Activation of smooth muscle contraction: relation between myosin phosphorylation and stiffness

Science ◽  
1986 ◽  
Vol 232 (4746) ◽  
pp. 80-82 ◽  
Author(s):  
KE Kamm ◽  
JT Stull
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Myosin Head ◽  
Isometric Force ◽  
Smooth Muscle Contraction ◽  
Myosin Phosphorylation ◽  
First Order ◽  
Cooperative Process ◽  
Pseudo First Order

Contraction and myosin light-chain phosphorylation were measured in electrically stimulated tracheal smooth muscle. Latencies for the onset of force, stiffness, and light-chain phosphorylation were 500 milliseconds. Myosin light chain was phosphorylated from 0.04 to 0.80 mole of phosphate per mole of light chain with a pseudo-first-order rate of 1.1 per second with no evidence of an ordered or negatively cooperative process. Following the period of latency, stiffness increased with phosphorylation and both increased more rapidly than isometric force. The linear relation between stiffness and phosphorylation during activation suggests independent attachment of each myosin head upon phosphorylation.

scholarly journals Tyrosine Kinase Pyk2 is Involved in Colonic Smooth Muscle Contraction via the RhoA/ROCK Pathway

2018 ◽  
pp. 89-98 ◽  
Author(s):  
Ling Tong ◽  
Jun-Ping Ao ◽  
Hong-Li Lu ◽  
Xu Huang ◽  
Jing-Yu Zang ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Tyrosine Kinase ◽  
Muscle Contraction ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Kinase Inhibitor ◽  
Isometric Force ◽  
Smooth Muscles ◽  
Smooth Muscle Contraction ◽  
Tyrosine Kinase 2

The contraction of gastrointestinal (GI) smooth muscles is regulated by both Ca(2+)-dependent and Ca(2+) sensitization mechanisms. Proline-rich tyrosine kinase 2 (Pyk2) is involved in the depolarization-induced contraction of vascular smooth muscle via a Ca(2+) sensitization pathway. However, the role of Pyk2 in GI smooth muscle contraction is unclear. The spontaneous contraction of colonic smooth muscle was measured by using isometric force transducers. Protein and phosphorylation levels were determined by using western blotting. Pyk2 protein was expressed in colonic tissue, and spontaneous colonic contractions were inhibited by PF-431396, a Pyk2 inhibitor, in the presence of tetrodotoxin (TTX). In cultured colonic smooth muscle cells (CSMCs), PF-431396 decreased the levels of myosin light chain (MLC20) phosphorylated at Ser19 and ROCK2 protein expression, but myosin light chain kinase (MLCK) expression was not altered. However, Y-27632, a Rho kinase inhibitor, increased phosphorylation of Pyk2 at Tyr402 and concomitantly decreased ROCK2 levels; the expression of MLCK in CSMCs did not change. The expression of P(Tyr402)-Pyk2 and ROCK2 was increased when CSMCs were treated with Ach. Pyk2 is involved in the process of colonic smooth muscle contraction through the RhoA/ROCK pathway. These pathways may provide very important targets for investigating GI motility disorders.

scholarly journals Effect of lithium on smooth muscle contraction and phosphorylation of myosin light chain by MLCK

2010 ◽  
pp. 919-926
Author(s):  
ZY Tang ◽  
ZN Liu ◽  
L Fu ◽  
DP Chen ◽  
QD Ai ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Atpase Activity ◽  
Muscle Contraction ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Contractile Activity ◽  
Smooth Muscle Contraction ◽  
Time Dependent ◽  
Dependent Manner ◽  
Myosin Phosphorylation

The aims of our study were to investigate into the effect of lithium on smooth muscle contraction and phosphorylation of myosin light chain (MLC20) by MLCK and to find out the clue of its mechanism. Isolated rabbit duodenum smooth muscle strips were used to study the effects of lithium on their contractile activity under the condition of Krebs’ solution by means of HW400S constant temperature smooth muscle trough. Myosin and MLCK were purified from the chicken gizzard smooth muscle. Myosin phosphorylation was determined by Glycerol-PAGE, myosin Mg2+-ATPase activity was measured by Pi liberation method. Lithium (10-40 mM) inhibited the contraction in duodenum in a dose-related and time-dependent manner. Lithium could also inhibit the extent of myosin phosphorylation in a dose-related and time-dependent manner, whereas it inhibited Mg2+-ATPase activity in a dose-related manner. Lithium inhibited smooth muscle contraction by inhibition of myosin phosphorylation and Mg2+-ATPase activity.

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.

