Address correspondence to Anders Arner, Department of Physiological Sciences, BMC F11, Tornavägen 10, SE-221 84 Lund, Sweden. Fax: (46) 46 222 7765; E-mail: Anders.Arner{at}mphy.lu.se
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ABSTRACT |
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Key Words: nonmuscle myosin urinary bladder ATP ADP phosphate
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INTRODUCTION |
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The velocity of actin over nonmuscle myosin is slow compared with muscle myosin in vitro (Umemoto and Sellers, 1990), suggesting differences in the kinetics of the cross-bridge interactions. In general, a relationship between the rate of the ADP release from the actinmyosinADP complex and the shortening velocity of muscle fibers has been proposed (Siemankowski et al., 1985
; Weiss et al., 2001
). Consistent with this, smooth muscle myosin has a higher ADP affinity in vitro (Cremo and Geeves, 1998
) and in muscles fibers (Arheden and Arner, 1992
; Nishiye et al., 1993
) compared with the faster skeletal muscle myosin. This correlation is also valid within the smooth muscle group; higher ADP affinity has been demonstrated in slow compared with fast smooth muscle types (Fuglsang et al., 1993
; Khromov et al., 1998
; Löfgren et al., 2001
). At present, information regarding the kinetics of the actinnonmuscle myosin interaction of the organized contractile system is very sparse.
Recently, a trangenic mouse strain lacking the smooth muscle myosin heavy chain (SM-MHC)* expression has been introduced (Morano et al., 2000). These animals die a few days after birth but their urinary bladder smooth muscle obtained immediately postnatally can contract by the action of the nonmuscle myosins. This contraction has a slow onset in response to stimulation and is characterized by a low shortening velocity. The smooth muscle of these animals provide a unique model for studies of regulation and contractile kinetics of nonmuscle myosin.
In the present study we have used the urinary bladder from SM-MHCdeficient mice to answer the following questions: (a) Is the slow onset of the contraction of intact smooth muscle preparations from the SM-MHCdeficient mice due to altered Ca2+ handling? (b) Can nonmuscle myosins form thick filaments in the smooth muscle cells? (c) What is the shortening velocity of fully activated organized nonmuscle myosin? (d) What are the binding characteristics of substrates and products (MgATP, MgADP, and inorganic phosphate) in the cross-bridge cycle of nonmuscle myosin?
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MATERIALS AND METHODS |
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Homozygous SM-MHCdeficient mice were identified by the thin-walled and distended urinary bladder and the lack of an initial force transient in intact preparations (Morano et al., 2000). This definition of the phenotype was confirmed by gelelectrophoresis showing a low total myosin heavy chain content and a lack of the 205-kD SM1-MHC band in these animals. It has been shown that the myosin content is essentially similar in the mice heterozygous and homozygous for the SM-MHC gene (Morano et al., 2000
) and these animals were analyzed as one group denoted SM-MHC expressing.
Measurements of Intracellular Calcium in Intact Preparations
Small strips (23 mm long, 500 µm wide) were prepared from the urinary bladder wall in the sagittal direction. The preparations were wrapped at each end with aluminum foil, and transferred to an oxygenated HEPES (N-(2-Hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid)) buffered solution containing, in mM: NaCl 118, KCl 5, Na2HPO4 1.2, MgCl2 1.2, HEPES 24 (pH 7.4), glucose 10, and CaCl2 1.5. Loading of Fura-2 was performed for 34 h at 22°C in the HEPES-buffered solution containing 10 µM Fura-2-AM (Molecular Probes), 1% DMSO, and 0.02% Pluronic F127. After an
30-min wash in the HEPES buffer the preparations were transferred to a 1-ml bath on the stage of an inverted microscope. The muscles were kept in a Krebs-Henseleit solution of the following composition (in mM): NaCl 119, glucose 12.2, KCl 4.6, NaHCO3 25, KH2PO4 1.2, MgSO4 1.2, CaCl2 2 at 37°C, gassed with 95% O2/5% CO2 giving a pH of 7.4. Recordings of force and intracellular free Ca2+ concentration were made in the Krebs-Henseleit solution as described previously (Lucius et al., 1998
) in the relaxed state (with and without 2 mM extracellular CaCl2) and after activation with 80 mM KCl. Force and fluorescence signals (at an emission wavelength of 510 nm) at excitation wavelengths 340 and 380 nm were recorded with a time resolution of 46 Hz. An internal calibration of the Ca2+ signal was made as described previously (Lucius et al., 1998
), using 50 µM ionomycin for permeabilization, 20 mM MnCl2 for quenching, and an apparent dissociation constant of the calciumfura-2 complex of 224 nM (Grynkiewicz et al., 1985
).
