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.

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.

Regulation of isotonic shortening velocity by second messengers in tracheal smooth muscle

1994 ◽  
Vol 266 (3) ◽  
pp. C684-C691 ◽  
Author(s):  
S. J. Gunst ◽  
M. H. al-Hassani ◽  
L. P. Adam
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Second Messengers ◽  
Time Dependent ◽  
Tracheal Smooth Muscle ◽  
Shortening Velocity ◽  
Active Stress ◽  
Muscle Tissues ◽  
Mlc Phosphorylation ◽  
Isotonic Shortening

Evidence suggests that the mechanical behavior of smooth muscle tissues is regulated by Ca(2+)-dependent changes in the phosphorylation of the 20,000-Da light chain of myosin (MLC). However, alternative mechanisms activated by specific kinases may be involved in regulating the shortening velocity in some smooth muscle tissues. To determine how the activation of protein kinases A or C affects the regulation of the shortening velocity in canine tracheal smooth muscle, we evaluated the effects of forskolin (10(-5) M) and phorbol 12,13-dibutyrate (PDBu, 3 x 10(-6) M) on active stress, intracellular Ca2+ ([Ca2+]i), MLC phosphorylation, and isotonic shortening velocity during contractions elicited by 60 mM KCl. Forskolin depressed and PDBu increased active stress, [Ca2+]i, MLC phosphorylation, and shortening velocity; thus the effects of these agents on the shortening velocity may result from changes in Ca(2+)-dependent MLC phosphorylation. In contrast, the decline in velocity that occurred with time during tonic contractions elicited by K+ depolarization was not associated with significant changes in MLC phosphorylation; thus the time-dependent changes in shortening velocity may be regulated by a mechanism other than MLC phosphorylation.

scholarly journals The Huxley crossbridge model as the basic mechanism for airway smooth muscle contraction

2019 ◽  
Vol 317 (2) ◽  
pp. L235-L246 ◽  
Author(s):  
Ling Luo ◽  
Lu Wang ◽  
Peter D. Paré ◽  
Chun Y. Seow ◽  
Pasquale Chitano
Keyword(s):  
Smooth Muscle ◽  
Muscle Contraction ◽  
Airway Smooth Muscle ◽  
Time Course ◽  
Striated Muscle ◽  
Isometric Force ◽  
Smooth Muscle Contraction ◽  
Time Dependent ◽  
Shortening Velocity ◽  
Mlc20 Phosphorylation

The cyclic interaction between myosin crossbridges and actin filaments underlies smooth muscle contraction. Phosphorylation of the 20-kDa myosin light chain (MLC20) is a crucial step in activating the crossbridge cycle. Our current understanding of smooth muscle contraction is based on observed correlations among MLC20 phosphorylation, maximal shortening velocity ( Vmax), and isometric force over the time course of contraction. However, during contraction there are changes in the extent of phosphorylation of many additional proteins as well as changes in activation of enzymes associated with the signaling pathways. As a consequence, the mechanical manifestation of muscle contraction is likely to change with time. To simplify the study of these relationships, we measured the mechanical properties of airway smooth muscle at different levels of MLC20 phosphorylation at a fixed time during contraction. A simple correlation emerged when time-dependent variables were fixed. MLC20 phosphorylation was found to be directly and linearly correlated with the active stress, stiffness, and power of the muscle; the observed weak dependence of Vmax on MLC20 phosphorylation could be explained by the presence of an internal load in the muscle preparation. These results can be entirely explained by the Huxley crossbridge model. We conclude that when the influence of time-dependent events during contraction is held constant, the basic crossbridge mechanism in smooth muscle is the same as that in striated muscle.

