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.

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.

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.

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.

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.

Regulation of vascular smooth muscle contraction: myosin light chain phosphorylation dependent and independent pathways

1994 ◽  
Vol 72 (11) ◽  
pp. 1386-1391 ◽  
Author(s):  
Yawen Zhang ◽  
Suzanne Moreland ◽  
Robert S. Moreland
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Muscle Contraction ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Smooth Muscle Contraction ◽  
Mitogen Activated Protein ◽  
Vascular Smooth Muscle Contraction ◽  
Triton X ◽  
Mlc Phosphorylation

Ca2+-dependent myosin light chain (MLC) phosphorylation is an important step in the initiation of smooth muscle contraction. However, MLC phosphorylation alone cannot account for all aspects of contractile regulation, suggesting the involvement of other elements. In this article we present evidence obtained from Triton X-100 detergent skinned and intact tissue which demonstrates that vascular smooth muscle contraction can be initiated by a Ca2+-dependent mechanism that does not require prior MLC phosphorylation. We show that Ca2+ can initiate contractions supported by cytidine triphosphate (CTP) and that these contractions are inhibited by calmodulin antagonists, suggesting a Ca2+–calmodulin dependence of force distinct from that for MLC phosphorylation. Evidence is presented to demonstrate that carotid medial fibers contain a mitogen-activated protein (MAP) kinase which is activated by Ca2+ and may catalyze caldesmon phosphorylation. Based in part on our results and those of other investigators, we propose that direct Ca2+–calmodulin binding to caldesmon or phosphorylation of caldesmon by a Ca2+-dependent MAP kinase disinhibits caldesmon. Disinhibition of caldesmon allows an inherent basal level of actin-activated myosin ATPase activity to be expressed. The result is the slow development of force.Key words: mitogen-activated protein kinase, caldesmon, Triton X-100, detergent-skinned fibers, cytidine triphosphate, calmodulin.

scholarly journals Rho-associated kinase plays a role in rabbit urethral smooth muscle contraction, but not via enhanced myosin light chain phosphorylation

2011 ◽  
Vol 300 (1) ◽  
pp. F73-F85 ◽  
Author(s):  
Michael P. Walsh ◽  
Keith Thornbury ◽  
William C. Cole ◽  
Gerard Sergeant ◽  
Mark Hollywood ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Muscle Contraction ◽  
Light Chain ◽  
Actin Polymerization ◽  
Myosin Light Chain ◽  
Smooth Muscle Contraction ◽  
Regulatory Light Chains ◽  
Kinase C ◽  
Protein Kinase C Inhibitor ◽  
Urethral Smooth Muscle

The involvement of Rho-associated kinase (ROK) in activation of rabbit urethral smooth muscle contraction was investigated by examining the effects of two structurally distinct inhibitors of ROK, Y27632 and H1152, on the contractile response to electric field stimulation, membrane depolarization with KCl, and α1-adrenoceptor stimulation with phenylephrine. Both compounds inhibited contractions elicited by all three stimuli. The protein kinase C inhibitor GF109203X, on the other hand, had no effect. Urethral smooth muscle strips were analyzed for phosphorylation of three potential direct or indirect substrates of ROK: 1) myosin regulatory light chains (LC20) at S19, 2) the myosin-targeting subunit of myosin light chain phosphatase (MYPT1) at T697 and T855, and 3) cofilin at S3. The following results were obtained: 1) under resting tension, LC20 was phosphorylated to 0.65 ± 0.02 mol Pi/mol LC20 ( n = 21) at S19; 2) LC20 phosphorylation did not change in response to KCl or phenylephrine; 3) ROK inhibition had no effect on LC20 phosphorylation in the absence or presence of contractile stimuli; 4) under resting conditions, MYPT1 was partially phosphorylated at T697 and T855 and cofilin at S3; 5) phosphorylation of MYPT1 and cofilin was unaffected by KCl or phenylephrine; and 6) KCl- and phenylephrine-induced contraction-relaxation cycles did not correlate with actin polymerization-depolymerization. We conclude that ROK plays an important role in urethral smooth muscle contraction, but not via inhibition of MLCP or polymerization of actin.

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.

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