INTRODUCTION

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WE PREVIOUSLY PROPOSED that some of the differences between smooth and striated muscle are due to the need for plastic structural arrangements that allow smooth muscle to adapt to a wide range of lengths (6). One corollary to this theory is that the well-known velocity slowing of smooth muscle during sustained activation (2, 3, 11, 12, 14, 17, 18) is due to thick filament lengthening, which places more cross bridges in parallel and reduces the number of cross bridges in series (6, 17). We recently showed that changes in the force-velocity curves during the early parts of a tetanus are consistent with this proposal (17). This "series-to-parallel" explanation is distinctly different from metabolic explanations suggesting that slowing is due to a reduction in the cross-bridge cycling rate. The most prevalent variant of these metabolic theories is the "latch-bridge" hypothesis (2, 3), positing that bridges slow when they become dephosphorylated. Support for this hypothesis derives from the finding that slowing is coincident with partial dephosphorylation of bridges. To test whether the slowing we attribute to thick filament lengthening could be coincident with dephosphorylation, we measured the level of myosin light chain (MLC) phosphorylation during the rise of force in electrically stimulated tetani.

The data analysis used here requires a knowledge of the activation level at each instant when force-velocity properties are assessed (17). The parameter used to signal activation was the maximum power value in the curves fitted to the instantaneous force-velocity curves (5, 17). A major conclusion of the present study is that maximum power varied in parallel with phosphorylation, at least when phosphorylation was changing slowly. Such a result is expected if the myofilaments were activated by myosin phosphorylation. The limits as well as the significance of this conclusion are discussed below. Another major finding of these experiments is that velocity declined most rapidly early in the tetanus when phosphorylation was still rising. Although this conclusion does not specifically support the series-to-parallel mechanism we have proposed, it does argue strongly against dephosphorylated bridges causing velocity slowing, at least under the conditions used here.

	METHODS

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Two parallel sets of experiments were done in muscles stimulated to generate tetani with bipolar pulses at 37°C. One was a measure of MLC phosphorylation in trachealis muscle frozen at various times during the rise and early plateau of the tetani. The other was a measure of the force-velocity properties in similar muscles at similar times.

Preparation

Solutions

Apparatus

Determination of MLC Phosphorylation

Nonphosphorylated and phosphorylated regulatory MLC were separated using a method modified from Hathaway and Haeberle (8) and Persechini et al. (15) for nondenaturing 1-mm minigels. At least two volumes of crude extract (e.g., 15 and 25 µl) of each sample were loaded in adjacent lanes on 3% acrylamide-urea stacking gels. Proteins were separated on 10% polyacrylamide-glycerol gels until 45 min after the dye front exited (total time ~4.5 h). Electrophoresis was performed at 6.0 mA/gel through the stacking gel (~1.0 h) and at 8 mA/gel through the separating gel (~3.5 h). Running buffer (upper and lower tanks) contained 100 mM glycine and 50 mM Tris base, pH 8.7; 2-mercaptoethanol (2.0 µl/ml buffer) was added to the upper tank.

A semidry technique at 400 mA for 45 min was used to transfer proteins to nitrocellulose sheets. Transfer buffer contained 25 mM Tris · HCl, 192 mM glycine, and 20% methanol at pH 8.3. Nitrocellulose sheets were blocked overnight at 4°C in Tris-buffered saline (TBS), pH 7.6, containing 0.1% Tween 20 (TBS-T) and 3.0% nonfat dry milk. On the next day, sheets were incubated at room temperature with 1:1,000 mouse monoclonal anti-MLC (Sigma Chemical) for 60 min in TBS-T containing 1.0% nonfat dry milk and then washed three times (5, 10, and 15 min) with TBS-T-1.0% milk. Sheets were then incubated with a biotin-conjugated, F(ab)-specific anti-mouse secondary antibody (Sigma Chemical; 1:2,500) for 40 min at room temperature in TBS-T-1.0% milk and washed three times (5, 10, and 15 min) with TBS-T. The blots were incubated for 40 min at room temperature with a streptavidin-horseradish peroxidase conjugate (Amersham; 1:5,000) and then rinsed twice (10 min each) in TBS-T and once (5 min) in TBS.

Excess TBS was removed from the blots, which were then exposed to a 1:1 dilution of distilled deionized water with ECL⁺Plus (Amersham) for 3 min. Chemilumigrams of the Western blots were obtained using Amersham Hyperfilm ECL. Only two spots, representing nonphosphorylated and phosphorylated MLC, were seen in each lane, and typical lumigrams for each time of measurement are shown in Fig. 1. The spot densities were measured using a Hewlett-Packard 4C scanner and ScanPlot software (20). This program allows the user to outline areas of the gel for analysis and then integrates the pixel density over the area outlined. Background was determined as the integral of an area of gel without spot, and the average pixel density in this area was subtracted from the average pixel density in the area containing the entire spot. Percent phosphorylation was determined as 100 times the ratio of the density of the spot containing phosphorylated light chain to the sum of the density of the spots for phosphorylated and nonphosphorylated protein in the same lane. A test for linearity of the assay was conducted by comparing the percent phosphorylation determined for the more heavily loaded sample with that for the less heavily loaded companion sample. Nonlinearity would produce a systematically greater level for one or the other of the loads. No systematic deviation was found, suggesting that the assay was very nearly linear.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Chemilumigrams of myosin light chain Western blots (left column) obtained from muscles at different times in tetanus (middle column). Lumigrams of the Western blots were scanned using ScanPlot software (20), and the ratio of phosphorylated myosin light chain expressed as percentage of the total light chain is given in right column. Two protein concentrations were loaded on the gels for each tissue frozen, typically 30 and 50 µg/lane. The upper lane for each of the chemilumigrams contains the greater protein load. The zero time point is for an unstimulated muscle frozen when stimulation would have begun, if the stimulator had not been switched off.

