Phosphorylation of smooth muscle regulatory myosin light chain (RLC) increases the mobility of myosin in native pyrophosphate acrylamide gel electrophoresis (PPi PAGE)

We tested the hypothesis that increases in force at a given cytosolic Ca2+ concentration (i.e., Ca2+ sensitization) produced by muscarinic stimulation of canine tracheal smooth muscle (CTSM) are produced in part by mechanisms independent of changes in regulatory myosin light chain (rMLC) phosphorylation. This was accomplished by comparing the relationship between rMLC phosphorylation and force in α-toxin-permeabilized CTSM in the absence and presence of acetylcholine (ACh). Forces were normalized to the contraction induced by 10 μM Ca2+ in each strip, and rMLC phosphorylation is expressed as a percentage of total rMLC. ACh (100 μM) plus GTP (1 μM) significantly shifted the Ca2+-force relationship curve to the left (EC50: 0.39 ± 0.06 to 0.078 ± 0.006 μM Ca2+) and significantly increased the maximum force (104.4 ± 4.8 to 120.2 ± 2.8%; n = 6 observations). The Ca2+-rMLC phosphorylation relationship curve was also shifted to the left (EC50: 1.26 ± 0.57 to 0.13 ± 0.04 μM Ca2+) and upward (maximum rMLC phosphorylation: 70.9 ± 7.9 to 88.5 ± 5.1%; n = 6 observations). The relationships between rMLC phosphorylation and force constructed from mean values at corresponding Ca2+concentrations were not different in the presence and absence of ACh. We find no evidence that muscarinic stimulation increases Ca2+ sensitivity in CTSM by mechanisms other than increases in rMLC phosphorylation.

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Effect of phorbol esters on Ca2+ sensitivity and myosin light-chain phosphorylation in airway smooth muscle

AJP Cell Physiology ◽

10.1152/ajpcell.1998.274.5.c1253 ◽

1998 ◽

Vol 274 (5) ◽

pp. C1253-C1260 ◽

Cited By ~ 34

Author(s):

Dorothee H. Bremerich ◽

Tetsuya Kai ◽

David O. Warner ◽

Keith A. Jones

Keyword(s):

Smooth Muscle ◽

Airway Smooth Muscle ◽

Light Chain ◽

Myosin Light Chain ◽

Phorbol Esters ◽

Isometric Force ◽

Calphostin C ◽

Regulatory Myosin Light Chain ◽

Kinase C ◽

The Relationship

We studied in β-escin-permeabilized canine tracheal smooth muscle (CTSM) the effect of the protein kinase C (PKC) agonist phorbol 12,13-dibutyrate (PDBu) on isometric force at a constant submaximal Ca2+ concentration (i.e., the effect on Ca2+ sensitivity) and regulatory myosin light-chain (rMLC) phosphorylation. PDBu increased Ca2+sensitivity, an increase associated with a concentration-dependent, sustained increase in rMLC phosphorylation. PDBu altered the relationship between rMLC phosphorylation and isometric force such that the increase in isometric force was less than that expected for the increase in rMLC phosphorylation observed. The effect of four PKC inhibitors [calphostin C, chelerythrine chloride, a pseudosubstrate inhibitor for PKC, PKC peptide-(19—31) (PSSI), and staurosporine] on PDBu-induced Ca2+ sensitization as well as the effect of calphostin C and PSSI on rMLC phosphorylation were determined. Whereas none of these compounds prevented or reversed the PDBu-induced increase in Ca2+sensitivity, the PDBu-induced increase in rMLC phosphorylation was inhibited. We conclude that PDBu increases rMLC phosphorylation by activation of PKC but that the associated PDBu-induced increases in Ca2+ sensitivity are mediated by mechanisms other than activation of PKC in permeabilized airway smooth muscle.

