Sample records for airways smooth muscle

  1. Mechanotransduction, asthma, and airway smooth muscle

    PubMed Central

    Fabry, Ben; Fredberg, Jeffrey J.

    2008-01-01

    Excessive force generation by airway smooth muscle is the main culprit in excessive airway narrowing during an asthma attack. The maximum force the airway smooth muscle can generate is exquisitely sensitive to muscle length fluctuations during breathing, and is governed by complex mechanotransduction events that can best be studied by a hybrid approach in which the airway wall is modeled in silico so as to set a dynamic muscle load comparable to that experienced in vivo. PMID:18836522

  2. Mechanisms of mechanical strain memory in airway smooth muscle.

    PubMed

    Kim, Hak Rim; Hai, Chi-Ming

    2005-10-01

    We evaluated the hypothesis that mechanical deformation of airway smooth muscle induces structural remodeling of airway smooth muscle cells, thereby modulating mechanical performance in subsequent contractions. This hypothesis implied that past experience of mechanical deformation was retained (or "memorized") as structural changes in airway smooth muscle cells, which modulated the cell's subsequent contractile responses. We termed this phenomenon mechanical strain memory. Preshortening has been found to induce attenuation of both force and isotonic shortening velocity in cholinergic receptor-activated airway smooth muscle. Rapid stretching of cholinergic receptor-activated airway smooth muscle from an initial length to a final length resulted in post-stretch force and myosin light chain phosphorylation that correlated significantly with initial length. Thus post-stretch muscle strips appeared to retain memory of the initial length prior to rapid stretch (mechanical strain memory). Cytoskeletal recruitment of actin- and integrin-binding proteins and Erk 1/2 MAPK appeared to be important mechanisms of mechanical strain memory. Sinusoidal length oscillation led to force attenuation during oscillation and in subsequent contractions in intact airway smooth muscle, and p38 MAPK appeared to be an important mechanism. In contrast, application of local mechanical strain to cultured airway smooth muscle cells induced local actin polymerization and cytoskeletal stiffening. It is conceivable that deep inspiration-induced bronchoprotection may be a manifestation of mechanical strain memory such that mechanical deformation from past breathing cycles modulated the mechanical performance of airway smooth muscle in subsequent cycles in a continuous and dynamic manner.

  3. Cystic Fibrosis Transmembrane Conductance Regulator in Sarcoplasmic Reticulum of Airway Smooth Muscle. Implications for Airway Contractility

    PubMed Central

    Cook, Daniel P.; Rector, Michael V.; Bouzek, Drake C.; Michalski, Andrew S.; Gansemer, Nicholas D.; Reznikov, Leah R.; Li, Xiaopeng; Stroik, Mallory R.; Ostedgaard, Lynda S.; Abou Alaiwa, Mahmoud H.; Thompson, Michael A.; Prakash, Y. S.; Krishnan, Ramaswamy; Meyerholz, David K.; Seow, Chun Y.

    2016-01-01

    Rationale: An asthma-like airway phenotype has been described in people with cystic fibrosis (CF). Whether these findings are directly caused by loss of CF transmembrane conductance regulator (CFTR) function or secondary to chronic airway infection and/or inflammation has been difficult to determine. Objectives: Airway contractility is primarily determined by airway smooth muscle. We tested the hypothesis that CFTR is expressed in airway smooth muscle and directly affects airway smooth muscle contractility. Methods: Newborn pigs, both wild type and with CF (before the onset of airway infection and inflammation), were used in this study. High-resolution immunofluorescence was used to identify the subcellular localization of CFTR in airway smooth muscle. Airway smooth muscle function was determined with tissue myography, intracellular calcium measurements, and regulatory myosin light chain phosphorylation status. Precision-cut lung slices were used to investigate the therapeutic potential of CFTR modulation on airway reactivity. Measurements and Main Results: We found that CFTR localizes to the sarcoplasmic reticulum compartment of airway smooth muscle and regulates airway smooth muscle tone. Loss of CFTR function led to delayed calcium reuptake following cholinergic stimulation and increased myosin light chain phosphorylation. CFTR potentiation with ivacaftor decreased airway reactivity in precision-cut lung slices following cholinergic stimulation. Conclusions: Loss of CFTR alters porcine airway smooth muscle function and may contribute to the airflow obstruction phenotype observed in human CF. Airway smooth muscle CFTR may represent a therapeutic target in CF and other diseases of airway narrowing. PMID:26488271

  4. Decreased airway narrowing and smooth muscle contraction in hyperresponsive pigs.

    PubMed

    Turner, Debra J; Noble, Peter B; Lucas, Matthew P; Mitchell, Howard W

    2002-10-01

    Increased smooth muscle contractility or reduced smooth muscle mechanical loads could account for the excessive airway narrowing and hyperresponsiveness seen in asthma. These mechanisms were investigated by using an allergen-induced porcine model of airway hyperresponsiveness. Airway narrowing to electric field stimulation was measured in isolated bronchial segments, over a range of transmural pressures (0-20 cmH(2)O). Contractile responses to ACh were measured in bronchial segments and in isolated tracheal smooth muscle strips isolated from control and test (ovalbumin sensitized and challenged) pigs. Test airways narrowed less than controls (P < 0.0001). Test pigs showed reduced contractility to ACh, both in isolated bronchi (P < 0.01) and smooth muscle strips (P < 0.01). Thus isolated airways from pigs exhibiting airway hyperresponsiveness in vivo are hyporesponsive in vitro. The decreased narrowing in bronchi from hyperresponsive pigs may be related to decreased smooth muscle contractility. These data suggest that mechanisms external to the airway wall may be important to the hyperresponsive nature of sensitized lungs.

  5. Length adaptation of airway smooth muscle.

    PubMed

    Bossé, Ynuk; Sobieszek, Apolinary; Paré, Peter D; Seow, Chun Y

    2008-01-01

    Many types of smooth muscle, including airway smooth muscle (ASM), are capable of generating maximal force over a large length range due to length adaptation, which is a relatively rapid process in which smooth muscle regains contractility after experiencing a force decrease induced by length fluctuation. Although the underlying mechanism is unclear, it is believed that structural malleability of smooth muscle cells is essential for the adaptation to occur. The process is triggered by strain on the cell cytoskeleton that results in a series of yet undefined biochemical and biophysical events leading to restructuring of the cytoskeleton and contractile apparatus and consequently optimization of the overlap between the myosin and actin filaments. Although length adaptability is an intrinsic property of smooth muscle, maladaptation of ASM could result in excessive constriction of the airways and the inability of deep inspirations to dilate them. In this article, we describe the phenomenon of length adaptation in ASM and some possible underlying mechanisms that involve the myosin filament assembly and disassembly. We discuss a possible role of maladaptation of ASM in the pathogenesis of asthma. We believe that length adaptation in ASM is mediated by specific proteins and their posttranslational regulations involving covalent modifications, such as phosphorylation. The discovery of these molecules and the processes that regulate their activity will greatly enhance our understanding of the basic mechanisms of ASM contraction and will suggest molecular targets to alleviate asthma exacerbation related to excessive constriction of the airways.

  6. Neutrophilic infiltration within the airway smooth muscle in patients with COPD

    PubMed Central

    Baraldo, S; Turato, G; Badin, C; Bazzan, E; Beghe, B; Zuin, R; Calabrese, F; Casoni, G; Maestrelli, P; Papi, A; Fabbri, L; Saetta, M

    2004-01-01

    Background: COPD is an inflammatory disorder characterised by chronic airflow limitation, but the extent to which airway inflammation is related to functional abnormalities is still uncertain. The interaction between inflammatory cells and airway smooth muscle may have a crucial role. Methods: To investigate the microlocalisation of inflammatory cells within the airway smooth muscle in COPD, surgical specimens obtained from 26 subjects undergoing thoracotomy (eight smokers with COPD, 10 smokers with normal lung function, and eight non-smoking controls) were examined. Immunohistochemical analysis was used to quantify the number of neutrophils, macrophages, mast cells, CD4+ and CD8+ cells localised within the smooth muscle of peripheral airways. Results: Smokers with COPD had an increased number of neutrophils and CD8+ cells in the airway smooth muscle compared with non-smokers. Smokers with normal lung function also had a neutrophilic infiltration in the airway smooth muscle, but to a lesser extent. When all the subjects were analysed as one group, neutrophilic infiltration was inversely related to forced expiratory volume in 1 second (% predicted). Conclusions: Microlocalisation of neutrophils and CD8+ cells in the airway smooth muscle in smokers with COPD suggests a possible role for these cells in the pathogenesis of smoking induced airflow limitation. PMID:15047950

  7. Matrix Metalloproteinase-1 Activation Contributes to Airway Smooth Muscle Growth and Asthma Severity

    PubMed Central

    Naveed, Shams-un-nisa; Clements, Debbie; Jackson, David J.; Philp, Christopher; Billington, Charlotte K.; Soomro, Irshad; Reynolds, Catherine; Harrison, Timothy W.; Johnston, Sebastian L.; Shaw, Dominick E.

    2017-01-01

    Rationale: Matrix metalloproteinase-1 (MMP-1) and mast cells are present in the airways of people with asthma. Objectives: To investigate whether MMP-1 could be activated by mast cells and increase asthma severity. Methods: Patients with stable asthma and healthy control subjects underwent spirometry, methacholine challenge, and bronchoscopy, and their airway smooth muscle cells were grown in culture. A second asthma group and control subjects had symptom scores, spirometry, and bronchoalveolar lavage before and after rhinovirus-induced asthma exacerbations. Extracellular matrix was prepared from decellularized airway smooth muscle cultures. MMP-1 protein and activity were assessed. Measurements and Main Results: Airway smooth muscle cells generated pro–MMP-1, which was proteolytically activated by mast cell tryptase. Airway smooth muscle treated with activated mast cell supernatants produced extracellular matrix, which enhanced subsequent airway smooth muscle growth by 1.5-fold (P < 0.05), which was dependent on MMP-1 activation. In asthma, airway pro–MMP-1 was 5.4-fold higher than control subjects (P = 0.002). Mast cell numbers were associated with airway smooth muscle proliferation and MMP-1 protein associated with bronchial hyperresponsiveness. During exacerbations, MMP-1 activity increased and was associated with fall in FEV1 and worsening asthma symptoms. Conclusions: MMP-1 is activated by mast cell tryptase resulting in a proproliferative extracellular matrix. In asthma, mast cells are associated with airway smooth muscle growth, MMP-1 levels are associated with bronchial hyperresponsiveness, and MMP-1 activation are associated with exacerbation severity. Our findings suggest that airway smooth muscle/mast cell interactions contribute to asthma severity by transiently increasing MMP activation, airway smooth muscle growth, and airway responsiveness. PMID:27967204

  8. Critical role of actin-associated proteins in smooth muscle contraction, cell proliferation, airway hyperresponsiveness and airway remodeling.

    PubMed

    Tang, Dale D

    2015-10-30

    Asthma is characterized by airway hyperresponsiveness and airway remodeling, which are largely attributed to increased airway smooth muscle contractility and cell proliferation. It is known that both chemical and mechanical stimulation regulates smooth muscle contraction. Recent studies suggest that contractile activation and mechanical stretch induce actin cytoskeletal remodeling in smooth muscle. However, the mechanisms that control actin cytoskeletal reorganization are not completely elucidated. This review summarizes our current understanding regarding how actin-associated proteins may regulate remodeling of the actin cytoskeleton in airway smooth muscle. In particular, there is accumulating evidence to suggest that Abelson tyrosine kinase (Abl) plays a critical role in regulating airway smooth muscle contraction and cell proliferation in vitro, and airway hyperresponsiveness and remodeling in vivo. These studies indicate that Abl may be a novel target for the development of new therapy to treat asthma.

  9. Myosin Light Chain Kinase Is Necessary for Tonic Airway Smooth Muscle Contraction*

    PubMed Central

    Zhang, Wen-Cheng; Peng, Ya-Jing; Zhang, Gen-Sheng; He, Wei-Qi; Qiao, Yan-Ning; Dong, Ying-Ying; Gao, Yun-Qian; Chen, Chen; Zhang, Cheng-Hai; Li, Wen; Shen, Hua-Hao; Ning, Wen; Kamm, Kristine E.; Stull, James T.; Gao, Xiang; Zhu, Min-Sheng

    2010-01-01

    Different interacting signaling modules involving Ca2+/calmodulin-dependent myosin light chain kinase, Ca2+-independent regulatory light chain phosphorylation, myosin phosphatase inhibition, and actin filament-based proteins are proposed as specific cellular mechanisms involved in the regulation of smooth muscle contraction. However, the relative importance of specific modules is not well defined. By using tamoxifen-activated and smooth muscle-specific knock-out of myosin light chain kinase in mice, we analyzed its role in tonic airway smooth muscle contraction. Knock-out of the kinase in both tracheal and bronchial smooth muscle significantly reduced contraction and myosin phosphorylation responses to K+-depolarization and acetylcholine. Kinase-deficient mice lacked bronchial constrictions in normal and asthmatic airways, whereas the asthmatic inflammation response was not affected. These results indicate that myosin light chain kinase acts as a central participant in the contractile signaling module of tonic smooth muscle. Importantly, contractile airway smooth muscles are necessary for physiological and asthmatic airway resistance. PMID:20018858

  10. What evidence implicates airway smooth muscle in the cause of BHR?

    PubMed

    Dulin, Nickolai O; Fernandes, Darren J; Dowell, Maria; Bellam, Shashi; McConville, John; Lakser, Oren; Mitchell, Richard; Camoretti-Mercado, Blanca; Kogut, Paul; Solway, Julian

    2003-02-01

    Bronchial hyperresponsiveness (BHR), the occurrence of excessive bronchoconstriction in response to relatively small constrictor stimuli, is a cardinal feature of asthma. Here, we consider the role that airway smooth muscle might play in the generation of BHR. The weight of evidence suggests that smooth muscle isolated from asthmatic tissues exhibits normal sensitivity to constrictor agonists when studied during isometric contraction, but the increased muscle mass within asthmatic airways might generate more total force than the lesser amount of muscle found in normal bronchi. Another salient difference between asthmatic and normal individuals lies in the effect of deep inhalation (DI) on bronchoconstriction. DI often substantially reverses induced bronchoconstriction in normals, while it often has much less effect on spontaneous or induced bronchoconstriction in asthmatics. It has been proposed that abnormal dynamic aspects of airway smooth muscle contraction velocity of contraction or plasticity- elasticity balance might underlie the abnormal DI response in asthma. We suggest a speculative model in which abnormally long actin filaments might account for abnormally increased elasticity of contracted airway smooth muscle.

  11. Endogenous gamma-aminobutyric acid modulates tonic guinea pig airway tone and propofol-induced airway smooth muscle relaxation.

    PubMed

    Gallos, George; Gleason, Neil R; Virag, Laszlo; Zhang, Yi; Mizuta, Kentaro; Whittington, Robert A; Emala, Charles W

    2009-04-01

    Emerging evidence indicates that an endogenous autocrine/paracrine system involving gamma-aminobutyric acid (GABA) is present in airways. GABAA channels, GABAB receptors, and the enzyme that synthesizes GABA have been identified in airway epithelium and smooth muscle. However, the endogenous ligand itself, GABA, has not been measured in airway tissues. The authors sought to demonstrate that GABA is released in response to contractile agonists and tonically contributes a prorelaxant component to contracted airway smooth muscle. The amount and cellular localization of GABA in upper guinea pig airways under resting and contracted tone was determined by high pressure liquid chromatography and immunohistochemistry, respectively. The contribution that endogenous GABA imparts on the maintenance of airway smooth muscle acetylcholine-induced contraction was assessed in intact guinea pig airway tracheal rings using selective GABAA antagonism (gabazine) under resting or acetylcholine-contracted conditions. The ability of an allosteric agent (propofol) to relax a substance P-induced relaxation in an endogenous GABA-dependent manner was assessed. GABA levels increased and localized to airway smooth muscle after contractile stimuli in guinea pig upper airways. Acetylcholine-contracted guinea pig tracheal rings exhibited an increase in contracted force upon addition of the GABAA antagonist gabazine that was subsequently reversed by the addition of the GABAA agonist muscimol. Propofol dose-dependently relaxed a substance P contraction that was blocked by gabazine. These studies demonstrate that GABA is endogenously present and increases after contractile stimuli in guinea pig upper airways and that endogenous GABA contributes a tonic prorelaxant component in the maintenance of airway smooth muscle tone.

  12. Endogenous γ-aminobutyric Acid Modulates Tonic Guinea Pig Airway Tone and Propofol-induced Airway Smooth Muscle Relaxation

    PubMed Central

    Gallos, George; Gleason, Neil R.; Virag, Laszlo; Zhang, Yi; Mizuta, Kentauro; Whittington, Robert A.; Emala, Charles W.

    2009-01-01

    Background Emerging evidence indicates that an endogenous autocrine/paracrine system involving γ-aminobutyric acid (GABA) is present in airways. GABAA channels, GABAB receptors and the enzyme that synthesizes GABA have been identified in airway epithelium and smooth muscle. However, the endogenous ligand itself, GABA, has not been measured in airway tissues. We sought to demonstrate that GABA is released in response to contractile agonists and tonically contributes a pro-relaxant component to contracted airway smooth muscle. Methods The amount and cellular localization of GABA in upper guinea pig airways under resting and contracted tone was determined by high pressure liquid chromatography and immunohistochemistry, respectively. The contribution that endogenous GABA imparts on the maintenance of airway smooth muscle acetylcholine-induced contraction was assessed in intact guinea pig airway tracheal rings using selective GABAA antagonism (gabazine) under resting or acetylcholine-contracted conditions. The ability of an allosteric agent (propofol) to relax a substance P-induced relaxation in an endogenous GABA-dependent manner was assessed. Results GABA levels increased and localized to airway smooth muscle following contractile stimuli in guinea pig upper airways. Acetylcholine-contracted guinea pig tracheal rings exhibited an increase in contracted force upon addition of the GABAA antagonist gabazine which was subsequently reversed by the addition of the GABAA agonist muscimol. Propofol dose-dependently relaxed a substance P contraction that was blocked by gabazine. Conclusion These studies demonstrate that GABA is endogenously present and increases following contractile stimuli in guinea pig upper airways and that endogenous GABA contributes a tonic pro-relaxant component in the maintenance of airway smooth muscle tone. PMID:19322939

  13. Chloride channel blockers promote relaxation of TEA-induced contraction in airway smooth muscle

    PubMed Central

    Yim, Peter D.; Gallos, George; Perez-zoghbi, Jose F.; Trice, Jacquelyn; Zhang, Yi; Siviski, Matthew; Sonett, Joshua; Emala, Charles W.

    2014-01-01

    Enhanced airway smooth muscle (ASM) contraction is an important component in the pathophysiology of asthma. We have shown that ligand gated chloride channels modulate ASM contractile tone during the maintenance phase of an induced contraction, however the role of chloride flux in depolarization-induced contraction remains incompletely understood. To better understand the role of chloride flux under these conditions, muscle force (human ASM, guinea pig ASM), peripheral small airway luminal area (rat ASM) and airway smooth muscle plasma membrane electrical potentials (human cultured ASM) were measured. We found ex vivo guinea pig airway rings, human ASM strips and small peripheral airways in rat lungs slices relaxed in response to niflumic acid following depolarization-induced contraction induced by K+ channel blockade with tetraethylammonium chloride (TEA). In isolated human airway smooth muscle cells TEA induce depolarization as measured by a fluorescent indicator or whole cell patch clamp and this depolarization was reversed by niflumic acid. These findings demonstrate that ASM depolarization induced contraction is dependent on chloride channel activity. Targeting of chloride channels may be a novel approach to relax hypercontractile airway smooth muscle in bronchoconstrictive disorders. PMID:24662476

  14. Chloride channel blockers promote relaxation of TEA-induced contraction in airway smooth muscle.

    PubMed

    Yim, Peter D; Gallos, George; Perez-Zoghbi, Jose F; Trice, Jacquelyn; Zhang, Yi; Siviski, Matthew; Sonett, Joshua; Emala, Charles W

    2013-01-01

    Enhanced airway smooth muscle (ASM) contraction is an important component in the pathophysiology of asthma. We have shown that ligand gated chloride channels modulate ASM contractile tone during the maintenance phase of an induced contraction, however the role of chloride flux in depolarization-induced contraction remains incompletely understood. To better understand the role of chloride flux under these conditions, muscle force (human ASM, guinea pig ASM), peripheral small airway luminal area (rat ASM) and airway smooth muscle plasma membrane electrical potentials (human cultured ASM) were measured. We found ex vivo guinea pig airway rings, human ASM strips and small peripheral airways in rat lungs slices relaxed in response to niflumic acid following depolarization-induced contraction induced by K(+) channel blockade with tetraethylammonium chloride (TEA). In isolated human airway smooth muscle cells TEA induce depolarization as measured by a fluorescent indicator or whole cell patch clamp and this depolarization was reversed by niflumic acid. These findings demonstrate that ASM depolarization induced contraction is dependent on chloride channel activity. Targeting of chloride channels may be a novel approach to relax hypercontractile airway smooth muscle in bronchoconstrictive disorders.

  15. Length oscillation induces force potentiation in infant guinea pig airway smooth muscle.

    PubMed

    Wang, Lu; Chitano, Pasquale; Murphy, Thomas M

    2005-12-01

    Deep inspiration counteracts bronchospasm in normal subjects but triggers further bronchoconstriction in hyperresponsive airways. Although the exact mechanisms for this contrary response by normal and hyperresponsive airways are unclear, it has been suggested that the phenomenon is related to changes in force-generating ability of airway smooth muscle after mechanical oscillation. It is known that healthy immature airways of both humans and animals exhibit hyperresponsiveness. We hypothesize that the profile of active force generation after mechanical oscillation changes with maturation and that this change contributes to the expression of airway hyperresponsiveness in juveniles. We examined the effect of an acute sinusoidal length oscillation on the force-generating ability of tracheal smooth muscle from 1 wk, 3 wk, and 2- to 3-mo-old guinea pigs. We found that the length oscillation produced 15-20% initial reduction in active force equally in all age groups. This was followed by a force recovery profile that displayed striking maturation-specific features. Unique to tracheal strips from 1-wk-old animals, active force potentiated beyond the maximal force generated before oscillation. We also found that actin polymerization was required in force recovery and that prostanoids contributed to the maturation-specific force potentiation in immature airway smooth muscle. Our results suggest a potentiated mechanosensitive contractile property of hyperresponsive airway smooth muscle. This can account for further bronchoconstriction triggered by deep inspiration in hyperresponsive airways.

  16. Activation of endogenous GABAA channels on airway smooth muscle potentiates isoproterenol-mediated relaxation.

    PubMed

    Gallos, George; Gleason, Neil R; Zhang, Yi; Pak, Sang-Woo; Sonett, J R; Yang, Jay; Emala, Charles W

    2008-12-01

    Reactive airway disease predisposes patients to episodes of acute smooth muscle mediated bronchoconstriction. We have for the first time recently demonstrated the expression and function of endogenous ionotropic GABA(A) channels on airway smooth muscle cells. We questioned whether endogenous GABA(A) channels on airway smooth muscle could augment beta-agonist-mediated relaxation. Guinea pig tracheal rings or human bronchial airway smooth muscles were equilibrated in organ baths with continuous digital tension recordings. After pretreatment with or without the selective GABA(A) antagonist gabazine (100 muM), airway muscle was contracted with acetylcholine or beta-ala neurokinin A, followed by relaxation induced by cumulatively increasing concentrations of isoproterenol (1 nM to 1 muM) in the absence or presence of the selective GABA(A) agonist muscimol (10-100 muM). In separate experiments, guinea pig tracheal rings were pretreated with the large conductance K(Ca) channel blocker iberiotoxin (100 nM) after an EC(50) contraction with acetylcholine but before cumulatively increasing concentrations of isoproterenol (1 nM to 1 uM) in the absence or presence of muscimol (100 uM). GABA(A) activation potentiated the relaxant effects of isoproterenol after an acetylcholine or tachykinin-induced contraction in guinea pig tracheal rings or an acetylcholine-induced contraction in human endobronchial smooth muscle. This muscimol-induced potentiation of relaxation was abolished by gabazine pretreatment but persisted after blockade of the maxi K(Ca) channel. Selective activation of endogenous GABA(A) receptors significantly augments beta-agonist-mediated relaxation of guinea pig and human airway smooth muscle, which may have important therapeutic implications for patients in severe bronchospasm.

  17. Length-dependent modulation of cytoskeletal remodeling and mechanical energetics in airway smooth muscle.

    PubMed

    Kim, Hak Rim; Liu, Katrina; Roberts, Thomas J; Hai, Chi-Ming

    2011-06-01

    Actin cytoskeletal remodeling is an important mechanism of airway smooth muscle (ASM) contraction. We tested the hypothesis that mechanical strain modulates the cholinergic receptor-mediated cytoskeletal recruitment of actin-binding and integrin-binding proteins in intact airway smooth muscle, thereby regulating the mechanical energetics of airway smooth muscle. We found that the carbachol-stimulated cytoskeletal recruitment of actin-related protein-3 (Arp3), metavinculin, and talin were up-regulated at short muscle lengths and down-regulated at long muscle lengths, suggesting that the actin cytoskeleton--integrin complex becomes enriched in cross-linked and branched actin filaments in shortened ASM. The mechanical energy output/input ratio during sinusoidal length oscillation was dependent on muscle length, oscillatory amplitude, and cholinergic activation. The enhancing effect of cholinergic stimulation on mechanical energy output/input ratio at short and long muscle lengths may be explained by the length-dependent modulation of cytoskeletal recruitment and crossbridge cycling, respectively. We postulate that ASM functions as a hybrid biomaterial, capable of switching between operating as a cytoskeleton-based mechanical energy store at short muscle lengths to operating as an actomyosin-powered mechanical energy generator at long muscle lengths. This postulate predicts that targeting the signaling molecules involved in cytoskeletal recruitment may provide a novel approach to dilating collapsed airways in obstructive airway disease.

  18. Dynamic equilibration of airway smooth muscle contraction during physiological loading.

    PubMed

    Latourelle, Jeanne; Fabry, Ben; Fredberg, Jeffrey J

    2002-02-01

    Airway smooth muscle contraction is the central event in acute airway narrowing in asthma. Most studies of isolated muscle have focused on statically equilibrated contractile states that arise from isometric or isotonic contractions. It has recently been established, however, that muscle length is determined by a dynamically equilibrated state of the muscle in which small tidal stretches associated with the ongoing action of breathing act to perturb the binding of myosin to actin. To further investigate this phenomenon, we describe in this report an experimental method for subjecting isolated muscle to a dynamic microenvironment designed to closely approximate that experienced in vivo. Unlike previous methods that used either time-varying length control, force control, or time-invariant auxotonic loads, this method uses transpulmonary pressure as the controlled variable, with both muscle force and muscle length free to adjust as they would in vivo. The method was implemented by using a servo-controlled lever arm to load activated airway smooth muscle strips with transpulmonary pressure fluctuations of increasing amplitude, simulating the action of breathing. The results are not consistent with classical ideas of airway narrowing, which rest on the assumption of a statically equilibrated contractile state; they are consistent, however, with the theory of perturbed equilibria of myosin binding. This experimental method will allow for quantitative experimental evaluation of factors that were previously outside of experimental control, including sensitivity of muscle length to changes of tidal volume, changes of lung volume, shape of the load characteristic, loss of parenchymal support and inflammatory thickening of airway wall compartments.

  19. Airway smooth muscle responsiveness from dogs with airway hyperresponsiveness after O/sub 3/ inhalation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jones, G.L.; O'Byrne, P.M.; Pashley, M.

    1988-07-01

    Airway hyperresponsiveness occurs after inhalation of O3 in dogs. The purpose of this study was to examine the responsiveness of trachealis smooth muscle in vitro to electrical field stimulation, exogenous acetylcholine, and potassium chloride from dogs with airway hyperresponsiveness after inhaled O3 in vivo and to compare this with the responsiveness of trachealis muscle from control dogs. In addition, excitatory junction potentials were measured with the use of single and double sucrose gap techniques in both groups of dogs to determine whether inhaled O3 affects the release of acetylcholine from parasympathetic nerves in trachealis muscle. Airway hyperresponsiveness developed in allmore » dogs after inhaled O3 (3 ppm for 30 min). The acetylcholine provocative concentration decreased from 4.11 mg/ml before O3 inhalation to 0.66 mg/ml after O3 (P less than 0.0001). The acetylcholine provocative concentration increased slightly after control inhalation of dry room air. Airway smooth muscle showed increased responses to both electrical field stimulation and exogenous acetylcholine but not to potassium chloride in preparations from dogs with airway hyperresponsiveness in vivo. The increased response to electrical field stimulation was not associated with a change in excitatory junctional potentials. These results suggest that a postjunctional alteration in trachealis muscle function occurs after inhaled O3 in dogs, which may account for airway hyperresponsiveness after O3 in vivo.« less

  20. Local small airway epithelial injury induces global smooth muscle contraction and airway constriction.

    PubMed

    Zhou, Jian; Alvarez-Elizondo, Martha B; Botvinick, Elliot; George, Steven C

    2012-02-01

    Small airway epithelial cells form a continuous sheet lining the conducting airways, which serves many functions including a physical barrier to protect the underlying tissue. In asthma, injury to epithelial cells can occur during bronchoconstriction, which may exacerbate airway hyperreactivity. To investigate the role of epithelial cell rupture in airway constriction, laser ablation was used to precisely rupture individual airway epithelial cells of small airways (<300-μm diameter) in rat lung slices (∼250-μm thick). Laser ablation of single epithelial cells using a femtosecond laser reproducibly induced airway contraction to ∼70% of the original cross-sectional area within several seconds, and the contraction lasted for up to 40 s. The airway constriction could be mimicked by mechanical rupture of a single epithelial cell using a sharp glass micropipette but not with a blunt glass pipette. These results suggest that soluble mediators released from the wounded epithelial cell induce global airway contraction. To confirm this hypothesis, the lysate of primary human small airway epithelial cells stimulated a similar airway contraction. Laser ablation of single epithelial cells triggered a single instantaneous Ca(2+) wave in the epithelium, and multiple Ca(2+) waves in smooth muscle cells, which were delayed by several seconds. Removal of extracellular Ca(2+) or decreasing intracellular Ca(2+) both blocked laser-induced airway contraction. We conclude that local epithelial cell rupture induces rapid and global airway constriction through release of soluble mediators and subsequent Ca(2+)-dependent smooth muscle shortening.

  1. Airway hyperresponsiveness; smooth muscle as the principal actor

    PubMed Central

    Lauzon, Anne-Marie; Martin, James G.

    2016-01-01

    Airway hyperresponsiveness (AHR) is a defining characteristic of asthma that refers to the capacity of the airways to undergo exaggerated narrowing in response to stimuli that do not result in comparable degrees of airway narrowing in healthy subjects. Airway smooth muscle (ASM) contraction mediates airway narrowing, but it remains uncertain as to whether the smooth muscle is intrinsically altered in asthmatic subjects or is responding abnormally as a result of the milieu in which it sits. ASM in the trachea or major bronchi does not differ in its contractile characteristics in asthmatics, but the more pertinent peripheral airways await complete exploration. The mass of ASM is increased in many but not all asthmatics and therefore cannot be a unifying hypothesis for AHR, although when increased in mass it may contribute to AHR. The inability of a deep breath to reverse or prevent bronchial narrowing in asthma may reflect an intrinsic difference in the mechanisms that lead to softening of contracted ASM when subjected to stretch. Cytokines such as interleukin-13 and tumor necrosis factor-α promote a more contractile ASM phenotype. The composition and increased stiffness of the matrix in which ASM is embedded promotes a more proliferative and pro-inflammatory ASM phenotype, but the expected dedifferentiation and loss of contractility have not been shown. Airway epithelium may drive ASM proliferation and/or molecular remodeling in ways that may lead to AHR. In conclusion, AHR is likely multifactorial in origin, reflecting the plasticity of ASM properties in the inflammatory environment of the asthmatic airway. PMID:26998246

  2. The relationship between bronchial hyperresponsiveness to methacholine and airway smooth muscle structure and reactivity.

    PubMed

    Armour, C L; Black, J L; Berend, N; Woolcock, A J

    1984-11-01

    The airway responsiveness of a group of 25 patients scheduled for lung resection was studied. 10 of 25 patients had a greater than or equal to 20% fall in FEV1 in response to inhaled methacholine (responders), with PD20 FEV1 values ranging from 0.6 to 7.3 mumol. Methacholine did not induce a 20% fall in FEV1 in 15 patients (non-responders). The sensitivity to carbachol and histamine of the bronchial smooth muscle resected from these patients was similar in tissue from responders and non-responders. There was no correlation between in vivo responsiveness to methacholine and in vitro sensitivity to carbachol or histamine. The volume of smooth muscle in some of these airway preparations was quantitated. There was a significant correlation between the maximum tension change in response to histamine and the volume of smooth muscle in each airway. There was no similar correlation for carbachol. The in vivo responsiveness to methacholine and in vitro sensitivity to histamine or carbachol was not related to the degree of inflammation in the airways studied. It is concluded that in vivo responsiveness cannot be explained in terms of smooth muscle sensitivity and that there may be differences between histamine and carbachol in the mechanism of contraction of airway smooth muscle.

  3. Rho Kinase (ROCK) collaborates with Pak to Regulate Actin Polymerization and Contraction in Airway Smooth Muscle.

    PubMed

    Zhang, Wenwu; Bhetwal, Bhupal P; Gunst, Susan J

    2018-05-10

    The mechanisms by which Rho kinase (ROCK) regulates airway smooth muscle contraction were determined in tracheal smooth muscle tissues. ROCK may mediate smooth muscle contraction by inhibiting myosin regulatory light chain (RLC) phosphatase. ROCK can also regulate F-actin dynamics during cell migration, and actin polymerization is critical for airway smooth muscle contraction. Our results show that ROCK does not regulate airway smooth muscle contraction by inhibiting myosin RLC phosphatase or by stimulating myosin RLC phosphorylation. We find that ROCK regulates airway smooth muscle contraction by activating the serine-threonine kinase Pak, which mediates the activation of Cdc42 and Neuronal-Wiskott-Aldrich Syndrome protein (N-WASp). N-WASP transmits signals from cdc42 to the Arp2/3 complex for the nucleation of actin filaments. These results demonstrate a novel molecular function for ROCK in the regulation of Pak and cdc42 activation that is critical for the processes of actin polymerization and contractility in airway smooth muscle. Rho kinase (ROCK), a RhoA GTPase effector, can regulate the contraction of airway and other smooth muscle tissues. In some tissues, ROCK can inhibit myosin regulatory light chain (RLC) phosphatase, which increases the phosphorylation of myosin RLC and promotes smooth muscle contraction. ROCK can also regulate cell motility and migration by affecting F-actin dynamics. Actin polymerization is stimulated by contractile agonists in airway smooth muscle tissues and is required for contractile tension development in addition to myosin RLC phosphorylation. We investigated the mechanisms by which ROCK regulates the contractility of tracheal smooth muscle tissues by expressing a kinase inactive mutant of ROCK, ROCK-K121G, in the tissues or by treating them with the ROCK inhibitor, H-1152P. Our results show no role for ROCK in the regulation of non-muscle or smooth muscle myosin RLC phosphorylation during contractile stimulation in this tissue

  4. Local small airway epithelial injury induces global smooth muscle contraction and airway constriction

    PubMed Central

    Zhou, Jian; Alvarez-Elizondo, Martha B.; Botvinick, Elliot

    2012-01-01

    Small airway epithelial cells form a continuous sheet lining the conducting airways, which serves many functions including a physical barrier to protect the underlying tissue. In asthma, injury to epithelial cells can occur during bronchoconstriction, which may exacerbate airway hyperreactivity. To investigate the role of epithelial cell rupture in airway constriction, laser ablation was used to precisely rupture individual airway epithelial cells of small airways (<300-μm diameter) in rat lung slices (∼250-μm thick). Laser ablation of single epithelial cells using a femtosecond laser reproducibly induced airway contraction to ∼70% of the original cross-sectional area within several seconds, and the contraction lasted for up to 40 s. The airway constriction could be mimicked by mechanical rupture of a single epithelial cell using a sharp glass micropipette but not with a blunt glass pipette. These results suggest that soluble mediators released from the wounded epithelial cell induce global airway contraction. To confirm this hypothesis, the lysate of primary human small airway epithelial cells stimulated a similar airway contraction. Laser ablation of single epithelial cells triggered a single instantaneous Ca2+ wave in the epithelium, and multiple Ca2+ waves in smooth muscle cells, which were delayed by several seconds. Removal of extracellular Ca2+ or decreasing intracellular Ca2+ both blocked laser-induced airway contraction. We conclude that local epithelial cell rupture induces rapid and global airway constriction through release of soluble mediators and subsequent Ca2+-dependent smooth muscle shortening. PMID:22114176

  5. The biophysics of asthmatic airway smooth muscle.

    PubMed

    Stephens, Newman L; Li, Weilong; Jiang, He; Unruh, H; Ma, Xuefei

    2003-09-16

    It is clear that significant advances have been made in the understanding of the physiology, biochemistry and molecular biology of airway smooth muscle (ASM) contraction and how the knowledge obtained from these approaches may be used to elucidate the pathogenesis of asthma. Not to belittle other theories of smooth muscle contraction extant in the field, perhaps the most outstanding development has been the formulation of plasticity theory. This may radically alter our understanding of smooth muscle contraction. Its message is that while shortening velocity and capacity are linear functions of length, active force is length independent. These changes are explained by the ability of thick filament protein to depolymerize at short lengths and to increase numbers of contractile units in series at lengths greater than optimal length or L(ref). Other advances are represented by the report that the major part of ASM shortening is complete within the initial first 20% of contraction time, that the nature and history of loading determine the extent of shortening and that these findings can be explained by the finding that the crossbridges are cycling four times faster than in the remaining time. Another unexpected finding is that late in the course of isotonic relaxation the muscle undergoes spontaneous activation which delays relaxation and smoothes it out; speculatively this could minimize turbulence of airflow. On the applied front evidence now shows the shortening ability of bronchial smooth muscle of human subjects of asthma is significantly increased. Measurements also indicate that increased smooth muscle myosin light chain kinase content, via increased actomyosin ATPase activity could be responsible for the changes in contractility.

  6. How the airway smooth muscle in cystic fibrosis reacts in proinflammatory conditions: implications for airway hyper-responsiveness and asthma in cystic fibrosis.

    PubMed

    McCuaig, Sarah; Martin, James G

    2013-04-01

    Among patients with cystic fibrosis there is a high prevalence (40-70%) of asthma signs and symptoms such as cough and wheezing and airway hyper-responsiveness to inhaled histamine or methacholine. Whether these abnormal airway responses are due to a primary deficiency in the cystic fibrosis transmembrane conductance regulator (CFTR) or are secondary to the inflammatory environment in the cystic fibrosis lungs is not clear. A role for the CFTR in smooth muscle function is emerging, and alterations in contractile signalling have been reported in CFTR-deficient airway smooth muscle. Persistent bacterial infection, especially with Pseudomonas aeruginosa, stimulates interleukin-8 release from the airway epithelium, resulting in neutrophilic inflammation. Increased neutrophilia and skewing of CFTR-deficient T-helper cells to type 2 helper T cells creates an inflammatory environment characterised by high concentrations of tumour necrosis factor α, interleukin-8, and interleukin-13, which might all contribute to increased contractility of airway smooth muscle in cystic fibrosis. An emerging role of interleukin-17, which is raised in patients with cystic fibrosis, in airway smooth muscle proliferation and hyper-responsiveness is apparent. Increased understanding of the molecular mechanisms responsible for the altered smooth muscle physiology in patients with cystic fibrosis might provide insight into airway dysfunction in this disease. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. A mathematical model of airway and pulmonary arteriole smooth muscle.

    PubMed

    Wang, Inga; Politi, Antonio Z; Tania, Nessy; Bai, Yan; Sanderson, Michael J; Sneyd, James

    2008-03-15

    Airway hyperresponsiveness is a major characteristic of asthma and is believed to result from the excessive contraction of airway smooth muscle cells (SMCs). However, the identification of the mechanisms responsible for airway hyperresponsiveness is hindered by our limited understanding of how calcium (Ca2+), myosin light chain kinase (MLCK), and myosin light chain phosphatase (MLCP) interact to regulate airway SMC contraction. In this work, we present a modified Hai-Murphy cross-bridge model of SMC contraction that incorporates Ca2+ regulation of MLCK and MLCP. A comparative fit of the model simulations to experimental data predicts 1), that airway and arteriole SMC contraction is initiated by fast activation by Ca2+ of MLCK; 2), that airway SMC, but not arteriole SMC, is inhibited by a slower activation by Ca2+ of MLCP; and 3), that the presence of a contractile agonist inhibits MLCP to enhance the Ca2+ sensitivity of airway and arteriole SMCs. The implication of these findings is that murine airway SMCs exploit a Ca2+-dependent mechanism to favor a default state of relaxation. The rate of SMC relaxation is determined principally by the rate of release of the latch-bridge state, which is predicted to be faster in airway than in arteriole. In addition, the model also predicts that oscillations in calcium concentration, commonly observed during agonist-induced smooth muscle contraction, cause a significantly greater contraction than an elevated steady calcium concentration.

  8. A Mathematical Model of Airway and Pulmonary Arteriole Smooth Muscle

    PubMed Central

    Wang, Inga; Politi, Antonio Z.; Tania, Nessy; Bai, Yan; Sanderson, Michael J.; Sneyd, James

    2008-01-01

    Airway hyperresponsiveness is a major characteristic of asthma and is believed to result from the excessive contraction of airway smooth muscle cells (SMCs). However, the identification of the mechanisms responsible for airway hyperresponsiveness is hindered by our limited understanding of how calcium (Ca2+), myosin light chain kinase (MLCK), and myosin light chain phosphatase (MLCP) interact to regulate airway SMC contraction. In this work, we present a modified Hai-Murphy cross-bridge model of SMC contraction that incorporates Ca2+ regulation of MLCK and MLCP. A comparative fit of the model simulations to experimental data predicts 1), that airway and arteriole SMC contraction is initiated by fast activation by Ca2+ of MLCK; 2), that airway SMC, but not arteriole SMC, is inhibited by a slower activation by Ca2+ of MLCP; and 3), that the presence of a contractile agonist inhibits MLCP to enhance the Ca2+ sensitivity of airway and arteriole SMCs. The implication of these findings is that murine airway SMCs exploit a Ca2+-dependent mechanism to favor a default state of relaxation. The rate of SMC relaxation is determined principally by the rate of release of the latch-bridge state, which is predicted to be faster in airway than in arteriole. In addition, the model also predicts that oscillations in calcium concentration, commonly observed during agonist-induced smooth muscle contraction, cause a significantly greater contraction than an elevated steady calcium concentration. PMID:18065464

  9. Modelling airway smooth muscle passive length adaptation via thick filament length distributions

    PubMed Central

    Donovan, Graham M.

    2013-01-01

    We present a new model of airway smooth muscle (ASM), which surrounds and constricts every airway in the lung and thus plays a central role in the airway constriction associated with asthma. This new model of ASM is based on an extension of sliding filament/crossbridge theory, which explicitly incorporates the length distribution of thick sliding filaments to account for a phenomenon known as dynamic passive length adaptation; the model exhibits good agreement with experimental data for ASM force–length behaviour across multiple scales. Principally these are (nonlinear) force–length loops at short timescales (seconds), parabolic force–length curves at medium timescales (minutes) and length adaptation at longer timescales. This represents a significant improvement on the widely-used cross-bridge models which work so well in or near the isometric regime, and may have significant implications for studies which rely on crossbridge or other dynamic airway smooth muscle models, and thus both airway and lung dynamics. PMID:23721681

  10. {beta}-Catenin regulates airway smooth muscle contraction.

    PubMed

    Jansen, Sepp R; Van Ziel, Anna M; Baarsma, Hoeke A; Gosens, Reinoud

    2010-08-01

    beta-Catenin is an 88-kDa member of the armadillo family of proteins that is associated with the cadherin-catenin complex in the plasma membrane. This complex interacts dynamically with the actin cytoskeleton to stabilize adherens junctions, which play a central role in force transmission by smooth muscle cells. Therefore, in the present study, we hypothesized a role for beta-catenin in the regulation of smooth muscle force production. beta-Catenin colocalized with smooth muscle alpha-actin (sm-alpha-actin) and N-cadherin in plasma membrane fractions and coimmunoprecipitated with sm-alpha-actin and N-cadherin in lysates of bovine tracheal smooth muscle (BTSM) strips. Moreover, immunocytochemistry of cultured BTSM cells revealed clear and specific colocalization of sm-alpha-actin and beta-catenin at the sites of cell-cell contact. Treatment of BTSM strips with the pharmacological beta-catenin/T cell factor-4 (TCF4) inhibitor PKF115-584 (100 nM) reduced beta-catenin expression in BTSM whole tissue lysates and in plasma membrane fractions and reduced maximal KCl- and methacholine-induced force production. These changes in force production were not accompanied by changes in the expression of sm-alpha-actin or sm-myosin heavy chain (MHC). Likewise, small interfering RNA (siRNA) knockdown of beta-catenin in BTSM strips reduced beta-catenin expression and attenuated maximal KCl- and methacholine-induced contractions without affecting sm-alpha-actin or sm-MHC expression. Conversely, pharmacological (SB-216763, LiCl) or insulin-induced inhibition of glycogen synthase kinase-3 (GSK-3) enhanced the expression of beta-catenin and augmented maximal KCl- and methacholine-induced contractions. We conclude that beta-catenin is a plasma membrane-associated protein in airway smooth muscle that regulates active tension development, presumably by stabilizing cell-cell contacts and thereby supporting force transmission between neighboring cells.

  11. Airway smooth muscle contraction - perspectives on past, present and future.

    PubMed

    Mitchell, H W

    2009-10-01

    Past and contemporary views of airway smooth muscle (ASM) have led to a high level of understanding of the control and intracellular regulation of force or shortening of ASM and of its possible role in airway disease. As well as the multitude of cellular mechanisms that regulate ASM contraction, a number of structural and mechanical factors, which are only present at the airway and lung level, provide overriding control over ASM. With new knowledge about the cellular physiology and biology of ASM, there is increasing need to understand how ASM contraction is regulated and expressed at these airway and system levels.

  12. A novel bronchial ring bioassay for the evaluation of small airway smooth muscle function in mice.

    PubMed

    Liu, John Q; Yang, Dennis; Folz, Rodney J

    2006-08-01

    Advances in our understanding of murine airway physiology have been hindered by the lack of suitable, ex vivo, small airway bioassay systems. In this study, we introduce a novel small murine airway bioassay system that permits the physiological and pharmacological study of intrapulmonary bronchial smooth muscle via a bronchial ring (BR) preparation utilizing BR segments as small as 200 microm in diameter. Using this ex vivo BR bioassay, we characterized small airway smooth muscle contraction and relaxation in the presence and absence of bronchial epithelium. In control BRs, the application of mechanical stretch is followed by spontaneous bronchial smooth muscle relaxation. BRs pretreated with methacholine (MCh) partially attenuate this stretch-induced relaxation by as much as 42% compared with control. MCh elicited a dose-dependent bronchial constriction with a maximal tension (E(max)) of 8.7 +/- 0.2 mN at an EC(50) of 0.33 +/- 0.02 microM. In the presence of nifedipine, ryanodine, 2-aminoethoxydiphenyl borate, and SKF-96365, E(max) to MCh was significantly reduced. In epithelium-denuded BRs, MCh-induced contraction was significantly enhanced to 11.4 +/- 1.0 mN with an EC(50) of 0.16 +/- 0.04 microM (P < 0.01). Substance P relaxed MCh-precontracted BR by 62.1%; however, this bronchial relaxation effect was completely lost in epithelium-denuded BRs. Papaverine virtually abolished MCh-induced constriction in both epithelium-intact and epithelium-denuded bronchial smooth muscle. In conclusion, this study introduces a novel murine small airway BR bioassay that allows for the physiological study of smooth muscle airway contractile responses that may aid in our understanding of the pathophysiology of asthma.

  13. Swine confinement buildings: effects of airborne particles and settled dust on airway smooth muscles.

    PubMed

    Demanche, Annick; Bonlokke, Jakob; Beaulieu, Marie-Josee; Assayag, Evelyne; Cormier, Yvon

    2009-01-01

    Swine confinement workers are exposed to various contaminants. These agents can cause airway inflammation and bronchoconstriction. This study was undertaken to evaluate if the bronchoconstrictive effects of swine barn air and settled dust are mediated by endotoxin, and if these effects are directly mediated on airway smooth muscles. Mouse tracheas where isolated and mounted isometrically in organ baths. Tracheas, with or without epithelium, were attached to a force transducer and tension was recorded. Concentrated swine building air at 68 EU/ml or settled dust extract at 0.01 g/ml were added for 20 minutes and tracheal smooth muscle contraction was measured. Direct role of LPS was assessed by removing it from air concentrates with an endotoxin affinity resin. Swine barn air and settled dust extract caused contraction of tracheal smooth muscle by 26 and 20%, respectively, of the maximal induced by methacholine. Removal of epithelium did not affect the contractile effects. LPS alone and LPS with peptidoglycans did not induce contraction. However, when endotoxin was removed from swine barn air concentrates, it lost 24% of its contractile effect. Concentrated swine barn air and settled dust have direct effects on airway smooth muscles. This effect is partially due to LPS but a synergy with other components of the environment of swine confinement buildings is required.

  14. Airway mechanics and methods used to visualize smooth muscle dynamics in vitro.

    PubMed

    Cooper, P R; McParland, B E; Mitchell, H W; Noble, P B; Politi, A Z; Ressmeyer, A R; West, A R

    2009-10-01

    Contraction of airway smooth muscle (ASM) is regulated by the physiological, structural and mechanical environment in the lung. We review two in vitro techniques, lung slices and airway segment preparations, that enable in situ ASM contraction and airway narrowing to be visualized. Lung slices and airway segment approaches bridge a gap between cell culture and isolated ASM, and whole animal studies. Imaging techniques enable key upstream events involved in airway narrowing, such as ASM cell signalling and structural and mechanical events impinging on ASM, to be investigated.

  15. A maturational model for the study of airway smooth muscle adaptation to mechanical oscillation.

    PubMed

    Wang, Lu; Chitano, Pasquale; Murphy, Thomas M

    2005-10-01

    It has been shown that mechanical stretches imposed on airway smooth muscle (ASM) by deep inspiration reduce the subsequent contractile response of the ASM. This passive maneuver of lengthening and retraction of the muscle is beneficial in normal subjects to counteract bronchospasm. However, it is detrimental to hyperresponsive airways because it triggers further bronchoconstriction. Although the exact mechanisms for this contrary response by normal and hyperresponsive airways are unclear, it has been suggested that the phenomenon is related to changes in ASM adaptability to mechanical oscillation. Healthy immature airways of both human and animal exhibit hyperresponsiveness, but whether the adaptative properties of hyperresponsive airway differ from normal is still unknown. In this article, we review the phenomenon of ASM adaptation to mechanical oscillation and its relevance and implication to airway hyperresponsiveness. We demonstrate that the age-specific expression of ASM adaptation is prominent using an established maturational animal model developed in our laboratory. Our data on immature ASM showed potentiated contractile force shortly after a length oscillation compared with the maximum force generated before oscillation. Several potential mechanisms such as myogenic response, changes in actin polymerization, or changes in the quantity of the cytoskeletal regulatory proteins plectin and vimentin, which may underlie this age-specific force potentiation, are discussed. We suggest a working model of the structure of smooth muscle associated with force transmission, which may help to elucidate the mechanisms responsible for the age-specific expression of smooth muscle adaptation. It is important to study the maturational profile of ASM adaptation as it could contribute to juvenile hyperresponsiveness.

  16. Epithelium-generated neuropeptide Y induces smooth muscle contraction to promote airway hyperresponsiveness.

    PubMed

    Li, Shanru; Koziol-White, Cynthia; Jude, Joseph; Jiang, Meiqi; Zhao, Hengjiang; Cao, Gaoyuan; Yoo, Edwin; Jester, William; Morley, Michael P; Zhou, Su; Wang, Yi; Lu, Min Min; Panettieri, Reynold A; Morrisey, Edward E

    2016-05-02

    Asthma is one of the most common chronic diseases globally and can be divided into presenting with or without an immune response. Current therapies have little effect on nonimmune disease, and the mechanisms that drive this type of asthma are poorly understood. Here, we have shown that loss of the transcription factors forkhead box P1 (Foxp1) and Foxp4, which are critical for lung epithelial development, in the adult airway epithelium evokes a non-Th2 asthma phenotype that is characterized by airway hyperresponsiveness (AHR) without eosinophilic inflammation. Transcriptome analysis revealed that loss of Foxp1 and Foxp4 expression induces ectopic expression of neuropeptide Y (Npy), which has been reported to be present in the airways of asthma patients, but whose importance in disease pathogenesis remains unclear. Treatment of human lung airway explants with recombinant NPY increased airway contractility. Conversely, loss of Npy in Foxp1- and Foxp4-mutant airway epithelium rescued the AHR phenotype. We determined that NPY promotes AHR through the induction of Rho kinase activity and phosphorylation of myosin light chain, which induces airway smooth muscle contraction. Together, these studies highlight the importance of paracrine signals from the airway epithelium to the underlying smooth muscle to induce AHR and suggest that therapies targeting epithelial induction of this phenotype may prove useful in treatment of noneosinophilic asthma.

  17. Disrupting actin-myosin-actin connectivity in airway smooth muscle as a treatment for asthma?

    PubMed

    Lavoie, Tera L; Dowell, Maria L; Lakser, Oren J; Gerthoffer, William T; Fredberg, Jeffrey J; Seow, Chun Y; Mitchell, Richard W; Solway, Julian

    2009-05-01

    Breathing is known to functionally antagonize bronchoconstriction caused by airway muscle contraction. During breathing, tidal lung inflation generates force fluctuations that are transmitted to the contracted airway muscle. In vitro, experimental application of force fluctuations to contracted airway smooth muscle strips causes them to relengthen. Such force fluctuation-induced relengthening (FFIR) likely represents the mechanism by which breathing antagonizes bronchoconstriction. Thus, understanding the mechanisms that regulate FFIR of contracted airway muscle could suggest novel therapeutic interventions to increase FFIR, and so to enhance the beneficial effects of breathing in suppressing bronchoconstriction. Here we propose that the connectivity between actin filaments in contracting airway myocytes is a key determinant of FFIR, and suggest that disrupting actin-myosin-actin connectivity by interfering with actin polymerization or with myosin polymerization merits further evaluation as a potential novel approach for preventing prolonged bronchoconstriction in asthma.

  18. Mechanical properties of asthmatic airway smooth muscle.

    PubMed

    Chin, Leslie Y M; Bossé, Ynuk; Pascoe, Chris; Hackett, Tillie L; Seow, Chun Y; Paré, Peter D

    2012-07-01

    Airway smooth muscle (ASM) is the major effector of excessive airway narrowing in asthma. Changes in some of the mechanical properties of ASM could contribute to excessive narrowing and have not been systematically studied in human ASM from nonasthmatic and asthmatic subjects. Human ASM strips (eight asthmatic and six nonasthmatic) were studied at in situ length and force was normalised to maximal force induced by electric field stimulation (EFS). Measurements included: passive and active force versus length before and after length adaptation, the force-velocity relationship, maximal shortening and force recovery after length oscillation. Force was converted to stress by dividing by cross-sectional area of muscle. The only functional differences were that the asthmatic tissue was stiffer at longer lengths (p<0.05) and oscillatory strain reduced isometric force in response to EFS by 19% as opposed to 36% in nonasthmatics (p<0.01). The mechanical properties of human ASM from asthmatic and nonasthmatic subjects are comparable except for increased passive stiffness and attenuated decline in force generation after an oscillatory perturbation. These data may relate to reduced bronchodilation induced by a deep inspiration in asthmatic subjects.

  19. Mechanism for substance P-induced relaxation of precontracted airway smooth muscle during development.

    PubMed

    Mhanna, M J; Dreshaj, I A; Haxhiu, M A; Martin, R J

    1999-01-01

    Release of substance P (SP) from sensory nerve endings of the tracheobronchial system modulates airway smooth muscle contraction and may cause relaxation of precontracted airways. We sought to elucidate the effect of postnatal maturation on SP-induced relaxation of precontracted airways and determine the roles of endogenously generated nitric oxide (NO) and prostaglandins (PGs). Cylindrical airway segments were isolated from the midtrachea of rats at four different ages, 1, 2, and 4 wk and 3 mo, and contracted to 50-75% of the maximum response induced by bethanechol. SP was then administered in the absence and presence of the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME), the PG inhibitor indomethacin, or both. Relaxation of airways with SP decreased significantly with advancing postnatal age. SP-induced tracheal relaxation was consistently attenuated by pretreatment with L-NAME, indomethacin, or both. In a different group of animals, L-NAME significantly attenuated the relaxant response of airways to PGE2 exposure, but indomethacin had no significant effect on the relaxant response to exogenous NO. We conclude that SP induces a relaxant effect on precontracted airway smooth muscle, which decreases with advancing age and is mediated via SP-induced release of NO and/or PG.

  20. RANTES release by human airway smooth muscle: effects of prostaglandin E(2) and fenoterol.

    PubMed

    Lazzeri, N; Belvisi, M G; Patel, H J; Chung, K F; Yacoub, M H; Mitchell, J A

    2001-12-21

    In human airway smooth muscle cells, the levels of RANTES were increased upon stimulation with interleukin-1beta together with tumour necrosis factor-alpha (TNF-alpha) (10 ng ml(-1) for each). In this study, we have assessed the effects of prostaglandin E(2) and the beta(2)-adrenoceptor agonist, fenoterol on RANTES (regulated upon activation, normal T cell expressed and secreted) release by these cells. The levels of RANTES released by human airway smooth muscle cells were measured after 24 h of treatment. Prostaglandin E(2) and fenoterol, only in presence of a cyclo-oxygenase inhibitor indomethacin (10(-6) M), provoked a concentration-dependent reduction in RANTES release. These data suggest that, in settings where cyclo-oxygenase activity is low, both drugs may relieve the symptoms of airway diseases by reducing RANTES production.

  1. Airway smooth muscle: a potential target for asthma therapy.

    PubMed

    Dowell, Maria L; Lavoie, Tera L; Solway, Julian; Krishnan, Ramaswamy

    2014-01-01

    Asthma is a major public health problem that afflicts nearly one in 20 people worldwide. Despite available treatments, asthma symptoms remain poorly controlled in a significant minority of asthma patients, especially those with severe disease. Accordingly, much ongoing effort has been directed at developing new therapeutic strategies; these efforts are described in detail below. Although mucus hypersecretion is an important component of asthma pathobiology, the primary mechanism of morbidity and mortality in asthma is excessive narrowing of the airway. The key end- effector of excessive airway narrowing is airway smooth muscle (ASM) contraction; overcoming ASM contraction is therefore a prominent therapeutic strategy. Here, we review exciting new advances aimed at ASM relaxation. Exciting advances in ASM biology have identified new therapeutic targets for the prevention or reversal of bronchoconstriction in asthma.

  2. Development and maintenance of force and stiffness in airway smooth muscle.

    PubMed

    Lan, Bo; Norris, Brandon A; Liu, Jeffrey C-Y; Paré, Peter D; Seow, Chun Y; Deng, Linhong

    2015-03-01

    Airway smooth muscle (ASM) plays a central role in the excessive narrowing of the airway that characterizes the primary functional impairment in asthma. This phenomenon is known as airway hyper-responsiveness (AHR). Emerging evidence suggests that the development and maintenance of ASM force involves dynamic reorganization of the subcellular filament network in both the cytoskeleton and the contractile apparatus. In this review, evidence is presented to support the view that regulation of ASM contraction extends beyond the classical actomyosin interaction and involves processes within the cytoskeleton and at the interfaces between the cytoskeleton, the contractile apparatus, and the extracellular matrix. These processes are initiated when the muscle is activated, and collectively they cause the cytoskeleton and the contractile apparatus to undergo structural transformation, resulting in a more connected and solid state that allows force generated by the contractile apparatus to be transmitted to the extracellular domain. Solidification of the cytoskeleton also serves to stiffen the muscle and hence the airway. Oscillatory strain from tidal breathing and deep inspiration is believed to be the counter balance that prevents hypercontraction and stiffening of ASM in vivo. Dysregulation of this balance could lead to AHR seen in asthma.

