Departments of 1 Pharmacology and Therapeutics, 2 Anatomy, and 3 Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3; 4 Krannert Institute of Cardiology, Indiana University, Indianapolis, Indiana 46202; and 5 The UBC McDonald Research Laboratories/The iCAPTURE Centre, St. Paul's Hospital/Providence Health Care, Vancouver, British Columbia, Canada V6Z 1Y6
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ABSTRACT |
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Phosphorylation of the 20-kDa regulatory myosin light chain (MLC) of smooth muscle is known to cause monomeric myosins in solution to self-assemble into thick filaments. The role of MLC phosphorylation in thick filament formation in intact muscle, however, is not clear. It is not known whether the phosphorylation is necessary to initiate thick filament assembly in vivo. Here we show, by using a potent inhibitor of MLC kinase (wortmannin), that the MLC phosphorylation and isometric force in trachealis muscle could be abolished without affecting calcium transients. By measuring cross-sectional densities of the thick filaments electron microscopically, we also show that inhibition of MLC phosphorylation alone did not cause disassembly of the filaments. The unphosphorylated thick filaments, however, partially dissolved when the muscle was subjected to oscillatory strains (which caused a 25% decrease in the thick filament density). The postoscillation filament density recovered to the preoscillation level only when wortmannin was removed and the muscle was stimulated. The data suggest that in vivo thick filament reassembly after mechanical perturbation is facilitated by the cyclic MLC phosphorylation associated with repeated stimulation.
airway smooth muscle; contraction; ultrastructure; biochemistry; calcium transients
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INTRODUCTION |
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IT IS GENERALLY AGREED that smooth muscle myosin thick filament is structurally less stable than its counterpart in striated muscle (for review see Ref. 1). The thick filaments in smooth muscle, however, do exist, even in the relaxed state (8, 24). It has been shown in some smooth muscles that assembly of the thick filaments (possibly from the phosphorylated monomeric myosin pool in the cell) occurs during contraction (8, 9, 30, 31). Recently, we observed that, by imposing length oscillation on airway smooth muscle in the relaxed state, partial dissolution of the thick filaments could be induced (14). The reduction in number and/or length of the thick filaments (reflected in the decrease of the cross-sectional density of the filaments in electron micrographs) was accompanied by a proportional decrease in isometric force; the density and force values returned to their preoscillation levels when the muscle was allowed to recover isometrically and subjected to periodic electrical stimulation (14). The mechanism governing thick filament assembly/disassembly in intact smooth muscle is poorly understood. In contrast, it has been well documented for isolated myosins in solution that, within the physiological range of ionic strength, phosphorylation of the 20-kDa regulatory myosin light chain (MLC) by MLC kinase (MLCK) converts a folded myosin monomer (10S) to an extended monomer (6S), which then combines with other extended monomers to form a thick filament (4, 19, 20, 26, 27). The present study is undertaken to examine the role of MLC phosphorylation in thick filament reassembly after mechanical perturbation in intact airway smooth muscle and to determine whether the mechanism that controls thick filament formation in vitro is also operative in vivo. An important issue to be addressed is whether the dynamic nature of the contractile filaments in smooth muscle could result in a mechanism of contraction that is fundamentally different from that of striated muscle. If the contraction-relaxation cycles in smooth muscle involve assembly/disassembly of the thick filaments and if phosphorylation of MLC plays a key role in the filamentogenesis, then we have to reevaluate the present theories of smooth muscle contraction that do not take into account the evanescence of myosin filaments.
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METHODS |
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Muscle preparation. Swine and canine tracheal smooth muscles were used in the experiments. The MLC phosphorylation and calcium transient studies were done using canine tissue in the laboratories of Ford and Burdyga in Indianapolis and Liverpool (UK), respectively; the electron-microscopic morphometric study was done using swine tissue in Seow's laboratory in Vancouver. Swine tracheas were obtained from a local abattoir near Vancouver. Canine tracheas were obtained from dogs after deep pentobarbital sodium anesthesia and euthanasia. The tracheas were put in ice-cold physiological saline solution (PSS) immediately after their removal from the animals.