Active and passive components in the length-dependent stiffness of tracheal smooth muscle during isotonic shortening

2005 ◽  
Vol 98 (1) ◽  
pp. 234-241 ◽  
Author(s):  
Richard A. Meiss ◽  
Ramana M. Pidaparti
Keyword(s):  
Smooth Muscle ◽  
Structural Model ◽  
Isometric Force ◽  
Isometric Tension ◽  
Tracheal Smooth Muscle ◽  
Shortening Velocity ◽  
Muscle Contractility ◽  
Smooth Muscle Tissue ◽  
Induced Changes ◽  
Isotonic Shortening

Contraction of smooth muscle tissue involves interactions between active and passive structures within the cells and in the extracellular matrix. This study focused on a defined mechanical behavior (shortening-dependent stiffness) of canine tracheal smooth muscle tissues to evaluate active and passive contributions to tissue behavior. Two approaches were used. In one, mechanical measurements were made over a range of temperatures to identify those functions whose temperature sensitivity (Q10) identified them as either active or passive. Isotonic shortening velocity and rate of isometric force development had high Q10 values (2.54 and 2.13, respectively); isometric stiffness showed Q10 values near unity. The shape of the curve relating stiffness to isotonic shortening lengths was unchanged by temperature. In the other approach, muscle contractility was reduced by applying a sudden shortening step during the rise of isometric tension. Control contractions began with the muscle at the stepped length so that properties were measured over comparable length ranges. Under isometric conditions, redeveloped isometric force was reduced, but the ratio between force and stiffness did not change. Under isotonic conditions beginning during force redevelopment at the stepped length, initial shortening velocity and the extent of shortening were reduced, whereas the rate of relaxation was increased. The shape of the curve relating stiffness to isotonic shortening lengths was unchanged, despite the step-induced changes in muscle contractility. Both sets of findings were analyzed in the context of a quasi-structural model describing the shortening-dependent stiffness of lightly loaded tracheal muscle strips.

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.

scholarly journals Determinants of mRNA stability in Dictyostelium discoideum amoebae: differences in poly(A) tail length, ribosome loading, and mRNA size cannot account for the heterogeneity of mRNA decay rates.

1988 ◽  
Vol 8 (5) ◽  
pp. 1957-1969 ◽  
Author(s):  
R A Shapiro ◽  
D Herrick ◽  
R E Manrow ◽  
D Blinder ◽  
A Jacobson
Keyword(s):  
Steady State ◽  
Dictyostelium Discoideum ◽  
Mrna Decay ◽  
Decay Rates ◽  
Time Dependent ◽  
Translational Efficiency ◽  
Cdna Clones ◽  
Dependent Manner ◽  
Significant Difference ◽  
Kinetics Of

As an approach to understanding the structures and mechanisms which determine mRNA decay rates, we have cloned and begun to characterize cDNAs which encode mRNAs representative of the stability extremes in the poly(A)+ RNA population of Dictyostelium discoideum amoebae. The cDNA clones were identified in a screening procedure which was based on the occurrence of poly(A) shortening during mRNA aging. mRNA half-lives were determined by hybridization of poly(A)+ RNA, isolated from cells labeled in a 32PO4 pulse-chase, to dots of excess cloned DNA. Individual mRNAs decayed with unique first-order decay rates ranging from 0.9 to 9.6 h, indicating that the complex decay kinetics of total poly(A)+ RNA in D. discoideum amoebae reflect the sum of the decay rates of individual mRNAs. Using specific probes derived from these cDNA clones, we have compared the sizes, extents of ribosome loading, and poly(A) tail lengths of stable, moderately stable, and unstable mRNAs. We found (i) no correlation between mRNA size and decay rate; (ii) no significant difference in the number of ribosomes per unit length of stable versus unstable mRNAs, and (iii) a general inverse relationship between mRNA decay rates and poly(A) tail lengths. Collectively, these observations indicate that mRNA decay in D. discoideum amoebae cannot be explained in terms of random nucleolytic events. The possibility that specific 3'-structural determinants can confer mRNA instability is suggested by a comparison of the labeling and turnover kinetics of different actin mRNAs. A correlation was observed between the steady-state percentage of a given mRNA found in polysomes and its degree of instability; i.e., unstable mRNAs were more efficiently recruited into polysomes than stable mRNAs. Since stable mRNAs are, on average, "older" than unstable mRNAs, this correlation may reflect a translational role for mRNA modifications that change in a time-dependent manner. Our previous studies have demonstrated both a time-dependent shortening and a possible translational role for the 3' poly(A) tracts of mRNA. We suggest, therefore, that the observed differences in the translational efficiency of stable and unstable mRNAs may, in part, be attributable to differences in steady-state poly(A) tail lengths.