Mechanical Studies

Protocol. Force-velocity data were obtained from muscles tetanized with bipolar electrical stimulation at 60 Hz, with the ends held constant until a predetermined time and then released to various isotonic loads. Velocity was measured as the slope of a tangent fitted to the length records from 50 to 115 ms after the release. One force-velocity point was obtained from each tetanus, and a family of eight to nine force-velocity points obtained from as many tetani were used to construct a single force-velocity curve. The time when tetanic force in the tetanus was judged to have reached its plateau was taken as the "reference" time. This time ranged from 11.0 to 12.5 s (mean 11.4 s) in the different muscles.

Data analysis. Force-velocity data were analyzed to determine the values of velocity and maximum power for the test curves relative to reference curves in the same experiment using a method previously developed (17). First, a nonlinear, least-squares method was used to fit the force-velocity points obtained at each time with a hyperbola (9). Maximum power in the fitted curves, determined as maximum value of the product of force times velocity, was used as a measure of activation (5, 17). The ratio of this value in each test curve to the value in its reference curve was used to signal the relative level of activation in the test curves and to adjust the test curve for the differences in activation before determination of the relative velocity in the test curve. The adjustment was made by dividing test isotonic forces by the ratio of maximum power values. A least-squares method was then used to find a common factor by which the reference curve could be scaled to superimpose on the adjusted test data. This common factor scaled force and velocity in opposite directions so that the reference curve fitted the test data. The scaling factor was then used to signal the difference in velocity at the test time relative to that at the reference time.

	RESULTS

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The results are summarized in Fig. 2, where the values of the several parameters are superimposed on the isometric force record obtained by signal averaging one reference tetanus from each of the seven muscles. This record and the percentage of MLC phosphorylation (means ± SE) assayed in muscles frozen at the times indicated are plotted in Fig. 2, A and B. The baseline level of phosphorylation (3.8 ± 1.4%) was assessed from six resting muscles run in with repetitive tetani at 5-min intervals for 1 h and frozen without stimulation at 5 min after the last stimulation.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Force, maximum velocity, maximum power, and myosin light chain phosphorylation at different times during electrically stimulated tetani. Isometric force and level of phosphorylation are plotted in A and B to be compared with maximum velocity in A and maximum power in B. Force, velocity, and power are plotted relative to the values when the muscles achieved their plateau value (). Phosphorylation values are percentages of total light chain phosphorylated, with scale adjusted so that the value at the reference time corresponds to the mechanical values at the reference time. Force record for times up to the reference is a signal average of 7 records obtained from each of the muscles in the first data set. Force value plotted at 25 s () is the mean of 8 muscles in the second set. Error bars indicating SE have been omitted where the plotted error is less than the size of the symbol and, for some points, have been omitted on one side to avoid clutter. Numbers above symbols in A indicate number of muscles averaged. The resting level of phosphorylation measured in 6 muscles run in by stimulating 12-s tetani every 5 min was measured at 5 min after the onset of the last stimulus and is plotted just before the zero time point. This resting level is also plotted as a horizontal dashed line to indicate the baseline value for the experiments.

Figure 2A superimposes on these data the mean values of the velocity scale factors at different times; Fig. 2B superimposes the maximum power values at the same times. As indicated in Fig. 2A, velocity rises to an early peak and falls most rapidly at a time when phosphorylation is still rising. By contrast, maximum power and phosphorylation (Fig. 2B) have very similar time courses. Although the changes in phosphorylation are relatively greater than the changes in maximum power, both parameters peak at 7-8 s after simulation and then fall progressively through the remainder of the tetanus.

A final point to be made from Fig. 2 is that the disparity between velocity and phosphorylation exists mainly at the outset of the tetanus. As shown in Fig. 2, all four parameters, force, velocity, power, and phosphorylation, fall to various extents after an initial peak. Thus the disparity between the decline in velocity and the rise in phosphorylation is greatest early in a tetanus and might be missed if these early times are not studied. The contractile parameter that correlated best with phosphorylation over the times studied was maximum power.

