scholarly journals Caldesmon is a Ca2+-regulatory component of native smooth-muscle thin filaments

1985 ◽  
Vol 231 (3) ◽  
pp. 517-522 ◽  
Author(s):  
S B Marston ◽  
W Lehman
Keyword(s):  
Smooth Muscle ◽  
Thin Filament ◽  
Myosin Light Chain ◽  
Potent Inhibitor ◽  
Peptide Mapping ◽  
Smooth Muscles ◽  
Physiological Mechanism ◽  
Thin Filaments ◽  
Muscle Types ◽  
Mgatpase Activity

Thin-filament preparations from four smooth muscle types (gizzard, stomach, trachea, aorta) all activate myosin MgATPase activity, are regulated by Ca2+, and contain actin, tropomyosin and a 120000-140000-Mr protein in the molar proportions 1:1/7:1/26. The 120000-140000-Mr protein from all sources is a potent inhibitor of actomyosin ATPase activity. Peptide-mapping and immunological evidence is presented showing that it is identical with caldesmon. Quantitative immunological data suggest that caldesmon is a component of all the thin filaments and that the thin-filament-bound caldesmon accounts for all the caldesmon in intact tissue. The myosin light-chain kinase content of thin-filament preparations was found to be negligible. We propose that caldesmon-based thin-filament Ca2+ regulation is a physiological mechanism in all smooth muscles.

scholarly journals Ca2+-calmodulin binding to caldesmon and the caldesmon-actin-tropomyosin complex. Its role in Ca2+ regulation of the activity of synthetic smooth-muscle thin filaments

1989 ◽  
Vol 257 (3) ◽  
pp. 839-843 ◽  
Author(s):  
K Pritchard ◽  
S B Marston
Keyword(s):  
Smooth Muscle ◽  
Thin Filament ◽  
Smooth Muscle Actin ◽  
Muscle Actin ◽  
Polarization Method ◽  
Thin Filaments ◽  
Calmodulin Binding ◽  
Muscle Thin Filament ◽  
Mgatpase Activity

We measured the concentration of calmodulin required to reverse inhibition by caldesmon of actin-activated myosin MgATPase activity, in a model smooth-muscle thin-filament system, reconstituted in vitro from purified vascular smooth-muscle actin, tropomyosin and caldesmon. At 37 degrees C in buffer containing 120 mM-KCl, 4 microM-Ca2+-calmodulin produced a half-maximal reversal of caldesmon inhibition, but more than 300 microM-Ca2+-calmodulin was necessary at 25 degrees C in buffer containing 60 mM-KCl. The binding affinity (K) of caldesmon for Ca2+-calmodulin was measured by a fluorescence-polarization method: K = 2.7 x 10(6) M-1 at 25 degrees C (60 mM-KCl); K = 1.4 x 10(6) M-1 at 37 degrees C in 70 mM-KCl-containing buffer; K = 0.35 x 10(6) M-1 at 37 degrees C in 120 mM-KCl- containing buffer (pH 7.0). At 37 degrees C/120 mM-KCl, but not at 25 degrees C/60 mM-KCl, Ca2+-calmodulin bound to caldesmon bound to actin-tropomyosin (K = 2.9 x 10(6) M-1). Ca2+ regulation in this system does not depend on a simple competition between Ca2+-calmodulin and actin for binding to caldesmon. Under conditions (37 degrees C/120 mM-KCl) where physiologically realistic concentrations of calmodulin can Ca2+-regulate synthetic thin filaments, Ca2+-calmodulin reverses caldesmon inhibition of actomyosin ATPase by forming a non-inhibited complex of Ca2+-calmodulin-caldesmon-(actin-tropomyosin).

