry as described below. Isotonic measurements. At 2 and 8 s, isometrically contracted muscle strips were quickly released to a set of randomly chosen different loads, which were lighter than the maximal tension (P,) that the muscle strips developed, so that the strips shortened to various extents. This resulted in a rapid transient due to shortening of the muscle’s series elastic component (SEC) followed by artifactual oscillations and a slow transient. The maximum slope of the slow transient (the maximum shortening velocity in the period between 180 and 220 ms after quick release) was computed and identified as the maximum shortening velocity of contractile element (CE) for the given load (Fig. 1). The velocities and their respective loads expressed as millinewtons per square millimeter of each muscle were fitted with the Hill equation (ll), which states that (P + a)(V + b) = (P, -t a)b, where P is load, P, is maximum isometric force developed by muscle, V is velocity, and a and b are constants with units of force and velocity, respectively. The maximum shortening velocities under zero load were obtained from the force-velocity curves. An F test for the goodness of fit was carried out; P values were set at co.05 for significance. Also, the means of velocities and their respective loads, expressed as fractions of their P, from each group, were plotted
(force-velocity
hyperbolic
curve).
The maximum shortening capacities (AL,,) of the muscles were directly measured from another set of isotonic shortenings under a load equal to predetermined resting tension of that particular muscle. To determine the proportion of shortening contributed by early normally cycling cross-bridges and latch bridges, respectively, the percent shortening developing within the first 2 s, with respect to maximum shortening, were measured from isotonic contraction curves of both groups. The reason for studying the shortening developing within the first 2 s is that in the trachealis 75% of the total shortening (AL,,,) is completed in this interval. This indicates that it is chiefly the normally cycling bridges that subserve the shortening function. It is also the normally cling bridges that were increased in the sensitized
cytra-
saert et al. (5) for cardiac muscle and was adapted for use with slower smooth muscle (20). The signal outputs from the lever were digitized and transmitted to an IBM computer, which analyzed the data and plotted it with a customized computer program. The lever control system allowed us to abruptly (within 3 ms) change the load on the muscle (load clamp) at any desired time. Isometric measurements. The muscle strips were allowed to contract isometrically at different lengths to
chealis. The properties of the normal SEC, which is the element that transmits force developed by the muscle’s con-
determine
that
the optimal
length
(I,) at which
the isotonic
experiments were to be carried out. The tensions developed at different lengths were plotted against their re-
tractile
element
(force
and shortening
generator)
to the
outside environment, have been reported by us for canine tracheal smooth muscle (29). Because alterations in these properties could affect the maximum velocity of muscle shortening, it became necessary to show that they were not altered in control and sensitized muscles and measured
estimates
of CE velocity
were valid.
In
this study the quick-release technique described above was used to measure the properties of the SEC (Fig. 1).
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SENSITIZED
Electrical
BRONCHIAL
SMOOTH
MUSCLE
41
stimulus
2
Time (set) FIG. 1. Original recordings of force, shortening, and derived velocity data obtained by quick release to various loads at 2 s in contraction. Top: force traces, electrical stimulation, and load clamp. Muscle strip was quickly released to 3 different loads (1,2, and 3). Middle: corresponding rapid length transient of series elastic component (SEC) recoil and shortenings of contractile eIement (CE) contraction. With a customized computer program, maximum shortening velocities of GE were computed directly by obtaining average of 20 data points 180420 ms after quick release when oscillation caused by quick release had ceased. Bottom: derived shortening velocity (VI, Vz, and V,) as a function of time, where maximum shortening velocities of strip under different loads are pointed out by arrows.
The active muscle was abruptly released at the selected time to a series of different isotonic loads. The rapid transient, as mentioned above, represented the elastic recoil of SEC. Because of the immediately following artifactual oscillations, the magnitude of the recoil could not be directly measured. However, the slow transient that followed and stemmed from contractile element shortening was graphically extrapolated backward to the point of intersection on the recoil trace. The magnitude of recoil so obtained was plotted against the isotonic load. The points for the data for the different loads delineated the length-tension curve for the SEC. To successfully complete a given experiment, a single muscle strip had to be stimulated -40 times at 6-min intervals. The stability of the muscle during the total duration of the experiment was crucial to obtaining accurate and precise values. Maximum isometric tension was measured every 30 min throughout the experiment to assess the stability of the muscle function. If P, differed >15% between the beginning and the end of the experiment, the data were discarded. There were three preparations rejected for this reason in the entire series.
tion grid with 100-pm resolution was placed in the muscle bath beside the muscle strip. The glass wall of the bath incorporated two ports of nondistorting glass allowing the width and thickness of the muscle strip to be clearly visualized. The total cross-sectional area of the strip was computed from the average of measurements of width and thickness made at the top, middle, and bottom of the muscle strip. The muscle strip was rectangular, provided care was taken in dissection and mounting. After the mechanical experiments were completed, bronchial smooth muscle strips from both groups were fixed at their optimal length with 10% Formalin fixative. Transverse sections, 5 pm thick, were made and stained with hematoxylin-eosin or van Giesson’s trichrome stain; the latter shows the smooth muscle outline quite well. The fraction of smooth muscle cross-sectional area in the total cross section of the strip was determined by planimetry of magnified projections of tissue transverse sections. This enabled us to determine whether the proportion of muscle in the tissue cross section was the same in bronchial smooth muscle strips from sensitized and littermate control dogs.
Morphological
Statistical
Measurements
A Sony AVC-D5 black and white video camera was used to capture images of muscle strips at I,. A calibra-
Analysis
An analysis of variance was performed on data obtained from the Hill equation. The least significant dif-
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42
SENSITIZED 12
-
10
-
G >
e--e
CBSM
n=13
O-C3
SBSM
n=12
BRONCHIAL
SMOOTH
MUSCLE
cant difference (P < 0.01) was also found in the proportion of isotonic shortening occurring within the first 2 s between control (0.51 t 0.049) and sensitized (0.81 t 0.032) muscles. There is no difference between the two l*‘s.
