Caidesmon and calponin phosphorylation in regulation of smooth muscle contraction

1994 ◽  
Vol 72 (11) ◽  
pp. 1410-1414 ◽  
Author(s):  
William T. Gerthoffer ◽  
Jennifer Pohl
Keyword(s):  
Smooth Muscle ◽  
Thin Filament ◽  
Transient Increase ◽  
Tracheal Smooth Muscle ◽  
Shortening Velocity ◽  
Myosin Phosphorylation ◽  
Actomyosin Atpase ◽  
Contractile System ◽  
Thin Filament Proteins

Recent studies of the smooth muscle contractile system indicate that Ca2+-dependent phosphorylation of the 20-kDa myosin light chains, modulation of phosphoprotein phosphatases, and phosphorylation of thin-filament proteins are all potential features of contractile system regulation. The thin-filament proteins caidesmon and calponin are known to inhibit actomyosin ATPase in vitro and actin sliding velocity in the in vitro motility assay. Inhibition of actomyosin ATPase is relieved by phosphorylation of caldesmon or calponin. The notion that caldesmon and calponin phosphorylation – dephosphorylation is important in the living smooth muscle cell was tested using canine tracheal smooth muscle strips labeled with 32P. We found that both caldesmon and calponin phosphorylation increased in response to stimulation with carbachol. Carbachol induced a biphasic increase in [Ca2+]i in canine tracheal smooth muscle, an early transient increase in myosin phosphorylation, which decayed to 0.4 mol Pi/mol light chain, and a rapid transient increase in tissue shortening velocity. Relative changes in caidesmon phosphorylation correlate best with force development and the [Ca2+]i transient, both of which follow a similar time course. Calponin phosphorylation appears to be a rapid transient event more similar to the transient increase in unloaded shortening velocity. Our results are consistent with a potential role for both caldesmon and calponin phosphorylation in regulating smooth muscle contraction.Key words: calcium, caldesmon, calponin, colon, myosin, phosphorylation, smooth muscle, trachea.

Myosin phosphorylation and regulation of cross-bridge cycle in tracheal smooth muscle

1983 ◽  
Vol 244 (3) ◽  
pp. C182-C187 ◽  
Author(s):  
W. T. Gerthoffer ◽  
R. A. Murphy
Keyword(s):  
Smooth Muscle ◽  
Tracheal Smooth Muscle ◽  
Shortening Velocity ◽  
Active Stress ◽  
Myosin Phosphorylation ◽  
Cross Bridge ◽  
Rapid Changes ◽  
Cross Bridges ◽  
Resting Muscle ◽  
Isotonic Shortening

We have tested the hypothesis that phosphorylation of the 20,000-dalton myosin light chains (LC 20) in rabbit tracheal smooth muscle modulates cross-bridge kinetics and isotonic shortening velocity. The thin muscle [190 +/- 10 (SE) microns] allowed detection of rapid changes in carbachol-induced active stress development, LC 20 phosphorylation, and isotonic shortening velocities. Phosphorylation of the LC 20 in resting muscle was 0.12 +/- 0.04 mol Pi/mol LC 20. Carbachol (10(-5) M) increased the level of phosphorylation to 0.46 +/- 0.03 mol Pi/mol LC 20 within 30 s. Phosphorylation then declined significantly as steady-state active stress was reached. A positive correlation was always found between LC 20 phosphorylation and shortening velocity. This result supports the hypothesis that the level of myosin phosphorylation was related to the mean cross-bridge cycling rate rather than the number of cross bridges contributing to the developed stress. Dephosphorylation of LC 20 occurred at about the same rate as the decline in shortening velocity and stress upon stimulus washout.

ATP hydrolysis during contraction of permeabilized airway smooth muscle

1999 ◽  
Vol 277 (2) ◽  
pp. L334-L342 ◽  
Author(s):  
Keith A. Jones ◽  
Robert R. Lorenz ◽  
Y. S. Prakash ◽  
Gary C. Sieck ◽  
David O. Warner
Keyword(s):  
Smooth Muscle ◽  
Steady State ◽  
Atp Hydrolysis ◽  
Isometric Force ◽  
Time Dependent ◽  
Tracheal Smooth Muscle ◽  
Hydrolysis Rate ◽  
Shortening Velocity ◽  
Actomyosin Atpase ◽  
Significant Difference