The biophysics and biochemistry of smooth muscle contraction

1992 ◽  
Vol 70 (4) ◽  
pp. 515-531 ◽  
Author(s):  
N. L. Stephens ◽  
C. Y. Seow ◽  
A. J. Halayko ◽  
H. Jiang
Keyword(s):  
Smooth Muscle ◽  
Muscle Contraction ◽  
Light Chain ◽  
Time Course ◽  
Myosin Light Chain ◽  
Isometric Force ◽  
Smooth Muscle Contraction ◽  
Cytoskeletal Proteins ◽  
Contractile Element ◽  
Force Velocity

In this review the biophysics and biochemistry of smooth muscle contraction are dealt with. We describe a new model for the study of bronchial smooth muscle, which facilitates study of cellular contractile mechanisms. A new concept emerging is that study of steady-state mechanical parameters such as maximal isometric force (Po) velocity is inadequate because two types of crossbridges (normally cycling (NBR) and latch) seem to be sequentially active during smooth muscle contraction. Thus quick-release techniques are required to characterize the force–velocity properties of the two types of bridges. Pathophysiological processes that affect the muscle's shortening ability seem to affect the early NBRs only. With respect to maximal shortening capacity of the smooth muscle, the role of loading is very important. The differences between isotonic, elastic, and viscous loading are considerable. Ultimately, the time course and magnitude of loading should exactly resemble that operative in vivo. Once again, it is the characteristic of loading in the early phase of contraction that is crucial, as most of the shortening in smooth muscle occurs early in the contraction. While the maximum force developed by smooth muscle per unit cross-sectional area is the same as for striated muscle, the velocity is 50 times less. The properties of the series and parallel elastic elements of smooth muscle are described. The latter, when in compression mode, acts as an internal resistance to shortening and probably limits it. Isotonic relaxation has therefore not been studied in smooth muscle. We have developed a shortening parameter that is independent of the load on the muscle and of the initial length of the muscle's contractile element. We report the novel observation that isotonically relaxing smooth muscle reactivates itself, resulting in terminal slowing of the relaxation process. With respect to the biochemistry of smooth muscle contraction, contractile (actin isoforms, myosin heavy and light chains and their isoforms), regulatory (calmodulin–4 Ca2+, myosin light chain kinase, myosin light chain and its phosphorylation, tropomyosin, caldesmon, and calponin), and cytoskeletal (chiefly desmin and vimentin) proteins are discussed. While the kinase activates the contractile system, caldesmon and calponin modulate the activity downward. The cytoskeletal proteins desmin, vimentin, and α-actinin could constitute the muscle cell's internal resistor.Key words: smooth muscle mechanics, force–velocity, smooth muscle, internal resistor, smooth muscle retardation, contractile proteins, regulatory proteins.

Calcium-dependent mechanisms of regulation of smooth muscle contraction

1991 ◽  
Vol 69 (12) ◽  
pp. 771-800 ◽  
Author(s):  
Michael P. Walsh
Keyword(s):  
Smooth Muscle ◽  
Muscle Contraction ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Binding Protein ◽  
Smooth Muscle Contraction ◽  
Light Chains ◽  
Contractile State ◽  
Calmodulin Binding

The contractile state of smooth muscle is regulated primarily by the sarcoplasmic (cytosolic) free Ca2+ concentration. A variety of stimuli that induce smooth muscle contraction (e.g., membrane depolarization, α-adrenergic and muscarinic agonists) trigger an increase in sarcoplasmic free [Ca2+] from resting levels of 120–270 to 500–700 nM. At the elevated [Ca2+], Ca2+ binds to calmodulin, the ubiquitous and multifunctional Ca2+-binding protein. The interaction of Ca2+ with CaM induces a conformational change in the Ca2+-binding protein with exposure of a site(s) of interaction with target proteins, the most important of which in the context of smooth muscle contraction is the enzyme myosin light chain kinase. The interaction of calmodulin with myosin light chain kinase results in activation of the kinase that catalyzes phosphorylation of myosin at serine-19 of each of the two 20-kDa light chains (native myosin is a hexamer composed of two heavy chains (230 kDa each) and two pairs of light chains (one pair of 20 kDa each and the other pair of 17 kDa each)). This simple phosphorylation reaction triggers cycling of myosin cross-bridges along actin filaments and the development of force. Relaxation of the muscle follows removal of Ca2+ from the sarcoplasm, whereupon calmodulin dissociates from myosin light chain kinase regenerating the inactive kinase; myosin is dephosphorylated by myosin light chain phosphatase(s), whereupon it dissociates and remains detached from the actin filament and the muscle relaxes. A substantial body of evidence has been accumulated in support of this central role of myosin phosphorylation–dephosphorylation in the regulation of smooth muscle contraction. However, a wide range of physiological and biochemical studies supports the existence of additional, secondary Ca2+-dependent mechanisms that can modulate or fine-tune the contractile state of the smooth muscle cell. Three such mechanisms have emerged: (i) the actin-, tropomyosin-, and calmodulin-binding protein, calponin; (ii) the actin-, myosin-, tropomyosin-, and calmodulin-binding protein, caldesmon; and (iii) the Ca2+- and phospholipid-dependent protein kinase (protein kinase C).Key words: smooth muscle, Ca2+, myosin phosphorylation, regulation of contraction.