Regulation of Contraction-Relaxation in Smooth Muscle

Physiology ◽  
1992 ◽  
Vol 7 (2) ◽  
pp. 59-64 ◽  
Author(s):  
DJ Hartshorne ◽  
T Kawamura
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Thin Filament ◽  
Myosin Light Chain ◽  
Regulatory Mechanisms ◽  
Regulation Of Contraction ◽  
Subsequent Activation

Contraction of smooth muscle requires phosphorylation of myosin. This is linked to intracellular Ca2+ transients by formation of the Ca2+-calmodulin complex and subsequent activation of myosin light chain kinase. Dephosphorylation is assumed to be unregulated. Other possible regulatory mechanisms include thin-filament-linked proteins caldesmon and calponin.

Increased Ca2+ and myosin phosphorylation, but not calmodulin activity in sensitized airway smooth muscles

1995 ◽  
Vol 268 (5) ◽  
pp. L739-L746 ◽  
Author(s):  
H. Jiang ◽  
K. Rao ◽  
X. Liu ◽  
G. Liu ◽  
N. L. Stephens
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Airway Hyperresponsiveness ◽  
Myosin Light Chain ◽  
Smooth Muscles ◽  
Total Activity ◽  
Shortening Velocity ◽  
Bronchial Smooth Muscle ◽  
And Control

The increased shortening velocity and capacity of airway smooth muscle (ASM) from ragweed pollen-sensitized dogs, which may be responsible for its in vivo airway hyperresponsiveness, have been shown to be associated with higher actomyosin adenosinetriphosphatase activity and greater level of phosphorylation of the 20-kDa myosin light-chain (MLC20) at rest and during contraction. Current studies show that the elevated level of phosphorylation may be the result of an increased myosin light-chain kinase (MLCK) activity due to excessive quantity of MLCK. There were no significant changes in total activity of calmodulin, a protein that binds and activates MLCK, in sensitized dog ASM (SASM) compared with control ASM (CASM). When normalized to the relative calmodulin content in the tissues, the specific calmodulin activities (means +/- SE) in sensitized tracheal smooth muscle (STSM) and sensitized bronchial smooth muscle (SBSM) and in their controls were not different (STSM 0.359 +/- 0.117, CTSM 0.339 +/- 0.136. SBSM 0.201 +/- 0.098, and control bronchial smooth muscle 0.213 +/- 0.056 nmol Pi.calmodulin content-1.min-1, respectively). Intracellular Ca2+ levels indicated by fura 2 fluorescent dye remained unaltered in SASM. We conclude that airway hyperresponsiveness may result from higher MLCK content in SASM rather than from changes in Ca(2+)-calmodulin activities, which is an example of alteration in Ca2- sensitivity of ASM.

Protein phosphorylation in cardiac and vascular smooth muscle

1981 ◽  
Vol 241 (2) ◽  
pp. H117-H128 ◽  
Author(s):  
M. Barany ◽  
K. Barany
Keyword(s):  
Smooth Muscle ◽  
Protein Phosphorylation ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Current Knowledge ◽  
Smooth Muscles ◽  
Arterial Smooth Muscle ◽  
Cardiac Sarcoplasmic Reticulum ◽  
The Many ◽  
Contraction Cycle

In the heart and arterial smooth muscles, several proteins are phosphorylated. This review summarizes our current knowledge about these phosphoproteins and their possible role in the function of these muscles. In the contractile apparatus, the phosphorylation of myosin light chain seems to be an integral part of the contraction cycle of arterial smooth muscle. However, in the heart the relationship between light chain phosphorylation-dehosphorylation and systolic-diastolic states remains open. In the heart, the phosphorylation of the inhibitory subunit of troponin, a myofibrillar protein, parallels the positive inotropic response induced by beta-adrenergic agonists. It seems likely that this phosphorylation is involved in the physiological stimulation of the heart by epinephrine. Cardiac sarcoplasmic reticulum contains a low-molecular-weight protein, phospholamban, the phosphorylation of which is required for Ca2+ transport. Ion fluxes through the heart sarcolemma may also be controlled through membrane protein phosphorylation. Key enzymes of the energy-yielding pathways in the heart, the pyruvate dehydrogenase multienzyme complex and phosphorylase, are turned on and off by phosphorylation-dephosphorylation mechanisms. Our understanding of protein phosphorylation in the heart has advanced greatly. In contrast, with the exception of the myosin light chain, much less is known about the many proteins phosphorylated in arterial smooth muscle.