For MLC phosphorylation experiments, thin strips of tracheal smooth muscle (~7.5 mm long, ~1.5-2.0 mm wide, ~0.1-0.2 mm thick) weighing ~2 mg were carefully cleaned and dissected. The ends were clamped in aluminum foil clips that maintained the preparations as flat sheets of muscle. One clip was slipped over a fixed hook at the bottom of the organ bath/freeze chamber, and the other clip was hooked to a force transducer by a short length of fine stainless steel wire. Tracheal smooth muscle preparations were placed horizontally and between platinum wire electrodes and bathed in HEPES-buffered Ringer solution (in mM: 140 NaCl, 20 HEPES, 4.0 KCl, 1.2 NaH2PO4, 2.0 MgSO4, 2.0 CaCl2, and 5.0 glucose) at 37°C in a flow-through organ bath. For calcium transient measurements, muscle strips were loaded with a membrane-permeant form of indo 1 (12 µM) for 2-3 h at room temperature on a rocking platform. This loading method has been shown to maximize the signal-to-noise ratio of the calcium transient record and minimize calcium buffering and, thus, minimally affect the rate of calcium transient changes (2). In addition, this method did not affect the electric field stimulation (EFS)-elicited force development measured before and after loading of indo 1 for smooth muscle preparations (2). The tissues were excited at a source light wavelength of 340 nm, and the fluorescence emission signals at 400 and 500 nm were recorded at 100 Hz. The ratio of these signals (F400/500) was used to indicate the time course and comparative amplitudes of the calcium transients. For electron-microscopic study, thin strips of smooth muscle bundles (~6 × 1.5 × 0.2 mm) were dissected from each porcine trachea, with the epithelial layer and the connective tissue of the adventitia carefully removed. Aluminum foil clips were attached to both ends of the muscle strip; one end of the muscle was connected to a stationary hook inside the muscle bath, and the other end was connected to a servo-controlled force/length lever. In the water-jacketed muscle bath, PSS (118 mM NaCl, 5 mM KCl, 1.2 mM NaH2PO4, 22.5 mM NaHCO3, 2 mM MgSO4, 2 mM CaCl2, and 2 g/l dextrose, pH 7.4) was bubbled with 5% CO2-95% O2 at 37°C. The apparatuses have been described previously (22, 29). An equilibration period of 1-1.5 h was allowed for each muscle preparation to adapt to the experimental condition. During the period of equilibration, the muscle was activated by electrical stimulation once every 5 min to produce brief (12-s) isometric tetani. Also during the equilibration period, a reference length (close to the in situ length) for the muscle (Lref) was determined for each preparation. At Lref, the passive tension of the preparation was ~1-2% of maximal isometric force. Wortmannin, an inhibitor of MLCK (17), was used in the experiments to inhibit MLC phosphorylation. Wortmannin was predissolved in DMSO in a 1 mM stock solution and mixed with PSS in 1:999 volume ratio to obtain the final concentration of 1 µM for the experiments.Quick-freezing of muscle tissue for MLC phosphorylation
determination.
Muscles with or without wortmannin treatment were flash frozen at
Lref 7 s after the onset of EFS using
acetone chilled to dry ice temperature (80°C). It has been shown in
one of our recent studies that peak phosphorylation of MLC occurred at
~7-8 s after stimulation (16). The quick-freezing
apparatus has been described in detail (15). Frozen
muscles were quickly transferred to similarly chilled acetone
containing 5% trichloroacetic acid (TCA) and 10 mM dithiothreitol
(DTT); tissues were kept in this solution in 1.5-ml microcentrifuge
tubes overnight at
80°C.
Determination of the ratio of MLC phosphorylation. Details for MLC phosphorylation measurement have been described recently by us (16). Briefly, after the freezing protocol, tissues were allowed to come to room temperature, and then the acetone-TCA-DTT solution was aspirated. The TCA was removed from the samples by washing twice at room temperature with 1.0 ml of acetone containing 10 mM DTT. After the second acetone-DTT wash, the tissues were cut from between the aluminum foil clips and placed in 7.5 M urea extraction buffer (200 µl/mg wet wt tissue). Samples were continually inverted on a rotator for 1.5 h at room temperature and then centrifuged at 15,000 g for 30 min. Nonphosphorylated and phosphorylated MLC were separated using a method modified from Hathaway and Haeberle (10) and Persechini et al. (21) for nondenaturing 1-mm minigels. Proteins were transferred using a semidry technique to nitrocellulose sheets at 400 mA for 45 min. The sheets were incubated with 1:1,000 mouse monoclonal anti-myosin light chain (Sigma) for 60 min and then with a biotin-conjugated F(ab)-specific anti-mouse secondary antibody (Sigma; 1:2,500) for 40 min. The blots were then incubated for 40 min with a streptavidin-horseradish peroxidase conjugate (Amersham; 1:5,000) and rinsed (twice for 10 min each) in Tris-buffered saline (TBS) with 0.1% Tween 20 and once (5 min) in TBS alone.