8-
Mwphological 5 l-
/ #
z-
f resting
0.2
w 0.4
! A
l/i’
/ 0
tension
* w
@&AA, 0.6
Q’
I 1.0
0.8
LENGTH
I 1.2
I 1.4
(I/lo)
FIG. 2. Length-tension
relationships of sensitized (SBSM) and control bronchial smooth muscle (CBSM). Active and resting tensions are shown. Both groups demonstrated similar length-tension relationships. Maximum active tensions (P,) are similar for both groups as well. Note that stresses were normalized with respect to muscle fraction in total tissue cross-sectional area. I,, Optimal length.
ference method was used to compare values between sensitized and control as well as between 2 and 8 s. The values of SEC and AL,,, from both groups were compared using an unpaired two-tailed t test with P set at 0.05.
Studies
Quantitative planimetry analysis showed no difference in the smooth muscle fraction in total tissue cross-sectional area of the strips. The proportions of muscle tissue in the total cross-sectional area were 0.301 t 0.02 and 0.311 t 0,03 for CBSM and SBSM, respectively. Hence, no correction for this factor was required in comparing tissue stresses developed by the CBSM and SBSM. No inflammation and cellular infiltration were found in the slides of bronchi from sensitized dogs. As shown in Fig. 5, the smooth muscle cells are arranged in a parallel fash0.5
A 1 V=b(Po-P)/(P+o) TIME: 2 set
0.4
0
SBSM
(n=13)
n
CBSM
(n=13)
t
hT
RESULTS
Isometric
Studies
Length-tension relationships of the two groups are shown in Fig. 2. No significant difference was found between the two when normalized with respect to smooth muscle cross-sectional area in the total cross section (Fig. 2). The values of P, for the two groups were not significantly different (Fig. 2). The slopes of the two resting tension curves are also similar, indicating operatively similar static properties between sensitized and control bronchial smooth muscles at rest. Isotonic
Studies
Force-velocity relationships. Figure 3 depicts the forcevelocity curves for sensitized and control bronchial smooth muscle (SBSM and CBSM, respectively) at 2 and 8 s. The force-velocity relationships were fitted with the Hill equation from which the desired muscle constants were obtained (Table 1). SBSM demonstrated greater maximum shortening velocity than the control at 2 s (P < 0.05). Both groups showed higher shortening velocities 2 s after the onset of stimulation than at 8 s (P < 0.05). Also the value of a/b, reported by us to be an index of internal resistance (29), is lower in SBSM than in CBSM. The maximum elongations of the SEC when the muscles developed their PO’swere 0.0873 t 0.004 and 0.087 t 0.004 (SE) of I, in CBSM and SBSM, respectively. No statistical difference was found between them. These are not statistically different (P > 0.05) from those we have previously reported for the trachealis (29). Maximum shortening capacity. The values of AL,,, under resting tension are displayed in Fig. 4. The AL,, for SBSM [0.67 t 0.038 I, (SE)] was significantly greater than that for CBSM (0.51 t 0.038, P < 0.01). A signifi-
0.0 0.0
0.2
0.4 LOAD
0.3
0.6 (fraction
of
Cl.8
1.0
0.8
1.0
PO)
B 1 V=b(Po-P)/(P+a) TIME: 8 set 0
0.0
SESM
0.2
(n=13)
H
0.4 LOAD
CBSM
0.6 (fraction
of
[n=13)
PO)
FIG. 3. Mean force-velocity data elicited from SBSM and CBSM by quick-release load clamping technique. Mean shortening velocities and their respective loads are expressed as fractions of PO’s 2 s (A) and 8 s (B) after onset of stimulation. At 2 s, sensitized groups showed greater maximum shortening velocity (V,) than controls, whereas PO’s were the same.
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SENSITIZED
BRONCHIAL
TABLE 1. Bronchial smooth muscle constants obtained from Hill equation SBSM(n 2s
=
13)
CBSM 8s
2s
0.35+0.02*+ 0.19t0.02 0.27t0.02" v,, I,ls I?,, mN/mm2 68.26t9.01 58.43t8.16 78.90t9.59" a,mN/mm2 13.25t1.81 10.03t1.4 16.96t1.98 b, lo/s 0.062t0.007 0.036t0.003 0.060t0.005 U/P, 0.193t0.007 0.173+0.007 0.219t0.009 a/b 209.6+30.58*+ 291.7+39.01+ 301.2t41.07* l/b 15.90~0.83" 30.21k2.53 17.80t1.44*
(n = 11) 8s
0.1&z0.01
68.24t8.29 13.84k1.83 0.039t0.005 0.201t0.007 374.4t43.62 30.13t4.53
Values are means t SE; n, no. of dogs. SBSM, sensitized bronchial smooth muscle; CBSM; control bronchial smooth muscle; V,, maximum shortening velocity; P,,, maximum isometric force developed by muscle; a and b, constants with units of force and velocity, respectively; 1,, optimal length. * P < 0.05 compared with values at 8 s. t P < 0.05 compared with control.
ion, which validates our interpretation cellular level.