  3. Nelumbo nucifera leaves extracts inhibit mouse airway smooth muscle contraction.

    PubMed

    Yang, Xiao; Xue, Lu; Zhao, Qingyang; Cai, Congli; Liu, Qing-Hua; Shen, Jinhua

    2017-03-20

    Alkaloids extracted from lotus leaves (AELL) can relax vascular smooth muscle. However, whether AELL has a similar relaxant role on airway smooth muscle (ASM) remains unknown. This study aimed to explore the relaxant property of AELL on ASM and the underlying mechanism. Alkaloids were extracted from dried lotus leaves using the high temperature rotary evaporation extraction method. The effects of AELL on mouse ASM tension were studied using force measuring and patch-clamp techniques. It was found that AELL inhibited the high K + or acetylcholine chloride (ACh)-induced precontraction of mouse tracheal rings by 64.8 ± 2.9%, or 48.8 ± 4.7%, respectively. The inhibition was statistically significant and performed in a dose-dependent manner. Furthermore, AELL-induced smooth muscle relaxation was partially mediated by blocking voltage-dependent Ca 2+ channels (VDCC) and non-selective cation channels (NSCC). AELL, which plays a relaxant role in ASM, might be a new complementary treatment to treat abnormal contractions of the trachea and asthma.

  4. Airway smooth muscle in airway reactivity and remodeling: what have we learned?

    PubMed Central

    2013-01-01

    It is now established that airway smooth muscle (ASM) has roles in determining airway structure and function, well beyond that as the major contractile element. Indeed, changes in ASM function are central to the manifestation of allergic, inflammatory, and fibrotic airway diseases in both children and adults, as well as to airway responses to local and environmental exposures. Emerging evidence points to novel signaling mechanisms within ASM cells of different species that serve to control diverse features, including 1) [Ca2+]i contractility and relaxation, 2) cell proliferation and apoptosis, 3) production and modulation of extracellular components, and 4) release of pro- vs. anti-inflammatory mediators and factors that regulate immunity as well as the function of other airway cell types, such as epithelium, fibroblasts, and nerves. These diverse effects of ASM “activity” result in modulation of bronchoconstriction vs. bronchodilation relevant to airway hyperresponsiveness, airway thickening, and fibrosis that influence compliance. This perspective highlights recent discoveries that reveal the central role of ASM in this regard and helps set the stage for future research toward understanding the pathways regulating ASM and, in turn, the influence of ASM on airway structure and function. Such exploration is key to development of novel therapeutic strategies that influence the pathophysiology of diseases such as asthma, chronic obstructive pulmonary disease, and pulmonary fibrosis. PMID:24142517

  5. Triptolide inhibits TGF-β1-induced cell proliferation in rat airway smooth muscle cells by suppressing Smad signaling

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chen, Ming; Lv, Zhiqiang; Huang, Linjie

    Background: We have reported that triptolide can inhibit airway remodeling in a murine model of asthma via TGF-β1/Smad signaling. In the present study, we aimed to investigate the effect of triptolide on airway smooth muscle cells (ASMCs) proliferation and the possible mechanism. Methods: Rat airway smooth muscle cells were cultured and made synchronized, then pretreated with different concentration of triptolide before stimulated by TGF-β1. Cell proliferation was evaluated by MTT assay. Flow cytometry was used to study the influence of triptolide on cell cycle and apoptosis. Signal proteins (Smad2, Smad3 and Smad7) were detected by western blotting analysis. Results: Triptolidemore » significantly inhibited TGF-β1-induced ASMC proliferation (P<0.05). The cell cycle was blocked at G1/S-interphase by triptolide dose dependently. No pro-apoptotic effects were detected under the concentration of triptolide we used. Western blotting analysis showed TGF-β1 induced Smad2 and Smad3 phosphorylation was inhibited by triptolide pretreatment, and the level of Smad7 was increased by triptolide pretreatment. Conclusions: Triptolide may function as an inhibitor of asthma airway remodeling by suppressing ASMCs proliferation via negative regulation of Smad signaling pathway. - Highlights: • In this study, rat airway smooth muscle cells were cultured and made synchronized. • Triptolide inhibited TGF-β1-induced airway smooth muscle cells proliferation. • Triptolide inhibited ASMCs proliferation via negative regulation of Smad signaling pathway.« less

  6. Gi-Coupled γ-Aminobutyric Acid–B Receptors Cross-Regulate Phospholipase C and Calcium in Airway Smooth Muscle

    PubMed Central

    Mizuta, Kentaro; Mizuta, Fumiko; Xu, Dingbang; Masaki, Eiji; Panettieri, Reynold A.

    2011-01-01

    γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system, and exerts its actions via both ionotropic (GABAA) and metabotropic (GABAB) receptors. Although the functional expression of GABAB receptors coupled to the Gi protein was reported for airway smooth muscle, the role of GABAB receptors in airway responsiveness remains unclear. We investigated whether Gi-coupled GABAB receptors cross-regulate phospholipase C (PLC), an enzyme classically regulated by Gq-coupled receptors in human airway smooth muscle cells. Both the GABAB-selective agonist baclofen and the endogenous ligand GABA significantly increased the synthesis of inositol phosphate, whereas GABAA receptor agonists, muscimol, and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol exerted no effect. The baclofen-induced synthesis of inositol phosphate and transient increases in [Ca2+]i were blocked by CGP35348 and CGP55845 (selective GABAB antagonists), pertussis toxin (PTX, which inactivates the Gi protein), gallein (a Gβγ signaling inhibitor), U73122 (an inhibitor of PLC-β), and xestospongin C, an inositol 1,4,5-triphosphate receptor blocker. Baclofen also potentiated the bradykinin-induced synthesis of inositol phosphate and transient increases in [Ca2+]i, which were blocked by CGP35348 or PTX. Moreover, baclofen potentiated the substance P–induced contraction of airway smooth muscle in isolated guinea pig tracheal rings. In conclusion, the stimulation of GABAB receptors in human airway smooth muscle cells rapidly mobilizes intracellular Ca2+ stores by the synthesis of inositol phosphate via the activation of PLC-β, which is stimulated by Gβγ protein liberated from Gi proteins coupled to GABAB receptors. Furthermore, crosstalk between GABAB receptors and Gq-coupled receptors potentiates the synthesis of inositol phosphate, transient increases in [Ca2+]i, and smooth muscle contraction through Gi proteins. PMID:21719794

  7. Assays for in vitro monitoring of human airway smooth muscle (ASM) and human pulmonary arterial vascular smooth muscle (VSM) cell migration.

    PubMed

    Goncharova, Elena A; Goncharov, Dmitry A; Krymskaya, Vera P

    2006-01-01

    Migration of human pulmonary vascular smooth muscle (VSM) cells contributes to vascular remodeling in pulmonary arterial hypertension and atherosclerosis. Evidence also indicates that, in part, migration of airway smooth muscle (ASM) cells may contribute to airway remodeling associated with asthma. Here we describe migration of VSM and ASM cells in vitro using Transwell or Boyden chamber assays. Because dissecting signaling mechanisms regulating cell migration requires molecular approaches, our protocol also describes how to assess migration of transfected VSM and ASM cells. Transwell or Boyden chamber assays can be completed in approximately 8 h and include plating of serum-deprived VSM or ASM cell suspension on membrane precoated with collagen, migration of cells toward chemotactic gradient and visual (Transwell) or digital (Boyden chamber) analysis of membrane. Although the Transwell assay is easy, the Boyden chamber assay requires hands-on experience; however, both assays are reliable cell-based approaches providing valuable information on how chemotactic and inflammatory factors modulate VSM and ASM migration.

  8. Sphingosine-1-phosphate induces pro-remodelling response in airway smooth muscle cells

    PubMed Central

    Fuerst, E; Foster, H R; Ward, J P T; Corrigan, C J; Cousins, D J; Woszczek, G

    2014-01-01

    Background Increased proliferation of airway smooth muscle (ASM) cells leading to hyperplasia and increased ASM mass is one of the most characteristic features of airway remodelling in asthma. A bioactive lipid, sphingosine-1-phosphate (S1P), has been suggested to affect airway remodelling by stimulation of human ASM cell proliferation. Objective To investigate the effect of S1P on signalling and regulation of gene expression in ASM cells from healthy and asthmatic individuals. Methods Airway smooth muscle cells grown from bronchial biopsies of healthy and asthmatic individuals were exposed to S1P. Gene expression was analysed using microarray, real-time PCR and Western blotting. Receptor signalling and function were determined by mRNA knockdown and intracellular calcium mobilization experiments. Results S1P potently regulated the expression of more than 80 genes in human ASM cells, including several genes known to be involved in the regulation of cell proliferation and airway remodelling (HBEGF, TGFB3, TXNIP, PLAUR, SERPINE1, RGS4). S1P acting through S1P2 and S1P3 receptors activated intracellular calcium mobilization and extracellular signal-regulated and Rho-associated kinases to regulate gene expression. S1P-induced responses were not inhibited by corticosteroids and did not differ significantly between ASM cells from healthy and asthmatic individuals. Conclusion S1P induces a steroid-resistant, pro-remodelling pathway in ASM cells. Targeting S1P or its receptors could be a novel treatment strategy for inhibiting airway remodelling in asthma. PMID:25041788

  9. Effects of sodium metabisulphite on guinea pig contractile airway smooth muscle responses in vitro.

    PubMed

    Sun, J; Sakamoto, T; Chung, K F

    1995-08-01

    Sodium metabisulphite (MBS) is known to induce bronchoconstriction in asthmatic patients. The effects of MBS on guinea pig airway smooth muscle and on neurally mediated contraction in vitro have been examined. Tracheal and bronchial airway segments were placed in oxygenated buffer solution and electrical field stimulation was performed in the presence of indomethacin (10(-5) M) and propranolol (10(-6) M) for the measurement of isometric tension. Atropine (10(-6) M) was added to bronchial tissues. Concentrations of MBS up to 10(-3) M had no direct effect on airway smooth muscle contraction and did not alter either tracheal smooth muscle contraction induced by electrical field stimulation at all frequencies or acetylcholine-induced tracheal smooth muscle contraction. There was a similar response in the absence of epithelium, except for potentiation of the response induced by electrical field stimulation at 0.5 Hz (24 (10)% increase). However, MBS (10(-5), 10(-6) and 10(-7) M) augmented neurally-mediated non-adrenergic non-cholinergic contractile responses in the bronchi (13.3 (3.2)%, 23.8 (9.6)%, and 6.4 (1.6)%, respectively). MBS had no effect on the contractile response induced by substance P, but at higher concentrations (10(-3) M and 10(-4) M) it caused a time-dependent attenuation of responses induced by either electrical field stimulation or exogenously applied acetylcholine or substance P. MBS had no direct contractile responses but enhanced bronchoconstriction induced by activation of non-cholinergic neural pathways in the bronchus, probably through increased release of neuropeptides. At high concentrations MBS inhibited contractile responses initiated by receptor or neural stimulation.

  10. Effects of sodium metabisulphite on guinea pig contractile airway smooth muscle responses in vitro.

    PubMed Central

    Sun, J.; Sakamoto, T.; Chung, K. F.

    1995-01-01

    BACKGROUND--Sodium metabisulphite (MBS) is known to induce bronchoconstriction in asthmatic patients. The effects of MBS on guinea pig airway smooth muscle and on neurally mediated contraction in vitro have been examined. METHODS--Tracheal and bronchial airway segments were placed in oxygenated buffer solution and electrical field stimulation was performed in the presence of indomethacin (10(-5) M) and propranolol (10(-6) M) for the measurement of isometric tension. Atropine (10(-6) M) was added to bronchial tissues. RESULTS--Concentrations of MBS up to 10(-3) M had no direct effect on airway smooth muscle contraction and did not alter either tracheal smooth muscle contraction induced by electrical field stimulation at all frequencies or acetylcholine-induced tracheal smooth muscle contraction. There was a similar response in the absence of epithelium, except for potentiation of the response induced by electrical field stimulation at 0.5 Hz (24 (10)% increase). However, MBS (10(-5), 10(-6) and 10(-7) M) augmented neurally-mediated non-adrenergic non-cholinergic contractile responses in the bronchi (13.3 (3.2)%, 23.8 (9.6)%, and 6.4 (1.6)%, respectively). MBS had no effect on the contractile response induced by substance P, but at higher concentrations (10(-3) M and 10(-4) M) it caused a time-dependent attenuation of responses induced by either electrical field stimulation or exogenously applied acetylcholine or substance P. CONCLUSIONS--MBS had no direct contractile responses but enhanced bronchoconstriction induced by activation of non-cholinergic neural pathways in the bronchus, probably through increased release of neuropeptides. At high concentrations MBS inhibited contractile responses initiated by receptor or neural stimulation. Images PMID:7570440

  11. Studies of the mechanism of passive anaphylaxis in human airway smooth muscle.

    PubMed

    Davis, C; Jones, T R; Daniel, E E

    1983-07-01

    This investigation was carried out to study allergic contraction of passively sensitized human airway smooth muscle in response to specific antigen challenge. We attempted to determine the role played by histamine, slow reaction substances (SRSs), and cyclooxygenase products in the mediation of this response in tracheal smooth muscle. Tissues were passively sensitized with serum from ragweed-sensitive patients (15 h, 4 degrees C). Subsequent challenge with ragweed antigen produced a slowly developing contraction. The peak contraction to a dose producing a maximal response was 37 +/- 6% of the carbachol maximum. Mepyramine (5 X 10(-6) M) did not alter the contraction. Methylprednisolone (2 X 10(-5) M) attenuated the response to antigen but had no significant effect on the contractile response to arachidonic acid. Indomethacin (5.6-28 X 10(-6) M) enhanced the peak antigen-induced contractions by 25 +/- 11% whereas 5,8,11,14-eicosatetraynoic acid (6.4 X 10(-5) M) selectively attenuated the antigen-induced contraction by 86 +/- 12%. Nordihydroguarietic acid (6-12 X 10(-6) M) attenuated both the antigen plus arachidonate induced responses. FPL-55712 (1-2 X 10(-6) M) antagonized the contractions to antigen. Compound 48/80 and goat antihuman immunoglobulin E produced similar slowly developing contractions in sensitized and in some nonsensitized tissues. These responses, except for an early component of the response to 48/80, were independent of histamine and were reversed by FPL-55712. These findings suggest that arachidonic acid metabolites mediate (slow reacting substances) and modulate (prostaglandins) allergic contraction of human airway smooth muscle while any histamine released contributes little or nothing to the contraction in the larger airways.

  12. The contribution of airway smooth muscle to airway narrowing and airway hyperresponsiveness in disease.

    PubMed

    Martin, J G; Duguet, A; Eidelman, D H

    2000-08-01

    Airway hyperresponsiveness (AHR), the exaggerated response to constrictor agonists in asthmatic subjects, is incompletely understood. Changes in either the quantity or properties of airway smooth muscle (ASM) are possible explanations for AHR. Morphometric analyses demonstrate structural changes in asthmatic airways, including subepithelial fibrosis, gland hyperplasia/hypertrophy, neovascularization and an increase in ASM mass. Mathematical modelling of airway narrowing suggests that, of all the changes in structure, the increase in ASM mass is the most probable cause of AHR. An increase in ASM mass in the large airways is more closely associated with a greater likelihood of dying from asthma than increases in ASM mass in other locations within the airway tree. ASM contraction is opposed by the elastic recoil of the lungs and airways, which appears to limit the degree of bronchoconstriction in vivo. The cyclical nature of tidal breathing applies stresses to the airway wall that enhance the bronchodilating influence of the lung tissues on the contracting ASM, in all probability by disrupting cross-bridges. However, the increase in ASM mass in asthma may overcome the limitation resulting from the impedances to ASM shortening imposed by the lung parenchyma and airway wall tissues. Additionally, ASM with the capacity to shorten rapidly may achieve shorter lengths and cause a greater degree of bronchoconstriction when stimulated to contract than slower ASM. Changes in ASM properties are induced by the process of sensitization and allergen-exposure such as enhancement of phospholipase C activity and inositol phosphate turnover, and increases in myosin light chain kinase activity. Whether changes in ASM mass or biochemical/biomechanical properties form the basis for asthma remains to be determined.

  13. Gi-coupled γ-aminobutyric acid-B receptors cross-regulate phospholipase C and calcium in airway smooth muscle.

    PubMed

    Mizuta, Kentaro; Mizuta, Fumiko; Xu, Dingbang; Masaki, Eiji; Panettieri, Reynold A; Emala, Charles W

    2011-12-01

    γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system, and exerts its actions via both ionotropic (GABA(A)) and metabotropic (GABA(B)) receptors. Although the functional expression of GABA(B) receptors coupled to the G(i) protein was reported for airway smooth muscle, the role of GABA(B) receptors in airway responsiveness remains unclear. We investigated whether G(i)-coupled GABA(B) receptors cross-regulate phospholipase C (PLC), an enzyme classically regulated by G(q)-coupled receptors in human airway smooth muscle cells. Both the GABA(B)-selective agonist baclofen and the endogenous ligand GABA significantly increased the synthesis of inositol phosphate, whereas GABA(A) receptor agonists, muscimol, and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol exerted no effect. The baclofen-induced synthesis of inositol phosphate and transient increases in [Ca(2+)](i) were blocked by CGP35348 and CGP55845 (selective GABA(B) antagonists), pertussis toxin (PTX, which inactivates the G(i) protein), gallein (a G(βγ) signaling inhibitor), U73122 (an inhibitor of PLC-β), and xestospongin C, an inositol 1,4,5-triphosphate receptor blocker. Baclofen also potentiated the bradykinin-induced synthesis of inositol phosphate and transient increases in [Ca(2+)](i), which were blocked by CGP35348 or PTX. Moreover, baclofen potentiated the substance P-induced contraction of airway smooth muscle in isolated guinea pig tracheal rings. In conclusion, the stimulation of GABA(B) receptors in human airway smooth muscle cells rapidly mobilizes intracellular Ca(2+) stores by the synthesis of inositol phosphate via the activation of PLC-β, which is stimulated by G(βγ) protein liberated from G(i) proteins coupled to GABA(B) receptors. Furthermore, crosstalk between GABA(B) receptors and G(q)-coupled receptors potentiates the synthesis of inositol phosphate, transient increases in [Ca(2+)](i), and smooth muscle contraction through G

  14. Iptakalim inhibits PDGF-BB-induced human airway smooth muscle cells proliferation and migration

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Liu, Wenrui; Kong, Hui; Zeng, Xiaoning

    Chronic airway diseases are characterized by airway remodeling which is attributed partly to the proliferation and migration of airway smooth muscle cells (ASMCs). ATP-sensitive potassium (K{sub ATP}) channels have been identified in ASMCs. Mount evidence has suggested that K{sub ATP} channel openers can reduce airway hyperresponsiveness and alleviate airway remodeling. Opening K{sup +} channels triggers K{sup +} efflux, which leading to membrane hyperpolarization, preventing Ca{sup 2+}entry through closing voltage-operated Ca{sup 2+} channels. Intracellular Ca{sup 2+} is the most important regulator of muscle contraction, cell proliferation and migration. K{sup +} efflux decreases Ca{sup 2+} influx, which consequently influences ASMCs proliferation andmore » migration. As a K{sub ATP} channel opener, iptakalim (Ipt) has been reported to restrain the proliferation of pulmonary arterial smooth muscle cells (PASMCs) involved in vascular remodeling, while little is known about its impact on ASMCs. The present study was designed to investigate the effects of Ipt on human ASMCs and the mechanisms underlying. Results obtained from cell counting kit-8 (CCK-8), flow cytometry and 5-ethynyl-2′-deoxyuridine (EdU) incorporation showed that Ipt significantly inhibited platelet-derived growth factor (PDGF)-BB-induced ASMCs proliferation. ASMCs migration induced by PDGF-BB was also suppressed by Ipt in transwell migration and scratch assay. Besides, the phosphorylation of Ca{sup 2+}/calmodulin-dependent kinase II (CaMKII), extracellular regulated protein kinases 1/2 (ERK1/2), protein kinase B (Akt), and cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) were as well alleviated by Ipt administration. Furthermore, we found that the inhibition of Ipt on the PDGF-BB-induced proliferation and migration in human ASMCs was blocked by glibenclamide (Gli), a selective K{sub ATP} channel antagonist. These findings provide a strong evidence to support that

  15. Oxygen dose responsiveness of human fetal airway smooth muscle cells.

    PubMed

    Hartman, William R; Smelter, Dan F; Sathish, Venkatachalem; Karass, Michael; Kim, Sunchin; Aravamudan, Bharathi; Thompson, Michael A; Amrani, Yassine; Pandya, Hitesh C; Martin, Richard J; Prakash, Y S; Pabelick, Christina M

    2012-10-15

    Maintenance of blood oxygen saturation dictates supplemental oxygen administration to premature infants, but hyperoxia predisposes survivors to respiratory diseases such as asthma. Although much research has focused on oxygen effects on alveoli in the setting of bronchopulmonary dysplasia, the mechanisms by which oxygen affects airway structure or function relevant to asthma are still under investigation. We used isolated human fetal airway smooth muscle (fASM) cells from 18-20 postconceptual age lungs (canalicular stage) to examine oxygen effects on intracellular Ca(2+) ([Ca(2+)](i)) and cellular proliferation. fASM cells expressed substantial smooth muscle actin and myosin and several Ca(2+) regulatory proteins but not fibroblast or epithelial markers, profiles qualitatively comparable to adult human ASM. Fluorescence Ca(2+) imaging showed robust [Ca(2+)](i) responses to 1 μM acetylcholine (ACh) and 10 μM histamine (albeit smaller and slower than adult ASM), partly sensitive to zero extracellular Ca(2+). Compared with adult, fASM showed greater baseline proliferation. Based on this validation, we assessed fASM responses to 10% hypoxia through 90% hyperoxia and found enhanced proliferation at <60% oxygen but increased apoptosis at >60%, effects accompanied by appropriate changes in proliferative vs. apoptotic markers and enhanced mitochondrial fission at >60% oxygen. [Ca(2+)](i) responses to ACh were enhanced for <60% but blunted at >60% oxygen. These results suggest that hyperoxia has dose-dependent effects on structure and function of developing ASM, which could have consequences for airway diseases of childhood. Thus detrimental effects on ASM should be an additional consideration in assessing risks of supplemental oxygen in prematurity.

  16. Mechanisms altering airway smooth muscle cell Ca+ homeostasis in two asthma models.

    PubMed

    Kellner, Julia; Tantzscher, Juliane; Oelmez, Hamza; Edelmann, Martin; Fischer, Rainald; Huber, Rudolf Maria; Bergner, Albrecht

    2008-01-01

    Asthma is characterized by airway remodeling, altered mucus production and airway smooth muscle cell (ASMC) contraction causing extensive airway narrowing. In particular, alterations of ASMC contractility seem to be of crucial importance. The elevation of the cytoplasmic Ca(2+) concentration is a key event leading to ASMC contraction and changes in the agonist-induced Ca(2+) increase in ASMC have been reported in asthma. The aim of this study was to investigate mechanisms underlying these changes. Murine tracheal smooth muscle cells (MTSMC) from T-bet KO mice and human bronchial smooth muscle cells (HBSMC) incubated with IL-13 and IL-4 served as asthma models. Acetylcholine-induced changes in the cytoplasmic Ca(2+) concentration were recorded using fluorescence microscopy and the expression of Ca(2+) homeostasis regulating proteins was investigated with Western blot analysis. Acetylcholine-induced Ca(2+) transients were elevated in both asthma models. This correlated with an increased Ca(2+) content of the sarcoplasmic reticulum (SR). In MTSMC from T-bet KO mice, the expression of the SR Ca(2+) buffers calreticulin and calsequestrin was higher compared to wild-type mice. In HBSMC incubated with IL-13 or IL-4, the expression of ryanodine receptors, inositol-3-phosphate receptors and sarcoplasmic/endoplasmic reticulum Ca(2+) ATPases 2 was increased compared to HBSMC without incubation with interleukins. The enlarged acetylcholine-induced Ca(2+) transients could be reversed by blocking inositol-3-phosphate receptors. We conclude that in the murine asthma model the SR Ca(2+) buffer capacity is increased, while in the human asthma model the expression of SR Ca(2+) channels is altered. The investigation of the Ca(2+) homeostasis of ASMC has the potential to provide new therapeutical options in asthma. Copyright 2008 S. Karger AG, Basel.

  17. Oxygen dose responsiveness of human fetal airway smooth muscle cells

    PubMed Central

    Hartman, William R.; Smelter, Dan F.; Sathish, Venkatachalem; Karass, Michael; Kim, Sunchin; Aravamudan, Bharathi; Thompson, Michael A.; Amrani, Yassine; Pandya, Hitesh C.; Martin, Richard J.; Prakash, Y. S.

    2012-01-01

    Maintenance of blood oxygen saturation dictates supplemental oxygen administration to premature infants, but hyperoxia predisposes survivors to respiratory diseases such as asthma. Although much research has focused on oxygen effects on alveoli in the setting of bronchopulmonary dysplasia, the mechanisms by which oxygen affects airway structure or function relevant to asthma are still under investigation. We used isolated human fetal airway smooth muscle (fASM) cells from 18–20 postconceptual age lungs (canalicular stage) to examine oxygen effects on intracellular Ca2+ ([Ca2+]i) and cellular proliferation. fASM cells expressed substantial smooth muscle actin and myosin and several Ca2+ regulatory proteins but not fibroblast or epithelial markers, profiles qualitatively comparable to adult human ASM. Fluorescence Ca2+ imaging showed robust [Ca2+]i responses to 1 μM acetylcholine (ACh) and 10 μM histamine (albeit smaller and slower than adult ASM), partly sensitive to zero extracellular Ca2+. Compared with adult, fASM showed greater baseline proliferation. Based on this validation, we assessed fASM responses to 10% hypoxia through 90% hyperoxia and found enhanced proliferation at <60% oxygen but increased apoptosis at >60%, effects accompanied by appropriate changes in proliferative vs. apoptotic markers and enhanced mitochondrial fission at >60% oxygen. [Ca2+]i responses to ACh were enhanced for <60% but blunted at >60% oxygen. These results suggest that hyperoxia has dose-dependent effects on structure and function of developing ASM, which could have consequences for airway diseases of childhood. Thus detrimental effects on ASM should be an additional consideration in assessing risks of supplemental oxygen in prematurity. PMID:22923637

  18. Tumor necrosis factor regulates NMDA receptor-mediated airway smooth muscle contractile function and airway responsiveness.

    PubMed

    Anaparti, Vidyanand; Pascoe, Christopher D; Jha, Aruni; Mahood, Thomas H; Ilarraza, Ramses; Unruh, Helmut; Moqbel, Redwan; Halayko, Andrew J

    2016-08-01

    We have shown that N-methyl-d-aspartate receptors (NMDA-Rs) are receptor-operated calcium entry channels in human airway smooth muscle (HASM) during contraction. Tumor necrosis factor (TNF) augments smooth muscle contractility by influencing pathways that regulate intracellular calcium flux and can alter NMDA-R expression and activity in cortical neurons and glial cells. We hypothesized that NMDA-R-mediated Ca(2+) and contractile responses of ASM can be altered by inflammatory mediators, including TNF. In cultured HASM cells, we assessed TNF (10 ng/ml, 48 h) effect on NMDA-R subunit abundance by quantitative PCR, confocal imaging, and immunoblotting. We observed dose- and time-dependent changes in NMDA-R composition: increased obligatory NR1 subunit expression and altered regulatory NR2 and inhibitory NR3 subunits. Measuring intracellular Ca(2+) flux in Fura-2-loaded HASM cultures, we observed that TNF exposure enhanced cytosolic Ca(2+) mobilization and changed the temporal pattern of Ca(2+) flux in individual myocytes induced by NMDA, an NMDA-R selective analog of glutamate. We measured airway responses to NMDA in murine thin-cut lung slices (TCLS) from allergen-naive animals and observed significant airway contraction. However, NMDA acted as a bronchodilator in TCLS from house dust mice-challenged mice and in allergen-naive TCLS subjected to TNF exposure. All contractile or bronchodilator responses were blocked by a selective NMDA-R antagonist, (2R)-amino-5-phosphonopentanoate, and bronchodilator responses were prevented by N(G)-nitro-l-arginine methyl ester (nitric oxide synthase inhibitor) or indomethacin (cyclooxygenase inhibitor). Collectively, we show that TNF augments NMDA-R-mediated Ca(2+) mobilization in HASM cells, whereas in multicellular TCLSs allergic inflammation and TNF exposure leads to NMDA-R-mediated bronchodilation. These findings reveal the unique contribution of ionotrophic NMDA-R to airway hyperreactivity. Copyright © 2016 the American

  19. The Dynamic Actin Cytoskeleton in Smooth Muscle.

    PubMed

    Tang, Dale D

    2018-01-01

    Smooth muscle contraction requires both myosin activation and actin cytoskeletal remodeling. Actin cytoskeletal reorganization facilitates smooth muscle contraction by promoting force transmission between the contractile unit and the extracellular matrix (ECM), and by enhancing intercellular mechanical transduction. Myosin may be viewed to serve as an "engine" for smooth muscle contraction whereas the actin cytoskeleton may function as a "transmission system" in smooth muscle. The actin cytoskeleton in smooth muscle also undergoes restructuring upon activation with growth factors or the ECM, which controls smooth muscle cell proliferation and migration. Abnormal smooth muscle contraction, cell proliferation, and motility contribute to the development of vascular and pulmonary diseases. A number of actin-regulatory proteins including protein kinases have been discovered to orchestrate actin dynamics in smooth muscle. In particular, Abelson tyrosine kinase (c-Abl) is an important molecule that controls actin dynamics, contraction, growth, and motility in smooth muscle. Moreover, c-Abl coordinates the regulation of blood pressure and contributes to the pathogenesis of airway hyperresponsiveness and vascular/airway remodeling in vivo. Thus, c-Abl may be a novel pharmacological target for the development of new therapy to treat smooth muscle diseases such as hypertension and asthma. © 2018 Elsevier Inc. All rights reserved.

  20. [Biomechanics and bio-energetics of smooth muscle contraction. Relation to bronchial hyperreactivity].

    PubMed

    Coirault, C; Blanc, F X; Chemla, D; Salmeron, S; Lecarpentier, Y

    2000-06-01

    Mechanical studies of isolated muscle and analysis of molecular actomyosin interactions have improved our understanding of the pathophysiology of airway smooth muscle. Mechanical properties of airway smooth muscle are similar to those of other smooth muscles. Airway smooth muscle exhibits spontaneous intrinsic tone and its maximum shortening velocity (Vmax) is 10-30 fold lower than in striated muscle. Smooth muscle myosin generates step size and elementary force per crossbridge interaction approximately similar to those of skeletal muscle myosin. Special slow cycling crossbridges, termed latch-bridges, have been attributed to myosin light chain dephosphorylation. From a mechanical point of view, it has been shown that airway hyperresponsiveness is characterized by an increased Vmax and an increased shortening capacity, with no significant change in the force-generating capacity.

  1. The Pivotal Role of Airway Smooth Muscle in Asthma Pathophysiology

    PubMed Central

    Ozier, Annaïg; Allard, Benoit; Bara, Imane; Girodet, Pierre-Olivier; Trian, Thomas; Marthan, Roger; Berger, Patrick

    2011-01-01

    Asthma is characterized by the association of airway hyperresponsiveness (AHR), inflammation, and remodelling. The aim of the present article is to review the pivotal role of airway smooth muscle (ASM) in the pathophysiology of asthma. ASM is the main effector of AHR. The mechanisms of AHR in asthma may involve a larger release of contractile mediators and/or a lower release of relaxant mediators, an improved ASM cell excitation/contraction coupling, and/or an alteration in the contraction/load coupling. Beyond its contractile function, ASM is also involved in bronchial inflammation and remodelling. Whereas ASM is a target of the inflammatory process, it can also display proinflammatory and immunomodulatory functions, through its synthetic properties and the expression of a wide range of cell surface molecules. ASM remodelling represents a key feature of asthmatic bronchial remodelling. ASM also plays a role in promoting complementary airway structural alterations, in particular by its synthetic function. PMID:22220184

  2. Neurokinin-neurotrophin interactions in airway smooth muscle

    PubMed Central

    Meuchel, Lucas W.; Stewart, Alecia; Smelter, Dan F.; Abcejo, Amard J.; Thompson, Michael A.; Zaidi, Syed I. A.; Martin, Richard J.

    2011-01-01

    Neurally derived tachykinins such as substance P (SP) play a key role in modulating airway contractility (especially with inflammation). Separately, the neurotrophin brain-derived neurotrophic factor (BDNF; potentially derived from nerves as well as airway smooth muscle; ASM) and its tropomyosin-related kinase receptor, TrkB, are involved in enhanced airway contractility. In this study, we hypothesized that neurokinins and neurotrophins are linked in enhancing intracellular Ca2+ concentration ([Ca2+]i) regulation in ASM. In rat ASM cells, 24 h exposure to 10 nM SP significantly increased BDNF and TrkB expression (P < 0.05). Furthermore, [Ca2+]i responses to 1 μM ACh as well as BDNF (30 min) effects on [Ca2+]i regulation were enhanced by prior SP exposure, largely via increased Ca2+ influx (P < 0.05). The enhancing effect of SP on BDNF signaling was blunted by the neurokinin-2 receptor antagonist MEN-10376 (1 μM, P < 0.05) to a greater extent than the neurokinin-1 receptor antagonist RP-67580 (5 nM). Chelation of extracellular BDNF (chimeric TrkB-Fc; 1 μg/ml), as well as tyrosine kinase inhibition (100 nM K252a), substantially blunted SP effects (P < 0.05). Overnight (24 h) exposure of ASM cells to 50% oxygen increased BDNF and TrkB expression and potentiated both SP- and BDNF-induced enhancement of [Ca2+]i (P < 0.05). These results suggest a novel interaction between SP and BDNF in regulating agonist-induced [Ca2+]i regulation in ASM. The autocrine mechanism we present here represents a new area in the development of bronchoconstrictive reflex response and airway hyperreactive disorders. PMID:21515660

  3. Characterization of primary cilia in human airway smooth muscle cells.

    PubMed

    Wu, Jun; Du, Hui; Wang, Xiangling; Mei, Changlin; Sieck, Gary C; Qian, Qi

    2009-08-01

    Considerable evidence indicates a key role for primary cilia of mammalian cells in mechanochemical sensing. Dysfunctions of primary cilia have been linked to the pathogenesis of several human diseases. However, cilia-related research has been limited to a few cell and tissue types; to our knowledge, no literature exists on primary cilia in airway smooth muscle (ASM). The aim of this study was to characterize primary cilia in human ASM. Primary cilia of human bronchial smooth muscle cells (HBSMCs) were examined using immunofluorescence confocal microscopy, and scanning and transmission electron microscopy. HBSMC migration and injury repair were examined by scratch-wound and epidermal growth factor (EGF)-induced migration assays. Cross-sectional images of normal human bronchi revealed that primary cilia of HBSMCs within each ASM bundle aggregated at the same horizontal level, forming a "cilium layer." Individual cilia of HBSMCs projected into extracellular matrix and exhibited varying degrees of deflection. Mechanochemical sensing molecules, polycystins, and alpha2-, alpha5-, and beta1-integrins were enriched in cilia, as was EGF receptor, known to activate jointly with integrins during cell migration. Migration assays demonstrated a ciliary contribution to HBSMC migration and wound repair. The primary cilia of ASM cells exert a role in sensing and transducing extracellular mechanochemical signals and in ASM injury repair. Defects in ASM ciliary function could potentially affect airway wall maintenance and/or remodeling, possibly relating to the genesis of bronchiectasis in autosomal dominant polycystic kidney disease, a disease of ciliopathy.

  4. Peripheral Airway Smooth Muscle, but Not the Trachealis, Is Hypercontractile in an Equine Model of Asthma.

    PubMed

    Matusovsky, Oleg S; Kachmar, Linda; Ijpma, Gijs; Bates, Genevieve; Zitouni, Nedjma; Benedetti, Andrea; Lavoie, Jean-Pierre; Lauzon, Anne-Marie

    2016-05-01

    Heaves is a naturally occurring equine disease that shares many similarities with human asthma, including reversible antigen-induced bronchoconstriction, airway inflammation, and remodeling. The purpose of this study was to determine whether the trachealis muscle is mechanically representative of the peripheral airway smooth muscle (ASM) in an equine model of asthma. Tracheal and peripheral ASM of heaves-affected horses under exacerbation, or under clinical remission of the disease, and control horses were dissected and freed of epithelium to measure unloaded shortening velocity (Vmax), stress (force/cross-sectional area), methacholine effective concentration at which 50% of the maximum response is obtained, and stiffness. Myofibrillar Mg(2+)-ATPase activity, actomyosin in vitro motility, and contractile protein expression were also measured. Horses with heaves had significantly greater Vmax and Mg(2+)-ATPase activity in peripheral airway but not in tracheal smooth muscle. In addition, a significant correlation was found between Vmax and the time elapsed since the end of the corticosteroid treatment for the peripheral airways in horses with heaves. Maximal stress and stiffness were greater in the peripheral airways of the horses under remission compared with controls and the horses under exacerbation, potentially due to remodeling. Actomyosin in vitro motility was not different between controls and horses with heaves. These data demonstrate that peripheral ASM is mechanically and biochemically altered in heaves, whereas the trachealis behaves as in control horses. It is therefore conceivable that the trachealis muscle may not be representative of the peripheral ASM in human asthma either, but this will require further investigation.

  5. Functional expression of the TMEM16 family of calcium-activated chloride channels in airway smooth muscle

    PubMed Central

    Remy, Kenneth E.; Danielsson, Jennifer; Funayama, Hiromi; Fu, Xiao Wen; Chang, Herng-Yu Sucie; Yim, Peter; Xu, Dingbang; Emala, Charles W.

    2013-01-01

    Airway smooth muscle hyperresponsiveness is a key component in the pathophysiology of asthma. Although calcium-activated chloride channel (CaCC) flux has been described in many cell types, including human airway smooth muscle (HASM), the true molecular identity of the channels responsible for this chloride conductance remains controversial. Recently, a new family of proteins thought to represent the true CaCCs was identified as the TMEM16 family. This led us to question whether members of this family are functionally expressed in native and cultured HASM. We further questioned whether expression of these channels contributes to the contractile function of HASM. We identified the mRNA expression of eight members of the TMEM16 family in HASM cells and show immunohistochemical evidence of TMEM16A in both cultured and native HASM. Functionally, we demonstrate that the classic chloride channel inhibitor, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), inhibited halide flux in cultured HASM cells. Moreover, HASM cells displayed classical electrophysiological properties of CaCCs during whole cell electrophysiological recordings, which were blocked by using an antibody selective for TMEM16A. Furthermore, two distinct TMEM16A antagonists (tannic acid and benzbromarone) impaired a substance P-induced contraction in isolated guinea pig tracheal rings. These findings demonstrate that multiple members of this recently described family of CaCCs are expressed in HASM cells, they display classic electrophysiological properties of CaCCs, and they modulate contractile tone in airway smooth muscle. The TMEM16 family may provide a novel therapeutic target for limiting airway constriction in asthma. PMID:23997176

  6. Functional expression of the TMEM16 family of calcium-activated chloride channels in airway smooth muscle.

    PubMed

    Gallos, George; Remy, Kenneth E; Danielsson, Jennifer; Funayama, Hiromi; Fu, Xiao Wen; Chang, Herng-Yu Sucie; Yim, Peter; Xu, Dingbang; Emala, Charles W

    2013-11-01

    Airway smooth muscle hyperresponsiveness is a key component in the pathophysiology of asthma. Although calcium-activated chloride channel (CaCC) flux has been described in many cell types, including human airway smooth muscle (HASM), the true molecular identity of the channels responsible for this chloride conductance remains controversial. Recently, a new family of proteins thought to represent the true CaCCs was identified as the TMEM16 family. This led us to question whether members of this family are functionally expressed in native and cultured HASM. We further questioned whether expression of these channels contributes to the contractile function of HASM. We identified the mRNA expression of eight members of the TMEM16 family in HASM cells and show immunohistochemical evidence of TMEM16A in both cultured and native HASM. Functionally, we demonstrate that the classic chloride channel inhibitor, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), inhibited halide flux in cultured HASM cells. Moreover, HASM cells displayed classical electrophysiological properties of CaCCs during whole cell electrophysiological recordings, which were blocked by using an antibody selective for TMEM16A. Furthermore, two distinct TMEM16A antagonists (tannic acid and benzbromarone) impaired a substance P-induced contraction in isolated guinea pig tracheal rings. These findings demonstrate that multiple members of this recently described family of CaCCs are expressed in HASM cells, they display classic electrophysiological properties of CaCCs, and they modulate contractile tone in airway smooth muscle. The TMEM16 family may provide a novel therapeutic target for limiting airway constriction in asthma.

  7. A 'Good' muscle in a 'Bad' environment: the importance of airway smooth muscle force adaptation to airway hyperresponsiveness.

    PubMed

    Bossé, Ynuk; Chapman, David G; Paré, Peter D; King, Gregory G; Salome, Cheryl M

    2011-12-15

    Asthma is characterized by airway inflammation, with a consequent increase in spasmogens, and exaggerated airway narrowing in response to stimuli, termed airway hyperresponsiveness (AHR). The nature of any relationship between inflammation and AHR is less clear. Recent ex vivo data has suggested a novel mechanism by which inflammation may lead to AHR, in which increased basal ASM-tone, due to the presence of spasmogens in the airways, may "strengthen" the ASM and ultimately lead to exaggerated airway narrowing. This phenomenon was termed "force adaptation" [Bossé, Y., Chin, L.Y., Paré, P.D., Seow, C.Y., 2009. Adaptation of airway smooth muscle to basal tone: relevance to airway hyperresponsiveness. Am. J. Respir. Cell Mol. Biol. 40, 13-18]. However, it is unknown whether the magnitude of the effect of force adaptation ex vivo could contribute to exaggerated airway narrowing in vivo. Our aim was to utilize a computational model of ASM shortening in order to quantify the potential effect of force adaptation on airway narrowing when all other mechanical factors were kept constant. The shortening in the model is dictated by a balance between physiological loads and ASM force-generating capacity at different lengths. The results suggest that the magnitude of the effect of force adaptation on ASM shortening would lead to substantially more airway narrowing during bronchial challenge at any given airway generation. We speculate that the increased basal ASM-tone in asthma, due to the presence of inflammation-derived spasmogens, produces an increase in the force-generating capacity of ASM, predisposing to AHR during subsequent challenge. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. Interleukin-4 activates large-conductance, calciumactivated potassium (BKCa) channels in human airway smooth muscle cells

    PubMed Central

    Martin, Gilles; O’Connell, Robert J.; Pietrzykowski, Andrzej Z.; Treistman, Steven N.; Ethier, Michael F.; Madison, J. Mark

    2014-01-01

    Large-conductance, calcium-activated potassium (BKCa) channels are regulated by voltage and near-membrane calcium concentrations and are determinants of membrane potential and excitability in airway smooth muscle cells. Since the T helper–2 (Th2) cytokine, interleukin (IL)-4, is an important mediator of airway inflammation, we investigated whether IL-4 rapidly regulated BKCa activity in normal airway smooth muscle cells. On-cell voltage clamp recordings were made on subconfluent, cultured human bronchial smooth muscle cells (HBSMC). Interleukin-4 (50 ng ml−1), IL-13 (50 ng ml−1) or histamine (10 μm) was added to the bath during the recordings. Immunofluorescence studies with selective antibodies against the α and β1 subunits of BKCa were also performed. Both approaches demonstrated that HBSMC membranes contained large-conductance channels (>200 pS) with both calcium and voltage sensitivity, all of which is characteristic of the BKCa channel. Histamine caused a rapid increase in channel activity, as expected. A new finding was that perfusion with IL-4 stimulated rapid, large increases in BKCa channel activity (77.2 ± 63.3-fold increase, P < 0.05, n = 18). This large potentiation depended on the presence of external calcium. In contrast, IL-13 (50 ng ml−1) had little effect on BKCa channel activity, but inhibited the effect of IL-4. Thus, HBSMC contain functional BKCa channels whose activity is rapidly potentiated by the cytokine, IL-4, but not by IL-13.These findings are consistent with a model in which IL-4 rapidly increases near-membrane calcium concentrations to regulate BKCa activity. PMID:18403443

  9. Nogo-B regulates migration and contraction of airway smooth muscle cells by decreasing ARPC 2/3 and increasing MYL-9 expression.

    PubMed

    Xu, Wujian; Hong, Weijun; Shao, Yan; Ning, Yunye; Cai, Zailong; Li, Qiang

    2011-01-21

    Abnormal proliferation, apoptosis, migration and contraction of airway smooth muscle (ASM) cells in airway remodeling in asthma are basically excessive repair responses to a network of inflammatory mediators such as PDGF, but the mechanisms of such responses remain unclear. Nogo-B, a member of the reticulum family 4(RTN4), is known to play a key role in arteriogenesis and tissue repair. Further studies are needed to elucidate the role of Nogo-B in airway smooth muscle abnormalities. A mouse model of chronic asthma was established by repeated OVA inhalation and subjected to Nogo-B expression analysis using immunohistochemistry and Western Blotting. Then, primary human bronchial smooth muscle cells (HBSMCs) were cultured in vitro and a siRNA interference was performed to knockdown the expression of Nogo-B in the cells. The effects of Nogo-B inhibition on PDGF-induced HBSMCs proliferation, migration and contraction were evaluated. Finally, a proteomic analysis was conducted to unveil the underlying mechanisms responsible for the function of Nogo-B. Total Nogo-B expression was approximately 3.08-fold lower in chronic asthmatic mice compared to naïve mice, which was obvious in the smooth muscle layer of the airways. Interference of Nogo-B expression by siRNA resulted nearly 96% reduction in mRNA in cultured HBSMCs. In addition, knockdown of Nogo-B using specific siRNA significantly decreased PDGF-induced migration of HBSMCs by 2.3-fold, and increased the cellular contraction by 16% compared to negative controls, but had limited effects on PDGF-induced proliferation. Furthermore, using proteomic analysis, we demonstrate that the expression of actin related protein 2/3 complex subunit 5 (ARPC 2/3) decreased and, myosin regulatory light chain 9 isoform a (MYL-9) increased after Nogo-B knockdown. These data define a novel role for Nogo-B in airway remodeling in chronic asthma. Endogenous Nogo-B, which may exert its effects through ARPC 2/3 and MYL-9, is necessary for the

  10. Relaxant effect of superimposed length oscillation on sensitized airway smooth muscle.

    PubMed

    Jo-Avila, Miguel; Al-Jumaily, Ahmed M; Lu, Jun

    2015-03-01

    Asthma is associated with reductions in the airway lumen and breathing difficulties that are attributed to airway smooth muscles (ASM) hyperconstriction. Pharmaceutical bronchodilators such as salbutamol and isoproterenol are normally used to alleviate this constriction. Deep inspirations and tidal oscillations (TO) have also been reported to relax ASM in healthy airways with less response in asthmatics. Little information is available on the effect of other forms of oscillation on asthmatic airways. This study investigates the effect of length oscillations (LO), with amplitude 1 and 1.5% in the frequency range 5-20 Hz superimposed on breathing equivalent LO, on contracted ASM dissected from sensitized mice. These mice are believed to show some symptoms such as airway hyperreactivity similar to those associated with asthma in humans. In the frequency range used in this work, this study shows an increase in ASM relaxation of an average of 10% for 1.5% amplitude when compared with TO, ISO, or the combination of both. No similar finding is observed with 1% amplitude. This suggests that superimposed length oscillation acting over the interaction of myosin and actin during contraction may lead to temporal rearrangement and disturbance of the cross-bridge process in asthmatic airways. Copyright © 2015 the American Physiological Society.

  11. Propofol preferentially relaxes neurokinin receptor-2-induced airway smooth muscle contraction in guinea pig trachea.

    PubMed

    Gleason, Neil R; Gallos, George; Zhang, Yi; Emala, Charles W

    2010-06-01

    Propofol is the anesthetic of choice for patients with reactive airway disease and is thought to reduce intubation- or irritant-induced bronchoconstriction by decreasing the cholinergic component of vagal nerve activation. However, additional neurotransmitters, including neurokinins, play a role in irritant-induced bronchoconstriction. We questioned the mechanistic assumption that the clinically recognized protective effect of propofol against irritant-induced bronchoconstriction during intubation was due to attenuation of airway cholinergic reflexes. Muscle force was continuously recorded from isolated guinea pig tracheal rings in organ baths. Rings were subjected to exogenous contractile agonists (acetylcholine, histamine, endothelin-1, substance P, acetyl-substance P, and neurokinin A) or to electrical field stimulation (EFS) to differentiate cholinergic or nonadrenergic, noncholinergic nerve-mediated contraction with or without cumulatively increasing concentrations of propofol, thiopental, etomidate, or ketamine. Propofol did not attenuate the cholinergic component of EFS-induced contraction at clinically relevant concentrations. In contrast, propofol relaxed nonadrenergic, noncholinergic-mediated EFS contraction at concentrations within the clinical range (20-100 mum, n = 9; P < 0.05), and propofol was more potent against an exogenous selective neurokinin-2 receptor versus neurokinin-1 receptor agonist contraction (n = 6, P < 0.001). Propofol, at clinically relevant concentrations, relaxes airway smooth muscle contracted by nonadrenergic, noncholinergic-mediated EFS and exogenous neurokinins but not contractions elicited by the cholinergic component of EFS. These findings suggest that the mechanism of protective effects of propofol against irritant-induced bronchoconstriction involves attenuation of tachykinins released from nonadrenergic, noncholinergic nerves acting at neurokinin-2 receptors on airway smooth muscle.

  12. p21-Activated kinase (Pak) regulates airway smooth muscle contraction by regulating paxillin complexes that mediate actin polymerization.

    PubMed

    Zhang, Wenwu; Huang, Youliang; Gunst, Susan J

    2016-09-01

    In airway smooth muscle, tension development caused by a contractile stimulus requires phosphorylation of the 20 kDa myosin light chain (MLC), which activates crossbridge cycling and the polymerization of a pool of submembraneous actin. The p21-activated kinases (Paks) can regulate the contractility of smooth muscle and non-muscle cells, and there is evidence that this occurs through the regulation of MLC phosphorylation. We show that Pak has no effect on MLC phosphorylation during the contraction of airway smooth muscle, and that it regulates contraction by mediating actin polymerization. We find that Pak phosphorylates the adhesion junction protein, paxillin, on Ser273, which promotes the formation of a signalling complex that activates the small GTPase, cdc42, and the actin polymerization catalyst, neuronal Wiskott-Aldrich syndrome protein (N-WASP). These studies demonstrate a novel role for Pak in regulating the contractility of smooth muscle by regulating actin polymerization. The p21-activated kinases (Pak) can regulate contractility in smooth muscle and other cell and tissue types, but the mechanisms by which Paks regulate cell contractility are unclear. In airway smooth muscle, stimulus-induced contraction requires phosphorylation of the 20 kDa light chain of myosin, which activates crossbridge cycling, as well as the polymerization of a small pool of actin. The role of Pak in airway smooth muscle contraction was evaluated by inhibiting acetylcholine (ACh)-induced Pak activation through the expression of a kinase inactive mutant, Pak1 K299R, or by treating tissues with the Pak inhibitor, IPA3. Pak inhibition suppressed actin polymerization and contraction in response to ACh, but it did not affect myosin light chain phosphorylation. Pak activation induced paxillin phosphorylation on Ser273; the paxillin mutant, paxillin S273A, inhibited paxillin Ser273 phosphorylation and inhibited actin polymerization and contraction. Immunoprecipitation analysis of

  13. Biphasic force response to iso-velocity stretch in airway smooth muscle.

    PubMed

    Norris, Brandon A; Lan, Bo; Wang, Lu; Pascoe, Christopher D; Swyngedouw, Nicholas E; Paré, Peter D; Seow, Chun Y

    2015-10-01

    Airway smooth muscle (ASM) in vivo is constantly subjected to oscillatory strain due to tidal breathing and deep inspirations. ASM contractility is known to be adversely affected by strains, especially those of large amplitudes. Based on the cross-bridge model of contraction, it is likely that strain impairs force generation by disrupting actomyosin cross-bridge interaction. There is also evidence that strain modulates muscle stiffness and force through induction of cytoskeletal remodeling. However, the molecular mechanism by which strain alters smooth muscle function is not entirely clear. Here, we examine the response of ASM to iso-velocity stretches to probe the components within the muscle preparation that give rise to different features in the force response. We found in ASM that force response to a ramp stretch showed a biphasic feature, with the initial phase associated with greater muscle stiffness compared with that in the later phase, and that the transition between the phases occurred at a critical strain of ∼3.3%. Only strains with amplitudes greater than the critical strain could lead to reduction in force and stiffness of the muscle in the subsequent stretches. The initial-phase stiffness was found to be linearly related to the degree of muscle activation, suggesting that the stiffness stems mainly from attached cross bridges. Both phases were affected by the degree of muscle activation and by inhibitors of myosin light-chain kinase, PKC, and Rho-kinase. Different responses due to different interventions suggest that cross-bridge and cytoskeletal stiffness is regulated differently by the kinases. Copyright © 2015 the American Physiological Society.

  14. Phosphodiesterases regulate airway smooth muscle function in health and disease.

    PubMed

    Krymskaya, Vera P; Panettieri, Reynold A

    2007-01-01

    On the basis of structure, regulation, and kinetic properties, phosphodiesterases (PDEs) represent a superfamily of enzymes divided into 11 subfamilies that catalyze cytosolic levels of 3',5'-cyclic adenosine monophosphate (cAMP) or 3',5'-cyclic guanosine monophosphate (cGMP) to 5'-AMP or 5'-GMP, respectively. PDE4 represents the major PDE expressed in inflammatory cells as well as airway smooth muscle (ASM), and selective PDE4 inhibitors provide a broad spectrum of anti-inflammatory effects such as abrogating cytokine and chemokine release from inflammatory cells and inhibiting inflammatory cell trafficking. Due to cell- and tissue-specific gene expression and regulation, PDEs modulate unique organ-based functions. New tools or compounds that selectively inhibit PDE subfamilies and genetically engineered mice deficient in selective isoforms have greatly enhanced our understanding of PDE function in airway inflammation and resident cell function. This chapter will focus on recent advances in our understanding of the role of PDE in regulating ASM function.

  15. Could an increase in airway smooth muscle shortening velocity cause airway hyperresponsiveness?

    PubMed Central

    Bullimore, Sharon R.; Siddiqui, Sana; Donovan, Graham M.; Martin, James G.; Sneyd, James; Bates, Jason H. T.

    2011-01-01

    Airway hyperresponsiveness (AHR) is a characteristic feature of asthma. It has been proposed that an increase in the shortening velocity of airway smooth muscle (ASM) could contribute to AHR. To address this possibility, we tested whether an increase in the isotonic shortening velocity of ASM is associated with an increase in the rate and total amount of shortening when ASM is subjected to an oscillating load, as occurs during breathing. Experiments were performed in vitro using 27 rat tracheal ASM strips supramaximally stimulated with methacholine. Isotonic velocity at 20% isometric force (Fiso) was measured, and then the load on the muscle was varied sinusoidally (0.33 ± 0.25 Fiso, 1.2 Hz) for 20 min, while muscle length was measured. A large amplitude oscillation was applied every 4 min to simulate a deep breath. We found that: 1) ASM strips with a higher isotonic velocity shortened more quickly during the force oscillations, both initially (P < 0.001) and after the simulated deep breaths (P = 0.002); 2) ASM strips with a higher isotonic velocity exhibited a greater total shortening during the force oscillation protocol (P < 0.005); and 3) the effect of an increase in isotonic velocity was at least comparable in magnitude to the effect of a proportional increase in ASM force-generating capacity. A cross-bridge model showed that an increase in the total amount of shortening with increased isotonic velocity could be explained by a change in either the cycling rate of phosphorylated cross bridges or the rate of myosin light chain phosphorylation. We conclude that, if asthma involves an increase in ASM velocity, this could be an important factor in the associated AHR. PMID:20971805

  16. A study of airway smooth muscle in asthmatic and non-asthmatic airways using PS-OCT (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Adams, David C.; Holz, Jasmin A.; Szabari, Margit V.; Hariri, Lida P.; Harris, R. Scott; Cho, Jocelyn L.; Hamilos, Daniel L.; Luster, Andrew D.; Medoff, Benjamin D.; Suter, Melissa J.

    2016-03-01

    Present understanding of the pathophysiological mechanisms of asthma has been severely limited by the lack of an imaging modality capable of assessing airway conditions of asthma patients in vivo. Of particular interest is the role that airway smooth muscle (ASM) plays in the development of asthma and asthma related symptoms. With standard Optical Coherence Tomography (OCT), imaging ASM is often not possible due to poor structural contrast between the muscle and surrounding tissues. A potential solution to this problem is to utilize additional optical contrast factors intrinsic to the tissue, such as birefringence. Due to its highly ordered structure, ASM is strongly birefringent. Previously, we demonstrated that Polarization Sensitive OCT(PS-OCT) has the potential to be used to visualize ASM as well as easily segment it from the surrounding (weakly) birefringent tissue by exploiting a property which allows it to discriminate the orientation of birefringent fibers. We have already validated our technology with a substantial set of histological comparisons made against data obtained ex vivo. In this work we present a comprehensive comparison of ASM distributions in asthmatic and non-asthmatic human volunteers. By isolating the ASM we parameterize its distribution in terms of both thickness and band width, calculated volumetrically over centimeters of airway. Using this data we perform analyses of the asthmatic and non-asthmatic airways using a broad number and variety and subjects.

  17. The Pro-Proliferative Effects of Nicotine and Its Underlying Mechanism on Rat Airway Smooth Muscle Cells

    PubMed Central

    He, Fang; Li, Bing; Zhao, Zhuxiang; Zhou, Yumin; Hu, Guoping; Zou, Weifeng; Hong, Wei; Zou, Yimin; Jiang, Changbin; Zhao, Dongxing; Ran, Pixin

    2014-01-01

    Recent studies have shown that nicotine, a major component of cigarette smoke, can stimulate the proliferation of non-neuronal cells. Cigarette smoking can promote a variety of pulmonary and cardiovascular diseases, such as chronic obstructive pulmonary disease (COPD), atherosclerosis, and cancer. A predominant feature of COPD is airway remodeling, which includes increased airway smooth muscle (ASM) mass. The mechanisms underlying ASM remodeling in COPD have not yet been fully elucidated. Here, we show that nicotine induces a profound and time-dependent increase in DNA synthesis in rat airway smooth muscle cells (RASMCs) in vitro. Nicotine also significantly increased the number of RASMCs, which was associated with the increased expression of Cyclin D1, phosphorylation of the retinoblastoma protein (RB) and was dependent on the activation of Akt. The activation of Akt by nicotine occurred within minutes and depended upon the nicotinic acetylcholine receptors (nAchRs). Activated Akt increased the phosphorylation of downstream substrates such as GSK3β. Our data suggest that the binding of nicotine to the nAchRs on RASMCs can regulate cellular proliferation by activating the Akt pathway. PMID:24690900

  18. Transforming growth factor‐β enhances Rho‐kinase activity and contraction in airway smooth muscle via the nucleotide exchange factor ARHGEF1

    PubMed Central

    Shaifta, Yasin; MacKay, Charles E.; Irechukwu, Nneka; O'Brien, Katie A.; Wright, David B.; Ward, Jeremy P. T.

