Correspondence to: Anders Arner, Department of Physiological Sciences, Lund University, Tornavägen 10, BMC F11, S-22184 Lund, Sweden. Fax:46-46-222-7765 E-mail:Anders.Arner{at}mphy.lu.se.
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Abstract |
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To explore the molecular mechanisms responsible for the variation in smooth muscle contractile kinetics, the influence of MgATP, MgADP, and inorganic phosphate (Pi) on force and shortening velocity in thiophosphorylated "fast" (taenia coli: maximal shortening velocity Vmax = 0.11 ML/s) and "slow" (aorta: Vmax = 0.015 ML/s) smooth muscle from the guinea pig were compared. Pi inhibited active force with minor effects on the Vmax. In the taenia coli, 20 mM Pi inhibited force by 25%. In the aorta, the effect was markedly less (<10%), suggesting differences between fast and slow smooth muscles in the binding of Pi or in the relative population of Pi binding states during cycling. Lowering of MgATP reduced force and Vmax. The aorta was less sensitive to reduction in MgATP (Km for Vmax: 80 µM) than the taenia coli (Km for Vmax: 350 µM). Thus, velocity is controlled by steps preceding the ATP binding and cross-bridge dissociation, and a weaker binding of ATP is not responsible for the lower Vmax in the slow muscle. MgADP inhibited force and Vmax. Saturating concentrations of ADP did not completely inhibit maximal shortening velocity. The effect of ADP on Vmax was observed at lower concentrations in the aorta compared with the taenia coli, suggesting that the ADP binding to phosphorylated and cycling cross-bridges is stronger in slow compared with fast smooth muscle.
Key Words: myosin isoforms, phosphate, ATP, ADP, force-velocity relation
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INTRODUCTION |
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The contractile apparatus in smooth muscle is characterized by lack of sarcomere units, slow contractile kinetics, and myosin-based regulation. Although smooth muscles share these properties, a large heterogeneity in contractile properties exists within the smooth muscle family. The smooth muscles have been divided into "visceral" and "multi-unit" types (
The molecular mechanisms responsible for the difference in cross-bridge kinetics between smooth muscles are not resolved. Alterations in the loop 1 at the 25/50-kD junction of the myosin II molecule alter the kinetics of myosin (
The actinmyosin interaction in smooth muscle is considered to occur according to the general kinetic scheme proposed for skeletal muscle, although several of the rate constants are slower in smooth muscle (
The finding that ADP inhibits the rate of relaxation from active contractions (
We have previously shown that ADP inhibits shortening velocity in Ca2+-activated skinned smooth muscle (
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MATERIALS AND METHODS |
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Muscle Preparations
The taenia coli and thoracic aorta were obtained from female guinea pigs. The animals weighed 400 g and were killed by cervical dislocation. The muscle tissues were cut out under a microscope and chemically skinned using Triton X-100 as described by
0.15 mm and a length of 24 mm. A 1.21.8-mm-long segment of the aorta was cut open and mounted with the circular muscle layer in the long axis of the preparation, which gave preparations of 23-mm length with the full thickness of the media layer (
0.05 mm).
Solutions
The solutions used for the skinned muscle preparations contained 30 mM N-Tris-(hydroxymethyl)methyl-2-aminoethane-sulfonic acid, 4 mM EGTA, and 2 mM free Mg2+. All solutions were adjusted to pH 6.9 with KOH and to an ionic strength of 150 mM using KCl. In the experiments, the concentrations of MgATP, MgADP, and inorganic phosphate (Pi)1 were varied. When ADP-depletion/ATP-generation was used, 12 mM phosphocreatine (PCr) and 0.5 mg/ml creatine kinase (CK) were added to solutions or, in the ATPase-determining experiments, 10 mM phosphoenol pyruvate and 20 U/ml pyruvate kinase. Experiments using ADP were performed in the presence of 0.2 mM of the myokinase inhibitor AP5A (
Quick-release Experiments
The muscle preparations were wrapped with small clips of aluminum foil at each end and mounted between a stainless steel pin attached to a force transducer (model AE 801; SensoNor a.s.) and an isotonic lever (5 ms. After each quick release, force and muscle length were recorded for 1 s using a sampling rate of 1 kHz on an RTI-800 Analogue Devices A/D board in a personal computer. The shortening velocity was determined at different points in time after the release by analysis of the length responses as described by
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(1) |
In Equation 1, a and b are constants, P the afterload, and Po the isometric force at each contraction. The maximal shortening velocity (Vmax) was then given by extrapolation of the fitted curve to P/Po = 0.
