Dynamic Assessment of Ca
            2+
            Sensitivity of Isometric Force in Intact Airway Smooth Muscle Using Phase Loop Plots

During agonist stimulation of airway smooth muscle (ASM), agonists such as ACh induce a transient increase in cytosolic Ca2+ concentration ([Ca2+]cyt), which leads to a contractile response (excitation-contraction (E-C) coupling). Previously, the sensitivity of the contractile response of ASM to elevated [Ca2+]cyt (Ca2+ sensitivity) was assessed as the ratio of maximum force to maximum [Ca2+]cyt. However, this static assessment of Ca2+ sensitivity overlooks the dynamic nature of E-C coupling in ASM. In this study, we simultaneously measured [Ca2+]cyt and isometric force responses to three concentrations of ACh (1, 2.6 and 10 μM). Both maximum [Ca2+]cyt and maximum force responses were ACh concentration-dependent, but force increased disproportionately, thereby increasing static Ca2+ sensitivity. The dynamic properties of E-C coupling were assessed in several ways. The temporal delay between the onset of ACh-induced [Ca2+]cyt and onset force responses was not affected by ACh concentration. The rates of rise of the ACh-induced [Ca2+]cyt and force responses increased with increasing ACh concentration. The integral of the phase-loop plot of [Ca2+]cyt and force from onset to steady-state also increased with increasing ACh concentration, whereas the rate of relaxation remained unchanged. Although these results suggest an ACh concentration-dependent increase in the rate of cross-bridge recruitment and in the rate of rise of [Ca2+]cyt, the extent of regulatory myosin light chain (rMLC20) phosphorylation was not dependent on ACh concentration. We conclude that the dynamic properties of [Ca2+]cyt and force responses in ASM are dependent on ACh concentration but reflect more than changes in the extent of rMLC20 phosphorylation.

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Myosin thick filament lability induced by mechanical strain in airway smooth muscle

Journal of Applied Physiology ◽

10.1152/jappl.2001.90.5.1811 ◽

2001 ◽

Vol 90 (5) ◽

pp. 1811-1816 ◽

Cited By ~ 78

Author(s):

Kuo-Hsing Kuo ◽

Lu Wang ◽

Peter D. Paré ◽

Lincoln E. Ford ◽

Chun Y. Seow

Keyword(s):

Smooth Muscle ◽

Airway Smooth Muscle ◽

Cross Sections ◽

Structural Changes ◽

Isometric Force ◽

Mechanical Strain ◽

Thick Filament ◽

Functional Changes ◽

Thick Filaments ◽

Length Changes

Airway smooth muscle adapts to different lengths with functional changes that suggest plastic alterations in the filament lattice. To look for structural changes that might be associated with this plasticity, we studied the relationship between isometric force generation and myosin thick filament density in cell cross sections, measured by electron microscope, after length oscillations applied to the relaxed porcine trachealis muscle. Muscles were stimulated regularly for 12 s every 5 min. Between two stimulations, the muscles were submitted to repeated passive ±30% length changes. This caused tetanic force and thick-filament density to fall by 21 and 27%, respectively. However, in subsequent tetani, both force and filament density recovered to preoscillation levels. These findings indicate that thick filaments in airway smooth muscle are labile, depolymerization of the myosin filaments can be induced by mechanical strain, and repolymerization of the thick filaments underlies force recovery after the oscillation. This thick-filament lability would greatly facilitate plastic changes of lattice length and explain why airway smooth muscle is able to function over a large length range.

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Specific reaginic antibody IgG1-induced changes of airway smooth muscle cells

Journal of Applied Physiology ◽

10.1152/jappl.1988.65.2.767 ◽

1988 ◽

Vol 65 (2) ◽

pp. 767-775 ◽

Cited By ~ 10

Author(s):

M. Souhrada ◽

J. F. Souhrada

Keyword(s):

Smooth Muscle ◽

Airway Smooth Muscle ◽

Contractile Response ◽

Isometric Force ◽

Airway Smooth Muscle Cells ◽

Base Line ◽

Dependent Relationship ◽

Reaginic Antibody ◽

Induced Changes ◽

Igg1 Concentration

It was found that 1) an administration of both immunoglobulin G1 (IgG1) or immunized serum caused an immediate depolarization and an increase in the isometric force of airway smooth muscle (ASM) cells, followed by a sustained hyperpolarization and a return of the tone to the base-line values; 2) an IgG1 concentration-dependent relationship was found between a peak depolarization, a peak hyperpolarization, and a peak isometric force; for these events 50% effective dose (ED50) was found to be 0.17, 0.14, and 0.25 microgram/ml of IgG1, respectively; 3) both electrical and contractile responses to ovalbumin of ASM cells sensitized with IgG1 were also dependent on the concentration of IgG1; the ED50 values of this relationship were 0.27 and 0.25 micrograms/ml of IgG1, respectively; 4) amiloride (10(-8) to 10(-5) M) pretreatment and a sodium-deficient environment attenuated sensitized-induced electrical and contractile changes as well as the response of ASM to ovalbumin (0.1%); and 5) pretreatment of ASM with diphenhydramine (10(-5) M) or FPL 55712 (10(-6) M) had no effect on sensitization-induced changes in membrane potential but attenuated electrical and contractile response of ASM to ovalbumin (0.1%).