    2017-01-01

    Key points Transforming growth‐factor‐β (TGF‐β) and RhoA/Rho‐kinase are independently implicated in the airway hyper‐responsiveness associated with asthma, but how these proteins interact is not fully understood.We examined the effects of pre‐treatment with TGF‐β on expression and activity of RhoA, Rho‐kinase and ARHGEF1, an activator of RhoA, as well as on bradykinin‐induced contraction, in airway smooth muscle.TGF‐β enhanced bradykinin‐induced RhoA translocation, Rho‐kinase‐dependent phosphorylation and contraction, but partially suppressed bradykinin‐induced RhoA activity (RhoA‐GTP content).TGF‐β enhanced the expression of ARHGEF1, while a small interfering RNA against ARHGEF1 and a RhoGEF inhibitor prevented the effects of TGF‐β on RhoA and Rho‐kinase activity and contraction, respectively.ARHGEF1 expression was also enhanced in airway smooth muscle from asthmatic patients and ovalbumin‐sensitized mice.ARHGEF1 is a key TGF‐β target gene, an important regulator of Rho‐kinase activity and therefore a potential therapeutic target for the treatment of asthmatic airway hyper‐responsiveness. Abstract Transforming growth factor‐β (TGF‐β), RhoA/Rho‐kinase and Src‐family kinases (SrcFK) have independently been implicated in airway hyper‐responsiveness, but how they interact to regulate airway smooth muscle contractility is not fully understood. We found that TGF‐β pre‐treatment enhanced acute contractile responses to bradykinin (BK) in isolated rat bronchioles, and inhibitors of RhoGEFs (Y16) and Rho‐kinase (Y27632), but not the SrcFK inhibitor PP2, prevented this enhancement. In cultured human airway smooth muscle cells (hASMCs), TGF‐β pre‐treatment enhanced the protein expression of the Rho guanine nucleotide exchange factor ARHGEF1, MLC20, MYPT‐1 and the actin‐severing protein cofilin, but not of RhoA, ROCK2 or c‐Src. In hASMCs, acute treatment with BK triggered subcellular translocation

  19. p21‐Activated kinase (Pak) regulates airway smooth muscle contraction by regulating paxillin complexes that mediate actin polymerization

    PubMed Central

    Zhang, Wenwu; Huang, Youliang

    2016-01-01

    Key points In airway smooth muscle, tension development caused by a contractile stimulus requires phosphorylation of the 20 kDa myosin light chain (MLC), which activates crossbridge cycling and the polymerization of a pool of submembraneous actin.The p21‐activated kinases (Paks) can regulate the contractility of smooth muscle and non‐muscle cells, and there is evidence that this occurs through the regulation of MLC phosphorylation.We show that Pak has no effect on MLC phosphorylation during the contraction of airway smooth muscle, and that it regulates contraction by mediating actin polymerization.We find that Pak phosphorylates the adhesion junction protein, paxillin, on Ser273, which promotes the formation of a signalling complex that activates the small GTPase, cdc42, and the actin polymerization catalyst, neuronal Wiskott–Aldrich syndrome protein (N‐WASP).These studies demonstrate a novel role for Pak in regulating the contractility of smooth muscle by regulating actin polymerization. Abstract The p21‐activated kinases (Pak) can regulate contractility in smooth muscle and other cell and tissue types, but the mechanisms by which Paks regulate cell contractility are unclear. In airway smooth muscle, stimulus‐induced contraction requires phosphorylation of the 20 kDa light chain of myosin, which activates crossbridge cycling, as well as the polymerization of a small pool of actin. The role of Pak in airway smooth muscle contraction was evaluated by inhibiting acetylcholine (ACh)‐induced Pak activation through the expression of a kinase inactive mutant, Pak1 K299R, or by treating tissues with the Pak inhibitor, IPA3. Pak inhibition suppressed actin polymerization and contraction in response to ACh, but it did not affect myosin light chain phosphorylation. Pak activation induced paxillin phosphorylation on Ser273; the paxillin mutant, paxillin S273A, inhibited paxillin Ser273 phosphorylation and inhibited actin polymerization and contraction

  20. Smooth muscle in human bronchi is disposed to resist airway distension.

    PubMed

    Gazzola, Morgan; Henry, Cyndi; Couture, Christian; Marsolais, David; King, Gregory G; Fredberg, Jeffrey J; Bossé, Ynuk

    2016-07-15

    Studying airway smooth muscle (ASM) in conditions that emulate the in vivo environment within which the bronchi normally operate may provide important clues regarding its elusive physiological function. The present study examines the effect of lengthening and shortening of ASM on tension development in human bronchial segments. ASM from each bronchial segment was set at a length approximating in situ length (Linsitu). Bronchial tension was then measured during a slow cyclical strain (0.004Hz, from 0.7Linsitu to 1.3Linsitu) in the relaxed state and at graded levels of activation by methacholine. In all cases, tension was greater at longer ASM lengths, and greater during lengthening than shortening. The threshold of methacholine concentration that was required for ASM to account for bronchial tension across the entire range of ASM lengths tested was on average smaller by 2.8 logs during lengthening than during shortening. The length-dependency of ASM tension, together with this lower threshold of methacholine concentration during lengthening versus shortening, suggest that ASM has a greater ability to resist airway dilation during lung inflation than to narrow the airways during lung deflation. More than serving to narrow the airway, as has long been thought, these data suggest that the main function of ASM contraction is to limit airway wall distension during lung inflation. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Mechanisms of Cigarette Smoke Effects on Human Airway Smooth Muscle.

    PubMed

    Wylam, Mark E; Sathish, Venkatachalem; VanOosten, Sarah Kay; Freeman, Michelle; Burkholder, David; Thompson, Michael A; Pabelick, Christina M; Prakash, Y S

    2015-01-01

    Cigarette smoke contributes to or exacerbates airway diseases such as asthma and COPD, where airway hyperresponsiveness and airway smooth muscle (ASM) proliferation are key features. While factors such as inflammation contribute to asthma in part by enhancing agonist-induced intracellular Ca(2+) ([Ca(2+)]i) responses of ASM, the mechanisms by which cigarette smoke affect ASM are still under investigation. In the present study, we tested the hypothesis that cigarette smoke enhances the expression and function of Ca(2+) regulatory proteins leading to increased store operated Ca(2+) entry (SOCE) and cell proliferation. Using isolated human ASM (hASM) cells, incubated in the presence and absence cigarette smoke extract (CSE) we determined ([Ca(2+)]i) responses and expression of relevant proteins as well as ASM proliferation, reactive oxidant species (ROS) and cytokine generation. CSE enhanced [Ca(2+)]i responses to agonist and SOCE: effects mediated by increased expression of TRPC3, CD38, STIM1, and/or Orai1, evident by attenuation of CSE effects when siRNAs against these proteins were used, particularly Orai1. CSE also increased hASM ROS generation and cytokine secretion. In addition, we found in the airways of patients with long-term smoking history, TRPC3 and CD38 expression were significantly increased compared to life-long never-smokers, supporting the role of these proteins in smoking effects. Finally, CSE enhanced hASM proliferation, an effect confirmed by upregulation of PCNA and Cyclin E. These results support a critical role for Ca(2+) regulatory proteins and enhanced SOCE to alter airway structure and function in smoking-related airway disease.

  2. Airway compliance and dynamics explain the apparent discrepancy in length adaptation between intact airways and smooth muscle strips.

    PubMed

    Dowie, Jackson; Ansell, Thomas K; Noble, Peter B; Donovan, Graham M

    2016-01-01

    Length adaptation is a phenomenon observed in airway smooth muscle (ASM) wherein over time there is a shift in the length-tension curve. There is potential for length adaptation to play an important role in airway constriction and airway hyper-responsiveness in asthma. Recent results by Ansell et al., 2015 (JAP 2014 10.1152/japplphysiol.00724.2014) have cast doubt on this role by testing for length adaptation using an intact airway preparation, rather than strips of ASM. Using this technique they found no evidence for length adaptation in intact airways. Here we attempt to resolve this apparent discrepancy by constructing a minimal mathematical model of the intact airway, including ASM which follows the classic length-tension curve and undergoes length adaptation. This allows us to show that (1) no evidence of length adaptation should be expected in large, cartilaginous, intact airways; (2) even in highly compliant peripheral airways, or at more compliant regions of the pressure-volume curve of large airways, the effect of length adaptation would be modest and at best marginally detectable in intact airways; (3) the key parameters which control the appearance of length adaptation in intact airways are airway compliance and the relaxation timescale. The results of this mathematical simulation suggest that length adaptation observed at the level of the isolated ASM may not clearly manifest in the normal intact airway. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. S100A8 protein attenuates airway hyperresponsiveness by suppressing the contraction of airway smooth muscle.

    PubMed

    Xu, Yu-Dong; Wang, Yu; Yin, Lei-Miao; Park, Gyoung-Hee; Ulloa, Luis; Yang, Yong-Qing

    2017-02-26

    Airway hyperresponsiveness (AHR) is a major clinical problem in allergic asthma mainly caused by the hypercontractility of airway smooth muscles (ASM). S100A8 is an important member of the S100 calcium-binding protein family with a potential to regulate cell contractility. Here, we analyze the potential of S100A8 to regulate allergen-induced AHR and ASM contraction. Treatment with recombinant S100A8 (rS100A8) diminished airway hyperresponsiveness in OVA-sensitized rats. ASM contraction assays showed that rS100A8 reduced hypercontractility in both isolated tracheal rings and primary ASM cells treated by acetylcholine. rS100A8 markedly rescued the phosphorylation level of myosin light chain induced by acetylcholine in ASM cells. These results show that rS100A8 plays a protective role in regulating AHR in asthma by inhibiting ASM contraction. These results support S100A8 as a novel therapeutic target to control ASM contraction in asthma. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  4. Effects of magnesium sulfate on airway smooth muscle contraction in rats.

    PubMed

    Betul Altinisik, Hatice; Kirdemir, Pakize; Altinisik, Ugur; Gokalp, Osman

    2016-08-01

    Aim To investigate the effect of magnesium sulfate (MgSO4) at different doses on isolated tracheal smooth muscle contraction in rats induced by different mechanisms. Methods Twelve rats' tracheas were placed into organ bath. Consecutively, acetylcholine (10-6,10-5,10-4 M), histamine(10-8,10-5,10-3 M) and KCl (30,60 mM) solutions was administered for contractions. MgSO4 from 10-4 to 10-1 M concentrations were subsequently administered after each constrictive agent and relaxation degrees were recorded. Results In the acetylcholine and KCl groups, dose dependent strong contractions were observed, but not in the histamine group and that group was excluded. Significant relaxation occurred with gradually increasing doses of MgSO4. In the high dose KCl group, a slight increase in contractions after the administration of 10-4 and 10-3 M MgSO4 was recorded. Conclusion We suggest that MgSO4 is effective in relaxing airway smooth muscle contractions caused by different factors; however, it must be considered that low doses of MgSO4 may only lead to a slight increase in contractions. Copyright© by the Medical Assotiation of Zenica-Doboj Canton.

  5. cAMP Regulation of Airway Smooth Muscle Function

    PubMed Central

    Billington, Charlotte K.; Ojo, Oluwaseun O.; Penn, Raymond B.; Ito, Satoru

    2013-01-01

    Agonists activating β2-adrenoceptors (β2ARs) on airway smooth muscle (ASM) are the drug of choice for rescue from acute bronchoconstriction in patients with both asthma and chronic obstructive pulmonary disease (COPD). Moreover, the use of long-acting β-agonists combined with inhaled corticosteroids constitutes an important maintenance therapy for these diseases. β-Agonists are effective bronchodilators due primarily to their ability to antagonize ASM contraction. The presumed cellular mechanism of action involves the generation of intracellular cAMP, which in turn can activate the effector molecules cAMP-dependent protein kinase (PKA) and Epac. Other agents such as prostaglandin E2 and phosphodiesterase inhibitors that also increase intracellular cAMP levels in ASM, can also antagonize ASM contraction, and inhibit other ASM functions including proliferation and migration. Therefore, β2ARs and cAMP are key players in combating the pathophysiology of airway narrowing and remodeling. However, limitations of β-agonist therapy due to drug tachyphylaxis related to β2AR desensitization, and recent findings regarding the manner in which β2ARs and cAMP signal, have raised new and interesting questions about these well-studied molecules. In this review we discuss current concepts regarding β2ARs and cAMP in the regulation of ASM cell functions and their therapeutic roles in asthma and COPD. PMID:22634112

  6. cAMP regulation of airway smooth muscle function.

    PubMed

    Billington, Charlotte K; Ojo, Oluwaseun O; Penn, Raymond B; Ito, Satoru

    2013-02-01

    Agonists activating β(2)-adrenoceptors (β(2)ARs) on airway smooth muscle (ASM) are the drug of choice for rescue from acute bronchoconstriction in patients with both asthma and chronic obstructive pulmonary disease (COPD). Moreover, the use of long-acting β-agonists combined with inhaled corticosteroids constitutes an important maintenance therapy for these diseases. β-Agonists are effective bronchodilators due primarily to their ability to antagonize ASM contraction. The presumed cellular mechanism of action involves the generation of intracellular cAMP, which in turn can activate the effector molecules cAMP-dependent protein kinase (PKA) and Epac. Other agents such as prostaglandin E(2) and phosphodiesterase inhibitors that also increase intracellular cAMP levels in ASM, can also antagonize ASM contraction, and inhibit other ASM functions including proliferation and migration. Therefore, β(2)ARs and cAMP are key players in combating the pathophysiology of airway narrowing and remodeling. However, limitations of β-agonist therapy due to drug tachyphylaxis related to β(2)AR desensitization, and recent findings regarding the manner in which β(2)ARs and cAMP signal, have raised new and interesting questions about these well-studied molecules. In this review we discuss current concepts regarding β(2)ARs and cAMP in the regulation of ASM cell functions and their therapeutic roles in asthma and COPD. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. Device for Investigation of Mechanical Tension of Isolated Smooth Muscle Vessels and Airway Segments of Animals

    NASA Astrophysics Data System (ADS)

    Aleinik, A.; Karpovich, N.; Turgunova, N.; Nosarev, A.

    2016-11-01

    For the purpose of testing and the search for new drug compounds, designed to heal many human diseases, it is necessary to investigate the deformation of experimental tissue samples under influence of these drugs. For this task a precision force sensor for measuring the mechanical tension, produced by isolated ring segments of blood vessels and airways was created. The hardware and software systems for the study of changes in contractile responses of the airway smooth muscles and blood vessels of experimental animals was developed.

  8. Human Rhinovirus Infection of Epithelial Cells Modulates Airway Smooth Muscle Migration.

    PubMed

    Shariff, Sami; Shelfoon, Christopher; Holden, Neil S; Traves, Suzanne L; Wiehler, Shahina; Kooi, Cora; Proud, David; Leigh, Richard

    2017-06-01

    Airway remodeling, a characteristic feature of asthma, begins in early life. Recurrent human rhinovirus (HRV) infections are a potential inciting stimulus for remodeling. One component of airway remodeling is an increase in airway smooth muscle cell (ASMC) mass with a greater proximity of the ASMCs to the airway epithelium. We asked whether human bronchial epithelial cells infected with HRV produced mediators that are chemotactic for ASMCs. ASMC migration was investigated using the modified Boyden Chamber and the xCELLigence Real-Time Cell Analyzer (ACEA Biosciences Inc., San Diego, CA). Multiplex bead analysis was used to measure HRV-induced epithelial chemokine release. The chemotactic effects of CCL5, CXCL8, and CXCL10 were also examined. Supernatants from HRV-infected epithelial cells caused ASMC chemotaxis. Pretreatment of ASMCs with pertussis toxin abrogated chemotaxis, as did treatment with formoterol, forskolin, or 8-bromo-cAMP. CCL5, CXCL8, and CXCL10 were the most up-regulated chemokines produced by HRV-infected airway epithelial cells. When recombinant CCL5, CXCL8, and CXCL10 were used at levels found in epithelial supernatants, they induced ASMC chemotaxis similar to that seen with epithelial cell supernatants. When examined individually, CCL5 was the most effective chemokine in causing ASMC migration, and treatment of supernatant from HRV-infected epithelial cells with anti-CCL5 antibodies significantly attenuated ASMC migration. These findings suggest that HRV-induced CCL5 can induce ASMC chemotaxis and thus may contribute to the pathogenesis of airway remodeling in patients with asthma.

  9. Smooth muscle myosin isoform expression and LC20 phosphorylation in innate rat airway hyperresponsiveness.

    PubMed

    Gil, Fulvio R; Zitouni, Nedjma B; Azoulay, Eric; Maghni, Karim; Lauzon, Anne-Marie

    2006-11-01

    Four smooth muscle myosin heavy chain (SMMHC) isoforms are generated by alternative mRNA splicing of a single gene. Two of these isoforms differ by the presence [(+)insert] or absence [(-)insert] of a 7-amino acid insert in the motor domain. The rate of actin filament propulsion of the (+)insert SMMHC isoform, as measured in the in vitro motility assay, is twofold greater than that of the (-)insert isoform. We hypothesized that a greater expression of the (+)insert SMMHC isoform and greater regulatory light chain (LC(20)) phosphorylation contribute to airway hyperresponsiveness. We measured airway responsiveness to methacholine in Fischer hyperresponsive and Lewis normoresponsive rats and determined SMMHC isoform mRNA and protein expression, as well as essential light chain (LC(17)) isoforms, h-caldesmon, and alpha-actin protein expression in their tracheae. We also measured tracheal muscle strip contractility in response to methacholine and corresponding LC(20) phosphorylation. We found Fischer rats have more (+)insert mRNA (69.4 +/- 2.0%) (mean +/- SE) than Lewis rats (53.0 +/- 2.4%; P < 0.05) and a 44% greater content of (+)insert isoform relative to total myosin protein. No difference was found for LC(17) isoform, h-caldesmon, and alpha-actin expression. The contractility experiments revealed a greater isometric force for Fischer trachealis segments (4.2 +/- 0.8 mN) than Lewis (1.9 +/- 0.4 mN; P < 0.05) and greater LC(20) phosphorylation level in Fischer (55.1 +/- 6.4) than in Lewis (41.4 +/- 6.1; P < 0.05) rats. These results further support the contention that innate airway hyperresponsiveness is a multifactorial disorder in which increased expression of the fast (+)insert SMMHC isoform and greater activation of LC(20) lead to smooth muscle hypercontractility.

  10. Human mast cell and airway smooth muscle cell interactions: implications for asthma.

    PubMed

    Page, S; Ammit, A J; Black, J L; Armour, C L

    2001-12-01

    Asthma is characterized by inflammation, hyperresponsiveness, and remodeling of the airway. Human mast cells (HMCs) play a central role in all of these changes by releasing mediators that cause exaggerated bronchoconstriction, induce human airway smooth muscle (HASM) cell proliferation, and recruit and activate inflammatory cells. Moreover, the number of HMCs present on asthmatic HASM is increased compared with that on nonasthmatic HASM. HASM cells also have the potential to actively participate in the inflammatory process by synthesizing cytokines and chemokines and expressing surface molecules, which have the capacity to perpetuate the inflammatory mechanisms present in asthma. This review specifically examines how the mediators of HMCs have the capacity to modulate many functions of HASM; how the synthetic function of HASM, particularly through the release and expression of stem cell factor, has the potential to influence HMC number and activation in an extraordinarily potent and proinflammatory manner; and how these interactions between HMCs and HASM have potential consequences for airway structure and inflammation relevant to the disease process of asthma.

  11. Emerging concepts in smooth muscle contributions to airway structure and function: implications for health and disease

    PubMed Central

    2016-01-01

    Airway structure and function are key aspects of normal lung development, growth, and aging, as well as of lung responses to the environment and the pathophysiology of important diseases such as asthma, chronic obstructive pulmonary disease, and fibrosis. In this regard, the contributions of airway smooth muscle (ASM) are both functional, in the context of airway contractility and relaxation, as well as synthetic, involving production and modulation of extracellular components, modulation of the local immune environment, cellular contribution to airway structure, and, finally, interactions with other airway cell types such as epithelium, fibroblasts, and nerves. These ASM contributions are now found to be critical in airway hyperresponsiveness and remodeling that occur in lung diseases. This review emphasizes established and recent discoveries that underline the central role of ASM and sets the stage for future research toward understanding how ASM plays a central role by being both upstream and downstream in the many interactive processes that determine airway structure and function in health and disease. PMID:27742732

  12. Direct evidence for functional smooth muscle myosin II in the 10S self-inhibited monomeric conformation in airway smooth muscle cells

    PubMed Central

    Milton, Deanna L.; Schneck, Amy N.; Ziech, Dominique A.; Ba, Mariam; Facemyer, Kevin C.; Halayko, Andrew J.; Baker, Jonathan E.; Gerthoffer, William T.; Cremo, Christine R.

    2011-01-01

    The 10S self-inhibited monomeric conformation of myosin II has been characterized extensively in vitro. Based upon its structural and functional characteristics, it has been proposed to be an assembly-competent myosin pool in equilibrium with filaments in cells. It is known that myosin filaments can assemble and disassemble in nonmuscle cells, and in some smooth muscle cells, but whether or not the disassembled pool contains functional 10S myosin has not been determined. Here we address this question using human airway smooth muscle cells (hASMCs). Using two antibodies against different epitopes on smooth muscle myosin II (SMM), two distinct pools of SMM, diffuse, and stress-fiber–associated, were visualized by immunocytochemical staining. The two SMM pools were functional in that they could be interconverted in two ways: (i) by exposure to 10S- versus filament-promoting buffer conditions, and (ii) by exposure to a peptide that shifts the filament-10S equilibrium toward filaments in vitro by a known mechanism that requires the presence of the 10S conformation. The effect of the peptide was not due to a trivial increase in SMM phosphorylation, and its specificity was demonstrated by use of a scrambled peptide, which had no effect. Based upon these data, we conclude that hASMCs contain a significant pool of functional SMM in the 10S conformation that can assemble into filaments upon changing cellular conditions. This study provides unique direct evidence for the presence of a significant pool of functional myosin in the 10S conformation in cells. PMID:21205888

  13. TNFα enhances force generation in airway smooth muscle

    PubMed Central

    Han, Young-Soo; Delmotte, Philippe

    2017-01-01

    Airway inflammation is a hallmark of asthma, triggering airway smooth muscle (ASM) hyperreactivity and airway remodeling. TNFα increases both agonist-induced cytosolic Ca2+ concentration ([Ca2+]cyt) and force in ASM. The effects of TNFα on ASM force may also be due to an increase in Ca2+ sensitivity, cytoskeletal remodeling, and/or changes in contractile protein content. We hypothesized that 24 h of exposure to TNFα increases ASM force by changing actin and myosin heavy chain (MyHC) content and/or polymerization. Porcine ASM strips were permeabilized with 10% Triton X-100, and force was measured in response to increasing concentrations of Ca2+ (pCa 9.0 to 4.0) in control and TNFα-treated groups. Relative phosphorylation of the regulatory myosin light chain (p-MLC) and total actin, MLC, and MyHC concentrations were quantified at pCa 9.0, 6.1, and 4.0. Actin polymerization was quantified by the ratio of filamentous to globular actin at pCa 9.0 and 4.0. For determination of total cross-bridge formation, isometric ATP hydrolysis rate at pCa 4.0 was measured using an enzyme-coupled NADH-linked fluorometric technique. Exposure to TNFα significantly increased force across the range of Ca2+ activation but did not affect the intrinsic Ca2+ sensitivity of force generation. The TNFα-induced increase in ASM force was associated with an increase in total actin, MLC, and MyHC content, as well as an increase in actin polymerization and an increase in maximum isometric ATP hydrolysis rate. The results of this study support our hypothesis that TNFα increases force generation in ASM by increasing the number of contractile units (actin-myosin content) contributing to force generation. PMID:28385814

  14. Molecular mechanisms underlying airway smooth muscle contraction and proliferation: implications for asthma.

    PubMed

    Pelaia, Girolamo; Renda, Teresa; Gallelli, Luca; Vatrella, Alessandro; Busceti, Maria Teresa; Agati, Sergio; Caputi, Mario; Cazzola, Mario; Maselli, Rosario; Marsico, Serafino A

    2008-08-01

    Airway smooth muscle (ASM) plays a key role in bronchomotor tone, as well as in structural remodeling of the bronchial wall. Therefore, ASM contraction and proliferation significantly participate in the development and progression of asthma. Many contractile agonists also behave as mitogenic stimuli, thus contributing to frame a hyperresponsive and hyperplastic ASM phenotype. In this review, the molecular mechanisms and signaling pathways involved in excitation-contraction coupling and ASM cell growth will be outlined. Indeed, the recent advances in understanding the basic aspects of ASM biology are disclosing important cellular targets, currently explored for the implementation of new, more effective anti-asthma therapies.

  15. Airway smooth muscle in asthma: linking contraction and mechanotransduction to disease pathogenesis and remodelling.

    PubMed

    Noble, Peter B; Pascoe, Chris D; Lan, Bo; Ito, Satoru; Kistemaker, Loes E M; Tatler, Amanda L; Pera, Tonio; Brook, Bindi S; Gosens, Reinoud; West, Adrian R

    2014-12-01

    Asthma is an obstructive airway disease, with a heterogeneous and multifactorial pathogenesis. Although generally considered to be a disease principally driven by chronic inflammation, it is becoming increasingly recognised that the immune component of the pathology poorly correlates with the clinical symptoms of asthma, thus highlighting a potentially central role for non-immune cells. In this context airway smooth muscle (ASM) may be a key player, as it comprises a significant proportion of the airway wall and is the ultimate effector of acute airway narrowing. Historically, the contribution of ASM to asthma pathogenesis has been contentious, yet emerging evidence suggests that ASM contractile activation imparts chronic effects that extend well beyond the temporary effects of bronchoconstriction. In this review article we describe the effects that ASM contraction, in combination with cellular mechanotransduction and novel contraction-inflammation synergies, contribute to asthma pathogenesis. Specific emphasis will be placed on the effects that ASM contraction exerts on the mechanical properties of the airway wall, as well as novel mechanisms by which ASM contraction may contribute to more established features of asthma such as airway wall remodelling. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Cysteinyl leukotrienes promote human airway smooth muscle migration.

    PubMed

    Parameswaran, Krishnan; Cox, Gerard; Radford, Katherine; Janssen, Luke J; Sehmi, Roma; O'Byrne, Paul M

    2002-09-01

    Cysteinyl leukotrienes promote airway smooth muscle (ASM) contraction and proliferation. Little is known about their role in ASM migration. We investigated this using cultured human ASMs (between the second and fifth passages) obtained from the large airways of resected nonasthmatic lung. Platelet-derived growth factor-BB (1 ng/ml) promoted significant (3.5-fold) ASM migration of myocytes across collagen-coated 8- micro m polycarbonate membranes in Transwell culture plates. Leukotriene E(4) (10(-7), 10(-8), 10(-9) M) did not demonstrate a chemotactic effect; it did promote chemokinesis. Priming by leukotriene E(4) (10(-7) M) significantly augmented the directional migratory response to platelet-derived growth factor (1.5-fold, p < 0.05). This was blocked by montelukast (10(-6) M), demonstrating the effect to be mediated by the cysteinyl leukotriene receptor. The "priming effect" was also partially attenuated by prostaglandin E(2) (10(-7) M). Whereas both the chemokinetic and the chemotactic "primed" responses were equally attenuated by a p38 mitogen-activated protein kinase inhibitor (SB203580, 25 micro M) and by a Rho-kinase inhibitor (Y27632, 10 micro M), the chemotactic response showed greater inhibition than chemokinesis by a phosphatidylinositol-3 kinase inhibitor (LY294002, 50 micro M). These experiments suggest that cysteinyl leukotrienes play an augmentary role in human ASM migration. The phosphatidylinositol-3 kinase pathway is a key signaling mechanism in the chemotactic migration of ASM cells in response to cysteinyl leukotrienes.

  17. 8-Bromo-cAMP decreases the Ca2+ sensitivity of airway smooth muscle contraction through a mechanism distinct from inhibition of Rho-kinase.

    PubMed

    Endou, Katsuaki; Iizuka, Kunihiko; Yoshii, Akihiro; Tsukagoshi, Hideo; Ishizuka, Tamotsu; Dobashi, Kunio; Nakazawa, Tsugio; Mori, Masatomo

    2004-10-01

    To clarify whether cyclic AMP (cAMP)/cAMP-dependent protein kinase (PKA) activation and Rho-kinase inhibition share a common mechanism to decrease the Ca2+ sensitivity of airway smooth muscle contraction, we examined the effects of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), a stable cAMP analog, and (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexane carboxamide dihydrochloride, monohydrate (Y-27632), a Rho-kinase inhibitor, on carbachol (CCh)-, guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS)-, 4beta-phorbol 12,13-dibutyrate (PDBu)-, and leukotriene D4 (LTD4)-induced Ca2+ sensitization in alpha-toxin-permeabilized rabbit tracheal and human bronchial smooth muscle. In rabbit trachea, CCh-induced smooth muscle contraction was inhibited by 8-BrcAMP and Y-27632 to a similar extent. However, GTPgammaS-induced smooth muscle contraction was resistant to 8-BrcAMP. In the presence of a saturating concentration of Y-27632, PDBu-induced smooth muscle contraction was completely reversed by 8-BrcAMP. Conversely, PDBu-induced smooth muscle contraction was resistant to Y-27632. In the presence of a saturating concentration of 8-BrcAMP, GTPgammaS-induced Ca2+ sensitization was also reversed by Y-27632. The 8-BrcAMP had no effect on the ATP-triggered contraction of tracheal smooth muscle that had been treated with calyculin A in rigor solutions. The 8-BrcAMP and Y-27632 additively accelerated the relaxation rate of PDBu- and GTPgammaS-treated smooth muscle under myosin light chain kinase-inhibited conditions. In human bronchus, LTD4-induced smooth muscle contraction was inhibited by both 8-BrcAMP and Y-27632. We conclude that cAMP/PKA-induced Ca2+ desensitization contains at least two mechanisms: 1) inhibition of the muscarinic receptor signaling upstream from Rho activation and 2) cAMP/PKA's preferential reversal of PKC-mediated Ca2+ sensitization in airway smooth muscle.

  18. Bitter tasting compounds dilate airways by inhibiting airway smooth muscle calcium oscillations and calcium sensitivity

    PubMed Central

    Tan, Xiahui; Sanderson, Michael J

    2014-01-01

    Background and Purpose While selective, bitter tasting, TAS2R agonists can relax agonist-contracted airway smooth muscle (ASM), their mechanism of action is unclear. However, ASM contraction is regulated by Ca2+ signalling and Ca2+ sensitivity. We have therefore investigated how the TAS2R10 agonists chloroquine, quinine and denotonium regulate contractile agonist-induced Ca2+ signalling and sensitivity. Experimental Approach Airways in mouse lung slices were contracted with either methacholine (MCh) or 5HT and bronchodilation assessed using phase-contrast microscopy. Ca2+ signalling was measured with 2-photon fluorescence microscopy of ASM cells loaded with Oregon Green, a Ca2+-sensitive indicator (with or without caged-IP3). Effects on Ca2+ sensitivity were assessed on lung slices treated with caffeine and ryanodine to permeabilize ASM cells to Ca2+. Key Results The TAS2R10 agonists dilated airways constricted by either MCh or 5HT, accompanied by inhibition of agonist-induced Ca2+ oscillations. However, in non-contracted airways, TAS2R10 agonists, at concentrations that maximally dilated constricted airways, did not evoke Ca2+ signals in ASM cells. Ca2+ increases mediated by the photolysis of caged-IP3 were also attenuated by chloroquine, quinine and denotonium. In Ca2+-permeabilized ASM cells, the TAS2R10 agonists dilated MCh- and 5HT-constricted airways. Conclusions and Implications TAS2R10 agonists reversed bronchoconstriction by inhibiting agonist-induced Ca2+ oscillations while simultaneously reducing the Ca2+ sensitivity of ASM cells. Reduction of Ca2+ oscillations may be due to inhibition of Ca2+ release through IP3 receptors. Further characterization of bronchodilatory TAS2R agonists may lead to the development of novel therapies for the treatment of bronchoconstrictive conditions. PMID:24117140

  19. Polo-like Kinase 1 Regulates Vimentin Phosphorylation at Ser-56 and Contraction in Smooth Muscle*

    PubMed Central

    Li, Jia; Wang, Ruping; Gannon, Olivia J.; Rezey, Alyssa C.; Jiang, Sixin; Gerlach, Brennan D.; Liao, Guoning

    2016-01-01

    Polo-like kinase 1 (Plk1) is a serine/threonine-protein kinase that has been implicated in mitosis, cytokinesis, and smooth muscle cell proliferation. The role of Plk1 in smooth muscle contraction has not been investigated. Here, stimulation with acetylcholine induced Plk1 phosphorylation at Thr-210 (an indication of Plk1 activation) in smooth muscle. Contractile stimulation also activated Plk1 in live smooth muscle cells as evidenced by changes in fluorescence resonance energy transfer signal of a Plk1 sensor. Moreover, knockdown of Plk1 in smooth muscle attenuated force development. Smooth muscle conditional knock-out of Plk1 also diminished contraction of mouse tracheal rings. Plk1 knockdown inhibited acetylcholine-induced vimentin phosphorylation at Ser-56 without affecting myosin light chain phosphorylation. Expression of T210A Plk1 inhibited the agonist-induced vimentin phosphorylation at Ser-56 and contraction in smooth muscle. However, myosin light chain phosphorylation was not affected by T210A Plk1. Ste20-like kinase (SLK) is a serine/threonine-protein kinase that has been implicated in spindle orientation and microtubule organization during mitosis. In this study knockdown of SLK inhibited Plk1 phosphorylation at Thr-210 and activation. Finally, asthma is characterized by airway hyperresponsiveness, which largely stems from airway smooth muscle hyperreactivity. Here, smooth muscle conditional knock-out of Plk1 attenuated airway resistance and airway smooth muscle hyperreactivity in a murine model of asthma. Taken together, these findings suggest that Plk1 regulates smooth muscle contraction by modulating vimentin phosphorylation at Ser-56. Plk1 activation is regulated by SLK during contractile activation. Plk1 contributes to the pathogenesis of asthma. PMID:27662907

  20. Calcium-activated chloride channel TMEM16A modulates mucin secretion and airway smooth muscle contraction.

    PubMed

    Huang, Fen; Zhang, Hongkang; Wu, Meng; Yang, Huanghe; Kudo, Makoto; Peters, Christian J; Woodruff, Prescott G; Solberg, Owen D; Donne, Matthew L; Huang, Xiaozhu; Sheppard, Dean; Fahy, John V; Wolters, Paul J; Hogan, Brigid L M; Finkbeiner, Walter E; Li, Min; Jan, Yuh-Nung; Jan, Lily Yeh; Rock, Jason R

    2012-10-02

    Mucous cell hyperplasia and airway smooth muscle (ASM) hyperresponsiveness are hallmark features of inflammatory airway diseases, including asthma. Here, we show that the recently identified calcium-activated chloride channel (CaCC) TMEM16A is expressed in the adult airway surface epithelium and ASM. The epithelial expression is increased in asthmatics, particularly in secretory cells. Based on this and the proposed functions of CaCC, we hypothesized that TMEM16A inhibitors would negatively regulate both epithelial mucin secretion and ASM contraction. We used a high-throughput screen to identify small-molecule blockers of TMEM16A-CaCC channels. We show that inhibition of TMEM16A-CaCC significantly impairs mucus secretion in primary human airway surface epithelial cells. Furthermore, inhibition of TMEM16A-CaCC significantly reduces mouse and human ASM contraction in response to cholinergic agonists. TMEM16A-CaCC blockers, including those identified here, may positively impact multiple causes of asthma symptoms.

  1. Prestretched airway smooth muscle response to length oscillation.

    PubMed

    Al-Jumaily, Ahmed M; Roos, Kevin; Bessaguet, Sandy; Jo Avila, Miguel

    2017-01-01

    Airway smooth muscle (ASM) hyperconstriction is the cause of many respiratory diseases including asthma. In vitro testing has demonstrated that the active forces of ASM are reduced by length oscillation (LO) mimicking tidal breathing. In a previous study, we demonstrated that this force reduction can be further enhanced when superimposing oscillations (with certain frequencies and amplitudes) on this LO In contrast, it has been reported that pressurizing the lung may help in relieving asthmatic airway constrictions. Ultimately, this pressurizing stretches the ASM and may disturb the acto-myosin cross-bridges in a manner similar to LO; however, it is of a static rather than dynamic nature. This research investigates the effect of combining both prestretch- and LO-applications on contracted porcine ASM Isolated porcine ASM relaxation was tested with a 0.56%, 2%, or 4% stretch of its reference length (L ref ) in addition to LO These oscillations are composed of a main wave mimicking the normal breathing (frequency of 0.33 Hz and amplitude of 4% L ref ) and superimposed oscillations (frequencies of 20, 30, 40, 60 and 80 Hz and amplitude of 1% L ref ). The oscillations were maintained for 10 min. The results demonstrate that a prestretch of 0.56% and 2% L ref does enhance the contracted ASM relaxation at certain superimposed length oscillations frequencies while of 4% L ref does not. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

  2. Airways in smooth muscle α-actin null mice experience a compensatory mechanism that modulates their contractile response.

    PubMed

    Shardonofsky, Felix R; Moore, Joan; Schwartz, Robert J; Boriek, Aladin M

    2012-03-01

    We hypothesized that ablation of smooth muscle α-actin (SM α-A), a contractile-cytoskeletal protein expressed in airway smooth muscle (ASM) cells, abolishes ASM shortening capacity and decreases lung stiffness. In both SM α-A knockout and wild-type (WT) mice, airway resistance (Raw) determined by the forced oscillation technique rose in response to intravenous methacholine (Mch). However, the slope of Raw (cmH(2)O·ml(-1)·s) vs. log(2) Mch dose (μg·kg(-1)·min(-1)) was lower (P = 0.007) in mutant (0.54 ± 0.14) than in WT mice (1.23 ± 0.19). RT-PCR analysis performed on lung tissues confirmed that mutant mice lacked SM α-A mRNA and showed that these mice had robust expressions of both SM γ-A mRNA and skeletal muscle (SKM) α-A mRNA, which were not expressed in WT mice, and an enhanced SM22 mRNA expression relative to that in WT mice. Compared with corresponding spontaneously breathing mice, mechanical ventilation-induced lung mechanical strain increased the expression of SM α-A mRNA in WT lungs; in mutant mice, it augmented the expressions of SM γ-A mRNA and SM22 mRNA and did not alter that of SKM α-A mRNA. In mutant mice, the expression of SM γ-A mRNA in the lung during spontaneous breathing and its enhanced expression following mechanical ventilation are consistent with the likely possibility that in the absence of SM α-A, SM γ-A underwent polymerization and interacted with smooth muscle myosin to produce ASM shortening during cholinergic stimulation. Thus our data are consistent with ASM in mutant mice experiencing compensatory mechanisms that modulated its contractile muscle capacity.

  3. Roxithromycin inhibits VEGF-induced human airway smooth muscle cell proliferation: Opportunities for the treatment of asthma

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pei, Qing-Mei, E-mail: 34713316@qq.com; Jiang, Ping, E-mail: jiangping@163.com; Yang, Min, E-mail: YangMin@163.com

    Asthma is a chronic respiratory disease characterized by reversible airway obstruction with persistent airway inflammation and airway remodelling, which is associated with increased airway smooth muscle (ASM) mass. Roxithromycin (RXM) has been widely used in asthma treatment; however, its mechanism of action is poorly understood. Vascular endothelial growth factor (VEGF) has been implicated in inflammatory and airway blood vessel remodelling in patients with asthma, and shown to promote ASM cell proliferation. Here, we investigated the effect of RXM on VEGF-induced ASM cell proliferation and attempted to elucidate the underlying mechanisms of action. We tested the effect of RXM on proliferationmore » and cell cycle progression, as well as on the expression of phospho-VEGF receptor 2 (VEGFR2), phospho-extracellular signal-regulated kinase 1/2 (ERK1/2), phospho-Akt, and caveolin-1 in VEGF-stimulated ASM cells. RXM inhibited VEGF-induced ASM cell proliferation and induced cell cycle arrest. Additionally, VEGF-induced ASM cell proliferation was suppressed by inhibiting the activity of ERK1/2, but not that of Akt. Furthermore, RXM treatment inhibits VEGF-induced activation of VEGFR2 and ERK and downregulation of caveolin-1 in a dose-dependent manner. RXM also inhibited TGF-β-induced VEGF secretion by ASM cells and BEAS-2B cells. Collectively, our findings suggest that RXM inhibits VEGF-induced ASM cell proliferation by suppression of VEGFR2 and ERK1/2 activation and caveolin-1 down-regulation, which may be involved in airway remodelling. Further elucidation of the mechanisms underlying these observations should enable the development of treatments for smooth muscle hyperplasia-associated diseases of the airway such as asthma. - Highlights: • RXM inhibited VEGF-induced ASM cell proliferation and induced cell cycle arrest. • VEGF-induced cell proliferation was suppressed by inhibiting the activity of ERK1/2. • RXM inhibits activation of VEGFR2 and ERK and

  4. Ryanodine receptors decant internal Ca2+ store in human and bovine airway smooth muscle.

    PubMed

    Tazzeo, T; Zhang, Y; Keshavjee, S; Janssen, L J

    2008-08-01

    Several putative roles for ryanodine receptors (RyR) were investigated in human and bovine airway smooth muscle. Changes in intracellular Ca2+ concentration ([Ca2+]i) and membrane current were investigated in single cells by confocal fluorimetry and patch-clamp electrophysiology, respectively, whereas mechanical activity was monitored in intact strips with force transducers. RyR released Ca2+ from the sarcoplasmic reticulum in a ryanodine- and chloroethyl phenol (CEP)-sensitive fashion. Neither ryanodine nor CEP inhibited responses to KCl, cholinergic agonists or serotonin, indicating no direct role for RyR in contraction; in fact, there was some augmentation of these responses. In tissues pre-contracted with carbachol, the concentration-response relationships for isoproterenol and salmeterol were unaffected by ryanodine; relaxations due to a nitric oxide donor were also largely unaffected. Finally, it was examined whether RyR were involved in regulating [Ca2+]i within the subplasmalemmal space using patch-clamp electrophysiology as well as Ca2+ fluorimetry: isoproterenol increased [Ca2+]i- and Ca2+-dependent K+ current activity in a ryanodine-sensitive fashion. In conclusion, ryanodine receptors in airway smooth muscle are not important in directly mediating contraction or relaxation. The current authors speculate instead that these allow the sarcoplasmic reticulum to release Ca2+ towards the plasmalemma (to unload an overly full Ca2+ store and/or increase the Ca2+-buffering capacity of the sarcoplasmic reticulum) without affecting bronchomotor tone.

  5. PKC-dependent regulation of Kv7.5 channels by the bronchoconstrictor histamine in human airway smooth muscle cells.

    PubMed

    Haick, Jennifer M; Brueggemann, Lioubov I; Cribbs, Leanne L; Denning, Mitchell F; Schwartz, Jeffrey; Byron, Kenneth L

    2017-06-01

    Kv7 potassium channels have recently been found to be expressed and functionally important for relaxation of airway smooth muscle. Previous research suggests that native Kv7 currents are inhibited following treatment of freshly isolated airway smooth muscle cells with bronchoconstrictor agonists, and in intact airways inhibition of Kv7 channels is sufficient to induce bronchiolar constriction. However, the mechanism by which Kv7 currents are inhibited by bronchoconstrictor agonists has yet to be elucidated. In the present study, native Kv7 currents in cultured human trachealis smooth muscle cells (HTSMCs) were observed to be inhibited upon treatment with histamine; inhibition of Kv7 currents was associated with membrane depolarization and an increase in cytosolic Ca 2+ ([Ca 2+ ] cyt ). The latter response was inhibited by verapamil, a blocker of L-type voltage-sensitive Ca 2+ channels (VSCCs). Protein kinase C (PKC) has been implicated as a mediator of bronchoconstrictor actions, although the targets of PKC are not clearly established. We found that histamine treatment significantly and dose-dependently suppressed currents through overexpressed wild-type human Kv7.5 (hKv7.5) channels in cultured HTSMCs, and this effect was inhibited by the PKC inhibitor Ro-31-8220 (3 µM). The PKC-dependent suppression of hKv7.5 currents corresponded with a PKC-dependent increase in hKv7.5 channel phosphorylation. Knocking down or inhibiting PKCα, or mutating hKv7.5 serine 441 to alanine, abolished the inhibitory effects of histamine on hKv7.5 currents. These findings provide the first evidence linking PKC activation to suppression of Kv7 currents, membrane depolarization, and Ca 2+ influx via L-type VSCCs as a mechanism for histamine-induced bronchoconstriction. Copyright © 2017 the American Physiological Society.

  6. Effect of fenoterol-induced constitutive beta(2)-adrenoceptor activity on contractile receptor function in airway smooth muscle.

    PubMed

    de Vries, B; Roffel, A F; Zaagsma, J; Meurs, H

    2001-11-23

    In the present study, we investigated the effect of fenoterol-induced constitutive beta(2)-adrenoceptor activity on muscarinic receptor agonist- and histamine-induced bovine tracheal smooth muscle contractions. Bovine tracheal smooth muscle strips were incubated with 10 microM fenoterol or vehicle for various periods of time (5, 30 min, 18 h) at 37 degrees C. After extensive washout (3 h, 37 degrees C), isometric contractions were measured to the full muscarinic receptor agonist methacholine, the partial muscarinic receptor agonist 4-(m-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium (McN-A-343) and histamine. Fenoterol treatment significantly reduced the sensitivity (pEC(50)) to methacholine in a time-dependent manner, without affecting maximal contraction (E(max)). Fenoterol treatment similarly reduced the pEC(50) of McN-A-343 and histamine; however, E(max) values were also reduced, to approximately 70% of control after 18-h treatment. The inverse agonist timolol, having no effect on control preparations, consistently restored the reduced pEC(50) and E(max) values of the contractile agonists. Remarkably, in the presence of timolol the pEC(50) values of McN-A-343 and histamine in fenoterol-treated airways were significantly enhanced compared to controls. In conclusion, fenoterol-induced constitutive beta(2)-adrenoceptor activity reduces muscarinic receptor agonist- and histamine-induced contractions of bovine tracheal smooth muscle, which can be reversed by the inverse agonist timolol. Moreover, after beta(2)-adrenoceptor agonist treatment, inverse agonism by beta-adrenoceptor antagonists may cause enhanced airway reactivity to contractile mediators.

  7. β-Agonist-mediated Relaxation of Airway Smooth Muscle Is Protein Kinase A-dependent*

    PubMed Central

    Morgan, Sarah J.; Deshpande, Deepak A.; Tiegs, Brian C.; Misior, Anna M.; Yan, Huandong; Hershfeld, Alena V.; Rich, Thomas C.; Panettieri, Reynold A.; An, Steven S.; Penn, Raymond B.

    2014-01-01

    Inhaled β-agonists are effective at reversing bronchoconstriction in asthma, but the mechanism by which they exert this effect is unclear and controversial. PKA is the historically accepted effector, although this assumption is made on the basis of associative and not direct evidence. Recent studies have asserted that exchange protein activated by cAMP (Epac), not PKA, mediates the relaxation of airway smooth muscle (ASM) observed with β-agonist treatment. This study aims to clarify the role of PKA in the prorelaxant effects of β-agonists on ASM. Inhibition of PKA activity via expression of the PKI and RevAB peptides results in increased β-agonist-mediated cAMP release, abolishes the inhibitory effect of isoproterenol on histamine-induced intracellular calcium flux, and significantly attenuates histamine-stimulated MLC-20 phosphorylation. Analyses of ASM cell and tissue contraction demonstrate that PKA inhibition eliminates most, if not all, β-agonist-mediated relaxation of contracted smooth muscle. Conversely, Epac knockdown had no effect on the regulation of contraction or procontractile signaling by isoproterenol. These findings suggest that PKA, not Epac, is the predominant and physiologically relevant effector through which β-agonists exert their relaxant effects. PMID:24973219

  8. Interleukin(IL)-1 Regulates Ozone-enhanced Tracheal Smooth Muscle Responsiveness by Increasing Substance P (SP) Production in Intrinsic Airway Neurons of Ferret

    PubMed Central

    Wu, Z.-X.; Barker, J. S.; Batchelor, T. P.; Dey, R.D.

    2008-01-01

    Exposure to ozone induces airway hyperresponsiveness (AHR) mediated partly by SP released from nerve terminals of intrinsic airway neurons. Our recent studies showed that IL-1, an important multifunctional proinflammatory cytokine, increases synthesis and release of SP from intrinsic airway neurons. The purpose of this study is to investigate the possible involvement of endogenous IL-1 in modulating neural responses associated with ozone-enhanced airway responsiveness. Ferrets were exposed to 2 ppm ozone or filtered air for 3 hrs. IL-1 in the bronchoalveolar lavage (BAL) fluid was significantly increased in ozone-exposed animals and responses of tracheal smooth muscle to methacholine (MCh) and electrical field stimulation (EFS) were elevated significantly. Both the SP nerve fiber density in tracheal smooth muscle and the number of SP-containing neurons in airway ganglia were significantly increased following ozone exposure. Pretreatment with IL-1 receptor antagonist (IL-1 Ra) significantly diminished ozone-enhanced airway responses to EFS as well as ozone-increased SP in the airway. To selectively investigate intrinsic airway neurons, segments of ferret trachea were maintained in culture conditions for 24 hrs to eliminate extrinsic contributions from sensory nerves. The segments were then exposed to 2 ppm ozone in vitro for 3 hrs. The changes of ozone-induced airway responses to MCh and EFS, and the SP levels in airway neurons paralleled those observed with in vivo ozone exposure. The ozone-enhanced airway responses and neuronal SP levels were inhibited by pretreatment with IL-1 Ra. These findings show that IL-1 is released during ozone exposure enhances airway responsiveness by modulating SP expression in airway neurons. PMID:18718561

  9. Prenatal Exposure to Respiratory Syncytial Virus Alters Postnatal Immunity and Airway Smooth Muscle Contractility during Early-Life Reinfections

    PubMed Central

    Harford, Terri J.; Agrawal, Vandana; Yen-Lieberman, Belinda; Rezaee, Fariba; Piedimonte, Giovanni

    2017-01-01

    Maternal viral infections can have pathological effects on the developing fetus which last long after birth. Recently, maternal-fetal transmission of respiratory syncytial virus (RSV) was shown to cause postnatal airway hyperreactivity (AHR) during primary early-life reinfection; however, the influence of prenatal exposure to RSV on offspring airway immunity and smooth muscle contractility during recurrent postnatal reinfections remains unknown. Therefore, we sought to determine whether maternal RSV infection impairs specific aspects of cell-mediated offspring immunity during early-life reinfections and the mechanisms leading to AHR. Red fluorescent protein-expressing recombinant RSV (rrRSV) was inoculated into pregnant rat dams at midterm, followed by primary and secondary postnatal rrRSV inoculations of their offspring at early-life time points. Pups and weanlings were tested for specific lower airway leukocyte populations by flow cytometry; serum cytokine/chemokine concentrations by multiplex ELISA and neurotrophins concentrations by standard ELISA; and ex vivo lower airway smooth muscle (ASM) contraction by physiological tissue bath. Pups born to RSV-infected mothers displayed elevated total CD3+ T cells largely lacking CD4+ and CD8+ surface expression after both primary and secondary postnatal rrRSV infection. Cytokine/chemokine analyses revealed reduced IFN-γ, IL-2, IL-12, IL-17A, IL-18, and TNF-α, as well as elevated nerve growth factor (NGF) expression. Prenatal exposure to RSV also increased ASM reactivity and contractility during early-life rrRSV infection compared to non-exposed controls. We conclude that maternal RSV infection can predispose offspring to postnatal lower airways dysfunction by altering immunity development, NGF signaling, and ASM contraction during early-life RSV reinfections. PMID:28178290

  10. Contribution of SRF, Elk-1, and myocardin to airway smooth muscle remodeling in heaves, an asthma-like disease of horses.

    PubMed

    Chevigny, Mylène; Guérin-Montpetit, Karine; Vargas, Amandine; Lefebvre-Lavoie, Josiane; Lavoie, Jean-Pierre

    2015-07-01

    Myocyte hyperplasia and hypertrophy contribute to the increased mass of airway smooth muscle (ASM) in asthma. Serum-response factor (SRF) is a transcription factor that regulates myocyte differentiation in vitro in vascular and intestinal smooth muscles. When SRF is associated with phosphorylated (p)Elk-1, it promotes ASM proliferation while binding to myocardin (MYOCD) leading to the expression of contractile elements in these tissues. The objective of this study was therefore to characterize the expression of SRF, pElk-1, and MYOCD in ASM cells from central and peripheral airways in heaves, a spontaneously occurring asthma-like disease of horses, and in controls. Six horses with heaves and five aged-matched controls kept in the same environment were studied. Nuclear protein expression of SRF, pElk-1, and MYOCD was evaluated in peripheral airways and endobronchial biopsies obtained during disease remission and after 1 and 30 days of naturally occurring antigenic exposure using immunohistochemistry and immunofluorescence techniques. Nuclear expression of SRF (P = 0.03, remission vs. 30 days) and MYOCD (P = 0.05, controls vs. heaves at 30 days) increased in the peripheral airways of horses with heaves during disease exacerbation, while MYOCD (P = 0.04, remission vs. 30 days) decreased in the central airways of control horses. No changes were observed in the expression of pElk-1 protein in either tissue. In conclusion, SRF and its cofactor MYOCD likely contribute to the hypertrophy of peripheral ASM observed in equine asthmatic airways, while the remodeling of the central airways is more static or involves different transcription factors. Copyright © 2015 the American Physiological Society.

  11. Phosphoinositide 3-kinase gamma regulates airway smooth muscle contraction by modulating calcium oscillations.

    PubMed

    Jiang, Haihong; Abel, Peter W; Toews, Myron L; Deng, Caishu; Casale, Thomas B; Xie, Yan; Tu, Yaping

    2010-09-01

    Phosphoinositide 3-kinase gamma (PI3Kgamma) has been implicated in the pathogenesis of asthma, but its mechanism has been considered indirect, through release of inflammatory cell mediators. Because airway smooth muscle (ASM) contractile hyper-responsiveness plays a critical role in asthma, the aim of the present study was to determine whether PI3Kgamma can directly regulate contractility of ASM. Immunohistochemistry staining indicated expression of PI3Kgamma protein in ASM cells of mouse trachea and lung, which was confirmed by Western blot analysis in isolated mouse tracheal ASM cells. PI3Kgamma inhibitor II inhibited acetylcholine (ACh)-stimulated airway contraction of cultured precision-cut mouse lung slices in a dose-dependent manner with 75% inhibition at 10 muM. In contrast, inhibitors of PI3Kalpha, PI3Kbeta, or PI3Kdelta, at concentrations 40-fold higher than their reported IC(50) values for their primary targets, had no effect. It is noteworthy that airways in lung slices pretreated with PI3Kgamma inhibitor II still exhibited an ACh-induced initial contraction, but the sustained contraction was significantly reduced. Furthermore, the PI3Kgamma-selective inhibitor had a small inhibitory effect on the ACh-stimulated initial Ca(2+) transient in ASM cells of mouse lung slices or isolated mouse ASM cells but significantly attenuated the sustained Ca(2+) oscillations that are critical for sustained airway contraction. This report is the first to show that PI3Kgamma directly controls contractility of airways through regulation of Ca(2+) oscillations in ASM cells. Thus, in addition to effects on airway inflammation, PI3Kgamma inhibitors may also exert direct effects on the airway contraction that contribute to pathologic airway hyper-responsiveness.

  12. Phosphoinositide 3-Kinase γ Regulates Airway Smooth Muscle Contraction by Modulating Calcium Oscillations

    PubMed Central

    Jiang, Haihong; Abel, Peter W.; Toews, Myron L.; Deng, Caishu; Casale, Thomas B.; Xie, Yan

    2010-01-01

    Phosphoinositide 3-kinase γ (PI3Kγ) has been implicated in the pathogenesis of asthma, but its mechanism has been considered indirect, through release of inflammatory cell mediators. Because airway smooth muscle (ASM) contractile hyper-responsiveness plays a critical role in asthma, the aim of the present study was to determine whether PI3Kγ can directly regulate contractility of ASM. Immunohistochemistry staining indicated expression of PI3Kγ protein in ASM cells of mouse trachea and lung, which was confirmed by Western blot analysis in isolated mouse tracheal ASM cells. PI3Kγ inhibitor II inhibited acetylcholine (ACh)-stimulated airway contraction of cultured precision-cut mouse lung slices in a dose-dependent manner with 75% inhibition at 10 μM. In contrast, inhibitors of PI3Kα, PI3Kβ, or PI3Kδ, at concentrations 40-fold higher than their reported IC50 values for their primary targets, had no effect. It is noteworthy that airways in lung slices pretreated with PI3Kγ inhibitor II still exhibited an ACh-induced initial contraction, but the sustained contraction was significantly reduced. Furthermore, the PI3Kγ-selective inhibitor had a small inhibitory effect on the ACh-stimulated initial Ca2+ transient in ASM cells of mouse lung slices or isolated mouse ASM cells but significantly attenuated the sustained Ca2+ oscillations that are critical for sustained airway contraction. This report is the first to show that PI3Kγ directly controls contractility of airways through regulation of Ca2+ oscillations in ASM cells. Thus, in addition to effects on airway inflammation, PI3Kγ inhibitors may also exert direct effects on the airway contraction that contribute to pathologic airway hyper-responsiveness. PMID:20501633

  13. β-Agonist-mediated relaxation of airway smooth muscle is protein kinase A-dependent.

    PubMed

    Morgan, Sarah J; Deshpande, Deepak A; Tiegs, Brian C; Misior, Anna M; Yan, Huandong; Hershfeld, Alena V; Rich, Thomas C; Panettieri, Reynold A; An, Steven S; Penn, Raymond B

    2014-08-15

    Inhaled β-agonists are effective at reversing bronchoconstriction in asthma, but the mechanism by which they exert this effect is unclear and controversial. PKA is the historically accepted effector, although this assumption is made on the basis of associative and not direct evidence. Recent studies have asserted that exchange protein activated by cAMP (Epac), not PKA, mediates the relaxation of airway smooth muscle (ASM) observed with β-agonist treatment. This study aims to clarify the role of PKA in the prorelaxant effects of β-agonists on ASM. Inhibition of PKA activity via expression of the PKI and RevAB peptides results in increased β-agonist-mediated cAMP release, abolishes the inhibitory effect of isoproterenol on histamine-induced intracellular calcium flux, and significantly attenuates histamine-stimulated MLC-20 phosphorylation. Analyses of ASM cell and tissue contraction demonstrate that PKA inhibition eliminates most, if not all, β-agonist-mediated relaxation of contracted smooth muscle. Conversely, Epac knockdown had no effect on the regulation of contraction or procontractile signaling by isoproterenol. These findings suggest that PKA, not Epac, is the predominant and physiologically relevant effector through which β-agonists exert their relaxant effects. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. IL-33 promotes mouse keratinocyte-derived chemokine, an IL-8 homologue, expression in airway smooth muscle cells in ovalbumin-sensitized mice.

    PubMed

    Wu, Wei; Xu, Yuzhu; He, Xinliang; Lu, Yan; Guo, Yali; Yin, Zhuoran; Xie, Jungang; Zhao, Jianping

    2014-12-01

    Although it is recognized that IL-33 plays a key role in the onset of asthma, it is currently unclear whether IL-33 acts on any other target cells besides mast cells and Th2 cells in asthma. We investigated that whether airway smooth muscle cells (ASMCs) could contribute to asthma via stimulation with IL-33. To create a mouse model of acute asthma, murine ASMCs were isolated and cultured in vitro with IL-33. The ASMCs were divided into two groups, ASMCs from normal mice and ASMCs from ovalbumin-sensitized mice. The release of mouse KC was analyzed by PCR and ELISA. Immunocytochemical Staining of murine ASMCs for ST2 and IL-1RAcP was performed. IL-33 promoted KC expression, both in terms of mRNA and protien levels, in ASMCs from ovalbumin-sensitized mice. ST2 and IL-1RAcP were expressed in the membrane of ASMCs in ovalbumin-sensitized mice. IL-33 may contribute to the inflammation in the airways by acting on airway smooth muscle cells. IL-33 and ST2 may play important roles in allergic bronchial asthma.

  15. Force maintenance and myosin filament assembly regulated by Rho-kinase in airway smooth muscle.

    PubMed

    Lan, Bo; Deng, Linhong; Donovan, Graham M; Chin, Leslie Y M; Syyong, Harley T; Wang, Lu; Zhang, Jenny; Pascoe, Christopher D; Norris, Brandon A; Liu, Jeffrey C-Y; Swyngedouw, Nicholas E; Banaem, Saleha M; Paré, Peter D; Seow, Chun Y

    2015-01-01

    Smooth muscle contraction can be divided into two phases: the initial contraction determines the amount of developed force and the second phase determines how well the force is maintained. The initial phase is primarily due to activation of actomyosin interaction and is relatively well understood, whereas the second phase remains poorly understood. Force maintenance in the sustained phase can be disrupted by strains applied to the muscle; the strain causes actomyosin cross-bridges to detach and also the cytoskeletal structure to disassemble in a process known as fluidization, for which the underlying mechanism is largely unknown. In the present study we investigated the ability of airway smooth muscle to maintain force after the initial phase of contraction. Specifically, we examined the roles of Rho-kinase and protein kinase C (PKC) in force maintenance. We found that for the same degree of initial force inhibition, Rho-kinase substantially reduced the muscle's ability to sustain force under static conditions, whereas inhibition of PKC had a minimal effect on sustaining force. Under oscillatory strain, Rho-kinase inhibition caused further decline in force, but again, PKC inhibition had a minimal effect. We also found that Rho-kinase inhibition led to a decrease in the myosin filament mass in the muscle cells, suggesting that one of the functions of Rho-kinase is to stabilize myosin filaments. The results also suggest that dissolution of myosin filaments may be one of the mechanisms underlying the phenomenon of fluidization. These findings can shed light on the mechanism underlying deep inspiration induced bronchodilation. Copyright © 2015 the American Physiological Society.