The preparations were initially mounted in a relaxing solution (1 nM free [Ca2+], pCa 9) and stretched to a passive force of 0.1 mN. Thereafter, the muscles were maximally activated using a repeated thiophosphorylation procedure (
-S. After a 5-min period in calcium-free rigor solution, a contraction was initiated by transfer to an ATP containing solution. When force had reached a plateau, the muscle fiber was transferred to fresh solution and a series of 1530 releases to different afterloads was performed. The isometric force was measured immediately before the beginning of each release series. Before the next contraction and force-velocity determination, the fiber was again treated with thiophosphorylation. A maximum of five (taenia coli) and six (aorta) contractions and force-velocity determinations were made on each preparation. In general, force and velocity data were normalized to values obtained in each fiber preparation during a standard reference contraction at saturating (3.2 mM) [MgATP] in the presence of PCr and CK. The different solutions were applied at random order. Six sets of experiments were performed: (1) varied [MgATP] in the presence of the PCr/CK system; (2) varied [MgATP] in the absence of the PCr/CK system; (3) varied [MgATP] in the presence of MgADP in the absence of the PCr/CK system (taenia coli only); (4) varied [MgADP] at constant [MgATP] (taenia coli: 1 and 6 mM; aorta: 10 mM); (5) 0 and 20 mM Pi at 3.2 mM MgATP in the presence of the PCr/CK system (aorta only); and (6) in rigor solutions (0 mM MgATP with 50 U/ml hexokinase and 10 mM glucose). In some of these experiments, apyrase (20 mg/ml) was included in rigor solutions.
For analysis of the [MgATP] dependence of the maximal shortening velocity (Vmax), a hyperbolic equation was used to determine the apparent binding constant (Km). Max denotes the Vmax at saturating [MgATP].
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(2) |
For analysis of [MgADP] dependent inhibition of velocity, the following equation was used to determine the apparent inhibition constant (Ki):
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(3) |
Isometric Force Experiments
In one series of experiments, the influence of varied Pi concentrations on active force was determined in the taenia coli and aorta. The preparations were mounted in 0.5- ml plastic baths for isometric force registration using AE 801 force transducers. Activation with thiophosphorylation was performed as described above. Four preparations were studied in parallel and force was measured at Pi concentrations in the range 040 mM in solutions with 3.2 mM [MgATP] and PCr/CK system. Pi was introduced in the contraction solution and force was determined at the plateau of each active contraction.
Determination of Tissue ATPase Activity
In a series of experiments performed to examine ATPase activity, skinned muscle fibers from the taenia coli and the aorta were mounted in 0.5-ml cups and attached to Grass FT03 force transducers. The experiments were performed essentially as described by
Determination of Myosin Isoforms
To examine the content of myosin essential light chain 17a and 17b in the skinned taenia coli and the aorta fibers (compare with
Statistics
All values are given as mean ± SEM with the number of observations within parenthesis. Curve fitting to the hyperbolic
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RESULTS |
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Fig 1 shows original recordings of force from quick-release experiments on a taenia coli and an aorta preparation. Contractions were elicited in the thiophosphorylated preparations at different MgADP concentrations at 1 and 10 mM MgATP for the taenia and the aorta, respectively. The different MgADP concentrations were applied at random order. Experiments with varied MgATP were performed in a similar way. We have previously shown (
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The force and velocity data from taenia coli and aorta preparations could be described by the
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The shortening response after a quick release in smooth muscle consists of an initial elastic recoil followed by a shortening with gradually decreasing velocity (
We have previously reported (20% of the basic essential myosin light chain (LC17b). This was confirmed in the present study (
25% LC17b). Previous studies have reported
60% LC17b in rat and rabbit aorta (
70% LC17b in the guinea pig aorta, which is consistent with the previous data from large elastic arteries.