scholarly journals Membrane depolarization-induced contraction of rat caudal arterial smooth muscle involves Rho-associated kinase

2002 ◽  
Vol 364 (2) ◽  
pp. 431-440 ◽  
Author(s):  
Mitsuo MITA ◽  
Hayato YANAGIHARA ◽  
Shigeru HISHINUMA ◽  
Masaki SAITO ◽  
Michael P. WALSH
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
State Level ◽  
Smooth Muscle Contraction ◽  
Membrane Depolarization ◽  
Arterial Smooth Muscle ◽  
Tonic Component ◽  
Phasic Component ◽  
Steady Level

Depolarization of the sarcolemma of smooth muscle cells activates voltage-gated Ca2+ channels, influx of Ca2+ and activation of cross-bridge cycling by phosphorylation of myosin catalysed by Ca2+/calmodulin-dependent myosin light-chain kinase (MLCK). Agonist stimulation of smooth muscle contraction often involves other kinases in addition to MLCK. In the present study, we address the hypothesis that membrane depolarization-induced contraction of rat caudal arterial smooth muscle may involve activation of Rho-associated kinase (ROK). Addition of 60mM K+ to de-endothelialized muscle strips in the presence of prazosin and propranolol induced a contraction that peaked rapidly and then declined to a steady level of force corresponding to approx. 30% of the peak contraction. This contractile response was abolished by the Ca2+-channel blocker nicardipine or the removal of extracellular Ca2+. An MLCK inhibitor (ML-9) inhibited both the phasic and tonic components of K+-induced contraction. On the other hand, the ROK inhibitors Y-27632 and HA-1077 abolished the tonic component of K+-induced contraction, and slightly reduced the phasic component. Phosphorylation levels of the 20-kDa light chain of myosin increased rapidly in response to 60mM K+ and subsequently declined to a steady-state level significantly greater than the resting level. Y-27632 abolished the sustained and reduced the phasic elevation of the phosphorylation of the 20-kDa light chain of myosin, without affecting the K+-induced elevation of cytosolic free Ca2+ concentration. These results indicate that ROK activation plays an important role in the sustained phase of K+-induced contraction of rat caudal arterial smooth muscle, but has little involvement in the phasic component of K+-induced contraction. Furthermore, these results are consistent with inhibition of myosin light-chain phosphatase by ROK, which would account for the sustained elevation of myosin phosphorylation and tension in response to membrane depolarization.

Communication between tyrosine kinase pathway and myosin light chain kinase pathway in smooth muscle

1996 ◽  
Vol 271 (4) ◽  
pp. H1348-H1355 ◽  
Author(s):  
N. Jin ◽  
R. A. Siddiqui ◽  
D. English ◽  
R. A. Rhoades
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Light Chain ◽  
Tyrosine Kinases ◽  
Myosin Light Chain ◽  
Smooth Muscle Contraction ◽  
Myosin Light Chain Phosphorylation ◽  
Kinase Pathway

Two separate signal transduction pathways exist in vascular smooth muscle: one for cell growth, proliferation, and differentiation and the other for contraction. Although activation of protein tyrosine kinases is intimately involved in the signaling pathway that induces cell growth, proliferation, and differentiation, activation of myosin light chain kinase (MLCK) is an important step in the pathway leading to smooth muscle contraction. Indirect evidence suggests that “cross talk” exists between these two signaling pathways, but the common intermediates are not well defined. The purpose of this study was to determine whether a vasoconstrictor and a mitogen initiate crossover signaling between the tyrosine kinase pathway and the MLCK pathway in vascular smooth muscle. Rat aorta and pulmonary arteries were isolated and stimulated with either fetal calf serum (FCS) or phenylephrine in the presence or absence of a tyrosine kinase inhibitor (genistein) or tyrosine phosphatase inhibitor [sodium o-vanadate (Na3 VO4)]. Isometric force was recorded as a function of time; myosin light chain phosphorylation, protein tyrosine phosphorylation, and mitogen-activated protein kinase (MAPK) mobility were determined by immunoblotting. The results demonstrate that FCS, which contains a variety of growth factors known to activate tyrosine kinases, induced myosin light chain phosphorylation and contraction in vascular smooth muscle. Phenylephrine, a vasoconstrictor known to activate MLCK, induced tyrosine phosphorylation of a 42-kDa protein identified as MAPK. Tyrosine phosphorylation of this protein was inhibited by genistein and enhanced by vanadate. Genistein significantly inhibited both serum- and phenylephrine-induced myosin light chain phosphorylation as well as the serum- and phenylephrine-induced force generation, whereas vanadate enhanced these responses. These data demonstrate interrelationship between activation of the tyrosine kinase pathway and the MLCK pathway in vascular smooth muscle. These interactions may influence smooth muscle contraction and be important in the regulation of smooth muscle cell proliferation.

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.

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