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 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.

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.

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.

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.

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.

Role of myosin light-chain phosphorylation in guinea pig gallbladder smooth muscle contraction

1994 ◽  
Vol 266 (3) ◽  
pp. G469-G474 ◽  
Author(s):  
R. J. Washabau ◽  
M. B. Wang ◽  
C. Dorst ◽  
J. P. Ryan
Keyword(s):  
Smooth Muscle ◽  
Steady State ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Smooth Muscle Contraction ◽  
Myosin Light Chain Phosphorylation ◽  
Shortening Velocity ◽  
Cross Bridges ◽  
Isotonic Shortening

In acetylcholine (ACh)-stimulated gallbladder smooth muscle, we have previously shown that phosphorylation of the 20,000-Da myosin light chains is necessary for the initiation of contraction, that myosin is stably phosphorylated at steady state, and that dephosphorylation of cross bridges is not necessary for the slowing of cross-bridge cycling rates during the period of steady-state isometric stress. The present studies were undertaken to determine whether 1) K+ (60 or 80 mM) or cholecystokinin (CCK, 10(-8) M) stimulation is accompanied by changes in myosin light-chain phosphorylation in gallbladder smooth muscle and 2) dephosphorylated noncycling cross bridges exist in K(+)- or CCK-stimulated gallbladder smooth muscle. Isometric stress, isotonic shortening velocity, and myosin light-chain phosphorylation were determined during contraction with K+ or CCK. Steady-state isometric stress was reached within 2.5 min of stimulation with K+ or CCK and was maintained for the duration of the stimulation. Stimulation with K+ or CCK was associated with rapid increases in myosin light-chain phosphorylation and maintenance of myosin light-chain phosphorylation during the stimulation. In contrast, isotonic shortening velocity was maximal at 1 min of stimulation with either K+ or CCK and then declined significantly to values that were only 26-32% of the peak velocity. These data, along with data from previous experiments with ACh, suggest that myosin light-chain phosphorylation is essential in the initiation of contraction in gallbladder smooth muscle, regardless of the source of Ca2+ or of the contractile agonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Passive sensitization of human bronchi augments smooth muscle shortening velocity and capacity

1994 ◽  
Vol 267 (2) ◽  
pp. L218-L222 ◽  
Author(s):  
R. W. Mitchell ◽  
E. Ruhlmann ◽  
H. Magnussen ◽  
A. R. Leff ◽  
K. F. Rabe
Keyword(s):  
Smooth Muscle ◽  
Human Serum ◽  
Immunoglobulin E ◽  
Isometric Force ◽  
Lung Resection ◽  
Electrical Field Stimulation ◽  
Shortening Velocity ◽  
Passive Sensitization ◽  
High Concentrations

We assessed whether incubation with human serum from atopic individuals containing high concentrations of immunoglobulin E (IgE) causes augmentation of maximal contraction of human bronchial smooth muscle from non-atopic subjects in vitro. Bronchi were obtained from eight patients undergoing lung resection, and force-velocity relationships were determined for eight pairs of epithelium-intact bronchial rings of generations 6-7 using an electromagnetic lever system, which allowed isotonic shortening when load-clamps [from 0 to maximal isometric force (P0)] were applied at specific times after onset of contraction. Contractions were elicited by supramaximal electrical field stimulation (50 Hz, 10 s train duration, 25 V). Optimal length (Lo) for each tissue was determined during equilibration. After resection, tissues were sensitized passively with human sera containing high titers (> 1,000 U/ml) of IgE by incubation for 16 h at 20 degrees C. Maximal shortening velocity (Vmax) was increased for passively sensitized bronchi [0.1150 +/- 0.0240 1/2 circumferences/s (1/2Cir/s)] compared with sham-sensitized bronchi [0.0731 +/- 0.0152 1/2Cir/s, P = 0.038]. Similarly, maximal shortening (delta Lmax) was augmented in sensitized bronchial rings (11.27 +/- 1.80 %Lo) compared with sham-sensitized tissues (8.19 +/- 1.39 %Lo, P = 0.012). However, P0 did not differ between sensitized (122.5 +/- 24.4 mN/cm2) compared with sham-sensitized tissues (138.4 +/- 32.1 mN/cm2, P = 0.642). Our data are the first demonstration that Vmax and delta Lmax are augmented in sensitized but not challenged human bronchial rings after passive sensitization using human serum containing high concentrations of IgE.

Myosin phosphorylation, force, and maximal shortening velocity in neurally stimulated tracheal smooth muscle

1985 ◽  
Vol 249 (3) ◽  
pp. C238-C247 ◽  
Author(s):  
K. E. Kamm ◽  
J. T. Stull
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Isometric Force ◽  
Muscle Length ◽  
Temporal Correlation ◽  
Tracheal Smooth Muscle ◽  
Shortening Velocity ◽  
Bathing Medium ◽  
Myosin Phosphorylation

Field stimulation of intrinsic nerves in bovine tracheal smooth muscle strips elicited atropine-sensitive contractions that were more rapid than those obtained by addition of carbamylcholine to the bathing medium. These stimulus conditions were used to improve estimates of maximal rates of activation as indicated by myosin light chain phosphorylation, maximal shortening velocity (Vo), and isometric force. Maximal values of Vo [0.25 X optimal muscle length X s-1] and light chain phosphorylation (0.65 mol phosphate/mol light chain) were attained after 5 s of stimulation and preceded maximal force (60 s). Force gradually fell to 0.85 times maximal values during 30 min of stimulation, while both light chain phosphorylation and Vo declined to 0.3 times the maximal value. The temporal correlation between light chain phosphorylation and Vo supports the hypothesis that myosin phosphorylation in smooth muscle functions in regulating cross-bridge cycling rates. Myosin was dephosphorylated during relaxation with a half time of 2.7 s. Calculated maximal cellular rates of light chain phosphorylation were similar to measured values, indicating that most of the kinase was activated on stimulation.

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