  16. Force maintenance and myosin filament assembly regulated by Rho-kinase in airway smooth muscle

    PubMed Central

    Lan, Bo; Deng, Linhong; Donovan, Graham M.; Chin, Leslie Y. M.; Syyong, Harley T.; Wang, Lu; Zhang, Jenny; Pascoe, Christopher D.; Norris, Brandon A.; Liu, Jeffrey C.-Y.; Swyngedouw, Nicholas E.; Banaem, Saleha M.; Paré, Peter D.

    2014-01-01

    Smooth muscle contraction can be divided into two phases: the initial contraction determines the amount of developed force and the second phase determines how well the force is maintained. The initial phase is primarily due to activation of actomyosin interaction and is relatively well understood, whereas the second phase remains poorly understood. Force maintenance in the sustained phase can be disrupted by strains applied to the muscle; the strain causes actomyosin cross-bridges to detach and also the cytoskeletal structure to disassemble in a process known as fluidization, for which the underlying mechanism is largely unknown. In the present study we investigated the ability of airway smooth muscle to maintain force after the initial phase of contraction. Specifically, we examined the roles of Rho-kinase and protein kinase C (PKC) in force maintenance. We found that for the same degree of initial force inhibition, Rho-kinase substantially reduced the muscle's ability to sustain force under static conditions, whereas inhibition of PKC had a minimal effect on sustaining force. Under oscillatory strain, Rho-kinase inhibition caused further decline in force, but again, PKC inhibition had a minimal effect. We also found that Rho-kinase inhibition led to a decrease in the myosin filament mass in the muscle cells, suggesting that one of the functions of Rho-kinase is to stabilize myosin filaments. The results also suggest that dissolution of myosin filaments may be one of the mechanisms underlying the phenomenon of fluidization. These findings can shed light on the mechanism underlying deep inspiration induced bronchodilation. PMID:25305246

  17. Matrix stiffness-modulated proliferation and secretory function of the airway smooth muscle cells.

    PubMed

    Shkumatov, Artem; Thompson, Michael; Choi, Kyoung M; Sicard, Delphine; Baek, Kwanghyun; Kim, Dong Hyun; Tschumperlin, Daniel J; Prakash, Y S; Kong, Hyunjoon

    2015-06-01

    Multiple pulmonary conditions are characterized by an abnormal misbalance between various tissue components, for example, an increase in the fibrous connective tissue and loss/increase in extracellular matrix proteins (ECM). Such tissue remodeling may adversely impact physiological function of airway smooth muscle cells (ASMCs) responsible for contraction of airways and release of a variety of bioactive molecules. However, few efforts have been made to understand the potentially significant impact of tissue remodeling on ASMCs. Therefore, this study reports how ASMCs respond to a change in mechanical stiffness of a matrix, to which ASMCs adhere because mechanical stiffness of the remodeled airways is often different from the physiological stiffness. Accordingly, using atomic force microscopy (AFM) measurements, we found that the elastic modulus of the mouse bronchus has an arithmetic mean of 23.1 ± 14 kPa (SD) (median 18.6 kPa). By culturing ASMCs on collagen-conjugated polyacrylamide hydrogels with controlled elastic moduli, we found that gels designed to be softer than average airway tissue significantly increased cellular secretion of vascular endothelial growth factor (VEGF). Conversely, gels stiffer than average airways stimulated cell proliferation, while reducing VEGF secretion and agonist-induced calcium responses of ASMCs. These dependencies of cellular activities on elastic modulus of the gel were correlated with changes in the expression of integrin-β1 and integrin-linked kinase (ILK). Overall, the results of this study demonstrate that changes in matrix mechanics alter cell proliferation, calcium signaling, and proangiogenic functions in ASMCs. Copyright © 2015 the American Physiological Society.

  18. Antimitogenic effect of bitter taste receptor agonists on airway smooth muscle cells

    PubMed Central

    Sharma, Pawan; Panebra, Alfredo; Pera, Tonio; Tiegs, Brian C.; Hershfeld, Alena; Kenyon, Lawrence C.

    2015-01-01

    Airway remodeling is a hallmark feature of asthma and chronic obstructive pulmonary disease. Clinical studies and animal models have demonstrated increased airway smooth muscle (ASM) mass, and ASM thickness is correlated with severity of the disease. Current medications control inflammation and reverse airway obstruction effectively but have limited effect on remodeling. Recently we identified the expression of bitter taste receptors (TAS2R) on ASM cells, and activation with known TAS2R agonists resulted in ASM relaxation and bronchodilation. These studies suggest that TAS2R can be used as new therapeutic targets in the treatment of obstructive lung diseases. To further establish their effectiveness, in this study we aimed to determine the effects of TAS2R agonists on ASM growth and promitogenic signaling. Pretreatment of healthy and asthmatic human ASM cells with TAS2R agonists resulted in a dose-dependent inhibition of ASM proliferation. The antimitogenic effect of TAS2R ligands was not dependent on activation of protein kinase A, protein kinase C, or high/intermediate-conductance calcium-activated K+ channels. Immunoblot analyses revealed that TAS2R agonists inhibit growth factor-activated protein kinase B phosphorylation without affecting the availability of phosphatidylinositol 3,4,5-trisphosphate, suggesting TAS2R agonists block signaling downstream of phosphatidylinositol 3-kinase. Furthermore, the antimitogenic effect of TAS2R agonists involved inhibition of induced transcription factors (activator protein-1, signal transducer and activator of transcription-3, E2 factor, nuclear factor of activated T cells) and inhibition of expression of multiple cell cycle regulatory genes, suggesting a direct inhibition of cell cycle progression. Collectively, these findings establish the antimitogenic effect of TAS2R agonists and identify a novel class of receptors and signaling pathways that can be targeted to reduce or prevent airway remodeling as well as

  19. Length oscillation mimicking periodic individual deep inspirations during tidal breathing attenuates force recovery and adaptation in airway smooth muscle.

    PubMed

    Raqeeb, Abdul; Solomon, Dennis; Paré, Peter D; Seow, Chun Y

    2010-11-01

    Airway smooth muscle (ASM) is able to generate maximal force under static conditions, and this isometric force can be maintained over a large length range due to length adaptation. The increased force at short muscle length could lead to excessive narrowing of the airways. Prolonged exposure of ASM to submaximal stimuli also increases the muscle's ability to generate force in a process called force adaptation. To date, the effects of length and force adaptation have only been demonstrated under static conditions. In the mechanically dynamic environment of the lung, ASM is constantly subjected to periodic stretches by the parenchyma due to tidal breathing and deep inspiration. It is not known whether force recovery due to muscle adaptation to a static environment could occur in a dynamic environment. In this study the effect of length oscillation mimicking tidal breathing and deep inspiration was examined. Force recovery after a length change was attenuated in the presence of length oscillation, except at very short lengths. Force adaptation was abolished by length oscillation. We conclude that in a healthy lung (with intact airway-parenchymal tethering) where airways are not allowed to narrow excessively, large stretches (associated with deep inspiration) may prevent the ability of the muscle to generate maximal force that would occur under static conditions irrespective of changes in mean length; mechanical perturbation on ASM due to tidal breathing and deep inspiration, therefore, is the first line of defense against excessive bronchoconstriction that may result from static length and force adaptation.

  20. Mounier-Kuhn syndrome: a case of tracheal smooth muscle remodeling.

    PubMed

    Cook, Daniel P; Adam, Ryan J; Abou Alaiwa, Mahmoud H; Eberlein, Michael; Klesney-Tait, Julia A; Parekh, Kalpaj R; Meyerholz, David K; Stoltz, David A

    2017-02-01

    Mounier-Kuhn syndrome is a rare clinical disorder characterized by tracheobronchial dilation and recurrent lower respiratory tract infections. While the etiology of the disease remains unknown, histopathological analysis of Mounier-Kuhn airways demonstrates that the disease is, in part, characterized by cellular changes in airway smooth muscle.

  1. Formalin produces depolarizations in human airway smooth muscle in vitro.

    PubMed

    Richards, Ira S; DeHate, Robin B

    2006-03-01

    Respiratory irritants may result in airway smooth muscle (ASM) depolarization and bronchoconstriction. We examined the effect of formalin on membrane potentials in human ASM in two types of in vitro preparations: strip preparations, which contain functional sensory and motor nerve endings and cultured cells, which lack these nerve endings due to the tissue dissociation process. Depolarizations occurred in atropine-treated strip preparations in response to formalin exposures, but not in similarly-treated cultured cells, suggesting a role for non-cholinergic mediators in formalin-induced depolarization. It is suggested that formalin may act as an irritant to produce bronchoconstriction that is mediated by the release of endogenous substance P (SP) from peripheral sensory nerve endings. This is supported by our observation that exogenous SP produced depolarizations of a magnitude similar to those produced by formalin in both strip preparations and cultured cells. In addition, capsaicin, which releases endogenous SP from nerve endings, produced depolarizations of a magnitude similar to formalin in strip preparations, but was without effect in cultured cells.

  2. Effect of endothelin-1 on the serotonin-induced contraction of smooth muscle in the guinea pig trachea.

    PubMed

    Yoshida, M; Aizawa, H; Hara, N

    1999-01-01

    Endothelin (ET), a potent constrictor of smooth muscle including that of the airways, may contribute to the development of airway hyperresponsiveness. To investigate the role of ET-1 on the airway smooth muscle, we examined the effects of ET-1 on the serotonin-induced contraction of guinea pig tracheal smooth muscle. The changes in isometric tension evoked by serotonin were measured before and after the application of a subthreshold dose (a dose which did not induce smooth muscle contraction by itself) of ET-1. Serotonin caused smooth muscle contraction in a dose-dependent manner. The subthreshold doses of ET-1 (1 pM) and sarafotoxin 6c (1 pM), a selective ETB receptor agonist, were found to potentiate significantly the contraction induced by serotonin. A potentiating effect of ET-1 was not altered by indomethacin or calphostin C, a protein kinase C inhibitor. These results suggest that a subthreshold concentration of ET-1 can potentiate serotonin-induced contraction of smooth muscle through the activation of ETB receptor, while in contrast cyclooxygenase and protein kinase C were found not to be involved in this mechanism.

  3. The association of cortactin with profilin-1 is critical for smooth muscle contraction.

    PubMed

    Wang, Ruping; Cleary, Rachel A; Wang, Tao; Li, Jia; Tang, Dale D

    2014-05-16

    Profilin-1 (Pfn-1) is an actin-regulatory protein that has a role in modulating smooth muscle contraction. However, the mechanisms that regulate Pfn-1 in smooth muscle are not fully understood. Here, stimulation with acetylcholine induced an increase in the association of the adapter protein cortactin with Pfn-1 in smooth muscle cells/tissues. Furthermore, disruption of the protein/protein interaction by a cell-permeable peptide (CTTN-I peptide) attenuated actin polymerization and smooth muscle contraction without affecting myosin light chain phosphorylation at Ser-19. Knockdown of cortactin by lentivirus-mediated RNAi also diminished actin polymerization and smooth muscle force development. However, cortactin knockdown did not affect myosin activation. In addition, cortactin phosphorylation has been implicated in nonmuscle cell migration. In this study, acetylcholine stimulation induced cortactin phosphorylation at Tyr-421 in smooth muscle cells. Phenylalanine substitution at this position impaired cortactin/Pfn-1 interaction in response to contractile activation. c-Abl is a tyrosine kinase that is necessary for actin dynamics and contraction in smooth muscle. Here, c-Abl silencing inhibited the agonist-induced cortactin phosphorylation and the association of cortactin with Pfn-1. Finally, treatment with CTTN-I peptide reduced airway resistance and smooth muscle hyperreactivity in a murine model of asthma. These results suggest that the interaction of cortactin with Pfn-1 plays a pivotal role in regulating actin dynamics, smooth muscle contraction, and airway hyperresponsiveness in asthma. The association of cortactin with Pfn-1 is regulated by c-Abl-mediated cortactin phosphorylation. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. The Association of Cortactin with Profilin-1 Is Critical for Smooth Muscle Contraction*

    PubMed Central

    Wang, Ruping; Cleary, Rachel A.; Wang, Tao; Li, Jia; Tang, Dale D.

    2014-01-01

    Profilin-1 (Pfn-1) is an actin-regulatory protein that has a role in modulating smooth muscle contraction. However, the mechanisms that regulate Pfn-1 in smooth muscle are not fully understood. Here, stimulation with acetylcholine induced an increase in the association of the adapter protein cortactin with Pfn-1 in smooth muscle cells/tissues. Furthermore, disruption of the protein/protein interaction by a cell-permeable peptide (CTTN-I peptide) attenuated actin polymerization and smooth muscle contraction without affecting myosin light chain phosphorylation at Ser-19. Knockdown of cortactin by lentivirus-mediated RNAi also diminished actin polymerization and smooth muscle force development. However, cortactin knockdown did not affect myosin activation. In addition, cortactin phosphorylation has been implicated in nonmuscle cell migration. In this study, acetylcholine stimulation induced cortactin phosphorylation at Tyr-421 in smooth muscle cells. Phenylalanine substitution at this position impaired cortactin/Pfn-1 interaction in response to contractile activation. c-Abl is a tyrosine kinase that is necessary for actin dynamics and contraction in smooth muscle. Here, c-Abl silencing inhibited the agonist-induced cortactin phosphorylation and the association of cortactin with Pfn-1. Finally, treatment with CTTN-I peptide reduced airway resistance and smooth muscle hyperreactivity in a murine model of asthma. These results suggest that the interaction of cortactin with Pfn-1 plays a pivotal role in regulating actin dynamics, smooth muscle contraction, and airway hyperresponsiveness in asthma. The association of cortactin with Pfn-1 is regulated by c-Abl-mediated cortactin phosphorylation. PMID:24700464

  5. Effects of acute ethanol exposure on cytokine production by primary airway smooth muscle cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kaphalia, Lata; Kalita, Mridul; Kaphalia, Bhupendra S.

    Both chronic and binge alcohol abuse can be significant risk factors for inflammatory lung diseases such as acute respiratory distress syndrome and chronic obstructive pulmonary disease. However, metabolic basis of alcohol-related lung disease is not well defined, and may include key metabolites of ethanol [EtOH] in addition to EtOH itself. Therefore, we investigated the effects of EtOH, acetaldehyde [ACE], and fatty acid ethyl esters [FAEEs] on oxidative stress, endoplasmic reticulum (ER) stress, AMP-activated protein kinase (AMPK) signaling and nuclear translocation of phosphorylated (p)-NF-κB p65 in primary human airway smooth muscle (HASM) cells stimulated to produce cytokines using LPS exposure. Bothmore » FAEEs and ACE induced evidence of cellular oxidative stress and ER stress, and increased p-NF-κB in nuclear extracts. EtOH and its metabolites decreased p-AMPKα activation, and induced expression of fatty acid synthase, and decreased expression of sirtuin 1. In general, EtOH decreased secretion of IP-10, IL-6, eotaxin, GCSF, and MCP-1. However, FAEEs and ACE increased these cytokines, suggesting that both FAEEs and ACE as compared to EtOH itself are proinflammatory. A direct effect of EtOH could be consistent with blunted immune response. Collectively, these two features of EtOH exposure, coupled with the known inhibition of innate immune response in our model might explain some clinical manifestations of EtOH exposure in the lung. - Highlights: • Metabolic basis for EtOH toxicity was studied in human airway smooth muscle (HASM) cells. • In HASM cells, EtOH metabolites were found to be relatively more toxic than EtOH itself. • EtOH metabolites mediate deactivation of AMPK via oxidative stress and ER stress. • EtOH metabolites were found to be more proinflammatory than EtOH itself in HASM cells.« less

  6. [Effects of dexamethasone on the expression of muscarinic receptor mRNA in asthmatic guinea pig airway smooth muscle and eosinophil infiltration in bronchoalveolar lavage fluid].

    PubMed

    Shi, Liang; Luo, Ya-ling; Lai, Wen-yan; Luo, Liang

    2005-08-01

    To investigate the effect of dexamethasone on the expression of muscarinic receptor (MR) mRNA in smooth muscle and infiltration of eosinophils (Eos) in the airway of asthmatic guinea pigs. Thirty healthy guinea pigs were randomized into 3 equal groups, the control group, asthmatic group and dexamethasone therapy group. Asthma was induced in the latter 2 groups with the asthma-inducing agents and received treatments as indicated. Bronchial alveolar lavage fluid(BALF) were collected subsequently from the guinea pigs for examining the total cell number and cell classification, and histopathologic examination of the lung tissue was performed. Semi-quantitative analysis with reverse transcriptional- polymerase chain reaction (RT-PCR) was performed for M(2) and M(3) receptor mRNA in airway smooth muscle. Compared with the control and the asthmatic group, the number of Eos in the BALF of dexamethasone therapy group was significantly lower (P<0.01). In spite of the presence of hyperemia and edema in the lung tissues of the dexamethasone therapy group, Eos infiltration was less severe than that in the asthmatic group. As found by RT-PCR, the quantity of M(2) receptor mRNA in the airway smooth muscle of the dexamethasone therapy group was significantly higher than those in both the control and asthmatic groups (P<0.01), and the quantity of M(3) receptor mRNA in the airway smooth muscle of dexamethasone therapy group was significantly higher than that in the asthmatic group, but did not significantly differ from that in the control group. The quantities of M(2) and M(3) receptor mRNAs in the control group were both significantly higher than that in asthmatic group (P<0.01). The expression of M(2) receptor is increased in antigen- challenged guinea pigs, and that of M(3) receptor decreased. Dexamethasone can treat asthma by inhibiting inflammatory action involving Eos infiltration, regulating the expressions of M(2) and M(3) receptors and restoring the function of M(2

  7. Evaluation of pharmacological relaxation effect of the natural product naringin on in vitro cultured airway smooth muscle cells and in vivo ovalbumin-induced asthma Balb/c mice

    PubMed Central

    Wang, Yue; Lu, Yun; Luo, Mingzhi; Shi, Xiaohao; Pan, Yan; Zeng, Huilong; Deng, Linhong

    2016-01-01

    Asthma has become a common chronic respiratory disease worldwide and its prevalence is predicted to continue increasing in the next decade, particularly in developing countries. A key component in asthma therapy is to alleviate the excessive bronchial airway narrowing ultimately due to airway smooth muscle contraction, which is often facilitated by a smooth muscle relaxant, such as the β2-adrenergic agonists. Recently, bitter taste receptor (TAS2R) agonists, including saccharin and chloroquine, have been found to potently relax the airway smooth muscle cells (ASMCs) via intracellular Ca2+ signaling. This inspires a great interest in screening the vast resource of natural bitter substances for potential bronchodilatory drugs. In the present study, the relaxation effect of naringin, a compound extracted from common grapefruit, on ASMCs cultured in vitro or bronchial airways of Balb/c mice in vivo was evaluated. The results demonstrated that, when exposed to increasing doses of naringin (0.125, 0.25, 0.5 and 1.0 mM), the traction force generated by the cultured ASMCs decreased progressively, while the intracellular calcium flux signaling in the ASMCs increased. When inhaled at increasing doses (15, 30 and 60 µg), naringin also dose-dependently reduced the bronchial airway resistance of the normal and ovalbumin-induced asthma Balb/c mice in response to challenge with methacholine. In conclusion, these findings indicate that naringin was able to effectively relax murine ASMCs in vitro and in vivo, thus suggesting that it is a promising drug agent to be further investigated in the development of novel bronchodilators for the treatment of asthma. PMID:28101344

  8. Acute administration of ivacaftor to people with cystic fibrosis and a G551D-CFTR mutation reveals smooth muscle abnormalities

    PubMed Central

    Adam, Ryan J.; Hisert, Katherine B.; Dodd, Jonathan D.; Grogan, Brenda; Launspach, Janice L.; Barnes, Janel K.; Gallagher, Charles G.; Sieren, Jered P.; Gross, Thomas J.; Fischer, Anthony J.; Cavanaugh, Joseph E.; Hoffman, Eric A.; Singh, Pradeep K.; Welsh, Michael J.; McKone, Edward F.; Stoltz, David A.

    2016-01-01

    BACKGROUND. Airflow obstruction is common in cystic fibrosis (CF), yet the underlying pathogenesis remains incompletely understood. People with CF often exhibit airway hyperresponsiveness, CF transmembrane conductance regulator (CFTR) is present in airway smooth muscle (ASM), and ASM from newborn CF pigs has increased contractile tone, suggesting that loss of CFTR causes a primary defect in ASM function. We hypothesized that restoring CFTR activity would decrease smooth muscle tone in people with CF. METHODS. To increase or potentiate CFTR function, we administered ivacaftor to 12 adults with CF with the G551D-CFTR mutation; ivacaftor stimulates G551D-CFTR function. We studied people before and immediately after initiation of ivacaftor (48 hours) to minimize secondary consequences of CFTR restoration. We tested smooth muscle function by investigating spirometry, airway distensibility, and vascular tone. RESULTS. Ivacaftor rapidly restored CFTR function, indicated by reduced sweat chloride concentration. Airflow obstruction and air trapping also improved. Airway distensibility increased in airways less than 4.5 mm but not in larger-sized airways. To assess smooth muscle function in a tissue outside the lung, we measured vascular pulse wave velocity (PWV) and augmentation index, which both decreased following CFTR potentiation. Finally, change in distensibility of <4.5-mm airways correlated with changes in PWV. CONCLUSIONS. Acute CFTR potentiation provided a unique opportunity to investigate CFTR-dependent mechanisms of CF pathogenesis. The rapid effects of ivacaftor on airway distensibility and vascular tone suggest that CFTR dysfunction may directly cause increased smooth muscle tone in people with CF and that ivacaftor may relax smooth muscle. FUNDING. This work was funded in part from an unrestricted grant from the Vertex Investigator-Initiated Studies Program. PMID:27158673

  9. Role of non-coding RNAs in maintaining primary airway smooth muscle cells

    PubMed Central

    2014-01-01

    Background The airway smooth muscle (ASM) cell maintains its own proliferative rate and contributes to the inflammatory response in the airways, effects that are inhibited by corticosteroids, used in the treatment of airways diseases. Objective We determined the differential expression of mRNAs, microRNAs (miRNAs) and long noncoding RNA species (lncRNAs) in primary ASM cells following treatment with a corticosteroid, dexamethasone, and fetal calf serum (FCS). Methods mRNA, miRNA and lncRNA expression was measured by microarray and quantitative real-time PCR. Results A small number of miRNAs (including miR-150, −371-5p, −718, −940, −1181, −1207-5p, −1915, and −3663-3p) were decreased following exposure to dexamethasone and FCS. The mRNA targets of these miRNAs were increased in expression. The changes in mRNA expression were associated with regulation of ASM actin cytoskeleton. We also observed changes in expression of lncRNAs, including natural antisense, pseudogenes, intronic lncRNAs, and intergenic lncRNAs following dexamethasone and FCS. We confirmed the change in expression of three of these, LINC00882, LINC00883, PVT1, and its transcriptional activator, c-MYC. We propose that four of these lincRNAs (RP11-46A10.4, LINC00883, BCYRN1, and LINC00882) act as miRNA ‘sponges’ for 4 miRNAs (miR-150, −371-5p, −940, −1207-5p). Conclusion This in-vitro model of primary ASM cell phenotype was associated with the regulation of several ncRNAs. Their identification allows for in-vitro functional experimentation to establish causality with the primary ASM phenotype, and in airway diseases such as asthma and chronic obstructive pulmonary disease (COPD). PMID:24886442

  10. Bitter taste receptors on airway smooth muscle bronchodilate by localized calcium signaling and reverse obstruction.

    PubMed

    Deshpande, Deepak A; Wang, Wayne C H; McIlmoyle, Elizabeth L; Robinett, Kathryn S; Schillinger, Rachel M; An, Steven S; Sham, James S K; Liggett, Stephen B

    2010-11-01

    Bitter taste receptors (TAS2Rs) on the tongue probably evolved to evoke signals for avoiding ingestion of plant toxins. We found expression of TAS2Rs on human airway smooth muscle (ASM) and considered these to be avoidance receptors for inhalants that, when activated, lead to ASM contraction and bronchospasm. TAS2R agonists such as saccharin, chloroquine and denatonium evoked increased intracellular calcium ([Ca²(+)](i)) in ASM in a Gβγ-, phospholipase Cβ (PLCβ)- and inositol trisphosphate (IP₃) receptor-dependent manner, which would be expected to evoke contraction. Paradoxically, bitter tastants caused relaxation of isolated ASM and dilation of airways that was threefold greater than that elicited by β-adrenergic receptor agonists. The relaxation induced by TAS2Rs is associated with a localized [Ca²(+)](i) response at the cell membrane, which opens large-conductance Ca²(+)-activated K(+) (BK(Ca)) channels, leading to ASM membrane hyperpolarization. Inhaled bitter tastants decreased airway obstruction in a mouse model of asthma. Given the need for efficacious bronchodilators for treating obstructive lung diseases, this pathway can be exploited for therapy with the thousands of known synthetic and naturally occurring bitter tastants.

  11. The Sophora flavescens flavonoid compound trifolirhizin inhibits acetylcholine induced airway smooth muscle contraction.

    PubMed

    Yang, Nan; Liang, Banghao; Srivastava, Kamal; Zeng, Jia; Zhan, Jixun; Brown, LaVerne; Sampson, Hugh; Goldfarb, Joseph; Emala, Charles; Li, Xiu-Min

    2013-11-01

    Asthma is a serious health problem worldwide, particularly in industrialized countries. Despite a better understanding of the pathophysiology of asthma, there are still considerable gaps in knowledge as well as a need for classes of drugs. ASHMI™ (Anti-asthma Herbal Medicine Intervention) is an aqueous extract of Ganoderma lucidum (Fr.) P. Karst (Ling Zhi), Sophora flavescens Aiton (Ku Shen) and Glycyrrhiza uralensis Fisch. ex DC (Gan Cao). It prevents allergic asthma airway hyper-reactivity in mice and inhibits acetylcholine (ACh) induced airway smooth muscle (ASM) contraction in tracheal rings from allergic asthmatic mice. The purpose of this research was to identify individual herb(s) and their active compound(s) that inhibit ASM contraction. It was found that S. flavescens, but not G. lucidum or G. uralensis aqueous extracts, inhibited ASM contraction in tracheal rings from asthmatic mice. Bioassay-guided isolation and identification of flavonoid fractions/compound(s) via methylene chloride extraction, preparative HPLC fractionation, and LC-MS and NMR spectroscopic analyses showed that trifolirhizin is an active constituent that inhibits acetylcholine mediated ASM contraction or directly relaxes pre-contracted ASM independent of β2-adrenoceptors. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. The Sophora Flavescens flavonoid compound trifolirhizin inhibits acetylcholine induced airway smooth muscle contraction

    PubMed Central

    Zeng, Jia; Zhan, Jixun; Brown, LaVerne; Sampson, Hugh; Goldfarb, Joseph; Emala, Charles; Li, Xiu-Min

    2014-01-01

    Asthma is a serious health problem worldwide, particularly in industrialized countries. Despite a better understanding of the pathophysiology of asthma, there are still considerable gaps in knowledge as well as a need for new classes of drugs. ASHMI™ (Anti-asthma Herbal Medicine Intervention) is an aqueous extract of Ganoderma lucidum (Fr.) P. Karst (Ling Zhi), Sophora flavescens Aiton (Ku Shen) and Glycyrrhiza uralensis Fisch. ex DC (Gan Cao). It prevents allergic asthma airway hyper-reactivity in mice and inhibits acetylcholine (ACh) induced airway smooth muscle (ASM) contraction in tracheal rings from allergic asthmatic mice. The purpose of this research was to identify individual herb(s) and their active compound(s) that inhibit ASM contraction. It was found that Sophora flavescens (S. flavescens), but not Ganoderma lucidum (G. lucidum) or Glycyrrhiza uralensis (G. uralensis) aqueous extracts, inhibited ASM contraction in tracheal rings from asthmatic mice. Bioassay-guided isolation and identification of flavonoid fractions/compound(s) via methylene chloride extraction, preparative HPLC fractionation, and LC-MS and NMR spectroscopic analyses showed that trifolirhizin is an active constituent that inhibits acetylcholine mediated ASM contraction or directly relaxes pre-contracted ASM independent of β2-adrenoceptors. PMID:23993294

  13. Targeting Phosphoinositide 3-Kinase γ in Airway Smooth Muscle Cells to Suppress Interleukin-13-Induced Mouse Airway Hyperresponsiveness

    PubMed Central

    Jiang, Haihong; Xie, Yan; Abel, Peter W.; Toews, Myron L.; Townley, Robert G.; Casale, Thomas B.

    2012-01-01

    We recently reported that phosphoinositide 3-kinase γ (PI3Kγ) directly regulates airway smooth muscle (ASM) contraction by modulating Ca2+ oscillations. Because ASM contraction plays a critical role in airway hyperresponsiveness (AHR) of asthma, the aim of the present study was to determine whether targeting PI3Kγ in ASM cells could suppress AHR in vitro and in vivo. Intranasal administration into mice of interleukin-13 (IL-13; 10 μg per mouse), a key pathophysiologic cytokine in asthma, induced AHR after 48 h, as assessed by invasive tracheostomy. Intranasal administration of a broad-spectrum PI3K inhibitor or a PI3Kγ-specific inhibitor 1 h before AHR assessment attenuated IL-13 effects. Airway responsiveness to bronchoconstrictor agonists was also examined in precision-cut mouse lung slices pretreated without or with IL-13 for 24 h. Acetylcholine and serotonin dose-response curves indicated that IL-13-treated lung slices had a 40 to 50% larger maximal airway constriction compared with controls. Furthermore, acetylcholine induced a larger initial Ca2+ transient and increased Ca2+ oscillations in IL-13-treated primary mouse ASM cells compared with control cells, correlating with increased cell contraction. As expected, PI3Kγ inhibitor treatment attenuated IL-13-augmented airway contractility of lung slices and ASM cell contraction. In both control and IL-13-treated ASM cells, small interfering RNA-mediated knockdown of PI3Kγ by 70% only reduced the initial Ca2+ transient by 20 to 30% but markedly attenuated Ca2+ oscillations and contractility of ASM cells by 50 to 60%. This report is the first to demonstrate that PI3Kγ in ASM cells is important for IL-13-induced AHR and that acute treatment with a PI3Kγ inhibitor can ameliorate AHR in a murine model of asthma. PMID:22543031

  14. Antimitogenic effect of bitter taste receptor agonists on airway smooth muscle cells.

    PubMed

    Sharma, Pawan; Panebra, Alfredo; Pera, Tonio; Tiegs, Brian C; Hershfeld, Alena; Kenyon, Lawrence C; Deshpande, Deepak A

    2016-02-15

    Airway remodeling is a hallmark feature of asthma and chronic obstructive pulmonary disease. Clinical studies and animal models have demonstrated increased airway smooth muscle (ASM) mass, and ASM thickness is correlated with severity of the disease. Current medications control inflammation and reverse airway obstruction effectively but have limited effect on remodeling. Recently we identified the expression of bitter taste receptors (TAS2R) on ASM cells, and activation with known TAS2R agonists resulted in ASM relaxation and bronchodilation. These studies suggest that TAS2R can be used as new therapeutic targets in the treatment of obstructive lung diseases. To further establish their effectiveness, in this study we aimed to determine the effects of TAS2R agonists on ASM growth and promitogenic signaling. Pretreatment of healthy and asthmatic human ASM cells with TAS2R agonists resulted in a dose-dependent inhibition of ASM proliferation. The antimitogenic effect of TAS2R ligands was not dependent on activation of protein kinase A, protein kinase C, or high/intermediate-conductance calcium-activated K(+) channels. Immunoblot analyses revealed that TAS2R agonists inhibit growth factor-activated protein kinase B phosphorylation without affecting the availability of phosphatidylinositol 3,4,5-trisphosphate, suggesting TAS2R agonists block signaling downstream of phosphatidylinositol 3-kinase. Furthermore, the antimitogenic effect of TAS2R agonists involved inhibition of induced transcription factors (activator protein-1, signal transducer and activator of transcription-3, E2 factor, nuclear factor of activated T cells) and inhibition of expression of multiple cell cycle regulatory genes, suggesting a direct inhibition of cell cycle progression. Collectively, these findings establish the antimitogenic effect of TAS2R agonists and identify a novel class of receptors and signaling pathways that can be targeted to reduce or prevent airway remodeling as well as

  15. Protective effects of anisodamine on cigarette smoke extract-induced airway smooth muscle cell proliferation and tracheal contractility

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Xu, Guang-Ni; Yang, Kai; Xu, Zu-Peng

    2012-07-01

    Anisodamine, an antagonist of muscarinic acetylcholine receptors (mAChRs), has been used therapeutically to improve smooth muscle function, including microvascular, intestinal and airway spasms. Our previous studies have revealed that airway hyper-reactivity could be prevented by anisodamine. However, whether anisodamine prevents smoking-induced airway smooth muscle (ASM) cell proliferation remained unclear. In this study, a primary culture of rat ASM cells was used to evaluate an ASM phenotype through the ability of the cells to proliferate and express contractile proteins in response to cigarette smoke extract (CSE) and intervention of anisodamine. Our results showed that CSE resulted in an increase in cyclinmore » D1 expression concomitant with the G0/G1-to-S phase transition, and high expression of M2 and M3. Functional studies showed that tracheal hyper-contractility accompanied contractile marker α-SMA high-expression. These changes, which occur only after CSE stimulation, were prevented and reversed by anisodamine, and CSE-induced cyclin D1 expression was significantly inhibited by anisodamine and the specific inhibitor U0126, BAY11-7082 and LY294002. Thus, we concluded that the protective and reversal effects and mechanism of anisodamine on CSE-induced events might involve, at least partially, the ERK, Akt and NF-κB signaling pathways associated with cyclin D1 via mAChRs. Our study validated that anisodamine intervention on ASM cells may contribute to anti-remodeling properties other than bronchodilation. -- Highlights: ► CSE induces tracheal cell proliferation, hyper-contractility and α-SMA expression. ► Anisodamine reverses CSE-induced tracheal hyper-contractility and cell proliferation. ► ERK, PI3K, and NF-κB pathways and cyclin D1 contribute to the reversal effect.« less

  16. Hyaluronic acid influence on platelet-induced airway smooth muscle cell proliferation.

    PubMed

    Svensson Holm, Ann-Charlotte B; Bengtsson, Torbjörn; Grenegård, Magnus; Lindström, Eva G

    2012-03-10

    Hyaluronic acid (HA) is one of the main components of the extracellular matrix (ECM) and is expressed throughout the body including the lung and mostly in areas surrounding proliferating and migrating cells. Furthermore, platelets have been implicated as important players in the airway remodelling process, e.g. due to their ability to induce airway smooth muscle cell (ASMC) proliferation. The aim of the present study was to investigate the role of HA, the HA-binding surface receptor CD44 and focal adhesion kinase (FAK) in platelet-induced ASMC proliferation. Proliferation of ASMC was measured using the MTS-assay, and we found that the CD44 blocking antibody and the HA synthase inhibitor 4-Methylumbelliferone (4-MU) significantly inhibited platelet-induced ASMC proliferation. The interaction between ASMC and platelets was studied by fluorescent staining of F-actin. In addition, the ability of ASMC to synthesise HA was investigated by fluorescent staining using biotinylated HA-binding protein and a streptavidin conjugate. We observed that ASMC produced HA and that a CD44 blocking antibody and 4-MU significantly inhibited platelet binding to the area surrounding the ASMC. Furthermore, the FAK-inhibitor PF 573228 inhibited platelet-induced ASMC proliferation. Co-culture of ASMC and platelets also resulted in increased phosphorylation of FAK as detected by Western blot analysis. In addition, 4-MU significantly inhibited the increased FAK-phosphorylation. In conclusion, our findings demonstrate that ECM has the ability to influence platelet-induced ASMC proliferation. Specifically, we propose that HA produced by ASMC is recognised by platelet CD44. The platelet/HA interaction is followed by FAK activation and increased proliferation of co-cultured ASMC. We also suggest that the mitogenic effect of platelets represents a potential important and novel mechanism that may contribute to airway remodelling. Copyright © 2011 Elsevier Inc. All rights reserved.

  17. An RGS4-mediated phenotypic switch of bronchial smooth muscle cells promotes fixed airway obstruction in asthma.

    PubMed

    Damera, Gautam; Druey, Kirk M; Cooper, Philip R; Krymskaya, Vera P; Soberman, Roy J; Amrani, Yassine; Hoshi, Toshinori; Brightling, Christopher E; Panettieri, Reynold A

    2012-01-01

    In severe asthma, bronchodilator- and steroid-insensitive airflow obstruction develops through unknown mechanisms characterized by increased lung airway smooth muscle (ASM) mass and stiffness. We explored the role of a Regulator of G-protein Signaling protein (RGS4) in the ASM hyperplasia and reduced contractile capacity characteristic of advanced asthma. Using immunocytochemical staining, ASM expression of RGS4 was determined in endobronchial biopsies from healthy subjects and those from subjects with mild, moderate and severe asthma. Cell proliferation assays, agonist-induced calcium mobilization and bronchoconstriction were determined in cultured human ASM cells and in human precision cut lung slices. Using gain- and loss-of-function approaches, the precise role of RGS proteins was determined in stimulating human ASM proliferation and inhibiting bronchoconstriction. RGS4 expression was restricted to a subpopulation of ASM and was specifically upregulated by mitogens, which induced a hyperproliferative and hypocontractile ASM phenotype similar to that observed in recalcitrant asthma. RGS4 expression was markedly increased in bronchial smooth muscle of patients with severe asthma, and expression correlated significantly with reduced pulmonary function. Whereas RGS4 inhibited G protein-coupled receptor (GPCR)-mediated bronchoconstriction, unexpectedly RGS4 was required for PDGF-induced proliferation and sustained activation of PI3K, a mitogenic signaling molecule that regulates ASM proliferation. These studies indicate that increased RGS4 expression promotes a phenotypic switch of ASM, evoking irreversible airway obstruction in subjects with severe asthma.

  18. Effects of Ginger and Its Constituents on Airway Smooth Muscle Relaxation and Calcium Regulation

    PubMed Central

    Siviski, Matthew E.; Zhang, Yi; Xu, Carrie; Hoonjan, Bhupinder; Emala, Charles W.

    2013-01-01

    The prevalence of asthma has increased in recent years, and is characterized by airway hyperresponsiveness and inflammation. Many patients report using alternative therapies to self-treat asthma symptoms as adjuncts to short-acting and long-acting β-agonists and inhaled corticosteroids (ICS). As many as 40% of patients with asthma use herbal therapies to manage asthma symptoms, often without proven efficacy or known mechanisms of action. Therefore, investigations of both the therapeutic and possible detrimental effects of isolated components of herbal treatments on the airway are important. We hypothesized that ginger and its active components induce bronchodilation by modulating intracellular calcium ([Ca2+]i) in airway smooth muscle (ASM). In isolated human ASM, ginger caused significant and rapid relaxation. Four purified constituents of ginger were subsequently tested for ASM relaxant properties in both guinea pig and human tracheas: [6]-gingerol, [8]-gingerol, and [6]-shogaol induced rapid relaxation of precontracted ASM (100–300 μM), whereas [10]-gingerol failed to induce relaxation. In human ASM cells, exposure to [6]-gingerol, [8]-gingerol, and [6]-shogaol, but not [10]-gingerol (100 μM), blunted subsequent Ca2+ responses to bradykinin (10 μM) and S-(−)-Bay K 8644 (10 μM). In A/J mice, the nebulization of [8]-gingerol (100 μM), 15 minutes before methacholine challenge, significantly attenuated airway resistance, compared with vehicle. Taken together, these novel data show that ginger and its isolated active components, [6]-gingerol, [8]-gingerol, and [6]-shogaol, relax ASM, and [8]-gingerol attenuates airway hyperresponsiveness, in part by altering [Ca2+]i regulation. These purified compounds may provide a therapeutic option alone or in combination with accepted therapeutics, including β2-agonists, in airway diseases such as asthma. PMID:23065130

  19. Fetal human airway smooth muscle cell production of leukocyte chemoattractants is differentially regulated by fluticasone.

    PubMed

    Pearson, Helen; Britt, Rodney D; Pabelick, Christine M; Prakash, Y S; Amrani, Yassine; Pandya, Hitesh C

    2015-12-01

    Adult human airway smooth muscle (ASM) produce cytokines involved in recruitment and survival of leukocytes within airway walls. Cytokine generation by adult ASM is glucocorticoid-sensitive. Whether developing lung ASM produces cytokines in a glucocorticoid-sensitive fashion is unknown. Cultured fetal human ASM cells stimulated with TNF-α (0-20 ng/ml) were incubated with TNF-α receptor-blocking antibodies, fluticasone (1 and 100 nm), or vehicle. Supernatants and cells were assayed for the production of CCL5, CXCL10, and CXCL8 mRNA and protein and glucocorticoid receptor phosphorylation. CCL5, CXCL10, and CXCL8 mRNA and protein production by fetal ASM cell was significantly and dose-dependently following TNF-α treatment. Cytokine mRNA and protein production were effectively blocked by TNF-α R1 and R2 receptor neutralizing antibodies but variably inhibited by fluticasone. TNF-α-induced TNF-R1 and R2 receptor mRNA expression was only partially attenuated by fluticasone. Glucocorticoid receptor phosphorylation at serine (Ser) 211 but not at Ser 226 was enhanced by fluticasone. Production of CCL5, CXCL10, and CXCL8 by fetal ASM appears to involve pathways that are both qualitatively and mechanistically distinct to those described for adult ASM. The findings imply developing ASM has potential to recruit leukocyte into airways and, therefore, of relevance to childhood airway diseases.

  20. Characterization of P2Y receptors mediating ATP induced relaxation in guinea pig airway smooth muscle: involvement of prostaglandins and K+ channels.

    PubMed

    Montaño, Luis M; Cruz-Valderrama, José E; Figueroa, Alejandra; Flores-Soto, Edgar; García-Hernández, Luz M; Carbajal, Verónica; Segura, Patricia; Méndez, Carmen; Díaz, Verónica; Barajas-López, Carlos

    2011-10-01

    In airway smooth muscle (ASM), adenosine 5'-triphosphate (ATP) induces a relaxation associated with prostaglandin production. We explored the role of K(+) currents (I (K)) in this relaxation. ATP relaxed the ASM, and this effect was abolished by indomethacin. Removal of airway epithelium slightly diminished the ATP-induced relaxation at lower concentration without modifying the responses to ATP at higher concentrations. ATPγS and UTP induced a concentration-dependent relaxation similar to ATP; α,β-methylene-ATP was inactive from 1 to 100 μM. Suramin or reactive blue 2 (RB2), P2Y receptor antagonists, did not modify the relaxation, but their combination significantly reduced this effect of ATP. The relaxation was also inhibited by N-ethylmaleimide (NEM; which uncouples G proteins). In myocytes, the ATP-induced I (K) increment was not modified by suramin or RB2 but the combination of both drugs abolished it. This increment in the I (K) was also completely nullified by NEM and SQ 22,536. 4-Amynopyridine or iberiotoxin diminished the ATP-induced I (K) increment, and the combination of both substances diminished ATP-induced relaxation. The presence of P2Y(2) and P2Y(4) receptors in smooth muscle was corroborated by Western blot and confocal images. In conclusion, ATP: (1) produces relaxation by inducing the production of bronchodilator prostaglandins in airway smooth muscle, most likely by acting on P2Y(4) and P2Y(2) receptors; (2) induces I (K) increment through activation of the delayed rectifier K(+) channels and the high-conductance Ca(2+)-dependent K(+) channels, therefore both channels are implicated in the ATP-induced relaxation; and (3) this I (K) increment is mediated by prostaglandin production which in turns increase cAMP signaling pathway.

  1. Pharmacologic effects of grain weevil extract on isolated guinea pig tracheal smooth muscle.

    PubMed

    Schachter, E Neil; Zuskin, Eugenija; Arumugam, Uma; Goswami, Satindra; Castranova, Vincent; Whitmer, Mike; Chiarelli, Angelo

    2008-01-01

    The grain weevil, an insect (pest) that infects grain, is a frequent contaminant of processed wheat, and its presence may contribute to respiratory abnormalities in grain workers. We studied the in vitro effects of an extract of grain weevil (GWE) on airway smooth muscle. Pharmacologic studies included in vitro challenge of guinea pig trachea with GWE, in parallel organ baths, pretreated with mediator-modifying agents or a control solution. Dose-related contractions of nonsensitized guinea pig trachea (GPT) were demonstrated using this extract. Pharmacologic studies were performed by pretreating guinea pig tracheal tissue with drugs known to modulate smooth muscle contraction: atropine, indomethacin, pyrilamine, acivicin, NDGA, BPB, TMB8, captopril, and capsaicin. Atropine, pyrilamine, BPB, and capsaicin significantly reduced the contractile effects of the extract at most of the challenge doses (p < 0.01 or p < 0.05). Inhibition of GWE-induced contraction by blocking of other mediators was less complete. We suggest that GWE causes dose-related airway smooth muscle constriction of the GPT by nonimmunologic mechanisms involving a variety of airway mediators and possibly cholinergic receptors.

  2. Comparison of gel contraction mediated by airway smooth muscle cells from patients with and without asthma.

    PubMed

    Matsumoto, Hisako; Moir, Lyn M; Oliver, Brian G G; Burgess, Janette K; Roth, Michael; Black, Judith L; McParland, Brent E

    2007-10-01

    Exaggerated bronchial constriction is the most significant and life threatening response of patients with asthma to inhaled stimuli. However, few studies have investigated the contractility of airway smooth muscle (ASM) from these patients. The purpose of this study was to establish a method to measure contraction of ASM cells by embedding them into a collagen gel, and to compare the contraction between subjects with and without asthma. Gel contraction to histamine was examined in floating gels containing cultured ASM cells from subjects with and without asthma following overnight incubation while unattached (method 1) or attached (method 2) to casting plates. Smooth muscle myosin light chain kinase protein levels were also examined. Collagen gels containing ASM cells reduced in size when stimulated with histamine in a concentration-dependent manner and reached a maximum at a mean (SE) of 15.7 (1.2) min. This gel contraction was decreased by inhibitors for phospholipase C (U73122), myosin light chain kinase (ML-7) and Rho kinase (Y27632). When comparing the two patient groups, the maximal decreased area of gels containing ASM cells from patients with asthma was 19 (2)% (n = 8) using method 1 and 22 (3)% (n = 6) using method 2, both of which were greater than that of cells from patients without asthma: 13 (2)% (n = 9, p = 0.05) and 10 (4)% (n = 5, p = 0.024), respectively. Smooth muscle myosin light chain kinase levels were not different between the two groups. The increased contraction of asthmatic ASM cells may be responsible for exaggerated bronchial constriction in asthma.

  3. Smooth muscle in the maintenance of increased airway resistance elicited by methacholine in humans.

    PubMed

    Chapman, David G; Pascoe, Chris D; Lee-Gosselin, Audrey; Couture, Christian; Seow, Chun Y; Paré, Peter D; Salome, Cheryl M; King, Gregory G; Bossé, Ynuk

    2014-10-15

    Airway narrowing is maintained for a prolonged period after acute bronchoconstriction in humans in the absence of deep inspirations (DIs). To determine whether maintenance of airway smooth muscle (ASM) shortening is responsible for the persistence of airway narrowing in healthy subjects following transient methacholine (MCh)-induced bronchoconstriction. On two separate visits, five healthy subjects underwent MCh challenges until respiratory system resistance (Rrs) had increased by approximately 1.5 cm H2O/L/s. Subjects took a DI either immediately after or 30 minutes after the last dose. The extent of renarrowing following the bronchodilator effect of DI was used to assess the continued action of MCh (calculated as percent change in Rrs from the pre-DI Rrs). We then used human bronchial rings to determine whether ASM can maintain shortening during a progressive decrease of carbachol concentration. The increased Rrs induced by MCh was maintained for 30 minutes despite waning of MCh concentration over that period, measured as attenuated renarrowing when the DI was taken 30 minutes after compared with immediately after the last dose (7 min post-DI, -36.2 ± 11.8 vs. 14.4 ± 13.2%; 12 min post-DI, -39.5 ± 9.8 vs. 15.2 ± 17.8%). Ex vivo, ASM shortening was largely maintained during a progressive decrease of carbachol concentration, even down to concentrations that would not be expected to induce shortening. The maintenance of airway narrowing despite MCh clearance in humans is attributed to an intrinsic ability of ASM to maintain shortening during a progressive decrease of contractile stimulation.

  4. Fetal human airway smooth muscle cell production of leukocyte chemoattractants is differentially regulated by fluticasone

    PubMed Central

    Pearson, Helen; Britt, Rodney D.; Pabelick, Christine M.; Prakash, Y.S.; Amrani, Yassine; Pandya, Hitesh C.

    2016-01-01

    Background Adult human airway smooth muscle (ASM) produce cytokines involved in recruitment and survival of leukocytes within airway walls. Cytokine generation by adult ASM is glucocorticoid-sensitive. Whether developing lung ASM produces cytokines in a glucocorticoid-sensitive fashion is unknown. Methods Cultured fetal human ASM cells stimulated with TNF-α (0–20 ng/ml) were incubated with TNF-α receptor-blocking antibodies, fluticasone (1 and 100 nm), or vehicle. Supernatants and cells were assayed for the production of CCL5, CXCL10, and CXCL8 mRNA and protein and glucocorticoid receptor phosphorylation. Results CCL5, CXCL10, and CXCL8 mRNA and protein production by fetal ASM cell was significantly and dose-dependently following TNF-α treatment. Cytokine mRNA and protein production were effectively blocked by TNF-α R1 and R2 receptor neutralizing antibodies but variably inhibited by fluticasone. TNF-α-induced TNF-R1 and R2 receptor mRNA expression was only partially attenuated by fluticasone. Glucocorticoid receptor phosphorylation at serine (Ser) 211 but not at Ser 226 was enhanced by fluticasone. Conclusion Production of CCL5, CXCL10, and CXCL8 by fetal ASM appears to involve pathways that are both qualitatively and mechanistically distinct to those described for adult ASM. The findings imply developing ASM has potential to recruit leukocyte into airways and, therefore, of relevance to childhood airway diseases. PMID:26331770

  5. Nicotine-Induced Airway Smooth Muscle Cell Proliferation Involves TRPC6-Dependent Calcium Influx Via α7 nAChR.

    PubMed

    Hong, Wei; Peng, Gongyong; Hao, Binwei; Liao, Baoling; Zhao, Zhuxiang; Zhou, Yumin; Peng, Fang; Ye, Xiuqin; Huang, Lingmei; Zheng, Mengning; Pu, Jinding; Liang, Chunxiao; Yi, Erkang; Peng, Huanhuan; Li, Bing; Ran, Pixin

    2017-01-01

    The proliferation of human bronchial smooth muscle cells (HBSMCs) is a key pathophysiological component of airway remodeling in chronic obstructive pulmonary disease (COPD) for which pharmacotherapy is limited, and only slight improvements in survival have been achieved in recent decades. Cigarette smoke is a well-recognized risk factor for COPD; however, the pathogenesis of cigarette smoke-induced COPD remains incompletely understood. This study aimed to investigate the mechanisms by which nicotine affects HBSMC proliferation. Cell viability was assessed with a CCK-8 assay. Proliferation was measured by cell counting and EdU immunostaining. Fluorescence calcium imaging was performed to measure intracellular Ca2+ concentration ([Ca2+]i). The results showed that nicotine promotes HBSMC proliferation, which is accompanied by elevated store-operated calcium entry (SOCE), receptor-operated calcium entry (ROCE) and basal [Ca2+]i in HBSMCs. Moreover, we also confirmed that canonical transient receptor potential protein 6 (TRPC6) and α7 nicotinic acetylcholine receptor (α7 nAChR) are involved in nicotine-induced upregulation of cell proliferation. Furthermore, we verified that activation of the PI3K/Akt signaling pathway plays a pivotal role in nicotine-enhanced proliferation and calcium influx in HBSMCs. Inhibition of α7 nAChR significantly decreased Akt phosphorylation levels, and LY294002 inhibited the protein expression levels of TRPC6. Herein, these data provide compelling evidence that calcium entry via the α7 nAChR-PI3K/Akt-TRPC6 signaling pathway plays an important role in the physiological regulation of airway smooth muscle cell proliferation, representing an important target for augmenting airway remodeling. © 2017 The Author(s). Published by S. Karger AG, Basel.

  6. Novel treatment strategies for smooth muscle disorders: Targeting Kv7 potassium channels.

    PubMed

    Haick, Jennifer M; Byron, Kenneth L

    2016-09-01

    Smooth muscle cells provide crucial contractile functions in visceral, vascular, and lung tissues. The contractile state of smooth muscle is largely determined by their electrical excitability, which is in turn influenced by the activity of potassium channels. The activity of potassium channels sustains smooth muscle cell membrane hyperpolarization, reducing cellular excitability and thereby promoting smooth muscle relaxation. Research over the past decade has indicated an important role for Kv7 (KCNQ) voltage-gated potassium channels in the regulation of the excitability of smooth muscle cells. Expression of multiple Kv7 channel subtypes has been demonstrated in smooth muscle cells from viscera (gastrointestinal, bladder, myometrial), from the systemic and pulmonary vasculature, and from the airways of the lung, from multiple species, including humans. A number of clinically used drugs, some of which were developed to target Kv7 channels in other tissues, have been found to exert robust effects on smooth muscle Kv7 channels. Functional studies have indicated that Kv7 channel activators and inhibitors have the ability to relax and contact smooth muscle preparations, respectively, suggesting a wide range of novel applications for the pharmacological tool set. This review summarizes recent findings regarding the physiological functions of Kv7 channels in smooth muscle, and highlights potential therapeutic applications based on pharmacological targeting of smooth muscle Kv7 channels throughout the body. Published by Elsevier Inc.

  7. Specificity of arrestin subtypes in regulating airway smooth muscle G protein-coupled receptor signaling and function.

    PubMed

    Pera, Tonio; Hegde, Akhil; Deshpande, Deepak A; Morgan, Sarah J; Tiegs, Brian C; Theriot, Barbara S; Choi, Yeon H; Walker, Julia K L; Penn, Raymond B

    2015-10-01

    Arrestins have been shown to regulate numerous G protein-coupled receptors (GPCRs) in studies employing receptor/arrestin overexpression in artificial cell systems. Which arrestin isoforms regulate which GPCRs in primary cell types is poorly understood. We sought to determine the effect of β-arrestin-1 or β-arrestin-2 inhibition or gene ablation on signaling and function of multiple GPCRs endogenously expressed in airway smooth muscle (ASM). In vitro [second messenger (calcium, cAMP generation)], ex vivo (ASM tension generation in suspended airway), and in vivo (invasive airway resistance) analyses were performed on human ASM cells and murine airways/whole animal subject to β-arrestin-1 or -2 knockdown or knockout (KO). In both human and murine model systems, knockdown or KO of β-arrestin-2 relative to control missense small interfering RNA or wild-type mice selectively increased (40-60%) β2-adrenoceptor signaling and function. β-arrestin-1 knockdown or KO had no effect on signaling and function of β2-adrenoceptor or numerous procontractile GPCRs, but selectively inhibited M3 muscarinic acetylcholine receptor signaling (∼50%) and function (∼25% ex vivo, >50% in vivo) without affecting EC50 values. Arrestin subtypes differentially regulate ASM GPCRs and β-arrestin-1 inhibition represents a novel approach to managing bronchospasm in obstructive lung diseases. © FASEB.

  8. The Expression of NOX4 in Smooth Muscles of Small Airway Correlates with the Disease Severity of COPD

    PubMed Central

    2016-01-01

    Airway smooth muscle (ASM) remodeling is a hallmark in chronic obstructive pulmonary disease (COPD), and nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases (NOXs) produced reactive oxygen species (ROS) play a crucial role in COPD pathogenesis. In the present study, the expression of NOX4 and its correlation with the ASM hypertrophy/hyperplasia, clinical pulmonary functions, and the expression of transforming growth factor β (TGF-β) in the ASM of COPD small airways were investigated by semiquantitative morphological and/or immunohistochemistry staining methods. The results showed that an elevated expression of NOX4 and TGF-β, along with an increased volume of ASM mass, was found in the ASM of small airways in COPD patients. The abundance of NOX4 protein in the ASM was increased with disease severity and inversely correlated with the pulmonary functions in COPD patients. In addition, the expression of NOX4 and ASM marker α-SMA was colocalized, and the increased NOX4 expression was found to accompany an upregulated expression of TGF-β in the ASM of small airways of COPD lung. These results indicate that NOX4 may be a key regulator in ASM remodeling of small airway, in part through a mechanism interacting with TGF-β signaling in the pathogenesis of COPD, which warrants further investigation. PMID:27656649

  9. Interleukin-1beta-induced airway hyperresponsiveness enhances substance P in intrinsic neurons of ferret airway.

    PubMed

    Wu, Z-X; Satterfield, B E; Fedan, J S; Dey, R D

    2002-11-01

    Interleukin (IL)-1beta causes airway inflammation, enhances airway smooth muscle responsiveness, and alters neurotransmitter expression in sensory, sympathetic, and myenteric neurons. This study examines the role of intrinsic airway neurons in airway hyperresponsiveness (AHR) induced by IL-1beta. Ferrets were instilled intratracheally with IL-1beta (0.3 microg/0.3 ml) or saline (0.3 ml) once daily for 5 days. Tracheal smooth muscle contractility in vitro and substance P (SP) expression in tracheal neurons were assessed. Tracheal smooth muscle reactivity to acetylcholine (ACh) and methacholine (MCh) and smooth muscle contractions to electric field stimulation (EFS) both increased after IL-1beta. The IL-1beta-induced AHR was maintained in tracheal segments cultured for 24 h, a procedure that depletes SP from sensory nerves while maintaining viability of intrinsic airway neurons. Pretreatment with CP-99994, an antagonist of neurokinin 1 receptor, attenuated the IL-1beta-induced hyperreactivity to ACh and MCh and to EFS in cultured tracheal segments. SP-containing neurons in longitudinal trunk, SP innervation of superficial muscular plexus neurons, and SP nerve fiber density in tracheal smooth muscle all increased after treatment with IL-1beta. These results show that IL-1beta-enhanced cholinergic airway smooth muscle contractile responses are mediated by the actions of SP released from intrinsic airway neurons.

  10. Effect of Loading History on Airway Smooth Muscle Cell-Matrix Adhesions.

    PubMed

    Irons, Linda; Owen, Markus R; O'Dea, Reuben D; Brook, Bindi S

    2018-06-05

    Integrin-mediated adhesions between airway smooth muscle (ASM) cells and the extracellular matrix (ECM) regulate how contractile forces generated within the cell are transmitted to its external environment. Environmental cues are known to influence the formation, size, and survival of cell-matrix adhesions, but it is not yet known how they are affected by dynamic fluctuations associated with tidal breathing in the intact airway. Here, we develop two closely related theoretical models to study adhesion dynamics in response to oscillatory loading of the ECM, representing the dynamic environment of ASM cells in vivo. Using a discrete stochastic-elastic model, we simulate individual integrin binding and rupture events and observe two stable regimes in which either bond formation or bond rupture dominate, depending on the amplitude of the oscillatory loading. These regimes have either a high or low fraction of persistent adhesions, which could affect the level of strain transmission between contracted ASM cells and the airway tissue. For intermediate loading, we observe a region of bistability and hysteresis due to shared loading between existing bonds; the level of adhesion depends on the loading history. These findings are replicated in a related continuum model, which we use to investigate the effect of perturbations mimicking deep inspirations (DIs). Because of the bistability, a DI applied to the high adhesion state could either induce a permanent switch to a lower adhesion state or allow a return of the system to the high adhesion state. Transitions between states are further influenced by the frequency of oscillations, cytoskeletal or ECM stiffnesses, and binding affinities, which modify the magnitudes of the stable adhesion states as well as the region of bistability. These findings could explain (in part) the transient bronchodilatory effect of a DI observed in asthmatics compared to a more sustained effect in normal subjects. Copyright © 2018 Biophysical

  11. Orosomucoid-like 3 (ORMDL3) upregulates airway smooth muscle proliferation, contraction, and Ca2+ oscillations in asthma.