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Androgens Induce Nongenomic Stimulation of Colonic Contractile Activity through Induction of Calcium Sensitization and Phosphorylation of LC20 and CPI-17

Molecular Endocrinology ◽

10.1210/me.2009-0472 ◽

2010 ◽

Vol 24 (5) ◽

pp. 1007-1023 ◽

Cited By ~ 13

Author(s):

María C. González-Montelongo ◽

Raquel Marín ◽

Tomás Gómez ◽

Jorge Marrero-Alonso ◽

Mario Díaz

Keyword(s):

Smooth Muscle ◽

Light Chain ◽

Myosin Light Chain ◽

Contractile Activity ◽

Rho Kinase ◽

Regulatory Subunit ◽

Calcium Sensitization ◽

Regulatory Myosin Light Chain ◽

Rapid Activation ◽

Stimulation Of

Abstract We show that androgens, testosterone and 5α-dihydrotestosterone (DHT), acutely (∼40 min) provoke the mechanical potentiation of spontaneous and agonist-induced contractile activity in mouse colonic longitudinal smooth muscle. The results using flutamide, finasteride, cycloheximide, and actinomycin D indicate that androgen-induced potentiation is dependent on androgen receptors, requires reduction of testosterone to DHT, and occurs independently of transcriptional and translational events. Using permeabilized colonic smooth muscle preparations, we could demonstrate that mechanical potentiation is entirely due to calcium sensitization of contractile machinery. In addition, DHT (10 nm) increased phosphorylation of both 20-kDa myosin light chain (LC20) [regulatory myosin light chain, (MLC)] and CPI-17 (an endogenous inhibitor of MLC phosphatase). Paralleling these findings, inhibition of Rho-associated Rho kinase (ROK) and/or protein kinase C (PKC) with, respectively, Y27632 and chelerythrine, prevented LC20 phosphorylation and abolished calcium sensitization. In addition, inhibition of ROK prevents CPI-17 phosphorylation, indicating that ROK is located upstream PKC-mediated CPI-17 modulation in the signalling cascade. Additionally, androgens induce a rapid activation of RhoA and its translocation to the plasma membrane to activate ROK. The results demonstrate that androgens induce sensitization of colonic smooth muscle to calcium through activation of ROK, which in turn, activates PKC to induce CPI-17 phosphorylation. Activation of this pathway induces a potent steady stimulation of LC20 by inhibiting MLC phosphatase and displacing the equilibrium of the regulatory subunit towards its phosphorylated state. This is the first demonstration that colonic smooth muscle is a physiological target for androgen hormones, and that androgens modulate force generation of smooth muscle contractile machinery through nongenomic calcium sensitization pathways.

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Dual Effects of Hexanol and Halothane on the Regulation of Calcium Sensitivity in Airway Smooth Muscle

Anesthesiology ◽

10.1097/00000542-200304000-00013 ◽

2003 ◽

Vol 98 (4) ◽

pp. 871-880 ◽

Cited By ~ 1

Author(s):

Hayashi Yoshimura ◽

Keith A. Jones ◽

William J. Perkins ◽

David O. Warner

Keyword(s):

Smooth Muscle ◽

G Protein ◽

Airway Smooth Muscle ◽

Light Chain ◽

Protein Function ◽

Myosin Light Chain ◽

Calcium Sensitivity ◽

Myosin Light Chain Phosphorylation ◽

Receptor Stimulation ◽

Regulatory Myosin Light Chain

Background Contraction of airway smooth muscle is regulated by receptor-coupled mechanisms that control the force developed for a given cytosolic calcium concentration (i.e., calcium sensitivity). Halothane antagonizes acetylcholine-induced increases in calcium sensitivity by inhibiting GTP-binding (G)-protein pathways. The authors tested the hypothesis that hexanol, like halothane, inhibits agonist-induced increases in calcium sensitivity in airway smooth muscle by inhibiting G-protein pathways. Methods Calcium sensitivity was assessed using alpha-toxin-permeabilized canine tracheal smooth muscle. In selected experiments, regulatory myosin light chain phosphorylation was also determined by Western blotting in the presence and absence of 10 mm hexanol and/or 100 microm acetylcholine. Results Hexanol (10 mm) and halothane (0.76 mm) attenuated acetylcholine-induced calcium sensitization by decreasing regulatory myosin light chain phosphorylation during receptor stimulation. Hexanol also inhibited increases in calcium sensitivity due to direct stimulation of heterotrimeric G-proteins with tetrafluoroaluminate but not with 3 microm GTPgammaS, consistent with prior results obtained with halothane. In contrast, in the absence of receptor stimulation, both compounds produced a small increase in calcium sensitivity by a G-protein-mediated increase in regulatory myosin light chain phosphorylation that was not affected by pertussis toxin treatment. Conclusions The authors noted dual effects of hexanol and halothane. In the presence of muscarinic receptor stimulation, hexanol, like halothane, decreases calcium sensitivity by interfering with heterotrimeric G-protein function. However, in the absence of muscarinic receptor stimulation, hexanol and halothane slightly increase calcium sensitivity by a G-protein-mediated process not sensitive to pertussis toxin. Hexanol may represent a useful experimental tool to study the effect of anesthetics on heterotrimeric G-protein function.