scholarly journals Mechanism of Catch Force: Tethering of Thick and Thin Filaments by Twitchin

2010 ◽  
Vol 2010 ◽  
pp. 1-20 ◽  
Author(s):  
Thomas M. Butler ◽  
Marion J. Siegman
Keyword(s):  
Protein Kinase ◽  
Intracellular Calcium ◽  
Protein Kinase A ◽  
Thin Filament ◽  
Smooth Muscles ◽  
Mechanical State ◽  
Thin Filaments ◽  
Force Maintenance ◽  
Basal Concentration ◽  
Kinase A

Catch is a mechanical state occurring in some invertebrate smooth muscles characterized by high force maintenance and resistance to stretch during extremely slow relaxation. During catch, intracellular calcium is near basal concentration and myosin crossbridge cyctng rate is extremely slow. Catch force is relaxed by a protein kinase A-mediated phosphorylation of sites near the N- and C- temini of the minititin twitchin (~526 kDa). Some catch force maintenance car also occur together with cycling myosin crossbridges at submaximal calcium concentrations, but not when the muscle is maximally activated. Additionally, the link responsible for catch can adjust during shortening of submaximally activated muscles and maintain catch force at the new shorter length. Twitchin binds to both thick and thin filaments, and the thin filament binding shown by both the N- and Cterminal portions of twitchin is decreased by phosphorylation of the sites that regulate catch. The data suggest that the twitchin molecule itself is the catch force beanng tether between thick and thin filaments. We present a model for the regulation of catch in which the twitchin tether can be displaced from thin filaments by both (a) the phosphorylation of twitchin and (b) the attachment of high force myosin crossbridges.

Expression and epitopic conservation of calponin in different smooth muscles and during development

1996 ◽  
Vol 74 (2) ◽  
pp. 187-196 ◽  
Author(s):  
Jian-Ping Jin ◽  
Michael P. Walsh ◽  
Mary E. Resek ◽  
Gail A. McMartin
Keyword(s):  
Smooth Muscle ◽  
Thin Filament ◽  
Smooth Muscles ◽  
Constitutive Expression ◽  
Young Chick ◽  
Adult Chicken ◽  
Muscle Tissues ◽  
Highly Sensitive ◽  
Cnbr Cleavage ◽  
Low Levels

Calponin is a thin filament associated protein found in smooth muscle as a potential modulator of contraction. Five mouse monoclonal antibodies (mAbs CP1, CP3, CP4, CP7, and CP8) were prepared against chicken gizzard α-calponin. The CP1 epitopic structure is conserved in smooth muscles across vertebrate phyla and is highly sensitive to CNBr cleavage in contrast with the chicken-specific CP4 and the avian–mammalian-specific CP8 epitopes that are resistant to CNBr fragmentation. Using this panel of mAbs against multiple epitopes, only α-calponin was detected in adult chicken smooth muscles and throughout development of the gizzard. Western blotting showed that the calponin content varied among different smooth muscle tissues and correlated with that of h-caldesmon. In contrast with the constitutive expression of calponin in phasic smooth muscle of the digestive tract, very low levels of calponin were detected in adult avian tracheas and no calponin expression was detected in embryonic and young chick tracheas. These results provide information on the structural conservation of calponins and suggest a relationship between calponin expression and smooth muscle functional states.Key words: smooth muscle calponin, caldesmon, expression, development, chicken trachea.