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Tissue elastance influences airway smooth muscle shortening: comparison of mechanical properties among different species

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y02-112 ◽

2002 ◽

Vol 80 (9) ◽

pp. 865-871 ◽

Cited By ~ 10

Author(s):

Anabelle M. Opazo Saez ◽

R Robert Schellenberg ◽

Mara S Ludwig ◽

Richard A Meiss ◽

Peter D Paré

Keyword(s):

Mechanical Properties ◽

Smooth Muscle ◽

Airway Smooth Muscle ◽

Isometric Force ◽

Published Data ◽

Passive Tension ◽

Active Force ◽

Muscle Shortening ◽

Human Airways ◽

Tissue Elastance

We have observed striking differences in the mechanical properties of airway smooth muscle preparations among different species. In this study, we provide a novel analysis on the influence of tissue elastance on smooth muscle shortening using previously published data from our laboratory. We have found that isolated human airways exhibit substantial passive tension in contrast to airways from the dog and pig, which exhibit little passive tension (<5% of maximal active force versus ~60% for human bronchi). In the dog and pig, airway preparations shorten up to 70% from Lmax (the length at which maximal active force occurs), whereas human airways shorten by only ~12% from Lmax. Isolated airways from the rabbit exhibit relatively low passive tension (~22% Fmax) and shorten by 60% from Lmax. Morphologic evaluation of airway cross sections revealed that 25-35% of the airway wall is muscle in canine, porcine, and rabbit airways in contrast to ~9% in human airway preparations. We postulate that the large passive tension needed to stretch the muscle to Lmax reflects the high connective tissue content surrounding the smooth muscle, which limits shortening during smooth muscle contraction by imposing an elastic load, as well as by causing radial constraint.Key words: isometric force, isotonic shortening, elastance.

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Effects of halothane on the relationship between cytosolic calcium and force in airway smooth muscle

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1994.266.2.l199 ◽

1994 ◽

Vol 266 (2) ◽

pp. L199-L204 ◽

Cited By ~ 6

Author(s):

K. A. Jones ◽

G. Y. Wong ◽

R. R. Lorenz ◽

D. O. Warner ◽

G. C. Sieck

Keyword(s):

Smooth Muscle ◽

Airway Smooth Muscle ◽

Isometric Force ◽

Cytosolic Calcium ◽

Simultaneous Recording ◽

Physiological Salt Solution ◽

Response Curves ◽

Fura 2 ◽

Contractile System ◽

Superfusion System

The mechanism of the direct relaxing effect of halothane on airway smooth muscle may involve a decrease in 1) cytosolic calcium concentration ([Ca2+]i) and/or 2) the force produced for a given [Ca2+]i (i.e., the “sensitivity” of the myofibrillar contractile system to Ca2+). This study was conducted to test the hypothesis that halothane reduces the sensitivity of the myofibrillar contractile system to Ca2+ during muscarinic receptor stimulation of canine tracheal smooth muscle. Isolated smooth muscle strips were mounted in a photometric superfusion system, stretched to their optimal length for force development, and loaded with the fluorescent Ca2+ indicator, fura 2, for simultaneous recording of fura 2 fluorescence and isometric force. Emission fluorescence intensities due to excitation at 340 (F340)- and 380 (F380)-nm wavelengths were measured and F340/F380 was used as an index of [Ca2+]i. After superfusion with Ca(2+)-free physiological salt solution (PSS) containing 1 or 100 microM acetylcholine (ACh), two consecutive cumulative concentration-response curves to CaCl2 (0.01–2.4 mM) were generated for each strip; one curve was generated in the presence of halothane. In strips stimulated with 1 (n = 6) or 100 (n = 6) microM ACh, the cumulative addition of CaCl2 to the Ca(2+)-free PSS caused concentration-dependent increases in both F340/F380 and force. In strips stimulated with 1 microM ACh, 2.4 +/- 0.3% halothane proportionally attenuated increases in both F340/F380 and force.(ABSTRACT TRUNCATED AT 250 WORDS)

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Latrunculin B increases force fluctuation-induced relengthening of ACh-contracted, isotonically shortened canine tracheal smooth muscle

Journal of Applied Physiology ◽

10.1152/japplphysiol.01378.2003 ◽

2005 ◽

Vol 98 (2) ◽

pp. 489-497 ◽

Cited By ~ 35

Author(s):

M. L. Dowell ◽

O. J. Lakser ◽

W. T. Gerthoffer ◽

J. J. Fredberg ◽

G. L. Stelmack ◽

...