We observed, when we determined the [MgATP] dependence of Vmax that the relation did not extrapolate to zero Vmax at zero [MgATP]. Therefore, we also performed quick-release experiments in rigor and found a significant shortening with an apparent Vmax under these conditions. The Vmax in rigor at 100 ms after release, relative to that at optimal [MgATP], was 39 ± 1.2% (n = 14) for the taenia coli and 55 ± 3.5% (n = 7) for the aorta. Quick-release experiments on preparations fixed with glutaraldehyde at the end of the experiments showed no shortening response, excluding that the shortening observed in rigor muscles was due to compliance of transducer, lever arm, or fiber attachment. A significant shortening response in rigor muscles was also observed in preparations where the ends had been fixed with cellulose acetate glue and using the slack test method. This shows that the response in rigor was not due to the aluminum clips or to the isotonic quick-release method. To ensure that the shortening in rigor was not due to ATP contamination in the solutions the rigor experiments were performed in solutions supplemented with glucose/hexokinase and apyrase as described in MATERIALS AND METHODS. To further ensure that shortening in rigor was not due to active cross-bridge cycling, we performed experiments in the presence of 1 mM vanadate, an inhibitor of active cross-bridge cycling in smooth muscle (
We have interpreted the shortening in rigor conditions as a result of viscous phenomena in the preparation. Since passive force in the relaxed state (pCa 9 solution) was low (taenia coli: 0.7 ± 0.5; aorta: 4.3 ± 1% of maximal active tension, n = 10), elastic or viscous elements in parallel with the contractile apparatus would not contribute to force or shortening. Therefore, we assumed a visco-elastic element in series with the contractile component. The shortening responses in rigor after a quick release consisted of an initial elastic recoil, and a subsequent "viscous" phase of slower shortening. The calculated maximal shortening velocity in rigor decreased with time in an approximately similar manner as in the active contraction (Vmax at 500 ms after release relative to that at 100 ms, taenia coli rigor: 20.1 ± 1.5; aorta rigor: 20.6 ± 3.2; taenia coli active: 31.9 ± 0.7; aorta active: 23.6 ± 1.7%, n = 6). Thus, the time constant of the visco-elastic element is approximately the same as that of the active response. Therefore, the visco-elastic component cannot be simply eliminated by measuring velocity at a different time after release. The amplitude of the viscous shortening phase in rigor, which reflects the properties of the spring in the visco-elastic component, was not linearly dependent on the amplitude of the force step. The resulting strain-force relationship was nonlinear, with an increasing stiffness at increasing strain. The behavior could be adequately described by an exponential spring similar to that proposed for series and parallel elastic components (
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The results in of Fig 3 A show the ATP dependence of Vmax for aorta and taenia coli preparations. The relation for the taenia coli was shifted towards higher [MgATP] compared with the aorta and the apparent Km (Equation 2) was about fourfold higher. A fit to the whole data set gave Km values of 351 ± 76 µM for the taenia coli and 84 ± 31 µM for the aorta. Fig 3 B shows the corresponding force values. Force was lower at reduced [MgATP], but appeared to be less influenced by a change in [MgATP] than Vmax. Force in rigor was 0.69 ± 0.03 (6) for the aorta and 0.42 ± 0.02 (14) for the taenia coli, relative to the corresponding values at optimal (3.2 mM) [MgATP]. In separate experiments, we determined active force normalized to cross-sectional area (determined by dividing preparation wet weight with length and density) and found that the force of the aorta preparations at maximal activation and optimal [MgATP] was lower than that of the taenia coli (4.5 ± 0.8 (6) vs. 59 ± 19 (4) mN/mm2). It should be noted that these values were not corrected for tissue content of smooth muscle myosin. The absolute forces that the preparations developed in these experiments were 6.4 ± 0.8 (4) mN (taenia coli) and 1.7 ± 0.3 (6) mN (aorta).
As shown in Fig 4 A, the Vmax of the skinned muscles was dependent on the presence of the phosphocreatine/creatine kinase system (PCr/CK). Removal of the backup system resulted in a significant reduction of the maximal shortening velocity at lower ATP concentrations. This effect was more pronounced in the aorta compared with the taenia, showing that lowered ATP/ADP ratios in the muscle fiber influence velocity more in the slow smooth muscle. The apparent Km for MgATP increased 1.8-fold in the taenia coli and
34-fold in the aorta.
