scholarly journals Inhibition of smooth-muscle myosin-light-chain phosphatase by Ruthenium Red

2000 ◽  
Vol 349 (3) ◽  
pp. 797-804 ◽  
Author(s):  
Aki YAMADA ◽  
Osamu SATO ◽  
Minoru WATANABE ◽  
Michael P. WALSH ◽  
Yasuo OGAWA ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Smooth Muscles ◽  
Direct Interaction ◽  
Smooth Muscle Contraction ◽  
Ruthenium Red ◽  
Dependent Manner ◽  
Myosin Light Chain Phosphatase ◽  
Concentration Dependent Manner

Ruthenium Red (RuR) is widely used as an inhibitor of ryanodine receptor Ca2+ release channels, but has additional effects such as the induction of Ca2+ sensitization of contraction of permeabilized smooth muscles. To address the mechanism underlying this process, we examined the effects of RuR on contractility in permeabilized guinea-pig ileum and on the activity of myosin-light-chain phosphatase (MP). RuR increased the force at submaximal [Ca2+] (pCa 6.3) approx. 4-fold. This effect was not observed after thiophosphorylation of MP. RuR also seemed capable of preventing the thiophosphorylation of MP, suggesting a direct interaction of RuR with MP. Consistent with this possibility, smooth-muscle MP was inhibited by RuR in a concentration-dependent manner (IC50 23µM). Exogenous calmodulin significantly increased RuR-induced contraction at pCa 6.3 but had little effect on contraction induced by microcystin at this [Ca2+]. Ca2+-independent contraction was induced by RuR (EC50 843µM) and by microcystin (EC50 59nM) but the maximal force induced by RuR was smaller than that induced by microcystin. The addition of 300µM RuR enhanced the contraction induced by 30nM microcystin but markedly decreased that induced by 1µM microcystin. Such a dual action of RuR on microcystin-induced effects was not observed in experiments using purified MP. We conclude that the RuR-induced Ca2+ sensitization of smooth-muscle contraction is due to the direct inhibition of MP by RuR.

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 The latch-bridge hypothesis of smooth muscle contraction

2005 ◽  
Vol 83 (10) ◽  
pp. 857-864 ◽  
Author(s):  
Richard A Murphy ◽  
Christopher M Rembold
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Striated Muscle ◽  
Smooth Muscle Contraction ◽  
Myosin Light Chain Phosphatase ◽  
Shortening Velocity ◽  
General Applicability ◽  
Cross Bridge ◽  
Mechanical Transduction

In contrast to striated muscle, both normalized force and shortening velocities are regulated functions of cross-bridge phosphorylation in smooth muscle. Physiologically this is manifested as relatively fast rates of contraction associated with transiently high levels of cross-bridge phosphorylation. In sustained contractions, Ca2+, cross-bridge phosphorylation, and ATP consumption rates fall, a phenomenon termed "latch". This review focuses on the Hai and Murphy (1988a) model that predicted the highly non-linear dependence of force on phosphorylation and a directly proportional dependence of shortening velocity on phosphorylation. This model hypothesized that (i) cross-bridge phosphorylation was obligatory for cross-bridge attachment, but also that (ii) dephosphorylation of an attached cross-bridge reduced its detachment rate. The resulting variety of cross-bridge cycles as predicted by the model could explain the observed dependencies of force and velocity on cross-bridge phosphorylation. New evidence supports modifications for more general applicability. First, myosin light chain phosphatase activity is regulated. Activation of myosin phosphatase is best demonstrated with inhibitory regulatory mechanisms acting via nitric oxide. The second modification of the model incorporates cooperativity in cross-bridge attachment to predict improved data on the dependence of force on phosphorylation. The molecular basis for cooperativity is unknown, but may involve thin filament proteins absent in striated muscle.Key words: chemo-mechanical transduction, activation-contraction coupling, cross-bridge, myosin light chain kinase, myosin light chain phosphatase, phosphorylation, cooperativity.

scholarly journals Phosphorylation of the myosin phosphatase inhibitors, CPI-17 and PHI-1, by integrin-linked kinase