    PubMed

    Chen, Jun; Miller, Marina; Unno, Hirotoshi; Rosenthal, Peter; Sanderson, Michael J; Broide, David H

    2017-09-07

    Airway hyperresponsiveness is a major feature of asthma attributed predominantly to an extrinsic immune/inflammatory response increasing airway smooth muscle (ASM) contractility. We investigated whether increased ASM expression of orosomucoid-like 3 (ORMDL3), a gene on chromosome 17q21 highly linked to asthma, induced increased ASM proliferation and contractility in vitro and influenced airway contractility and calcium flux in ASM in precision-cut lung slices (PCLSs) from wild-type and hORMDL3 Zp3-Cre mice (which express increased levels of human ORMDL3 [hORMDL3]). Levels of ASM proliferation and contraction were assessed in ASM cells transfected with ORMDL3 in vitro. In addition, airway contractility and calcium oscillations were quantitated in ASM cells in PCLSs derived from naive wild-type and naive hORMDL3 Zp3-Cre mice, which do not have a blood supply. Increased ASM expression of ORMDL3 in vitro resulted in increased ASM proliferation and contractility. PCLSs derived from naive hORMDL3 Zp3-Cre mice, which do not have airway inflammation, exhibit increased airway contractility with increased calcium oscillations in ASM cells. Increased ASM ORMDL3 expression increases levels of ASM sarcoplasmic reticulum Ca 2+ ATPase 2b (SERCA2b), which increases ASM proliferation and contractility. Overall, these studies provide evidence that an intrinsic increase in ORMDL3 expression in ASM can induce increased ASM proliferation and contractility, which might contribute to increased airway hyperresponsiveness in the absence of airway inflammation in asthmatic patients. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  12. Cooling-induced contraction in ovine airways smooth muscle.

    PubMed

    Mustafa, S M; Pilcher, C W; Williams, K I

    1999-02-01

    The mechanism of cold-induced bronchoconstriction is poorly understood. This prompted the present study whose aim was to determine the step-wise direct effect of cooling on smooth muscle of isolated ovine airways and analyse the role of calcium in the mechanisms involved. Isolated tracheal strips and bronchial segments were suspended in organ baths containing Krebs' solution for isometric tension recording. Tissue responses during stepwise cooling from 37 to 5 degrees C were examined. Cooling induced a rapid and reproducible contraction proportional to cooling temperature in ovine tracheal and bronchial preparations which was epithelium-independent. On readjustment to 37 degrees C the tone returned rapidly to basal level. Maximum contraction was achieved at a temperature of 5 degrees C for trachea and 15 degrees C for bronchiole. Cooling-induced contractions (CIC) was resistant to tetrodotoxin (1; 10 micrometer), and not affected by the muscarinic antagonist atropine (1 micrometer) or the alpha-adrenergic antagonist phentolamine (1 micrometer), or the histamine H1-antagonist mepyramine (1 micrometer) or indomethacin (1 micrometer). Ca2+ antagonists (nifedipine and verapamil) and Mn2+ raised tracheal but not bronchiolar tone and augmented CIC. Incubation in Ca2+-free, EGTA-containing Krebs' solution for 5 min had no effect on CIC, although it significantly reduced KCl-induced contraction by up to 75%. Cooling inhibited Ca2+ influx measured using 45Ca2+ uptake. Caffeine (100 micrometer) significantly inhibited CIC. The results show that cooling-induced contractions do not appear to involve activation of nerve endings, all surface reception systems or Ca2+ influx. However, CIC is mainly dependent on release of intracellular Ca2+. Copyright 1999 The Italian Pharmacological Society.

  13. Ketamine relaxes airway smooth muscle contracted by endothelin.

    PubMed

    Sato, T; Matsuki, A; Zsigmond, E K; Rabito, S F

    1997-04-01

    Endothelins (ETs) are synthesized not only in vascular endothelial cells but also in airway epithelial cells. Increased ET-1 has been demonstrated in bronchial epithelium of asthmatic patients, and, in severe asthma attacks, ET-1 increases in plasma and bronchoalveolar lavage fluid. In this study, we investigated whether ketamine (KET) relaxes ET-induced tracheal contractions. Female guinea pigs were killed with an overdose of pentobarbital. The trachea was removed and cut spirally into two strips that were mounted in an organ bath filled with Krebs-bicarbonate buffer. The response of each strip to 10(-7) M carbachol was taken as 100% contraction to which the response to ET was referred. The contribution of the epithelium to the relaxant effect of KET was studied in denuded tracheae or in the presence of 5 x 10(-5) M indomethacin. ET-1 (3 x 10(-8) M) induced contractions that were 76 +/- 3% of those induced by carbachol. KET reversed the response to ET-1 in a dose-dependent fashion. Similarly, ET-2 (3 x 10(-8) M) induced contractions that were 74 +/- 5% of those induced by carbachol, and KET also reversed this response in a dose-dependent manner. In epithelium-denuded strips, ET-1 induced contractions that were 104 +/- 3% of those induced by carbachol, and KET still reversed this response. The tonic phase of the response to ET-1 was equal (100 +/- 6%) to the response to carbachol, and KET did not affect it significantly. In the presence of ryanodine, KET reduced the ET-1-induced contraction from 67 +/- 2% to 36 +/- 3.%, P < 0.01. In the presence of nicardipine, KET also inhibited the ET-1-induced contraction. We conclude that KET relaxes the tracheal smooth muscle contracted by ETs via a mechanism that is independent of the tracheal epithelium. The relaxant effect of KET on the ET-induced contraction of the trachealis muscle is not dependent upon blockade of 1) sarcolemma influx of Ca2+ through the dihydropyridine Ca2+ channel or 2) the release of intracellular Ca2

  14. Th1 cytokine-induced syndecan-4 shedding by airway smooth muscle cells is dependent on mitogen-activated protein kinases.

    PubMed

    Tan, Xiahui; Khalil, Najwa; Tesarik, Candice; Vanapalli, Karunasri; Yaputra, Viki; Alkhouri, Hatem; Oliver, Brian G G; Armour, Carol L; Hughes, J Margaret

    2012-04-01

    In asthma, airway smooth muscle (ASM) chemokine secretion can induce mast cell recruitment into the airways. The functions of the mast cell chemoattractant CXCL10, and other chemokines, are regulated by binding to heparan sulphates such as syndecan-4. This study is the first demonstration that airway smooth muscle cells (ASMC) from people with and without asthma express and shed syndecan-4 under basal conditions. Syndecan-4 shedding was enhanced by stimulation for 24 h with the Th1 cytokines interleukin-1β (IL-1β) or tumor necrosis factor-α (TNF-α), but not interferon-γ (IFNγ), nor the Th2 cytokines IL-4 and IL-13. ASMC stimulation with IL-1β, TNF-α, and IFNγ (cytomix) induced the highest level of syndecan-4 shedding. Nonasthmatic and asthmatic ASM cell-associated syndecan-4 protein expression was also increased by TNF-α or cytomix at 4-8 h, with the highest levels detected in cytomix-stimulated asthmatic cells. Cell-associated syndecan-4 levels were decreased by 24 h, whereas shedding remained elevated at 24 h, consistent with newly synthesized syndecan-4 being shed. Inhibition of ASMC matrix metalloproteinase-2 did not prevent syndecan-4 shedding, whereas inhibition of ERK MAPK activation reduced shedding from cytomix-stimulated ASMC. Although ERK inhibition had no effect on syndecan-4 mRNA levels stimulated by cytomix, it did cause an increase in cell-associated syndecan-4 levels, consistent with the shedding being inhibited. In conclusion, ASMC produce and shed syndecan-4 and although this is increased by the Th1 cytokines, the MAPK ERK only regulates shedding. ASMC syndecan-4 production during Th1 inflammatory conditions may regulate chemokine activity and mast cell recruitment to the ASM in asthma.

  15. Stress and strain in the contractile and cytoskeletal filaments of airway smooth muscle.

    PubMed

    Deng, Linhong; Bosse, Ynuk; Brown, Nathan; Chin, Leslie Y M; Connolly, Sarah C; Fairbank, Nigel J; King, Greg G; Maksym, Geoffrey N; Paré, Peter D; Seow, Chun Y; Stephen, Newman L

    2009-10-01

    Stress and strain are omnipresent in the lung due to constant lung volume fluctuation associated with respiration, and they modulate the phenotype and function of all cells residing in the airways including the airway smooth muscle (ASM) cell. There is ample evidence that the ASM cell is very sensitive to its physical environment, and can alter its structure and/or function accordingly, resulting in either desired or undesired consequences. The forces that are either conferred to the ASM cell due to external stretching or generated inside the cell must be borne and transmitted inside the cytoskeleton (CSK). Thus, maintaining appropriate levels of stress and strain within the CSK is essential for maintaining normal function. Despite the importance, the mechanisms regulating/dysregulating ASM cytoskeletal filaments in response to stress and strain remained poorly understood until only recently. For example, it is now understood that ASM length and force are dynamically regulated, and both can adapt over a wide range of length, rendering ASM one of the most malleable living tissues. The malleability reflects the CSK's dynamic mechanical properties and plasticity, both of which strongly interact with the loading on the CSK, and all together ultimately determines airway narrowing in pathology. Here we review the latest advances in our understanding of stress and strain in ASM cells, including the organization of contractile and cytoskeletal filaments, range and adaptation of functional length, structural and functional changes of the cell in response to mechanical perturbation, ASM tone as a mediator of strain-induced responses, and the novel glassy dynamic behaviors of the CSK in relation to asthma pathophysiology.

  16. Overexpression of human Hsp27 inhibits serum-induced proliferation in airway smooth muscle myocytes and confers resistance to hydrogen peroxide cytotoxicity.

    PubMed

    Salinthone, Sonemany; Ba, Mariam; Hanson, Lisa; Martin, Jody L; Halayko, Andrew J; Gerthoffer, William T

    2007-11-01

    Airway smooth muscle (ASM) hypertrophy and hyperplasia are characteristics of asthma that lead to thickening of the airway wall and obstruction of airflow. Very little is known about mechanisms underlying ASM remodeling, but in vascular smooth muscle, it is known that progression of atherosclerosis depends on the balance of myocyte proliferation and cell death. Small heat shock protein 27 (Hsp27) is antiapoptotic in nonmuscle cells, but its role in ASM cell survival is unknown. Our hypothesis was that phosphorylation of Hsp27 may regulate airway remodeling by modifying proliferation, cell survival, or both. To test this hypothesis, adenoviral vectors were used to overexpress human Hsp27 in ASM cells. Cells were infected with empty vector (Ad5) or wild-type Hsp27 (AdHsp27 WT), and proliferation and death were assessed. Overexpressing Hsp27 WT caused a 50% reduction in serum-induced proliferation and increased cell survival after exposure to 100 microM hydrogen peroxide (H(2)O(2)) compared with mock-infected controls. Overexpression studies utilizing an S15A, S78A, and S82A non-phosphorylation mutant (AdHsp27 3A) and an S15D, S78D, and S82D pseudo-phosphorylation mutant (AdHsp27 3D) showed phosphorylation of Hsp27 was necessary for regulation of ASM proliferation, but not survival. Hsp27 provided protection against H(2)O(2)-induced cytotoxicity by upregulating cellular glutathione levels and preventing necrotic cell death, but not apoptotic cell death. The results support the notion that ASM cells can be stimulated to undergo proliferation and death and that Hsp27 may regulate these processes, thereby contributing to airway remodeling in asthmatics.

  17. Expression and coupling of neurokinin receptor subtypes to inositol phosphate and calcium signaling pathways in human airway smooth muscle cells.

    PubMed

    Mizuta, Kentaro; Gallos, George; Zhu, Defen; Mizuta, Fumiko; Goubaeva, Farida; Xu, Dingbang; Panettieri, Reynold A; Yang, Jay; Emala, Charles W

    2008-03-01

    Neuropeptide tachykinins (substance P, neurokinin A, and neurokinin B) are present in peripheral terminals of sensory nerve fibers within the respiratory tract and cause airway contractile responses and hyperresponsiveness in humans and most mammalian species. Three subtypes of neurokinin receptors (NK1R, NK2R, and NK3R) classically couple to Gq protein-mediated inositol 1,4,5-trisphosphate (IP3) synthesis and liberation of intracellular Ca2+, which initiates contraction, but their expression and calcium signaling mechanisms are incompletely understood in airway smooth muscle. All three subtypes were identified in native and cultured human airway smooth muscle (HASM) and were subsequently overexpressed in HASM cells using a human immunodeficiency virus-1-based lentivirus transduction system. Specific NKR agonists {NK1R, [Sar9,Met(O2)11]-substance P; NK2R, [beta-Ala8]-neurokinin A(4-10); NK3R, senktide} stimulated inositol phosphate synthesis and increased intracellular Ca2+ concentration ([Ca2+]i) in native HASM cells and in HASM cells transfected with each NKR subtype. These effects were blocked by NKR-selective antagonists (NK1R, L-732138; NK2R, GR-159897; NK3R, SB-222200). The initial transient and sustained phases of increased [Ca2+]i were predominantly inhibited by the IP3 receptor antagonist 2-aminoethoxydiphenyl borate (2-APB) or the store-operated Ca2+ channel antagonist SKF-96365, respectively. These results show that all three subtypes of NKRs are expressed in native HASM cells and that IP3 levels are the primary mediators of NKR-stimulated initial [Ca2+]i increases, whereas store-operated Ca2+ channels mediate the sustained phase of the [Ca2+]i increase.

  18. Effects of lipopolysaccharide from Pseudomonas aeruginosa on airway smooth muscle functions in guinea pigs.

    PubMed

    Yamawaki, I; Tamaoki, J; Kanemura, T; Horii, S; Takizawa, T

    1990-01-01

    To elucidate the mechanisms of airway hyperreactivity induced by lipopolysaccharide (LPS), we studied isolated tracheal segments from guinea pigs under isometric conditions in vitro. Guinea pigs were injected intraperitoneally with endotoxin (1 mg/kg; LPS from Pseudomonas aeruginosa, serotype 10) for 4 days, and animals treated with sterile nonpyrogenic saline served as controls. Histological examination of trachea revealed moderate structural damage of epithelial layer in the LPS-treated group. Treatment with LPS potentiated the contractile responses of tracheal smooth muscle to acetylcholine, causing a leftward displacement of dose-response curves so that the EC50 values decreased from 1.1 +/- 3.7 x 10(-5) to 4.4 +/- 3.7 x 10(-7) M (mean +/- SE, p less than 0.01). Likewise, LPS shifted the dose-response curves for histamine and substance P to lower concentrations by approximately 0.5-1.0 log U. Each of these potentiations was not affected by pretreatment of tissues with indomethacin or propranolol. Addition of isoproterenol to tracheal segments precontracted with acetylcholine caused concentration-dependent relaxation, an effect that was significantly greater in controls than in the LPS-treated group. These results suggest that airway hyperreactivity induced by LPS in guinea pigs may be attributed to a decreased ability of respiratory epithelial cells to generate a relaxing factor.

  19. Exploiting the relationship between birefringence and force to measure airway smooth muscle contraction with PS-OCT (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Adams, David C.; Hariri, Lida P.; Holz, Jasmin A.; Szabari, Margit V.; Harris, R. Scott; Cho, Jocelyn L.; Hamilos, Daniel L.; Luster, Andrew D.; Medoff, Benjamin D.; Suter, Melissa J.

    2016-03-01

    The ability to observe airway dynamics is fundamental to forming a complete understanding of pulmonary diseases such as asthma. We have previously demonstrated that Optical Coherence Tomography (OCT) can be used to observe structural changes in the airway during bronchoconstriction, but standard OCT lacks the contrast to discriminate airway smooth muscle (ASM) bands- ASM being responsible for generating the force that drives airway constriction- from the surrounding tissue. Since ASM in general exhibits a greater degree of birefringence than the surrounding tissue, a potential solution to this problem lies in the implementation of polarization sensitivity (PS) to the OCT system. By modifying the OCT system so that it is sensitive to the birefringence of tissue under inspection, we can visualize the ASM with much greater clarity and definition. In this presentation we show that the force of contraction can be indirectly measured by an associated increase in the birefringence signal of the ASM. We validate this approach by attaching segments of swine trachea to an isometric force transducer and stimulating contraction, while simultaneously measuring the exerted force and imaging the segment with PS-OCT. We then show how our results may be used to extrapolate the force of contraction of closed airways in absence of additional measurement devices. We apply this technique to assess ASM contractility volumetrically and in vivo, in both asthmatic and non-asthmatic human volunteers.

  20. Bitter taste receptors in the wrong place: novel airway smooth muscle targets for treating asthma.

    PubMed

    Liggett, Stephen B

    2014-01-01

    There is a need to expand the classes of drugs used to treat obstructive lung diseases to achieve better outcomes. With only one class of direct bronchodilators (β-agonists), we sought to find receptors on human airway smooth muscle (ASM) that act via a unique mechanism to relax the muscle, have a diverse agonist binding profile to enhance the probability of finding new therapeutics, and relax ASM with equal or greater efficacy than β-agonists. We have found that human and mouse ASM express six bitter taste receptor (TAS2R) subtypes, previously thought only to exist in taste buds of the tongue. Agonists acting at TAS2Rs evoke profound bronchodilation via a Ca(2+)-dependent mechanism. TAS2R function is not altered in asthma models, undergoes minimal tachyphylaxis upon repetitive dosing, and relaxes even under extreme desensitization of relaxation by β-agonists. Taken together, TAS2Rs on ASM represent a novel pathway to consider for development of agonists in the treatment of asthma and chronic obstructive lung disease.

  1. Role of canonical transient receptor potential channel-3 in acetylcholine-induced mouse airway smooth muscle cell proliferation.

    PubMed

    Chen, Xiao-Xu; Zhang, Jia-Hua; Pan, Bin-Hua; Ren, Hui-Li; Feng, Xiu-Ling; Wang, Jia-Ling; Xiao, Jun-Hua

    2017-10-15

    Canonical transient receptor potential channel-3 (TRPC3)-encoded Ca 2+ -permeable nonselective cation channel (NSCC) has been proven to be an important native constitutively active channel in airway smooth muscle cell (ASMC), which plays significant roles in physiological and pathological conditions by controlling Ca 2+ homeostasis in ASMC. Acetylcholine (ACh) is generally accepted as a contractile parasympathetic neurotransmitter in the airway. Recently studies have revealed the pathological role of ACh in airway remodeling, however, the mechanisms remain unclear. Here, we investigated the role of TRPC3 in ACh-induced ASMC proliferation. Primary mouse ASMCs were cultured with or without ACh treatment, then cell viability, TRPC3 expression, NSCC currents and [Ca 2+ ] i changes were examined by MTT assay, cell counting, Western blotting, standard whole-cell patch clamp recording and calcium imaging, respectively. Small interfering RNA (siRNA) technology was used to confirm the contribution of TRPC3 to ACh-induced ASMC proliferation. TRPC3 blocker Gd 3+ , antibody or siRNA largely inhibited ACh-induced up-regulation of TRPC3 protein, enhancement of NSCC currents, resting [Ca 2+ ] i and KCl-induced changes in [Ca 2+ ] i , eventually inhibiting ACh-induced ASMC proliferation. Our data suggested ACh could induce ASMC proliferation, and TRPC3 may be involved in ACh-induced ASMC proliferation that occurs with airway remodeling. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Airway structure and function in Eisenmenger's syndrome.

    PubMed

    McKay, K O; Johnson, P R; Black, J L; Glanville, A R; Armour, C L

    1998-10-01

    The responsiveness of airways from patients with Eisenmenger's syndrome (n = 5) was compared with that in airways from organ donors (n = 10). Enhanced contractile responses to cholinergic stimulation were found in airways from patients with Eisenmenger's syndrome. The maximal responses to acetylcholine, carbachol, and parasympathetic nerve stimulation in airway tissue from these patients were 221%, 139%, and 152%, respectively, of the maximal responses obtained in donor tissue. Further, relaxation responses to isoproterenol and levocromakalim were absent (n = 2) or markedly impaired (n = 3) in airways from patients with Eisenmenger's syndrome. This attenuated relaxation response was nonspecific in that it was also absent after vasoactive intestinal peptide, sodium nitroprusside, papaverine, and electrical field application. These observations can most likely be explained by a decrease in intrinsic smooth muscle tone, as precontraction of airways revealed relaxation responses that were equivalent to those obtained in donor tissues. Morphometric analysis of tissues used for the functional studies revealed no differences in the airway dimensions (internal perimeter) or airway wall components (e.g., smooth muscle, cartilage) or total area to explain these observations. Although the mechanism for this observed decrease in intrinsic airway smooth muscle tone is not certain, it may be due to alteration in the substructure of the airway wall or, alternatively, may result from the continued release of depressant factors in the vicinity of the smooth muscle which permanently alters smooth muscle responsiveness.

  3. Does smooth muscle in an intact airway undergo length adaptation during a sustained change in transmural pressure?

    PubMed

    Ansell, Thomas K; McFawn, Peter K; McLaughlin, Robert A; Sampson, David D; Eastwood, Peter R; Hillman, David R; Mitchell, Howard W; Noble, Peter B

    2015-03-01

    In isolated airway smooth muscle (ASM) strips, an increase or decrease in ASM length away from its current optimum length causes an immediate reduction in force production followed by a gradual time-dependent recovery in force, a phenomenon termed length adaptation. In situ, length adaptation may be initiated by a change in transmural pressure (Ptm), which is a primary physiological determinant of ASM length. The present study sought to determine the effect of sustained changes in Ptm and therefore, ASM perimeter, on airway function. We measured contractile responses in whole porcine bronchial segments in vitro before and after a sustained inflation from a baseline Ptm of 5 cmH2O to 25 cmH2O, or deflation to -5 cmH2O, for ∼50 min in each case. In one group of airways, lumen narrowing and stiffening in response to electrical field stimulation (EFS) were assessed from volume and pressure signals using a servo-controlled syringe pump with pressure feedback. In a second group of airways, lumen narrowing and the perimeter of the ASM in situ were determined by anatomical optical coherence tomography. In a third group of airways, active tension was determined under isovolumic conditions. Both inflation and deflation reduced the contractile response to EFS. Sustained Ptm change resulted in a further decrease in contractile response, which returned to baseline levels upon return to the baseline Ptm. These findings reaffirm the importance of Ptm in regulating airway narrowing. However, they do not support a role for ASM length adaptation in situ under physiological levels of ASM lengthening and shortening. Copyright © 2015 the American Physiological Society.

  4. Chloride channel blockade relaxes airway smooth muscle and potentiates relaxation by β-agonists

    PubMed Central

    Yim, Peter; Rinderspacher, Alison; Fu, Xiao Wen; Zhang, Yi; Landry, Donald W.; Emala, Charles W.

    2014-01-01

    Severe bronchospasm refractory to β-agonists continues to cause significant morbidity and mortality in asthmatic patients. We questioned whether chloride channels/transporters are novel targets for the relaxation of airway smooth muscle (ASM). We have screened a library of compounds, derivatives of anthranilic and indanyloxyacetic acid, that were originally developed to antagonize chloride channels in the kidney. We hypothesized that members of this library would be novel calcium-activated chloride channel blockers for the airway. The initial screen of this compound library identified 4 of 20 compounds that relaxed a tetraethylammonium chloride-induced contraction in guinea pig tracheal rings. The two most effective compounds, compounds 1 and 13, were further studied for their potential to either prevent the initiation of or relax the maintenance phase of an acetylcholine (ACh)-induced contraction or to potentiate β-agonist-mediated relaxation. Both relaxed an established ACh-induced contraction in human and guinea pig ex vivo ASM. In contrast, the prevention of an ACh-induced contraction required copretreatment with the sodium-potassium-chloride cotransporter blocker bumetanide. The combination of compound 13 and bumetanide also potentiated relaxation by the β-agonist isoproterenol in guinea pig tracheal rings. Compounds 1 and 13 hyperpolarized the plasma cell membrane of human ASM cells and blocked spontaneous transient inward currents, a measure of chloride currents in these cells. These functional and electrophysiological data suggest that modulating ASM chloride flux is a novel therapeutic target in asthma and other bronchoconstrictive diseases. PMID:24879056

  5. Nitric oxide and vasoactive intestinal peptide as co-transmitters of airway smooth-muscle relaxation: analysis in neuronal nitric oxide synthase knockout mice.

    PubMed

    Hasaneen, Nadia A; Foda, Hussein D; Said, Sami I

    2003-09-01

    Both vasoactive intestinal peptide (VIP) and nitric oxide (NO) relax airway smooth muscle and are potential co-transmitters of neurogenic airway relaxation. The availability of neuronal NO synthase (nNOS) knockout mice (nNOS-/-) provides a unique opportunity for evaluating NO. To evaluate the relative importance of NO, especially that generated by nNOS, and VIP as transmitters of the inhibitory nonadrenergic, noncholinergic (NANC) system. In this study, we compared the neurogenic (tetrodotoxin-sensitive) NANC relaxation of tracheal segments from nNOS-/- mice and control wild-type mice (nNOS(+/+)), induced by electrical field stimulation (EFS). We also examined the tracheal contractile response to methacholine and its relaxant response to VIP. EFS (at 60 V for 2 ms, at 10, 15, or 20 Hz) dose-dependently reduced tracheal tension, and the relaxations were consistently smaller (approximately 40%) in trachea from nNOS-/- mice than from control wild-type mice (p < 0.001). VIP (10(- 8) to 10(-6) mol/L) induced concentration-dependent relaxations that were approximately 50% smaller in nNOS-/- tracheas than in control tracheas. Methacholine induced concentration-dependent contractions that were consistently higher in the nNOS-/- tracheas relative to wild-type mice tracheas (p > 0.05). Our data suggest that, in mouse trachea, NO is probably responsible for mediating a large (approximately 60%) component of neurogenic NANC relaxation, and a similar (approximately 50%) component of the relaxant effect of VIP. The results imply that NO contributes significantly to neurogenic relaxation of mouse airway smooth muscle, whether due to neurogenic stimulation or to the neuropeptide VIP.

  6. Expression and coupling of neurokinin receptor subtypes to inositol phosphate and calcium signaling pathways in human airway smooth muscle cells

    PubMed Central

    Mizuta, Kentaro; Gallos, George; Zhu, Defen; Mizuta, Fumiko; Goubaeva, Farida; Xu, Dingbang; Panettieri, Reynold A.; Yang, Jay; Emala, Charles W.

    2013-01-01

    Neuropeptide tachykinins (substance P, neurokinin A, and neurokinin B) are present in peripheral terminals of sensory nerve fibers within the respiratory tract and cause airway contractile responses and hyperresponsiveness in humans and most mammalian species. Three subtypes of neurokinin receptors (NK1R, NK2R, and NK3R) classically couple to Gq protein-mediated inositol 1,4,5-trisphosphate (IP3) synthesis and liberation of intracellular Ca2+, which initiates contraction, but their expression and calcium signaling mechanisms are incompletely understood in airway smooth muscle. All three subtypes were identified in native and cultured human airway smooth muscle (HASM) and were subsequently overexpressed in HASM cells using a human immunodeficiency virus-1-based lentivirus transduction system. Specific NKR agonists {NK1R, [Sar9,Met(O2)11]-substance P; NK2R, [β-Ala8]-neurokinin A(4–10); NK3R, senktide} stimulated inositol phosphate synthesis and increased intracellular Ca2+ concentration ([Ca2+]i) in native HASM cells and in HASM cells transfected with each NKR subtype. These effects were blocked by NKR-selective antagonists (NK1R, L-732138; NK2R, GR-159897; NK3R, SB-222200). The initial transient and sustained phases of increased [Ca2+]i were predominantly inhibited by the IP3 receptor antagonist 2-aminoethoxydiphenyl borate (2-APB) or the store-operated Ca2+ channel antagonist SKF-96365, respectively. These results show that all three subtypes of NKRs are expressed in native HASM cells and that IP3 levels are the primary mediators of NKR-stimulated initial [Ca2+]i increases, whereas store-operated Ca2+ channels mediate the sustained phase of the [Ca2+]i increase. PMID:18203813

  7. Dense-body aggregates as plastic structures supporting tension in smooth muscle cells.

    PubMed

    Zhang, Jie; Herrera, Ana M; Paré, Peter D; Seow, Chun Y

    2010-11-01

    The wall of hollow organs of vertebrates is a unique structure able to generate active tension and maintain a nearly constant passive stiffness over a large volume range. These properties are predominantly attributable to the smooth muscle cells that line the organ wall. Although smooth muscle is known to possess plasticity (i.e., the ability to adapt to large changes in cell length through structural remodeling of contractile apparatus and cytoskeleton), the detailed structural basis for the plasticity is largely unknown. Dense bodies, one of the most prominent structures in smooth muscle cells, have been regarded as the anchoring sites for actin filaments, similar to the Z-disks in striated muscle. Here, we show that the dense bodies and intermediate filaments formed cable-like structures inside airway smooth muscle cells and were able to adjust the cable length according to cell length and tension. Stretching the muscle cell bundle in the relaxed state caused the cables to straighten, indicating that these intracellular structures were connected to the extracellular matrix and could support passive tension. These plastic structures may be responsible for the ability of smooth muscle to maintain a nearly constant tensile stiffness over a large length range. The finding suggests that the structural plasticity of hollow organs may originate from the dense-body cables within the smooth muscle cells.

  8. Bronchial inflammation induced PKCζ over-expression: involvement in mechanical properties of airway smooth muscle.

    PubMed

    Morin, Caroline; Fortin, Samuel; Rousseau, Eric

    2012-02-01

    Protein kinase C variants (PKCs) have been involved in the control of airway smooth muscle (ASM) tone, and abnormalities in PKC-dependent signaling have been associated with respiratory diseases such as asthma. In this study, the role of atypical PKCζ in airway hyperresponsiveness was investigated, using an in-vitro model of TNFα-treated human bronchi and an in vivo guinea pig model of chronic asthma. Our results demonstrated that PKCζ-specific inhibition produced a significant increase in isoproterenol sensitivity in TNFα-treated bronchi and ovalbumin (OVA)-sensitized guinea pig bronchi. The role of epoxy-eicosanoids, known to exert anti-inflammatory effects in lung, on PKCζ expression and activity in these models was evaluated. An enhanced PKCζ protein expression was delineated in TNFα-treated bronchi when compared with control (untreated) and epoxy-eicosanoid-treated bronchi. Measurements of Ca(2+) sensitivity, performed in TNFα-treated bronchi, demonstrated that treatment with myristoylated (Myr) PKCζ peptide inhibitor resulted in significant reductions of pCa-induced tension. Epoxy-eicosanoid treatments had similar effects on Ca(2+) sensitivity in TNFα-treated bronchi. In control and epoxy-eicosanoid-treated bronchi, the phosphorylated forms of p38MAPK and CPI-17 were significantly decreased compared with the TNFα-treated bronchi. An enhanced expression of PKCζ was ascertained in our in-vivo model of allergic asthma. Hence an increased Ca(2+) sensitivity could be explained by the phosphorylation of p38-MAPK, which in turn leads to phosphorylation and activation of the CPI-17 regulatory protein. This process was reversed upon treatment with the Myr-PKCζ-peptide inhibitor. The present data provide relevant evidence regarding the role of PKCζ in human and rodent models of airways inflammation.

  9. Bitter taste receptor agonists alter mitochondrial function and induce autophagy in airway smooth muscle cells.

    PubMed

    Pan, Shi; Sharma, Pawan; Shah, Sushrut D; Deshpande, Deepak A

    2017-07-01

    Airway remodeling, including increased airway smooth muscle (ASM) mass, is a hallmark feature of asthma and COPD. We previously identified the expression of bitter taste receptors (TAS2Rs) on human ASM cells and demonstrated that known TAS2R agonists could promote ASM relaxation and bronchodilation and inhibit mitogen-induced ASM growth. In this study, we explored cellular mechanisms mediating the antimitogenic effect of TAS2R agonists on human ASM cells. Pretreatment of ASM cells with TAS2R agonists chloroquine and quinine resulted in inhibition of cell survival, which was largely reversed by bafilomycin A1, an autophagy inhibitor. Transmission electron microscope studies demonstrated the presence of double-membrane autophagosomes and deformed mitochondria. In ASM cells, TAS2R agonists decreased mitochondrial membrane potential and increased mitochondrial ROS and mitochondrial fragmentation. Inhibiting dynamin-like protein 1 (DLP1) reversed TAS2R agonist-induced mitochondrial membrane potential change and attenuated mitochondrial fragmentation and cell death. Furthermore, the expression of mitochondrial protein BCL2/adenovirus E1B 19-kDa protein-interacting protein 3 (Bnip3) and mitochondrial localization of DLP1 were significantly upregulated by TAS2R agonists. More importantly, inhibiting Bnip3 mitochondrial localization by dominant-negative Bnip3 significantly attenuated cell death induced by TAS2R agonist. Collectively the TAS2R agonists chloroquine and quinine modulate mitochondrial structure and function, resulting in ASM cell death. Furthermore, Bnip3 plays a central role in TAS2R agonist-induced ASM functional changes via a mitochondrial pathway. These findings further establish the cellular mechanisms of antimitogenic effects of TAS2R agonists and identify a novel class of receptors and pathways that can be targeted to mitigate airway remodeling as well as bronchoconstriction in obstructive airway diseases. Copyright © 2017 the American Physiological

  10. Attenuation of airway smooth muscle contractility via flavonol-mediated inhibition of phospholipase-Cβ

    PubMed Central

    Brown, Amy; Danielsson, Jennifer; Townsend, Elizabeth A.; Zhang, Yi; Perez-Zoghbi, Jose F.; Emala, Charles W.

    2016-01-01

    Enhanced contractility of airway smooth muscle (ASM) is a major pathophysiological characteristic of asthma. Expanding the therapeutic armamentarium beyond β-agonists that target ASM hypercontractility would substantially improve treatment options. Recent studies have identified naturally occurring phytochemicals as candidates for acute ASM relaxation. Several flavonoids were evaluated for their ability to acutely relax human and murine ASM ex vivo and murine airways in vivo and were evaluated for their ability to inhibit procontractile signaling pathways in human ASM (hASM) cells. Two members of the flavonol subfamily, galangin and fisetin, significantly relaxed acetylcholine-precontracted murine tracheal rings ex vivo (n = 4 and n = 5, respectively, P < 0.001). Galangin and fisetin also relaxed acetylcholine-precontracted hASM strips ex vivo (n = 6–8, P < 0.001). Functional respiratory in vivo murine studies demonstrated that inhaled galangin attenuated the increase in lung resistance induced by inhaled methacholine (n = 6, P < 0.01). Both flavonols, galangin and fisetin, significantly inhibited purified phosphodiesterase-4 (PDE4) (n = 7, P < 0.05; n = 7, P < 0.05, respectively), and PLCβ enzymes (n = 6, P < 0.001 and n = 6, P < 0.001, respectively) attenuated procontractile Gq agonists' increase in intracellular calcium (n = 11, P < 0.001), acetylcholine-induced increases in inositol phosphates, and CPI-17 phosphorylation (n = 9, P < 0.01) in hASM cells. The prorelaxant effect retained in these structurally similar flavonols provides a novel pharmacological method for dual inhibition of PLCβ and PDE4 and therefore may serve as a potential treatment option for acute ASM constriction. PMID:26773068

  11. Attenuation of airway smooth muscle contractility via flavonol-mediated inhibition of phospholipase-Cβ.

    PubMed

    Brown, Amy; Danielsson, Jennifer; Townsend, Elizabeth A; Zhang, Yi; Perez-Zoghbi, Jose F; Emala, Charles W; Gallos, George

    2016-04-15

    Enhanced contractility of airway smooth muscle (ASM) is a major pathophysiological characteristic of asthma. Expanding the therapeutic armamentarium beyond β-agonists that target ASM hypercontractility would substantially improve treatment options. Recent studies have identified naturally occurring phytochemicals as candidates for acute ASM relaxation. Several flavonoids were evaluated for their ability to acutely relax human and murine ASM ex vivo and murine airways in vivo and were evaluated for their ability to inhibit procontractile signaling pathways in human ASM (hASM) cells. Two members of the flavonol subfamily, galangin and fisetin, significantly relaxed acetylcholine-precontracted murine tracheal rings ex vivo (n = 4 and n = 5, respectively, P < 0.001). Galangin and fisetin also relaxed acetylcholine-precontracted hASM strips ex vivo (n = 6-8, P < 0.001). Functional respiratory in vivo murine studies demonstrated that inhaled galangin attenuated the increase in lung resistance induced by inhaled methacholine (n = 6, P < 0.01). Both flavonols, galangin and fisetin, significantly inhibited purified phosphodiesterase-4 (PDE4) (n = 7, P < 0.05; n = 7, P < 0.05, respectively), and PLCβ enzymes (n = 6, P < 0.001 and n = 6, P < 0.001, respectively) attenuated procontractile Gq agonists' increase in intracellular calcium (n = 11, P < 0.001), acetylcholine-induced increases in inositol phosphates, and CPI-17 phosphorylation (n = 9, P < 0.01) in hASM cells. The prorelaxant effect retained in these structurally similar flavonols provides a novel pharmacological method for dual inhibition of PLCβ and PDE4 and therefore may serve as a potential treatment option for acute ASM constriction. Copyright © 2016 the American Physiological Society.

  12. Selective inhibition of histamine-evoked Ca2+ signals by compartmentalized cAMP in human bronchial airway smooth muscle cells.

    PubMed

    Dale, Philippa; Head, Victoria; Dowling, Mark R; Taylor, Colin W

    2018-05-01

    Intracellular Ca 2+ and cAMP typically cause opposing effects on airway smooth muscle contraction. Receptors that stimulate these pathways are therapeutic targets in asthma and chronic obstructive pulmonary disease. However, the interactions between different G protein-coupled receptors (GPCRs) that evoke cAMP and Ca 2+ signals in human bronchial airway smooth muscle cells (hBASMCs) are poorly understood. We measured Ca 2+ signals in cultures of fluo-4-loaded hBASMCs alongside measurements of intracellular cAMP using mass spectrometry or [ 3 H]-adenine labeling. Interactions between the signaling pathways were examined using selective ligands of GPCRs, and inhibitors of Ca 2+ and cAMP signaling pathways. Histamine stimulated Ca 2+ release through inositol 1,4,5-trisphosphate (IP 3 ) receptors in hBASMCs. β 2 -adrenoceptors, through cAMP and protein kinase A (PKA), substantially inhibited histamine-evoked Ca 2+ signals. Responses to other Ca 2+ -mobilizing stimuli were unaffected by cAMP (carbachol and bradykinin) or minimally affected (lysophosphatidic acid). Prostaglandin E 2 (PGE 2 ), through EP 2 and EP 4 receptors, stimulated formation of cAMP and inhibited histamine-evoked Ca 2+ signals. There was no consistent relationship between the inhibition of Ca 2+ signals and the amounts of intracellular cAMP produced by different stimuli. We conclude that β-adrenoceptors, EP 2 and EP 4 receptors, through cAMP and PKA, selectively inhibit Ca 2+ signals evoked by histamine in hBASMCs, suggesting that PKA inhibits an early step in H 1 receptor signaling. Local delivery of cAMP within hyperactive signaling junctions mediates the inhibition. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  13. Spatial and temporal traction response in human airway smooth muscle cells

    NASA Technical Reports Server (NTRS)

    Tolic-Norrelykke, Iva Marija; Butler, James P.; Chen, Jianxin; Wang, Ning

    2002-01-01

    Tractions that cells exert on their substrates are essential in cell spreading, migration, and contraction. These tractions can be determined by plating the cells on a flexible gel and measuring the deformation of the gel by using fluorescent beads embedded just below the surface of the gel. In this article we describe the image correlation method (ICM) optimized for determining the displacement field of the gel under a contracting cell. For the calculation of the traction field from the displacement field we use the recently developed method of Fourier transform traction cytometry (FTTC). The ICM and FTTC methods are applied to human airway smooth muscle cells during stimulation with the contractile agonist histamine or the relaxing agonist isoproterenol. The overall intensity of the cell contraction (the median traction magnitude, the energy transferred from the cell to the gel, and the net contractile moment) increased after activation with histamine, and decreased after treatment with isoproterenol. Cells exhibited regional differences in the time course of traction during the treatment. Both temporal evolution and magnitude of traction increase induced by histamine varied markedly among different cell protrusions, whereas the nuclear region showed the smallest response. These results suggest that intracellular mediators of cell adhesion and contraction respond to contractile stimuli with different rates and intensities in different regions of the cell.

  14. Inducible nitric oxide synthase evoked nitric oxide counteracts capsaicin-induced airway smooth muscle contraction, but exacerbates plasma extravasation.

    PubMed

    Li, Ping-Chia; Shaw, Chen-Fu; Kuo, Tin-Fan; Chien, Chiang-Ting

    2005-04-18

    The contribution of nitric oxide (NO) to capsaicin-evoked airway responses was investigated in rats. The measurement of plasma NO level, airway dynamics, airway smooth muscle electromyogram, and plasma extravasation by India ink and Evans blue leakage technique was adapted. Capsaicin-evoked hypotension, bronchoconstriction, trachea plasma extravasation as well as increases in plasma NO level in a dose-dependent manner. L-732138 (NK1 receptor antagonist) or SR-48968 (NK2 receptor antagonist) pretreatment reduced capsaicin-enhanced hypotension, bronchoconstriction, plasma extravasation, and plasma NO level. N(G)-nitro-L-Arginine methyl ester (L-NAME, 10 mg/kg, i.v.), a non-selective NO synthase (NOS) inhibitor, or aminoguanidine (10 mg/kg, i.v.), a selective inducible NOS (iNOS) inhibitor, reduced capsaicin-induced increases in plasma NO level and protected against capsaicin-induced plasma extravasation, whereas L-arginine (150 mg/kg, i.v.), a NO precursor, enhanced capsaicin-evoked plasma NO level and plasma extravasation. L-Arginine pretreatment ameliorated capsaicin-induced bronchoconstriction, whereas L-NAME and aminoguanidine exaggerated capsaicin-induced bronchoconstriction. In summary, NK1 and NK2 receptors and iNOS play a role in NO formation and on capsaicin-induced bronchoconstriction and plasma extravasation. NO generated by iNOS counteracts tachykinin-mediated bronchoconstriction, but exacerbates tachykinin-mediated plasma extravasation.

  15. Glucocorticoid- and Protein Kinase A–Dependent Transcriptome Regulation in Airway Smooth Muscle

    PubMed Central

    Misior, Anna M.; Deshpande, Deepak A.; Loza, Matthew J.; Pascual, Rodolfo M.; Hipp, Jason D.; Penn, Raymond B.

    2009-01-01

    Glucocorticoids (GCs) and protein kinase A (PKA)–activating agents (β-adrenergic receptor agonists) are mainstream asthma therapies based on their ability to prevent or reverse excessive airway smooth muscle (ASM) constriction. Their abilities to regulate another important feature of asthma—excessive ASM growth—are poorly understood. Recent studies have suggested that GCs render agents of inflammation such as IL-1β and TNF-α mitogenic to ASM, via suppression of (antimitogenic) induced cyclooxygenase-2–dependent PKA activity. To further explore the mechanistic basis of these observations, we assessed the effects of epidermal growth factor and IL-1β stimulation, and the modulatory effects of GC treatment and PKA inhibition, on the ASM transcriptome by microarray analysis. Results demonstrate that ASM stimulated with IL-1β, in a manner that is often cooperative with stimulation with epidermal growth factor, exhibit a profound capacity to function as immunomodulatory cells. Moreover, results implicate an important role for induced autocrine/paracrine factors (many whose regulation was minimally affected by GCs or PKA inhibition) as regulators of both airway inflammation and ASM growth. Induction of numerous chemokines, in conjunction with regulation of proteases and agents of extracellular matrix remodeling, is suggested as an important mechanism promoting upregulated G protein–coupled receptor signaling capable of stimulating ASM growth. Additional functional assays suggest that intracellular PKA plays a critical role in suppressing the promitogenic effects of induced autocrine factors in ASM. Finally, identification and comparison of GC- and PKA-sensitive genes in ASM provide insight into the complementary effects of β-agonist/GC combination therapies, and suggest specific genes as important targets for guiding the development of new generations of GCs and adjunct asthma therapies. PMID:19059887

  16. Airways and vascular smooth muscles relaxant activities of Gaultheria trichophylla.

    PubMed

    Alam, Fiaz; Saqib, Qazi Najumus; Shah, Abdul Jabbar

    2017-01-01

    The aim of this experimental work was to explore the potential pharmacological activities of Gaultheria trichophylla Royle in hyperactive respiratory and vascular conditions. Gaultheria trichophylla was extracted with solvents, phytochemical detection tests were performed, and rabbit trachea and aorta strips were used to evaluate its effects on airways and vascular smooth muscles. Qualitative phytochemical tests showed the presence of flavonoids, alkaloids, anthraquinones, saponins, terpenoids, and condensed tannins. The methanol extract caused inhibition (EC 50 values of 3.12 mg/mL) of carbachol (1 μM) and partial relaxation of K + (80 mM) caused contractions in tracheal strips. The chloroform extract was comparatively more potent against carbachol than K+ induced contraction with EC 50 values of 0.64 and 2.26 mg/mL, respectively. However, the n-hexane extract showed more potency against K + than cabachol induced contractions, as in case with verapamil, with EC 50 values of 0.61 and 6.58 mg/mL, respectively. In isolated prepared trachea, the extracts displaced the carbachol concentration response curves and maximum response was suppressed. In rabbit aorta preparations, methanol and n-hexane extracts partially relaxed phenylephrine (1 μM) and K + induced vasoconstrictions. However, the chloroform extract inhibited phenylephrine induced contractions and exhibited a vasoconstrictor effect at lower concentrations and a relaxant effect at higher concentrations against K + precontractions. The data indicates that, in addition to others, the extracts of G .trichophylla possess verapamil like Ca ++ channel blocking components which explain the possible role of this plant in respiratory and vascular conditions.

  17. Inflammation alters regional mitochondrial Ca²+ in human airway smooth muscle cells.

    PubMed

    Delmotte, Philippe; Yang, Binxia; Thompson, Michael A; Pabelick, Christina M; Prakash, Y S; Sieck, Gary C

    2012-08-01

    Regulation of cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) in airway smooth muscle (ASM) is a key aspect of airway contractility and can be modulated by inflammation. Mitochondria have tremendous potential for buffering [Ca(2+)](cyt), helping prevent Ca(2+) overload, and modulating other intracellular events. Here, compartmentalization of mitochondria to different cellular regions may subserve different roles. In the present study, we examined the role of Ca(2+) buffering by mitochondria and mitochondrial Ca(2+) transport mechanisms in the regulation of [Ca(2+)](cyt) in enzymatically dissociated human ASM cells upon exposure to the proinflammatory cytokines TNF-α and IL-13. Cells were loaded simultaneously with fluo-3 AM and rhod-2 AM, and [Ca(2+)](cyt) and mitochondrial Ca(2+) concentration ([Ca(2+)](mito)) were measured, respectively, using real-time two-color fluorescence microscopy in both the perinuclear and distal, perimembranous regions of cells. Histamine induced a rapid increase in both [Ca(2+)](cyt) and [Ca(2+)](mito), with a significant delay in the mitochondrial response. Inhibition of the mitochondrial Na(+)/Ca(2+) exchanger (1 μM CGP-37157) increased [Ca(2+)](mito) responses in perinuclear mitochondria but not distal mitochondria. Inhibition of the mitochondrial uniporter (1 μM Ru360) decreased [Ca(2+)](mito) responses in perinuclear and distal mitochondria. CGP-37157 and Ru360 significantly enhanced histamine-induced [Ca(2+)](cyt). TNF-α and IL-13 both increased [Ca(2+)](cyt), which was associated with decreased [Ca(2+)](mito) in the case of TNF-α but not IL-13. The effects of TNF-α on both [Ca(2+)](cyt) and [Ca(2+)](mito) were affected by CGP-37157 but not by Ru360. Overall, these data demonstrate that in human ASM cells, mitochondria buffer [Ca(2+)](cyt) after agonist stimulation and its enhancement by inflammation. The differential regulation of [Ca(2+)](mito) in different parts of ASM cells may serve to locally regulate Ca(2+) fluxes from

  18. Nerve growth factor-enhanced airway responsiveness involves substance P in ferret intrinsic airway neurons.

    PubMed

    Wu, Z-X; Dey, R D

    2006-07-01

    Nerve growth factor (NGF), a member of the neurotrophin family, enhances synthesis of neuropeptides in sensory and sympathetic neurons. The aim of this study was to examine the effect of NGF on airway responsiveness and determine whether these effects are mediated through synthesis and release of substance P (SP) from the intrinsic airway neurons. Ferrets were instilled intratracheally with NGF or saline. Tracheal smooth muscle contractility to methacholine and electrical field stimulation (EFS) was assessed in vitro. Contractions of isolated tracheal smooth muscle to EFS at 10 and 30 Hz were significantly increased in the NGF treatment group (10 Hz: 33.57 +/- 2.44%; 30 Hz: 40.12 +/- 2.78%) compared with the control group (10 Hz: 27.24 +/- 2.14%; 30 Hz: 33.33 +/- 2.31%). However, constrictive response to cholinergic agonist was not significantly altered between the NGF treatment group and the control group. The NGF-induced modulation of airway smooth muscle to EFS was maintained in tracheal segments cultured for 24 h, a procedure that causes a significant anatomic and functional loss of SP-containing sensory fibers while maintaining viability of intrinsic airway neurons. The number of SP-containing neurons in longitudinal trunk and superficial muscular plexus and SP nerve fiber density in tracheal smooth muscle all increased significantly in cultured trachea treated with NGF. Pretreatment with CP-99994, an antagonist of neurokinin 1 receptor, attenuated the NGF-induced increased contraction to EFS in cultured segments but had no effect in saline controls. These results show that the NGF-enhanced airway smooth muscle contractile responses to EFS are mediated by the actions of SP released from intrinsic airway neurons.

  19. Emergence of airway smooth muscle mechanical behavior through dynamic reorganization of contractile units and force transmission pathways

    PubMed Central

    2014-01-01

    Airway hyperresponsiveness (AHR) in asthma remains poorly understood despite significant research effort to elucidate relevant underlying mechanisms. In particular, a significant body of experimental work has focused on the effect of tidal fluctuations on airway smooth muscle (ASM) cells, tissues, lung slices, and whole airways to understand the bronchodilating effect of tidal breathing and deep inspirations. These studies have motivated conceptual models that involve dynamic reorganization of both cytoskeletal components as well as contractile machinery. In this article, a biophysical model of the whole ASM cell is presented that combines 1) crossbridge cycling between actin and myosin; 2) actin-myosin disconnectivity, under imposed length changes, to allow dynamic reconfiguration of “force transmission pathways”; and 3) dynamic parallel-to-serial transitions of contractile units within these pathways that occur through a length fluctuation. Results of this theoretical model suggest that behavior characteristic of experimentally observed force-length loops of maximally activated ASM strips can be explained by interactions among the three mechanisms. Crucially, both sustained disconnectivity and parallel-to-serial transitions are necessary to explain the nature of hysteresis and strain stiffening observed experimentally. The results provide strong evidence that dynamic rearrangement of contractile machinery is a likely mechanism underlying many of the phenomena observed at timescales associated with tidal breathing. This theoretical cell-level model captures many of the salient features of mechanical behavior observed experimentally and should provide a useful starting block for a bottom-up approach to understanding tissue-level mechanical behavior. PMID:24481961

  20. Extracellular acidification induces connective tissue growth factor production through proton-sensing receptor OGR1 in human airway smooth muscle cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Matsuzaki, Shinichi; Ishizuka, Tamotsu, E-mail: tamotsui@showa.gunma-u.ac.jp; Yamada, Hidenori

    Highlights: {yields} The involvement of extracellular acidification in airway remodeling was investigated. {yields} Extracellular acidification alone induced CTGF production in human ASMCs. {yields} Extracellular acidification enhanced TGF-{beta}-induced CTGF production in human ASMCs. {yields} Proton-sensing receptor OGR1 was involved in acidic pH-stimulated CTGF production. {yields} OGR1 may play an important role in airway remodeling in asthma. -- Abstract: Asthma is characterized by airway inflammation, hyper-responsiveness and remodeling. Extracellular acidification is known to be associated with severe asthma; however, the role of extracellular acidification in airway remodeling remains elusive. In the present study, the effects of acidification on the expression of connectivemore » tissue growth factor (CTGF), a critical factor involved in the formation of extracellular matrix proteins and hence airway remodeling, were examined in human airway smooth muscle cells (ASMCs). Acidic pH alone induced a substantial production of CTGF, and enhanced transforming growth factor (TGF)-{beta}-induced CTGF mRNA and protein expression. The extracellular acidic pH-induced effects were inhibited by knockdown of a proton-sensing ovarian cancer G-protein-coupled receptor (OGR1) with its specific small interfering RNA and by addition of the G{sub q/11} protein-specific inhibitor, YM-254890, or the inositol-1,4,5-trisphosphate (IP{sub 3}) receptor antagonist, 2-APB. In conclusion, extracellular acidification induces CTGF production through the OGR1/G{sub q/11} protein and inositol-1,4,5-trisphosphate-induced Ca{sup 2+} mobilization in human ASMCs.« less

  1. Vitamin D deficiency causes airway hyperresponsiveness, increases airway smooth muscle mass, and reduces TGF‐β expression in the lungs of female BALB/c mice

    PubMed Central

    Foong, Rachel E.; Shaw, Nicole C.; Berry, Luke J.; Hart, Prue H.; Gorman, Shelley; Zosky, Graeme R.

    2014-01-01

    Abstract Vitamin D deficiency is associated with disease severity in asthma. We tested whether there is a causal association between vitamin D deficiency, airway smooth muscle (ASM) mass, and the development of airway hyperresponsiveness (AHR). A physiologically relevant mouse model of vitamin D deficiency was developed by raising BALB/c mice on vitamin D‐deficient or ‐replete diets. AHR was assessed by measuring lung function responses to increasing doses of inhaled methacholine. Five‐micron sections from formalin‐fixed lungs were used for ASM measurement and assessment of lung structure using stereological methods. Transforming growth factor (TGF)‐β levels were measured in bronchoalveolar lavage fluid (BALF). Lungs were dissected from embryonic day (E) 17.5 vitamin D‐deficient and ‐replete fetal mice for quantification of ASM density and relative gene expression of TGF‐β signaling pathway molecules. Eight‐week‐old adult vitamin D‐deficient female mice had significantly increased airway resistance and ASM in the large airways compared with controls. Vitamin D‐deficient female mice had a smaller lung volume, volume of parenchyma, and alveolar septa. Both vitamin D‐deficient male and female mice had reduced TGF‐β levels in BALF. Vitamin D deficiency did not have an effect on ASM density in E17.5 mice, however, expression of TGF‐β1 and TGF‐β receptor I was downregulated in vitamin D‐deficient female fetal mice. Decreased expression of TGF‐β1 and TGF‐β receptor I during early lung development in vitamin D‐deficient mice may contribute to airway remodeling and AHR in vitamin D‐deficient adult female mice. This study provides a link between vitamin D deficiency and respiratory symptoms in chronic lung disease. PMID:24760528

  2. Quercetin acutely relaxes airway smooth muscle and potentiates β-agonist-induced relaxation via dual phosphodiesterase inhibition of PLCβ and PDE4

    PubMed Central

    Emala, Charles W.

    2013-01-01

    Asthma is a disease of the airways with symptoms including exaggerated airway narrowing and airway inflammation. Early asthma therapies used methylxanthines to relieve symptoms, in part, by inhibiting cyclic nucleotide phosphodiesterases (PDEs), the enzyme responsible for degrading cAMP. The classification of tissue-specific PDE subtypes and the clinical introduction of PDE-selective inhibitors for chronic obstructive pulmonary disease (i.e., roflumilast) have reopened the possibility of using PDE inhibition in the treatment of asthma. Quercetin is a naturally derived PDE4-selective inhibitor found in fruits, vegetables, and tea. We hypothesized that quercetin relaxes airway smooth muscle via cAMP-mediated pathways and augments β-agonist relaxation. Tracheal rings from male A/J mice were mounted in myographs and contracted with acetylcholine (ACh). Addition of quercetin (100 nM-1 mM) acutely and concentration-dependently relaxed airway rings precontracted with ACh. In separate studies, pretreatment with quercetin (100 μM) prevented force generation upon exposure to ACh. In additional studies, quercetin (50 μM) significantly potentiated isoproterenol-induced relaxations. In in vitro assays, quercetin directly attenuated phospholipase C activity, decreased inositol phosphate synthesis, and decreased intracellular calcium responses to Gq-coupled agonists (histamine or bradykinin). Finally, nebulization of quercetin (100 μM) in an in vivo model of airway responsiveness significantly attenuated methacholine-induced increases in airway resistance. These novel data show that the natural PDE4-selective inhibitor quercetin may provide therapeutic relief of asthma symptoms and decrease reliance on short-acting β-agonists. PMID:23873842

  3. Rho-kinase inhibitors augment the inhibitory effect of propofol on rat bronchial smooth muscle contraction.

    PubMed

    Hanazaki, Motohiko; Yokoyama, Masataka; Morita, Kiyoshi; Kohjitani, Atsushi; Sakai, Hiroyasu; Chiba, Yoshihiko; Misawa, Miwa

    2008-06-01

    Airway smooth muscle contraction is not caused by the increase in intracellular Ca(2+) ([Ca(2+)](i)) alone because agonist stimulation increases tension at the same [Ca(2+)](i) (increase in Ca(2+) sensitivity). The small G protein Rho A and Rho-kinase (ROCK) play important roles in the regulation of Ca(2+) sensitivity. In this study, we investigated the effects of three ROCK inhibitors (fasudil, Y-27632, and H-1152) on rat airway smooth muscle contraction and the effects of ROCK inhibitors on propofol-induced bronchodilatory effects. Ring strips from intrapulmonary bronchus of male Wistar rats were placed in 400-microL organ baths containing Krebs-Henseleit solution. After obtaining stable contraction with 30 microM acetylcholine, (1) propofol (1 microM-1 mM) was cumulatively applied; (2) cumulative doses of Y-27632 (0.01-300 microM), fasudil (0.01-100 microM), or H-1152 (0.01-100 microM) were applied; (3) propofol (1 microM-1 mM), with Y-27632, fasudil or H-1152 (0.03 microM or 0.1 microM), was cumulatively applied. (1) Propofol produced concentration-dependent relaxation of rat bronchial smooth muscle. (2) All ROCK inhibitors produced concentration-dependent relaxation. (3) 0.03 microM Y-27632 and fasudil had no significant effect on the concentration-response curve for propofol, while 0.1 microM of both agents significantly shifted concentration-response curves to the left and decreased EC(50). H-1152 (both 0.03 microM and 0.1 microM) significantly sifted the concentration-response curve for propofol to the left and decreased EC(50). ROCK inhibitors, especially H-1152, can attenuate the contraction of rat airway smooth muscle. The combined use of ROCK inhibitors and propofol causes greater relaxation.

  4. Caveolin-1: Functional Insights into Its Role in Muscarine- and Serotonin-Induced Smooth Muscle Constriction in Murine Airways

    PubMed Central

    Keshavarz, Maryam; Schwarz, Heike; Hartmann, Petra; Wiegand, Silke; Skill, Melanie; Althaus, Mike; Kummer, Wolfgang; Krasteva-Christ, Gabriela

    2017-01-01

    An increased bronchoconstrictor response is a hallmark in the progression of obstructive airway diseases. Acetylcholine and 5-hydroxytryptamine (5-HT, serotonin) are the major bronchoconstrictors. There is evidence that both cholinergic and serotonergic signaling in airway smooth muscle (ASM) involve caveolae. We hypothesized that caveolin-1 (cav-1), a structural protein of caveolae, plays an important regulatory role in ASM contraction. We analyzed airway contraction in different tracheal segments and extra- and intrapulmonary bronchi in cav-1 deficient (cav-1−/−) and wild-type mice using organ bath recordings and videomorphometry of methyl-beta-cyclodextrin (MCD) treated and non-treated precision-cut lung slices (PCLS). The presence of caveolae was investigated by electron microscopy. Receptor subtypes driving 5-HT-responses were studied by RT-PCR and videomorphometry after pharmacological inhibition with ketanserin. Cav-1 was present in tracheal epithelium and ASM. Muscarine induced a dose dependent contraction in all airway segments. A significantly higher Emax was observed in the caudal trachea. Although, caveolae abundancy was largely reduced in cav-1−/− mice, muscarine-induced airway contraction was maintained, albeit at diminished potency in the middle trachea, in the caudal trachea and in the bronchus without changes in the maximum efficacy. MCD-treatment of PLCS from cav-1−/− mice reduced cholinergic constriction by about 50%, indicating that cholesterol-rich plasma domains account for a substantial portion of the muscarine-induced bronchoconstriction. Notably, cav-1-deficiency fully abrogated 5-HT-induced contraction of extrapulmonary airways. In contrast, 5-HT-induced bronchoconstriction was fully maintained in cav-1-deficient intrapulmonary bronchi, but desensitization upon repetitive stimulation was enhanced. RT-PCR analysis revealed 5-HT1B, 5-HT2A, 5-HT6, and 5-HT7 receptors as the most prevalent subtypes in the airways. The 5-HT

  5. Allergen and ozone exacerbate serotonin-induced increases in airway smooth muscle contraction in a model of childhood asthma.