Preparation of Permeabilized Tissue
Strips (23 mm long, 500 µm wide) of urinary bladder were pinned to silica gel and permeabilized with 1% Triton X-100 as described previously (Arner and Hellstrand, 1985
), and thereafter kept in a glycerol containing solution at -20°C and used for quick-release experiments and isometric force registrations, as described below, within a month. All experiments on skinned muscle preparations were performed at room temperature (22°C).
Quick-release Experiments
Maximal unloaded shortening velocity (Vmax) at maximal activation was examined with the isotonic quick-release method (compare Arner and Hellstrand, 1985). The preparations were attached with aluminum clips between a force transducer (AE 801; SensoNor) and an isotonic lever arm. The experimental procedure was executed as described previously (Löfgren et al., 2001
). The preparations were kept maximally activated using repeated incubation with ATP-
-S (Arheden et al., 1988
). Contractions were elicited by transfer to an ATP-containing solution (see below) and at the plateau of each contraction, a series of 1525 releases to different afterloads was performed. After each release, the length and afterload were recorded for 1 s. Velocity was determined 100 ms after release. Vmax was calculated by fitting the Hill equation (Hill, 1938
; Eq. 1) to the force-velocity data and extrapolating to zero afterload:
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In this equation, V is the velocity, Po is isometric force, P is afterload, and a and b are constants. Two series of experiments were performed: (a) varied [MgATP] in the presence of an ATP-regenerating system and (b) varied [MgADP] at constant [MgATP] in the absence of an ATP-regenerating system. The ATP and ADP concentrations were applied at random order. At the end of the experiments in the first series, a contraction in 3.2 mM MgATP was followed by a 40 min long incubation in rigor solution (0 MgATP and 0 phosphocreatine and creatine kinase with 50 U/ml hexokinase and 10 mM glucose) before a series of releases was performed, in order to determine the apparent Vmax in rigor (Löfgren et al., 2001).
Measurements of Isometric Force
Skinned preparations were mounted between a force transducer (AE 801; SensoNor) and a stainless steel pin and maximally activated using ATP--S (Arheden et al., 1988
). In the series of experiments where the effects of inorganic phosphate on isometric force was determined, the inorganic phosphate was introduced in the ATP-containing activating solution and the different concentrations of inorganic phosphate were applied at random order. In a separate series of experiments, the maximal force generation at 3.2 mM MgATP was determined. These preparations were fixed in 1% glutaraldehyde, embedded in Epon, and sectioned for light microscopy to determine preparation cross-sectional area.
Solutions Used in Experiments on Skinned Preparations
For the experiments on skinned preparations, a solution of the following composition was used: 30 mM TES, 4 mM EGTA, and 2 mM free Mg2+. The ionic strength and the pH were adjusted to 150 mM and 6.9, respectively, using KCl and KOH. The standard ATP-containing solution contained 3.2 mM MgATP and an ATP-regenerating system with 12 mM of phosphocreatine and 0.5 mg/ml of creatine kinase. In the ADP-containing solutions, the ATP-regenerating system was not used and 0.2 mM of the myokinase inhibitor AP5A was added (Feldhaus et al., 1975).
Western Blotting and Immunohistochemistry
Pieces of whole bladder wall were quickly frozen in liquid N2 and kept at -80°C. The samples and SDS gels were prepared essentially as described by Wede et al. (2002). Samples were loaded on three gels. One gel was stained with Coomassie blue and the other two were used for Western blot using a polyclonal rabbit antibody against nonmuscle myosin heavy chain A (NM-MHC-A), a gift from Dr. R. Adelstein (Kelley et al., 1996
), or a polyclonal rabbit antibody against NM-MHC-B (Sjuve et al., 2001
). Immunoreactivity was detected using EnhancedChemiLuminescence (ECL; Amersham Biosciences) and visualized with a Fluo-S Max (Bio-Rad Laboratories). For immunohistochemistry, urinary bladders were cut open, pinned to pieces of cork, fixed in 2% formaldehyde containing 0.2% picric acid in 0.1 M phosphate buffer (pH 7.2) over night, rinsed in a Tyrode solution with 10% sucrose, and embedded for cryosectioning (Sjuve et al., 1998
). Sections were stained with NM-MHC-A or NM-MHC-B antibodies. Double staining with a monoclonal antibody raised in mouse against smooth muscle
-actin (Cy3 conjugated; C6198, Sigma-Aldrich) was used to determine the colocalization of the proteins in cells/regions.