Phosphorylation of caldesmon by ERK MAP kinases in smooth muscle

2000 ◽  
Vol 278 (4) ◽  
pp. C718-C726 ◽  
Author(s):  
Jason C. Hedges ◽  
Brian C. Oxhorn ◽  
Michael Carty ◽  
Leonard P. Adam ◽  
Ilia A. Yamboliev ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Map Kinase ◽  
Map Kinases ◽  
Isometric Force ◽  
Phosphorylation Site ◽  
Smooth Muscle Contraction ◽  
Tracheal Smooth Muscle ◽  
Muscarinic Agonists

Phosphorylation of h-caldesmon has been proposed to regulate airway smooth muscle contraction. Both extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinases phosphorylate h-caldesmon in vitro. To determine whether both enzymes phosphorylate caldesmon in vivo, phosphorylation-site-selective antibodies were used to assay phosphorylation of MAP kinase consensus sites. Stimulation of cultured tracheal smooth muscle cells with ACh or platelet-derived growth factor increased caldesmon phosphorylation at Ser789 by about twofold. Inhibiting ERK MAP kinase activation with 50 μM PD-98059 blocked agonist-induced caldesmon phosphorylation completely. Inhibiting p38 MAP kinases with 25 μM SB-203580 had no effect on ACh-induced caldesmon phosphorylation. Carbachol stimulation increased caldesmon phosphorylation at Ser789 in intact tracheal smooth muscle, which was blocked by the M2 antagonist AF-DX 116 (1 μM). AF-DX 116 inhibited carbachol-induced isometric contraction by 15 ± 1.4%, thus dissociating caldesmon phosphorylation from contraction. Activation of M2 receptors leads to activation of ERK MAP kinases and phosphorylation of caldesmon with little or no functional effect on isometric force. P38 MAP kinases are also activated by muscarinic agonists, but they do not phosphorylate caldesmon in vivo.

Velocity-length-time relations in canine tracheal smooth muscle

1988 ◽  
Vol 64 (5) ◽  
pp. 2053-2057 ◽  
Author(s):  
C. Y. Seow ◽  
N. L. Stephens
Keyword(s):  
Smooth Muscle ◽  
Goodness Of Fit ◽  
Time Course ◽  
Muscle Length ◽  
Tracheal Smooth Muscle ◽  
Minimum Length ◽  
Shortening Velocity ◽  
Contracting Muscle ◽  
Parabolic Function ◽  
The Moment

Zero-load velocity (V0) as a function of the length of canine tracheal smooth muscle was obtained by applying zero-load clamps to isotonically contracting muscle under various loads. The load clamps were applied at a specific time after onset of contraction. The magnitude of the isotonic load therefore determines the length of the muscle at the moment of release or at the moment the unloaded shortening velocity was measured. A family of such V0-muscle length (L) curves was obtained at 1-s intervals in the time course of contraction. The V0-L curve was fitted by a parabolic function with satisfactory goodness of fit. The maximum shortening velocity at optimum muscle length varied with time, but the minimum length at which V0 diminished to zero was time independent.

