BIOCHEMICAL MECHANISMS BY WHICH REACTIVE OXIDATIVE SPECIES MODULATE CALCIUM SENSITIVITY IN AIRWAY SMOOTH MUSCLE

Cytosolic calcium is a key determinant of the contractile state of airway smooth muscle (ASM). To investigate the mechanisms by which histamine affects cytosolic calcium, we measured changes in inositol 1,4,5-trisphosphate (IP3) following the addition of histamine to cultured canine ASM cells. The effect of phorbol 12-myristate 13-acetate (PMA) on IP3 formation was investigated under conditions previously shown to abolish histamine-induced calcium release. In both intact cells and ASM membranes, histamine produced a significant increase in IP3 formation, which was inhibited by PMA. The site of this blockade was investigated by examining the effect of PMA on guanine nucleotide-stimulated IP3 formation and on phosphoinositide-specific phospholipase C (PI-PLC) activity in ASM membranes. Guanine nucleotide-stimulated IP3 formation was inhibited by PMA pretreatment. Membrane-associated PI-PLC activity was also decreased, an effect that was not due simply to a shift in the calcium sensitivity of the enzyme. We conclude that in cultured canine ASM cells, PMA blocks histamine-induced IP3 formation and that this inhibition is caused, in part, by a postreceptor site of action of protein kinase C, possibly via a direct effect on PI-PLC.

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

Journal of Applied Physiology ◽

10.1152/jappl.2000.89.5.2092 ◽

2000 ◽

Vol 89 (5) ◽

pp. 2092-2098 ◽

Cited By ~ 40

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.

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Halothane Does Not Inhibit the Functional Coupling between the β2-Adrenergic Receptor and the GαsHeterotrimeric G Protein

Anesthesiology ◽

10.1097/00000542-200604000-00020 ◽

2006 ◽

Vol 104 (4) ◽

pp. 754-762

Author(s):

Masao Hayashi ◽

Sumedha G. Penheiter ◽

Tetsuzo Nakayama ◽

Alan R. Penheiter ◽

David O. Warner ◽

...

Keyword(s):

Smooth Muscle ◽

Airway Smooth Muscle ◽

Adrenergic Receptor ◽

Minimum Alveolar Concentration ◽

Calcium Sensitivity ◽

Functional Coupling ◽

Nucleotide Exchange ◽

Β2 Adrenergic Receptor ◽

In Vivo Animal Models

Background This study investigated whether halothane affects the functional coupling between the beta2 adrenergic receptor and the alpha subunit of its cognate stimulatory heterotrimeric guanosine-5'-triphosphate (GTP)-binding protein (Galphas). The authors hypothesized that halothane does not affect isoproterenol-promoted guanosine nucleotide exchange at Galphas and hence would not affect isoproterenol-induced relaxation of airway smooth muscle. Methods Halothane effects on isoproterenol-induced inhibition of calcium sensitivity were measured in permeabilized porcine airway smooth muscle. Galphas nucleotide exchange was measured in crude membranes prepared from COS-7 cells transfected to transiently coexpress the human beta1 or beta2 receptor each with human short Galphas. A radioactive, nonhydrolyzable analog of GTP, [S]GTPgammaS, was used as the reporter for nucleotide exchange at Galphas. Results Halothane (0.75 mm, approximately 2.8 minimum alveolar concentration [MAC] in pigs) did not affect isoproterenol-induced inhibition of calcium sensitivity. Isoproterenol caused a time- and concentration-dependent increase in Galphas nucleotide exchange. Halothane, even at concentrations of 1.5 mm (approximately 5.6 MAC), had no effect on basal Galphas nucleotide exchange in the absence of isoproterenol, whereas halothane inhibited isoproterenol-promoted Galphas nucleotide exchange in both the beta1-Galphas and beta2-Galphas expressing membranes. However, the effect was significantly greater on beta1-Galphas coupling compared with beta2-Galphas coupling, with no effect on beta2-Galphas coupling at 2.8 MAC halothane. Conclusion Halothane does not inhibit the biochemical coupling between the beta2 receptor and Galphas and hence does not affect the inhibition of calcium sensitivity induced by isoproterenol. Therefore, halothane should not affect the efficacy of beta2 agonists, as suggested by studies of in vivo animal models of asthma.

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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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Mfge8 Prevents Airway Constriction In Asthma By Opposing The Effect Of IL-13 On Airway Smooth Muscle Calcium Sensitivity

10.1164/ajrccm-conference.2012.185.1_meetingabstracts.a4146 ◽

2012 ◽

Author(s):

Makoto Kudo ◽

Yuk Yin Cheung ◽

Stephen Sakuma ◽

William McKleroy ◽

Katherine Huang ◽

...

Keyword(s):

Smooth Muscle ◽

Airway Smooth Muscle ◽

Calcium Sensitivity ◽

Airway Constriction ◽

Muscle Calcium

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

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00159.2002 ◽

2003 ◽

Vol 284 (2) ◽

pp. L324-L332 ◽

Cited By ~ 9

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.

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Effects of Intravenous Anesthetics on Ca2+ Sensitivity in Canine Tracheal Smooth Muscle

Anesthesiology ◽

10.1097/00000542-200001000-00024 ◽

2000 ◽

Vol 92 (1) ◽

pp. 133-133 ◽

Cited By ~ 13

Author(s):

Motohiko Hanazaki ◽

Keith A. Jones ◽

David O. Warner

Keyword(s):

Smooth Muscle ◽

Muscarinic Receptor ◽

Airway Smooth Muscle ◽

Common Property ◽

Calcium Sensitivity ◽

Volatile Anesthetics ◽

Tracheal Smooth Muscle ◽

Receptor Stimulation ◽

Intravenous Anesthetics ◽

Anesthetic Drugs

Background Halothane and other volatile anesthetics relax airway smooth muscle in part by decreasing the amount of force produced for a particular intracellular calcium concentration (the Ca2+ sensitivity) during muscarinic receptor stimulation. In this study, ketamine, propofol, and midazolam were evaluated to determine whether the inhibitory effect of volatile anesthetics on this signal transduction pathway is a general property of other types of anesthetic drugs. Methods A beta-escin permeabilized canine tracheal smooth muscle preparation was used. Ketamine, propofol, and midazolam, in concentrations producing near-maximal relaxation in intact airway smooth muscle (200 microM, 270 microM, and 100 microM, respectively), were applied to permeabilized muscles stimulated with calcium in either the absence or the presence of muscarinic receptor stimulation provided by acetylcholine. The effect of halothane also was evaluated. Results Confirming previous studies, halothane (0.75 mM) decreased calcium sensitivity during muscarinic receptor stimulation. None of the intravenous anesthetics studied affected Ca2+ sensitivity, either in the absence or the presence of muscarinic receptor stimulation. Conclusions Intravenous anesthetics in high concentrations directly relax canine tracheal smooth muscle without affecting Ca2+ sensitivity. The inhibition of agonist-induced increases in Ca2+ sensitivity of canine tracheal smooth is not a common property of anesthetics, but is unique to volatile agents.

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