Electron Microscopy
Strips of urinary bladder tissue were pinned to silica gel and fixed using glutaraldehyde and paraformaldehyde as described previously (Sjuve et al., 1998). The preparations were stored at 4°C in cacodylate buffer (0.125 M) with 0.1% glutaraldehyde. Post fixation was in 1% OsO2, followed by contrasting with uranyl acetate and Reynolds' lead citrate, and embedding in Epon. Sections, 50-nm thick, were examined using a Jeol JEM 1230 microscope equipped with a Gatan Multiscan CCD camera Model 791.
Statistics
Values are given as means ± SEM. Curve fitting was performed using routines implemented in Sigma Plot (SPSS Science). Statistical comparisons were made using Student's t test or repeated measures analysis of variance (using SPSS 10.1.3).
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RESULTS |
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To ensure that the preparations were fully activated with the ATP--S thiophosphorylation procedure, we performed experiments (n = 2 in each group) where new-born SM-MHCexpressing and SM-MHCdeficient tissues were compared. The preparations were subsequently (a) suboptimally activated with 3 min ATP-
-S treatment, (b) activated for 15 min with ATP-
-S according to our standard protocol and (c) activated for 30 min with ATP-
-S in the presence of 30 nM microcystin to fully inhibit the phosphatase. The force after 3 min thiophosphorylation (SM-MHCdeficient: 0.49 ± 0.08; SM-MHCexpressing: 0.58 ± 0.03) and after 30 min (SM-MHCdeficient: 1.12 ± 0.03; SM-MHCexpressing: 1.15 ± 0.04) relative to that after the 15-min thiophosphorylation period was similar in the two groups. These results suggest that no difference in the rate of Ca2+-induced light chain phosphorylation is present between the two groups. Experiments in which free Ca2+ level was increased (pCa 94.5) in the presence of calmodulin at the plateau of contractions after the 15- and 30-min thiophosphorylatyion periods were also performed. No further increase in force was detected, showing that phosphorylation was maximal and sufficient to fully activate the tissues.
Nucleotide Dependence of Maximal Shortening Velocity
To examine the dependence of Vmax on nucleotides, separate experiments in order to construct concentration-response curves of MgATP and of MgADP (at 6 and 10 mM [MgATP]) were performed on the SM-MHCdeficient and SM-MHCexpressing tissues. We observed, in accordance with our previous study on the guinea pig aorta and taenia coli, that a significant shortening velocity could be recorded in ATP-free rigor solution (Löfgren et al., 2001). The mechanism behind this phenomenon is unknown, but most likely it reflects a visco-elastic property of the tissue. To correct for the influence of this process, the apparent Vmax at zero MgATP was determined on each muscle preparation at the end of the experiment. The mean value of this velocity was subtracted from each Vmax value as described previously (Löfgren et al., 2001
). These velocities were 26.2 ± 2.4% (new-born SM-MHC expressing, n = 6), 27.0 ± 1.5% (3-wk-old SM-MHCexpressing, n = 6) and 41.7 ± 7.0% (SM-MHCdeficient, n = 6) of the Vmax at saturating MgATP. Fig. 3
shows the influence of varied concentrations of MgATP (Fig. 3 A) and MgADP (Fig. 3 B) on Vmax. The data at different MgATP were fitted to a hyperbolic equation (Eq. 2), giving an apparent binding constant (Km) for MgATP and Vmax.