Blebbistatin, a myosin II inhibitor, suppresses contraction and disrupts contractile filaments organization of skinned taenia cecum from guinea pig

2010 ◽  
Vol 298 (5) ◽  
pp. C1118-C1126 ◽  
Author(s):  
Masaru Watanabe ◽  
Masatoshi Yumoto ◽  
Hideyuki Tanaka ◽  
Hon Hui Wang ◽  
Takeshi Katayama ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Muscle Contraction ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Myosin Ii ◽  
Smooth Muscle Contraction ◽  
Myosin Light Chain Phosphorylation ◽  
Thin Filaments ◽  
Tension Development

To explore the precise mechanisms of the inhibitory effects of blebbistatin, a potent inhibitor of myosin II, on smooth muscle contraction, we studied the blebbistatin effects on the mechanical properties and the structure of contractile filaments of skinned (cell membrane permeabilized) preparations from guinea pig taenia cecum. Blebbistatin at 10 μM or higher suppressed Ca2+-induced tension development at any given Ca2+ concentration but had little effects on the Ca2+-induced myosin light chain phosphorylation. Blebbistatin also suppressed the 10 and 2.75 mM Mg2+-induced, “myosin light chain phosphorylation-independent” tension development at more than 10 μM. Furthermore, blebbistatin induced conformational change of smooth muscle myosin (SMM) and disrupted arrangement of SMM and thin filaments, resulting in inhibition of actin-SMM interaction irrespective of activation with Ca2+. In addition, blebbistatin partially inhibited Mg2+-ATPase activity of native actomyosin from guinea pig taenia cecum at around 10 μM. These results suggested that blebbistatin suppressed skinned smooth muscle contraction through disruption of structure of SMM by the agent.

scholarly journals Inhibition of smooth-muscle myosin-light-chain phosphatase by Ruthenium Red

2000 ◽  
Vol 349 (3) ◽  
pp. 797-804 ◽  
Author(s):  
Aki YAMADA ◽  
Osamu SATO ◽  
Minoru WATANABE ◽  
Michael P. WALSH ◽  
Yasuo OGAWA ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Smooth Muscles ◽  
Direct Interaction ◽  
Smooth Muscle Contraction ◽  
Ruthenium Red ◽  
Dependent Manner ◽  
Myosin Light Chain Phosphatase ◽  
Concentration Dependent Manner

Ruthenium Red (RuR) is widely used as an inhibitor of ryanodine receptor Ca2+ release channels, but has additional effects such as the induction of Ca2+ sensitization of contraction of permeabilized smooth muscles. To address the mechanism underlying this process, we examined the effects of RuR on contractility in permeabilized guinea-pig ileum and on the activity of myosin-light-chain phosphatase (MP). RuR increased the force at submaximal [Ca2+] (pCa 6.3) approx. 4-fold. This effect was not observed after thiophosphorylation of MP. RuR also seemed capable of preventing the thiophosphorylation of MP, suggesting a direct interaction of RuR with MP. Consistent with this possibility, smooth-muscle MP was inhibited by RuR in a concentration-dependent manner (IC50 23µM). Exogenous calmodulin significantly increased RuR-induced contraction at pCa 6.3 but had little effect on contraction induced by microcystin at this [Ca2+]. Ca2+-independent contraction was induced by RuR (EC50 843µM) and by microcystin (EC50 59nM) but the maximal force induced by RuR was smaller than that induced by microcystin. The addition of 300µM RuR enhanced the contraction induced by 30nM microcystin but markedly decreased that induced by 1µM microcystin. Such a dual action of RuR on microcystin-induced effects was not observed in experiments using purified MP. We conclude that the RuR-induced Ca2+ sensitization of smooth-muscle contraction is due to the direct inhibition of MP by RuR.

scholarly journals Cytoplasmic free calcium, myosin light chain phosphorylation, and force in phasic and tonic smooth muscle.