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Halothane inhibits agonist-induced potentiation of rMLC phosphorylation in permeabilized airway smooth muscle

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1997.273.1.l80 ◽

1997 ◽

Vol 273 (1) ◽

pp. L80-L85 ◽

Cited By ~ 1

Author(s):

K. A. Jones ◽

A. Hirasaki ◽

D. H. Bremerich ◽

C. Jankowski ◽

D. O. Warner

Keyword(s):

Smooth Muscle ◽

Steady State ◽

Vascular Smooth Muscle ◽

Light Chain ◽

Myosin Light Chain ◽

Isometric Force ◽

State Level ◽

Steady State Level ◽

Regulatory Myosin Light Chain ◽

The Relationship

Agonist-induced increases in CA2+ sensitivity are mediated in part by mechanisms that increase phosphorylation of the regulatory myosin light chain (rMLC) at constant cytosolic Ca2+ concentration ([Ca2+]i). The current study tested the hypothesis that halothane inhibits acetylcholine (ACh)-induced potentiation of rMLC phosphorylation in beta-escin-permeabilized canine tracheal smooth muscle. ACh plus GTP significantly potentiated the increase in isometric force and rMLC phosphorylation induced by 0.8 microM free Ca2+. However, whereas the potentiation of isometric force was sustained, the potentiation of rMLC phosphorylation was biphasic, peaking at 0.5 min and then declining by approximately 10 min to a steady-state level significantly above that induced by 0.8 microM free Ca2+ alone. This finding suggests that mechanisms in addition to changes in rMLC phosphorylation may mediate ACh-induced Ca2+ sensitization, as has been reported for vascular smooth muscle. Halothane (0.91 +/- 0.10 mM) significantly inhibited ACh plus GTP-induced potentiation of rMLC phosphorylation and isometric force after 2 (peak rMLC phosphorylation) and 15 (steady-state rMLC phosphorylation) min of stimulation. However, the effect of halothane on the potentiation of isometric force was significantly less than that expected from its effect on rMLC phosphorylation (i.e., halothane changed the relationship between rMLC phosphorylation and isometric force). These results demonstrate that halothane inhibits the ACh-induced increase in Ca2+ sensitivity by inhibiting the membrane receptor-coupled mechanisms that increase rMLC phosphorylation at constant submaximal [Ca2+]i. Possible additional effects of halothane on rMLC phosphorylation-independent mechanisms cannot be ruled out.

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A myosin phosphatase targeting subunit isoform transition defines a smooth muscle developmental phenotypic switch

AJP Cell Physiology ◽

10.1152/ajpcell.2000.278.3.c589 ◽

2000 ◽

Vol 278 (3) ◽

pp. C589-C600 ◽

Cited By ~ 56

Author(s):

Wessel P. Dirksen ◽

Franjo Vladic ◽

Steven A. Fisher

Keyword(s):