of the data at the
DISCuSSION
Status asthmaticus patients have been reported to demonstrate bronchial smooth muscle hypertrophy (9, 13, 31); however, one study (26) reported that bronchial smooth muscle content was not significantly increased in asthmatic patients who had died from nonasthmatic causes. In the studies where smooth muscle hypertrophy was reported, the effective factor was likely the incidence of repeated asthmatic attacks. We have never noted hypertrophy in either tracheal or bronchial smooth muscle of our dogs, probably because these animals have only been sensitized and never challenged. Inflammation and the concomitant cellular infiltration are not seen either. This suggests, in turn, that although inflammation may be responsible for the chronic asthmatic response, it likely plays almost no role in the acute response. Furthermore these animals are very young and likely to show only the primary disease changes; this should facilitate elucidation of primary causes. Because the flow-limiting segment of the respiratory tree is the third to seventh generations of the bronchial tree (21) and because it has been suggested that the acute asthmatic response to antigen challenge likely involves constriction of the central airways (8), it is evident that it is the smooth muscle from this segment that must be investigated. Therefore we developed a bronchial preparation from which the cartilage plates and connective tissue were removed while leaving smooth muscle structurally and functionally intact (15). With this preparation, the length-tension relationships of SBSM and CBSM were delineated. The P, in sensitized dogs was unchanged, indicating that isometric parameters are insensitive indicators of disease. Isometric properties of airway smooth muscle relate to the stiffness of the bronchial wall only, and their role in regulating resistance is minor. Inasmuch as the resistance to flow in a tube depends on its diameter, flow regulation in airways or blood vessels, which are directly controlled by shortening or elongating rather than stiffening of smooth muscle, is best studied in vitro by
SMOOTH
43
MUSCLE
evaluation of isotonic parameters such as the force-velocity relationship and AL,,. However, very few studies of these parameters (28) have been carried out. We found an increase of 31.4% in maximum shortening capacity in SBSM. This is significant with respect to allergic bronchoconstriction, because computations using Poiseuille’s equation, which applies here because the flow in this portion of the respiratory tree is laminar, indicate that this increase in shortening would translate into a 386.1% increase in airflow resistance in the sensitized model (assuming that all other variables held constant). Because most of the shortening occurred within the first 2 s, the normally cycling cross-bridges are more important to study as far as isotonic shortening is concerned. The increased shortening velocity and amount of shortening at 2 s in SBSM suggest that normally cycling cross-bridges are responsible for the increased AL,,,. That is, an increase in the rate of normally cycling crossbridges would decrease the time that is needed for achievement of maximum isotonic shortening. These early cross-bridges are activated by phosphorylation of myosin light chain (6,18). A concomitant increase in activity of myofibrillar adenosinetriphosphatase has been shown in the sensitized tracheal, as well as in bronchial and pulmonary arterial, smooth muscle (19, 23). The level of myosin light chain (20 kDa) phosphorylation has been found to be increased in sensitized trachealis (17). It, therefore, is likely that the greater maximum shortening velocities found in SBSM are the result of increased myosin adenosinetriphosphatase activation brought about by an elevated level of myosin light chain phosphorylation. Initial experiments in this laboratory have indicated an increased cellular content of myosin light chain kinase in sensitized airway smooth muscle (23). Another factor that could affect the magnitude of shortening is the compliance of the so-called internal resistor. The evidence that such a phenomenon exists is straightforward. If a resting muscle, stretched to beyond I,, is released, it returns to I,. Furthermore, if stimulation is removed from a maximally shortened muscle, the muscle reelongates to its original length. It is as if there were an elastic resistance to shortening and stretching within the cell. In the shortening phase, this resistor is compressed and stores potential energy. When the stimulus 1 .o
0.8 z 5 -
0.6
i2 z F
0.4
I
CBSM
n=13
m
SBSM
n=13
E 5 0.2
0.0
FIG. 4. Maximum shortening capacities of SBSM and CBSM. SBSM showed a greater shortening capacity (AL-) than CBSM. is greater in SBSM than in CBSM. *Values for AL,, at 2 s (L& SBSM are significantly higher (P < 0.01) than for CBSM.
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44
SENSITIZED
BRONCHIAL
SMOOTH
MUSCLE
FIG. 5. Appearance of 5th generation bronchial smooth muscle and its neighboring structures from sensitized (S) and control (C) dogs. Parallel arrangement of smooth muscle fibers, as judged from orientation of nuclei, can be seen. Neither smooth muscle hypertrophy nor abnormal changes to other structures were found. Magnification x200. Sm, smooth muscle; Ca, cartilage; Ep, epithelium.
is turned off, the resistor reexpands and restores the muscle to its original length. We have also reported that the internal resistance to shortening, given by a/b (30) (or parallel elastic component) of the sensitized tracheal smooth muscle is more compliant than that of the control (28). This could account for the increased shortening in sensitized muscle, all other factors being equal. The mechanism for increased compliance of the internal re-
sistor in sensitized muscle is still unknown. Changes in properties of collagen, elastin, and other structures in the extracellular and extrafascicular spaces are strong contenders. However, the finding that the passive properties (resting tensions) from both groups were the same does not support the idea that changes in collagen and elastin are responsible. The cytoskeleton is thus another contender. It has been reported that low doses of ionizing
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SENSITIZED
BRONCHIAL
radiation of skinned skeletal muscle cells induced changes in two cytoskeletal proteins, nebulin and titin, resulting in decreased passive and active tensions in response to calcium (12). Although titin and nebulin have not been found in smooth muscle, there are a large number of other smaller-molecular-weight cytoskeletal proteins present in smooth muscle (24) such as filamin, desmin, vimentin, vinculin, plectin, cu-actinin, and synemin, all of which could contribute to the structure of the internal resistor. Additionally, Rasmussen et al. (24) have shown that these proteins are phosphorylated late in contraction. The purpose of this phosphorylation is not clear; however, it could alter the mechanical properties of the cytoskeletal network and, therefore, change the properties of the internal resistor. The SEC data showed no significant difference in mechanical properties between sen.sitized and con trol muscles. Thi .s is perhaps of no great importance in our studies, because isotonic velocity measured is truly that of the muscle’s CE. However, in Viva, where contractions may be auxotonic, changes in the properties of the SEC would affect measured velocities considerably because of the changing length of the SEC. This project was supported by an operating grant from The Medical Research Council of Canada. H. Jiang is the recipient of a fellowship from The Medical Research Council of Canada. Address for reprint requests: H. Jiang, Dept. of Physiology, University of Manitoba, 770 Bannatyne Ave., Winnipeg, Manitoba R3E OW3, Canada. Received 24 September
1990; accepted in final form 21 August 1991.