Excess TBS was removed from the blots, which were then exposed to a 1:1 dilution of distilled deionized water with enhanced chemiluminescence (ECL Plus, Amersham) for 3 min. Lumigrams of the Western blots for nonphosphorylated and phosphorylated MLC were obtained on Amersham Hyperfilm ECL, scanned, and analyzed using ScanPlot software (28). Values are expressed as percent phosphorylated MLC by dividing the volume of phosphorylated MLC by the total volume of nonphosphorylated and phosphorylated MLC and multiplying by 100.Fixation of muscle tissue for electron microscopy.
Five muscle strips were dissected from each trachea. After
equilibration in the muscle bath, each of the five strips was fixed in
one of the five time points during the course of the experiment (Fig.
1). The experiment was completed using
four tracheas; therefore, the total number of muscle strips fixed for
electron-microscopic examination was 20.
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Electron microscopy. Details of the methods have been described by us recently (14). Briefly, the fixing solution contained 1.5% glutaraldehyde, 1.5% paraformaldehyde, and 2% tannic acid in 0.1 M sodium cacodylate buffer that was prewarmed to the same temperature as the bathing solution (37°C). Muscle preparations were fixed for 15 min while they were still attached to the experimental apparatus. Care was taken not to disrupt the muscle mechanically during the initial fixation. The strip was then removed from the apparatus and cut into small blocks (~1 × 0.5 × 0.2 mm) and put in the same fixing solution for 2 h at 4°C on a shaker. The blocks were then washed three times in 0.1 M sodium cacodylate (30 min). In the process of secondary fixation, the blocks were put in 1% OsO4-0.1 M sodium cacodylate buffer for 2 h and then washed three times with distilled water (30 min). The blocks were further treated with 1% uranyl acetate for 1 h (en bloc staining) and washed with distilled water. Increasing concentrations of ethanol (50, 70, 80, 90, and 95%) were used (10 min each) in the process of dehydration. For the final process of dehydration, 100% ethanol and propylene oxide were used (three 10-min washes each). The blocks were left overnight in the resin (TAAB 812 mix, medium hardness) and then embedded in molds and placed in an oven at 60°C for 8 h. The embedded blocks were sectioned on a microtome using a diamond knife. The ~100-nm-thick sections were then placed on 400-mesh cooper grids and stained with 1% uranyl acetate and Reynolds lead citrate for 4 and 3 min, respectively. Images of the cross sections of the muscle cells were obtained with a Phillips 300 electron microscope.
Morphometric analysis. Fifteen pictures were taken for each of the muscle samples fixed, for a total of 300 pictures. All pictures contained one whole cell cross section. Among the 15 pictures from each muscle sample, 5 contained nuclei, 5 contained a central cluster of mitochondria, and 5 contained scattered mitochondria and other organelles. This sampling criterion was used to ensure an even sampling of different segments of the cells. Myosin thick filaments were identified by eye and manually counted for the whole cell cross section. To eliminate possible bias in the analysis, the samples were coded so that the analysis was carried out in a "blind" manner. The codes were revealed only after all samples had been analyzed. The cell cross-sectional area was measured using a morphometric measurement device (Carl Zeiss). The thick filament density was obtained by dividing the number of thick filaments counted for the whole cell cross section by the area of the cell cross section minus areas occupied by the nucleus, mitochondria, and other organelles. The thick filament density measured was therefore the cytoplasmic density.
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RESULTS |
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Wortmannin inhibition of isometric force and MLC phosphorylation in
canine trachealis muscle.
We recently showed that MLC phosphorylation in canine trachealis muscle
reached a peak at ~7 s after the onset of EFS (16). In
the present study, the effect of wortmannin on MLC phosphorylation was
therefore examined by freezing the tissue at rest and 7 s after
the onset of EFS. Examples of the chemilumigrams are shown in Fig.
2. Isometric force and MLC
phosphorylation were totally abolished by 1.0 µM wortmannin.
Phosphorylation level at 7 s of EFS in the absence of wortmannin
was 46.3 ± 3.7%; this was reduced in the presence of wortmannin
to 4.5 ± 2.4%, which was not different from the baseline
phosphorylation level in relaxed trachealis muscle (4.3 ± 2.1%).