Smooth Muscle ◽

Alternative Splicing ◽

Light Chain ◽

Myosin Light Chain ◽

Muscle Relaxation ◽

Smooth Muscle Relaxation ◽

Myosin Phosphatase ◽

Mrna Isoforms ◽

Regulatory Myosin Light Chain ◽

Muscle Tissues

Smooth muscle myosin phosphatase dephosphorylates the regulatory myosin light chain and thus mediates smooth muscle relaxation. The activity of this myosin phosphatase is dependent upon its myosin-targeting subunit (MYPT1). Isoforms of MYPT1 have been identified, but how they are generated and their relationship to smooth muscle phenotypes is not clear. Cloning of the middle section of chicken and rat MYPT1 genes revealed that each gene gave rise to isoforms by cassette-type alternative splicing of exons. In chicken, a 123-nucleotide exon was included or excluded from the mature mRNA, whereas in rat two exons immediately downstream were alternative. MYPT1 isoforms lacking the alternative exon were only detected in mature chicken smooth muscle tissues that display phasic contractile properties, but the isoform ratios were variable. The patterns of expression of rat MYPT1 mRNA isoforms were more complex, with three major and two minor isoforms present in all smooth muscle tissues at varying stoichiometries. Isoform switching was identified in the developing chicken gizzard, in which the exon-skipped isoform replaced the exon-included isoform around the time of hatching. This isoform switch occurred after transitions in myosin heavy chain and myosin light chain (MLC17) isoforms and correlated with a severalfold increase in the rate of relaxation. The developmental switch of MYPT1 isoforms is a good model for determining the mechanisms and significance of alternative splicing in smooth muscle.

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S-nitrosoglutathione-induced decrease in calcium sensitivity of airway smooth muscle

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.2000.278.3.l521 ◽

2000 ◽

Vol 278 (3) ◽

pp. L521-L527 ◽

Cited By ~ 10

Author(s):

Christina M. Pabelick ◽

David O. Warner ◽

William J. Perkins ◽

Keith A. Jones

Keyword(s):

Smooth Muscle ◽

Light Chain ◽

Myosin Light Chain ◽

Muscle Protein ◽

Isometric Force ◽

Calcium Sensitivity ◽

Nitric Oxide Donor ◽

Regulatory Myosin Light Chain ◽

Intracellular Ca ◽

The Relationship

The purpose of this study was to examine whether the nitric oxide donor S-nitrosoglutathione (GSNO) relaxes canine tracheal smooth muscle (CTSM) strips by decreasing Ca2+sensitivity [i.e., the amount of force for a given intracellular Ca2+ concentration ([Ca2+]i)]. We further investigated whether GSNO decreases Ca2+ sensitivity by altering the relationship between regulatory myosin light chain (rMLC) phosphorylation and [Ca2+]i and the relationship between force and rMLC phosphorylation. GSNO (100 μM) relaxed intact CTSM strips contracted with 45 mM KCl by decreasing Ca2+ sensitivity in comparison to control strips without significantly decreasing [Ca2+]i. GSNO reduced the amount of rMLC phosphorylation for a given [Ca2+]i but did not affect the relationship between isometric force and rMLC phosphorylation. These results show that in CTSM strips contracted with KCl, GSNO decreases Ca2+ sensitivity by affecting the level of rMLC phosphorylation for a given [Ca2+]i, suggesting that myosin light chain kinase is inhibited or that smooth muscle protein phosphatases are activated by GSNO.

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Hydrogen peroxide decreases Ca2+ sensitivity in airway smooth muscle by inhibiting rMLC phosphorylation

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1999.277.4.l816 ◽

1999 ◽

Vol 277 (4) ◽

pp. L816-L822 ◽

Cited By ~ 6

Author(s):

Robert R. Lorenz ◽

David O. Warner ◽

Keith A. Jones

Keyword(s):

Hydrogen Peroxide ◽

Smooth Muscle ◽

Light Chain ◽

Myosin Light Chain ◽

Muscle Protein ◽

Intracellular Camp ◽

Myosin Light Chain Phosphorylation ◽

Regulatory Myosin Light Chain ◽

Intracellular Cgmp ◽

Intracellular Ca

The purpose of this study was to determine the mechanism by which hydrogen peroxide (H2O2), an important inflammatory mediator, relaxes canine tracheal smooth muscle (CTSM). H2O2caused concentration-dependent relaxations of CTSM strips contracted with ACh or isotonic KCl [EC50 of 0.24 ± 0.04 (SE) and 0.23 ± 0.04 mM, respectively]. Indomethacin (10 μM) decreased the sensitivity of both KCl- and ACh-contracted strips to H2O2. H2O2increased intracellular cAMP levels, an increase that was abolished by indomethacin. H2O2did not affect intracellular cGMP levels. In strips treated with indomethacin and contracted with ACh or isotonic KCl, H2O2-evoked relaxations were accompanied by increases in intracellular Ca2+ concentration and decreases in regulatory myosin light chain phosphorylation. We conclude that H2O2decreases Ca2+ sensitivity in CTSM by decreasing regulatory myosin light chain phosphorylation through inhibition of myosin light chain kinase and/or activation of smooth muscle protein phosphatases.

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