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The parameter "Max" denotes the estimated maximal shortening velocity at saturating [MgATP]. This parameter was in the curve fitting not different from the measured maximal velocities at 3.2 and 10 mM MgATP for each group. The mean Km values for MgATP and Vmax were for the new-born SM-MHCexpressing: 0.013 ± 0.004, for the 3-wk-old SM-MHCexpressing: 0.024 ± 0.009, and for the SM-MHCdeficient mice: 0.023 ± 0.017 mM, n = 56. In view of the low velocities, especially in the SM-MHCdeficient tissue, the number of data points below maximal were limited and therefore we cannot exclude a small systematic error in the determination of Km. However, based on the values from the individual experiments presented above and the mean data presented in Fig. 3 A, we conclude that the Km values for the three groups were similar.
Experiments with varied concentrations of MgADP have to be performed in the absence of an ATP-regenerating system. High MgATP concentrations (6 and 10 mM) were used in these experiments, to ensure that the ATP level was saturating even in the absence of ATP-regenerating system. The Vmax at 6 and 10 mM without ATP-regenerating system were for all groups between 98 and 112% of Vmax at 10 mM MgATP with ATP regeneration. As seen in Fig. 3, B and C, MgADP inhibited shortening velocity. The effects of MgADP were dependent on the [MgATP] (compare Fig. 3, B and C), suggesting a competition between ATP and ADP. For the urinary bladders from SM-MHCexpressing mice, minimal inhibition of shortening velocity was observed at 10 mM MgATP (Fig. 3 C) whereas at 6 mM MgATP (Fig. 3 B), velocity was inhibited in a dose-dependent manner by MgADP. In the SM-MHCdeficient tissue the dependence of Vmax on MgADP was very steep at 6 mM MgATP. At 10 mM MgATP for the SM-MHCdeficient tissues and at 6 mM MgATP for the SM-MHCexpressing tissues, a graded inhibition by MgADP was observed. As seen in Fig. 3, the maximal effect of MgADP seemed to level off at an inhibition of Vmax to 50% of maximal. This is similar to previous observations in fast and slow smooth muscle from the guinea-pig (Löfgren et al., 2001
). Since velocity could not be completely inhibited by MgADP, Michaelis-Menten's kinetics for complete inhibition in its simplest form could not be applied. Since the data suggest an inhibition to
50% we used a modified equation (Eq. 3) to describe the inhibition of Vmax by MgADP at constant [MgATP].
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Using the Km values for MgATP (compare text above and Fig. 3 A) we fitted Eq. 3 to the to the mean data in Fig. 3, B and C, to obtain an estimate of the apparent Ki values for MgADP. Best fits were obtained at 6 mM MgATP for the SM-MHCexpressing groups and at 10 mM MgATP for the SM-MHCdeficient tissue. The mean values for the Ki were for the new-born SM-MHCexpressing: 5.8; for the 3-wk-old SM-MHCexpressing: 8.8 and for the SM-MHCdeficient mice: 0.64 µM (n = 56), showing an 10-fold lower Ki in the SM-MHCdeficient compared with the SM-MHCexpressing tissues. If the data were analyzed using a standard Michaelis-Menten's equation for competitive inhibition using the lowest concentrations of MgADP only, an
10fold lower Ki for MgADP for the SM-MHCdeficient compared with the SM-MHCexpressing tissue was also observed.
Phosphate Dependence of Force
The effects of varied concentrations of inorganic phosphate (Pi) on active force of skinned preparations are shown in Fig. 4 . Force was inhibited by Pi in a dose-dependent manner in all preparations. The 3-wk-old SM-MHCexpressing group was most sensitive to Pi, with 50% inhibition of force at 40 mM Pi. The SM-MHCdeficient group was markedly less sensitive compared with the SM-MHCexpressing groups.
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Ultrastructure
Transmission electron microscopy was performed on transverse sections of the urinary bladder preparations. The main focus of these experiments was to identify myosin filaments in SM-MHCdeficient smooth muscle cells. Structures with sizes corresponding to cross sectioned actin and myosin filaments were observed. The measured diameter of the latter was 14.3 ± 0.41 nm, n = 36. In smooth muscle cells from SM-MHCexpressing mice regions with comparatively abundant thick filaments could easily be identified. Regions with thick filaments (Fig. 6
B) were observed also in smooth muscle cells of the SM-MHCdeficient mice; however, such regions were very sparse and contained fewer thick filaments compared with those in SM-MHCexpressing tissue.