    PubMed

    Moore, Brian D; Hyde, Dallas; Miller, Lisa; Wong, Emily; Frelinger, Jessica; Schelegle, Edward S

    2012-01-01

    Serotonin (5-HT) modulates cholinergic neurotransmission and exacerbates airway smooth muscle (ASM) contraction in normal animal and nonasthmatic human tissue. Exposure to house dust mite allergen (HDMA) and ozone (O(3)) leads to airway hyperreactivity and 5-HT-positive cells in the airway epithelium of infant rhesus monkeys. Research shows that concomitant exposure in allergic animals has an additive effect on airway hyperreactivity. In this study, the hypothesis is that the exposure of allergic infant rhesus monkeys to HDMA, O(3) and in combination, acting through 5-HT receptors, enhances 5-HT modulation of postganglionic cholinergic ASM contraction. Twenty-four HDMA-sensitized infant monkeys were split into 4 groups at the age of 1 month, and were exposed to filtered air (FA), HDMA, O(3) or in combination (HDMA+O(3)). At the age of 6 months, airway rings were harvested and postganglionic, and parasympathetic-mediated ASM contraction was evaluated using electrical-field stimulation (EFS). 5-HT exacerbated the EFS response within all exposure groups, but had no effect in the FA group. 5-HT(2), 5-HT(3) and 5-HT(4) receptor agonists exacerbated the response. 5-HT concentration-response curves performed after incubation with specific receptor antagonists confirmed the involvement of 5-HT(2), 5-HT(3) and 5-HT(4) receptors. Conversely, a 5-HT(1) receptor agonist attenuated the tension across all groups during EFS, and in ASM contracted via exogenous acetylcholine. HDMA, O(3) and HDMA+O(3) exposure in a model of childhood allergic asthma enhances 5-HT exacerbation of EFS-induced ASM contraction through 5-HT(2), 5-HT(3) and 5-HT(4) receptors. A nonneurogenic inhibitory pathway exists, unaffected by exposure, mediated by 5-HT(1) receptors located on ASM. Copyright © 2012 S. Karger AG, Basel.

  6. CXCL1 inhibits airway smooth muscle cell migration through the decoy receptor Duffy antigen receptor for chemokines.

    PubMed

    Al-Alwan, Laila A; Chang, Ying; Rousseau, Simon; Martin, James G; Eidelman, David H; Hamid, Qutayba

    2014-08-01

    Airway smooth muscle cell (ASMC) migration is an important mechanism postulated to play a role in airway remodeling in asthma. CXCL1 chemokine has been linked to tissue growth and metastasis. In this study, we present a detailed examination of the inhibitory effect of CXCL1 on human primary ASMC migration and the role of the decoy receptor, Duffy AgR for chemokines (DARC), in this inhibition. Western blots and pathway inhibitors showed that this phenomenon was mediated by activation of the ERK-1/2 MAPK pathway, but not p38 MAPK or PI3K, suggesting a biased selection in the signaling mechanism. Despite being known as a nonsignaling receptor, small interference RNA knockdown of DARC showed that ERK-1/2 MAPK activation was significantly dependent on DARC functionality, which, in turn, was dependent on the presence of heat shock protein 90 subunit α. Interestingly, DARC- or heat shock protein 90 subunit α-deficient ASMCs responded to CXCL1 stimulation by enhancing p38 MAPK activation and ASMC migration through the CXCR2 receptor. In conclusion, we demonstrated DARC's ability to facilitate CXCL1 inhibition of ASMC migration through modulation of the ERK-1/2 MAPK-signaling pathway. Copyright © 2014 by The American Association of Immunologists, Inc.

  7. The contribution of Ca2+ signaling and Ca2+ sensitivity to the regulation of airway smooth muscle contraction is different in rats and mice.

    PubMed

    Bai, Yan; Sanderson, Michael J

    2009-06-01

    To determine the relative contributions of Ca(2+) signaling and Ca(2+) sensitivity to the contractility of airway smooth muscle cells (SMCs), we compared the contractile responses of mouse and rat airways with the lung slice technique. Airway contraction was measured by monitoring changes in airway lumen area with phase-contrast microscopy, whereas changes in intracellular calcium concentration ([Ca(2+)](i)) of the SMCs were recorded with laser scanning microscopy. In mice and rats, methacholine (MCh) or serotonin induced concentration-dependent airway contraction and Ca(2+) oscillations in the SMCs. However, rat airways demonstrated greater contraction compared with mice, in response to agonist-induced Ca(2+) oscillations of a similar frequency. Because this indicates that rat airway SMCs have a higher Ca(2+) sensitivity compared with mice, we examined Ca(2+) sensitivity with Ca(2+)-permeabilized airway SMCs in which the [Ca(2+)](i) was experimentally controlled. In the absence of agonists, high [Ca(2+)](i) induced a sustained contraction in rat airways but only a transient contraction in mouse airways. This sustained contraction of rat airways was relaxed by Y-23672, a Rho kinase inhibitor, but not affected by GF-109203X, a PKC inhibitor. The subsequent exposure of Ca(2+)-permeabilized airway SMCs, with high [Ca(2+)](i), to MCh elicited a further contraction of rat airways and initiated a sustained contraction of mouse airways, without changing the [Ca(2+)](i) of the SMCs. Collectively, these results indicate that airway SMCs of rats have a substantially higher innate Ca(2+) sensitivity than mice and that this strongly influences the transduction of the frequency of Ca(2+) oscillations into the contractility of airway SMCs.

  8. Airway basement membrane perimeter in human airways is not a constant; potential implications for airway remodeling in asthma.

    PubMed

    McParland, Brent E; Paré, Peter D; Johnson, Peter R A; Armour, Carol L; Black, Judith L

    2004-08-01

    Many studies that demonstrate an increase in airway smooth muscle in asthmatic patients rely on the assumption that bronchial internal perimeter (P(i)) or basement membrane perimeter (P(bm)) is a constant, i.e., not affected by fixation pressure or the degree of smooth muscle shortening. Because it is the basement membrane that has been purported to be the indistensible structure, this study examines the assumption that P(bm) is not affected by fixation pressure. P(bm) was determined for the same human airway segment (n = 12) fixed at distending pressures of 0 cmH(2)O and 21 cmH(2)O in the absence of smooth muscle tone. P(bm) for the segment fixed at 0 cmH(2)O was determined morphometrically, and the P(bm) for the same segment, had the segment been fixed at 21 cmH(2)O, was predicted from knowing the luminal volume and length of the airway when distended to 21 cmH(2)O (organ bath-derived P(i)). To ensure an accurate transformation of the organ bath-derived P(i) value to a morphometry-derived P(bm) value, had the segment been fixed at 21 cmH(2)O, the relationship between organ bath-derived P(i) and morphometry-derived P(bm) was determined for five different bronchial segments distended to 21 cmH(2)O and fixed at 21 cmH(2)O (r(2) = 0.99, P < 0.0001). Mean P(bm) for bronchial segments fixed at 0 cmH(2)O was 9.4 +/- 0.4 mm, whereas mean predicted P(bm), had the segments been fixed at 21 cmH(2)O, was 14.1 +/- 0.5 mm (P < 0.0001). This indicates that P(bm) is not a constant when isolated airway segments without smooth muscle tone are fixed distended to 21 cmH(2)O. The implication of these results is that the increase in smooth muscle mass in asthma may have been overestimated in some previous studies. Therefore, further studies are required to examine the potential artifact using whole lungs with and without abolition of airway smooth muscle tone and/or inflation.

  9. Estrogen receptor beta signaling inhibits PDGF induced human airway smooth muscle proliferation.

    PubMed

    Ambhore, Nilesh Sudhakar; Katragadda, Rathnavali; Raju Kalidhindi, Rama Satyanarayana; Thompson, Michael A; Pabelick, Christina M; Prakash, Y S; Sathish, Venkatachalem

    2018-04-20

    Airway smooth muscle (ASM) cell hyperplasia driven by persistent inflammation is a hallmark feature of remodeling in asthma. Sex steroid signaling in the lungs is of considerable interest, given epidemiological data showing more asthma in pre-menopausal women and aging men. Our previous studies demonstrated that estrogen receptor (ER) expression increases in asthmatic human ASM; however, very limited data are available regarding differential roles of ERα vs. ERβ isoforms in human ASM cell proliferation. In this study, we evaluated the effect of selective ERα and ERβ modulators on platelet-derived growth factor (PDGF)-stimulated ASM proliferation and the mechanisms involved. Asthmatic and non-asthmatic primary human ASM cells were treated with PDGF, 17β-estradiol, ERα-agonist and/or ERβ-agonist and/or G-protein-coupled estrogen receptor 30 (GPR30/GPER) agonist and proliferation was measured using MTT and CyQuant assays followed by cell cycle analysis. Transfection of small interfering RNA (siRNA) ERα and ERβ significantly altered the human ASM proliferation. The specificity of siRNA transfection was confirmed by Western blot analysis. Gene and protein expression of cell cycle-related antigens (PCNA and Ki67) and C/EBP were measured by RT-PCR and Western analysis, along with cell signaling proteins. PDGF significantly increased ASM proliferation in non-asthmatic and asthmatic cells. Treatment with PPT showed no significant effect on PDGF-induced proliferation, whereas WAY interestingly suppressed proliferation via inhibition of ERK1/2, Akt, and p38 signaling. PDGF-induced gene expression of PCNA, Ki67 and C/EBP in human ASM was significantly lower in cells pre-treated with WAY. Furthermore, WAY also inhibited PDGF-activated PCNA, C/EBP, cyclin-D1, and cyclin-E. Overall, we demonstrate ER isoform-specific signaling in the context of ASM proliferation. Activation of ERβ can diminish remodeling in human ASM by inhibiting pro-proliferative signaling pathways

  10. Suppression of Eosinophil Integrins Prevents Remodeling of Airway Smooth Muscle in Asthma

    PubMed Central

    Januskevicius, Andrius; Gosens, Reinoud; Sakalauskas, Raimundas; Vaitkiene, Simona; Janulaityte, Ieva; Halayko, Andrew J.; Hoppenot, Deimante; Malakauskas, Kestutis

    2017-01-01

    Background: Airway smooth muscle (ASM) remodeling is an important component of the structural changes to airways seen in asthma. Eosinophils are the prominent inflammatory cells in asthma, and there is some evidence that they contribute to ASM remodeling via released mediators and direct contact through integrin–ligand interactions. Eosinophils express several types of outer membrane integrin, which are responsible for cell–cell and cell–extracellular matrix interactions. In our previous study we demonstrated that asthmatic eosinophils show increased adhesion to ASM cells and it may be important factor contributing to ASM remodeling in asthma. According to these findings, in the present study we investigated the effects of suppression of eosinophil integrin on eosinophil-induced ASM remodeling in asthma. Materials and Methods: Individual combined cell cultures of immortalized human ASM cells and eosinophils from peripheral blood of 22 asthmatic patients and 17 healthy controls were prepared. Eosinophil adhesion was evaluated using eosinophil peroxidase activity assay. Genes expression levels in ASM cells and eosinophils were measured using quantitative real-time PCR. ASM cell proliferation was measured using alamarBlue® solution. Eosinophil integrins were blocked by incubating with Arg-Gly-Asp-Ser peptide. Results: Eosinophils from the asthma group showed increased outer membrane α4β1 and αMβ2 integrin expression, increased adhesion to ASM cells, and overexpression of TGF-β1 compared with eosinophils from the healthy control group. Blockade of eosinophil RGD-binding integrins by Arg-Gly-Asp-Ser peptide significantly reduced adhesion of eosinophils to ASM cells in both groups. Integrin-blocking decreased the effects of eosinophils on TGF-β1, WNT-5a, and extracellular matrix protein gene expression in ASM cells and ASM cell proliferation in both groups. These effects were more pronounced in the asthma group compared with the control group. Conclusion

  11. Bulgarian propolis induces analgesic and anti-inflammatory effects in mice and inhibits in vitro contraction of airway smooth muscle.

    PubMed

    Paulino, Niraldo; Dantas, Andreia Pires; Bankova, Vassya; Longhi, Daniela Taggliari; Scremin, Amarilis; de Castro, Solange Lisboa; Calixto, João Batista

    2003-11-01

    Propolis is a bee product, which has long been used in folk medicine for the management of different diseases. In this study we evaluated the analgesic and anti-inflammatory effects of a standard ethanolic extract of Bulgarian propolis (Et-Blg) in mice and its in vitro effect on airway smooth muscle. Et-Blg inhibited acetic acid-induced abdominal contortions with an ID(50) = 7.4 +/- 0.7 mg. kg(-1). In the formalin test, the extract caused a significant reduction in pain in mice treated with 100 mg. kg(-1) Et-Blg during the neurogenic phase and for the inflammatory phase with all doses of the extract, with an ID(50) = 2.5 +/- 0.4 mg. kg(-1). Et-Blg inhibited also the capsaicin-induced ear edema in mice; however, this extract was ineffective when assessed in the tail-flick and hot-plate thermal assays. The analgesic effect of Et-Blg was associated with the inhibition of inflammatory responses and not to a simple irritation of nervous terminals. In vitro, this extract inhibited the contraction of trachea smooth muscle induced by histamine (IC(50) = 50 +/- 5 microg. mL(-1)), capsaicin (IC(50) = 26.8 +/- 3 microg. mL(-1)), 80 mM KCl (IC(50) = 27.8 +/- 3 microg. mL(-1)), and carbachol (IC(50) = 54 +/- 2 microg. mL(-1)).

  12. Exogenous S100A8 protein inhibits PDGF-induced migration of airway smooth muscle cells in a RAGE-dependent manner.

    PubMed

    Xu, Yu-Dong; Wei, Ying; Wang, Yu; Yin, Lei-Miao; Park, Gyoung-Hee; Liu, Yan-Yan; Yang, Yong-Qing

    2016-03-25

    S100A8 is an important member of the S100 protein family, which is involved in intracellular and extracellular regulatory activities. We previously reported that the S100A8 protein was differentially expressed in the asthmatic respiratory tracts. To understand the potential role of S100A8 in asthma, we investigated the effect of recombinant S100A8 protein on the platelet-derived growth factor (PDGF)-induced migration of airway smooth muscle cells (ASMCs) and the underlying molecular mechanism by using multiple methods, such as impedance-based xCELLigence migration assay, transwell migration assays and wound-healing assays. We found that exogenous S100A8 protein significantly inhibited PDGF-induced ASMC migration. Furthermore, the migration inhibition effect of S100A8 was blocked by neutralizing antibody against the receptor for advanced glycation end-products (RAGE), a potential receptor for the S100A8 protein. These findings provide direct evidence that exogenous S100A8 protein inhibits the PDGF-induced migration of ASMCs through the membrane receptor RAGE. Our study highlights a novel role of S100A8 as a potential means of counteracting airway remodeling in chronic airway diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Microfluidic lung airway-on-a-chip with arrayable suspended gels for studying epithelial and smooth muscle cell interactions.

    PubMed

    Humayun, Mouhita; Chow, Chung-Wai; Young, Edmond W K

    2018-05-01

    Chronic lung diseases (CLDs) are regulated by complex interactions between many different cell types residing in lung airway tissues. Specifically, interactions between airway epithelial cells (ECs) and airway smooth muscle cells (SMCs) have been shown in part to play major roles in the pathogenesis of CLDs, but the underlying molecular mechanisms are not well understood. To advance our understanding of lung pathophysiology and accelerate drug development processes, new innovative in vitro tissue models are needed that can reconstitute the complex in vivo microenvironment of human lung tissues. Organ-on-a-chip technologies have recently made significant strides in recapitulating physiological properties of in vivo lung tissue microenvironments. However, novel advancements are still needed to enable the study of airway SMC-EC communication with matrix interactions, and to provide higher throughput capabilities and manufacturability. We have developed a thermoplastic-based microfluidic lung airway-on-a-chip model that mimics the lung airway tissue microenvironment, and in particular, the interactions between SMCs, ECs, and supporting extracellular matrix (ECM). The microdevice is fabricated from acrylic using micromilling and solvent bonding techniques, and consists of three vertically stacked microfluidic compartments with a bottom media reservoir for SMC culture, a middle thin hydrogel layer, and an upper microchamber for achieving air-liquid interface (ALI) culture of the epithelium. A unique aspect of the design lies in the suspended hydrogel with upper and lower interfaces for EC and SMC culture, respectively. A mixture of type I collagen and Matrigel was found to promote EC adhesion and monolayer formation, and SMC adhesion and alignment. Optimal culturing protocols were established that enabled EC-SMC coculture for more than 31 days. Epithelial monolayers displayed common morphological markers including ZO-1 tight junctions and F-actin cell cortices, while

  14. Leiomodin and tropomodulin in smooth muscle

    NASA Technical Reports Server (NTRS)

    Conley, C. A.

    2001-01-01

    Evidence is accumulating to suggest that actin filament remodeling is critical for smooth muscle contraction, which implicates actin filament ends as important sites for regulation of contraction. Tropomodulin (Tmod) and smooth muscle leiomodin (SM-Lmod) have been found in many tissues containing smooth muscle by protein immunoblot and immunofluorescence microscopy. Both proteins cofractionate with tropomyosin in the Triton-insoluble cytoskeleton of rabbit stomach smooth muscle and are solubilized by high salt. SM-Lmod binds muscle tropomyosin, a biochemical activity characteristic of Tmod proteins. SM-Lmod staining is present along the length of actin filaments in rat intestinal smooth muscle, while Tmod stains in a punctate pattern distinct from that of actin filaments or the dense body marker alpha-actinin. After smooth muscle is hypercontracted by treatment with 10 mM Ca(2+), both SM-Lmod and Tmod are found near alpha-actinin at the periphery of actin-rich contraction bands. These data suggest that SM-Lmod is a novel component of the smooth muscle actin cytoskeleton and, furthermore, that the pointed ends of actin filaments in smooth muscle may be capped by Tmod in localized clusters.

  15. Interleukin-4 upregulates RhoA protein via an activation of STAT6 in cultured human bronchial smooth muscle cells.

    PubMed

    Chiba, Yoshihiko; Todoroki, Michiko; Misawa, Miwa

    2010-02-01

    Interleukin-4 (IL-4) is believed to play a role in allergic bronchial asthma, and has been suggested to cause hyperresponsiveness of airway smooth muscle. In the present study, the effects of IL-4 on the expression of RhoA protein, a monomeric GTP-binding protein that contributes to the contraction of smooth muscle, were determined in cultured human bronchial smooth muscle cells (hBSMCs). Incubation of hBSMCs with IL-4 (100ng/mL) caused a distinct phosphorylation of signal transducer and activator of transcription 6 (STAT6), a major signal transducer activated by IL-4, indicating that IL-4 is capable of activating signal transduction in the hBSMCs directly. IL-4 also caused a significant increase in the expression level of RhoA protein: the peak of the upregulation of RhoA protein was observed at 12-24h after the IL-4 treatment. Both the phosphorylation of STAT6 and the upregulation of RhoA protein induced by IL-4 were inhibited by the co-incubation with AS1517499, a selective inhibitor of STAT6, in a concentration-dependent fashion. These findings suggest that IL-4 is capable of inducing an upregulation of RhoA via an activation of STAT6 in cultured hBSMCs. The RhoA upregulation induced by IL-4, one of the Th2 cytokines upregulated in the airways of allergic bronchial asthmatics, might result in an augmentation of bronchial smooth muscle contractility, that is one of the causes of airway hyperresponsiveness. Copyright 2009 Elsevier Ltd. All rights reserved.

  16. Phenotype and Functional Features of Human Telomerase Reverse Transcriptase Immortalized Human Airway Smooth Muscle Cells from Asthmatic and Non-Asthmatic Donors.

    PubMed

    Burgess, J K; Ketheson, A; Faiz, A; Limbert Rempel, K A; Oliver, B G; Ward, J P T; Halayko, A J

    2018-01-16

    Asthma is an obstructive respiratory disease characterised by chronic inflammation with airway hyperresponsiveness. In asthmatic airways, there is an increase in airway smooth muscle (ASM) cell bulk, which differs from non-asthmatic ASM in characteristics. This study aimed to assess the usefulness of hTERT immortalisation of human ASM cells as a research tool. Specifically we compared proliferative capacity, inflammatory mediator release and extracellular matrix (ECM) production in hTERT immortalised and parent primary ASM cells from asthmatic and non-asthmatic donors. Our studies revealed no significant differences in proliferation, IL-6 and eotaxin-1 production, or CTGF synthesis between donor-matched parent and hTERT immortalised ASM cell lines. However, deposition of ECM proteins fibronectin and fibulin-1 was significantly lower in immortalised ASM cells compared to corresponding primary cells. Notably, previously reported differences in proliferation and inflammatory mediator release between asthmatic and non-asthmatic ASM cells were retained, but excessive ECM protein deposition in asthmatic ASM cells was lost in hTERT ASM cells. This study shows that hTERT immortalised ASM cells mirror primary ASM cells in proliferation and inflammatory profile characteristics. Moreover, we demonstrate both strengths and weaknesses of this immortalised cell model as a representation of primary ASM cells for future asthma pathophysiological research.

  17. Substance P released from intrinsic airway neurons contributes to ozone-enhanced airway hyperresponsiveness in ferret trachea.

    PubMed

    Wu, Zhong-Xin; Satterfield, Brian E; Dey, Richard D

    2003-08-01

    Exposure to ozone (O3) induces airway hyperresponsiveness mediated partly through the release of substance P (SP) from nerve terminals in the airway wall. Although substantial evidence suggests that SP is released by sensory nerves, SP is also present in neurons of airway ganglia. The purpose of this study was to investigate the role of intrinsic airway neurons in O3-enhanced airway responsiveness in ferret trachea. To remove the effects of sensory innervation, segments of ferret trachea were maintained in culture conditions for 24 h before in vitro exposure to 2 parts/million of O3 or air for 1 h. Sensory nerve depletion was confirmed by showing that capsaicin did not affect tracheal smooth muscle responsiveness to cholinergic agonist or contractility responses to electrical field stimulation (EFS). Contractions of isolated tracheal smooth muscle to EFS were significantly increased after in vitro O3 exposure, but the constrictor response to cholinergic agonist was not altered. Pretreatment with CP-99994, an antagonist of the neurokinin 1 receptor, attenuated the increased contraction to EFS after O3 exposure but had no effect in the air exposure group. The number of SP-positive neurons in longitudinal trunk ganglia, the extent of SP innervation to superficial muscular plexus nerve cell bodies, and SP nerve fiber density in tracheal smooth muscle all increased significantly after O3 exposure. The results show that release of SP from intrinsic airway neurons contributes to O3-enhanced tracheal smooth muscle responsiveness by facilitating acetylcholine release from cholinergic nerve terminals.

  18. Transcriptional profiling identifies the long noncoding RNA plasmacytoma variant translocation (PVT1) as a novel regulator of the asthmatic phenotype in human airway smooth muscle.

    PubMed

    Austin, Philip J; Tsitsiou, Eleni; Boardman, Charlotte; Jones, Simon W; Lindsay, Mark A; Adcock, Ian M; Chung, Kian Fan; Perry, Mark M

    2017-03-01

    The mechanism underlying nonsevere and severe asthma remains unclear, although it is commonly associated with increased airway smooth muscle (ASM) mass. Long noncoding RNAs (lncRNAs) are known to be important in regulating healthy primary airway smooth muscle cells (ASMCs), whereas changed expression has been observed in CD8 T cells from patients with severe asthma. Primary ASMCs were isolated from healthy subjects (n = 9) and patients classified as having nonsevere (n = 9) or severe (n = 9) asthma. ASMCs were exposed to dexamethasone and FCS. mRNA and lncRNA expression was measured by using a microarray and quantitative real-time PCR. Bioinformatic analysis was used to examine relevant biological pathways. Finally, the lncRNA plasmacytoma variant translocation 1 (PVT1) was inhibited by transfection of primary ASMCs with small interfering RNAs, and the effect on ASMC phenotype was examined. The mRNA expression profile was significantly different between patient groups after exposure to dexamethasone and FCS, and these were associated with biological pathways that might be relevant to the pathogenesis of asthma, including cellular proliferation and pathways associated with glucocorticoid activity. We also observed a significant change in lncRNA expression, yet the expression of only one lncRNA (PVT1) is decreased in patients with corticosteroid-sensitive nonsevere asthma and increased in patients with corticosteroid-insensitive severe asthma. Subsequent targeting studies demonstrated the importance of this lncRNA in controlling both proliferation and IL-6 release in ASMCs from patients with severe asthma. lncRNAs are associated with the aberrant phenotype observed in ASMCs from asthmatic patients. Targeting PVT1 might be effective in reducing airway remodeling in asthmatic patients. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  19. Smooth muscle length adaptation and actin filament length: a network model of the cytoskeletal dysregulation.

    PubMed

    Silveira, Paulo S P; Fredberg, Jeffrey J

    2005-10-01

    Length adaptation of the airway smooth muscle cell is attributable to cytoskeletal remodeling. It has been proposed that dysregulated actin filaments may become longer in asthma, and that such elongation would prevent a parallel-to-series transition of contractile units, thus precluding the well-known beneficial effects of deep inspirations and tidal breathing. To test the potential effect that actin filament elongation could have in overall muscle mechanics, we present an extremely simple model. The cytoskeleton is represented as a 2-D network of links (contractile filaments) connecting nodes (adhesion plaques). Such a network evolves in discrete time steps by forming and dissolving links in a stochastic fashion. Links are formed by idealized contractile units whose properties are either those from normal or elongated actin filaments. Oscillations were then imposed on the network to evaluate both the effects of breathing and length adaptation. In response to length oscillation, a network with longer actin filaments showed smaller decreases of force, smaller increases in compliance, and higher shortening velocities. Taken together, these changes correspond to a network that is refractory to the effects of breathing and therefore approximates an asthmatic scenario. Thus, an extremely simple model seems to capture some relatively complex mechanics of airway smooth muscle, supporting the idea that dysregulation of actin filament length may contribute to excessive airway narrowing.

  20. Development and characterization of a 3D multicell microtissue culture model of airway smooth muscle.

    PubMed

    West, Adrian R; Zaman, Nishat; Cole, Darren J; Walker, Matthew J; Legant, Wesley R; Boudou, Thomas; Chen, Christopher S; Favreau, John T; Gaudette, Glenn R; Cowley, Elizabeth A; Maksym, Geoffrey N

    2013-01-01

    Airway smooth muscle (ASM) cellular and molecular biology is typically studied with single-cell cultures grown on flat 2D substrates. However, cells in vivo exist as part of complex 3D structures, and it is well established in other cell types that altering substrate geometry exerts potent effects on phenotype and function. These factors may be especially relevant to asthma, a disease characterized by structural remodeling of the airway wall, and highlights a need for more physiologically relevant models of ASM function. We utilized a tissue engineering platform known as microfabricated tissue gauges to develop a 3D culture model of ASM featuring arrays of ∼0.4 mm long, ∼350 cell "microtissues" capable of simultaneous contractile force measurement and cell-level microscopy. ASM-only microtissues generated baseline tension, exhibited strong cellular organization, and developed actin stress fibers, but lost structural integrity and dissociated from the cantilevers within 3 days. Addition of 3T3-fibroblasts dramatically improved survival times without affecting tension development or morphology. ASM-3T3 microtissues contracted similarly to ex vivo ASM, exhibiting reproducible responses to a range of contractile and relaxant agents. Compared with 2D cultures, microtissues demonstrated identical responses to acetylcholine and KCl, but not histamine, forskolin, or cytochalasin D, suggesting that contractility is regulated by substrate geometry. Microtissues represent a novel model for studying ASM, incorporating a physiological 3D structure, realistic mechanical environment, coculture of multiple cells types, and comparable contractile properties to existing models. This new model allows for rapid screening of biochemical and mechanical factors to provide insight into ASM dysfunction in asthma.

  1. Smooth muscle sphincteroplasty in colostomy.

    PubMed

    Kostov, Daniel V; Temelkov, Temelko D; Dragnev, Nedyalko A; Kobakov, Georgi L; Ivanov, Krasimir D

    2004-04-01

    The present work elaborated on Schmidt's idea of an effective smooth muscle sphincteroplasty. The aim of the study was to analyze the effects on the patients with a lower quadrant colostomy constructed after abdominoperineal extirpation of a modified smooth muscle sphincteroplasty combined with colon irrigations. Seventy-two rectal cancer patients (39 men and 33 women, median age, 54.5 years) with smooth muscle sphincteroplasty and 20 controls with conventional colostomy using colon irrigations (11 men and 9 women, median age, 63.2 years) were examined. A modified smooth muscle wrap of the colostomy with a free graft of a 4-cm-long colon segment without mucosa was applied. In this precolostomy segment a high intraluminal pressure was achieved. The functional capacity and anatomic integrity of the transplanted smooth muscle graft were examined manometrically, electromyographically, and histomorphologically. The functional activity of the colostomy was assessed by periodic recording of the number of "spontaneous" and "directed" defecations.RESULTS. In the patients with smooth muscle sphincteroplasty, the basal intraluminal pressure of the precolostomy segment two years after operation measured 29.7 mmHg. After dilatation of the transplant, these pressures reached up to 43 mmHg ( P < 0.001). The weekly "spontaneous" stools were 3 to 5 times less frequent than in the controls ( P < 0.001). The modified smooth muscle sphincteroplasty offers operative-technical opportunities for increasing intraluminal pressure in the precolostomy colon segment. Its combination with colonic irrigations facilitates control of the evacuatory rhythm and "spontaneous" stools in colostomy patients, thus improving their quality of life.

  2. IL-17A Mediates a Selective Gene Expression Profile in Asthmatic Human Airway Smooth Muscle Cells

    PubMed Central

    Dragon, Stéphane; Hirst, Stuart J.; Lee, Tak H.

    2014-01-01

    Airway smooth muscle (ASM) cells are thought to contribute to the pathogenesis of allergic asthma by orchestrating and perpetuating airway inflammation and remodeling responses. In this study, we evaluated the IL-17RA signal transduction and gene expression profile in ASM cells from subjects with mild asthma and healthy individuals. Human primary ASM cells were treated with IL-17A and probed by the Affymetrix GeneChip array, and gene targets were validated by real-time quantitative RT-PCR. Genomic analysis underlined the proinflammatory nature of IL-17A, as multiple NF-κB regulatory factors and chemokines were induced in ASM cells. Transcriptional regulators consisting of primary response genes were overrepresented and displayed dynamic expression profiles. IL-17A poorly enhanced IL-1β or IL-22 gene responses in ASM cells from both subjects with mild asthma and healthy donors. Interestingly, protein modifications to the NF-κB regulatory network were not observed after IL-17A stimulation, although oscillations in IκBε expression were detected. ASM cells from subjects with mild asthma up-regulated more genes with greater overall variability in response to IL-17A than from healthy donors. Finally, in response to IL-17A, ASM cells displayed rapid activation of the extracellular signal–regulated kinase/ribosomal S6 kinase signaling pathway and increased nuclear levels of phosphorylated extracellular signal–regulated kinase. Taken together, our results suggest that IL-17A mediated modest gene expression response, which, in cooperation with the NF-κB signaling network, may regulate the gene expression profile in ASM cells. PMID:24393021

  3. The anti-proliferative and anti-inflammatory response of COPD airway smooth muscle cells to hydrogen sulfide.

    PubMed

    Perry, Mark M; Tildy, Bernadett; Papi, Alberto; Casolari, Paolo; Caramori, Gaetano; Rempel, Karen Limbert; Halayko, Andrew J; Adcock, Ian; Chung, Kian Fan

    2018-05-09

    COPD is a common, highly debilitating disease of the airways, primarily caused by smoking. Chronic inflammation and structural remodelling are key pathological features of this disease caused, in part, by the aberrant function of airway smooth muscle (ASM). We have previously demonstrated that hydrogen sulfide (H 2 S) can inhibit ASM cell proliferation and CXCL8 release, from cells isolated from non-smokers. We examined the effect of H 2 S upon ASM cells from COPD patients. ASM cells were isolated from non-smokers, smokers and patients with COPD (n = 9). Proliferation and cytokine release (IL-6 and CXCL8) of ASM was induced by FCS, and measured by bromodeoxyuridine incorporation and ELISA, respectively. Exposure of ASM to H 2 S donors inhibited FCS-induced proliferation and cytokine release, but was less effective upon COPD ASM cells compared to the non-smokers and smokers. The mRNA and protein expression of the enzymes responsible for endogenous H 2 S production (cystathionine-β-synthase [CBS] and 3-mercaptopyruvate sulphur transferase [MPST]) were inhibited by H 2 S donors. Finally, we report that exogenous H 2 S inhibited FCS-stimulated phosphorylation of ERK-1/2 and p38 mitogen activated protein kinases (MAPKs), in the non-smoker and smoker ASM cells, with little effect in COPD cells. H 2 S production provides a novel mechanism for the repression of ASM proliferation and cytokine release. The ability of COPD ASM cells to respond to H 2 S is attenuated in COPD ASM cells despite the presence of the enzymes responsible for H 2 S production.

  4. Arachidonate-Regulated Ca2+ Influx in Human Airway Smooth Muscle

    PubMed Central

    Thompson, Michael A.; Prakash, Y. S.

    2014-01-01

    Plasma membrane Ca2+ influx, especially store-operated Ca2+ entry triggered by sarcoplasmic reticulum (SR) Ca2+ release, is a key component of intracellular calcium concentration ([Ca2+]i) regulation in airway smooth muscle (ASM). Agonist-induced Ca2+ oscillations in ASM that involve both influx and SR mechanisms have been previously demonstrated. In nonexcitable cells, [Ca2+]i oscillations involve Ca2+ influx via arachidonic acid (AA) –stimulated channels, which show similarities to store-operated Ca2+ entry, although their molecular identity remains undetermined. Little is known about AA-regulated Ca2+ channels or their regulation in ASM. In enzymatically dissociated human ASM cells loaded with the Ca2+ indicator, fura-2, AA (1–10 μM) triggered [Ca2+]i oscillations that were inhibited by removal of extracellular Ca2+. Other fatty acids, such as the diacylglycerol analog, 1-oleoyl-2-acetyl-SN-glycerol, oleic acid, and palmitic acid (10 μM each), failed to elicit similar [Ca2+]i responses. Preincubation with LaCl3 (1 μM or 1 mM) inhibited AA-induced oscillations. Inhibition of receptor-operated channels (SKF96,365 [10 μM]), lipoxygenase (zileuton [10 μM]), or cyclooxygenase (indomethacin [10 μM]) did not affect oscillation parameters. Inhibition of SR Ca2+ release (ryanodine [10 μM] or inositol 1,4,5-trisphosphate receptor inhibitor, xestospongin C [1 μM]) decreased [Ca2+]i oscillation frequency and amplitude. Small interfering RNA against caveolin-1, stromal interaction molecule 1, or Orai3 (20 nM each) reduced the frequency and amplitude of AA-induced [Ca2+]i oscillations. In ASM cells derived from individuals with asthma, AA increased oscillation amplitude, but not frequency. These results are highly suggestive of a novel AA-mediated Ca2+–regulatory mechanism in human ASM, reminiscent of agonist-induced oscillations. Given the role of AA in ASM intracellular signaling, especially with inflammation, AA-regulated Ca2+ channels could potentially

  5. Intracellular interactions of umeclidinium and vilanterol in human airway smooth muscle.

    PubMed

    Shaikh, Nooreen; Johnson, Malcolm; Hall, David A; Chung, Kian Fan; Riley, John H; Worsley, Sally; Bhavsar, Pankaj K

    2017-01-01

    Intracellular mechanisms of action of umeclidinium (UMEC), a long-acting muscarinic receptor antagonist, and vilanterol (VI), a long-acting β 2 -adrenoceptor (β 2 R) agonist, were investigated in target cells: human airway smooth-muscle cells (ASMCs). ASMCs from tracheas of healthy lung-transplant donors were treated with VI, UMEC, UMEC and VI combined, or control compounds (salmeterol, propranolol, ICI 118.551, or methacholine [MCh]). Cyclic adenosine monophosphate (cAMP) was measured using an enzyme-linked immunosorbent assay, intracellular free calcium ([Ca 2+ ] i ) using a fluorescence assay, and regulator of G-protein signaling 2 (RGS2) messenger RNA using real-time quantitative polymerase chain reaction. VI and salmeterol (10 -12 -10 -6 M) induced cAMP production from ASMCs in a concentration-dependent manner, which was greater for VI at all concentrations. β 2 R antagonism by propranolol or ICI 118.551 (10 -12 -10 -4 M) resulted in concentration-dependent inhibition of VI-induced cAMP production, and ICI 118.551 was more potent. MCh (5×10 -6 M, 30 minutes) attenuated VI-induced cAMP production ( P <0.05), whereas pretreatment with UMEC (10 -8 M, 1 hour) restored the magnitude of VI-induced cAMP production. ASMC stimulation with MCh (10 -11 -5×10 -6 M) resulted in a concentration-dependent increase in [Ca 2+ ] i , which was attenuated with UMEC pretreatment. Reduction of MCh-induced [Ca 2+ ] i release was greater with UMEC + VI versus UMEC. UMEC enhanced VI-induced RGS2 messenger RNA expression. These data indicate that UMEC reverses cholinergic inhibition of VI-induced cAMP production, and is a more potent muscarinic receptor antagonist when in combination with VI versus either alone.

  6. Intracellular interactions of umeclidinium and vilanterol in human airway smooth muscle

    PubMed Central

    Shaikh, Nooreen; Johnson, Malcolm; Hall, David A; Chung, Kian Fan; Riley, John H; Worsley, Sally; Bhavsar, Pankaj K

    2017-01-01

    Background Intracellular mechanisms of action of umeclidinium (UMEC), a long-acting muscarinic receptor antagonist, and vilanterol (VI), a long-acting β2-adrenoceptor (β2R) agonist, were investigated in target cells: human airway smooth-muscle cells (ASMCs). Materials and methods ASMCs from tracheas of healthy lung-transplant donors were treated with VI, UMEC, UMEC and VI combined, or control compounds (salmeterol, propranolol, ICI 118.551, or methacholine [MCh]). Cyclic adenosine monophosphate (cAMP) was measured using an enzyme-linked immunosorbent assay, intracellular free calcium ([Ca2+]i) using a fluorescence assay, and regulator of G-protein signaling 2 (RGS2) messenger RNA using real-time quantitative polymerase chain reaction. Results VI and salmeterol (10−12–10−6 M) induced cAMP production from ASMCs in a concentration-dependent manner, which was greater for VI at all concentrations. β2R antagonism by propranolol or ICI 118.551 (10−12–10−4 M) resulted in concentration-dependent inhibition of VI-induced cAMP production, and ICI 118.551 was more potent. MCh (5×10−6 M, 30 minutes) attenuated VI-induced cAMP production (P<0.05), whereas pretreatment with UMEC (10−8 M, 1 hour) restored the magnitude of VI-induced cAMP production. ASMC stimulation with MCh (10−11–5×10−6 M) resulted in a concentration-dependent increase in [Ca2+]i, which was attenuated with UMEC pretreatment. Reduction of MCh-induced [Ca2+]i release was greater with UMEC + VI versus UMEC. UMEC enhanced VI-induced RGS2 messenger RNA expression. Conclusion These data indicate that UMEC reverses cholinergic inhibition of VI-induced cAMP production, and is a more potent muscarinic receptor antagonist when in combination with VI versus either alone. PMID:28721035

  7. Sexual Dimorphism in the Regulation of Estrogen, Progesterone, and Androgen Receptors by Sex Steroids in the Rat Airway Smooth Muscle Cells

    PubMed Central

    Zarazúa, Abraham; González-Arenas, Aliesha; Ramírez-Vélez, Gabriela; Bazán-Perkins, Blanca; Guerra-Araiza, Christian; Campos-Lara, María G.

    2016-01-01

    The role of sex hormones in lung is known. The three main sex steroid receptors, estrogen, progesterone, and androgen, have not been sufficiently studied in airway smooth muscle cells (ASMC), and the sex hormone regulation on these receptors is unknown. We examined the presence and regulation of sex hormone receptors in female and male rat ASMC by Western blotting and flow cytometry. Gonadectomized rats were treated with 17β-estradiol, progesterone, 17β-estradiol + progesterone, or testosterone. ASMC were enzymatically isolated from tracheas and bronchi. The experiments were performed with double staining flow cytometry (anti-α-actin smooth muscle and antibodies to each hormone receptor). ERα, ERβ, tPR, and AR were detected in females or males. ERα was upregulated by E2 and T and downregulated by P4 in females; in males, ERα was downregulated by P4, E + P, and T. ERβ was downregulated by each treatment in females, and only by E + P and T in males. tPR was downregulated by P4, E + P, and T in females. No hormonal regulation was observed in male receptors. AR was downregulated in males treated with E + P and T. We have shown the occurrence of sex hormone receptors in ASMC and their regulation by the sex hormones in female and male rats. PMID:27110242

  8. Non-muscle (NM) myosin heavy chain phosphorylation regulates the formation of NM myosin filaments, adhesome assembly and smooth muscle contraction.

    PubMed

    Zhang, Wenwu; Gunst, Susan J

    2017-07-01

    Non-muscle (NM) and smooth muscle (SM) myosin II are both expressed in smooth muscle tissues, however the role of NM myosin in SM contraction is unknown. Contractile stimulation of tracheal smooth muscle tissues stimulates phosphorylation of the NM myosin heavy chain on Ser1943 and causes NM myosin filament assembly at the SM cell cortex. Expression of a non-phosphorylatable NM myosin mutant, NM myosin S1943A, in SM tissues inhibits ACh-induced NM myosin filament assembly and SM contraction, and also inhibits the assembly of membrane adhesome complexes during contractile stimulation. NM myosin regulatory light chain (RLC) phosphorylation but not SM myosin RLC phosphorylation is regulated by RhoA GTPase during ACh stimulation, and NM RLC phosphorylation is required for NM myosin filament assembly and SM contraction. NM myosin II plays a critical role in airway SM contraction that is independent and distinct from the function of SM myosin. The molecular function of non-muscle (NM) isoforms of myosin II in smooth muscle (SM) tissues and their possible role in contraction are largely unknown. We evaluated the function of NM myosin during contractile stimulation of canine tracheal SM tissues. Stimulation with ACh caused NM myosin filament assembly, as assessed by a Triton solubility assay and a proximity ligation assay aiming to measure interactions between NM myosin monomers. ACh stimulated the phosphorylation of NM myosin heavy chain on Ser1943 in tracheal SM tissues, which can regulate NM myosin IIA filament assembly in vitro. Expression of the non-phosphorylatable mutant NM myosin S1943A in SM tissues inhibited ACh-induced endogenous NM myosin Ser1943 phosphorylation, NM myosin filament formation, the assembly of membrane adhesome complexes and tension development. The NM myosin cross-bridge cycling inhibitor blebbistatin suppressed adhesome complex assembly and SM contraction without inhibiting NM myosin Ser1943 phosphorylation or NM myosin filament assembly. Rho

  9. Substance P relaxes rat bronchial smooth muscle via epithelial prostanoid synthesis.

    PubMed

    Bodelsson, M; Blomquist, S; Caverius, K; Törnebrandt, K

    1999-01-01

    Substance P is present in bronchial nerve fibres. The physiological actions of substance P are mediated via tachykinin NK(1) receptors. Immunochemical studies have demonstrated tachykinin NK(1) receptors in the rat airway epithelium. To elucidate how epithelial tachykinin NK(1) receptors affect smooth muscle response to substance P. Contractile response of isolated rat bronchial trunk with or without epithelium was recorded. In intact segments precontracted by 5-hydroxytryptamine, relaxation was induced by substance P and the nitric oxide donor, sodium nitroprusside. Removal of the epithelium abolished relaxation induced by substance P but did not affect relaxation induced by sodium nitroprusside. The cyclo-oxygenase inhibitor, indomethacin, but not the nitric oxide synthase inhibitor, L-N(G)-monomethylarginine, reduced the relaxation in response to substance P. Epithelial tachykinin NK(1) receptors mediate substance-P-induced relaxation of rat bronchial smooth muscle via release of prostanoids but not nitric oxide.

  10. Nuclear fusion-independent smooth muscle differentiation of human adipose-derived stem cells induced by a smooth muscle environment.

    PubMed

    Zhang, Rong; Jack, Gregory S; Rao, Nagesh; Zuk, Patricia; Ignarro, Louis J; Wu, Benjamin; Rodríguez, Larissa V

    2012-03-01

    Human adipose-derived stem cells hASC have been isolated and were shown to have multilineage differentiation capacity. Although both plasticity and cell fusion have been suggested as mechanisms for cell differentiation in vivo, the effect of the local in vivo environment on the differentiation of adipose-derived stem cells has not been evaluated. We previously reported the in vitro capacity of smooth muscle differentiation of these cells. In this study, we evaluate the effect of an in vivo smooth muscle environment in the differentiation of hASC. We studied this by two experimental designs: (a) in vivo evaluation of smooth muscle differentiation of hASC injected into a smooth muscle environment and (b) in vitro evaluation of smooth muscle differentiation capacity of hASC exposed to bladder smooth muscle cells. Our results indicate a time-dependent differentiation of hASC into mature smooth muscle cells when these cells are injected into the smooth musculature of the urinary bladder. Similar findings were seen when the cells were cocultured in vitro with primary bladder smooth muscle cells. Chromosomal analysis demonstrated that microenvironment cues rather than nuclear fusion are responsible for this differentiation. We conclude that cell plasticity is present in hASCs, and their differentiation is accomplished in the absence of nuclear fusion. Copyright © 2011 AlphaMed Press.

  11. Mast cell migration to Th2 stimulated airway smooth muscle from asthmatics

    PubMed Central

    Sutcliffe, A; Kaur, D; Page, S; Woodman, L; Armour, C L; Baraket, M; Bradding, P; Hughes, J M; Brightling, C E

    2006-01-01

    Background Mast cell microlocalisation within the airway smooth muscle (ASM) bundle is an important determinant of the asthmatic phenotype. We hypothesised that mast cells migrate towards ASM in response to ASM derived chemokines. Methods Primary ASM cultures from subjects with and without asthma were stimulated with interleukin (IL)‐1β, IL‐4, and IL‐13 alone and in combination. Mast cell chemotaxis towards these ASM supernatants was investigated, and the chemotaxins mediating migration by using specific blocking antibodies for stem cell factor (SCF) and the chemokine receptors CCR3, CXCR1, 3 and 4 as well as the Gi inhibitor pertussis toxin and the tyrosine kinase inhibitor genistein were defined. The concentrations of CCL11, CXCL8, CXCL10, TGF‐β, and SCF in the supernatants were measured and the effect of non‐asthmatic ASM supernatants on the mast cell chemotactic activity of asthmatic ASM was examined. Results Human lung mast cells and HMC‐1 cells migrated towards Th2 stimulated ASM from asthmatics but not non‐asthmatics. Mast cell migration was mediated through the combined activation of CCR3 and CXCR1. CCL11 and CXCL8 expression by ASM increased markedly after stimulation, but was similar in those with and without asthma. ASM supernatants from non‐asthmatics inhibited mast cell migration towards the asthmatic ASM supernatant. Conclusion Th2 stimulated ASM from asthmatics is chemotactic for mast cells. Non‐asthmatic ASM releases a mediator or mediators that inhibit mast cell migration towards stimulated asthmatic ASM. Specifically targeting mast cell migration into the ASM bundle may provide a novel treatment for asthma. PMID:16601090

  12. The dopamine D1 receptor is expressed and facilitates relaxation in airway smooth muscle.

    PubMed

    Mizuta, Kentaro; Zhang, Yi; Xu, Dingbang; Mizuta, Fumiko; D'Ovidio, Frank; Masaki, Eiji; Emala, Charles W

    2013-09-02

    Dopamine signaling is mediated by Gs protein-coupled "D1-like" receptors (D1 and D5) and Gi-coupled "D2-like" receptors (D2-4). In asthmatic patients, inhaled dopamine induces bronchodilation. Although the Gi-coupled dopamine D2 receptor is expressed and sensitizes adenylyl cyclase activity in airway smooth muscle (ASM) cells, the Gs-coupled dopamine D1-like receptor subtypes have never been identified on these cells. Activation of Gs-coupled receptors stimulates cyclic AMP (cAMP) production through the stimulation of adenylyl cyclase, which promotes ASM relaxation. We questioned whether the dopamine D1-like receptor is expressed on ASM, and modulates its function through Gs-coupling. The mRNA and protein expression of dopamine D1-like receptor subtypes in both native human and guinea pig ASM tissue and cultured human ASM (HASM) cells was measured. To characterize the stimulation of cAMP through the dopamine D1 receptor, HASM cells were treated with dopamine or the dopamine D1-like receptor agonists (A68930 or SKF38393) before cAMP measurements. To evaluate whether the activation of dopamine D1 receptor induces ASM relaxation, guinea pig tracheal rings suspended under isometric tension in organ baths were treated with cumulatively increasing concentrations of dopamine or A68930, following an acetylcholine-induced contraction with or without the cAMP-dependent protein kinase (PKA) inhibitor Rp-cAMPS, the large-conductance calcium-activated potassium (BKCa) channel blocker iberiotoxin, or the exchange proteins directly activated by cAMP (Epac) antagonist NSC45576. Messenger RNA encoding the dopamine D1 and D5 receptors were detected in native human ASM tissue and cultured HASM cells. Immunoblots confirmed the protein expression of the dopamine D1 receptor in both native human and guinea pig ASM tissue and cultured HASM cells. The dopamine D1 receptor was also immunohistochemically localized to both human and guinea pig ASM. The dopamine D1-like receptor agonists

  13. Interaction between endoplasmic/sarcoplasmic reticulum stress (ER/SR stress), mitochondrial signaling and Ca(2+) regulation in airway smooth muscle (ASM).

    PubMed

    Delmotte, Philippe; Sieck, Gary C

    2015-02-01

    Airway inflammation is a key aspect of diseases such as asthma. Several inflammatory cytokines (e.g., TNFα and IL-13) increase cytosolic Ca(2+) ([Ca(2+)]cyt) responses to agonist stimulation and Ca(2+) sensitivity of force generation, thereby enhancing airway smooth muscle (ASM) contractility (hyper-reactive state). Inflammation also induces ASM proliferation and remodeling (synthetic state). In normal ASM, the transient elevation of [Ca(2+)]cyt induced by agonists leads to a transient increase in mitochondrial Ca(2+) ([Ca(2+)]mito) that may be important in matching ATP production with ATP consumption. In human ASM (hASM) exposed to TNFα and IL-13, the transient increase in [Ca(2+)]mito is blunted despite enhanced [Ca(2+)]cyt responses. We also found that TNFα and IL-13 induce reactive oxidant species (ROS) formation and endoplasmic/sarcoplasmic reticulum (ER/SR) stress (unfolded protein response) in hASM. ER/SR stress in hASM is associated with disruption of mitochondrial coupling with the ER/SR membrane, which relates to reduced mitofusin 2 (Mfn2) expression. Thus, in hASM it appears that TNFα and IL-13 result in ROS formation leading to ER/SR stress, reduced Mfn2 expression, disruption of mitochondrion-ER/SR coupling, decreased mitochondrial Ca(2+) buffering, mitochondrial fragmentation, and increased cell proliferation.

  14. Regeneration and Maintenance of Intestinal Smooth Muscle Phenotypes

    NASA Astrophysics Data System (ADS)

    Walthers, Christopher M.

    Tissue engineering is an emerging field of biomedical engineering that involves growing artificial organs to replace those lost to disease or injury. Within tissue engineering, there is a demand for artificial smooth muscle to repair tissues of the digestive tract, bladder, and vascular systems. Attempts to develop engineered smooth muscle tissues capable of contracting with sufficient strength to be clinically relevant have so far proven unsatisfactory. The goal of this research was to develop and sustain mature, contractile smooth muscle. Survival of implanted SMCs is critical to sustain the benefits of engineered smooth muscle. Survival of implanted smooth muscle cells was studied with layered, electrospun polycaprolactone implants with lasercut holes ranging from 0--25% porosity. It was found that greater angiogenesis was associated with increased survival of implanted cells, with a large increase at a threshold between 20% and 25% porosity. Heparan sulfate coatings improved the speed of blood vessel infiltration after 14 days of implantation. With these considerations, thicker engineered tissues may be possible. An improved smooth muscle tissue culture technique was utilized. Contracting smooth muscle was produced in culture by maintaining the native smooth muscle tissue organization, specifically by sustaining intact smooth muscle strips rather than dissociating tissue in to isolated smooth muscle cells. Isolated cells showed a decrease in maturity and contained fewer enteric neural and glial cells. Muscle strips also exhibited periodic contraction and regular fluctuation of intracellular calclium. The muscle strip maturity persisted after implantation in omentum for 14 days on polycaprolactone scaffolds. A low-cost, disposable bioreactor was developed to further improve maturity of cultured smooth muscle cells in an environment of controlled cyclical stress.The bioreactor consistently applied repeated mechanical strain with controllable inputs for strain

  15. The contribution of inositol 1,4,5-trisphosphate and ryanodine receptors to agonist-induced Ca(2+) signaling of airway smooth muscle cells.

    PubMed

    Bai, Yan; Edelmann, Martin; Sanderson, Michael J

    2009-08-01

    The relative contribution of inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) and ryanodine receptors (RyRs) to agonist-induced Ca(2+) signaling in mouse airway smooth muscle cells (SMCs) was investigated in lung slices with phase-contrast or laser scanning microscopy. At room temperature (RT), methacholine (MCh) or 5-hydroxytryptamine (5-HT) induced Ca(2+) oscillations and an associated contraction in small airway SMCs. The subsequent exposure to an IP(3)R antagonist, 2-aminoethoxydiphenyl borate (2-APB), inhibited the Ca(2+) oscillations and induced airway relaxation in a concentration-dependent manner. 2-APB also inhibited Ca(2+) waves generated by the photolytic release of IP(3). However, the RyR antagonist ryanodine had no significant effect, at any concentration, on airway contraction or agonist- or IP(3)-induced Ca(2+) oscillations or Ca(2+) wave propagation. By contrast, a second RyR antagonist, tetracaine, relaxed agonist-contracted airways and inhibited agonist-induced Ca(2+) oscillations in a concentration-dependent manner. However, tetracaine did not affect IP(3)-induced Ca(2+) release or wave propagation nor the Ca(2+) content of SMC Ca(2+) stores as evaluated by Ca(2+)-release induced by caffeine. Conversely, both ryanodine and tetracaine completely blocked agonist-independent slow Ca(2+) oscillations induced by KCl. The inhibitory effects of 2-APB and absence of an effect of ryanodine on MCh-induced airway contraction or Ca(2+) oscillations of SMCs were also observed at 37 degrees C. In Ca(2+)-permeable SMCs, tetracaine inhibited agonist-induced contraction without affecting intracellular Ca(2+) levels indicating that relaxation also resulted from a reduction in Ca(2+) sensitivity. These results indicate that agonist-induced Ca(2+) oscillations in mouse small airway SMCs are primary mediated via IP(3)Rs and that tetracaine induces relaxation by both decreasing Ca(2+) sensitivity and inhibiting agonist-induced Ca(2+) oscillations via an IP(3

  16. Heparin and structurally related polymers attenuate eotaxin-1 (CCL11) release from human airway smooth muscle.

    PubMed

    Kanabar, V; Page, C P; Simcock, D E; Karner, C; Mahn, K; O'Connor, B J; Hirst, S J

    2008-06-01

    The glycosaminoglycan heparin has anti-inflammatory activity and is exclusively found in mast cells, which are localized within airway smooth muscle (ASM) bundles of asthmatic airways. Interleukin (IL)-13 induces the production of multiple inflammatory mediators from ASM including the eosinophil chemoattractant chemokine, eotaxin-1. Heparin and related glycosaminoglycan polymers having structurally heterogeneous polysaccharide side chains that varied in molecular weight, sulphation and anionic charge were used to identify features of the heparin molecule linked to anti-inflammatory activity. Cultured human ASM cells were stimulated with interleukin (IL)-13 in the absence or presence of heparin and related polymers. Eotaxin-1 was quantified using chemokine antibody arrays and ELISA. Unfractionated heparin attenuated IL-13-dependent eotaxin-1 production and this effect was reproduced with low molecular weight heparins (3 and 6 kDa), demonstrating a minimum activity fragment of at least 3 kDa. N-desulphated, 20% re-N-acetylated heparin (anticoagulant) was ineffective against IL-13-dependent eotaxin-1 production compared with 90% re-N-acetylated (anticoagulant) or O-desulphated (non-anticoagulant) heparin, suggesting a requirement for N-sulphation independent of anticoagulant activity. Other sulphated molecules with variable anionic charge and molecular weight exceeding 3 kDa (dextran sulphate, fucoidan, chondroitin sulphate B) inhibited IL-13-stimulated eotaxin-1 release to varying degrees. However, non-sulphated dextran had no effect. Inhibition of IL-13-dependent eotaxin-1 release by heparin involved but did not depend upon sulphation, though loss of N-sulphation reduced the attenuating activity, which could be restored by N-acetylation. This anti-inflammatory effect was also partially dependent on anionic charge, but independent of molecular size above 3 kDa and the anticoagulant action of heparin.

  17. Decrease of airway smooth muscle contractility induced by simulated breathing maneuvers is not simply proportional to strain.

    PubMed

    Pascoe, Chris D; Seow, Chun Y; Paré, Peter D; Bossé, Ynuk

    2013-02-01

    The lung is a dynamic organ and the oscillating stress applied to the airway wall during breathing maneuvers can decrease airway smooth muscle (ASM) contractility. However, it is unclear whether it is the stress or the attendant strain that is responsible for the decline of ASM force associated with breathing maneuvers, and whether tone can prevent the decline of force by attenuating the strain. To investigate these questions, ovine tracheal strips were subjected to oscillating stress that simulates breathing maneuvers, and the resulting strain and decline of force were measured in the absence or presence of different levels of tone elicited by acetylcholine. In relaxed ASM, high stress simulating 20 cm H(2)O-transpulmonary pressure excursions strained ASM strips by 20.7% and decreased force by 17.1%. When stress oscillations were initiated during measurement of ACh concentration-response curves, tone almost abrogated strain at an ACh concentration of 10(-6) M (1.1%) but the decline of force was not affected (18.9%). When stress oscillations were initiated after ACh-induced contraction had reached its maximal force, strain was almost abrogated at an ACh concentration of 10(-6) M (0.9%) and the decline of force was attenuated (10.1%). However, even at the highest ACh concentration (10(-4) M), substantial decline of force (6.1%) was still observed despite very small strain (0.7%). As expected, the results indicate that tone attenuated the strain experienced by ASM during breathing maneuver simulations. More surprisingly, the reduction of strain induced by tone was not proportional to its effect on the decline of force induced by simulated breathing maneuvers.

  18. Kinematic MRI study of upper-airway biomechanics using electrical muscle stimulation

    NASA Astrophysics Data System (ADS)

    Brennick, Michael J.; Margulies, Susan S.; Ford, John C.; Gefter, Warren B.; Pack, Allan I.

    1997-05-01

    We have developed a new and powerful method to study the movement and function of upper airway muscles. Our method is to use direct electrical stimulation of individual upper airway muscles, while performing state of the art high resolution magnetic resonance imaging (MRI). We have adapted a paralyzed isolated UA cat model so that positive or negative static pressure in the UA can be controlled at specific levels while electrical muscle stimulation is applied during MRI. With these techniques we can assess the effect of muscle stimulation on airway cross-sectional area compliance and soft tissue motion. We are reporting the preliminary results and MRI techniques which have enabled us to examine changes in airway dimensions which result form electrical stimulation of specific upper airway dilator muscles. The results of this study will be relevant to the development of new clinical treatments for obstructive sleep apnea by providing new information as to exactly how upper airway muscles function to dilate the upper airway and the strength of stimulation required to prevent the airway obstruction when overall muscle tone may not be sufficient to maintain regular breathing.