Resting tension was not affected by addition of wortmannin. These
results are summarized in Table 1.
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Effect of wortmannin on calcium transients.
Wortmannin had no significant effect on calcium transients as measured
by F400/500 using indo 1 (Fig.
3), whereas force of contraction elicited
by EFS was significantly and progressively attenuated in the presence
of 1.0 µM wortmannin (n = 5 preparations from 5 animals). This indicates that the processes upstream from the
generation of calcium signals were intact or at least functional in
these muscle preparations. It was noted from the force-calcium loops
that the intracellular calcium transient rose before the induction of
force in these canine tracheal smooth muscle preparations (Fig.
3B).
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Effect of wortmannin on cross-sectional density of myosin thick filaments. The thick filament density was examined under five different conditions as illustrated in Fig. 1. Muscle preparations fixed at time 1 (immediately after the equilibration period, Fig. 1) were the controls. At time 2, muscles had been exposed to wortmannin (1 µM) for ~30 min and the electrically elicited isometric force had diminished to nearly zero. At time 3, muscles had been subjected to a 5-min period of length oscillation, with a frequency of 0.5 Hz and a stretch amplitude of 30% Lref in the presence of wortmannin. The preparations were fixed immediately after the oscillation (time 3). At time 4, muscle preparations were fixed 30 min after the oscillation. During this period, muscles were allowed to "recover" in the absence of mechanical perturbation but in the presence of wortmannin. The muscles were also stimulated electrically once every 5 min to monitor force recovery. At time 5, muscles were fixed after a period of recovery (50 min) in the absence of wortmannin. Electrical stimulations were applied to the muscles to monitor force recovery. The recovery of force was incomplete even after 10 stimulations (50 min).
Electron micrographs of cross sections of trachealis muscle fixed under the control condition (Fig. 1, time 1) and a test condition (time 4) where MLC phosphorylation (when the muscle was stimulated) was inhibited by wortmannin are shown in Fig. 4. Figure 5 summarizes the results. Isometric force produced by the control group of muscles was 171 ± 14 kPa. In the presence of 1 µM wortmannin (times 2-4), the force was reduced to nearly zero (Fig. 5). The force recovered partially (71%) after removal of wortmannin from the PSS. Full recovery of the force was not attained after incubation of the muscle in zero-wortmannin PSS for 50 min with repeated washes (Fig. 5, time 5). The thick filament density was reduced slightly (9%, not significant) in the presence of wortmannin before mechanical agitation (Fig. 5, time 2). After the imposed length of oscillation, the density decreased significantly to 75% of the control value (Fig. 5, time 3). No recovery of force and thick filament density was observed as long as wortmannin was present in the solution, despite repeated stimulations in the isometric state (Fig. 5, time 4). The thick filament density recovered fully after removal of wortmannin, whereas isometric force recovered only partially (Fig. 5, time 5). Densities assessed from the three different cell longitudinal segments (central, near central, and near ends) under the same condition were not different; the results were therefore pooled. The densities measured from 15 cell cross sections in each muscle sample were averaged, and the mean of this average was averaged with the means from three other muscle samples fixed under the same experimental condition. One-way ANOVA showed that the variation in density associated with the five experimental conditions was significant (P < 0.05). The density variation between times 1, 2, and 5, however, was not significant, nor was the density difference among times 3 and 4 (Fig. 5). One-way ANOVA also showed that the variation in force was significant (P < 0.05).
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Control observations. Because DMSO was used as solvent for wortmannin (see METHODS), the effect of DMSO alone on isometric force and thick filament density was investigated. When 10 µl of DMSO (the same amount used as vehicle for wortmannin in actual experiments) were added to the 10-ml muscle bath, no change in active isometric force or resting tension could be detected in the pair of muscle preparations examined. There was no change in thick filament density (P > 0.5, t-test): 65.8 ± 5.2 and 63.2 ± 2.9 filaments/µm2 for the control and test (1 µl/ml DMSO) preparations, respectively. Eight cell cross sections for each preparation were used to determine the thick filament density.
Table 2 lists the averaged number of thick filaments per cell cross section and averaged cross-sectional area of the cells. The cross-sectional density of the thick filaments was calculated from the measurements described above.