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MUSCLE
45
TANEMURA. Distribution of smooth muscles along the bronchial tree. A morphometric study of ordinary autopsy lungs. Am. Rev. Respir. Dis. 141: 1322-1326, 1990. 11. HILL, A. V. The heat of shortening and the dynamic constants of muscle. Proc. R. Sot. Lond. Ser. B 126: 136-195, 1938. 12. HOROWITS, R., E. S. KEMPNER, M. E. BISHOR, AND R. J. PoDOLSKY. A physiological role for titin and nebulin in skeletal muscle. Nature Lond. 323: 160-164, 1986. 13. HWBER, H. L., AND K, K. KOESSLER. The pathology of bronchial asthma. Arch. Intern. Med. 30: 689-760, 1922. l4 JIANG, H., A. J. HALAYKO, K. RAO, AND N. L. STEPHENS. Normal’ ization of force generated by canine tracheal and bronchial smooth muscle. Am. J. Physiol. 260 (Lung Cell. MOL. Physiol. 4): L522-L529, 1991. JIANG, H., AND N, L. STEPHENS. Contractile of bron15*chial smooth muscle with and without cartilage.properties J. AppZ. Physiol. 69: 120-126, 1990. 16. KEPRON, W., J. M. JAMES, B. KIRK, A. H. SEHON, AND K. S. TSE. A canine model for reaginic hypersensitivity and allergic bronchoconstriction. J. Allergy CZin. Immunol. 59: 64-69, 1977. 17. KONG, S., A. J. HALAYKO, AND N. L. STEPHENS. Increased myosin phosphorylation in sensitized canine tracheal smooth muscle. Am. J. Physiol. 259 (Lung CeZZ.Mol. Physiol. 3): L53-L56, 1990. 18. KONG, S. K., R. P. C. SHIU, AND N. L. STEPHENS. Role of myosin light chain phosphorylation in canine tracheal smooth muscle con19 traction (Abstract). Federation Proc. 43: 427A, 1984. . KONG, S. K., R. P. C. SHIU, AND N. L. STEPHENS. Studies of myofibrillar ATPase in ragweed-sensitized canine pulmonary smooth muscle. J. Appl. Physiol. 60: 92-96, 1986. 20 MITCHELL, R. W., AND N. L. STEPHENS. Maximum shortening velocity of smooth muscle: zero load-clamp vs. afterloaded method. J. AppZ. Physiol. 55: 1630-1633, 1983. 21 PEDLEY, T. J., R. C. SCHROTER, AND M. F. SUDLOW. The prediction of pressure drop and variation of resistance within the human bronchial airways. Respir. PhysioZ. 9: 387-405, 1970. 22* PINCKAFID, R. N., M. HOLONEN, AND A. L. MENG. Preferential expression of antibovine serum albumin IgE homocytotropic antibody synthesis and anaphylactic sensitivity in the neonatal rabbit. J. Allergy CZin. Immunol. 49: 301-310, 1972. 23. RAO, K., H. JIANG, A. J. HALAYKO, N. PAN, AND N. L. STEPHENS. Increased ATPase activity and myosin light chain kinase (MLCK) content in airway smooth muscle from sensitized dogs. In: Regulation of Smooth Mu&e: Progress in Solving the Puzzle, edited by R. S. Moreland. New York: Plenum. In press. 24. RASMUSSEN, H., Y. TAKUWA, AND S. PARK. Protein kinase C in the regulation of smooth muscle contraction. FASEB J. 1: 177-185, 1987. 25. SIGURDSSON, S. B., H. JIANG, W. KEPRON, AND N. L. STEPHENS. Sensitized canine bronchial smooth muscle as a model for allergic bronchospasm (Abstract). FASEB J. 4: A616,1990. 26. SOBONYA, R. E. Quantitative structural alterations in long-standing allergic asthma. Am. Rev. Respir. Dis. 130: 289-292, 1990. 27. STEPHENS, N. L. Airway smooth muscle: physiology, bronchomotor tone, pharmacology and relation to asthma. In: Bronchial Asthma, edited by E. 3. Weiss, M. S. Segal, and M. Stein. Boston, MA: Little, Brown, 1985, p. 96-110. 28. STEPHENS, N. L., S. K. KONG, AND C. Y. SEOW. Mechanisms of increased shortening of sensitized airway smooth muscle. In: Mechanisms of Asthma: Pharmacology, Physiology, and Management, edited by C. L. Armour and J. L. Black. New York: Liss, 1988, p. 231-254. 29. STEPHENS, N. L., AND U. KROMER. Series elastic component of tracheal smooth muscle. Am. J. Physiol. 220: 1890-1895, 1971. 30. STEPHENS,N. L.,V. NATHANIEL, W. KEPRON, R.W. MITCHELL, C. Y. SEOW, AND S. K. KONG. Anatomy and physiology of respiratory smooth muscle tone. In: Drug Therapy for Asthma: Research and Cliniccal Practice, edited by J. W. Jenne and S. Murphy. New York: Dekker, 1987, vol. 31, p. l-66. (Lung Biol. Health Dis. Ser.) 31. TAKIZAWA, T., AND W. M. THURLBECK. Muscle and mucous gland size in the major bronchi of patients with chronic bronchitis, asthma, and asthmatic bronchitis. Am. Rev. Respir. Dis. 104: 331336,197l. l
l
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bronchial smooth muscle hyperresponsiveness; ragweed-sensitized canine asthma model; mechanism for airway hyperresponsiveness ALTHOUGHTHEPATHOPHYSIOLOGICAL changesleading to asthma are not yet well known, increased airway responsiveness to a number of allergic and nonallergic stim-
uli is an important aspect of the disease (7). Sensitized canine tracheal smooth muscle has been shown to undergo nonspecific alterations in mechanical properties (1, 2, 4, 28), which, along with the observation of Cock0161-7567/92
$2.00 Copyright
croft et al. (7), suggests that the essential
defect in aller-
gic bronchial hyperresponsiveness might be at muscle cell level. The role of airway smooth muscle as the effector in allergic bronchial hyperresponsiveness has been considerably investigated (27). Studies in our laboratory have focused on the mechanical properties of tracheal smooth muscle, which we believe to be a mechanical model for airways down to those generations of bronchi where allergic bronchial hyperresponsiveness occurs. However, the tracheal smooth muscle is not a perfect model for bronchial smooth muscle, because we have shown that although the bronchial smooth muscle possesses the same qualitative
mechanical
properties
as the
