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.

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

1997 ◽  
Vol 273 (1) ◽  
pp. L80-L85 ◽  
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.

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.

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.

cGMP modulation of Ca2+sensitivity in airway smooth muscle

1999 ◽  
Vol 276 (1) ◽  
pp. L35-L40 ◽  
Author(s):  
Keith A. Jones ◽  
Gilbert Y. Wong ◽  
Christopher J. Jankowski ◽  
Masaki Akao ◽  
David O. Warner
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Isometric Force ◽  
Membrane Receptor ◽  
Tracheal Smooth Muscle ◽  
Response Curves ◽  
Smooth Muscle Preparation ◽  
Regulatory Myosin Light Chain ◽  
The Relationship ◽  
Concentration Response Curve

A β-escin-permeabilized canine tracheal smooth muscle preparation was used to test the hypothesis that cGMP decreases Ca2+ sensitivity in airway smooth muscle primarily by inhibiting the membrane receptor-coupled mechanisms that regulate Ca2+ sensitivity and not by inhibiting Ca2+/calmodulin activation of the contractile proteins. 8-Bromo-cGMP (100 μM) had no effect on the free Ca2+concentration-response curves generated in the absence of muscarinic receptor stimulation. In the presence of 100 μM ACh plus 10 μM GTP, 8-bromo-cGMP (100 μM) caused a rightward shift of the free Ca2+ concentration-response curve, significantly increasing the EC50for free Ca2+ from 0.35 ± 0.03 to 0.75 ± 0.06 μM; this effect of 8-bromo-cGMP was concentration dependent from 1 to 100 μM. 8-Bromo-cGMP (100 μM) decreased the level of regulatory myosin light chain (rMLC) phosphorylation for a given cytosolic Ca2+ concentration but had no effect on the amount of isometric force produced for a given level of rMLC phosphorylation. These findings suggest that cGMP decreases Ca2+ sensitivity in canine tracheal smooth muscle primarily by inhibiting the membrane receptor-coupled mechanisms that modulate the relationship between cytosolic Ca2+ concentration and rMLC phosphorylation.

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.

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.

Phorbol ester-induced contraction in chemically skinned vascular smooth muscle

1986 ◽  
Vol 251 (3) ◽  
pp. C356-C361 ◽  
Author(s):  
M. Chatterjee ◽  
M. Tejada
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Vascular Smooth Muscle ◽  
Phorbol Ester ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Phorbol Esters ◽  
Myosin Light Chain Phosphorylation ◽  
Kinase C

We studied the contractile response to phorbol esters and its relationship to myosin light chain phosphorylation in intact and Triton X-100-skinned porcine carotid preparations. Muscle contraction was activated by phorbol 12,13-dibutyrate (PDBu) and phorbol 12,13-didecanoate (PDD). Dose-dependent contractions to PDBu were obtained both in the intact and skinned preparations. The maximal values of stress in response to PDBu were 1.11 +/- 0.10 X 10(5) N/m2 (n = 7) in the intact and 5.72 +/- 0.59 X 10(4) N/m2 (n = 10) in the skinned muscles. The skinned tissues responded to PDD, which has been shown to activate protein kinase C, but not to the inactive isomer 4 alpha-PDD, thus ruling out nonspecific phorbol effects. The phorbol ester response exhibited a Ca2+ dependence. High stresses in the skinned muscles (5.53 +/- 0.69 X 10(4) N/m2, n = 8) were associated with low values of myosin light chain phosphorylation (0.18 +/- 0.01 mol Pi/mol light chain, n = 8). Thus phorbol esters can contract vascular smooth muscle by a mechanism that is not proportional to myosin light chain phosphorylation and that may involve activation of protein kinase C.

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.

Acetylcholine-induced phosphorylation of CPI-17 in rat bronchial smooth muscle: the roles of Rho-kinase and protein kinase C

2005 ◽  
Vol 83 (4) ◽  
pp. 375-381 ◽  
Author(s):  
Hiroyasu Sakai ◽  
Tomona Hirano ◽  
Hisao Takeyama ◽  
Yoshihiko Chiba ◽  
Miwa Misawa
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Dependent Manner ◽  
Rock Inhibitor ◽  
Bronchial Smooth Muscle ◽  
Calphostin C ◽  
Kinase C

It has been demonstrated that CPI-17 provokes an inhibition of myosin light chain phosphatase to increase myosin light chain phosphorylaton and Ca2+ sensitivity during contraction of vascular smooth muscle. However, expression and agonist-mediated regulation of CPI-17 in bronchial smooth muscle have not been documented. Thus, expression and phosphorylation of CPI-17 mediated by PKC and ROCK were investigated using rat bronchial preparations. Acetylcholine (ACh)-induced contraction and Ca2+ sensitization were both attenuated by 10–6 mol Y-27632 /L, a ROCK inhibitor, 10–6 mol calphostin C/L, a PKC inhibitor, and their combination. A PKC activator, PDBu, induced a Ca2+ sensitization in α-toxin-permeabilized bronchial smooth muscle. In this case, the Ca2+ sensitizing effect was significantly inhibited by caphostin C but not by Y-27632. An immunoblot study demonstrated CPI-17 expression in the rat bronchial smooth muscle. Acetylcholine induced a phosphorylation of CPI-17 in a concentration-dependent manner, which was significantly inhibited by Y-27632 and calphostin C. In conclusion, these data suggest that both PKC and ROCK are involved in force development, Ca2+ sensitization, and CPI-17 phosphorylation induced by ACh stimulation in rat bronchial smooth muscle. As such, RhoA/ROCK, PKC/CPI-17, and RhoA/ROCK/CPI pathways may play important roles in the ACh-induced Ca2+ sensitization of bronchial smooth muscle contraction.Key words: CPI-17, bronchial smooth muscle, acetylcholine, ROCK, protein kinase C.

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