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.

Isoproterenol attenuates myosin phosphorylation and contraction of tracheal muscle

1989 ◽  
Vol 66 (5) ◽  
pp. 2017-2022 ◽  
Author(s):  
K. Obara ◽  
P. de Lanerolle
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Isometric Force ◽  
Smooth Muscles ◽  
Inhibitory Effect ◽  
Isometric Tension ◽  
Myosin Light Chain Phosphorylation ◽  
Response Curves ◽  
Shortening Velocity ◽  
Myosin Phosphorylation

The effects of isoproterenol on isometric force, unloaded shortening velocity, and myosin phosphorylation were examined in thin muscle bundles (0.1–0.2 mm diam) dissected from lamb tracheal smooth muscle. Methacholine (10(-6) M) induced rapid increases in isometric force and in phosphorylation of the 20,000-Da myosin light chain. Myosin phosphorylation remained elevated during steady-state maintenance of isometric force. The shortening velocity peaked at 15 s after stimulation with methacholine and then declined to approximately 45% of the maximal value by 3 min. Isoproterenol pretreatment inhibited methacholine-stimulated myosin light chain phosphorylation, shortening velocity, and force during the early stages of force generation. However, the inhibitory effect of isoproterenol on force and myosin phosphorylation is proportionally greater than that on shortening velocity. Isoproterenol pretreatment also caused a rightward non-parallel shift in the methacholine dose-response curves for both isometric tension and myosin light chain phosphorylation. These data demonstrate that isoproterenol attenuates the contractile properties of airway smooth muscles by affecting the rate and extent of myosin light chain phosphorylation, perhaps through a mechanism that involves the synergistic interaction of myosin light chain kinase phosphorylation and Ca2+ metabolism.

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.

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 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)

A radioimmunoblotting method for measuring myosin light chain phosphorylation levels in smooth muscle

1985 ◽  
Vol 249 (3) ◽  
pp. C345-C351 ◽  
Author(s):  
D. R. Hathaway ◽  
J. R. Haeberle
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Light Chains ◽  
Myosin Light Chain Phosphorylation ◽  
Electrophoretic Method ◽  
Coomassie Blue ◽  
Antibody Complex

A method for measuring the molar stoichiometry of myosin light chain phosphorylation in intact smooth muscle has been developed. Antiserum to the 20,000-Da light chains of bovine aortic smooth muscle was harvested from rabbits and used to label light chains by a radioimmunoblotting procedure. In the initial characterization it was found that the 20,000-Da light chains could be transferred by electroblotting from polyacrylamide gels to nitrocellulose paper with an efficiency of approximately 80% over a protein range of 0.1-5.0 micrograms. At a dilution of 1:500, the unpurified light chain antiserum required approximately 10-12 h at 22 degrees C to reach equilibrium binding to the transferred light chains. Moreover, equilibrium labeling of the light chain-antibody complex with 125I-protein A required 4-6 h of incubation at 22 degrees C. By using these conditions, a radioimmunoassay for the 20,000-Da light chains was developed that was linear over a protein range of 0.1-5.0 micrograms (5-250 pmol). As little as 20 ng of light chains could be measured if a second antibody procedure (goat anti-rabbit immunoglobulin G Fab fragments) was used. Phosphorylated and unphosphorylated myosin light chains were separated by glycerol-urea polyacrylamide gel electrophoresis. This procedure, combined with radioimmunoblot, gave similar estimates of phosphorylation levels when compared with direct assay for phosphate or scanning of Coomassie blue-stained gels. Moreover, when applied to intact uterine smooth muscle, the glycerol-urea gel radioimmunoblot gave values of myosin light chain phosphorylation for relaxed and contracted muscles that were not statistically different from those obtained with a two-dimensional electrophoretic method.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypertrophy of colonic smooth muscle: contractile proteins, shortening velocity, and regulation

1997 ◽  
Vol 272 (6) ◽  
pp. G1571-G1580 ◽  
Author(s):  
M. J. Siegman ◽  
T. M. Butler ◽  
S. U. Mooers ◽  
L. Trinkle-Mulcahy ◽  
S. Narayan ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Contractile Proteins ◽  
Simple Relationship ◽  
Myosin Light Chain Phosphorylation ◽  
Normal Muscle ◽  
Shortening Velocity ◽  
Significant Difference ◽  
Muscle Contractile

Smooth muscle in megacolon was studied in the lethal spotted mouse and its normal sibling. In megacolon, structural remodeling and a very large increase in total protein content are associated with some changes in the contractile protein isoform composition. 1) There is a higher actin concentration in megacolon, primarily caused by a larger proportion of gamma-isoforms. 2) There was no difference in myosin concentration or in SM1/SM2 heavy chains in megacolon and normal muscle contractile proteins. 3) Only LC17a essential light chain is present in both normal and megacolon. 4) The 25- to 50-kDa 5'-insert occurs in 15-20% of the myosin in normal colon, compared with 5- to 10-fold lower amounts in megacolon. In permeabilized muscles there was no significant difference in unloaded shortening velocity (Vo) with maximal thiophosphorylation of the 20-kDa light chains, nor was there significant difference in the force vs. Ca2+ and force vs. myosin light chain phosphorylation relationships. At approximately 60% myosin light chain phosphorylation after Ca2+ activation, Vo of megacolon was approximately two times faster than Vo of normal muscle. These cellular changes largely account for the higher propulsive velocity of the colon in situ. The distribution of myosin and actin isoforms and the lack of a simple relationship between myosin light chain phosphorylation and Vo point to the operation of additional regulatory processes.

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