Effects of calponin on isometric force and shortening velocity in permeabilized taenia coli smooth muscle

1996 ◽  
Vol 270 (2) ◽  
pp. C481-C487 ◽  
Author(s):  
K. Obara ◽  
P. T. Szymanski ◽  
T. Tao ◽  
R. J. Paul
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Isometric Force ◽  
Dependent Manner ◽  
Taenia Coli ◽  
Shortening Velocity ◽  
Muscle Contractility ◽  
Smooth Muscle Contractility ◽  
Contractile System ◽  
Cross Bridges

Calponin, a thin filament-associated protein, inhibits actomyosin adenosinetriphosphatase in solution and has been suggested to modulate smooth muscle contractility. We used permeabilized guinea pig taenia coli smooth muscle to investigate whether calponin can modulate actin-myosin interaction in a more organized contractile system. Fibers were permeabilized with Triton X-100 and glycerol, which permit access of large macromolecules to the contractile apparatus. For contractures elicited by Ca2+ (6.6 microM + 0.1 microM calmodulin), the recombinant alpha-isoform of chicken gizzard calponin (CaP) decreased isometric force (Fo) and unloaded shortening velocity (Vus) in a dose-dependent manner; 1 microM CaP had minimal effects on force (< 10%) but reduced Vus by approximately 50% and 10 microM CaP reduced Fo to 27% of control and Vus to near zero levels. To eliminate any effects of the binding of calmodulin by CaP and consequent inhibition of myosin light chain kinase activity, we also studied fibers activated by thiophosphorylation of the myosin regulatory light chain. Fo was only moderately inhibited, remaining at approximately 75% of control in the presence of CaP (10 microM), whereas Vus was reduced to 32% of control. A similar inhibition was obtained with a mutant (CaPcys175) that retains the ability to bind to actin. CaP phosphorylated by protein kinase C and CaPcys175 mutant labeled with 1,5-IAEDANS, which bind actin poorly, were not effective inhibitors. Our results indicate that 1) CaP more strongly inhibits Vus (approximately cross-bridge cycle rate) than Fo (approximately number of activated cross bridges) and 2) the effects of CaP are related to its binding to actin. Thus the function of CaP in regulation of smooth muscle contractility may be more strongly related to its function as a modulator of velocity, as related to the "latch state," than as an "on-off" switch.

Vanadate oxidation activates contraction in skinned smooth muscle without myosin light chain phosphorylation

1997 ◽  
Vol 272 (1) ◽  
pp. C278-C288 ◽  
Author(s):  
M. J. Lalli ◽  
K. Obara ◽  
R. J. Paul
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Muscle Activation ◽  
Isometric Force ◽  
Total Force ◽  
Shortening Velocity ◽  
Irreversible Damage ◽  
Initial Control ◽  
High Concentrations ◽  
Cross Bridges

Phosphorylation of the myosin regulatory light chain (LC20-P1) is the major route of smooth muscle activation. However, after prior exposure to vanadate, permeabilized guinea pig taenia coli smooth muscle contracts in the absence of LC20-P1. We characterized the vanadate-induced contraction and investigated the mechanism of this novel activation pathway. Addition of vanadate to a control contracture (6.6 microM Ca2+) inhibits force (effective dose for 50% response was approximately 100 microM). In contrast, preincubation with high concentrations of vanadate (threshold at 1-2 mM) elicited a contraction on subsequent transfer of the fiber to a vanadate-free, Ca(2+)-free solution. Maximum isometric force of approximately 60% of control was obtained in fibers preincubated in 4 mM vanadate for 10 min. Addition of Ca2+ to a vanadate-induced contracture increased force, but the total force never exceeded the initial control. After maximal thiophosphorylation of LC20 with adenosine 5'-O-(3-thiotriphosphate), treatment with vanadate did not increase force. Unloaded shortening velocity (Vmax) was similar in Ca2+ and vanadate contractures and was additive. After thiophosphorylation, preincubation in vanadate had no effect on Vmax, suggesting that vanadate affected the number of activated bridges and not cycle rate. Vanadate mechanisms likely involve oxidation, since preincubation with 4 mM vanadate and 25 mM dithiothreitol (DTT) did not produce force. DTT could reverse a vanadate-induced contracture in 30-60 min. Subsequently, fibers demonstrated control contraction/relaxation cycles. Thus vanadate treatment did not cause irreversible damage, such as the extraction of proteins. Potential oxidation sites are proteins at 17 kDa and between 30 and 40 kDa, which were not alkylated by N-ethylmaleimide if they were treated in the presence of vanadate or in the rigor state. Vanadate-induced contractures are likely mediated by a reversible oxidation that activates cross bridges similarly to that of LC20-Pi and may play an important role in oxidant injury.

