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

2003 ◽  
Vol 98 (4) ◽  
pp. 871-880 ◽  
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.

Selected Contribution: Mechanical strain increases force production and calcium sensitivity in cultured airway smooth muscle cells

2000 ◽  
Vol 89 (5) ◽  
pp. 2092-2098 ◽  
Author(s):  
Paul G. Smith ◽  
Chaity Roy ◽  
Steven Fisher ◽  
Qi-Quan Huang ◽  
Frank Brozovich
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Airway Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Force Production ◽  
Muscle Cells ◽  
Calcium Sensitivity ◽  
Airway Smooth Muscle Cells ◽  
Maximal Force

Cultured airway smooth muscle cells subjected to cyclic deformational strain have increased cell content of myosin light chain kinase (MLCK) and myosin and increased formation of actin filaments. To determine how these changes may increase cell contractility, we measured isometric force production with changes in cytosolic calcium in individual permeabilized cells. The pCa for 50% maximal force production was 6.6 ± 0.4 in the strain cells compared with 5.9 ± 0.3 in control cells, signifying increased calcium sensitivity in strain cells. Maximal force production was also greater in strain cells (8.6 ± 2.9 vs. 5.7 ± 3.1 μN). The increased maximal force production in strain cells persisted after irreversible thiophosphorylation of myosin light chain, signifying that increased force could not be explained by differences in myosin light chain phosphorylation. Cells strained for brief periods sufficient to increase cytoskeletal organization but insufficient to increase contractile protein content also produced more force, suggesting that strain-induced cytoskeletal reorganization also increases force production.

Relationship between force and regulatory myosin light chain phosphorylation in airway smooth muscle

2000 ◽  
Vol 279 (1) ◽  
pp. L52-L58 ◽  
Author(s):  
Tetsuya Kai ◽  
Hayashi Yoshimura ◽  
Keith A. Jones ◽  
David O. Warner
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Myosin Light Chain Phosphorylation ◽  
Mean Values ◽  
Regulatory Myosin Light Chain ◽  
Muscarinic Stimulation ◽  
The Relationship ◽  
Force Relationship ◽  
Stimulation Of

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.

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

1998 ◽  
Vol 274 (5) ◽  
pp. C1253-C1260 ◽  
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.

A novel mechanism by which hydrogen peroxide decreases calcium sensitivity in airway smooth muscle

2003 ◽  
Vol 284 (2) ◽  
pp. L324-L332 ◽  
Author(s):  
William J. Perkins ◽  
Robert R. Lorenz ◽  
Michelle Bogoger ◽  
David O. Warner ◽  
Christine R. Cremo ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Myosin Light Chain ◽  
Calcium Sensitivity ◽  
Heavy Meromyosin ◽  
Regulatory Myosin Light Chain ◽  
Myosin Subfragment ◽  
Intracellular Ca ◽  
Triton X ◽  
Dependent Mechanism

The purpose of this study was to test the hypothesis that H2O2decreases the amount of force produced by a given intracellular Ca2+ concentration (i.e., the Ca2+ sensitivity) in airway smooth muscle (ASM) in part by mechanisms independent of changes in regulatory myosin light chain (rMLC) phosphorylation. A new preparation was developed and validated in which canine ASM strips were first exposed to H2O2 and then permeabilized with 10% Triton X-100 to assess the persistent effects of H2O2 on Ca2+ sensitivity. Experiments in which H2O2 was administered before permeabilization revealed a novel mechanism that contributed to reduced Ca2+ sensitivity independently of changes in rMLC phosphorylation, in addition to an rMLC phosphorylation-dependent mechanism. The mechanism depended on factors not available in the permeabilized ASM strip or in the buffer to which the strip was exposed, since there was no effect when H2O2was added to permeabilized strips. H2O2treatment of a maximally thiophosphorylated purified myosin subfragment (heavy meromyosin) significantly reduced actomyosin ATPase activity, suggesting one mechanism by which the phosphorylation-independent reduction in Ca2+ sensitivity may occur.

Force and myosin light chain phosphorylation in dog airway smooth muscle activated in different ways

2003 ◽  
Vol 137 (2-3) ◽  
pp. 141-149 ◽  
Author(s):  
Theodor Burdyga ◽  
Richard W Mitchell ◽  
Joseph Ragozzino ◽  
Lincoln E Ford
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Myosin Light Chain Phosphorylation

scholarly journals Decreased Phosphatase Activity, Increased Ca2+ Sensitivity, and Myosin Light Chain Phosphorylation in Urinary Bladder Smooth Muscle of Newborn Mice

2005 ◽  
Vol 125 (2) ◽  
pp. 187-196 ◽  
Author(s):  
Mari Ekman ◽  
Katarina Fagher ◽  
Mia Wede ◽  
Karolina Stakeberg ◽  
Anders Arner
Keyword(s):  
Smooth Muscle ◽  
Urinary Bladder ◽  
Phosphatase Activity ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Calcium Sensitivity ◽  
Myosin Light Chain Phosphorylation ◽  
Nonmuscle Myosin ◽  
Bladder Smooth Muscle ◽  
Urinary Bladder Smooth Muscle

Developmental changes in the regulation of smooth muscle contraction were examined in urinary bladder smooth muscle from mice. Maximal active stress was lower in newborn tissue compared with adult, and it was correlated with a lower content of actin and myosin. Sensitivity to extracellular Ca2+ during high-K+ contraction, was higher in newborn compared with 3-wk-old and adult bladder strips. Concentrations at half maximal tension (EC50) were 0.57 ± 0.01, 1.14 ± 0.12, and 1.31 ± 0.08 mM. Force of the newborn tissue was inhibited by ∼45% by the nonmuscle myosin inhibitor Blebbistatin, whereas adult tissue was not affected. The calcium sensitivity in newborn tissue was not affected by Blebbistatin, suggesting that nonmuscle myosin is not a primary cause for increased calcium sensitivity. The relation between intracellular [Ca2+] and force was shifted toward lower [Ca2+] in the newborn bladders. This increased Ca2+ sensitivity was also found in permeabilized muscles (EC50: 6.10 ± 0.07, 5.77 ± 0.08, and 5.55 ± 0.02 pCa units, in newborn, 3-wk-old, and adult tissues). It was associated with an increased myosin light chain phosphorylation and a decreased rate of dephosphorylation. No difference was observed in the myosin light chain phosphorylation rate, whereas the rate of myosin light chain phosphatase–induced relaxation was about twofold slower in the newborn tissue. The decreased rate was associated with a lower expression of the phosphatase regulatory subunit MYPT-1 in newborn tissue. The results show that myosin light chain phosphatase activity can be developmentally regulated in mammalian urinary bladders. The resultant alterations in Ca2+ sensitivity may be of importance for the nervous and myogenic control of the newborn bladders.

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

2000 ◽  
Vol 278 (3) ◽  
pp. L521-L527 ◽  
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.

scholarly journals Inhibition of myosin light-chain phosphorylation inverts the birefringence response of porcine airway smooth muscle

2007 ◽  
Vol 578 (2) ◽  
pp. 563-568 ◽  
Author(s):  
Alexander V. Smolensky ◽  
Susan H. Gilbert ◽  
Margaret Harger-Allen ◽  
Lincoln E. Ford
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Myosin Light Chain Phosphorylation

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

1999 ◽  
Vol 277 (4) ◽  
pp. L816-L822 ◽  
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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