2002 ◽  
Vol 367 (2) ◽  
pp. 517-524 ◽  
Author(s):  
Jing Ti DENG ◽  
Cindy SUTHERLAND ◽  
David L. BRAUTIGAN ◽  
Masumi ETO ◽  
Michael P. WALSH
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Smooth Muscle Contraction ◽  
Kinase Assay ◽  
Myosin Light Chain Phosphatase ◽  
Phosphatase Inhibitors ◽  
Phosphatase Inhibitor ◽  
Integrin Linked Kinase ◽  
Phosphopeptide Mapping

Integrin-linked kinase (ILK) has been implicated in Ca2+- independent contraction of smooth muscle via its ability to phosphorylate myosin. We investigated the possibility that this kinase might also phosphorylate and regulate the myosin light-chain phosphatase inhibitor proteins CPI-17 [protein kinase C (PKC)-dependent phosphatase inhibitor of 17kDa] and PHI-1 (phosphatase holoenzyme inhibitor-1), known substrates of PKC. Both phosphatase inhibitors were phosphorylated by ILK in an in-gel kinase assay and in solution. A Thr→Ala mutation at Thr38 of CPI-17 and Thr57 of PHI-1 eliminated phosphorylation by ILK. Phosphopeptide mapping, phospho amino acid analysis and immunoblotting using phospho-specific antibodies indicated that ILK predominantly phosphorylated the site critical for potent inhibition, i.e. Thr38 of CPI-17 or Thr57 of PHI-1. CPI-17 and PHI-1 thiophosphorylated by ILK at Thr38 or Thr57 respectively inhibited myosin light-chain phosphatase (MLCP) activity bound to myosin, whereas the site-specific mutants CPI-17-Thr38Ala and PHI-1-Thr57Ala, treated with ILK under identical conditions, like the untreated wild-type proteins had no effect on the phosphatase. Consistent with these effects, both thiophospho-CPI-17 and -PHI-1 induced Ca2+ sensitization of contraction of Triton X-100-demembranated rat-tail arterial smooth muscle, whereas CPI-17-Thr38Ala and PHI-1-Thr57Ala treated with ILK in the presence of adenosine 5′-[γ-thio]triphosphate failed to evoke a contractile response. We conclude that ILK may activate smooth-muscle contraction both directly, via phosphorylation of myosin, and indirectly, via phosphorylation and activation of CPI-17 and PHI-1, leading to inhibition of MLCP.

Polylysine activates smooth muscle myosin ATPase activity via induction of a 10S to 6S transition

1993 ◽  
Vol 265 (2) ◽  
pp. C379-C386 ◽  
Author(s):  
P. T. Szymanski ◽  
D. G. Ferguson ◽  
R. J. Paul
Keyword(s):  
Smooth Muscle ◽  
Atpase Activity ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Myosin Atpase ◽  
Solution Viscosity ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Myosin Atpase Activity ◽  
Muscle Myosin

Polylysine (10-13 kDa) stimulates contraction in smooth muscle skinned fibers and activates actomyosin adenosinetriphosphatase (ATPase) activity in the absence of myosin light chain phosphorylation [P. T. Szymanski and R. J. Paul. Adv. Exp. Med. 304: 363-368, 1991; P. T. Szymanski, J. D. Strauss, G. Doerman, J. DiSalvo, and R. J. Paul. Am J. Physiol. 262 (Cell Physiol. 31): C1445-C1455, 1992]. To provide further information on the mechanism of polylysine action on contractility in smooth muscle, we investigated its effect on ATPase activity and conformation of purified gizzard myosin. We report here that polylysine directly stimulates myosin ATPase activity in a concentration-dependent manner. This stimulation could be completely abolished with the addition of heparin, a negatively charged heteropolysaccharide. Polylysine (10 microM) increases myosin ATPase activity to a level similar to that of myosin phosphorylation. Addition of 10 microM polylysine to phosphorylated myosin [with myosin light chain kinase and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), to approximately 1.9 mol P/mol myosin], however, did not further stimulate ATPase activity. At 0.2 M KCl (the salt concentration at which myosin exists primary in the 10S form), the addition of polylysine increases myosin ATPase activity to a level comparable to that of untreated myosin in 0.3 M KCl. These changes parallel the increase in solution viscosity elicited by polylysine. These results suggest that polylysine induces a transition in myosin conformation from the 10S to the 6S form, and this was confirmed by electron microscopy.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Analysis of phosphorylation of the myosin-targeting subunit of myosin light chain phosphatase by Phos-tag SDS-PAGE

