Smooth muscle phosphatases: structure, regulation, and function

1994 ◽  
Vol 72 (11) ◽  
pp. 1427-1433 ◽  
Author(s):  
M. D. Pato ◽  
A. G. Tulloch ◽  
M. P. Walsh ◽  
E. Kerc
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Protein Phosphatases ◽  
Smooth Muscles ◽  
Light Chains ◽  
Myosin Light Chains ◽  
Heavy Meromyosin ◽  
Smooth Muscle Contractility

Smooth muscle contraction is regulated primarily by the reversible phosphorylation of myosin by myosin light chain kinase. Secondary mechanisms that might modulate contractility are phosphorylation–dephosphorylation of myosin light chain kinase and thin-filament proteins, caldesmon and calponin. Purification of several protein phosphatases that are active toward myosin light chains and (or) myosin and heavy meromyosin from smooth muscles has been reported. All the cytosolic turkey gizzard smooth muscle phosphatases, termed SMP-I, -II, -III, and -IV, dephosphorylate myosin light chains rapidly, but only SMP-III and -IV are active toward myosin and heavy meromyosin, suggesting that SMP-III and -IV might be directly involved in the relaxation of smooth muscle. SMP-III and -IV exhibit properties typical of type 1 protein phosphatases following tryptic digestion. These enzymes appear to share structural similarity with myofibrillar phosphatase PP1M. Purified calponin phosphatase and caldesmon phosphatase from chicken gizzards are structurally and immunologically identical with SMP-I, a type 2A protein phosphatase. SMP-I dephosphorylates calponin faster than it does caldesmon, and has much higher activity toward these substrates than SMP-II, -III, and -IV. Thus, one role for SMP-I might be to regulate the activities of caldesmon and calponin. Since SMP-I is active toward myosin light chain kinase, it might also modulate this enzyme.Key words: smooth muscle, contractility, protein phosphatases, smooth muscle phosphatases.

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 Embryonic chicken skeletal, cardiac, and smooth muscles express a common embryo-specific myosin light chain.

1985 ◽  
Vol 100 (6) ◽  
pp. 2025-2030 ◽  
Author(s):  
H Takano-Ohmuro ◽  
T Obinata ◽  
M Kawashima ◽  
T Masaki ◽  
T Tanaka
Keyword(s):  
Light Chain ◽  
Gel Electrophoresis ◽  
Myosin Light Chain ◽  
Smooth Muscles ◽  
Light Chains ◽  
Myosin Light Chains ◽  
Striated Muscles ◽  
Two Dimensional Gel Electrophoresis ◽  
Embryonic Chicken ◽  
Muscle Myosin

It has been demonstrated that embryonic chicken gizzard smooth muscle contains a unique embryonic myosin light chain of 23,000 mol wt, called L23 (Katoh, N., and S. Kubo, 1978, Biochem. Biophys. Acta, 535:401-411; Takano-Ohmuro, H., T. Obinata, T. Mikawa, and T. Masaki, 1983, J. Biochem. (Tokyo), 93:903-908). When we examined myosins in developing chicken ventricular and pectoralis muscles by two-dimensional gel electrophoresis, the myosin light chain (Le) that completely comigrates with L23 was detected in both striated muscles at early developmental stages. Two monoclonal antibodies, MT-53f and MT-185d, were applied to characterize the embryonic light chain Le of striated muscles. Both monoclonal antibodies were raised to fast skeletal muscle myosin light chains; the former antibody is specific to fast muscle myosin light chains 1 and 3, whereas the latter recognizes not only fast muscle myosin light chains but also the embryonic smooth muscle light chain L23. The immunoblots combined with both one- and two-dimensional gel electrophoresis showed that Le reacts with MT-185d but not with MT-53f. These results strongly indicate that Le is identical to L23 and that embryonic chicken skeletal, cardiac, and smooth muscles express a common embryo-specific myosin light chain.

scholarly journals Myosin light chain kinase-regulated endothelial cell contraction: the relationship between isometric tension, actin polymerization, and myosin phosphorylation.

1995 ◽  
Vol 130 (3) ◽  
pp. 613-627 ◽  
Author(s):  
Z M Goeckeler ◽  
R B Wysolmerski
Keyword(s):  
Endothelial Cell ◽  
Light Chain ◽  
Isometric Contraction ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Myosin Ii ◽  
Isometric Tension ◽  
Stress Fibers ◽  
Light Chains ◽  
Myosin Light Chains

