Halothane Increases Smooth Muscle Protein Phosphatase in Airway Smooth Muscle

2001 ◽  
Vol 94 (1) ◽  
pp. 129-136 ◽  
Author(s):  
Motohiko Hanazaki ◽  
Keith A. Jones ◽  
William J. Perkins ◽  
David O. Warner
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Light Chain ◽  
Protein Phosphatase ◽  
Myosin Light Chain ◽  
Muscle Protein ◽  
Isometric Force ◽  
Activity Assay ◽  
Muscarinic Stimulation ◽  
Intracellular Ca

Background Halothane relaxes airway smooth muscle, in part, by decreasing the force produced for a given intracellular [Ca(2+)] (i.e., Ca(2+) sensitivity) during muscarinic stimulation, an effect produced by a decrease in regulatory myosin light-chain (rMLC) phosphorylation. The authors tested the hypothesis that halothane reduces rMLC phosphorylation during muscarinic stimulation at constant intracellular [Ca(2+)] by increasing smooth muscle protein phosphatase (SMPP) activity, without changing myosin light-chain kinase (MLCK) activity. Methods Enzyme activities were assayed in beta-escin permeabilized strips of canine tracheal smooth muscle. Under conditions of constant intracellular [Ca(2+)], the rate of rMLC phosphorylation was measured by Western blotting during inhibition of SMPP with microcystin-LR (to assay MLCK activity) or during inhibition of MLCK by wortmannin and adenosine triphosphate depletion (to assay SMPP activity). The effect of halothane (0.8 mm) on enzyme activities and isometric force during stimulation with 0.6 microm Ca(2+) and 10 microm acetylcholine was determined. Results Halothane produced a 14 +/- 8% (mean +/- SD) decrease in isometric force by significantly reducing rMLC phosphorylation (from 32 +/- 9% to 28 +/- 9%). Halothane had no significant effect on any parameter of a monoexponential relation fit to the data for the MLCK activity assay. In contrast, halothane significantly decreased the half-time for rMLC dephosphorylation in the SMPP activity assay (from 0.74 +/- 0.28 min to 0.44 +/- 0.10 min), indicating that it increased SMPP activity. Conclusions Halothane decreases Ca(2+) sensitivity and rMLC phosphorylation in airway smooth muscle during muscarinic receptor stimulation by increasing SMPP activity, without affecting MLCK, probably by disrupting receptor G-protein signaling pathways that inhibit SMPP.

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.

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.

Sensitized airway smooth muscle plasticity and hyperreactivity: a reviewThis article is one of a selection of papers published in the Special Issue on Recent Advances in Asthma Research.

2007 ◽  
Vol 85 (7) ◽  
pp. 679-685 ◽  
Author(s):  
N. L. Stephens ◽  
Z.-Q. Cheng ◽  
A. Fust
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Isometric Force ◽  
Total Activity ◽  
Smooth Muscle Myosin ◽  
Significant Difference ◽  
Muscle Myosin ◽  
Isotonic Shortening

To help elucidate the mechanisms underlying asthmatic bronchospasm, the goal of our research has been to determine whether airway smooth muscle (ASM) hyperreactivity was the responsible factor. We reported that in a canine model of asthma, the shortening capacity (ΔLmax) and velocity (Vo) of in vitro sensitized muscle were significantly increased. This increase was of sufficient magnitude to account for 75% narrowing of the in vivo airway, but maximal isometric force was unchanged. This last feature has been reported by others. Under lightly loaded conditions, ASM completes 75% of its isotonic shortening within the first 2 s. Furthermore, 90% of the increased shortening of ragweed pollen-sensitized ASM (SASM), compared with control (CASM), is complete within the first 2 s. The study of shortening beyond this period will apparently not yield much useful information, and studies of isotonic shortening should be focused on this interval. Although both CASM and SASM showed plasticity and adaptation with respect to isometric force, neither muscle type showed a difference in the force developed in these phases. During isotonic shortening, no evidence of plasticity was seen, but the equilibrated SASM showed increased ΔLmax and Vo of shortening. Molecular mechanisms of changes in Vo could result from changes in the kinetics of the myosin heavy chain ATPase. Motility assay, however, showed no changes between CASM and SASM in the ability of the purified myosin molecule (SF1) to translocate a marker actin filament. On the other hand, we found that the state of activation of the ATPase by phosphorylation of smooth muscle myosin light chain (molecular mass 20 000 Da) was greater in the SASM. This would account for the increased Vo. Investigating the signalling pathway, we found that whereas [Ca2+]i increased in both isometric and isotonic contraction, there was no significant difference between CASM and SASM. The content and activity of calmodulin were also not different between the 2 muscles. Nevertheless, we did find that content and total activity of smooth muscle myosin light chain kinase (smMLCK) and the abundance of its message were greater; this would explain the increased MLC20 phosphorylation. The binding affinity between Ca2+ and calmodulin and between 4 Ca2+ calmodulin and smMLCK remains to be studied. We conclude that SASM shows increased isotonic shortening capacity and velocity. It also shows increased content and total activity of smMLCK, which is consistent with the increased shortening. Plasticity produced by oscillation is not seen in the shortening muscle, although it is seen with respect to force development. It did not modulate the behaviour of the sensitized muscle.