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DISCUSSION |
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We interpret the contractions of bladder muscle from SM-MHCdeficient mice to reflect active force generation from a contractile apparatus containing smooth muscle actin and NM-MHC. It should, however, be noted that a very small number of interstitial cells expressing NM-MHC and most likely nonmuscle actin isoforms are present in the tissue. We do not have data suggesting myosin filaments in these cells and cannot at present determine if they contribute to force generation and shortening of the tissue. Different actin isoforms (skeletal and smooth) have been shown to have similar function in the actinmyosin interaction (Mossakowska and Strzelecka-Golaszewska, 1985; Harris and Warshaw, 1993
) and cytoplasmic and smooth muscle specific actin isoforms can coexist in the same thin filament (Drew and Murphy, 1997
). This suggests that our data, independent of cell type, reflect properties of the nonmuscle myosin.
The nonmuscle myosin IIs are considered to be regulated by myosin light chain phosphorylation (Bresnick, 1999), mainly via myosin light chain kinase (MLCK) activated by Ca2+-calmodulin. We show that the activation by depolarization was associated with a transient in intracellular [Ca2+], exhibiting an initial peak and a plateau during the sustained phase of the contraction. The transients in [Ca2+] after activation were similar in SM-MHCexpressing and SM-MHCdeficient muscles. In the SM-MHCexpressing muscles the force responses to high K+ showed an initial transient and a sustained tonic phase while in the SM-MHCdeficient tissue the initial force transient is absent (compare Morano et al., 2000
). This difference in force responses cannot be explained by differences in Ca2+ transients, but rather reflect differences in cellular activation processes or in properties of the contractile myosin.
We examined cross-bridge kinetics of organized nonmuscle myosin in the SM-MHCdeficient tissue. Comparisons were made with the SM-MHCexpressing tissue with low relative contents of nonmuscle myosin. Skinned preparations were used to achieve maximal activation with thiophosphorylation of the regulatory light chains and to control substrate and product levels. Isometric force of the skinned preparations from SM-MHCdeficient tissue was 79-fold lower that than of the SM-MHCexpressing tissues. This difference was also present in intact tissue if the maximal forces were compared. The lower force could theoretically reflect shorter contractile filament length (altered parallel coupling), lowered intrinsic force generation of the contractile proteins, or lower content of contractile proteins. Large alterations in filament length seems to be excluded by the shortening velocity data and by reports that unitary cross-bridge force differs little between muscle myosins (Guilford et al., 1997). We found a low total myosin content in the SM-MHCdeficient bladders and the most likely explanation for the lower maximal force compared with the SM-MHCexpressing tissue is therefore a lower total myosin content. The lower myosin content would not, however, primarily affect the maximal shortening velocity, which reflects cross-bridge turnover under unloaded conditions.
The maximal shortening velocity (Vmax) of the SM-MHCdeficient tissue was 30% of that in the SM-MHCexpressing tissue. In the in vitro motility assay the NM-MHC-A gives a higher actin filament sliding velocity and ATPase activity compared with the NM-MHC-B isoform (Kelley et al., 1996
). In a study by Umemoto and Sellers (1990)
, cytoplasmic (platelet, nonmuscle A) and smooth (turkey gizzad) muscle myosins were compared using in vitro motility assays at 25°C. They showed that the nonmuscle myosin had a velocity (0.054 µm/s) which is
2025% of that of the smooth muscle myosin (0.237 µm/s). The MgATPase activity of these two myosins is similar, which could reflect that the filament translocation velocity in vitro and ATPase are limited by different cross-bridge reactions (Sellers et al., 1981
; Umemoto and Sellers, 1990
). The difference in filament velocity is similar to the difference recorded in the present study of the filamentous forms of nonmuscle myosins. If we use the in vitro motility data, in combination with our determinations of Vmax, we can estimate the length of the contractile unit in the smooth muscle (compare half sarcomere length in striated muscle) to
1.7 µm in control and 1.2 µm in SM-MHCdeficient tissue. Although these values are very crude estimates, they seem reasonable in view of the estimated thick filament length (2.2 µm) in smooth muscle (Ashton et al., 1975
).