This study determined whether the time-dependent decline in the rate of ATP hydrolysis by actomyosin ATPase during sustained isometric force can occur in the absence of a time-dependent decline in regulatory myosin light chain (rMLC) phosphorylation in Triton X-100-permeabilized canine tracheal smooth muscle. Maximal activation with 10 μM Ca2+ induced sustained increases in isometric force, stiffness, and rMLC phosphorylation; however, the increase in the ATP hydrolysis rate was initially high but then declined to a steady-state level above that of the unstimulated muscle (basal 31.8 ± 5.8 nmol ⋅ cm−3 ⋅ s−1; peak 81.4 ± 11.3 nmol ⋅ cm−3 ⋅ s−1; steady-state 62.2 ± 9.1 nmol ⋅ cm−3 ⋅ s−1). Activation of strips in which the rMLC was irreversibly and maximally thiophosphorylated with adenosine 5′- O-(3-thiotriphosphate) also induced sustained increases in isometric force and stiffness but a nonsustained increase in ATP hydrolysis rate. There was no significant difference in the peak or steady-state isometric force, stiffness, or ATP hydrolysis rate or in the steady-state maximum unloaded shortening velocity between strips activated by 10 μM Ca2+ or rMLC thiophosphorylation (0.058 ± 0.016 and 0.047 ± 0.011 muscle lengths/s, respectively). Mechanisms other than changes in rMLC phosphorylation contribute to the time-dependent decline in actomyosin ATPase activity during sustained activation of canine tracheal smooth muscle.

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.

Regulation of the contractile element of airway smooth muscle

1991 ◽  
Vol 261 (2) ◽  
pp. L15-L28 ◽  
Author(s):  
W. T. Gerthoffer
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Thin Filament ◽  
Muscle Tone ◽  
Smooth Muscles ◽  
Membrane Receptors ◽  
Contractile Element ◽  
Myosin Phosphorylation ◽  
Contractile System ◽  
Protein Components

Smooth muscle of the mammalian airways controls airway diameter and resistance to airflow. Smooth muscle tone is in turn controlled by a variety of external signals that are transduced to useful work by contractile proteins. The protein components of the contractile element of airway smooth muscle are similar to those found in other smooth muscles and include actin, myosin, tropomyosin, caldesmon, and calponin. There has been significant recent progress in studies of contractile system regulation of airway smooth muscle. Regulation of myosin light chain kinase, identification of the sites phosphorylated on the regulatory myosin light chains, and the effect of myosin phosphorylation on stress development and crossbridge cycling rates have all been studied in some detail. We infer from these studies that besides myosin phosphorylation there is an important role for a thin filament Ca(2+)-dependent regulatory mechanism. The potentially important thin filament proteins caldesmon and calponin are present in tracheal smooth muscle and may be phosphorylated during contraction. The use of intracellular Ca2+ indicators to estimate changes in intracellular Ca2+ ([Ca2+]i) and the development of several skinned fiber preparations have broadened the scope of physiological studies with airway smooth muscle and have suggested that the contractile element sensitivity to Ca2+ is not fixed but might be modulated by undefined messengers or excitation-contraction pathways. This adds an additional challenge to the continuing effort to define the messengers and regulatory proteins that couple activation of membrane receptors to the contractile element in airway smooth muscle.

Characterization and confocal imaging of calponin in gastrointestinal smooth muscle

1993 ◽  
Vol 265 (5) ◽  
pp. C1371-C1378 ◽  
Author(s):  
M. P. Walsh ◽  
J. D. Carmichael ◽  
G. J. Kargacin
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Smooth Muscle Cells ◽  
Thin Filament ◽  
Muscle Cells ◽  
Biochemical Properties ◽  
Confocal Imaging ◽  
Isolated Cells ◽  
Independent Manner

Calponin isolated from chicken gizzard smooth muscle binds in vitro to actin in a Ca(2+)-independent manner and thereby inhibits the actin-activated Mg(2+)-adenosinetriphosphatase of smooth muscle myosin. This inhibition is relieved when calponin is phosphorylated by protein kinase C or Ca2+/calmodulin-dependent protein kinase II, suggesting that calponin is involved in thin filament-associated regulation of smooth muscle contraction. To further examine this possibility, calponin was isolated from toad stomach smooth muscle, characterized biochemically, and localized in intact isolated cells. Toad stomach calponin had the same basic biochemical properties as calponin from other sources. Confocal immunofluorescence microscopy revealed that calponin in intact smooth muscle cells was localized to long filamentous structures that were colabeled by antibodies to actin or tropomyosin. Preservation of the basic biochemical properties of calponin from species to species suggests that these properties are relevant for its in vivo function. Its colocalization with actin and tropomyosin indicates that calponin is associated with the thin filament in intact smooth muscle cells.