trachealis, there are quantitative differences, as well as differences in pharmacology (25). Therefore it was necessary to employ the bronchial smooth muscle preparation (15) itself for mechanical study if elucidation of the pathogenesis of asthma was to be successful. This had not been attempted in the past because of the belief that attempts to remove cartilaginous plaques, a sine qua non if isotonic studies were to be undertaken, would seriously damage the attached smooth muscle. However, in an examination of histological slides of the bronchi, we noted that the muscle was not attached directly to cartilage at any point. Attachment was via connective tissue. We demonstrated that removal of the cartilage and provision of an intact muscle was feasible (15). The present study was undertaken to determine whether the mechanical properties of bronchial smooth muscle from dogs are altered after ragweed pollen sensitization. METHODS
In Vivo Sensitization Dogs were sensitized according to the method of Kepron et al. (16). Briefly, newborn mongrel dogs received intraperitoneal injections of 500 pg of ragweed pollen extract in 30 mg of an aluminum hydroxide adjuvant within 24 h of birth. The booster injections were repeated weekly for 8 wk and at biweekly intervals thereafter. This method of immunization (16, 22) has been shown to induce prolonged immunoglobulin E antibody production of high titers against the ragweed pollen extract. Randomly selected littermates of these sensitized dogs received, at the same time, injections of the adjuvant alone and were used as controls. Sensitization to ragweed was determined by the homologous passive cutaneous ana-
0 1992 the American
Physiological
Society
39
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40
SENSITIZED
BRONCHIAL
phylaxis test. A sensitized dog demonstrating passive cutaneous anaphylaxis titers > 1:256 and its nonsensitized littermate control were selected randomly on a given day for the in vitro studies. In previous in vivo studies (16), all sensitized littermates with passive cutaneous anaphylaxis titers A:64 developed marked increases in specific airway resistance on bronchoprovocation with ragweed extract aerosol compared with controls similarly challenged. In addition, sensitized dogs were fourfold more responsive to acetylcholine than their littermate controls (3). In Vitro Mechanical
Studies
BronchiaL smooth muscle preparation. After induction of anesthesia with pentobarbital sodium (30 mg/kg body wt iv), 13 sensitized and 13 control dogs of both sexes were killed with intravenous injections of saturated KCl. The lungs were promptly removed and placed into icecold Krebs-Henseleit solution aerated with 95% O,-5% CO,. The branches of the bronchi were dissected in gassed Krebs-Henseleit solution, and segments of nonbranching fifth-generation bronchi (0.8-1.2 mm diam) were isolated, because the orientation of the muscle fibres in such regions is parallel (10,15). The rings were cut open into rectangular strips, and the connective tissues and cartilage plates were removed under the dissecting microscope, as described previously (15). The lower end of the muscle strip was anchored by a clamp at the bottom of the organ bath (50 ml), and the upper end was connected to an electromagnetic lever with 7-O braided surgical thread. The muscle strips were equilibrated for M-2 h in the muscle bath, which was maintained at a PO, of 600 Torr, a PCO, of 40 Torr, and pH of 7.40, at a temperature of 37OC. The Krebs-Henseleit solution in the bath was replaced every 30 min. The tissue baths in which the muscle strips were immersed contained a fixed volume (30 ml) of Krebs-Henseleit solution of the following composition (in mM): 115 NaCl, 25 NaHCO,, 1.38 NaH,PO,, 2.51 KCl, 2.46 MgSO, +7H,O, 1.91 CaC1,, and 5.56 dextrose. Mechanical data acquisition system. The muscle strips were stimulated every 6 min with an electrical stimulator that generated a 60-Hz 300-mA/cm2 current. The duration of the stimulus was 10 s. The supramaximal voltage (110% of the voltage that resulted in maximal response, 18 V) was used to elicit maximal activation of the contractile elements. The contractions of the muscle strips were obtained by the lever system that was originally
developed
by Brut-
SMOOTH
MUSCLE
spective lengths to obtain the length-tension relationships for sensitized and control bronchial smooth muscle. Tension was expressed in newtons per square meter of smooth muscle tissue in the whole cross-sectional area of the strips (14,15). The cross-sectional areas of muscle strips were computed from images captured by a video camera system described in Morphological Measurements. The fraction of the muscle tissue cross-sectional area in the whole strip cross-sectional area was determined by planimetry as described below. Isotonic measurements. At 2 and 8 s, isometrically contracted muscle strips were quickly released to a set of randomly chosen different loads, which were lighter than the maximal tension (P,) that the muscle strips developed, so that the strips shortened to various extents. This resulted in a rapid transient due to shortening of the muscle’s series elastic component (SEC) followed by artifactual oscillations and a slow transient. The maximum slope of the slow transient (the maximum shortening velocity in the period between 180 and 220 ms after quick release) was computed and identified as the maximum shortening velocity of contractile element (CE) for the given load (Fig. 1). The velocities and their respective loads expressed as millinewtons per square millimeter of each muscle were fitted with the Hill equation (ll), which states that (P + a)(V + b) = (P, -t a)b, where P is load, P, is maximum isometric force developed by muscle, V is velocity, and a and b are constants with units of force and velocity, respectively. The maximum shortening velocities under zero load were obtained from the force-velocity curves. An F test for the goodness of fit was carried out; P values were set at co.05 for significance. Also, the means of velocities and their respective loads, expressed as fractions of their P, from each group, were plotted
(force-velocity
hyperbolic
curve).