Effects of microtubule disruption on force, velocity, stiffness and [Ca2+]i in porcine coronary arteries

2000 ◽  
Vol 279 (5) ◽  
pp. H2493-H2501 ◽  
Author(s):  
Richard J. Paul ◽  
Peggy Sue Bowman ◽  
Michael S. Kolodney
Keyword(s):  
Smooth Muscle ◽  
Coronary Arteries ◽  
Isometric Force ◽  
Shortening Velocity ◽  
Cultured Fibroblasts ◽  
Microtubule Disruption ◽  
Highly Sensitive ◽  
Artery Stiffness ◽  
Force Velocity ◽  
Microtubule Stabilizer

Force generated by smooth muscle cells is believed to result from the interaction of actin and myosin filaments and is regulated through phosphorylation of the myosin regulatory light chain (LC20). The role of other cytoskeleton filaments, such as microtubules and intermediate filaments, in determining the mechanical output of smooth muscle is unclear. In cultured fibroblasts, microtubule disruption results in large increases in force similar to contractions associated with LC20 phosphorylation (15). One hypothesis, the “tensegrity” or “push-pull” model, attributes this increase in force to the disruption of microtubules functioning as rigid struts to resist force generated by actin-myosin interaction (9). In porcine coronary arteries, the disruption of microtubules by nocodazole (11 μM) also elicited moderate but significant increases in isometric force (10–40% of a KCl contracture), which could be blocked or reversed by taxol (a microtubule stabilizer). We tested whether this nocodazole-induced force was accompanied by changes in coronary artery stiffness or unloaded shortening velocity, parameters likely to be highly sensitive to microtubule resistance elements. Few changes were seen, ruling out push-pull mechanisms for the increase in force by nocodazole. In contrast, the intracellular calcium concentration, measured by fura 2 in the intact artery, was increased by nocodazole in parallel with force, and this was inhibited and/or reversed by taxol. Our results indicate that microtubules do not significantly contribute to vascular smooth muscle mechanical characteristics but, importantly, may play a role in modulation of Ca2+ signal transduction.

Effect of airway inflammation on smooth muscle shortening and contractility in guinea pig trachealis

1993 ◽  
Vol 265 (6) ◽  
pp. L549-L554 ◽  
Author(s):  
R. W. Mitchell ◽  
I. M. Ndukwu ◽  
K. Arbetter ◽  
J. Solway ◽  
A. R. Leff
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Neurogenic Inflammation ◽  
Isometric Force ◽  
Electrical Field Stimulation ◽  
Shortening Velocity ◽  
Lever System ◽  
Force Velocity

We studied the effect of either 1) immunogenic inflammation caused by aerosolized ovalbumin or 2) neurogenic inflammation caused by aerosolized capsaicin in vivo on guinea pig tracheal smooth muscle (TSM) contractility in vitro. Force-velocity relationships were determined for nine epithelium-intact TSM strips from ovalbumin-sensitized (OAS) vs. seven sham-sensitized controls and TSM strips for seven animals treated with capsaicin aerosol (Cap-Aer) vs. eight sham controls. Muscle strips were tethered to an electromagnetic lever system, which allowed isotonic shortening when load clamps [from 0 to maximal isometric force (Po)] were applied at specific times after onset of contraction. Contractions were elicited by supramaximal electrical field stimulation (60 Hz, 10-s duration, 18 V). Optimal length for each muscle was determined during equilibration. Maximal shortening velocity (Vmax) was increased in TSM from OAS (1.72 +/- 0.46 mm/s) compared with sham-sensitized animals (0.90 +/- 0.15 mm/s, P < 0.05); Vmax for TSM from Cap-Aer (0.88 +/- 0.11 mm/s) was not different from control TSM (1.13 +/- 0.08 mm/s, P = NS). Similarly, maximal shortening (delta max) was augmented in TSM from OAS (1.01 +/- 0.15 mm) compared with sham-sensitized animals (0.72 +/- 0.14 mm, P < 0.05); delta max for TSM from Cap-Aer animals (0.65 +/- 0.11 mm) was not different from saline aerosol controls (0.71 +/- 0.15 mm, P = NS). We demonstrate Vmax and delta max are augmented in TSM after ovalbumin sensitization; in contrast, neurogenic inflammation caused by capsaicin has no effect on isolated TSM contractility in vitro. These data suggest that airway hyperresponsiveness in vivo that occurs in association with immunogenic or neurogenic inflammation may result from different effects of these types of inflammation on airway smooth muscle.

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