Abstract 958: Cardiac MyBP-C Modulates Actomyosin Interactions: Evidence From Cardiac MyBP-c−/ − Mice

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Yves Lecarpentier ◽  
Nicolas Vignier ◽  
Patricia Oliviero ◽  
Miguel Cortes-Morichetti ◽  
Lucie Carrier ◽  
...  
Keyword(s):  
Thin Filament ◽  
Isometric Force ◽  
Binding Protein ◽  
Critical Role ◽  
Cardiac Contractility ◽  
Left Ventricular ◽  
Actin Binding ◽  
Power Stroke ◽  
Shortening Velocity

The precise role of cardiac myosin binding protein C (cMyBP-C) on actomyosin interaction (AMI) remains unknown. We hypothesized that the lack of cMyBP-C impaired cardiac AMI. Experiments were performed on 16 weeks old cMyBP-C −/− (KO) and age-matched wild-type (WT) mice (n=20/group). In vitro mechanical and energetics properties were performed on left ventricular (LV) papillary muscles and Huxley’s equations were used to characterize AMI. In vitro motility assays were performed using myosin purified from LV. Myosin-based sliding velocities of actin filaments were analyzed at baseline, after pretreatment of the myosin solution with 10 umol of the catalytic subunit of PKA and/or in the presence of increasing amount of α-actinin, an actin-binding protein that acts as an internal load thereby providing an index of relative isometric force. Western-blot analysis was used to quantify cMyBP-C and phosphorylated cMyBP-C in myosin solutions. Compared to WT, both total tension and maximum shortening velocity were lower in KO (p<0.001). The probability for myosin to be weakly bound to actin was higher in KO than in WT (8.6±0.3 vs. 5.4±0.2%, p<0.05), whereas the number of strongly bound, high-force generated state cross-bridges was lower in KO (6.4±0.9 vs. 11.6±1.0 10 9 /mm 2 , p<0.001). The unitary force per AMI was lower in KO than in WT (p<0.01). At baseline, myosin-based velocities of actin were slower in KO than in WT (1.65±0.01 vs. 1.98±0.01 um/s, p<0.01). The minimum amount of α-actinin needed to completely arrest the thin filament motility was significantly higher in WT than in KO (73.3±1.1 vs 29.1±0.1 ug/l, p<0.001). As expected, cMyBP-C was present in WT myosin solution whereas cMyBP-C was not detected in KO. In WT, PKA induced a 1.6-fold increased in cMyBP-C phosphorylation (p<0.01) associated with a 53±1% increase in the amount of α-actinin required to arrest thin filament motility (p<0.001). PKA did not modify sliding velocity in WT. In KO, PKA had no effect on myosin sliding. We conclude that cMyBP-C regulates AMI by limiting inefficient cross-bridge formation and by enhancing the power stroke step. Phosphorylation status of cMyBP-C appears to play a critical role on cardiac contractility through a direct effect on the myosin molecular motor.

Effect of transpulmonary pressure on airway closure in immature and mature rabbits

1995 ◽  
Vol 78 (2) ◽  
pp. 505-512 ◽  
Author(s):  
R. S. Tepper ◽  
S. J. Gunst ◽  
C. M. Doerschuk ◽  
X. Shen ◽  
W. Bray
Keyword(s):  
Smooth Muscle ◽  
Small Volume ◽  
Transpulmonary Pressure ◽  
Lung Parenchyma ◽  
Tracheal Smooth Muscle ◽  
Airway Closure ◽  
Maximal Stimulation ◽  
In Vitro Sensitivity ◽  
Airway Opening

The transpulmonary pressures (Ptp values) at which airway closure occurred during maximal stimulation with methacholine were compared in 10 mature and 9 immature rabbit lungs by using an alveolar capsule technique to assess airway closure. After maximal constriction, airway opening and alveolar capsule pressures were recorded during small volume oscillations as Ptp was lowered from 12 to 4 cmH2O. At each Ptp, the proportion of alveolar capsules indicating airway closure was greater for the immature than for the mature lungs (P < 0.025). At Ptp of 4 cmH2O, only 20% of alveolar capsules indicated airway closure in the mature lungs in contrast to 85% indicating closure in the immature lungs (P < 0.001). The in vitro sensitivity of tracheal smooth muscle to acetylcholine and histamine was greater in tissues from immature than from mature rabbits. We conclude that the more frequent airway closure observed in immature rabbits could reflect maturational differences in the structure of the bronchi or lung parenchyma or differences in the coupling between the parenchyma and the airways.