Effects of myosin kinase inhibiting peptide on contractility and LC20 phosphorylation in skinned smooth muscle

1992 ◽  
Vol 262 (6) ◽  
pp. C1437-C1445 ◽  
Author(s):  
J. D. Strauss ◽  
P. de Lanerolle ◽  
R. J. Paul
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Kinase Inhibitor ◽  
Isometric Force ◽  
Polyacrylamide Gel Electrophoresis ◽  
Competitive Inhibitor ◽  
Myosin Light Chain Phosphorylation ◽  
Taenia Coli ◽  
Shortening Velocity

A peptide inhibitor, myosin kinase inhibitor (MKI), of myosin light chain kinase (MLCK) was tested for its effects on contractility and myosin light chain phosphorylation in Triton X-100 skinned guinea pig taenia coli. MKI is based on the amino acid sequence of the myosin light chain (residues 11-19 LC20) and is a competitive inhibitor [inhibitory constant (Ki) congruent to 10 microM] of purified MLCK with respect to myosin light chain (LC20). MKI inhibited unloaded shortening velocity (V(us)) and the calcium-sensitive ATPase activity of the skinned fibers but had no significant effect on steady-state isometric force or myosin light chain phosphorylation, as measured by IEF-polyacrylamide gel electrophoresis analysis. MKI had no significant effect on V(us) of thiophosphorylated fibers in the absence of calcium. MKI inhibited MLCK activity in protein extracts from taenia coli, as measured by radioactive phosphate incorporation into LC20. Surprisingly, MKI also inhibited the phosphatase activity of these same extracts. This peptide slowed the rate and extent of relaxation of calcium-contracted fibers and elicited a contraction in relaxed fibers. These results are consistent with the hypothesis that MKI may be a phosphatase inhibitor as well as an inhibitor of MLCK. Our data further suggest that the rate of phosphorylation-dephosphorylation turnover may be important in regulating V(us) in smooth muscle.

scholarly journals Substrate and Product Dependence of Force and Shortening in Fast and Slow Smooth Muscle

2001 ◽  
Vol 117 (5) ◽  
pp. 407-418 ◽  
Author(s):  
Mia Löfgren ◽  
Ulf Malmqvist ◽  
Anders Arner
Keyword(s):  
Smooth Muscle ◽  
Molecular Mechanisms ◽  
Smooth Muscles ◽  
Slow Muscle ◽  
Taenia Coli ◽  
Active Force ◽  
Shortening Velocity ◽  
Relative Population ◽  
Cross Bridges ◽  
Muscle Contractile

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 (&lt;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.

Myosin light chain phosphorylation and contraction of guinea pig gallbladder smooth muscle

1991 ◽  
Vol 261 (6) ◽  
pp. G952-G957
Author(s):  
R. J. Washabau ◽  
M. B. Wang ◽  
J. P. Ryan
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Light Chain ◽  
Myosin Light Chain ◽  
State Level ◽  
Myosin Light Chain Phosphorylation ◽  
Shortening Velocity ◽  
Cross Bridges ◽  
Contraction And Relaxation ◽  
Isotonic Shortening

