Differential signaling by muscarinic m3 and m2 receptors determines sustained myosin light chain (MLC) phosphorylation and smooth muscle contraction

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.

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Diabetes decreases rabbit bladder smooth muscle contraction while increasing levels of myosin light chain phosphorylation

AJP Renal Physiology ◽

10.1152/ajprenal.00027.2004 ◽

2004 ◽

Vol 287 (4) ◽

pp. F690-F699 ◽

Cited By ~ 23

Author(s):

Xiaoling Su ◽

Arun Changolkar ◽

Samuel Chacko ◽

Robert S. Moreland

Keyword(s):

Diabetes Mellitus ◽

Smooth Muscle ◽

Muscle Contraction ◽

Light Chain ◽

Myosin Light Chain ◽

Smooth Muscle Contraction ◽

Bladder Smooth Muscle ◽

Animal Groups ◽

Urinary Bladder Smooth Muscle ◽

Mlc Phosphorylation

The effect of diabetes mellitus on the regulation of urinary bladder smooth muscle contraction was studied. Diabetes was induced in the rabbit by alloxan injection followed by 16 wk of housing. The bladder was harvested and strips of wall devoid of both mucosa and serosa were examined. Intact strips of bladder smooth muscle from diabetic animals produced less stress in response to membrane depolarization than muscle from control animals; sensitivity to KCl was not changed. Carbachol responses were similar in muscle strips from the two animal groups. Basal myosin light chain (MLC) phosphorylation levels were significantly elevated in response to most stimuli in muscle strips from diabetic animals, although levels of stress were either unchanged or lower. α-Toxin-permeabilized strips that allow for control of the intracellular environment while maintaining excitation-contraction coupling showed increased levels of MLC phosphorylation but decreased sensitivity to activator Ca2+ in smooth muscle from diabetic animals. MLC phosphatase contents were similar in smooth muscle from the two animal groups; however, MLC phosphatase activity was greater in muscle from control compared with diabetic animals. These results suggest that diabetes mellitus uncouples basal MLC phosphorylation from force in the bladder smooth muscle cell.

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Calcium-dependent mechanisms of regulation of smooth muscle contraction

Biochemistry and Cell Biology ◽

10.1139/o91-119 ◽

1991 ◽

Vol 69 (12) ◽

pp. 771-800 ◽

Cited By ~ 104

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.

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Role of non-kinase activity of myosin light-chain kinase in regulating smooth muscle contraction, a review dedicated to Dr. Setsuro Ebashi

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2007.11.096 ◽

2008 ◽

Vol 369 (1) ◽

pp. 135-143 ◽

Cited By ~ 16

Author(s):

Akio Nakamura ◽

Ce Xie ◽

Yue Zhang ◽

Ying Gao ◽

Hong-Hui Wang ◽

...

Keyword(s):

Smooth Muscle ◽

Muscle Contraction ◽

Light Chain ◽

Myosin Light Chain Kinase ◽

Myosin Light Chain ◽

Kinase Activity ◽

Smooth Muscle Contraction

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

AJP Renal Physiology ◽

10.1152/ajprenal.00011.2010 ◽

2011 ◽

Vol 300 (1) ◽

pp. F73-F85 ◽

Cited By ~ 21

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.

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Blebbistatin, a myosin II inhibitor, suppresses contraction and disrupts contractile filaments organization of skinned taenia cecum from guinea pig

AJP Cell Physiology ◽

10.1152/ajpcell.00269.2009 ◽

2010 ◽

Vol 298 (5) ◽

pp. C1118-C1126 ◽

Cited By ~ 15

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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