  19. Oxidative stress–induced mitochondrial dysfunction drives inflammation and airway smooth muscle remodeling in patients with chronic obstructive pulmonary disease

    PubMed Central

    Wiegman, Coen H.; Michaeloudes, Charalambos; Haji, Gulammehdi; Narang, Priyanka; Clarke, Colin J.; Russell, Kirsty E.; Bao, Wuping; Pavlidis, Stelios; Barnes, Peter J.; Kanerva, Justin; Bittner, Anton; Rao, Navin; Murphy, Michael P.; Kirkham, Paul A.; Chung, Kian Fan; Adcock, Ian M.; Brightling, Christopher E.; Davies, Donna E.; Finch, Donna K.; Fisher, Andrew J.; Gaw, Alasdair; Knox, Alan J.; Mayer, Ruth J.; Polkey, Michael; Salmon, Michael; Singh, David

    2015-01-01

    Background Inflammation and oxidative stress play critical roles in patients with chronic obstructive pulmonary disease (COPD). Mitochondrial oxidative stress might be involved in driving the oxidative stress–induced pathology. Objective We sought to determine the effects of oxidative stress on mitochondrial function in the pathophysiology of airway inflammation in ozone-exposed mice and human airway smooth muscle (ASM) cells. Methods Mice were exposed to ozone, and lung inflammation, airway hyperresponsiveness (AHR), and mitochondrial function were determined. Human ASM cells were isolated from bronchial biopsy specimens from healthy subjects, smokers, and patients with COPD. Inflammation and mitochondrial function in mice and human ASM cells were measured with and without the presence of the mitochondria-targeted antioxidant MitoQ. Results Mice exposed to ozone, a source of oxidative stress, had lung inflammation and AHR associated with mitochondrial dysfunction and reflected by decreased mitochondrial membrane potential (ΔΨm), increased mitochondrial oxidative stress, and reduced mitochondrial complex I, III, and V expression. Reversal of mitochondrial dysfunction by the mitochondria-targeted antioxidant MitoQ reduced inflammation and AHR. ASM cells from patients with COPD have reduced ΔΨm, adenosine triphosphate content, complex expression, basal and maximum respiration levels, and respiratory reserve capacity compared with those from healthy control subjects, whereas mitochondrial reactive oxygen species (ROS) levels were increased. Healthy smokers were intermediate between healthy nonsmokers and patients with COPD. Hydrogen peroxide induced mitochondrial dysfunction in ASM cells from healthy subjects. MitoQ and Tiron inhibited TGF-β–induced ASM cell proliferation and CXCL8 release. Conclusions Mitochondrial dysfunction in patients with COPD is associated with excessive mitochondrial ROS levels, which contribute to enhanced inflammation and cell

  20. Oxidative stress-induced mitochondrial dysfunction drives inflammation and airway smooth muscle remodeling in patients with chronic obstructive pulmonary disease.

    PubMed

    Wiegman, Coen H; Michaeloudes, Charalambos; Haji, Gulammehdi; Narang, Priyanka; Clarke, Colin J; Russell, Kirsty E; Bao, Wuping; Pavlidis, Stelios; Barnes, Peter J; Kanerva, Justin; Bittner, Anton; Rao, Navin; Murphy, Michael P; Kirkham, Paul A; Chung, Kian Fan; Adcock, Ian M

    2015-09-01

    Inflammation and oxidative stress play critical roles in patients with chronic obstructive pulmonary disease (COPD). Mitochondrial oxidative stress might be involved in driving the oxidative stress-induced pathology. We sought to determine the effects of oxidative stress on mitochondrial function in the pathophysiology of airway inflammation in ozone-exposed mice and human airway smooth muscle (ASM) cells. Mice were exposed to ozone, and lung inflammation, airway hyperresponsiveness (AHR), and mitochondrial function were determined. Human ASM cells were isolated from bronchial biopsy specimens from healthy subjects, smokers, and patients with COPD. Inflammation and mitochondrial function in mice and human ASM cells were measured with and without the presence of the mitochondria-targeted antioxidant MitoQ. Mice exposed to ozone, a source of oxidative stress, had lung inflammation and AHR associated with mitochondrial dysfunction and reflected by decreased mitochondrial membrane potential (ΔΨm), increased mitochondrial oxidative stress, and reduced mitochondrial complex I, III, and V expression. Reversal of mitochondrial dysfunction by the mitochondria-targeted antioxidant MitoQ reduced inflammation and AHR. ASM cells from patients with COPD have reduced ΔΨm, adenosine triphosphate content, complex expression, basal and maximum respiration levels, and respiratory reserve capacity compared with those from healthy control subjects, whereas mitochondrial reactive oxygen species (ROS) levels were increased. Healthy smokers were intermediate between healthy nonsmokers and patients with COPD. Hydrogen peroxide induced mitochondrial dysfunction in ASM cells from healthy subjects. MitoQ and Tiron inhibited TGF-β-induced ASM cell proliferation and CXCL8 release. Mitochondrial dysfunction in patients with COPD is associated with excessive mitochondrial ROS levels, which contribute to enhanced inflammation and cell hyperproliferation. Targeting mitochondrial ROS represents

  1. The dopamine D1 receptor is expressed and facilitates relaxation in airway smooth muscle

    PubMed Central

    2013-01-01

    Background Dopamine signaling is mediated by Gs protein-coupled “D1-like” receptors (D1 and D5) and Gi-coupled “D2-like” receptors (D2-4). In asthmatic patients, inhaled dopamine induces bronchodilation. Although the Gi-coupled dopamine D2 receptor is expressed and sensitizes adenylyl cyclase activity in airway smooth muscle (ASM) cells, the Gs-coupled dopamine D1-like receptor subtypes have never been identified on these cells. Activation of Gs-coupled receptors stimulates cyclic AMP (cAMP) production through the stimulation of adenylyl cyclase, which promotes ASM relaxation. We questioned whether the dopamine D1-like receptor is expressed on ASM, and modulates its function through Gs-coupling. Methods The mRNA and protein expression of dopamine D1-like receptor subtypes in both native human and guinea pig ASM tissue and cultured human ASM (HASM) cells was measured. To characterize the stimulation of cAMP through the dopamine D1 receptor, HASM cells were treated with dopamine or the dopamine D1-like receptor agonists (A68930 or SKF38393) before cAMP measurements. To evaluate whether the activation of dopamine D1 receptor induces ASM relaxation, guinea pig tracheal rings suspended under isometric tension in organ baths were treated with cumulatively increasing concentrations of dopamine or A68930, following an acetylcholine-induced contraction with or without the cAMP-dependent protein kinase (PKA) inhibitor Rp-cAMPS, the large-conductance calcium-activated potassium (BKCa) channel blocker iberiotoxin, or the exchange proteins directly activated by cAMP (Epac) antagonist NSC45576. Results Messenger RNA encoding the dopamine D1 and D5 receptors were detected in native human ASM tissue and cultured HASM cells. Immunoblots confirmed the protein expression of the dopamine D1 receptor in both native human and guinea pig ASM tissue and cultured HASM cells. The dopamine D1 receptor was also immunohistochemically localized to both human and guinea pig ASM. The

  2. Smooth muscle of telokin-deficient mice exhibits increased sensitivity to Ca2+ and decreased cGMP-induced relaxation.

    PubMed

    Khromov, A S; Wang, H; Choudhury, N; McDuffie, M; Herring, B P; Nakamoto, R; Owens, G K; Somlyo, A P; Somlyo, A V

    2006-02-14

    Cyclic nucleotides can relax smooth muscle without a change in [Ca2+]i, a phenomenon termed Ca2+ desensitization, contributing to vasodilation, gastrointestinal motility, and airway resistance. The physiological importance of telokin, a 17-kDa smooth muscle-specific protein and target for cyclic nucleotide-induced Ca2+ desensitization, was determined in telokin null mice bred to a congenic background. Telokin null ileal smooth muscle homogenates compared to wild type exhibited an approximately 30% decrease in myosin light-chain phosphatase (MLCP) activity, which was reflected in a significant leftward shift (up to 2-fold at pCa 6.3) of the Ca2+ force relationship accompanied by an increase in myosin light-chain phosphorylation. No difference in the Ca2+ force relationship occurred in telokin WT and knockout (KO) aortas, presumably reflecting the normally approximately 5-fold lower telokin content in aorta vs. ileum smooth muscle. Ca2+ desensitization of contractile force by 8-Br-cGMP was attenuated by 50% in telokin KO intestinal smooth muscle. The rate of force relaxation reflecting MLCP activity, in the presence of 50 microM 8-Br-cGMP, was also significantly slowed in telokin KO vs. WT ileum and was rescued by recombinant telokin. Normal thick filaments in telokin KO smooth muscles indicate that telokin is not required for filament formation or stability. Results indicate that a primary role of telokin is to modulate force through increasing MLCP activity and that this effect is further potentiated through phosphorylation by cGMP in telokin-rich smooth tissues.

  3. Effect of airway acidosis and alkalosis on airway vascular smooth muscle responsiveness to albuterol.

    PubMed

    Cancado, Jose E; Mendes, Eliana S; Arana, Johana; Horvath, Gabor; Monzon, Maria E; Salathe, Matthias; Wanner, Adam

    2015-04-02

    In vitro and animal experiments have shown that the transport and signaling of β2-adrenergic agonists are pH-sensitive. Inhaled albuterol, a hydrophilic β2-adrenergic agonist, is widely used for the treatment of obstructive airway diseases. Acute exacerbations of obstructive airway diseases can be associated with changes in ventilation leading to either respiratory acidosis or alkalosis thereby affecting albuterol responsiveness in the airway. The purpose of this study was to determine if airway pH has an effect on albuterol-induced vasodilation in the airway. Ten healthy volunteers performed the following respiratory maneuvers: quiet breathing, hypocapnic hyperventilation, hypercapnic hyperventilation, and eucapnic hyperventilation (to dissociate the effect of pH from the effect of ventilation). During these breathing maneuvers, exhaled breath condensate (EBC) pH and airway blood flow response to inhaled albuterol (ΔQ̇aw) were assessed. Mean ± SE EBC pH (units) and ΔQ̇aw (μl.min(-1).mL(-1)) were 6.4 ± 0.1 and 16.8 ± 1.9 during quiet breathing, 6.3 ± 0.1 and 14.5 ± 2.4 during eucapnic hyperventilation, 6.6 ± 0.2 and -0.2 ± 1.8 during hypocapnic hyperventilation (p = 0.02 and <0.01 vs. quiet breathing), and 5.9 ± 0.1 and 2.0 ± 1.5 during hypercapnic hyperventilation (p = 0.02 and <0.02 vs quiet breathing). Albuterol responsiveness in the airway as assessed by ΔQ̇aw is pH sensitive. The breathing maneuver associated with decreased and increased EBC pH both resulted in a decreased responsiveness independent of the level of ventilation. These findings suggest an attenuated response to hydrophilic β2-adrenergic agonists during airway disease exacerbations associated with changes in pH. Registered at clinicaltrials.gov: NCT01216748 .

  4. Inhibition of myosin light-chain phosphorylation inverts the birefringence response of porcine airway smooth muscle

    PubMed Central

    Smolensky, Alexander V; Gilbert, Susan H; Harger-Allen, Margaret; Ford, Lincoln E

    2007-01-01

    Muscle birefringence, caused mainly by parallel thick filaments, increases in smooth muscle during stimulation, signalling thick filament formation upon activation. The reverse occurs in skeletal muscle, where a decrease in birefringence has been correlated with crossbridge movement away from the thick filaments. When force generation by trachealis muscle was inhibited with wortmannin, which inhibits myosin light-chain phosphorylation and thick-filament formation, but not the calcium increase caused by stimulation, the birefringence response inverted, suggesting crossbridge movement similar to that of skeletal muscle. Resistance to quick stretches was much greater in stimulated muscle than in unstimulated muscle before wortmannin treatment and no different in stimulated and unstimulated muscle after force inhibition by wortmannin. Before wortmannin treatment, stimulation reduced thick-filament cross-sectional areas in electron micrographs by 44%. After force inhibition by wortmannin, filament areas were not significantly different in stimulated and unstimulated muscle and not significantly different from those of relaxed muscle without wortmannin treatment. These results suggest that myofibrillar-space calcium causes crossbridges to move away from the thick filaments without firmly attaching to thin filaments. PMID:17095560

  5. MicroRNA-142 Inhibits Proliferation and Promotes Apoptosis in Airway Smooth Muscle Cells During Airway Remodeling in Asthmatic Rats via the Inhibition of TGF-β -Dependent EGFR Signaling Pathway.

    PubMed

    Wang, Jing; Wang, Hu-Shan; Su, Zhen-Bo

    2018-06-27

    Asthma is a heterogeneous disease characterized by chronic airway inflammation resulting from airway hyper-responsiveness to diverse stimuli. In this study, we investigated whether microRNA-142 (miR-142) expression affects proliferation and apoptosis in airway smooth muscle cells (ASMCs) during airway remodeling in asthmatic rats. Thirty six Wistar rats were randomly classified into a control group and an model group. miR-142 mimics and inhibitors were constructed, and ASMCs were transfected using liposomes according to the following groups: blank, negative control (NC), miR-142 mimics, miR-142 inhibitors, si-TGF-β and miR-142 inhibitors + si-TGF-β. We verified that miR-142 targets TGF-β using a dual-luciferase reporter assay. The expression levels of miR-142, TGF-β, EGFR and apoptosis signaling pathway-related genes were determined using RT-qPCR and western blotting. Changes in cell proliferation, cell cycle progression and apoptosis were analyzed using MTT assays and flow cytometry. Rats with asthma had higher expression levels of EGFR and Akt and lower miR-142 levels. miR-142 was negatively correlated with TGF-β expression. In ASMCs, the expression of TGF-β, EGFR, Akt, phosphorylated-Akt (p-Akt), Bcl-2 and Bcl-xl and the rate of early apoptosis were decreased while expression of Bax and p21 and the proliferation rate were elevated with the upregulation of miR-142. The opposite results were observed with the downregulation of miR-142. Finally, the proliferative rate was decreased while the apoptosis rate was increased and expression levels of EGFR, Akt, p-Akt, Bcl-2 and Bcl-xl were reduced while Bax and p21 were elevated in the ASMCs transfected with miR-142 inhibitors and si-TGF-β. The results of our study suggest that miR-142 inhibits proliferation and promotes apoptosis in ASMCs during airway remodeling in asthmatic rats by inhibiting TGF-β expression via a mechanism involving the EGFR signaling pathway. © 2018 The Author(s). Published by S. Karger AG

  6. Compartmental and temporal dynamics of chronic inflammation and airway remodelling in a chronic asthma mouse model.

    PubMed

    Alrifai, Mohammed; Marsh, Leigh M; Dicke, Tanja; Kılıç, Ayse; Conrad, Melanie L; Renz, Harald; Garn, Holger

    2014-01-01

    Allergic asthma is associated with chronic airway inflammation and progressive airway remodelling. However, the dynamics of the development of these features and their spontaneous and pharmacological reversibility are still poorly understood. We have therefore investigated the dynamics of airway remodelling and repair in an experimental asthma model and studied how pharmacological intervention affects these processes. Using BALB/c mice, the kinetics of chronic asthma progression and resolution were characterised in absence and presence of inhaled corticosteroid (ICS) treatment. Airway inflammation and remodelling was assessed by the analysis of bronchoalveolar and peribronichal inflammatory cell infiltrate, goblet cell hyperplasia, collagen deposition and smooth muscle thickening. Chronic allergen exposure resulted in early (goblet cell hyperplasia) and late remodelling (collagen deposition and smooth muscle thickening). After four weeks of allergen cessation eosinophilic inflammation, goblet cell hyperplasia and collagen deposition were resolved, full resolution of lymphocyte inflammation and smooth muscle thickening was only observed after eight weeks. ICS therapy when started before the full establishment of chronic asthma reduced the development of lung inflammation, decreased goblet cell hyperplasia and collagen deposition, but did not affect smooth muscle thickening. These effects of ICS on airway remodelling were maintained for a further four weeks even when therapy was discontinued. Utilising a chronic model of experimental asthma we have shown that repeated allergen exposure induces reversible airway remodelling and inflammation in mice. Therapeutic intervention with ICS was partially effective in inhibiting the transition from acute to chronic asthma by reducing airway inflammation and remodelling but was ineffective in preventing smooth muscle hypertrophy.

  7. Involvement of the Tyr kinase/JNK pathway in carbachol-induced bronchial smooth muscle contraction in the rat.

    PubMed

    Sakai, Hiroyasu; Watanabe, Yu; Honda, Mai; Tsuiki, Rika; Ueda, Yusuke; Nagai, Yuki; Narita, Minoru; Misawa, Miwa; Chiba, Yoshihiko

    2013-05-01

    Tyrosine (Tyr) kinases and mitogen-activated protein kinases have been thought to participate in the contractile response in various smooth muscles. The aim of the current study was to investigate the involvement of the Tyr kinase pathway in the contraction of bronchial smooth muscle. Ring preparations of bronchi isolated from rats were suspended in an organ bath. Isometric contraction of circular smooth muscle was measured. Immunoblotting was used to examine the phosphorylation of c-Jun N-terminal kinasess (JNKs) in bronchial smooth muscle. To examine the role of mitogen-activated protein kinase(s) in bronchial smooth muscle contraction, the effects of MPAK inhibitors were investigated in this study. The contraction induced by carbachol (CCh) was significantly inhibited by pretreatment with selective Tyr kinase inhibitors (genistein and ST638, n = 6, respectively), and a JNK inhibitor (SP600125, n = 6). The contractions induced by high K depolarization (n = 4), orthovanadate (a potent Tyr phosphatase inhibitor) and sodium fluoride (a G protein activator; NaF) were also significantly inhibited by selective Tyr kinase inhibitors and a JNK inhibitor (n = 4, respectively). However, the contraction induced by calyculin-A was not affected by SP600125. On the other hand, JNKs were phosphorylated by CCh (2.2 ± 0,4 [mean±SEM] fold increase). The JNK phosphorylation induced by CCh was significantly inhibited by SP600125 (n = 4). These findings suggest that the Tyr kinase/JNK pathway may play a role in bronchial smooth muscle contraction. Strategies to inhibit JNK activation may represent a novel therapeutic approach for diseases involving airway obstruction, such as asthma and chronic obstructive pulmonary disease.

  8. Vitamin D Modulates Expression of the Airway Smooth Muscle Transcriptome in Fatal Asthma

    PubMed Central

    Johnson, Martin; Nikolos, Christina; Jester, William; Klanderman, Barbara; Litonjua, Augusto A.; Tantisira, Kelan G.; Truskowski, Kevin; MacDonald, Kevin; Panettieri, Reynold A.; Weiss, Scott T.

    2015-01-01

    Globally, asthma is a chronic inflammatory respiratory disease affecting over 300 million people. Some asthma patients remain poorly controlled by conventional therapies and experience more life-threatening exacerbations. Vitamin D, as an adjunct therapy, may improve disease control in severe asthma patients since vitamin D enhances glucocorticoid responsiveness and mitigates airway smooth muscle (ASM) hyperplasia. We sought to characterize differences in transcriptome responsiveness to vitamin D between fatal asthma- and non-asthma-derived ASM by using RNA-Seq to measure ASM transcript expression in five donors with fatal asthma and ten non-asthma-derived donors at baseline and with vitamin D treatment. Based on a Benjamini-Hochberg corrected p-value <0.05, 838 genes were differentially expressed in fatal asthma vs. non-asthma-derived ASM at baseline, and vitamin D treatment compared to baseline conditions induced differential expression of 711 and 867 genes in fatal asthma- and non-asthma-derived ASM, respectively. Functional gene categories that were highly represented in all groups included extracellular matrix, and responses to steroid hormone stimuli and wounding. Genes differentially expressed by vitamin D also included cytokine and chemokine activity categories. Follow-up qPCR and individual analyte ELISA experiments were conducted for four cytokines (i.e. CCL2, CCL13, CXCL12, IL8) to measure TNFα-induced changes by asthma status and vitamin D treatment. Vitamin D inhibited TNFα-induced IL8 protein secretion levels to a comparable degree in fatal asthma- and non-asthma-derived ASM even though IL8 had significantly higher baseline levels in fatal asthma-derived ASM. Our findings identify vitamin D-specific gene targets and provide transcriptomic data to explore differences in the ASM of fatal asthma- and non-asthma-derived donors. PMID:26207385

  9. Asthmatic airway smooth muscle CXCL10 production: mitogen-activated protein kinase JNK involvement

    PubMed Central

    Alrashdan, Yazan A.; Alkhouri, Hatem; Chen, Emily; Lalor, Daniel J.; Poniris, Maree; Henness, Sheridan; Brightling, Christopher E.; Burgess, Janette K.; Armour, Carol L.; Ammit, Alaina J.

    2012-01-01

    CXCL10 (IP10) is involved in mast cell migration to airway smooth muscle (ASM) bundles in asthma. We aimed to investigate the role of cytokine-induced MAPK activation in CXCL10 production by ASM cells from people with and without asthma. Confluent growth-arrested ASM cells were treated with inhibitors of the MAPKs ERK, p38, and JNK and transcription factor NF-κB, or vehicle, and stimulated with IL-1β, TNF-α, or IFN-γ, alone or combined (cytomix). CXCL10 mRNA and protein, JNK, NF-κB p65 phosphorylation, and Iκ-Bα protein degradation were assessed using real-time PCR, ELISA, and immunoblotting, respectively. Cytomix, IL-1β, and TNF-α induced CXCL10 mRNA expression more rapidly in asthmatic than nonasthmatic ASM cells. IL-1β and/or TNF-α combined with IFN-γ synergistically increased asthmatic ASM cell CXCL10 release. Inhibitor effects were similar in asthmatic and nonasthmatic cells, but cytomix-induced release was least affected, with only JNK and NF-κB inhibitors halving it. Notably, JNK phosphorylation was markedly less in asthmatic compared with nonasthmatic cells. However, in both, the JNK inhibitor SP600125 reduced JNK phosphorylation and CXCL10 mRNA levels but did not affect CXCL10 mRNA stability or Iκ-Bα degradation. Together, the JNK and NF-κB inhibitors completely inhibited their CXCL10 release. We concluded that, in asthmatic compared with nonasthmatic ASM cells, JNK activation was reduced and CXCL10 gene expression was more rapid following cytomix stimulation. However, in both, JNK activation did not regulate early events leading to NF-κB activation. Thus JNK and NF-κB provide independent therapeutic targets for limiting CXCL10 production and mast cell migration to the ASM in asthma. PMID:22387292

  10. Asthmatic airway smooth muscle CXCL10 production: mitogen-activated protein kinase JNK involvement.

    PubMed

    Alrashdan, Yazan A; Alkhouri, Hatem; Chen, Emily; Lalor, Daniel J; Poniris, Maree; Henness, Sheridan; Brightling, Christopher E; Burgess, Janette K; Armour, Carol L; Ammit, Alaina J; Hughes, J Margaret

    2012-05-15

    CXCL10 (IP10) is involved in mast cell migration to airway smooth muscle (ASM) bundles in asthma. We aimed to investigate the role of cytokine-induced MAPK activation in CXCL10 production by ASM cells from people with and without asthma. Confluent growth-arrested ASM cells were treated with inhibitors of the MAPKs ERK, p38, and JNK and transcription factor NF-κB, or vehicle, and stimulated with IL-1β, TNF-α, or IFN-γ, alone or combined (cytomix). CXCL10 mRNA and protein, JNK, NF-κB p65 phosphorylation, and Iκ-Bα protein degradation were assessed using real-time PCR, ELISA, and immunoblotting, respectively. Cytomix, IL-1β, and TNF-α induced CXCL10 mRNA expression more rapidly in asthmatic than nonasthmatic ASM cells. IL-1β and/or TNF-α combined with IFN-γ synergistically increased asthmatic ASM cell CXCL10 release. Inhibitor effects were similar in asthmatic and nonasthmatic cells, but cytomix-induced release was least affected, with only JNK and NF-κB inhibitors halving it. Notably, JNK phosphorylation was markedly less in asthmatic compared with nonasthmatic cells. However, in both, the JNK inhibitor SP600125 reduced JNK phosphorylation and CXCL10 mRNA levels but did not affect CXCL10 mRNA stability or Iκ-Bα degradation. Together, the JNK and NF-κB inhibitors completely inhibited their CXCL10 release. We concluded that, in asthmatic compared with nonasthmatic ASM cells, JNK activation was reduced and CXCL10 gene expression was more rapid following cytomix stimulation. However, in both, JNK activation did not regulate early events leading to NF-κB activation. Thus JNK and NF-κB provide independent therapeutic targets for limiting CXCL10 production and mast cell migration to the ASM in asthma.

  11. Tachykinin receptors and the airways.

    PubMed

    Frossard, N; Advenier, C

    1991-01-01

    The tachykinins, substance P, neurokinin A and neurokinin B, belong to a structural family of peptides. In mammalian airways, substance P and neurokinin A are colocalized to afferent C-fibres. Substance P-containing fibres are close to bronchial epithelium, smooth muscle, mucus glands and blood vessels. Sensory neuropeptides may be released locally, possibly as a result of a local reflex, and produce bronchial obstruction through activation of specific receptors on these various tissues. Three types of tachykinin receptors, namely NK-1, NK-2 and NK-3 receptors, have been characterized by preferential activation by substance P, neurokinin A and neurokinin B respectively. NK-1 and NK-2 receptors were recently cloned. The determination of receptor types involved in the effects of tachykinins in the airways has been done with synthetic agonists and antagonists binding specifically to NK-1, NK-2 and NK-3 receptors. Although the existence of species differences, the conclusion that bronchial smooth muscle contraction is mainly related to activation of NK-2 receptors on bronchial smooth muscle cell has been drawn. The hypothesis of a NK-2 receptor subclassification has been proposed with NK-2A receptor subtype in the guinea-pig airways. Other effects in the airways are related to stimulation of NK-1 receptors on mucus cells, vessels, epithelium and inflammatory cells. A non-receptor-mediated mechanism is also involved in the effect of substance P on inflammatory cells and mast cells.

  12. The Role of TNF Family Molecules Light in Cellular Interaction Between Airway Smooth Muscle Cells and T Cells During Chronic Allergic Inflammation.

    PubMed

    Shi, Fei; Xiong, Yi; Zhang, Yarui; Qiu, Chen; Li, Manhui; Shan, Aijun; Yang, Ying; Li, Binbin

    2018-06-01

    Interaction between T cells and airway smooth muscle (ASM) cells has been identified as an important factor in the development of asthma. LIGHT (known as TNFSF14) -mediated signaling likely contributes to various inflammatory disorders and airway remodeling. The objective of this study was to investigate the roles of LIGHT-mediated pathways in the interaction between ASM cells and T cells during chronic allergic inflammation. Mice were sensitized and challenged by ovalbumin (OVA) to induce chronic airway allergic inflammation. The control group received PBS only. The histological features and LIGHT expressions in lungs were assessed in vivo. Furthermore, T cells and ASM cells derived from the model mice were co-cultured both in the presence and absence of anti-LIGHT Ab for 72 h. The effects of LIGHT blockade on expressions of downstream signaling molecules, proliferation, and apoptosis of ASM cells, differentiation of T cells, and inflammatory cytokines release were evaluated. We demonstrated that LIGHT blockade strikingly inhibited the mRNA and protein expressions of HVEM, c-JUN, and NFκB. Additionally, LIGHT blockade resulted in decreased proliferation and increased apoptosis of ASM cells. Moreover, depletion of LIGHT dramatically reduced the differentiation of CD4 + T cells into Th1, Th2, and Th17 cells, as well as inhibited inflammatory cytokines release including IL-13, TGF-β, and IFN-γ, which are associated with CD4 + T cell differentiation and ASM cell proliferation. LIGHT plays an important role in the interaction between T cells and ASM cells in chronic allergic asthma. Blockade of LIGHT markedly suppressed ASM hyperplasia and inflammatory responses, which might be modulated through HVEM-NFκB or c-JUN pathways. Therefore, targeting LIGHT is a promising therapeutic strategy for airway inflammation and remodeling in chronic allergic asthma.

  13. Interaction between endoplasmic/sarcoplasmic reticulum stress (ER/SR stress), mitochondrial signaling and Ca2+ regulation in airway smooth muscle (ASM)1

    PubMed Central

    Delmotte, Philippe; Sieck, Gary C.

    2015-01-01

    Airway inflammation is a key aspect of diseases such as asthma. Several inflammatory cytokines (e.g., TNFα and IL-13) increase cytosolic Ca2+ ([Ca2+]cyt) responses to agonist stimulation and Ca2+ sensitivity of force generation, thereby enhancing airway smooth muscle (ASM) contractility (hyper-reactive state). Inflammation also induces ASM proliferation and remodeling (synthetic state). In normal ASM, the transient elevation of [Ca2+]cyt induced by agonists leads to a transient increase in mitochondrial Ca2+ ([Ca2+]mito) that may be important in matching ATP production with ATP consumption. In human ASM (hASM) exposed to TNFα and IL-13, the transient increase in [Ca2+]mito is blunted despite enhanced [Ca2+]cyt responses. We also found that TNFα and IL-13 induce reactive oxidant species (ROS) formation and endoplasmic/sarcoplasmic reticulum (ER/SR) stress (unfolded protein response) in hASM. ER/SR stress in hASM is associated with disruption of mitochondrial coupling with the ER/SR membrane, which relates to reduced mitofusin 2 (Mfn2) expression. Thus, in hASM it appears that TNFα and IL-13 result in ROS formation leading to ER/SR stress, reduced Mfn2 expression, disruption of mitochondrion–ER/SR coupling, decreased mitochondrial Ca2+ buffering, mitochondrial fragmentation, and increased cell proliferation. PMID:25506723

  14. The Role of Inflammation Resolution Speed in Airway Smooth Muscle Mass Accumulation in Asthma: Insight from a Theoretical Model

    PubMed Central

    Chernyavsky, Igor L.; Croisier, Huguette; Chapman, Lloyd A. C.; Kimpton, Laura S.; Hiorns, Jonathan E.; Brook, Bindi S.; Jensen, Oliver E.; Billington, Charlotte K.; Hall, Ian P.; Johnson, Simon R.

    2014-01-01

    Despite a large amount of in vitro data, the dynamics of airway smooth muscle (ASM) mass increase in the airways of patients with asthma is not well understood. Here, we present a novel mathematical model that describes qualitatively the growth dynamics of ASM cells over short and long terms in the normal and inflammatory environments typically observed in asthma. The degree of ASM accumulation can be explained by an increase in the rate at which ASM cells switch between non-proliferative and proliferative states, driven by episodic inflammatory events. Our model explores the idea that remodelling due to ASM hyperplasia increases with the frequency and magnitude of these inflammatory events, relative to certain sensitivity thresholds. It highlights the importance of inflammation resolution speed by showing that when resolution is slow, even a series of small exacerbation events can result in significant remodelling, which persists after the inflammatory episodes. In addition, we demonstrate how the uncertainty in long-term outcome may be quantified and used to design an optimal low-risk individual anti-proliferative treatment strategy. The model shows that the rate of clearance of ASM proliferation and recruitment factors after an acute inflammatory event is a potentially important, and hitherto unrecognised, target for anti-remodelling therapy in asthma. It also suggests new ways of quantifying inflammation severity that could improve prediction of the extent of ASM accumulation. This ASM growth model should prove useful for designing new experiments or as a building block of more detailed multi-cellular tissue-level models. PMID:24632688

  15. Aerobic metabolism on muscle contraction in porcine gastric smooth muscle.

    PubMed

    Kanda, Hidenori; Kaneda, Takeharu; Nagai, Yuta; Urakawa, Norimoto; Shimizu, Kazumasa

    2018-05-18

    Exposure to chronic hypoxic conditions causes various gastric diseases, including gastric ulcers. It has been suggested that gastric smooth muscle contraction is associated with aerobic metabolism. However, there are no reports on the association between gastric smooth muscle contraction and aerobic metabolism, and we have investigated this association in the present study. High K + - and carbachol (CCh)-induced muscle contractions involved increasing O 2 consumption. Aeration with N 2 (hypoxia) and NaCN significantly decreased high K + - and CCh-induced muscle contraction and O 2 consumption. In addition, hypoxia and NaCN significantly decreased creatine phosphate (PCr) contents in the presence of high K + . Moreover, decrease in CCh-induced contraction and O 2 consumption was greater than that of high K + . Our results suggest that hypoxia and NaCN inhibit high K + - and CCh-induced contractions in gastric fundus smooth muscles by decreasing O 2 consumption and intracellular PCr content. However, the inhibition of CCh-induced muscle contraction was greater than that of high K + -induced muscle contraction.

  16. Nonmuscle myosin is regulated during smooth muscle contraction.

    PubMed

    Yuen, Samantha L; Ogut, Ozgur; Brozovich, Frank V

    2009-07-01

    The participation of nonmuscle myosin in force maintenance is controversial. Furthermore, its regulation is difficult to examine in a cellular context, as the light chains of smooth muscle and nonmuscle myosin comigrate under native and denaturing electrophoresis techniques. Therefore, the regulatory light chains of smooth muscle myosin (SM-RLC) and nonmuscle myosin (NM-RLC) were purified, and these proteins were resolved by isoelectric focusing. Using this method, intact mouse aortic smooth muscle homogenates demonstrated four distinct RLC isoelectric variants. These spots were identified as phosphorylated NM-RLC (most acidic), nonphosphorylated NM-RLC, phosphorylated SM-RLC, and nonphosphorylated SM-RLC (most basic). During smooth muscle activation, NM-RLC phosphorylation increased. During depolarization, the increase in NM-RLC phosphorylation was unaffected by inhibition of either Rho kinase or PKC. However, inhibition of Rho kinase blocked the angiotensin II-induced increase in NM-RLC phosphorylation. Additionally, force for angiotensin II stimulation of aortic smooth muscle from heterozygous nonmuscle myosin IIB knockout mice was significantly less than that of wild-type littermates, suggesting that, in smooth muscle, activation of nonmuscle myosin is important for force maintenance. The data also demonstrate that, in smooth muscle, the activation of nonmuscle myosin is regulated by Ca(2+)-calmodulin-activated myosin light chain kinase during depolarization and a Rho kinase-dependent pathway during agonist stimulation.

  17. PM2.5 promotes human bronchial smooth muscle cell migration via the sonic hedgehog signaling pathway.

    PubMed

    Ye, Xiuqin; Hong, Wei; Hao, Binwei; Peng, Gongyong; Huang, Lingmei; Zhao, Zhuxiang; Zhou, Yumin; Zheng, Mengning; Li, Chenglong; Liang, Chunxiao; Yi, Erkang; Pu, Jinding; Li, Bing; Ran, Pixin

    2018-03-02

    The contribution of airway remodeling in chronic obstructive pulmonary disease (COPD) has been well documented, with airway smooth muscle cell proliferation and migration playing a role in the remodeling process. Here, we aimed to verify the effects of fine particulate matter (PM2.5) on human bronchial smooth muscle cell (HBSMC) migration and to explore the underlying signaling pathways. HBSMC apoptosis, proliferation and migration were measured using flow cytometry, cell counting and transwell migration assays, respectively. The role of the hedgehog pathway in cell migration was assessed by western blotting to measure the expression of Sonic hedgehog (Shh), Gli1 and Snail. Furthermore, siRNA was used to knock down Gli1 or Snail expression. PM2.5 induced HBSMC apoptosis in a dose-dependent manner, although certain concentrations of PM2.5 did not induce HBSMC proliferation or apoptosis. Interestingly, cell migration was stimulated by PM2.5 doses far below those that induced apoptosis. Additional experiments revealed that these PM2.5 doses enhanced the expression of Shh, Gli1 and Snail in HBSMCs. Furthermore, PM2.5-induced cell migration and protein expression were enhanced by recombinant Shh and attenuated by cyclopamine. Similar results were obtained by knocking down Gli1 or Snail. These findings suggest that PM2.5, which may exert its effects through the Shh signaling pathway, is necessary for the migration of HBSMCs. These data define a novel role for PM2.5 in airway remodeling in COPD.

  18. Interstitial Cells: Regulators of Smooth Muscle Function

    PubMed Central

    Sanders, Kenton M.; Ward, Sean M.; Koh, Sang Don

    2014-01-01

    Smooth muscles are complex tissues containing a variety of cells in addition to muscle cells. Interstitial cells of mesenchymal origin interact with and form electrical connectivity with smooth muscle cells in many organs, and these cells provide important regulatory functions. For example, in the gastrointestinal tract, interstitial cells of Cajal (ICC) and PDGFRα+ cells have been described, in detail, and represent distinct classes of cells with unique ultrastructure, molecular phenotypes, and functions. Smooth muscle cells are electrically coupled to ICC and PDGFRα+ cells, forming an integrated unit called the SIP syncytium. SIP cells express a variety of receptors and ion channels, and conductance changes in any type of SIP cell affect the excitability and responses of the syncytium. SIP cells are known to provide pacemaker activity, propagation pathways for slow waves, transduction of inputs from motor neurons, and mechanosensitivity. Loss of interstitial cells has been associated with motor disorders of the gut. Interstitial cells are also found in a variety of other smooth muscles; however, in most cases, the physiological and pathophysiological roles for these cells have not been clearly defined. This review describes structural, functional, and molecular features of interstitial cells and discusses their contributions in determining the behaviors of smooth muscle tissues. PMID:24987007

  19. Bronchodilatory and B-adrenergic effects of methanolic and aqueous extracts of Althaea root on isolated tracheobronchial smooth rat muscle.

    PubMed

    Alani, Behrang; Zare, Mohammad; Noureddini, Mahdi

    2015-01-01

    The smooth muscle contractions of the tracheobronchial airways are mediated through the balance of adrenergic, cholinergic and peptidergic nervous mechanisms. This research was designed to determine the bronchodilatory and B-adrenergic effects of methanolic and aqueous extracts of root Althaea on the isolated tracheobronchial smooth muscle of the rat. In this experimental study, 116 tracheobronchial sections (5 mm) from 58 healthy male Sprague-Dawley rats were dissected and divided into 23 groups. The effect of methanolic and aqueous extracts of the root Althaea was assayed at different concentrations (0.2, 0.6, 2.6, 6.6, 14.6 μg/ml) and epinephrine (5 μm) in the presence and absence of propranolol (1 μM) under one g tension based on the isometric method. This assay was recorded in an organ bath containing Krebs-Henseleit solution for tracheobronchial smooth muscle contractions using potassium chloride (KCl) (60 mM) induction. Epinephrine (5 μm) alone and root methanolic and aqueous extract concentrations (0.6-14.6 μg/ml) reduced tracheobronchial smooth muscle contractions induced using KCl (60 mM) in a dose dependent manner. Propranolol inhibited the antispasmodic effect of epinephrine on tracheobronchial smooth muscle contractions, but could not reduce the antispasmodic effect of the root extract concentrations. The methanolic and aqueous extracts of Althaea root inhibited the tracheobronchial smooth muscle contractions of rats in a dose dependent manner, but B-adrenergic receptors do not appear to engage in this process. Understanding the mechanism of this process can be useful in the treatment of pulmonary obstructive diseases like asthma.

  20. Thiazolidinediones inhibit airway smooth muscle release of the chemokine CXCL10: in vitro comparison with current asthma therapies

    PubMed Central

    2012-01-01

    Background Activated mast cells are present within airway smooth muscle (ASM) bundles in eosinophilic asthma. ASM production of the chemokine CXCL10 plays a role in their recruitment. Thus the effects of glucocorticoids (fluticasone, budesonide), long-acting β2-agonists (salmeterol, formoterol) and thiazolidinediones (ciglitazone, rosiglitazone) on CXCL10 production by ASM cells (ASMC) from people with and without asthma were investigated in vitro. Methods Confluent serum-deprived cells were treated with the agents before and during cytokine stimulation for 0-24 h. CXCL10 protein/mRNA, IκB-α levels and p65 activity were measured using ELISA, RT PCR, immunoblotting and p65 activity assays respectively. Data were analysed using ANOVA followed by Fisher’s post-hoc test. Results Fluticasone and/or salmeterol at 1 and 100 nM inhibited CXCL10 release induced by IL-1β and TNF-α, but not IFNγ or all three cytokines (cytomix). The latter was also not affected by budesonide and formoterol. In asthmatic ASMC low salmeterol, but not formoterol, concentrations increased cytomix-induced CXCL10 release and at 0.01 nM enhanced NF-κB activity. Salmeterol 0.1nM together with fluticasone 0.1 and 10 nM still increased CXCL10 release. The thiazolidinediones ciglitazone and rosiglitazone (at 25 and 100 μM) inhibited cytomix-induced CXCL10 release but these inhibitory effects were not prevented by the PPAR-g antagonist GW9662. Ciglitazone did not affect early NF-κB activity and CXCL10 mRNA production. Conclusions Thus the thiazolidinediones inhibited asthmatic ASMC CXCL10 release under conditions when common asthma therapies were ineffective or enhanced it. They may provide an alternative strategy to reduce mast cell-ASM interactions and restore normal airway physiology in asthma. PMID:23034049

  1. Thiazolidinediones inhibit airway smooth muscle release of the chemokine CXCL10: in vitro comparison with current asthma therapies.

    PubMed

    Seidel, Petra; Alkhouri, Hatem; Lalor, Daniel J; Burgess, Janette K; Armour, Carol L; Hughes, J Margaret

    2012-10-04

    Activated mast cells are present within airway smooth muscle (ASM) bundles in eosinophilic asthma. ASM production of the chemokine CXCL10 plays a role in their recruitment. Thus the effects of glucocorticoids (fluticasone, budesonide), long-acting β2-agonists (salmeterol, formoterol) and thiazolidinediones (ciglitazone, rosiglitazone) on CXCL10 production by ASM cells (ASMC) from people with and without asthma were investigated in vitro. Confluent serum-deprived cells were treated with the agents before and during cytokine stimulation for 0-24 h. CXCL10 protein/mRNA, IκB-α levels and p65 activity were measured using ELISA, RT PCR, immunoblotting and p65 activity assays respectively. Data were analysed using ANOVA followed by Fisher's post-hoc test. Fluticasone and/or salmeterol at 1 and 100 nM inhibited CXCL10 release induced by IL-1β and TNF-α, but not IFNγ or all three cytokines (cytomix). The latter was also not affected by budesonide and formoterol. In asthmatic ASMC low salmeterol, but not formoterol, concentrations increased cytomix-induced CXCL10 release and at 0.01 nM enhanced NF-κB activity. Salmeterol 0.1 nM together with fluticasone 0.1 and 10 nM still increased CXCL10 release. The thiazolidinediones ciglitazone and rosiglitazone (at 25 and 100 μM) inhibited cytomix-induced CXCL10 release but these inhibitory effects were not prevented by the PPAR-g antagonist GW9662. Ciglitazone did not affect early NF-κB activity and CXCL10 mRNA production. Thus the thiazolidinediones inhibited asthmatic ASMC CXCL10 release under conditions when common asthma therapies were ineffective or enhanced it. They may provide an alternative strategy to reduce mast cell-ASM interactions and restore normal airway physiology in asthma.

  2. Active Components of Ginger Potentiate β-Agonist–Induced Relaxation of Airway Smooth Muscle by Modulating Cytoskeletal Regulatory Proteins

    PubMed Central

    Zhang, Yi; Xu, Carrie; Wakita, Ryo; Emala, Charles W.

    2014-01-01

    β-Agonists are the first-line therapy to alleviate asthma symptoms by acutely relaxing the airway. Purified components of ginger relax airway smooth muscle (ASM), but the mechanisms are unclear. By elucidating these mechanisms, we can explore the use of phytotherapeutics in combination with traditional asthma therapies. The objectives of this study were to: (1) determine if 6-gingerol, 8-gingerol, or 6-shogaol potentiate β-agonist–induced ASM relaxation; and (2) define the mechanism(s) of action responsible for this potentiation. Human ASM was contracted in organ baths. Tissues were relaxed dose dependently with β-agonist, isoproterenol, in the presence of vehicle, 6-gingerol, 8-gingerol, or 6-shogaol (100 μM). Primary human ASM cells were used for cellular experiments. Purified phosphodiesterase (PDE) 4D or phospholipase C β enzyme was used to assess inhibitory activity of ginger components using fluorescent assays. A G-LISA assay was used to determine the effects of ginger constituents on Ras homolog gene family member A activation. Significant potentiation of isoproterenol-induced relaxation was observed with each of the ginger constituents. 6-Shogaol showed the largest shift in isoproterenol half-maximal effective concentration. 6-Gingerol, 8-gingerol, or 6-shogaol significantly inhibited PDE4D, whereas 8-gingerol and 6-shogaol also inhibited phospholipase C β activity. 6-Shogaol alone inhibited Ras homolog gene family member A activation. In human ASM cells, these constituents decreased phosphorylation of 17-kD protein kinase C–potentiated inhibitory protein of type 1 protein phosphatase and 8-gingerol decreased myosin light chain phosphorylation. Isolated components of ginger potentiate β-agonist–induced relaxation in human ASM. This potentiation involves PDE4D inhibition and cytoskeletal regulatory proteins. Together with β-agonists, 6-gingerol, 8-gingerol, or 6-shogaol may augment existing asthma therapy, resulting in relief of symptoms

  3. Store-operated Ca2+ entry is involved in transforming growth factor-β1 facilitated proliferation of rat airway smooth muscle cells.

    PubMed

    Gao, Ya-Dong; Zheng, Jun-Wen; Li, Ping; Cheng, Ming; Yang, Jiong

    2013-06-01

    To investigate the role and underlying mechanisms of store-operated Ca(2+) entry (SOCE) in mediating the promoting effect of transforming growth factor (TGF)-β1 on the proliferation of airway smooth muscle cells (ASMCs). Rat bronchial smooth muscle cells were cultured as we described previously. The intracellular Ca(2+) concentration ([Ca(2+)]i) of ASMCs was measured by laser confocal microscope Ca(2+) fluorescence imaging with Fluo-3/AM. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and p27 expression assay were used to determine the proliferation rate of ASMCs. We demonstrated that TGF-β1 (10 ng/ml) increased basal (Ca(2+)]i) level, [Ca(2+)]i rise induced by thapsigargin-induced Ca(2+) release and SOCE in rat ASMCs. This effect of TGF-β1 on SOCE was not inhibited by glucocorticoid dexamethasone (DXM, 100 nM), antioxidant α-tocopherol (100 μM), and intermediate-conductance Ca(2+)-activated K(+) channels (IKCa) inhibitor charybdotoxin (100 nM), suggesting that reactive oxygen species and IKCa channels might not mediate the effect of TGF-β1. TGF-β1 slightly increased the expression of Orai1 and STIM1, two important molecules involved in the molecule component and regulation of SOC channels, in the presence of 10% fetal bovine serum (FBS). The proliferation of ASMC stimulated with 2.5% FBS was promoted by TGF-β1, and partly inhibited by non-specific Ca(2+) channel blocker SKF-96365 (10 μM) and Ni(2+) (100 μM). DXM, α-tocopherol, and charybdotoxin had no effect on the proliferation promoted by TGF-β1. TGF-β1 promotes ASMC proliferation partly through increasing the expression and activity of SOC channels.

  4. Anti-CTGF single-chain variable fragment dimers inhibit human airway smooth muscle (ASM) cell proliferation by down-regulating p-Akt and p-mTOR levels.

    PubMed

    Gao, Wei; Cai, Liting; Xu, Xudong; Fan, Juxiang; Xue, Xiulei; Yan, Xuejiao; Qu, Qinrong; Wang, Xihua; Zhang, Chen; Wu, Guoqiu

    2014-01-01

    Connective tissue growth factor (CTGF) contributes to airway smooth muscle (ASM) cell hyperplasia in asthma. Humanized single-chain variable fragment antibody (scFv) was well characterized as a CTGF antagonist in the differentiation of fibroblast into myofibroblast and pulmonary fibrosis in our previous studies. To further improve the bioactivity of scFv, we constructed a plasmid to express scFv-linker-matrilin-6×His fusion proteins that could self-assemble into the scFv dimers by disulfide bonds in matrilin under non-reducing conditions. An immunoreactivity assay demonstrated that the scFv dimer could highly bind to CTGF in a concentration-dependent manner. The MTT and EdU assay results revealed that CTGF (≥10 ng/mL) promoted the proliferation of ASM cells, and this effect was inhibited when the cells were treated with anti-CTGF scFv dimer. The western blot analysis results showed that increased phosphorylation of Akt and mTOR induced by CTGF could be suppressed by this scFv dimer. Based on these findings, anti-CTGF scFv dimer may be a potential agent for the prevention of airway remodeling in asthma.

  5. Anti-CTGF Single-Chain Variable Fragment Dimers Inhibit Human Airway Smooth Muscle (ASM) Cell Proliferation by Down-Regulating p-Akt and p-mTOR Levels

    PubMed Central

    Xu, Xudong; Fan, Juxiang; Xue, Xiulei; Yan, Xuejiao; Qu, Qinrong; Wang, Xihua; Zhang, Chen; Wu, Guoqiu

    2014-01-01

    Connective tissue growth factor (CTGF) contributes to airway smooth muscle (ASM) cell hyperplasia in asthma. Humanized single-chain variable fragment antibody (scFv) was well characterized as a CTGF antagonist in the differentiation of fibroblast into myofibroblast and pulmonary fibrosis in our previous studies. To further improve the bioactivity of scFv, we constructed a plasmid to express scFv-linker-matrilin-6×His fusion proteins that could self-assemble into the scFv dimers by disulfide bonds in matrilin under non-reducing conditions. An immunoreactivity assay demonstrated that the scFv dimer could highly bind to CTGF in a concentration-dependent manner. The MTT and EdU assay results revealed that CTGF (≥10 ng/mL) promoted the proliferation of ASM cells, and this effect was inhibited when the cells were treated with anti-CTGF scFv dimer. The western blot analysis results showed that increased phosphorylation of Akt and mTOR induced by CTGF could be suppressed by this scFv dimer. Based on these findings, anti-CTGF scFv dimer may be a potential agent for the prevention of airway remodeling in asthma. PMID:25478966

  6. Effects of epithelium removal on relaxation of airway smooth muscle induced by vasoactive intestinal peptide and electrical field stimulation.

    PubMed Central

    Farmer, S. G.; Togo, J.

    1990-01-01

    1. We have studied the effect of epithelium removal on relaxation of guinea-pig isolated tracheal smooth muscle induced by vasoactive intestinal peptide (VIP) or stimulation of non-adrenergic, non-cholinergic (NANC) inhibitory nerves. Also examined were the effects of inhibitors of neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE). 2. Epithelium removal produced a 3.6 +/- 0.4 fold leftward shift in the VIP concentration-response curve. The supersensitivity to VIP, following epithelium removal was abolished by phosphoramidon or thiorphan (NEP inhibitors), but unaffected by captopril (an ACE inhibitor). In intact trachea, the NEP inhibitors produced leftward shifts in the VIP curves similar to those produced by epithelium removal. 3. In contrast to responses to exogenous VIP, neurogenic NANC inhibitory responses to electrical field stimulation were affected neither by epithelial denudation nor by the peptidase inhibitors. 4. As in previous studies, epithelium removal increased tracheal sensitivity to isoprenaline. This was not altered by pretreatment with a cocktail of peptidase inhibitors. Thus, the effect of the NEP inhibitors on responses to VIP appears to be relatively specific. 5. These data indicate that exogenous VIP is a substrate for airway NEP, since inhibition of the enzyme potentiates the peptide. This is further evidence that the airway epithelium provides a source for the metabolism of mediators. 6. In guinea-pig trachea the NEP responsible for cleaving VIP may be located largely in the epithelial layer, since NEP inhibition was without effect on sensitivity to VIP in epithelium-denuded preparations. If VIP is a NANC inhibitory neurotransmitter in this tissue its degradation endogenously does not appear to involve epithelial NEP. PMID:2196967

  7. Porcine Stomach Smooth Muscle Force Depends on History-Effects.

    PubMed

    Tomalka, André; Borsdorf, Mischa; Böl, Markus; Siebert, Tobias

    2017-01-01

    The stomach serves as food reservoir, mixing organ and absorption area for certain substances, while continually varying its position and size. Large dimensional changes during ingestion and gastric emptying of the stomach are associated with large changes in smooth muscle length. These length changes might induce history-effects, namely force depression (FD) following active muscle shortening and force enhancement (FE) following active muscle stretch. Both effects have impact on the force generating capacity of the stomach, and thus functional relevance. However, less is known about history-effects and active smooth muscle properties of stomach smooth muscle. Thus, the aim of this study was to investigate biomechanical muscle properties as force-length and force-velocity relations (FVR) of porcine stomach smooth muscle strips, extended by the analysis of history-effects on smooth muscle force. Therefore, in total n = 54 tissue strips were dissected in longitudinal direction from the ventral fundus of porcine stomachs. Different isometric, isotonic, and isokinetic contraction protocols were performed during electrical muscle stimulation. Cross-sectional areas (CSA) of smooth muscles were determined from cryo-histological sections stained with Picrosirius Red. Results revealed that maximum smooth muscle tension was 10.4 ± 2.6 N/cm 2 . Maximum shortening velocity ( V max ) and curvature factor ( curv ) of the FVR were 0.04 ± 0.01 [optimum muscle length/s] and 0.36 ± 0.15, respectively. The findings of the present study demonstrated significant ( P < 0.05) FD [up to 32% maximum muscle force ( F im )] and FE (up to 16% F im ) of gastric muscle tissue, respectively. The FE- and FD-values increased with increasing ramp amplitude. This outstanding muscle behavior is not accounted for in existing models so far and strongly supports the idea of a holistic reflection of distinct stomach structure and function. For the first time this study provides a comprehensive set of

  8. Porcine Stomach Smooth Muscle Force Depends on History-Effects

    PubMed Central

    Tomalka, André; Borsdorf, Mischa; Böl, Markus; Siebert, Tobias

    2017-01-01

    The stomach serves as food reservoir, mixing organ and absorption area for certain substances, while continually varying its position and size. Large dimensional changes during ingestion and gastric emptying of the stomach are associated with large changes in smooth muscle length. These length changes might induce history-effects, namely force depression (FD) following active muscle shortening and force enhancement (FE) following active muscle stretch. Both effects have impact on the force generating capacity of the stomach, and thus functional relevance. However, less is known about history-effects and active smooth muscle properties of stomach smooth muscle. Thus, the aim of this study was to investigate biomechanical muscle properties as force-length and force-velocity relations (FVR) of porcine stomach smooth muscle strips, extended by the analysis of history-effects on smooth muscle force. Therefore, in total n = 54 tissue strips were dissected in longitudinal direction from the ventral fundus of porcine stomachs. Different isometric, isotonic, and isokinetic contraction protocols were performed during electrical muscle stimulation. Cross-sectional areas (CSA) of smooth muscles were determined from cryo-histological sections stained with Picrosirius Red. Results revealed that maximum smooth muscle tension was 10.4 ± 2.6 N/cm2. Maximum shortening velocity (Vmax) and curvature factor (curv) of the FVR were 0.04 ± 0.01 [optimum muscle length/s] and 0.36 ± 0.15, respectively. The findings of the present study demonstrated significant (P < 0.05) FD [up to 32% maximum muscle force (Fim)] and FE (up to 16% Fim) of gastric muscle tissue, respectively. The FE- and FD-values increased with increasing ramp amplitude. This outstanding muscle behavior is not accounted for in existing models so far and strongly supports the idea of a holistic reflection of distinct stomach structure and function. For the first time this study provides a comprehensive set of stomach

  9. Androgens are bronchoactive drugs that act by relaxing airway smooth muscle and preventing bronchospasm.

    PubMed

    Montaño, Luis M; Espinoza, Julia; Flores-Soto, Edgar; Chávez, Jaime; Perusquía, Mercedes

    2014-07-01

    Changes in the androgen levels in asthmatic men may be associated with the severity of asthma. Androgens induce a nongenomic relaxation in airway smooth muscle, but the underlying mechanisms remain unclear. The aim of this study was to investigate the potential bronchorelaxing action of testosterone (TES) and its metabolites (5α- and 5β-dihydrotestosterone (DHT). A preventive effect on ovalbumin (OVA)-induced bronchospasm was observed in sensitized guinea pigs for each androgen. Androgens were studied in response to bronchoconstrictors: carbachol (CCh) and KCl in isolated trachea rings with and without epithelium from non-sensitized and sensitized animals as well as on OVA-induced contraction. Androgens concentration-dependently abolished the contraction in response to CCh, KCl, and OVA. There were significant differences in the sensitivity to the relaxation induced by each androgen. 5β-DHT was more potent for relaxing KCl-induced contraction, while TES and 5α-DHT were more potent for CCh- and OVA-induced contraction. No differences were found in preparations with and without epithelium or in the presence of a nitric oxide (NO) synthase inhibitor or an inhibitor of K(+) channels. These data indicate the absence of involvement of the epithelium-, NO- and K(+) channels-dependent pathway in androgen-induced relaxation. However, in dissociated tracheal myocytes loaded with the calcium-binding fluorescent dye Fura -2, physiological concentrations of androgens decreased the KCl-induced [Ca(2+)]i increment. 5β-DHT was the most potent at decreasing KCl-induced [Ca(2+)]i increment and preventing bronchospasm. We suggest that androgen-induced brochorelaxation was mediated via decreased Ca(2+) influx through L-type Ca(2+)channels but additional Ca(2+) entry blockade may be involved. Molecular changes in androgen structure may determine its preferential site of action. © 2014 Society for Endocrinology.

  10. A-type potassium currents in smooth muscle.

    PubMed

    Amberg, Gregory C; Koh, Sang Don; Imaizumi, Yuji; Ohya, Susumu; Sanders, Kenton M

    2003-03-01

    A-type currents are voltage-gated, calcium-independent potassium (Kv) currents that undergo rapid activation and inactivation. Commonly associated with neuronal and cardiac cell-types, A-type currents have also been identified and characterized in vascular, genitourinary, and gastrointestinal smooth muscle cells. This review examines the molecular identity, biophysical properties, pharmacology, regulation, and physiological function of smooth muscle A-type currents. In general, this review is intended to facilitate the comparison of A-type currents present in different smooth muscles by providing a comprehensive report of the literature to date. This approach should also aid in the identification of areas of research requiring further attention.

  11. Prostaglandins, oxygen tension and smooth muscle tone

    PubMed Central

    Eckenfels, A.; Vane, J. R.

    1972-01-01

    1. By using indomethacin to inhibit their intramural synthesis, we have investigated the contribution of prostaglandins to the maintenance of (a) the intrinsic tone of isolated smooth muscle preparations and (b) contractions produced by drugs or high oxygen concentration. 2. When treated with indomethacin, the rat stomach strip and chick rectum preparation slowly relaxed, whether they were bathed in Krebs solution or blood. Although their sensitivity to added prostaglandin was somewhat enhanced, they became insensitive to changes in oxygen or glucose concentration. However, another smooth muscle preparation, the rat colon, was neither relaxed by indomethacin nor contracted by high oxygen concentration. 3. These results support the hypothesis that intramural generation of prostaglandin maintains the tone of some smooth muscle preparations. 4. Contractions of the guinea-pig isolated colon were induced by histamine. These contractions were normally well maintained but in Krebs solution lacking either oxygen or glucose, only the initial spike contraction remained. In the presence of indomethacin the histamine contraction was also poorly sustained, but maintenance was restored by a low concentration of prostaglandin E2. 5. Thus, the effects on smooth muscle of oxygen or glucose lack may also be mediated by reduction in the synthesis or effects of an intramural prostaglandin. Extension of this hypothesis to intestinal and vascular smooth muscle in vivo is discussed. PMID:5072227

  12. Mitochondrial motility and vascular smooth muscle proliferation.

    PubMed

    Chalmers, Susan; Saunter, Christopher; Wilson, Calum; Coats, Paul; Girkin, John M; McCarron, John G

    2012-12-01

    Mitochondria are widely described as being highly dynamic and adaptable organelles, and their movement is thought to be vital for cell function. Yet, in various native cells, including those of heart and smooth muscle, mitochondria are stationary and rigidly structured. The significance of the differences in mitochondrial behavior to the physiological function of cells is unclear and was studied in single myocytes and intact resistance-sized cerebral arteries. We hypothesized that mitochondrial dynamics is controlled by the proliferative status of the cells. High-speed fluorescence imaging of mitochondria in live vascular smooth muscle cells shows that the organelle undergoes significant reorganization as cells become proliferative. In nonproliferative cells, mitochondria are individual (≈ 2 μm by 0.5 μm), stationary, randomly dispersed, fixed structures. However, on entering the proliferative state, mitochondria take on a more diverse architecture and become small spheres, short rod-shaped structures, long filamentous entities, and networks. When cells proliferate, mitochondria also continuously move and change shape. In the intact pressurized resistance artery, mitochondria are largely immobile structures, except in a small number of cells in which motility occurred. When proliferation of smooth muscle was encouraged in the intact resistance artery, in organ culture, the majority of mitochondria became motile and the majority of smooth muscle cells contained moving mitochondria. Significantly, restriction of mitochondrial motility using the fission blocker mitochondrial division inhibitor prevented vascular smooth muscle proliferation in both single cells and the intact resistance artery. These results show that mitochondria are adaptable and exist in intact tissue as both stationary and highly dynamic entities. This mitochondrial plasticity is an essential mechanism for the development of smooth muscle proliferation and therefore presents a novel therapeutic

  13. A deterministic model predicts the properties of stochastic calcium oscillations in airway smooth muscle cells.

    PubMed

    Cao, Pengxing; Tan, Xiahui; Donovan, Graham; Sanderson, Michael J; Sneyd, James

    2014-08-01

    The inositol trisphosphate receptor ([Formula: see text]) is one of the most important cellular components responsible for oscillations in the cytoplasmic calcium concentration. Over the past decade, two major questions about the [Formula: see text] have arisen. Firstly, how best should the [Formula: see text] be modeled? In other words, what fundamental properties of the [Formula: see text] allow it to perform its function, and what are their quantitative properties? Secondly, although calcium oscillations are caused by the stochastic opening and closing of small numbers of [Formula: see text], is it possible for a deterministic model to be a reliable predictor of calcium behavior? Here, we answer these two questions, using airway smooth muscle cells (ASMC) as a specific example. Firstly, we show that periodic calcium waves in ASMC, as well as the statistics of calcium puffs in other cell types, can be quantitatively reproduced by a two-state model of the [Formula: see text], and thus the behavior of the [Formula: see text] is essentially determined by its modal structure. The structure within each mode is irrelevant for function. Secondly, we show that, although calcium waves in ASMC are generated by a stochastic mechanism, [Formula: see text] stochasticity is not essential for a qualitative prediction of how oscillation frequency depends on model parameters, and thus deterministic [Formula: see text] models demonstrate the same level of predictive capability as do stochastic models. We conclude that, firstly, calcium dynamics can be accurately modeled using simplified [Formula: see text] models, and, secondly, to obtain qualitative predictions of how oscillation frequency depends on parameters it is sufficient to use a deterministic model.