2016 ◽  
Vol 310 (8) ◽  
pp. C681-C691 ◽  
Author(s):  
Cindy Sutherland ◽  
Justin A. MacDonald ◽  
Michael P. Walsh
Keyword(s):  
Smooth Muscle ◽  
Muscle Contraction ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Coiled Coil ◽  
Smooth Muscle Contraction ◽  
Myosin Light Chain Phosphatase ◽  
Tissue Samples ◽  
Phosphatase Inhibitor ◽  
Sds Page

Phosphorylation of the myosin-targeting subunit 1 of myosin light chain phosphatase (MYPT1) plays an important role in the regulation of smooth muscle contraction, and several sites of phosphorylation by different protein Ser/Thr kinases have been identified. Furthermore, in some instances, phosphorylation at specific sites affects phosphorylation at neighboring sites, with functional consequences. Characterization of the complex phosphorylation of MYPT1 in tissue samples at rest and in response to contractile and relaxant stimuli is, therefore, challenging. We have exploited Phos-tag SDS-PAGE in combination with Western blotting using antibodies to MYPT1, including phosphospecific antibodies, to separate multiple phosphorylated MYPT1 species and quantify MYPT1 phosphorylation stoichiometry using purified, full-length recombinant MYPT1 phosphorylated by Rho-associated coiled-coil kinase (ROCK) and cAMP-dependent protein kinase (PKA). This approach confirmed that phosphorylation of MYPT1 by ROCK occurs at Thr697 and Thr855, PKA phosphorylates these two sites and the neighboring Ser696 and Ser854, and prior phosphorylation at Thr697 and Thr855 by ROCK precludes phosphorylation at Ser696 and Ser854 by PKA. Furthermore, phosphorylation at Thr697 and Thr855 by ROCK exposes two other sites of phosphorylation by PKA. Treatment of Triton-skinned rat caudal arterial smooth muscle strips with the membrane-impermeant phosphatase inhibitor microcystin or treatment of intact tissue with the membrane-permeant phosphatase inhibitor calyculin A induced slow, sustained contractions that correlated with phosphorylation of MYPT1 at 7 to ≥10 sites. Phos-tag SDS-PAGE thus provides a suitable and convenient method for analysis of the complex, multisite MYPT1 phosphorylation events involved in the regulation of myosin light chain phosphatase activity and smooth muscle contraction.

Receptors and signaling pathway underlying relaxations to isoprostanes in canine and porcine airway smooth muscle

2002 ◽  
Vol 283 (5) ◽  
pp. L1151-L1159 ◽  
Author(s):  
Adriana Catalli ◽  
Dawei Zhang ◽  
Luke J. Janssen
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Phosphodiesterase Inhibitor ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Type Iv ◽  
F Ring

Using muscle bath techniques, we examined the inhibitory activities of several E- and F-ring isoprostanes in canine and porcine airway smooth muscle. 8-Isoprostaglandin E1 and 8-isoprostaglandin E2 (8-iso PGE2) reversed cholinergic tone in a concentration-dependent manner, whereas the F-ring isoprostanes were ineffective. Desensitization with 8-iso-PGE2 and PGE2 implicated isoprostane activity at the PGE2 receptor (EP). We found that the inhibitory E-ring isoprostane responses were significantly augmented by rolipram (a type IV phosphodiesterase inhibitor), while 1 H-[1,2,4]-oxadiazolo[4,3- a]quinoxalin-1-one (a guanylate cyclase inhibitor) had no effect, suggesting a role for cAMP in isoprostane-mediated relaxations. 8-Iso-PGE2 did not reverse KCl tone, suggesting that voltage-dependent Ca2+ influx and myosin light chain kinase are not suppressed by isoprostanes. Patch-clamp studies showed marked suppression of K+ currents by 8-iso-PGE2. We conclude that E-ring isoprostanes exert PGE2receptor-directed, cAMP-dependent relaxations in canine and porcine airway smooth muscle. This activity is not dependent on K+channel activation or the direct inhibition of voltage-operated Ca2+ influx or myosin light chain kinase.

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.

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