The maximum shortening capacities (AL,,) of the muscles were directly measured from another set of isotonic shortenings under a load equal to predetermined resting tension of that particular muscle. To determine the proportion of shortening contributed by early normally cycling cross-bridges and latch bridges, respectively, the percent shortening developing within the first 2 s, with respect to maximum shortening, were measured from isotonic contraction curves of both groups. The reason for studying the shortening developing within the first 2 s is that in the trachealis 75% of the total shortening (AL,,,) is completed in this interval. This indicates that it is chiefly the normally cycling bridges that subserve the shortening function. It is also the normally cling bridges that were increased in the sensitized
cytra-
saert et al. (5) for cardiac muscle and was adapted for use with slower smooth muscle (20). The signal outputs from the lever were digitized and transmitted to an IBM computer, which analyzed the data and plotted it with a customized computer program. The lever control system allowed us to abruptly (within 3 ms) change the load on the muscle (load clamp) at any desired time. Isometric measurements. The muscle strips were allowed to contract isometrically at different lengths to
chealis. The properties of the normal SEC, which is the element that transmits force developed by the muscle’s con-
determine
that
the optimal
length
(I,) at which
the isotonic
experiments were to be carried out. The tensions developed at different lengths were plotted against their re-
tractile
element
(force
and shortening
generator)
to the
outside environment, have been reported by us for canine tracheal smooth muscle (29). Because alterations in these properties could affect the maximum velocity of muscle shortening, it became necessary to show that they were not altered in control and sensitized muscles and measured
estimates
of CE velocity
were valid.
In
this study the quick-release technique described above was used to measure the properties of the SEC (Fig. 1).
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SENSITIZED
Electrical
BRONCHIAL
SMOOTH
MUSCLE
41
stimulus
2
Time (set) FIG. 1. Original recordings of force, shortening, and derived velocity data obtained by quick release to various loads at 2 s in contraction. Top: force traces, electrical stimulation, and load clamp. Muscle strip was quickly released to 3 different loads (1,2, and 3). Middle: corresponding rapid length transient of series elastic component (SEC) recoil and shortenings of contractile eIement (CE) contraction. With a customized computer program, maximum shortening velocities of GE were computed directly by obtaining average of 20 data points 180420 ms after quick release when oscillation caused by quick release had ceased. Bottom: derived shortening velocity (VI, Vz, and V,) as a function of time, where maximum shortening velocities of strip under different loads are pointed out by arrows.
The active muscle was abruptly released at the selected time to a series of different isotonic loads. The rapid transient, as mentioned above, represented the elastic recoil of SEC. Because of the immediately following artifactual oscillations, the magnitude of the recoil could not be directly measured. However, the slow transient that followed and stemmed from contractile element shortening was graphically extrapolated backward to the point of intersection on the recoil trace. The magnitude of recoil so obtained was plotted against the isotonic load. The points for the data for the different loads delineated the length-tension curve for the SEC. To successfully complete a given experiment, a single muscle strip had to be stimulated -40 times at 6-min intervals. The stability of the muscle during the total duration of the experiment was crucial to obtaining accurate and precise values. Maximum isometric tension was measured every 30 min throughout the experiment to assess the stability of the muscle function. If P, differed >15% between the beginning and the end of the experiment, the data were discarded. There were three preparations rejected for this reason in the entire series.
tion grid with 100-pm resolution was placed in the muscle bath beside the muscle strip. The glass wall of the bath incorporated two ports of nondistorting glass allowing the width and thickness of the muscle strip to be clearly visualized. The total cross-sectional area of the strip was computed from the average of measurements of width and thickness made at the top, middle, and bottom of the muscle strip. The muscle strip was rectangular, provided care was taken in dissection and mounting. After the mechanical experiments were completed, bronchial smooth muscle strips from both groups were fixed at their optimal length with 10% Formalin fixative. Transverse sections, 5 pm thick, were made and stained with hematoxylin-eosin or van Giesson’s trichrome stain; the latter shows the smooth muscle outline quite well. The fraction of smooth muscle cross-sectional area in the total cross section of the strip was determined by planimetry of magnified projections of tissue transverse sections. This enabled us to determine whether the proportion of muscle in the tissue cross section was the same in bronchial smooth muscle strips from sensitized and littermate control dogs.
Morphological
Statistical
Measurements
A Sony AVC-D5 black and white video camera was used to capture images of muscle strips at I,. A calibra-
Analysis
An analysis of variance was performed on data obtained from the Hill equation. The least significant dif-
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42
SENSITIZED 12
-
10
-
G >
e--e
CBSM
n=13
O-C3
SBSM
n=12
BRONCHIAL
SMOOTH
MUSCLE
cant difference (P < 0.01) was also found in the proportion of isotonic shortening occurring within the first 2 s between control (0.51 t 0.049) and sensitized (0.81 t 0.032) muscles. There is no difference between the two l*‘s.