	DISCUSSION

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We previously proposed that the early velocity slowing in airway smooth muscle is due to thick filament lengthening (17), and not to metabolic slowing, as has been postulated to be caused by dephosphorylation of cross bridges (2, 3). The present experiments were undertaken to determine whether the velocity slowing was at all correlated with a fall in MLC phosphorylation. The results show that most of the slowing occurs over a period when phosphorylation is rising, at least during the phase of the tetanus when force is rising. We interpret this finding as arguing strongly against dephosphorylated "latch bridges" being the cause of the early slowing measured here.

Our previous study (17) showed that force and velocity change in opposite directions during the rise and early plateau of a tetanus. When a correction was made for variations in the level of activation, the reference force-velocity curve could be superimposed on test curves obtained at different times by using a common factor to scale force and velocity points in opposite directions. Here we use the same technique to compare velocities in the test and reference curves, and we use the scale factor as a measure of changes in velocity. Although the rationale for this method derives from a specific model, the method has the advantage of using the entire force-velocity curve to assess velocity changes. A simple determination of maximum velocity as the zero force intercept of the fitted curve would give results similar to those presented here but is subject to the inaccuracy of having the values dominated by a few low-force points in each curve.

This analysis requires a knowledge of the level of activation at each time. As in our earlier study (17), we use maximum power to signal the level of activation. This practice derives from the consideration that activated cross bridges contribute to power, irrespective of whether they are arrayed in series or in parallel. When loading conditions are optimized so that the activated bridges produce maximum power, then the level of power will reflect the number of activated bridges (5). The phosphorylation data support this use of maximum power to signal changes in activation. In addition, the correlation of maximum power with phosphorylation suggests that the only effect of phosphorylation on mechanical activity is to activate individual cross bridges without otherwise altering their contractile kinetics.

It is well known that smooth muscle is activated by phosphorylation of MLC (1, 7, 19), and the level of activation is likely to be determined by the extent of phosphorylation. The finding that maximum power and light chain phosphorylation vary together suggests that both indicate the level of activation. It should also be emphasized that this correlation is likely to be true only when levels of phosphorylation are changing slowly. If events must occur between the chemical change and the onset of contractile activity, such that there is a lag between changes in light chain phosphorylation and changes in shortening or force production, then the biochemical change will precede the mechanical manifestation of activation. Such events would certainly include cross-bridge attachment to thin filaments followed by chemical reactions that might be necessary to allow cross-bridge movement through the power stroke and, possibly, other events such as thick filament formation. Similarly, if deactivation results from dephosphorylation and attached bridges are dephosphorylated, then there would be a lag between dephosphorylation and the fall of mechanical activity. An example of such a lag is shown in our development of the method for rapidly freezing muscles (13). Phosphorylation begins to rise during the latency before the onset of force development, rises rapidly, and is substantial before the muscle is capable of any contractile activity.

The possibility must be considered that the measured level of phosphorylation represents something in addition to the protein change required for activation. The use of antibodies to detect the different forms of light chain almost certainly excludes other proteins migrating with light chain as a source of spurious changes in spot density. It is known, however, that there are at least two phosphorylation sites on MLC (15), and it is not clear how these relate to each other or to the physiological processes investigated. Under more extreme conditions than those used here, we have seen three separate forms of light chain with different mobilities, probably representing non-, mono-, and diphosphorylated forms of light chain. The absence of a third spot in any of the lumigrams seen here suggests that there were no diphosphorylated light chains under the present physiological conditions. The experiments do not, however, exclude the possibility that the single protein spots seen on our gels contain two forms of monophosphorylated light chain, with dephosphorylation of only one of these needed to cause velocity slowing. Such a mechanism seems unlikely, however, because it requires that the relevant site first become phosphorylated, to activate contraction and to allow rapid shortening, and then become dephosphorylated, to cause velocity slowing, while the other site becomes phosphorylated, to maintain activation, without appreciable diphosphorylation. In addition, phosphorylation rose substantially as velocity declined, and this would require substantial phosphorylation of the second site as the first site became dephosphorylated.

Relationship to Earlier Work

At least two separate laboratories have studied tracheal muscles stimulated electrically. The first such experiments, in bovine tracheal muscle, suggested that velocity and phosphorylation fell together during the rise of tetanic force, but the time resolution would not have revealed the separation found here. Velocity and phosphorylation were measured at 2, 5, and 15 s, with an additional phosphorylation level measured at 10 s, and the highest levels of both were seen at 2 s. These experiments could not have distinguished an earlier velocity peak at 2 s and a later phosphorylation peak at 6-8 s. In addition, a later study from the same group showed a dissociation of velocity and dephosphorylation in vascular smooth muscle activated chemically with different agonists (13).

The other group that studied phosphorylation and velocity did not report the results together. In one study of the effect of ragweed sensitization and shortening, they showed that, for isometric contractions of muscles from unsensitized dogs, phosphorylation peaked at 7.5 s (Fig. 1B in Ref. 10). In another study, they showed that velocity peaked at 2.5 s (18). These results are in exact agreement with those presented here. Thus much of the published evidence for tracheal muscle stimulated electrically is in agreement, or at least not in disagreement, with that presented here.

Conclusion