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DISCUSSION |
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Results from the present study indicate that MLC phosphorylation played an important role in myosin thick filament reassembly after partial dissolution induced by oscillatory strains in intact airway smooth muscle. Although the thick filament density was measured only in the resting state, the finding that the density is dependent on the history of MLC phosphorylation indicates that thick filament formation in vivo could be controlled by the transient light chain phosphorylation associated with stimulation and suggests that the mechanism described for the MLC phosphorylation-dependent thick filament assembly/disassembly in solution studies (4, 19, 20, 26, 27) may be applicable to the regulation of thick filament formation and contraction in intact cells.
Calcium handling in intact airway smooth muscle appears to be unaffected by wortmannin (Fig. 3). The change in the myosin thick filament density observed in the presence of wortmannin, therefore, is not likely due to calcium-dependent regulatory pathways upstream of MLC phosphorylation. Our recent study showed that calcium played an important role in the maintenance of integrity of the thick filaments in trachealis muscle (11). The influence of calcium on thick filament formation probably goes beyond the participation of calcium in the calcium/calmodulin-MLCK activation pathway that controls MLC phosphorylation; the resting level of intracellular calcium in unphosphorylated trachealis muscle appeared to be important also for the maintenance of thick filaments (11, 16), because by lowering the resting calcium concentration through incubation of muscle in zero external calcium + EGTA, the cross-sectional density of the thick filaments was reduced by 35% (11). Because there is no evidence that wortmannin altered the resting calcium or resting MLC phosphorylation level, the change in thick filament density observed in this study is not likely related to changes in resting intracellular calcium level.
Although wortmannin is a potent MLCK inhibitor, it is not specific.
Wortmannin has been shown to inhibit phosphatidylinositol 3-kinase-
and mitogen-activated protein kinases (32), which in turn
modulate smooth muscle contraction via a pathway distinct from the
calcium/calmodulin-MLCK pathway (33). Interpretation of
the present results is based only on the observable effects of
wortmannin on MLC phosphorylation.
Isometric force production in electrically stimulated airway smooth muscle appears to be totally dependent on MLC phosphorylation. Formation of myosin thick filaments appears to be partially dependent on MLC phosphorylation (Fig. 5). At least some of the thick filaments were preserved in resting muscle where MLC phosphorylation was found to be nearly zero; this level of phosphorylation was not changed by addition of wortmannin (Table 1). It was therefore not surprising to see that addition of wortmannin did not produce a significant change in the cross-sectional density of the thick filaments, even though the ability of the muscle to generate force was abolished (because of the lack of MLC phosphorylation on muscle stimulation; Fig. 2 and Fig. 5, time 2). However, in the unphosphorylated state, the thick filaments were not stable; a significant reduction in the cross-sectional density of the thick filaments was observed after the muscle was subjected to a brief period of length oscillation (Fig. 5, time 3). The same oscillation protocol was used in one of our recent studies (14), in which a similar degree of reduction in myosin thick filament density was shown in muscles subjected to the oscillation in the relaxed state without wortmannin. The thick filament instability, therefore, was not due to the presence of wortmannin but likely to the fact that the level of MLC phosphorylation was low in the resting state. We did not examine the thick filament stability in the activated state; it is therefore not known whether the same oscillatory strains applied to activated trachealis muscle would produce the same degree of thick filament disassembly observed in the relaxed state. In muscles not exposed to wortmannin, the reduction in filament density was transient: the density recovered fully after a few cycles of contraction and relaxation in isometric mode (14). The present results showed that, in the presence of wortmannin, the density did not recover, even after repeated stimulation in the isometric state (Fig. 5, time 4). We interpret this as evidence of the need for cyclic MLC phosphorylation (associated with the repeated stimulation without wortmannin) for thick filament reassembly after the filaments have been partially disassembled by mechanical agitation. The effect of wortmannin appeared to be reversible. Although the force did not recover fully after removal of the inhibitor, the thick filament density recovered to the preoscillation level (Fig. 5, time 5). The incomplete force recovery could be explained by an incomplete removal of wortmannin from the intracellular space, with some MLCK still inhibited by the residual wortmannin. The complete recovery of the thick filament density may indicate that thick filament formation does not depend on activation of all available MLCK.