These experiments were designed to determine 1) whether acetylcholine (ACh) stimulation is accompanied by changes in myosin light chain phosphorylation in gallbladder smooth muscle and 2) whether dephosphorylated noncycling cross bridges (latch bridges) exist in gallbladder smooth muscle. Isometric stress, isotonic shortening velocity, and myosin light chain phosphorylation were determined under conditions of contraction and relaxation in ACh-stimulated guinea pig gallbladder smooth muscle. Unstimulated muscle contained 6.8 +/- 2.0% phosphorylated myosin light chain. ACh stimulation (5 x 10(-5) or 10(-4) M) was associated with a rapid increase in myosin light chain phosphorylation to a value that was maintained throughout the tonic contraction. In contrast, isotonic shortening velocity was maximal at 30 s of stimulation and then declined over time to a steady-state level that was 25-30% of the peak velocity. Upon agonist washout (relaxation), dephosphorylation of the myosin light chain occurred at about the same rate as the decline in shortening velocity and preceded the decline in isometric stress. These data suggest that ACh stimulation is accompanied by changes in myosin light chain phosphorylation but that dephosphorylation of cross bridges is not necessary for the slowing of cross-bridge cycling rates in gallbladder smooth muscle.

Role of magnesium in activation of smooth muscle

1988 ◽  
Vol 255 (4) ◽  
pp. C465-C472 ◽  
Author(s):  
R. J. Paul ◽  
J. C. Ruegg
Keyword(s):  
Smooth Muscle ◽  
Regulatory Mechanism ◽  
Isometric Force ◽  
Taenia Coli ◽  
Shortening Velocity ◽  
Myosin Phosphorylation ◽  
Skinned Smooth Muscle ◽  
Myosin Interaction ◽  
Gamma S

We studied the effects of Mg2+-free solutions on isometric force (F0) and unloaded shortening velocity (Vus) in contractions elicited by Ca2+ or by ATP after thiophosphorylation by adenosine 5'-O-(3-thiotriphosphate (ATP gamma S) in chemically skinned guinea pig taenia coli smooth muscle. In Mg2+-free solutions, increasing Ca2+ did not increase Fo above resting levels. At the peak of a control contraction elicited by Ca2+, transfer to Mg2+-free (but Ca2+-containing) solutions resulted in a rapid relaxation and concomitant dephosphorylation of myosin. After ATP gamma S, a contracture required neither Mg2+ nor Ca2+ in the solutions for control levels of Fo. Vus in the Mg2+-free solutions after ATP gamma S was approximately 50% of control and could be restored to near control levels by addition of Mg2+ but not Ca2+. After ATP gamma S, pretreatment with 4 mM EDTA and contracture in 0.1 mM EDTA-containing solutions decreased Fo to 70-80% of control and Vus to 50-60% of control. Our results suggest that the relatively high requirement for Mg2+ for contraction in skinned smooth muscle largely reflects the Mg2+ dependence of myosin kinase and not for actin-myosin interaction. The dependence of Fo on Mg2+ (in the presence of excess ATP) in taenia coli is less than that reported for skeletal muscle. Appreciable force can be maintained with no added Mg2+ in the presence of 4 mMEDTA, and thus it appears that ATP4- can be a substrate for contraction after ATP gamma S treatment. In addition, our data imply that any Ca2+-dependent regulatory mechanism that does not involve myosin phosphorylation/dephosphorylation, if present, requires Mg2+ for expression.

scholarly journals Could an increase in airway smooth muscle shortening velocity cause airway hyperresponsiveness?

2011 ◽  
Vol 300 (1) ◽  
pp. L121-L131 ◽  
Author(s):  
Sharon R. Bullimore ◽  
Sana Siddiqui ◽  
Graham M. Donovan ◽  
James G. Martin ◽  
James Sneyd ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Airway Hyperresponsiveness ◽  
Isometric Force ◽  
Muscle Length ◽  
Shortening Velocity ◽  
Bridge Model ◽  
Generating Capacity ◽  
Cross Bridges