scholarly journals Tumour necrosis factor-α induces hyperreactivity in tracheal smooth muscle of the guinea-pig in vitro

1998 ◽  
Vol 12 (1) ◽  
pp. 45-49 ◽  
Author(s):  
H-J. Pennings ◽  
K. Kramer ◽  
A. Bast ◽  
W.A. Buurman ◽  
E.F.M. Wouters
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Tumour Necrosis Factor ◽  
Tumour Necrosis ◽  
Tracheal Smooth Muscle ◽  
Factor I ◽  
Necrosis Factor

Mechanical properties of human bronchial smooth muscle in vitro

1992 ◽  
Vol 73 (4) ◽  
pp. 1481-1485 ◽  
Author(s):  
K. Ishida ◽  
P. D. Pare ◽  
J. Hards ◽  
R. R. Schellenberg
Keyword(s):  
Mechanical Properties ◽  
Smooth Muscle ◽  
Electrical Field Stimulation ◽  
Tracheal Smooth Muscle ◽  
Cross Sectional ◽  
Velocity Of Shortening ◽  
Muscle Shortening ◽  
Tension Generation ◽  
Very High

The in vitro mechanical properties of smooth muscle strips from 10 human main stem bronchi obtained immediately after pneumonectomy were evaluated. Maximal active isometric and isotonic responses were obtained at varying lengths by use of electrical field stimulation (EFS). At the length (Lmax) producing maximal force (Pmax), resting tension was very high (60.0 +/- 8.8% Pmax). Maximal fractional muscle shortening was 25.0 +/- 9.0% at a length of 75% Lmax, whereas less shortening occurred at Lmax (12.2 +/- 2.7%). The addition of increasing elastic loads produced an exponential decrease in the shortening and velocity of shortening but increased tension generation of muscle strips stimulated by EFS. Morphometric analysis revealed that muscle accounted for 8.7 +/- 1.5% of the total cross-sectional tissue area. Evaluation of two human tracheal smooth muscle preparations revealed mechanics similar to the bronchial preparations. Passive tension at Lmax was 10-fold greater and maximal active shortening was threefold less than that previously demonstrated for porcine trachealis by us of the same apparatus. We attribute the limited shortening of human bronchial and tracheal smooth muscle to the larger load presumably provided by a connective tissue parallel elastic component within the evaluated tissues, which must be overcome for shortening to occur. We suggest that a decrease in airway wall elastance could increase smooth muscle shortening, leading to excessive responses to contractile agonists, as seen in airway hyperresponsiveness.

Myosin phosphorylation and contraction of feline esophageal smooth muscle

1985 ◽  
Vol 249 (1) ◽  
pp. C9-C14 ◽  
Author(s):  
N. W. Weisbrodt ◽  
R. A. Murphy
Keyword(s):  
Smooth Muscle ◽  
Mechanical Activation ◽  
Smooth Muscles ◽  
Circular Muscle ◽  
Electrical Field Stimulation ◽  
Muscle Layer ◽  
Field Stimulation ◽  
Shortening Velocity ◽  
Myosin Phosphorylation ◽  
Isotonic Shortening

We tested the hypothesis that phosphorylation of the 20,000-Da light chain of myosin (LC 20) is related to mechanical activation of esophageal smooth muscle. Circular muscle layer strips of cat esophagus were taken from the lower esophageal sphincter (LES) and the distal esophageal body (EB). The LES strips developed tone spontaneously, and the EB strips were tonically contracted with carbachol. Both tissues relaxed in response to electrical-field stimulation. Phosphorylation of the LC 20 was determined in tissues quick-frozen during relaxation and during stress redevelopment after cessation of field stimulation. Stress and phosphorylation levels were low after 30 s of field stimulation, and a rapid contraction followed field stimulation. Phosphorylation in the LES increased from 0.043 +/- 0.029 to 0.328 +/- 0.043 mol Pi/mol LC 20 within 10 s after stimulation of the inhibitory nerves was terminated, while stress was still rising rapidly. Phosphorylation in the LES then declined to a steady-state value of 0.162 +/- 0.034 mol Pi/mol LC 20 after 10 min. Isotonic shortening velocities at a constant afterload following a quick release showed changes with time that were proportional to the level of phosphorylation. This was also true for values of maximal shortening velocity estimated for zero external load and for the rate of stress redevelopment after a step shortening. Comparable measurements were made in the carbachol-contracted EB. These results indicate that visceral smooth muscles, which normally function tonically (LES) or phasically (EB), exhibit an initial rapid mechanical activation associated with myosin phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

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