Keyword(s):

Smooth Muscle ◽

Airway Smooth Muscle ◽

Actin Filament ◽

Isometric Force ◽

Muscle Length ◽

Tracheal Smooth Muscle ◽

Filament Length ◽

Latrunculin B ◽

Force Fluctuation ◽

Reference Length

We hypothesized that differences in actin filament length could influence force fluctuation-induced relengthening (FFIR) of contracted airway smooth muscle and tested this hypothesis as follows. One-hundred micromolar ACh-stimulated canine tracheal smooth muscle (TSM) strips set at optimal reference length ( Lref) were allowed to shorten against 32% maximal isometric force (Fmax) steady preload, after which force oscillations of ±16% Fmax were superimposed. Strips relengthened during force oscillations. We measured hysteresivity and calculated FFIR as the difference between muscle length before and after 20-min imposed force oscillations. Strips were relaxed by ACh removal and treated for 1 h with 30 nM latrunculin B (sequesters G-actin and promotes depolymerization) or 500 nM jasplakinolide (stabilizes actin filaments and opposes depolymerization). A second isotonic contraction protocol was then performed; FFIR and hysteresivity were again measured. Latrunculin B increased FFIR by 92.2 ± 27.6% Lref and hysteresivity by 31.8 ± 13.5% vs. pretreatment values. In contrast, jasplakinolide had little influence on relengthening by itself; neither FFIR nor hysteresivity was significantly affected. However, when jasplakinolide-treated tissues were then incubated with latrunculin B in the continued presence of jasplakinolide for 1 more h and a third contraction protocol performed, latrunculin B no longer substantially enhanced TSM relengthening. In TSM treated with latrunculin B + jasplakinolide, FFIR increased by only 3.03 ± 5.2% Lref and hysteresivity by 4.14 ± 4.9% compared with its first (pre-jasplakinolide or latrunculin B) value. These results suggest that actin filament length, in part, determines the relengthening of contracted airway smooth muscle.

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cGMP modulation of Ca2+sensitivity in airway smooth muscle

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1999.276.1.l35 ◽

1999 ◽

Vol 276 (1) ◽

pp. L35-L40 ◽

Cited By ~ 14

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.

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Respiratory epithelium modulates the responses of canine bronchi to cooling

Journal of Applied Physiology ◽

10.1152/jappl.1993.74.5.2421 ◽

1993 ◽

Vol 74 (5) ◽

pp. 2421-2425 ◽

Cited By ~ 3

Author(s):

Y. Gao ◽

P. M. Vanhoutte

Keyword(s):

Arachidonic Acid ◽

Smooth Muscle ◽

Airway Smooth Muscle ◽

Isometric Force ◽

Nordihydroguaiaretic Acid ◽

Respiratory Epithelium ◽

Resting Tension ◽

Cytochrome P 450 ◽

Evoked Contractions

The present study was designed to investigate the effect of cooling on the modulatory role of the respiratory epithelium on the underlying smooth muscle. Canine bronchial rings and segments (with or without epithelium) were suspended in organ chambers and perfused with modified Krebs-Ringer bicarbonate solution, respectively. Isometric force was recorded. Cooling did not affect the resting tension of the bronchi. During contractions to carbachol, cooling evoked contractions in bronchi with epithelium but relaxations in those without epithelium. In the presence of indomethacin, cooling induced contractions in both preparations with and without epithelium. The contractions in bronchi with epithelium were significantly larger than those in bronchi without epithelium. After treatment with indomethacin, exogenous arachidonic acid potentiated the cooling-induced contractions in preparations with epithelium but not in those without epithelium. This potentiation was not affected by nordihydroguaiaretic acid. SKF 525-A and metyrapone, inhibitors of cytochrome P-450 monooxygenases, converted the cooling-induced contractions of preparations with epithelium to relaxations and had no significant effects on the responses of preparations without epithelium. These observations suggest that cooling induces from the epithelium the release of a cytochrome P-450-derived eicosanoid that potentiates contractions of the underlying airway smooth muscle to carbachol.

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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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Halothane Increases Smooth Muscle Protein Phosphatase in Airway Smooth Muscle

Anesthesiology ◽

10.1097/00000542-200101000-00023 ◽

2001 ◽

Vol 94 (1) ◽

pp. 129-136 ◽

Cited By ~ 7

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.

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