The phosphorylation of regulatory myosin light chains by the Ca2+/calmodulin-dependent enzyme myosin light chain kinase (MLCK) has been shown to be essential and sufficient for initiation of endothelial cell retraction in saponin permeabilized monolayers (Wysolmerski, R. B. and D. Lagunoff. 1990. Proc. Natl. Acad. Sci. USA. 87:16-20). We now report the effects of thrombin stimulation on human umbilical vein endothelial cell (HUVE) actin, myosin II and the functional correlate of the activated actomyosin based contractile system, isometric tension development. Using a newly designed isometric tension apparatus, we recorded quantitative changes in isometric tension from paired monolayers. Thrombin stimulation results in a rapid sustained isometric contraction that increases 2- to 2.5-fold within 5 min and remains elevated for at least 60 min. The phosphorylatable myosin light chains from HUVE were found to exist as two isoforms, differing in their molecular weights and isoelectric points. Resting isometric tension is associated with a basal phosphorylation of 0.54 mol PO4/mol myosin light chain. After thrombin treatment, phosphorylation rapidly increases to 1.61 mol PO4/mol myosin light chain within 60 s and remains elevated for the duration of the experiment. Myosin light chain phosphorylation precedes the development of isometric tension and maximal phosphorylation is maintained during the sustained phase of isometric contraction. Tryptic phosphopeptide maps from both control and thrombin-stimulated cultures resolve both monophosphorylated Ser-19 and diphosphorylated Ser-19/Thr-18 peptides indicative of MLCK activation. Changes in the polymerization of actin and association of myosin II correlate temporally with the phosphorylation of myosin II and development of isometric tension. Activation results in a 57% increase in F-actin content within 90 s and 90% of the soluble myosin II associates with the reorganizing F-actin. Furthermore, the disposition of actin and myosin II undergoes striking reorganization. F-actin initially forms a fine network of filaments that fills the cytoplasm and then reorganizes into prominent stress fibers. Myosin II rapidly forms discrete aggregates associated with the actin network and by 2.5 min assumes a distinct periodic distribution along the stress fibers.

Effect of muscle length on isometric stress and myosin light chain phosphorylation in gallbladder smooth muscle

1991 ◽  
Vol 260 (6) ◽  
pp. G920-G924 ◽  
Author(s):  
R. J. Washabau ◽  
M. B. Wang ◽  
C. L. Dorst ◽  
J. P. Ryan
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Muscle Length ◽  
Stress Sensitivity ◽  
Light Chains ◽  
Myosin Light Chains ◽  
Myosin Light Chain Phosphorylation ◽  
Myofilament Activation

These experiments were designed to characterize the effect of muscle length on isometric stress, sensitivity to stimulation, and phosphorylation of the 20,000-Da myosin light chains in guinea pig gallbladder smooth muscle. Basal, active, and total isometric stress were determined in acetylcholine- or K(+)-treated (10(-4) M ACh, 80 mM KCl) muscle strips at 0.6-1.3 times the optimal muscle length (Lo) for isometric stress development. The effect of muscle length on the sensitivity to ACh and K+ was determined in cumulative dose-response experiments (10(-8) to 10(-4) M ACh, 10-80 mM KCl) at 0.7, 1.0, and 1.3 Lo. The effect of muscle length on myosin light chain phosphorylation was determined in ACh- or K(+)-treated (10(-4) M ACh, 80 mM KCl) muscle strips at 0.7, 1.0, and 1.3 Lo. In gallbladder smooth muscle, 1) active isometric stresses at 0.7 and 1.3 Lo were less than active isometric stress at 1.0 Lo; 2) the sensitivity of developed stress was similar at 1.0 and 1.3 Lo but decreased at 0.7 Lo; 3) the decline in isometric stress and sensitivity at 0.7 Lo was associated with reduced levels of phosphorylated myosin light chain; and 4) the decline in isometric stress at 1.3 Lo was not associated with reduced amounts of phosphorylated myosin light chain. These results suggest that the decline in active stress and sensitivity at short muscle lengths (L less than Lo) in gallbladder smooth muscle is due, at least in part, to decreases in the activation of the myofilaments. The decline in active isometric stress at long muscle lengths (L greater than Lo) is not due to changes in myofilament activation.

Cooperative activation of myosin by light chain phosphorylation in permeabilized smooth muscle

1992 ◽  
Vol 263 (1) ◽  
pp. C210-C219 ◽  
Author(s):  
T. B. Vyas ◽  
S. U. Mooers ◽  
S. R. Narayan ◽  
J. C. Witherell ◽  
M. J. Siegman ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Quantitative Relationship ◽  
Small Degree ◽  
Light Chains ◽  
Myosin Light Chains ◽  
Myosin Light Chain Phosphorylation ◽  
Maximum Increase ◽  
Rabbit Portal Vein