8-
Mwphological 5 l-
/ #
z-
f resting
0.2
w 0.4
! A
l/i’
/ 0
tension
* w
@&AA, 0.6
Q’
I 1.0
0.8
LENGTH
I 1.2
I 1.4
(I/lo)
FIG. 2. Length-tension
relationships of sensitized (SBSM) and control bronchial smooth muscle (CBSM). Active and resting tensions are shown. Both groups demonstrated similar length-tension relationships. Maximum active tensions (P,) are similar for both groups as well. Note that stresses were normalized with respect to muscle fraction in total tissue cross-sectional area. I,, Optimal length.
ference method was used to compare values between sensitized and control as well as between 2 and 8 s. The values of SEC and AL,,, from both groups were compared using an unpaired two-tailed t test with P set at 0.05.
Studies
Quantitative planimetry analysis showed no difference in the smooth muscle fraction in total tissue cross-sectional area of the strips. The proportions of muscle tissue in the total cross-sectional area were 0.301 t 0.02 and 0.311 t 0,03 for CBSM and SBSM, respectively. Hence, no correction for this factor was required in comparing tissue stresses developed by the CBSM and SBSM. No inflammation and cellular infiltration were found in the slides of bronchi from sensitized dogs. As shown in Fig. 5, the smooth muscle cells are arranged in a parallel fash0.5
A 1 V=b(Po-P)/(P+o) TIME: 2 set
0.4
0
SBSM
(n=13)
n
CBSM
(n=13)
t
hT
RESULTS
Isometric
Studies
Length-tension relationships of the two groups are shown in Fig. 2. No significant difference was found between the two when normalized with respect to smooth muscle cross-sectional area in the total cross section (Fig. 2). The values of P, for the two groups were not significantly different (Fig. 2). The slopes of the two resting tension curves are also similar, indicating operatively similar static properties between sensitized and control bronchial smooth muscles at rest. Isotonic
Studies
Force-velocity relationships. Figure 3 depicts the forcevelocity curves for sensitized and control bronchial smooth muscle (SBSM and CBSM, respectively) at 2 and 8 s. The force-velocity relationships were fitted with the Hill equation from which the desired muscle constants were obtained (Table 1). SBSM demonstrated greater maximum shortening velocity than the control at 2 s (P < 0.05). Both groups showed higher shortening velocities 2 s after the onset of stimulation than at 8 s (P < 0.05). Also the value of a/b, reported by us to be an index of internal resistance (29), is lower in SBSM than in CBSM. The maximum elongations of the SEC when the muscles developed their PO’swere 0.0873 t 0.004 and 0.087 t 0.004 (SE) of I, in CBSM and SBSM, respectively. No statistical difference was found between them. These are not statistically different (P > 0.05) from those we have previously reported for the trachealis (29). Maximum shortening capacity. The values of AL,,, under resting tension are displayed in Fig. 4. The AL,, for SBSM [0.67 t 0.038 I, (SE)] was significantly greater than that for CBSM (0.51 t 0.038, P < 0.01). A signifi-
0.0 0.0
0.2
0.4 LOAD
0.3
0.6 (fraction
of
Cl.8
1.0
0.8
1.0
PO)
B 1 V=b(Po-P)/(P+a) TIME: 8 set 0
0.0
SESM
0.2
(n=13)
H
0.4 LOAD
CBSM
0.6 (fraction
of
[n=13)
PO)
FIG. 3. Mean force-velocity data elicited from SBSM and CBSM by quick-release load clamping technique. Mean shortening velocities and their respective loads are expressed as fractions of PO’s 2 s (A) and 8 s (B) after onset of stimulation. At 2 s, sensitized groups showed greater maximum shortening velocity (V,) than controls, whereas PO’s were the same.
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SENSITIZED
BRONCHIAL
TABLE 1. Bronchial smooth muscle constants obtained from Hill equation SBSM(n 2s
=
13)
CBSM 8s
2s
0.35+0.02*+ 0.19t0.02 0.27t0.02" v,, I,ls I?,, mN/mm2 68.26t9.01 58.43t8.16 78.90t9.59" a,mN/mm2 13.25t1.81 10.03t1.4 16.96t1.98 b, lo/s 0.062t0.007 0.036t0.003 0.060t0.005 U/P, 0.193t0.007 0.173+0.007 0.219t0.009 a/b 209.6+30.58*+ 291.7+39.01+ 301.2t41.07* l/b 15.90~0.83" 30.21k2.53 17.80t1.44*
(n = 11) 8s
0.1&z0.01
68.24t8.29 13.84k1.83 0.039t0.005 0.201t0.007 374.4t43.62 30.13t4.53
Values are means t SE; n, no. of dogs. SBSM, sensitized bronchial smooth muscle; CBSM; control bronchial smooth muscle; V,, maximum shortening velocity; P,,, maximum isometric force developed by muscle; a and b, constants with units of force and velocity, respectively; 1,, optimal length. * P < 0.05 compared with values at 8 s. t P < 0.05 compared with control.
ion, which validates our interpretation cellular level.