In general, differences in shortening velocity between muscles are considered to reflect properties of the myosin molecules. Shortening velocity is considered to be rate-limited by the rate of cross-bridge dissociation (Weiss et al., 2001). In smooth muscle cells, the second-order rate constant of the ATP-induced dissociation of the rigor complex has been reported to be lower than that of skeletal muscle (Nishiye et al., 1993
) and lower in slow compared with fast smooth muscles (Somlyo et al., 1988
; Khromov et al., 1996
). The Km values for ATP and Vmax of the SM-MHCdeficient and SM-MHCexpressing urinary bladders were approximately equal. The ratio between Vmax and Km for ATP are correlated with the binding constant for ATP to the actinmyosin complex (Stienen et al., 1988
). Since the Vmax differed by a factor of
3 at similar Km, this could suggest that the binding of MgATP to the NM-MHC is weaker compared with that of the SM-MHC. However, the ATP-induced dissociation is fast at saturating MgATP and is thus most likely not limiting the maximal velocity of sliding in smooth muscles.
The ADP-release reaction preceding the cross-bridge dissociation is considered to be important for the velocity of filament sliding (Siemankowski et al., 1985; Weiss et al., 2001
). In the slow smooth muscle, the binding of ADP is generally stronger than for the faster skeletal muscle (Arheden and Arner, 1992
; Nishiye et al., 1993
; Cremo and Geeves, 1998
). This relation between velocity and ADP binding is also observed within the smooth muscle group, where the fast and slow muscle types exhibit differences in binding of ADP to both the rigor state (Fuglsang et al., 1993
) and to phosphorylated cycling cross-bridges (Löfgren et al., 2001
). Here we present data on the effects of ADP on organized nonmuscle myosins. ADP inhibited the Vmax in both SM-MHCexpressing and SM-MHCdeficient tissue. Interestingly, the velocity could not be completely inhibited with ADP within the range studied, but the effect saturated at a relative velocity of
50% of that in the absence of ADP, a phenomenon also observed in fast and slow smooth muscles of the guinea-pig (Löfgren et al., 2001
). One possible interpretation of this is that the incomplete inhibition is due to differential effects of ADP on the two heads in the myosin molecule during cycling (Löfgren et al., 2001
). Due to the complexity of the incomplete inhibition by MgADP, an apparent inhibition constant (Ki) could not be directly derived using Michaelis-Menten's kinetics. Using a modified model assuming 50% maximal inhibition, a 10-fold lower Ki in the SM-MHCdeficient tissues was found. This suggests that the maximally phosphorylated NM-MHC has a stronger ADP binding during cross-bridge cycling.
Fast and slow smooth muscles also differ significantly regarding the inhibitory effect of inorganic phosphate (Pi) on isometric force. Force of smooth muscle is less sensitive to Pi than force of skeletal muscle (Österman and Arner, 1995) and slow smooth muscle is less influenced by Pi than fast smooth muscle (Löfgren et al., 2001
). Our data from the SM-MHCdeficient mice show that the force of nonmuscle myosin has a lower sensitivity to Pi compared with the smooth muscle myosin. This shows that the NM-MHC in comparison to the SM-MHC, not only differs in ADP and cross-bridge dissociation reactions, but also in reactions associated with force generation. The Pi effects also provide a measure of cross-bridge kinetics different from the shortening velocity parameter. The results also argue that the difference in Vmax is not only due to structural differences in, for example, filament length.
NM-MHCs have important functions in several nonmuscle contractile systems and experiments on the organized NM-MHC in the smooth muscle tissue can provide a tool for analysis of the general contractile function of this myosin type. In summary, our results show that the NM-MHC can form filaments in smooth muscle cells and that the NM-MHC has a lower shortening velocity, and has different kinetics of reactions involved in cross-bridge dissociation and in force generation compared with the smooth muscle myosin.
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FOOTNOTES |
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ACKNOWLEDGMENTS |
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The study was supported by grants from Swedish Medical Research Council (A. Arner: 04x-8268, EE: 04x-13406), Medical Faculty Lund University, Crafoord foundation, Swedish Heart Lung Foundation, and Deutsche Forschungsgemeinschaft (I. Morano: Mo. 362/16-2).
Olaf S. Andersen served as editor.
Submitted: 24 September 2002
Revised: 27 February 2003
Accepted: 28 February 2003
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REFERENCES |
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