Regulation of force and shortening velocity by calcium and myosin phosphorylation in chemically skinned smooth muscle

1996 ◽  
Vol 433 (1-2) ◽  
pp. 42-48 ◽  
Author(s):  
U. Malmqvist ◽  
A. Arner
Keyword(s):  
Smooth Muscle ◽  
Shortening Velocity ◽  
Myosin Phosphorylation ◽  
Skinned 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.

Effects of Ca2+, Mg2+, and myosin phosphorylation on skinned smooth muscle fibers

1987 ◽  
Vol 252 (5) ◽  
pp. C543-C554 ◽  
Author(s):  
R. J. Barsotti ◽  
M. Ikebe ◽  
D. J. Hartshorne
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Isometric Tension ◽  
Skinned Fibers ◽  
Myosin Light Chain Phosphorylation ◽  
Shortening Velocity ◽  
Myosin Phosphorylation ◽  
Chicken Gizzard ◽  
Skinned Smooth Muscle

Isometric tension, unloaded shortening velocity (Vus), and myosin light chain phosphorylation were measured with skinned chicken gizzard fibers at various Ca2+ concentrations and at two concentrations of free Mg2+, 0.7 and 2.2 mM. At low free Mg2+, an increase in Ca2+ from pCa 8.0 to 6.4 resulted in an increase of all three parameters. Between pCa 6.4 and 5.0, isometric tension and phosphorylation remained constant but Vus continued to increase. At low free Mg2+, therefore, Vus showed a dependence both on phosphorylation and on Ca2+. At high free Mg2+, tension and Vus increased as phosphorylation increased and both were maximum at pCa 6.4, where phosphorylation became constant. Therefore, at high free Mg2+, Vus was dependent only on phosphorylation and did not show an additional Ca2+ dependence. Incubation of the Ca2+-independent kinase (approximately 3 microM) with skinned fibers under various conditions resulted in a constant level of phosphorylation (49-58%). At high free Mg2+ plus the Ca2+-independent kinase Vus was independent of Ca2+, whereas at low free Mg2+ Vus increased from pCa 6.4 to 5.0. These data are consistent with the hypothesis that Ca2+ binding to the Ca2+-Mg2+ sites of myosin increase Vus and that this occurs at Ca2+ concentrations higher than those necessary to saturate calmodulin.

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)

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.

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 calcium in tonic and phasic contractions of colonic smooth muscle

1991 ◽  
Vol 260 (6) ◽  
pp. G958-G964 ◽  
Author(s):  
W. T. Gerthoffer ◽  
K. A. Murphey ◽  
J. Mangini ◽  
S. Boman ◽  
F. A. Lattanzio
Keyword(s):  
Smooth Muscle ◽  
Time Dependence ◽  
Fluorescence Intensity ◽  
Light Chain ◽  
Proximal Colon ◽  
Shortening Velocity ◽  
Myosin Phosphorylation ◽  
Quick Release ◽  
Circular Smooth Muscle ◽  
Cross Bridge

The time dependence of lightly loaded shortening velocity, myosin phosphorylation, and changes in myoplasmic Ca2+ concentration ([Ca2+]i) were measured during tonic and phasic contractions of circular smooth muscle from the proximal colon of the dog. Shortening velocity was measured by quick release to a 10% afterload. Myosin phosphorylation was measured by an immunoblot method, and changes in [Ca2+]i were estimated by measuring fluorescence intensity at 550 nm in muscle strips loaded with fluo-3. During tonic contractions induced by 60 mM K+, phosphorylation increased monotonically from 0.11 +/- 0.011 to 0.29 +/- 0.015 mol Pi/mol light chain at 10 min. In contrast, lightly loaded shortening velocity increased rapidly within 10 s to 0.042 +/- 0.003 lengths/s and decreased exponentially to 0.013 +/- 0.001 lengths/s at 15 min. During transient contractions induced by 100 microM acetylcholine, phosphorylation increased from 0.16 +/- 0.03 to 0.30 +/- 0.06 mol Pi/mol light chain at 19 s. In contrast, shortening velocity increased to 0.068 +/- 0.015 lengths/s within 2.4 s and decreased significantly to 0.027 +/- 0.009 lengths/s at 22 s. Fluo-3 fluorescence increased in parallel with force during both tonic and transient contractions. In a smooth muscle that is able to contract both tonically and phasically we observed transient increases in shortening velocity without concurrent phosphorylation or [Ca2+]i transients. Therefore, there are factors in addition to myosin phosphorylation or changes in [Ca2+]i that regulate cross-bridge cycling rates in both tonic and phasic contractions.

Calcium dependence of myosin phosphorylation and airway smooth muscle contraction and relaxation

1986 ◽  
Vol 250 (4) ◽  
pp. C597-C604 ◽  
Author(s):  
W. T. Gerthoffer
Keyword(s):  
Smooth Muscle ◽  
Steady State ◽  
Dose Response ◽  
Time Course ◽  
Physiological Salt Solution ◽  
Shortening Velocity ◽  
Myosin Phosphorylation ◽  
Calcium Dependence ◽  
Contraction And Relaxation ◽  
Isotonic Shortening

The time course and the steady-state calcium dependence of myosin phosphorylation and isotonic shortening velocity were studied during contraction and relaxation of canine tracheal smooth muscle. Dephosphorylation of myosin coincided with the decay of isotonic shortening velocity during rapid relaxation following agonist washout. However, the decay of shortening velocity preceded dephosphorylation during a slow relaxation induced by Ca2+-free physiological salt solution (PSS). Carbachol dose-response curves for isometric stress development and myosin phosphorylation were superimposable but shifted to the left of the shortening velocity dose-response. The steady-state Ca2+ dependence of myosin phosphorylation was defined using carbachol and K+ as agonists. There was a significant dissociation of dephosphorylation and relaxation following a stepwise reduction of extracellular CaCl2 concentration. This result was related to muscarinic activation because the dissociation of relaxation and dephosphorylation was reduced by atropine in muscles stimulated with K+. Myosin phosphorylation was completely dissociated from contraction when muscles were stimulated with carbachol in Ca2+-free PSS and contracted by readmission of CaCl2. Mechanisms in addition to myosin phosphorylation appear to regulate airway muscle tone and shortening velocity, and two possibilities are discussed.

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