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In experimentation without backup system, diffusion of ATP and ADP in the preparations becomes important. Most likely the change in ATP dependence of Vmax, observed when the backup system is removed (Fig 4) is due to a change in the ADP/ATP ratio in the interior of the fiber preparation. To exclude that the more pronounced dependence on the backup system in the aorta preparations was due to greater changes in ADP/ATP in the tissue due to higher tissue ATPase, we examined the tissue ATPase activity in the presence of phosphoenol pyruvate. In the maximally thiophosphorylated preparations, the ATPase was 0.51 ± 0.12 (4) µmol min-1 g-1 for the taenia coli and 0.31 ± 0.02 (6) µmol min-1 g-1 for the aorta. These values reflect the total ATPase in the active muscle and most likely include contribution from both actinmyosin interaction and noncontractile, possibly ecto-, ATPases in the tissue. Assuming a cylindrical geometry and a diffusion constant of 2 x 10-7 cm2/s (
To investigate the effects of ADP on the ATP dependence of Vmax, force-velocity relations were determined at different ATP concentrations in the presence of 2.66 and 5.32 mM MgADP in the taenia coli preparation (Fig 5). The ATP dependence of Vmax (Fig 5 A) was shifted towards higher [MgATP] in the presence of ADP. Addition of 2.66 mM [MgADP] did influence the extrapolated Vmax at saturating [MgATP] to a minor extent and the apparent Km for MgATP increased to 0.811 mM (fit to Equation 2). The inhibition of Vmax at the high MgADP concentrations (5.32 mM) could not be reversed by increased [MgATP], suggesting noncompetitive effects of ADP at higher concentrations. Increasing [MgADP] resulted in a decrease in active force (Fig 5 B).
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Experiments using addition of ADP have to be performed in the absence of a backup system. Since Vmax of the aorta was markedly influenced by removal of the backup system (Fig 4 A), it is not possible to examine the effects of varied [MgATP] at constant [MgADP] in this preparation as was performed for the taenia coli (Fig 5). Instead, we chose concentrations of MgATP (6 mM for the taenia coli and 10 mM for the aorta) where removal of the backup system did not influence the maximal velocity or force (Fig 4). At these MgATP concentrations, we varied the [MgADP]. Fig 6 shows the effects of ADP on the maximal shortening velocity and force of aorta and taenia coli preparations. Addition of ADP inhibited Vmax in both tissues, but the effects occurred at much lower concentrations in the aorta. Note that [MgATP] was higher in the experiments on the aorta, showing that the inhibition of velocity occurred at lower ADP/ATP ratios. The velocity did not approach zero even at high [MgADP], but approached a value of 50% of maximal. This behavior was observed both in the aorta and the taenia coli (Fig 6). Even at lower [MgATP] (1 mM, data not shown), addition of ADP did not reduce velocity to zero in the taenia coli; Vmax was inhibited at saturating [MgADP] to
50% of the value at zero MgADP. The data for the whole range of ADP concentrations in Fig 6 could not be directly described using simple Michaelis-Menten's kinetics since velocity was not inhibited to zero at saturating ADP. If we only use the initial part of the data at nonsaturating ADP concentrations (where Lineweaver-Burke plots were linear) fitting to Equation 3 gave apparent Ki values for ADP of
10 µM in the aorta and
360 µM in the taenia coli. Although the Ki values at present cannot be directly interpreted, the analysis show that a pronounced difference in the ADP binding exists between the two muscles. In the aorta, force was slightly inhibited in the highest MgADP concentration interval, but was essentially unchanged in the range of MgADP concentrations where the inhibition of velocity occurred. Thus, velocity could be decreased by ADP by
30%, at essentially unchanged force in the aorta.
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Fig 7 shows the effects of Pi on active force and shortening velocity. Force was inhibited by Pi in a dose-dependent manner in both aorta and taenia coli. The effects of phosphate were more pronounced in the taenia coli preparations. The relation between force and log10([Pi]) was almost linear. The effects of phosphate on maximal shortening velocity were investigated in the aorta preparations. In the presence of 20 mM Pi, where force was inhibited by 15%, velocity was slightly, but not significantly, increased. Maximal shortening velocities of aorta preparations at 3.2 mM MgATP and PCr/CK were with 20 mM Pi 0.017 ± 0.002 ML/s and without Pi 0.014 ± 0.001 ML/s.
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DISCUSSION |
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The aim of the present study was to examine in detail the substrate and product dependence of the force-velocity relation in two smooth muscles that represent the near extremes in the distribution of smooth muscles contractile properties. The guinea pig aorta belongs to the slow group of muscles with high content of essential light chain b (LC17b) and low or lacking heavy chain insert (
Our quick-release experiments on muscles in rigor revealed an important technical aspect of experiments on smooth muscle preparations, i.e., a significant contribution of viscous elements on the shortening responses. A more detailed analysis revealed that the viscous responses in rigor could be described by a series-coupled element with a nonlinear spring and a viscous element. This model showed a weak dependence of viscous maximal velocity on isometric tension in rigor and, therefore, we subtracted a constant velocity from the active contractions. After this correction, the dependence of Vmax on [MgATP] could be adequately described by a hyperbolic equation. At present, we do not have any data regarding the nature of the viscous component, it might reside outside of the contractile machinery, in the cytoskeleton, in the cellcell interactions, or be a part of the cross-bridge interaction.