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.

scholarly journals FFAR1 activation attenuates histamine-induced myosin light chain phosphorylation and cortical tension development in human airway smooth muscle cells

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Shengjie Xu ◽  
Anthony Schwab ◽  
Nikhil Karmacharya ◽  
Gaoyuan Cao ◽  
Joanna Woo ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Airway Hyperreactivity ◽  
Human Airway Smooth Muscle ◽  
Tension Development ◽  
Cortical Tension ◽  
Human Airway ◽  
Mlc Phosphorylation

Abstract Background Activation of free fatty acid receptors (FFAR1 and FFAR4) which are G protein-coupled receptors (GPCRs) with established (patho)physiological roles in a variety of obesity-related disorders, induce human airway smooth muscle (HASM) cell proliferation and shortening. We reported amplified agonist-induced cell shortening in HASM cells obtained from obese lung donors. We hypothesized that FFAR1 modulate excitation–contraction (EC) coupling in HASM cells and play a role in obesity-associated airway hyperresponsiveness. Methods In HASM cells pre-treated (30 min) with FFAR1 agonists TAK875 and GW9508, we measured histamine-induced Ca2+ mobilization, myosin light chain (MLC) phosphorylation, and cortical tension development with magnetic twisting cytometry (MTC). Phosphorylation of MLC phosphatase and Akt also were determined in the presence of the FFAR1 agonists or vehicle. In addition, the effects of TAK875 on MLC phosphorylation were measured in HASM cells desensitized to β2AR agonists by overnight salmeterol treatment. The inhibitory effect of TAK875 on MLC phosphorylation was compared between HASM cells from age and sex-matched non-obese and obese human lung donors. The mean measurements were compared using One-Way ANOVA with Dunnett’s test for multiple group comparisons or Student’s t-test two-group comparison. For cortical tension measurements by magnetic twisted cytometry, mixed effect model using SAS V.9.2 was applied. Means were considered significant when p ≤ 0.05. Results Unexpectedly, we found that TAK875, a synthetic FFAR1 agonist, attenuated histamine-induced MLC phosphorylation and cortical tension development in HASM cells. These physiological outcomes were unassociated with changes in histamine-evoked Ca2+ flux, protein kinase B (AKT) activation, or MLC phosphatase inhibition. Of note, TAK875-mediated inhibition of MLC phosphorylation was maintained in β2AR-desensitized HASM cells and across obese and non-obese donor-derived HASM cells. Conclusions Taken together, our findings identified the FFAR1 agonist TAK875 as a novel bronchoprotective agent that warrants further investigation to treat difficult-to-control asthma and/or airway hyperreactivity in obesity.

Mechanical strain increases velocity and extent of shortening in cultured airway smooth muscle cells

1999 ◽  
Vol 277 (2) ◽  
pp. L343-L348 ◽  
Author(s):  
Paul G. Smith ◽  
Chaity Roy ◽  
Jamie Dreger ◽  
Frank Brozovich
Keyword(s):  
Smooth Muscle ◽  
Mechanical Stress ◽  
Airway Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Mechanical Strain ◽  
Airway Smooth Muscle Cells ◽  
Maximal Velocity ◽  
Cell Contractility ◽  
Cell Shortening

Abnormal mechanical stress on lung tissue is associated with increased mass and contractility of airway smooth muscle (ASM). We have reported that cultured ASM cells subjected to cyclic strain exhibit increased myosin light chain kinase (MLCK) and stress filaments. Increased MLCK may increase contractile velocity, whereas increased stress filaments could impede cell shortening by increasing the cell’s internal load. To study strain-induced changes in cell contractility, the time course of shortening of individual cells exposed to 90 mM KCl was recorded. Length vs. time plots revealed significantly greater maximal velocity of shortening in strain cells than control (no strain). This correlated with an increase in MLCK and myosin light chain phosphorylation measured in strain cells in separate experiments. The extent of cell shortening tended to be greater in the strain cells so that increased impedance to shortening was not detected. Mechanical stress may therefore increase the contractility of ASM by increasing the content of MLCK.