of the data at the
DISCuSSION
Status asthmaticus patients have been reported to demonstrate bronchial smooth muscle hypertrophy (9, 13, 31); however, one study (26) reported that bronchial smooth muscle content was not significantly increased in asthmatic patients who had died from nonasthmatic causes. In the studies where smooth muscle hypertrophy was reported, the effective factor was likely the incidence of repeated asthmatic attacks. We have never noted hypertrophy in either tracheal or bronchial smooth muscle of our dogs, probably because these animals have only been sensitized and never challenged. Inflammation and the concomitant cellular infiltration are not seen either. This suggests, in turn, that although inflammation may be responsible for the chronic asthmatic response, it likely plays almost no role in the acute response. Furthermore these animals are very young and likely to show only the primary disease changes; this should facilitate elucidation of primary causes. Because the flow-limiting segment of the respiratory tree is the third to seventh generations of the bronchial tree (21) and because it has been suggested that the acute asthmatic response to antigen challenge likely involves constriction of the central airways (8), it is evident that it is the smooth muscle from this segment that must be investigated. Therefore we developed a bronchial preparation from which the cartilage plates and connective tissue were removed while leaving smooth muscle structurally and functionally intact (15). With this preparation, the length-tension relationships of SBSM and CBSM were delineated. The P, in sensitized dogs was unchanged, indicating that isometric parameters are insensitive indicators of disease. Isometric properties of airway smooth muscle relate to the stiffness of the bronchial wall only, and their role in regulating resistance is minor. Inasmuch as the resistance to flow in a tube depends on its diameter, flow regulation in airways or blood vessels, which are directly controlled by shortening or elongating rather than stiffening of smooth muscle, is best studied in vitro by
SMOOTH
43
MUSCLE
evaluation of isotonic parameters such as the force-velocity relationship and AL,,. However, very few studies of these parameters (28) have been carried out. We found an increase of 31.4% in maximum shortening capacity in SBSM. This is significant with respect to allergic bronchoconstriction, because computations using Poiseuille’s equation, which applies here because the flow in this portion of the respiratory tree is laminar, indicate that this increase in shortening would translate into a 386.1% increase in airflow resistance in the sensitized model (assuming that all other variables held constant). Because most of the shortening occurred within the first 2 s, the normally cycling cross-bridges are more important to study as far as isotonic shortening is concerned. The increased shortening velocity and amount of shortening at 2 s in SBSM suggest that normally cycling cross-bridges are responsible for the increased AL,,,. That is, an increase in the rate of normally cycling crossbridges would decrease the time that is needed for achievement of maximum isotonic shortening. These early cross-bridges are activated by phosphorylation of myosin light chain (6,18). A concomitant increase in activity of myofibrillar adenosinetriphosphatase has been shown in the sensitized tracheal, as well as in bronchial and pulmonary arterial, smooth muscle (19, 23). The level of myosin light chain (20 kDa) phosphorylation has been found to be increased in sensitized trachealis (17). It, therefore, is likely that the greater maximum shortening velocities found in SBSM are the result of increased myosin adenosinetriphosphatase activation brought about by an elevated level of myosin light chain phosphorylation. Initial experiments in this laboratory have indicated an increased cellular content of myosin light chain kinase in sensitized airway smooth muscle (23). Another factor that could affect the magnitude of shortening is the compliance of the so-called internal resistor. The evidence that such a phenomenon exists is straightforward. If a resting muscle, stretched to beyond I,, is released, it returns to I,. Furthermore, if stimulation is removed from a maximally shortened muscle, the muscle reelongates to its original length. It is as if there were an elastic resistance to shortening and stretching within the cell. In the shortening phase, this resistor is compressed and stores potential energy. When the stimulus 1 .o
0.8 z 5 -
0.6
i2 z F
0.4
I
CBSM
n=13
m
SBSM
n=13
E 5 0.2
0.0
FIG. 4. Maximum shortening capacities of SBSM and CBSM. SBSM showed a greater shortening capacity (AL-) than CBSM. is greater in SBSM than in CBSM. *Values for AL,, at 2 s (L& SBSM are significantly higher (P < 0.01) than for CBSM.
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44
SENSITIZED
BRONCHIAL
SMOOTH
MUSCLE
FIG. 5. Appearance of 5th generation bronchial smooth muscle and its neighboring structures from sensitized (S) and control (C) dogs. Parallel arrangement of smooth muscle fibers, as judged from orientation of nuclei, can be seen. Neither smooth muscle hypertrophy nor abnormal changes to other structures were found. Magnification x200. Sm, smooth muscle; Ca, cartilage; Ep, epithelium.
is turned off, the resistor reexpands and restores the muscle to its original length. We have also reported that the internal resistance to shortening, given by a/b (30) (or parallel elastic component) of the sensitized tracheal smooth muscle is more compliant than that of the control (28). This could account for the increased shortening in sensitized muscle, all other factors being equal. The mechanism for increased compliance of the internal re-
sistor in sensitized muscle is still unknown. Changes in properties of collagen, elastin, and other structures in the extracellular and extrafascicular spaces are strong contenders. However, the finding that the passive properties (resting tensions) from both groups were the same does not support the idea that changes in collagen and elastin are responsible. The cytoskeleton is thus another contender. It has been reported that low doses of ionizing
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SENSITIZED
BRONCHIAL
radiation of skinned skeletal muscle cells induced changes in two cytoskeletal proteins, nebulin and titin, resulting in decreased passive and active tensions in response to calcium (12). Although titin and nebulin have not been found in smooth muscle, there are a large number of other smaller-molecular-weight cytoskeletal proteins present in smooth muscle (24) such as filamin, desmin, vimentin, vinculin, plectin, cu-actinin, and synemin, all of which could contribute to the structure of the internal resistor. Additionally, Rasmussen et al. (24) have shown that these proteins are phosphorylated late in contraction. The purpose of this phosphorylation is not clear; however, it could alter the mechanical properties of the cytoskeletal network and, therefore, change the properties of the internal resistor. The SEC data showed no significant difference in mechanical properties between sen.sitized and con trol muscles. Thi .s is perhaps of no great importance in our studies, because isotonic velocity measured is truly that of the muscle’s CE. However, in Viva, where contractions may be auxotonic, changes in the properties of the SEC would affect measured velocities considerably because of the changing length of the SEC. This project was supported by an operating grant from The Medical Research Council of Canada. H. Jiang is the recipient of a fellowship from The Medical Research Council of Canada. Address for reprint requests: H. Jiang, Dept. of Physiology, University of Manitoba, 770 Bannatyne Ave., Winnipeg, Manitoba R3E OW3, Canada. Received 24 September
1990; accepted in final form 21 August 1991.
REFERENCES 1. ANTONISSEN, L. A., R. W. MITCHELL, E. A. KROEGER, W. KEPRON, K. S. TSE, ANI) N. L. STEPHENS. Mechanical alterations of
2.
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