The phosphate (Pi) release is considered to be associated with force generation in skeletal and smooth muscles (
The apparent Km for the ATP effects on Vmax in the slow aorta muscle was about fourfold lower than that of the fast taenia coli smooth muscle, with apparent Km values of 84 and 351 µM, respectively. This finding is consistent with results from slow and fast rabbit skeletal muscles, where the Km for MgATP was lower in the slow muscle (semimembranous, 18 µM; psoas, 150 µM;
Actually a comparison of the ratios Vmax/Km between muscles gives information on the difference in apparent second-order rate constant for ATP-induced dissociation, assuming similar sarcomere equivalent lengths and cross-bridge attachment ranges. This ratio was 50% lower in the aorta, which could be consistent with a slightly lower dissociation constant for ATP. The second- order rate constant for the ATP-induced dissociation from rigor has been suggested to be lower in smooth muscle fiber preparations compared with skeletal muscle (
Previous studies on skinned smooth muscles in rigor have shown a strong MgADP binding, with a Kd of 1 µM (
60 µM in skeletal muscle (
Even though the inhibition of Vmax by MgADP occurred at low concentrations, the velocity was only inhibited to 50% of maximal at saturating [MgADP] in the presence of MgATP (Fig 6). This behavior is clearly different from the inhibition of velocity when ATP was reduced, where Vmax approached zero (i.e., the apparent Vmax in rigor). We assume that addition of ADP generates a population of A-M-ADP states that oppose shortening. It could be possible that the situation at saturating [MgADP] is a rigorlike state with altered viscous properties giving an apparent velocity that is higher than that in rigor. This seems very unlikely since we found that addition of MgADP to rigor did not increase velocity. A second possible explanation could be that the MgADP binding is weaker at higher MgADP concentrations, a situation where velocity is decreased. This finding is difficult to explain since a lower velocity would shift the distribution of cross-bridge strain towards lower strain in the negative direction, which according to general models of muscle contraction would increase the binding affinity of MgADP (
50% might be consistent with a model where only one of the two myosin heads can initiate and perform the power stroke reactions, and where a subsequent attachment of the second head promotes ADP release of the leading head.
The interpretation of the Ki values is not straight forward since we could not completely inhibit shortening velocity and we cannot at this stage present a complete model to analyze the behavior. However, if we analyze the initial part of the ADP inhibition data (Fig 6) we find an apparent Ki value of 10 µM in the aorta and 360 µM for the taenia coli. This suggests that the binding of ADP to cycling cross-bridges differs with a factor of
40 between the slow and fast smooth muscle types. This result is consistent with the finding of a fourfold difference in binding of ADP to rigor cross-bridges between a fast/phasic (Kd = 4.9 µM rabbit bladder) and a slow/tonic (Kd = 1.1 µM rabbit femoral artery) smooth muscle (
In the in vitro motility assay, the velocity of actin over smooth muscle myosin is influenced by ATP and ADP concentrations. It has been shown that the Km for ATP is 40 µM and the Ki for ADP is 0.24 mM using turkey gizzard myosin at 30°C (
In the intact smooth muscle, intracellular [ADP] has been shown to be in the submillimolar range in the relaxed state and to increase during active contraction and metabolic inhibition (
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Footnotes |
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1 Abbreviations used in this paper: A-M, actin-myosin; A-M-ADP, actin-myosin-ADP; CK, creatine kinase; ML, muscle lengths; PCr, phosphocreatine; Pi, inorganic phosphate.
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Acknowledgements |
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We are grateful for the excellent technical assistance of Mrs. Christina Persson. We also thank Dr. Juris Galvanovskis for the help with solving mathematical problems associated with the nonlinear elastic model.
This work was supported by grants from the Swedish Medical Research Council (No. 04X-12584 to U. Malmqvist and No. 04X-8268 to A. Arner) and the Medical Faculty Lund University.
Submitted: 4 December 2000
Revised: 6 March 2001
Accepted: 27 March 2001
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