Isotonic relaxation of control and sensitized airway smooth muscle

2005 ◽  
Vol 83 (10) ◽  
pp. 941-951 ◽  
Author(s):  
N L Stephens ◽  
A Fust ◽  
H Jiang ◽  
W Li ◽  
X Ma
Keyword(s):  
Smooth Muscle ◽  
Intracellular Calcium ◽  
Airway Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Phase Iii ◽  
Contractile Element ◽  
Second Phase ◽  
Half Time ◽  
Phase Phase

Smooth muscle relaxation has most often been studied in isometric mode. However, this only tells us about the stiffness properties of the bronchial wall and thus only about wall capacitative properties. It tells us little about airflow. To study the latter, which of course is the meaningful parameter in regulation of ventilation and in asthma, we studied isotonic shortening of bronchial smooth muscle (BSM) strips. Failure of BSM to relax could be another important factor in maintaining high airway resistance. To analyze relaxation curves, we developed an index of isotonic relaxation, t1/2(P, lCE), which is the half-time for relaxation that is independent of muscle load (P) and of initial contractile element length (lCE). This index was measured in curves of relaxation initiated at 2 s (normally cycling crossbridges) and at 10 s (latch-bridges). At 10 s no difference was seen for adjusted t1/2(P, lCE) between curves obtained from control and sensitized BSM, (8.38 ± 0.92 s vs. 7.78 ± 0.93 s, respectively). At 2 s the half-time was almost doubled in the sensitized BSM (6.98 ± 0.01 s (control) vs. 12.74 ± 2.5 s (sensitized)). Thus, changes in isotonic relaxation are only seen during early contraction. Using zero load clamps, we monitored the time course of velocity during relaxation and noted that it varied according to 3 phases. The first phase (phase i) immediately followed cessation of electrical field stimulation (EFS) at 10 s and showed almost the same velocity as during the latter 1/3 of shortening; the second phase (phase ii) was linear in shape and is associated with zero load velocity, we speculate it could stem from elastic recoil of the cells' internal resistor; and the third phase (phase iii) was convex downwards. The zero load velocities in phase iii showed a surprising spontaneous increase suggesting reactivation of the muscle. Measurements of intracellular calcium (Fura-2 study) and of phosphorylation of the 20 kDa myosin light chain showed simultaneous increments, indicating phase iii represented an active process. Studies are under way to determine what changes occur in these 3 phases in a sensitized muscle. And of course, in the context of this conference, just what role the plastic properties of the muscle play in relaxation requires serious consideration.Key words: airway smooth muscle, sensitized smooth muscle, isotonic relaxation, intracellular calcium transients, myosin light chain (20 kDa) phosphorylation.

Inhibitory effect of calyculin A, a Ser/Thr protein phosphatase type I inhibitor, on renin secretion

1998 ◽  
Vol 275 (5) ◽  
pp. F664-F670 ◽  
Author(s):  
Chun Sik Park ◽  
Mi Hyun Kim ◽  
Chae Hun Leem ◽  
Yeon Jin Jang ◽  
Hae Won Kim ◽  
...  
Keyword(s):  
Okadaic Acid ◽  
Light Chain ◽  
Protein Phosphatase ◽  
Myosin Light Chain ◽  
Renin Secretion ◽  
Type I ◽  
Dependent Manner ◽  
Calyculin A ◽  
Selective Inhibitors ◽  
Intracellular Ca

We have recently shown that several putative selective inhibitors of Ca2+-calmodulin-dependent myosin light chain kinase (MLCK), such as ML-9 [1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine], reversibly stimulate renin secretion [C. S. Park, S.-H. Chang, H. S. Lee, S.-H. Kim, J. W. Chang, and C. D. Hong. Am. J. Physiol. 271 ( Cell Physiol. 40): C242–C247, 1996]. We hypothesized that Ca2+ inhibits renin secretion, via phosphorylation of 20-kDa myosin light chain (MLC20), by activating MLCK. In the present studies, we have investigated the types of protein phosphatase (PP) involved in the control of renin secretion through inhibition of MLC dephosphorylation using inhibitors of various types of serine/threonine-specific protein phosphatases. Cyclosporin A, a putative inhibitor of PP type 2 (calcineurin), was without effect. Calyculin A and okadaic acid, putative selective inhibitors of both PP type 1 (PP1) and type 2A (PP2A), significantly inhibited renin secretion under control conditions. Calyculin A had inhibitory effects at least 10-fold more potent than okadaic acid, suggesting that PP1, rather than PP2A, is involved in the control of renin secretion. Furthermore, calyculin A blocked the reversal of renin secretion preinhibited by raised intracellular Ca2+ concentrations in a concentration-dependent manner. Calyculin A (10−6 M) significantly inhibited renin secretion stimulated by lowering intracellular Ca2+ concentrations and blocked the stimulatory effect of ML-9 on renin secretion. Taking all of these results into consideration, we hypothesize that dephosphorylation of MLC20 by Ca2+-independent PP1 stimulates renin secretion, whereas phosphorylation of MLC20 by Ca2+-calmodulin-dependent MLCK inhibits it. This hypothesized regulatory model of renin secretion predicts that the rate of renin secretion at a given time is determined by the ratio of phosphorylated to dephosphorylated MLC20, which is, in turn, determined by the dynamic balance between activity of MLCK and MLC phosphatase.

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