The purpose of this study was to determine the quantitative relationship between the number of myosin molecules that increase their ATPase activity and the degree of myosin light chain phosphorylation in smooth muscle. Single turnover experiments on the nucleotide bound to myosin were performed in the permeabilized rabbit portal vein. In the resting muscle, the rate of exchange of bound nucleoside diphosphate was biphasic and complete in approximately 30 min. When approximately 80% of the myosin light chain was thiophosphorylated, the nucleoside diphosphate exchange occurred at a much faster rate and was almost complete in 2 min. Thiophosphorylation of 10% of the myosin light chains caused an increase in the rate of ADP exchange from much more than 10% of the myosin subfragment-1. Less than 20% thiophosphorylation of the total myosin light chains resulted in the maximum increase in ADP exchanged in 2 min. It appears that a small degree of myosin light chain phosphorylation cooperatively turns on the maximum number of myosin molecules. Interestingly, even though less than 20% thiophosphorylation of the myosin light chain caused the maximum exchange of ADP within 2 min, higher degrees of thiophosphorylation were associated with further increases in the ATPase rates. We conclude that a small degree of myosin light chain thiophosphorylation cooperatively activates the maximum number of myosin molecules, and a higher degree of thiophosphorylation makes the myosin cycle faster. A kinetic model is proposed in which the rate constant for attachment of unphosphorylated cross bridges varies as a function of myosin light chain phosphorylation.

Ca2+ sensitization of smooth muscle contractility induced by ruthenium red

1999 ◽  
Vol 276 (3) ◽  
pp. C566-C575 ◽  
Author(s):  
Aki Yamada ◽  
Susumu Ohya ◽  
Masaru Hirano ◽  
Minoru Watanabe ◽  
Michael P. Walsh ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Smooth Muscles ◽  
Ruthenium Red ◽  
Skinned Fibers ◽  
Dependent Manner ◽  
Smooth Muscle Contractility ◽  
Concentration Dependent Manner

The effects of ruthenium red (RuR) on contractility were examined in skinned fibers of guinea pig smooth muscles, where sarcoplasmic reticulum function was destroyed by treatment with A-23187. Contractions of skinned fibers of the urinary bladder were enhanced by RuR in a concentration-dependent manner (EC50 = 60 μM at pCa 6.0). The magnitude of contraction at pCa 6.0 was increased to 320% of control by 100 μM RuR. Qualitatively, the same results were obtained in skinned fibers prepared from the ileal longitudinal smooth muscle layer and mesenteric artery. The maximal contraction induced by pCa 4.5 was not affected significantly by RuR. The enhanced contraction by RuR was not reversed by the addition of guanosine 5′- O-(2-thiodiphosphate) or a peptide inhibitor of protein kinase C [PKC-(19—31)]. The application of microcystin, a potent protein phosphatase inhibitor, induced a tonic contraction of skinned smooth muscle at low Ca2+ concentration ([Ca2+]; pCa > 8.0). RuR had a dual effect on the microcystin-induced contraction-to- enhancement ratio at low concentrations and suppression at high concentrations. The relaxation following the decrease in [Ca2+] from pCa 5.0 to >8.0 was significantly slowed down by an addition of RuR. Phosphorylation of the myosin light chain at pCa 6.3 was significantly increased by RuR in skinned fibers of the guinea pig ileum. These results indicate that RuR markedly increases the Ca2+ sensitivity of the contractile system, at least in part via inhibition of myosin light chain phosphatase.

scholarly journals Search of the binding site of the calix[4]arene with the myosin light-chain kinase by molecular dynamics method

2019 ◽  
Vol 78 (2) ◽  
pp. 57-61
Author(s):  
A. Zaremba ◽  
M. Platonov
Keyword(s):  
Molecular Dynamics ◽  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Catalytic Domain ◽  
Smooth Muscles ◽  
Molecular Dynamics Method ◽  
Intercellular Interaction ◽  
Dynamics Method

Disruptions of the functional activity of human smooth muscle are associated with a significant number of pathological conditions of the human body. The myosin light-chain kinase is the key enzyme of the signaling cascade of neurohumoral signals in smooth muscle cells. Especially it is important in the long-term tonic contraction. Disruption of its kinase activity can lead to a weakening of the intercellular interaction of the epithelial and endothelial cells, disruption of functioning of the intestinal smooth muscles and vessels, complication of labor activity. At the moment the search for effectors of this enzyme is being carried out. The problem is that most drugs are removed at the general body level due to toxic effects on other tissues (organs) or adverse chemical and physical properties. Such substances require adapters (carriers) devoid of these defects and inert in vivo. The most promising are calixarenes. In this study, the molecular dynamics method was used to determine the stability of the calix [4] arenetetrazulphate complex and the myosin light-chain kinase catalytic domain. Initially, by means of docking, the most favorable position of calixaren was determined; it turned out to be a catalytic kinase pocket. After that, the molecular-dynamic experiment was conducted to determine the energy of interaction. It turned out that the total energy of the interaction is about -300 cJ/mol. This indicates the high stability of the complex. Due the location of the ligand, its effect on the enzymatic activity of the kinase can be assumed, therefore, the use of this calixarene as a drug delivery system seems inappropriate.

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