1988 ◽  
Vol 92 (6) ◽  
pp. 713-729 ◽  
Author(s):  
B Himpens ◽  
G Matthijs ◽  
A V Somlyo ◽  
T M Butler ◽  
A P Somlyo
Keyword(s):  
Smooth Muscle ◽  
Pulmonary Artery ◽  
Light Chain ◽  
Time Course ◽  
Myosin Light Chain ◽  
Muscle Tension ◽  
Smooth Muscles ◽  
Free Calcium ◽  
Myosin Light Chain Phosphorylation ◽  
Tonic Smooth Muscle

The time course of [Ca2+]i, tension, and myosin light chain phosphorylation were determined during prolonged depolarization with high K+ in intact tonic (rabbit pulmonary artery) and phasic (longitudinal layer of guinea pig ileum) smooth muscles. [Ca2+]i was monitored with the 340 nm/380 nm signal ratio of the fluorescent indicator fura-2. The fluorescence ratio had a similar time course in both muscle types during depolarization with 109 mM [K+]o; after a transient peak, there was a decline to 70% of its peak value in tonic smooth muscle, and to 60% in phasic smooth muscle. Tension, however, continued to increase in the pulmonary artery, while in the ileum it declined in parallel with the [Ca2+]i. On changing [K+]o from 109 to 20 mM, tension and [Ca2+]i either remained unchanged or declined in parallel in the pulmonary artery. Phosphorylation of the 20-kD myosin light chain, measured during stimulation of muscle strips with 109 mM [K+]o in another set of experiments, increased from 3% to a peak of 50% in the intact pulmonary artery, and then declined to a steady state value of 23%. In the intact ileum, a very rapid, early transient phosphorylation (up to 50%) at 2-3 s was seen. This transient declined by 30 s to a value that was close to the resting level (7%), while tension remained at 55% of its peak force. A quick release during maintained stimulation induced no detectable change in the [Ca2+]i in either type of smooth muscle. We discuss the possibility that the slowly rising tonic tension in pulmonary artery could be due to cooperativity between phosphorylated and nonphosphorylated crossbridges.

scholarly journals Calcium ion-regulated thin filaments from vascular smooth muscle

1980 ◽  
Vol 185 (2) ◽  
pp. 355-365 ◽  
Author(s):  
S B Marston ◽  
R M Trevett ◽  
M Walters
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Atpase Activity ◽  
Thin Filament ◽  
Troponin I ◽  
Troponin C ◽  
Myosin Atpase ◽  
Protein G ◽  
Thin Filaments ◽  
Myosin Atpase Activity

Myosin and actin competition tests indicated the presence of both thin-filament and myosin-linked Ca2+-regulatory systems in pig aorta and turkey gizzard smooth-muscle actomyosin. A thin-filament preparation was obtained from pig aortas. The thin filaments had no significant ATPase activity [1.1 +/- 2.6 nmol/mg per min (mean +/- S.D.)], but they activated skeletal-muscle myosin ATPase up to 25-fold [500 nmol/mg of myosin per min (mean +/- S.D.)] in the presence of 10(-4) M free Ca2+. At 10(-8) M-Ca2+ the thin filaments activated myosin ATPase activity only one-third as much. Thin-filament activation of myosin ATPase activity increased markedly in the range 10(-6)-10(-5) M-Ca2+ and was half maximal at 2.7 × 10(-6) M (pCa2+ 5.6). The skeletal myosin-aorta-thin-filament mixture gave a biphasic ATPase-rate-versus-ATP-concentration curve at 10(-8) M-Ca2+ similar to the curve obtained with skeletal-muscle thin filaments. Thin filaments bound up to 9.5 mumol of Ca2+/g in the presence of MgATP2-. In the range 0.06-27 microM-Ca2+ binding was hyperbolic with an estimated binding constant of (0.56 +/- 0.07) x 10(6) M-1 (mean +/- S.D.) and maximum binding of 8.0 +/- 0.8 mumol/g (mean +/- S.D.). Significantly less Ca2+ bound in the absence of ATP. The thin filaments contained actin, tropomyosin and several other unidentified proteins. 6 M-Urea/polyacrylamide-gel electrophoresis at pH 8.3 showed proteins that behaved like troponin I and troponin C. This was confirmed by forming interspecific complexes between radioactive skeletal-muscle troponin I and troponin C and the aorta thin-filament proteins. The thin filaments contained at least 1.4 mumol of a troponin C-like protein/g and at least 1.1 mumol of a troponin I-like protein/g.

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