Airway hyperresponsiveness (AHR) is a characteristic feature of asthma. It has been proposed that an increase in the shortening velocity of airway smooth muscle (ASM) could contribute to AHR. To address this possibility, we tested whether an increase in the isotonic shortening velocity of ASM is associated with an increase in the rate and total amount of shortening when ASM is subjected to an oscillating load, as occurs during breathing. Experiments were performed in vitro using 27 rat tracheal ASM strips supramaximally stimulated with methacholine. Isotonic velocity at 20% isometric force (Fiso) was measured, and then the load on the muscle was varied sinusoidally (0.33 ± 0.25 Fiso, 1.2 Hz) for 20 min, while muscle length was measured. A large amplitude oscillation was applied every 4 min to simulate a deep breath. We found that: 1) ASM strips with a higher isotonic velocity shortened more quickly during the force oscillations, both initially ( P < 0.001) and after the simulated deep breaths ( P = 0.002); 2) ASM strips with a higher isotonic velocity exhibited a greater total shortening during the force oscillation protocol ( P < 0.005); and 3) the effect of an increase in isotonic velocity was at least comparable in magnitude to the effect of a proportional increase in ASM force-generating capacity. A cross-bridge model showed that an increase in the total amount of shortening with increased isotonic velocity could be explained by a change in either the cycling rate of phosphorylated cross bridges or the rate of myosin light chain phosphorylation. We conclude that, if asthma involves an increase in ASM velocity, this could be an important factor in the associated AHR.

Potentiation of smooth muscle contractility after rapid changes in isotonic force

1996 ◽  
Vol 271 (2) ◽  
pp. C511-C523 ◽  
Author(s):  
R. A. Meiss
Keyword(s):  
Smooth Muscle ◽  
Time Course ◽  
Muscle Length ◽  
Muscle Contractility ◽  
Smooth Muscle Contractility ◽  
Contractile System ◽  
Rapid Changes ◽  
Step Duration ◽  
Cytoskeletal Elements ◽  
Isotonic Shortening

The sudden application of step increases in afterload (0.4-3.0 s in duration) arrested the isotonic shortening of electrically stimulated ovarian ligament smooth muscle strips from rabbits. Force steps were chosen to produce, after initial rapid yielding, a quasi-steady state in which muscle length and force remained constant. Removal of the extra afterload allowed renewed shortening that began with a velocity transiently greater than that measured before the force step (at same muscle length). The rate of force redevelopment was also transiently potentiated under isometric conditions after the removal of the extra load. Both types of potentiation depended on force-step duration, and the transients decayed exponentially with a time constant of approximately 0.25 s. The stiffness of the muscle during the force step was initially depressed but then increased along an exponential time course while force and length remained constant. These observations are consistent with an initial detachment of a portion of the cross-bridge array, which then reattached during the course of the force step, with potentiation being due to either a transient increase in cycling rate or a time-dependent reconfiguration of cytoskeletal elements supporting the contractile system.

Pharmacological modulation of the mechanical response of airway smooth muscle to length oscillation

1997 ◽  
Vol 83 (3) ◽  
pp. 739-745 ◽  
Author(s):  
X. Shen ◽  
M. F. Wu ◽  
R. S. Tepper ◽  
S. J. Gunst
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Mechanical Response ◽  
Isometric Force ◽  
Shortening Velocity ◽  
Airway Reactivity ◽  
Pharmacological Modulation ◽  
Activation Mechanisms ◽  
Cross Bridges

Shen, X., M. F. Wu, R. S. Tepper, and S. J. Gunst. Pharmacological modulation of the mechanical response of airway smooth muscle to length oscillation. J. Appl. Physiol. 83(3): 739–745, 1997.—Stretch and retraction of the airways caused by changes in lung volume may play an important role in regulating airway reactivity. We studied the effects of different pharmacological stimuli on airway smooth muscle to determine whether the muscle behavior during length oscillation can be modulated pharmacologically and to evaluate the role of different activation mechanisms in determining its behavior during the oscillation. Active force decreased below the static isometric force during the shortening phase of length oscillation, resulting in an overall depression of force during the length oscillation cycle. This pattern of response was unaffected by the contractile stimulus or level of activation, suggesting that it was caused by a mechanism that is independent of the level of activation of cross bridges. The normalized area of the length-force hysteresis loop (hysteresivity) differed depending on the stimulus used for contraction. Effects of different stimuli on hysteresivity were not correlated with their effects on isotonic shortening velocity or isometric force, suggesting that the pharmacological modulation of the behavior of airway smooth muscle during length oscillation at these amplitudes cannot be accounted for by the effects on the cross-bridge cycling rate.

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