It is not known whether greater amplitudes of length oscillation will cause a total disappearance of the thick filaments in muscles with unphosphorylated MLC. Our previous studies in resting airway smooth muscle (29) have shown that the amount of decrease in isometric force after oscillation was dependent on the oscillation amplitude. It is likely that the postoscillation thick filament density is also dependent on the applied oscillation amplitude (14); that is, the greater the amplitude of oscillatory strain, the more likely it is that the thick filaments will disassemble. In the present study, the stretch amplitude was limited to 30% Lref, which is about the same degree of stretch experienced by airway smooth muscle in vivo during a deep inspiration (5). This amplitude obviously did not result in complete disassembly of the thick filaments in intact, relaxed smooth muscle (Fig. 5). It is possible that a minimal amount of thick filaments will always be maintained in intact smooth muscle cells, no matter how rigorously the muscle is mechanically perturbed. Kendrick-Jones et al. (13) showed that, under in vitro conditions, once a critical concentration of myosins is exceeded, thick filaments will form automatically, even among unphosphorylated myosins.
The thick filament density found in the present study differed significantly from that found in one of our previous studies (14). The underlying cause is not entirely clear. The cross-sectional areas of muscle cells in our previous study (14) were, on average, 20-30% larger than those in the present study, suggesting that we fixed the muscle strip at a longer (more stretched) length in the present study (if it is assumed that the cell volume is conserved at different cell lengths). Interestingly, we consistently found that cells fixed at longer lengths have higher thick filament density (unpublished observation), especially in the activated state, suggesting that thick filament content is increased in muscles accommodated at longer lengths. The unstable thick filaments in the resting (unphosphorylated) state could be another factor contributing to the variability in the density measurements. Although care was taken not to disturb the muscle (mechanically) in the fixing process, it was not possible to eliminate mechanical disturbance totally. Our studies were therefore designed in a way that a control was always included in a set of test experiments to eliminate variabilities such as those associated with different animals, different ways the tissues were handled in each set of experiments (by different experimenters), and different batches of chemicals used in the tissue fixation.
Needham and Shoenberg (18) proposed a controversial mechanism for smooth muscle contraction based on the observation that myosin thick filaments in smooth muscle were extremely labile compared with those in striated muscle. They postulated that the thick filaments were formed only after the muscle was activated and the filaments were dissolved when the muscle was relaxed. The hypothesis was discarded by most investigators in the field because of the findings that thick filaments were present in relaxed smooth muscles with unphosphorylated MLC (3, 6, 7, 24, 25) and that, at least in chicken gizzard smooth muscle, there was no evidence that monomeric myosins were being incorporated into thick filaments when the muscle was activated (12). More recently, it was found that although thick filaments did exist in relaxed anococcygeus muscle, they lengthened on activation (8, 9, 30, 31). Those same studies also found that such myosin evanescence did not exist in taenia coli. It appears that myosin filament lability is smooth muscle type specific and that the existence of thick filaments in the relaxed state does not mean that the filament is nonlabile. We found in a recent study (11) that the cross-sectional density of the thick filaments in the trachealis muscle increased dramatically (144%) when the muscle was activated. Functional studies also suggest that in the trachealis muscle the thick filaments lengthen during the time course of an isometric contraction, which could account for the velocity decrease and force increase observed during the same time period (23). The present study has shed additional light on this subject: thick filaments in airway smooth muscle are structurally unstable; partial dissolution of the filaments occurs when the relaxed muscle is strained; and inhibition of the light chain phosphorylation by wortmannin prevents reassembly of the myosins into filaments.
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ACKNOWLEDGEMENTS |
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The authors thank Pitt Meadows Meats (Pitt Meadows, BC, Canada) for the supply of fresh porcine tracheas in kind support for this research project and Cathy Pollock (Canada Food Inspection Agency, Establishment 362) for help in obtaining the tracheas.
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FOOTNOTES |
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This study is supported by Canadian Institutes of Health Research Operating Grant MOP-13271 to C. Y. Seow.
Present addresses: R. W. Mitchell, Sect. of Pulmonary and Critical Care Medicine, Dept. of Medicine, University of Chicago, Chicago, IL 60637; T. Burdyga, Dept. of Physiology, University of Liverpool, Liverpool L69 3BX, UK.
Address for reprint requests and other correspondence: C. Y. Seow, Dept. of Pharmacology and Therapeutics, University of British Columbia, 2176 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3 (E-mail: cseow{at}interchange.ubc.ca).
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.
First published January 16, 2002;10.1152/ajpcell.00554.2001
Received 18 November 2001; accepted in final form 10 January 2002.
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