  14. Vardenafil inhibiting parasympathetic function of tracheal smooth muscle.

    PubMed

    Lee, Fei-Peng; Chao, Pin-Zhir; Wang, Hsing-Won

    2018-07-01

    Levitra, a phosphodiesterase-5 (PDE5) inhibitor, is the trade name of vardenafil. Nowadays, it is applied to treatment of erectile dysfunction. PDE5 inhibitors are employed to induce dilatation of the vascular smooth muscle. The effect of Levitra on impotency is well known; however, its effect on the tracheal smooth muscle has rarely been explored. When administered for sexual symptoms via oral intake or inhalation, Levitra might affect the trachea. This study assessed the effects of Levitra on isolated rat tracheal smooth muscle by examining its effect on resting tension of tracheal smooth muscle, contraction caused by 10 -6  M methacholine as a parasympathetic mimetic, and electrically induced tracheal smooth muscle contractions. The results showed that adding methacholine to the incubation medium caused the trachea to contract in a dose-dependent manner. Addition of Levitra at doses of 10 -5  M or above elicited a significant relaxation response to 10 -6  M methacholine-induced contraction. Levitra could inhibit electrical field stimulation-induced spike contraction. It alone had minimal effect on the basal tension of the trachea as the concentration increased. High concentrations of Levitra could inhibit parasympathetic function of the trachea. Levitra when administered via oral intake might reduce asthma attacks in impotent patients because it might inhibit parasympathetic function and reduce methacholine-induced contraction of the tracheal smooth muscle. Copyright © 2018. Published by Elsevier Taiwan LLC.

  15. Gα12 facilitates shortening in human airway smooth muscle by modulating phosphoinositide 3-kinase-mediated activation in a RhoA-dependent manner.

    PubMed

    Yoo, Edwin J; Cao, Gaoyuan; Koziol-White, Cynthia J; Ojiaku, Christie A; Sunder, Krishna; Jude, Joseph A; Michael, James V; Lam, Hong; Pushkarsky, Ivan; Damoiseaux, Robert; Di Carlo, Dino; Ahn, Kwangmi; An, Steven S; Penn, Raymond B; Panettieri, Reynold A

    2017-12-01

    PI3K-dependent activation of Rho kinase (ROCK) is necessary for agonist-induced human airway smooth muscle cell (HASMC) contraction, and inhibition of PI3K promotes bronchodilation of human small airways. The mechanisms driving agonist-mediated PI3K/ROCK axis activation, however, remain unclear. Given that G 12 family proteins activate ROCK pathways in other cell types, their role in M 3 muscarinic acetylcholine receptor-stimulated PI3K/ROCK activation and contraction was examined. Gα 12 coupling was evaluated using co-immunoprecipitation and serum response element (SRE)-luciferase reporter assays. siRNA and pharmacological approaches, as well as overexpression of a regulator of G-protein signaling (RGS) proteins were applied in HASMCs. Phosphorylation levels of Akt, myosin phosphatase targeting subunit-1 (MYPT1), and myosin light chain-20 (MLC) were measured. Contraction and shortening were evaluated using magnetic twisting cytometry (MTC) and micro-pattern deformation, respectively. Human precision-cut lung slices (hPCLS) were utilized to evaluate bronchoconstriction. Knockdown of M 3 receptors or Gα 12 attenuated activation of Akt, MYPT1, and MLC phosphorylation. Gα 12 coimmunoprecipitated with M 3 receptors, and p115RhoGEF-RGS overexpression inhibited carbachol-mediated induction of SRE-luciferase reporter. p115RhoGEF-RGS overexpression inhibited carbachol-induced activation of Akt, HASMC contraction, and shortening. Moreover, inhibition of RhoA blunted activation of PI3K. Lastly, RhoA inhibitors induced dilation of hPCLS. Gα 12 plays a crucial role in HASMC contraction via RhoA-dependent activation of the PI3K/ROCK axis. Inhibition of RhoA activation induces bronchodilation in hPCLS, and targeting Gα 12 signaling may elucidate novel therapeutic targets in asthma. These findings provide alternative approaches to the clinical management of airway obstruction in asthma. © 2017 The British Pharmacological Society.

  16. β2-Agonist Induced cAMP Is Decreased in Asthmatic Airway Smooth Muscle Due to Increased PDE4D

    PubMed Central

    Trian, Thomas; Burgess, Janette K.; Niimi, Kyoko; Moir, Lyn M.; Ge, Qi; Berger, Patrick; Liggett, Stephen B.; Black, Judith L.; Oliver, Brian G.

    2011-01-01

    Background and Objective Asthma is associated with airway narrowing in response to bronchoconstricting stimuli and increased airway smooth muscle (ASM) mass. In addition, some studies have suggested impaired β-agonist induced ASM relaxation in asthmatics, but the mechanism is not known. Objective To characterize the potential defect in β-agonist induced cAMP in ASM derived from asthmatic in comparison to non-asthmatic subjects and to investigate its mechanism. Methods We examined β2-adrenergic (β2AR) receptor expression and basal β-agonist and forskolin (direct activator of adenylyl cyclase) stimulated cAMP production in asthmatic cultured ASM (n = 15) and non-asthmatic ASM (n = 22). Based on these results, PDE activity, PDE4D expression and cell proliferation were determined. Results In the presence of IBMX, a pan PDE inhibitor, asthmatic ASM had ∼50% lower cAMP production in response to isoproterenol, albuterol, formoterol, and forskolin compared to non-asthmatic ASM. However when PDE4 was specifically inhibited, cAMP production by the agonists and forskolin was normalized in asthmatic ASM. We then measured the amount and activity of PDE4, and found ∼2-fold greater expression and activity in asthmatic ASM compared to non-asthmatic ASM. Furthermore, inhibition of PDE4 reduced asthmatic ASM proliferation but not that of non-asthmatic ASM. Conclusion Decreased β-agonist induced cAMP in ASM from asthmatics results from enhanced degradation due to increased PDE4D expression. Clinical manifestations of this dysregulation would be suboptimal β-agonist-mediated bronchodilation and possibly reduced control over increasing ASM mass. These phenotypes appear to be “hard-wired” into ASM from asthmatics, as they do not require an inflammatory environment in culture to be observed. PMID:21611147

  17. Influence of parainfluenza-1 respiratory tract viral infection on endothelin receptor-effector systems in mouse and rat tracheal smooth muscle.

    PubMed Central

    Knott, P. G.; Henry, P. J.; McWilliam, A. S.; Rigby, P. J.; Fernandes, L. B.; Goldie, R. G.

    1996-01-01

    1. In this study we have compared the effects of parainfluenza-1 respiratory tract viral infection on the density and function of ETA and ETB receptors in rat and mouse tracheal airway smooth muscle. 2. The bronchoconstrictor effect of inhaled methacholine was significantly enhanced in virus-infected rats, at both 4 and 12 days post-inoculation. That is, the concentration of methacholine causing an increase in resistance of 100% (PC100 methacholine) was significantly lower in virus-infected animals at both 4 and 12 days post-inoculation (n = 6-8; P < 0.05). 3. Total specific binding of [125I]-endothelin-1 and the relative proportions of ETA and ETB binding sites for [125I]-endothelin-1 were assessed in tracheal airway smooth muscle in parainfluenza-1-infected rats and mice at days 2, 4 and 12 post-inoculation using the ligands BQ-123 (1 microM; ETA receptor-selective) and sarafotoxin S6c (100 nM; ETB receptor-selective). Total specific binding in mice was significantly reduced at day 2 post-inoculation (n = 5; P < 0.05) but not at days 4 and 12 post-inoculation (n = 5). In control mice, the proportions of ETA and ETB binding sites were 53%:47% at day 2 and 43%:57% at day 4 and these were significantly altered by parainfluenza-1 infection such that, the ratios were 81%:19% at day 2 and 89%:11% at day 4 (P < 0.05). By day 12 post-inoculation, the proportion of ETA and ETB binding sites in tracheal smooth muscle from mice infected with parainfluenza-1 was not significantly different from control. In rat tracheal airway smooth muscle, neither total specific binding nor the ETA and ETB binding site ratio (64%:36%) were significantly altered in virus-inoculated rats at days 2, 4 or 12 post-inoculation (n = 5). 4. Parainfluenza-1 infection in mice had no effect on the sensitivity or maximal contractile effect of endothelin-1 in tracheal smooth muscle at days 2, 4 or 12 post-inoculation (n = 4). In contrast, contraction in response to the ETB receptor-selective agonist

  18. Mechanisms of Vascular Smooth Muscle Contraction and the Basis for Pharmacologic Treatment of Smooth Muscle Disorders

    PubMed Central

    Brozovich, F.V.; Nicholson, C.J.; Degen, C.V.; Gao, Yuan Z.; Aggarwal, M.

    2016-01-01

    The smooth muscle cell directly drives the contraction of the vascular wall and hence regulates the size of the blood vessel lumen. We review here the current understanding of the molecular mechanisms by which agonists, therapeutics, and diseases regulate contractility of the vascular smooth muscle cell and we place this within the context of whole body function. We also discuss the implications for personalized medicine and highlight specific potential target molecules that may provide opportunities for the future development of new therapeutics to regulate vascular function. PMID:27037223

  19. Disturbance of smooth muscle regulatory function by Eisenia foetida toxin lysenin: insight into the mechanism of smooth muscle contraction.

    PubMed

    Czuryło, Edward A; Kulikova, Natalia; Sobota, Andrzej

    2008-05-01

    Lysenin, a toxin present in the coelomic fluid of the earthworm Eisenia foetida, is known to cause a long-lasting contraction of rat aorta smooth muscle strips. We addressed the mechanisms underlying its action on smooth muscle cells and present the first report demonstrating a completely new property of lysenin unrelated to its basic sphingomyelin-binding ability. Here we report lysenin enhancement effect on smooth muscle actomyosin ATPase activity and the ability of networking the actin filaments. The maximum enhancement of the ATPase activity of actomyosin at 120 mM KCl was observed at a molar ratio of lysenin to actin of about 1:10(5), while at 70 mM KCl at the ratio of about 1:10(6). The effect of lysenin became most pronounced only when both smooth muscle regulatory proteins, tropomyosin and caldesmon, were present. Co-sedimentation experiments indicated that lysenin did not displace neither tropomyosin nor caldesmon from the thin filament. Thus, the lysenin-dependent abolishment of the inhibitory effect of caldesmon on the ATPase activity was related rather to the modification of the filament structure. The ability of the toxin to exert its stimulatory effect at extremely low concentrations (as low as one molecule of lysenin per 10(6) actin molecules) may result from the long-range cooperative transitions in the entire thin filament with an involvement of smooth muscle tropomyosin, while the role of caldesmon may be limited exclusively to the inhibition of ATPase activity.

  20. Stimulation of aortic smooth muscle cell mitogenesis by serotonin

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nemecek, G.M.; Coughlin, S.R.; Handley, D.A.

    1986-02-01

    Bovine aortic smooth muscle cells in vitro responded to 1 nM to 10 ..mu..M serotonin with increased incorporation of (/sup 3/H)thymidine into DNA. The mitogenic effect of serotonin was half-maximal at 80 nM and maximal above 1 ..mu..M. At a concentration of 1 ..mu..M, serotonin stimulated smooth muscle cell mitogenesis to the same extent as human platelet-derived growth factor (PDGF) at 12 ng/ml. Tryptamine was approx. = 1/10th as potent as serotonin as a mitogen for smooth muscle cells. Other indoles that are structurally related to serotonin (D- and L-tryptophan, 5-hydroxy-L-tryptophan, N-acetyl-5-hydroxytryptamine, melatonin, 5-hydroxyindoleacetic acid, and 5-hydroxytryptophol) and quipazine weremore » inactive. The stimulatory effect of serotonin on smooth muscle cell DNA synthesis required prolonged (20-24 hr) exposure to the agonist and was attenuated in the presence of serotonin D receptor antagonists. When smooth muscle cells were incubated with submaximal concentrations of serotonin and PDGF, synergistic rather than additive mitogenic responses were observed. These data indicate that serotonin has a significant mitogenic effect on smooth muscle cells in vitro, which appears to be mediated by specific plasma membrane receptors.« less

  1. Tracheal smooth muscle responses to substance P and neurokinin A in the piglet.

    PubMed

    Haxhiu-Poskurica, B; Haxhiu, M A; Kumar, G K; Miller, M J; Martin, R J

    1992-03-01

    The tachykinins substance P (SP) and neurokinin A (NKA) have been shown to induce airway smooth muscle contraction in mature animals, and the enzyme neutral endopeptidase (NEP) modulates this effect. We evaluated maturation of SP- and NKA-induced tracheal smooth muscle contraction and modulation of their effects by NEP in anesthetized, paralyzed, and artificially ventilated piglets less than 4 days, 2-3 wk, and 10 wk of age. Tracheal smooth muscle tension was measured in vivo from an open tracheal segment by use of a force transducer. Intravenous SP caused a dose-dependent increase in tracheal tension in all three age groups; however, the response in less than 4-day-old piglets was significantly weaker than in 2- to 3- and 10-wk-old piglets. NKA caused a dose-dependent increase in tracheal tension only in 2- to 3- and 10-wk-old piglets. The response of tracheal tension to NKA was weaker than the response to SP in all age groups. Atropine (2 mg/kg) significantly diminished the responses of tracheal tension to SP and NKA, indicating a cholinergic contribution to these responses at all ages. Intravenous thiorphan, a known NEP inhibitor, potentiated the effects of SP only in 2- to 3- and 10-wk-old piglets and did not affect the response of tracheal tension to NKA at any age. Biochemical analyses demonstrated a significant increase in tracheal NEP activity in comparably aged piglets over the first 10 wk of life.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Cisapride stimulates contraction of idiopathic megacolonic smooth muscle in cats.

    PubMed

    Hasler, A H; Washabau, R J

    1997-01-01

    We have previously shown that cisapride, a substituted piperidinyl benzamide, stimulates contraction of healthy feline colonic smooth muscle. The purpose of the present investigation was to determine the effect of cisapride on feline idiopathic megacolonic smooth muscle function. Longitudinal smooth muscle strips from ascending and descending colon were obtained from cats with idiopathic megacolon, suspended in a 1.5 mM Ca(2+)-HEPES buffer solution (37 degrees C, 100% O2, pH 7.4), attached to isometric force transducers, and stretched to optimal muscle length (Lo). Control responses were obtained at each muscle site with acetylcholine (10(-8) to 10(-4) M), substance P (10(-11) to 10(-7) M), or potassium chloride (10 to 80 mM). Muscles were then stimulated with cumulative (10(-9) to 10(-6) M) doses of cisapride in the absence or presence of tetrodotoxin (10(-6) M) and atropine (10(-6) M), or in a 0 calcium HEPES buffer solution. In cats with idiopathic megacolon, cisapride stimulated contractions of longitudinal smooth muscle from both the ascending and the descending colon. Cisapride-induced contractions were similar in magnitude to those induced by substance P and acetylcholine in the ascending colon, but were less than those observed in the descending colon. Cisapride-induced contractions in megacolonic smooth muscle were only partially inhibited by tetrodotoxin and atropine, but were virtually abolished by removal of extracellular calcium. We concluded that cisapride-induced contractions of feline megacolonic smooth muscle are largely smooth muscle mediated and dependent on influx of extracellular calcium. Cisapride-induced contractions in megacolonic smooth muscle are only partially dependent on enteric cholinergic nerves. Thus, cisapride may be useful in the treatment of cats with idiopathic megacolon.

  3. Importance of contraction history on muscle force of porcine urinary bladder smooth muscle.

    PubMed

    Menzel, Robin; Böl, Markus; Siebert, Tobias

    2017-02-01

    The purpose of this study was to provide a comprehensive dataset of porcine urinary bladder smooth muscle properties. Particularly, the history dependence of force production, namely force depression (FD) following shortening and force enhancement (FE) following stretch, was analysed. During active micturition, the circumference of the urinary bladder changes enormously. Thus, FD might be an important phenomenon during smooth muscle contraction. Electrically stimulated, intact urinary bladder strips from pigs (n = 10) were suspended in an aerated-filled organ bath, and different isometric, isotonic, and isokinetic contraction protocols were performed to determine the force-length and the force-velocity relation. FD and FE were assessed in concentric and eccentric contractions with different ramp lengths and ramp velocities. Bladder smooth muscles exhibit considerable amounts of FD and FE. The amount of FD increased significantly with ramp length, while FE did not change. However, FE and FD were independent of ramp velocity. The results imply that smooth muscle bladder strips exhibit similar muscle properties and history-dependent behaviour compared to striated muscles. The provided dataset of muscle properties is important for bladder modelling as well as for the analyses and interpretation of dynamic bladder filling and voiding.

  4. Ureter smooth muscle cell orientation in rat is predominantly longitudinal.

    PubMed

    Spronck, Bart; Merken, Jort J; Reesink, Koen D; Kroon, Wilco; Delhaas, Tammo

    2014-01-01

    In ureter peristalsis, the orientation of the contracting smooth muscle cells is essential, yet current descriptions of orientation and composition of the smooth muscle layer in human as well as in rat ureter are inconsistent. The present study aims to improve quantification of smooth muscle orientation in rat ureters as a basis for mechanistic understanding of peristalsis. A crucial step in our approach is to use two-photon laser scanning microscopy and image analysis providing objective, quantitative data on smooth muscle cell orientation in intact ureters, avoiding the usual sectioning artifacts. In 36 rat ureter segments, originating from a proximal, middle or distal site and from a left or right ureter, we found close to the adventitia a well-defined longitudinal smooth muscle orientation. Towards the lamina propria, the orientation gradually became slightly more disperse, yet the main orientation remained longitudinal. We conclude that smooth muscle cell orientation in rat ureter is predominantly longitudinal, though the orientation gradually becomes more disperse towards the proprial side. These findings do not support identification of separate layers. The observed longitudinal orientation suggests that smooth muscle contraction would rather cause local shortening of the ureter, than cause luminal constriction. However, the net-like connective tissue of the ureter wall may translate local longitudinal shortening into co-local luminal constriction, facilitating peristalsis. Our quantitative, minimally invasive approach is a crucial step towards more mechanistic insight into ureter peristalsis, and may also be used to study smooth muscle cell orientation in other tube-like structures like gut and blood vessels.

  5. Modeling the dispersion effects of contractile fibers in smooth muscles

    NASA Astrophysics Data System (ADS)

    Murtada, Sae-Il; Kroon, Martin; Holzapfel, Gerhard A.

    2010-12-01

    Micro-structurally based models for smooth muscle contraction are crucial for a better understanding of pathological conditions such as atherosclerosis, incontinence and asthma. It is meaningful that models consider the underlying mechanical structure and the biochemical activation. Hence, a simple mechanochemical model is proposed that includes the dispersion of the orientation of smooth muscle myofilaments and that is capable to capture available experimental data on smooth muscle contraction. This allows a refined study of the effects of myofilament dispersion on the smooth muscle contraction. A classical biochemical model is used to describe the cross-bridge interactions with the thin filament in smooth muscles in which calcium-dependent myosin phosphorylation is the only regulatory mechanism. A novel mechanical model considers the dispersion of the contractile fiber orientations in smooth muscle cells by means of a strain-energy function in terms of one dispersion parameter. All model parameters have a biophysical meaning and may be estimated through comparisons with experimental data. The contraction of the middle layer of a carotid artery is studied numerically. Using a tube the relationships between the internal pressure and the stretches are investigated as functions of the dispersion parameter, which implies a strong influence of the orientation of smooth muscle myofilaments on the contraction response. It is straightforward to implement this model in a finite element code to better analyze more complex boundary-value problems.

  6. On the thermodynamics of smooth muscle contraction

    NASA Astrophysics Data System (ADS)

    Stålhand, Jonas; McMeeking, Robert M.; Holzapfel, Gerhard A.

    2016-09-01

    Cell function is based on many dynamically complex networks of interacting biochemical reactions. Enzymes may increase the rate of only those reactions that are thermodynamically consistent. In this paper we specifically treat the contraction of smooth muscle cells from the continuum thermodynamics point of view by considering them as an open system where matter passes through the cell membrane. We systematically set up a well-known four-state kinetic model for the cross-bridge interaction of actin and myosin in smooth muscle, where the transition between each state is driven by forward and reverse reactions. Chemical, mechanical and energy balance laws are provided in local forms, while energy balance is also formulated in the more convenient temperature form. We derive the local (non-negative) production of entropy from which we deduce the reduced entropy inequality and the constitutive equations for the first Piola-Kirchhoff stress tensor, the heat flux, the ion and molecular flux and the entropy. One example for smooth muscle contraction is analyzed in more detail in order to provide orientation within the established general thermodynamic framework. In particular the stress evolution, heat generation, muscle shorting rate and a condition for muscle cooling are derived.

  7. Loop diuretics inhibit cholinergic and noncholinergic nerves in guinea pig airways.

    PubMed

    Elwood, W; Lötvall, J O; Barnes, P J; Chung, K F

    1991-06-01

    Furosemide, a loop diuretic, is known to inhibit the response to a variety of indirect bronchial challenges in humans but does not inhibit bronchoconstriction induced by inhaled methacholine or histamine. We have investigated the effects of the two loop diuretics, furosemide (10(-6) to 10(-3) M) and bumetanide (10(-7) to 10(-4) M), on airway smooth muscle contraction in vitro induced by electrical field stimulation (EFS), or exogenously applied acetylcholine (ACh) or substance P (SP) in guinea pig tracheal and bronchial smooth muscle strips pretreated with indomethacin (10(-5) M) and propranolol (10(-6) M). Both furosemide and bumetanide caused a concentration-dependent inhibition of cholinergically mediated neural contraction in the trachea. The effect of furosemide was not influenced by the presence of airway epithelium. Furthermore, both furosemide and bumetanide inhibited in a concentration-dependent fashion nonadrenergic, noncholinergic (NANC) contraction induced by electrical field stimulation of bronchi pretreated with atropine (10(-5) M). Neither drug at the highest concentration inhibited the responses to exogenous acetylcholine (10(-8) to 10(-2) M) or substance P (10(-9) to 10(-5) M). Thus loop diuretics inhibit the neurally induced contraction of guinea pig airways without a direct effect on airway smooth muscle. We conclude that loop diuretics inhibit both cholinergic and excitatory NANC neurotransmission in guinea pig airways and that this effect may be related to their inhibitory effects on the sodium-potassium-chloride cotransporter.

  8. S100A8 inhibits PDGF-induced proliferation of airway smooth muscle cells dependent on the receptor for advanced glycation end-products.

    PubMed

    Xu, Yu-Dong; Wang, Yu; Yin, Lei-Miao; Peng, Ling-Ling; Park, Gyoung-Hee; Yang, Yong-Qing

    2017-06-21

    Airway remodeling is a key feature of asthma, characterized by increased proliferation of airway smooth muscle cells (ASMCs). S100A8 is a calcium-binding protein with a potential to regulate cell proliferation. Here, the effect of exogenous S100A8 protein on the proliferation of ASMCs induced by platelet-derived growth factor (PDGF) and the underlying molecular mechanism was investigated. Rat ASMCs were cultured with or without a neutralizing antibody to the receptor for advanced glycation end-products (RAGE), a potential receptor for S100A8 protein. Purified recombinant rat S100A8 protein was then added into the cultured cells, and the proliferation of ASMCs induced by PDGF was detected by colorimetric-based WST-8 assay and ampedance-based xCELLigence proliferation assay. The expression levels of RAGE in ASMCs were analyzed using western blotting assay. Results showed that exogenous S100A8 inhibited the PDGF-induced proliferation of rat ASMCs in a dose-dependent manner with the maximal effect at 1 μg/ml in vitro. Furthermore, when ASMCs was pre-treated with anti-RAGE neutralizing antibody, the inhibitory effect of S100A8 on PDGF-induced proliferation was significantly suppressed. In addition, neither the treatment with S100A8 or PDGF alone nor the pre-treatment with rS100A8 followed by PDGF stimulation affected the expression levels of RAGE. Our study demonstrated that S100A8 inhibits PDGF-induced ASMCs proliferation in a manner dependent on membrane receptor RAGE.

  9. Overexpression of soluble ADAM33 promotes a hypercontractile phenotype of the airway smooth muscle cell in rat

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Duan, Yiyuan; Long, Jiaoyue; Chen, Jun

    A disintegrin and metalloproteinase 33 (ADAM33) has been identified as a susceptibility gene for asthma, but details of the causality are not fully understood. We hypothesize that soluble ADAM33 (sADAM33) overexpression can alter the mechanical behaviors of airway smooth muscle cells (ASMCs) via regulation of the cell's contractile phenotype, and thus contributes to airway hyperresponsiveness (AHR) in asthma. To test this hypothesis, we either overexpressed or knocked down the sADAM33 in rat ASMCs by transfecting the cells with sADAM33 coding sequence or a small interfering RNA (siRNA) that specifically targets the ADAM33 disintegrin domain, and subsequently assessed the cells formore » stiffness, contractility and traction force, together with the expression level of contractile and proliferative phenotype markers. We also investigated whether these changes were dependent on Rho/ROCK pathway by culturing the ASMCs either in the absence or presence of ROCK inhibitor (H1152). The results showed that the ASMCs with sADAM33 overexpression were stiffer and more contractile, generated greater traction force, exhibited increased expression levels of contractile phenotype markers and markedly enhanced Rho activation. Furthermore these changes were largely attenuated when the cells were cultured in the presence of H-1152. However, the knock-down of ADAM33 seemed insufficient to influence majority of the mechanical behaviors of the ASMCs. Taken together, we demonstrated that sADAM33 overexpression altered the mechanical behaviors of ASMCs in vitro, which was most likely by promoting a hypercontractile phenotype transition of ASMCs through Rho/ROCK pathway. This revelation may establish the previously missing link between ADAM33 expression and AHR, and also provide useful insight for targeting sADAM33 in asthma prevention and therapy. - Highlights: • sADAM33 overexpression enhances the stiffness, traction force and contractility of ASMCs. • sADAM33 overexpression

  10. Nonparametric Model of Smooth Muscle Force Production During Electrical Stimulation.

    PubMed

    Cole, Marc; Eikenberry, Steffen; Kato, Takahide; Sandler, Roman A; Yamashiro, Stanley M; Marmarelis, Vasilis Z

    2017-03-01

    A nonparametric model of smooth muscle tension response to electrical stimulation was estimated using the Laguerre expansion technique of nonlinear system kernel estimation. The experimental data consisted of force responses of smooth muscle to energy-matched alternating single pulse and burst current stimuli. The burst stimuli led to at least a 10-fold increase in peak force in smooth muscle from Mytilus edulis, despite the constant energy constraint. A linear model did not fit the data. However, a second-order model fit the data accurately, so the higher-order models were not required to fit the data. Results showed that smooth muscle force response is not linearly related to the stimulation power.

  11. Endogenous Cardiac Troponin T Modulates Ca2+-Mediated Smooth Muscle Contraction

    PubMed Central

    Kajioka, Shunichi; Takahashi-Yanaga, Fumi; Shahab, Nouval; Onimaru, Mitsuho; Matsuda, Miho; Takahashi, Ryosuke; Asano, Haruhiko; Morita, Hiromitsu; Morimoto, Sachio; Yonemitsu, Yoshikazu; Hayashi, Maya; Seki, Narihito; Sasaguri, Toshiuyki; Hirata, Masato; Nakayama, Shinsuke; Naito, Seiji

    2012-01-01

    Mechanisms linked to actin filaments have long been thought to cooperate in smooth muscle contraction, although key molecules were unclear. We show evidence that cardiac troponin T (cTnT) substantially contributes to Ca2+-mediated contraction in a physiological range of cytosolic Ca2+ concentration ([Ca2+]i). cTnT was detected in various smooth muscles of the aorta, trachea, gut and urinary bladder, including in humans. Also, cTnT was distributed along with tropomyosin in smooth muscle cells, suggesting that these proteins are ready to cause smooth muscle contraction. In chemically permeabilised smooth muscle of cTnT+/− mice in which cTnT reduced to ~50%, the Ca2+-force relationship was shifted toward greater [Ca2+]i, indicating a sizeable contribution of cTnT to smooth muscle contraction at [Ca2+]i < 1 μM. Furthermore, addition of supplemental TnI and TnC reconstructed a troponin system to enhance contraction. The results indicated that a Tn/Tn-like system on actin-filaments cooperates together with the thick-filament pathway. PMID:23248744

  12. Upper Airway Collapsibility (Pcrit) and Pharyngeal Dilator Muscle Activity are Sleep Stage Dependent

    PubMed Central

    Carberry, Jayne C.; Jordan, Amy S.; White, David P.; Wellman, Andrew; Eckert, Danny J.

    2016-01-01

    Study Objectives: An anatomically narrow/highly collapsible upper airway is the main cause of obstructive sleep apnea (OSA). Upper airway muscle activity contributes to airway patency and, like apnea severity, can be sleep stage dependent. Conversely, existing data derived from a small number of participants suggest that upper airway collapsibility, measured by the passive pharyngeal critical closing pressure (Pcrit) technique, is not sleep stage dependent. This study aimed to determine the effect of sleep stage on Pcrit and upper airway muscle activity in a larger cohort than previously tested. Methods: Pcrit and/or muscle data were obtained from 72 adults aged 20–64 y with and without OSA.Pcrit was determined via transient reductions in continuous positive airway pressure (CPAP) during N2, slow wave sleep (SWS) and rapid eye movement (REM) sleep. Genioglossus and tensor palatini muscle activities were measured: (1) awake with and without CPAP, (2) during stable sleep on CPAP, and (3) in response to the CPAP reductions used to quantify Pcrit. Results: Pcrit was 4.9 ± 1.4 cmH2O higher (more collapsible) during REM versus SWS (P = 0.012), 2.3 ± 0.6 cmH2O higher during REM versus N2 (P < 0.001), and 1.6 ± 0.7 cmH2O higher in N2 versus SWS (P = 0.048). Muscle activity decreased from wakefulness to sleep and from SWS to N2 to REM sleep for genioglossus but not for tensor palatini. Pharyngeal muscle activity increased by ∼50% by breath 5 following CPAP reductions. Conclusions: Upper airway collapsibility measured via the Pcrit technique and genioglossus muscle activity vary with sleep stage. These findings should be taken into account when performing and interpreting “passive” Pcrit measurements. Citation: Carberry JC, Jordan AS, White DP, Wellman A, Eckert DJ. Upper airway collapsibility (Pcrit) and pharyngeal dilator muscle activity are sleep stage dependent. SLEEP 2016;39(3):511–521. PMID:26612386

  13. Human urotensin-II is a potent spasmogen of primate airway smooth muscle

    PubMed Central

    Hay, Douglas W P; Luttmann, Mark A; Douglas, Stephen A

    2000-01-01

    The contractile profile of human urotensin-II (hU-II) was examined in primate airway and pulmonary vascular tissues. hU-II contracted tissues from different airway regions with similar potencies (pD2s from 8.6 to 9.2). However, there were regional differences in the efficacy of hU-II, with a progressive increase in the maximum contraction from trachea to smaller airway regions (from 9 to 41% of the contraction to 10 μM carbachol). hU-II potently contracted pulmonary artery tissues from different regions with similar potencies and efficacies: pD2s=8.7 to 9.3 and maximal contractions=79 to 86% of 60 mM KCl. hU-II potently contracted pulmonary vein preparations taken proximal to the atria, but had no effect in tissues from distal to the atria. This is the first report describing the contractile activity of hU-II in airways and suggests that the potential pathophysiological role of this peptide in lung diseases warrants investigation. PMID:10960062

  14. Vinpocetine Attenuates the Osteoblastic Differentiation of Vascular Smooth Muscle Cells

    PubMed Central

    Chen, Xiu-Juan; Wang, Na; Yi, Peng-Fei; Song, Min; Zhang, Bo; Wang, Yu-Zhong; Liang, Qiu-Hua

    2016-01-01

    Vascular calcification is an active process of osteoblastic differentiation of vascular smooth muscle cells; however, its definite mechanism remains unknown. Vinpocetine, a derivative of the alkaloid vincamine, has been demonstrated to inhibit the high glucose-induced proliferation of vascular smooth muscle cells; however, it remains unknown whether vinpocetine can affect the osteoblastic differentiation of vascular smooth muscle cells. We hereby investigated the effect of vinpocetine on vascular calcification using a beta-glycerophosphate-induced cell model. Our results showed that vinpocetine significantly reduced the osteoblast-like phenotypes of vascular smooth muscle cells including ALP activity, osteocalcin, collagen type I, Runx2 and BMP-2 expression as well as the formation of mineralized nodule. Vinpocetine, binding to translocation protein, induced phosphorylation of extracellular signal-related kinase and Akt and thus inhibited the translocation of nuclear factor-kappa B into the nucleus. Silencing of translocator protein significantly attenuated the inhibitory effect of vinpocetine on osteoblastic differentiation of vascular smooth muscle cells. Taken together, vinpocetine may be a promising candidate for the clinical therapy of vascular calcification. PMID:27589055

  15. Vinpocetine Attenuates the Osteoblastic Differentiation of Vascular Smooth Muscle Cells.

    PubMed

    Ma, Yun-Yun; Sun, Lin; Chen, Xiu-Juan; Wang, Na; Yi, Peng-Fei; Song, Min; Zhang, Bo; Wang, Yu-Zhong; Liang, Qiu-Hua

    2016-01-01

    Vascular calcification is an active process of osteoblastic differentiation of vascular smooth muscle cells; however, its definite mechanism remains unknown. Vinpocetine, a derivative of the alkaloid vincamine, has been demonstrated to inhibit the high glucose-induced proliferation of vascular smooth muscle cells; however, it remains unknown whether vinpocetine can affect the osteoblastic differentiation of vascular smooth muscle cells. We hereby investigated the effect of vinpocetine on vascular calcification using a beta-glycerophosphate-induced cell model. Our results showed that vinpocetine significantly reduced the osteoblast-like phenotypes of vascular smooth muscle cells including ALP activity, osteocalcin, collagen type I, Runx2 and BMP-2 expression as well as the formation of mineralized nodule. Vinpocetine, binding to translocation protein, induced phosphorylation of extracellular signal-related kinase and Akt and thus inhibited the translocation of nuclear factor-kappa B into the nucleus. Silencing of translocator protein significantly attenuated the inhibitory effect of vinpocetine on osteoblastic differentiation of vascular smooth muscle cells. Taken together, vinpocetine may be a promising candidate for the clinical therapy of vascular calcification.

  16. Immunomodulation of afferent neurons in guinea-pig isolated airway.

    PubMed

    Riccio, M M; Myers, A C; Undem, B J

    1996-03-01

    1. The trachea, larynx and main bronchi with the right vagus nerve and nodose ganglion were isolated from guinea-pigs passively immunized 24 h previously with serum containing anti-ovalbumin antibody. 2. The airways were placed in one compartment of a Perspex chamber for recording of isometric tension while the nodose ganglion and attached vagus nerve were pulled into another compartment. Action potentials arriving from single airway afferent nerve endings were monitored extracellularly using a glass microelectrode positioned near neuronal cell bodies in the ganglion. Mechanosensitivity of the nerve endings was quantified using calibrated von Frey filaments immediately before and after exposure to antigen (10 micrograms ml-1 ovalbumin). 3. Ten endings responded to the force exerted by the lowest filament (0.078 mN) and were not further investigated. In airways from thirteen immunized guinea-pigs, the mechanical sensitivity of A delta afferent fibres (conduction velocity = 4.3 +/- 0.6 m s-1) was enhanced 4.1 +/- 0.9-fold following airway exposure to antigen (P < 0.005). Mechanical sensitivities of afferent fibres (conduction velocity = 4.3 +/- 0.6 m s-1) from non-immunized control guinea-pig airways were unaffected by antigen (n = 13). 4. Antigen did not overtly cause action potential generation except in one instance when the receptive field was located over the smooth muscle. This ending also responded to methacholine suggesting that spatial changes in the receptive field, induced by muscle contraction, were responsible for the activation. 5. The mediators responsible for these effects are unknown, although histamine, prostaglandins, leukotrienes and tachykinins do not appear to be essential. The increase in mechanical responsiveness was not associated with the smooth muscle contraction since leukotriene C4, histamine and tachykinins, which all caused a similar contraction to antigen, did not affect mechanical thresholds. Moreover, the antigen-induced increases in

  17. [Intrarenal smooth muscle: histology of a complex urodymamic machine].

    PubMed

    Arias, L F; Ortiz-Arango, N

    2013-03-01

    To know better the microscopic arrangement of the bundles of smooth muscle in the human renal parenchyma, their distribution and anatomical relationships, trying to make a reconstruction of this muscular system. Five adult human kidneys and one fetal kidney were processed "in toto" with cross sections every 300μm. In the histological sections we identify the smooth muscle fibers trying to determine its insertion, course and anatomical relationship with other structures of the kidney tissue. There are bundles of smooth muscle fibers of variable thickness parallel to the edges of the medullary pyramids, bundles that surrounding the medulla in a spiral course, and bundles that accompany arcuate vessels, the latter being the most abundant and easy to identify. These groups of muscle fibers do not have a precise or constant insertion site, their periodicity is not homogeneous and they are not a direct extension of the muscle of the renal pelvis, although some bundles are in contact with it. There are also unusual and inconstant small muscle fibers no associated to vessels in the interstitium of the cortex and, exceptionally, in the medulla. There is a complex microscopic system of smooth muscle fibers that partially surround the renal medulla and are related to renal pelvic muscles without a direct continuity with them. Although this small muscular system is under-recognized, could be very important in urodynamics. Copyright © 2012 AEU. Published by Elsevier Espana. All rights reserved.

  18. Induction of apoptosis by pyrrolidinedithiocarbamate and N-acetylcysteine in vascular smooth muscle cells.

    PubMed

    Tsai, J C; Jain, M; Hsieh, C M; Lee, W S; Yoshizumi, M; Patterson, C; Perrella, M A; Cooke, C; Wang, H; Haber, E; Schlegel, R; Lee, M E

    1996-02-16

    Pyrrolidinedithiocarbamate (PDTC) and N-acetylcysteine (NAC) have been used as antioxidants to prevent apoptosis in lymphocytes, neurons, and vascular endothelial cells. We report here that PDTC and NAC induce apoptosis in rat and human smooth muscle cells. In rat aortic smooth muscle cells, PDTC induced cell shrinkage, chromatin condensation, and DNA strand breaks consistent with apoptosis. In addition, overexpression of Bcl-2 suppressed vascular smooth muscle cell death caused by PDTC and NAC. The viability of rat aortic smooth muscle cells decreased within 3 h of treatment with PDTC and was reduced to 30% at 12 h. The effect of PDTC and NAC on smooth muscle cells was not species specific because PDTC and NAC both caused dose-dependent reductions in viability in rat and human aortic smooth muscle cells. In contrast, neither PDTC nor NAC reduced viability in human aortic endothelial cells. The use of antioxidants to induce apoptosis in vascular smooth muscle cells may help prevent their proliferation in arteriosclerotic lesions.

  19. Cardiac, skeletal, and smooth muscle mitochondrial respiration: are all mitochondria created equal?

    PubMed Central

    Park, Song-Young; Gifford, Jayson R.; Andtbacka, Robert H. I.; Trinity, Joel D.; Hyngstrom, John R.; Garten, Ryan S.; Diakos, Nikolaos A.; Ives, Stephen J.; Dela, Flemming; Larsen, Steen; Drakos, Stavros

    2014-01-01

    Unlike cardiac and skeletal muscle, little is known about vascular smooth muscle mitochondrial respiration. Therefore, the present study examined mitochondrial respiratory rates in smooth muscle of healthy human feed arteries and compared with that of healthy cardiac and skeletal muscles. Cardiac, skeletal, and smooth muscles were harvested from a total of 22 subjects (53 ± 6 yr), and mitochondrial respiration was assessed in permeabilized fibers. Complex I + II, state 3 respiration, an index of oxidative phosphorylation capacity, fell progressively from cardiac to skeletal to smooth muscles (54 ± 1, 39 ± 4, and 15 ± 1 pmol·s−1·mg−1, P < 0.05, respectively). Citrate synthase (CS) activity, an index of mitochondrial density, also fell progressively from cardiac to skeletal to smooth muscles (222 ± 13, 115 ± 2, and 48 ± 2 μmol·g−1·min−1, P < 0.05, respectively). Thus, when respiration rates were normalized by CS (respiration per mitochondrial content), oxidative phosphorylation capacity was no longer different between the three muscle types. Interestingly, complex I state 2 normalized for CS activity, an index of nonphosphorylating respiration per mitochondrial content, increased progressively from cardiac to skeletal to smooth muscles, such that the respiratory control ratio, state 3/state 2 respiration, fell progressively from cardiac to skeletal to smooth muscles (5.3 ± 0.7, 3.2 ± 0.4, and 1.6 ± 0.3 pmol·s−1·mg−1, P < 0.05, respectively). Thus, although oxidative phosphorylation capacity per mitochondrial content in cardiac, skeletal, and smooth muscles suggest all mitochondria are created equal, the contrasting respiratory control ratio and nonphosphorylating respiration highlight the existence of intrinsic functional differences between these muscle mitochondria. This likely influences the efficiency of oxidative phosphorylation and could potentially alter ROS production. PMID:24906913

  20. Cardiac, skeletal, and smooth muscle mitochondrial respiration: are all mitochondria created equal?

    PubMed

    Park, Song-Young; Gifford, Jayson R; Andtbacka, Robert H I; Trinity, Joel D; Hyngstrom, John R; Garten, Ryan S; Diakos, Nikolaos A; Ives, Stephen J; Dela, Flemming; Larsen, Steen; Drakos, Stavros; Richardson, Russell S

    2014-08-01

    Unlike cardiac and skeletal muscle, little is known about vascular smooth muscle mitochondrial respiration. Therefore, the present study examined mitochondrial respiratory rates in smooth muscle of healthy human feed arteries and compared with that of healthy cardiac and skeletal muscles. Cardiac, skeletal, and smooth muscles were harvested from a total of 22 subjects (53 ± 6 yr), and mitochondrial respiration was assessed in permeabilized fibers. Complex I + II, state 3 respiration, an index of oxidative phosphorylation capacity, fell progressively from cardiac to skeletal to smooth muscles (54 ± 1, 39 ± 4, and 15 ± 1 pmol·s(-1)·mg(-1), P < 0.05, respectively). Citrate synthase (CS) activity, an index of mitochondrial density, also fell progressively from cardiac to skeletal to smooth muscles (222 ± 13, 115 ± 2, and 48 ± 2 μmol·g(-1)·min(-1), P < 0.05, respectively). Thus, when respiration rates were normalized by CS (respiration per mitochondrial content), oxidative phosphorylation capacity was no longer different between the three muscle types. Interestingly, complex I state 2 normalized for CS activity, an index of nonphosphorylating respiration per mitochondrial content, increased progressively from cardiac to skeletal to smooth muscles, such that the respiratory control ratio, state 3/state 2 respiration, fell progressively from cardiac to skeletal to smooth muscles (5.3 ± 0.7, 3.2 ± 0.4, and 1.6 ± 0.3 pmol·s(-1)·mg(-1), P < 0.05, respectively). Thus, although oxidative phosphorylation capacity per mitochondrial content in cardiac, skeletal, and smooth muscles suggest all mitochondria are created equal, the contrasting respiratory control ratio and nonphosphorylating respiration highlight the existence of intrinsic functional differences between these muscle mitochondria. This likely influences the efficiency of oxidative phosphorylation and could potentially alter ROS production.

  1. Blood sinuses in the submucosa of the large airways of the sheep.

    PubMed Central

    Hill, P; Goulding, D; Webber, S E; Widdicombe, J G

    1989-01-01

    We have studied the airway vasculature in sheep using light and transmission electron microscopy, as well as arterial and venous (retrograde) injections of anatomical corrosion compound and latex. Vascular casts were viewed by scanning electron microscopy. There is a complex network of blood sinuses of large diameter (up to 500 microns) in the submucosa of the large airways. The vessels have thin walls formed by a single layer of flattened endothelium with tight junctions and without pericytes or smooth muscle cells. Characteristically the sinuses lie between the cartilage and lamina propria of the trachea or between cartilage and smooth muscle in the bronchi. Sinuses of greater than 50 microns transverse diameter are not found in airways less than 1.0 mm across. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 7 PMID:2808119

  2. A texture analysis method for MR images of airway dilator muscles: a feasibility study

    PubMed Central

    Järnstedt, J; Sikiö, M; Viik, J; Dastidar, P; Peltomäki, T; Eskola, H

    2014-01-01

    Objectives: Airway dilator muscles play an important role in the analysis of breathing-related symptoms, such as obstructive sleep apnoea. Texture analysis (TA) provides a new non-invasive method for analysing airway dilator muscles. In this study, we propose a TA methodology for airway dilator muscles and prove the robustness of this method. Methods: 15 orthognathic surgery patients underwent 3-T MRI. Computerized TA was performed on 20 regions of interest (ROIs) in the patients' airway dilator muscles. 53 texture parameters were calculated for all ROIs. The robustness of the TA method was analysed by altering the locations, sizes and shapes of the ROIs. Results: Our study shows that there is significant difference in TA results as the size or shape of ROI changes. The change of location of the ROI inside the studied muscle does not affect the TA results. Conclusions: The TA method is valid for airway dilator muscles. We propose a methodology in which the number of co-occurrence parameters is reduced by using mean values from four different directions (0°, 45°, 90° and 135°) with pixel spacing of 1 pixel. PMID:24773626

  3. Smooth muscle tumors of soft tissue and non-uterine viscera: biology and prognosis.

    PubMed

    Miettinen, Markku

    2014-01-01

    Smooth muscle tumors are here considered an essentially dichotomous group composed of benign leiomyomas and malignant leiomyosarcomas. Soft tissue smooth muscle tumors with both atypia and mitotic activity are generally diagnosed leiomyosarcomas acknowledging potential for metastasis. However, lesions exist that cannot be comfortably placed in either category, and in such cases the designation 'smooth muscle tumor of uncertain biologic potential' is appropriate. The use of this category is often necessary with limited sampling, such as needle core biopsies. Benign smooth muscle tumors include smooth muscle hamartoma and angioleiomyoma. A specific category of leiomyomas are estrogen-receptor positive ones in women. These are similar to uterine leiomyomas and can occur anywhere in the abdomen and abdominal wall. Leiomyosarcomas can occur at any site, although are more frequent in the retroperitoneum and proximal extremities. They are recognized by likeness to smooth muscle cells but can undergo pleomorphic evolution ('dedifferentiation'). Presence of smooth muscle actin is nearly uniform and desmin-positivity usual. This and the lack of KIT expression separate leiomyosarcoma from GIST, an important problem in abdominal soft tissues. EBV-associated smooth muscle tumors are a specific subcategory occurring in AIDS or post-transplant patients. These tumors can have incomplete smooth muscle differentiation but show nuclear EBER as a diagnostic feature. In contrast to many other soft tissue tumors, genetics of smooth muscle tumors are poorly understood and such diagnostic testing is not yet generally applicable in this histogenetic group. Leiomyosarcomas are known to be genetically complex, often showing 'chaotic' karyotypes including aneuploidy or polyploidy, and no recurrent tumor-specific translocations have been detected.

  4. Dexmedetomidine's inhibitory effects on acetylcholine release from cholinergic nerves in guinea pig trachea: a mechanism that accounts for its clinical benefit during airway irritation.

    PubMed

    Mikami, Maya; Zhang, Yi; Kim, Benjamin; Worgall, Tilla S; Groeben, Harald; Emala, Charles W

    2017-03-29

    Airway instrumentation can evoke upper airway reflexes including bronchoconstriction and cough which can cause serious complications including airway trauma, laryngospasm or bronchospasm which may in turn lead to difficulty with ventilation and hypoxemia. These airway events are mediated in part by irritant-induced neuronal modulation of airway tone and cough responses. We investigated whether the commonly used anesthetic agents dexmedetomidine, lidocaine or remifentanil attenuated neuronal and airway smooth muscle responses in the upper airways of guinea pigs. The ability of dexmedetomidine, lidocaine or remifentanil to attenuate direct cholinergic nerve stimulation, C-fiber stimulation or direct smooth muscle contraction were studied using isolated tracheal rings from male guinea pigs under four paradigms; (1) the magnitude of contractile force elicited by cholinergic electrical field stimulation (EFS); (2) the amount of acetylcholine released during cholinergic EFS; (3) the direct airway smooth muscle relaxation of a sustained acetylcholine-induced contraction and (4) the magnitude of C-fiber mediated contraction. Dexmedetomidine (1-100 μM) and lidocaine (1 mM) attenuated cholinergic 30Hz EFS-induced tracheal ring contraction while remifentanil (10 μM) had no effect. Dexmedetomidine at 10 μM (p = 0.0047) and 100 μM (p = 0.01) reduced cholinergic EFS-induced acetylcholine release while lidocaine (10 μM-1 mM) and remifentanil (0.1-10 μM) did not. Tracheal ring muscle force induced by the exogenous addition of the contractile agonist acetylcholine or by a prototypical C-fiber analogue of capsaicin were also attenuated by 100 μM dexmedetomidine (p = 0.0061 and p = 0.01, respectively). The actual tracheal tissue concentrations of dexmedetomidine achieved (0.54-26 nM) following buffer application of 1-100 μM of dexmedetomidine were within the range of clinically achieved plasma concentrations (12 nM). The α2 adrenoceptor agonist

  5. Overexpression of microRNA-375 impedes platelet-derived growth factor-induced proliferation and migration of human fetal airway smooth muscle cells by targeting Janus kinase 2.

    PubMed

    Ji, Yamei; Yang, Xin; Su, Huixia

    2018-02-01

    The abnormal proliferation and migration of airway smooth muscle (ASM) cells play a critical role in airway remodeling during the development of asthma. MicroRNAs (miRNAs) have emerged as critical regulators of ASM cell proliferation and migration in airway remodeling. In this study, we aimed to investigate the potential role of miR-375 in the regulation of platelet-derived growth factor (PDGF)-induced fetal ASM cell proliferation and migration. Our results showed that miR-375 expression was significantly decreased in fetal ASM cells that were treated with PDGF. Functional data showed that overexpression of miR-375 inhibited the proliferation and migration of fetal ASM cells, whereas inhibition of miR-375 enhanced the proliferation and migration of fetal ASM cells. The results of bioinformatics analysis and a dual-luciferase reporter assay showed that miR-375 binds directly to the 3'-untranslated region of Janus kinase 2 (JAK2). Further data confirmed that miR-375 negatively regulates the expression of JAK2 in fetal ASM cells. Moreover, miR-375 also impeded the PDGF-induced activation of signal transducer and activator of transcription 3 (STAT3) in fetal ASM cells. However, restoration of JAK2 expression partially reversed the inhibitory effect of miR-375 on fetal ASM cell proliferation and migration. Overall, our results demonstrate that miR-375 inhibits fetal ASM cell proliferation and migration by targeting JAK2/STAT3 signaling. Our study provides a potential therapeutic target for the development of novel treatment strategies for pediatric asthma. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  6. Vasodilator-stimulated Phosphoprotein (VASP) Regulates Actin Polymerization and Contraction in Airway Smooth Muscle by a Vinculin-dependent Mechanism*

    PubMed Central

    Wu, Yidi; Gunst, Susan J.

    2015-01-01

    Vasodilator-stimulated phosphoprotein (VASP) can catalyze actin polymerization by elongating actin filaments. The elongation mechanism involves VASP oligomerization and its binding to profilin, a G-actin chaperone. Actin polymerization is required for tension generation during the contraction of airway smooth muscle (ASM); however, the role of VASP in regulating actin dynamics in ASM is not known. We stimulated ASM cells and tissues with the contractile agonist acetylcholine (ACh) or the adenylyl cyclase activator, forskolin (FSK), a dilatory agent. ACh and FSK stimulated VASP Ser157 phosphorylation by different kinases. Inhibition of VASP Ser157 phosphorylation by expression of the mutant VASP S157A in ASM tissues suppressed VASP phosphorylation and membrane localization in response to ACh, and also inhibited contraction and actin polymerization. ACh but not FSK triggered the formation of VASP-VASP complexes as well as VASP-vinculin and VASP-profilin complexes at membrane sites. VASP-VASP complex formation and the interaction of VASP with vinculin and profilin were inhibited by expression of the inactive vinculin mutant, vinculin Y1065F, but VASP phosphorylation and membrane localization were unaffected. We conclude that VASP phosphorylation at Ser157 mediates its localization at the membrane, but that VASP Ser157 phosphorylation and membrane localization are not sufficient to activate its actin catalytic activity. The interaction of VASP with activated vinculin at membrane adhesion sites is a necessary prerequisite for VASP-mediated molecular processes necessary for actin polymerization. Our results show that VASP is a critical regulator of actin dynamics and tension generation during the contractile activation of ASM. PMID:25759389

  7. Role of ROCK expression in gallbladder smooth muscle contraction.

    PubMed

    Wang, Bin; Ding, You-Ming; Wang, Chun-Tao; Wang, Wei-Xing

    2015-08-01

    Cholelithiasis is a common medical condition whose incidence rate is increasing yearly, while its pathogenesis has yet to be elucidated. The present study assessed the expression of Rho-kinase (ROCK) in gallbladder smooth muscles and its effect on the contractile function of gallbladder smooth muscles during gallstone formation. Thirty male guinea pigs were randomly divided into three groups: The control group, the gallstone model group and the fasudil interference group. The fasting volume (FV) and bile capacity of the gallbladder (FB) as well as the total cholesterol (TC) and triglyceride (TG) contents of the gallbladder bile were determined. In addition, the gallbladder was dissected to identify whether any gallstones had formed. Part of the gallbladder tissue specimens were used for immunohistochemical analysis of ROCK expression in gallbladder smooth muscles. The results showed that four guinea pigs in the model group and eight in the fasudil group displayed gallstone formation, while there was no gallstone formation in the control group. The FV and FB were significantly increased in the model and fasudil groups. Similarly, the TC and TG contents of gallbladder bile were increased in these groups. The positive expression rate of ROCK in gallbladder smooth muscles in the model and fasudil groups was significantly reduced compared with that in the control group (P<0.05). The results of the present study indicated that the reduction of ROCK expression in guinea pig gallbladder smooth muscles weakened gallbladder contraction and thereby promoted gallstone formation.

  8. Glucocorticoids inhibit sulfur mustard-induced airway muscle hyperresponsiveness to substance P.

    PubMed

    Calvet, J H; D'Ortho, M P; Jarreau, P H; Levame, M; Harf, A; Macquin-Mavier, I

    1994-11-01

    To explore the mechanisms of airway hyperreactivity to aerosolized substance P observed in guinea pigs 14 days after intratracheal injection of sulfur mustard (SM), we studied the effects of epithelium removal and inhibition of neutral endopeptidase (NEP) activity on airway muscle responsiveness. Tracheal rings from SM-intoxicated guinea pigs expressed a greater contractile response to substance P than rings from nonintoxicated guinea pigs. After epithelium removal or incubation with the NEP inhibitor phosphoramidon, the contractile responses of tracheal rings to substance P did not differ in guinea pigs injected with SM or ethanol (SM solvent). Treatment of the guinea pigs with betamethasone for 7 days before measurement abolished the airway muscle hyperresponsiveness observed in untreated SM-intoxicated guinea pigs and partially restored tracheal epithelium NEP activity. In addition, the tracheal epithelium height and cell density of SM-intoxicated guinea pigs treated with betamethasone were significantly greater than in those without betamethasone. These results demonstrate that SM intoxication induces airway muscle hyperresponsiveness to substance P by reducing tracheal epithelial NEP activity and that glucocorticoids might inhibit this hyperresponsiveness by increasing this activity.

  9. Histochemical characteristics of a tonic smooth muscle.

    PubMed

    Gilloteaux, J; Stalmans-Falys, M

    1979-09-01

    It is suggested that ABRM, smooth muscle of Mytilus edulis L. and Mytilus galloprovincialis Lmk. (Mollusca Pelecypoda), is composed of one histochemical fibre type. The fibres are characterized by a low myofibrillar ATPase activity. Succinic and nicotinamide adenine dinucleotide oxidoreductase activities are distributed in a reverse pattern than that of the ATPase activity. Glycogen phosphorylase is richly represented in ABRM fibres and this detection is in opposition with the negative detection of alkaline phosphatase activity. These preliminary histochemical observations are similar to those found in some vertebrate smooth muscles. Mitochondrial glycerol-3-phosphate, 6-phosphogluconate, lactate and octopine dehydrogenases are not detected in muscle fibres whereas glio-interstitial tissues show weak but distinct reactivity. These last results especially characterize Mytilus catch fibres and are briefly discussed in relationship with previous physiological, biochemical and morphological observations.

  10. Fibronectin Matrix Polymerization Regulates Smooth Muscle Cell Phenotype through a Rac1 Dependent Mechanism

    PubMed Central

    Shi, Feng; Long, Xiaochun; Hendershot, Allison; Miano, Joseph M.; Sottile, Jane

    2014-01-01

    Smooth muscle cells are maintained in a differentiated state in the vessel wall, but can be modulated to a synthetic phenotype following injury. Smooth muscle phenotypic modulation is thought to play an important role in the pathology of vascular occlusive diseases. Phenotypically modulated smooth muscle cells exhibit increased proliferative and migratory properties that accompany the downregulation of smooth muscle cell marker proteins. Extracellular matrix proteins, including fibronectin, can regulate the smooth muscle phenotype when used as adhesive substrates. However, cells produce and organize a 3-dimensional fibrillar extracellular matrix, which can affect cell behavior in distinct ways from the protomeric 2-dimensional matrix proteins that are used as adhesive substrates. We previously showed that the deposition/polymerization of fibronectin into the extracellular matrix can regulate the deposition and organization of other extracellular matrix molecules in vitro. Further, our published data show that the presence of a fibronectin polymerization inhibitor results in increased expression of smooth muscle cell differentiation proteins and inhibits vascular remodeling in vivo. In this manuscript, we used an in vitro cell culture system to determine the mechanism by which fibronectin polymerization affects smooth muscle phenotypic modulation. Our data show that fibronectin polymerization decreases the mRNA levels of multiple smooth muscle differentiation genes, and downregulates the levels of smooth muscle α-actin and calponin proteins by a Rac1-dependent mechanism. The expression of smooth muscle genes is transcriptionally regulated by fibronectin polymerization, as evidenced by the increased activity of luciferase reporter constructs in the presence of a fibronectin polymerization inhibitor. Fibronectin polymerization also promotes smooth muscle cell growth, and decreases the levels of actin stress fibers. These data define a Rac1-dependent pathway wherein

  11. Role of Telokin in Regulating Murine Gastric Fundus Smooth Muscle Tension

    PubMed Central

    An, Changlong; Bhetwal, Bhupal P.; Sanders, Kenton M.; Somlyo, Avril V.; Perrino, Brian A.

    2015-01-01

    Telokin phosphorylation by cyclic GMP-dependent protein kinase facilitates smooth muscle relaxation. In this study we examined the relaxation of gastric fundus smooth muscles from basal tone, or pre-contracted with KCl or carbachol (CCh), and the phosphorylation of telokin S13, myosin light chain (MLC) S19, MYPT1 T853, T696, and CPI-17 T38 in response to 8-Bromo-cGMP, the NO donor sodium nitroprusside (SNP), or nitrergic neurotransmission. We compared MLC phosphorylation and the contraction and relaxation responses of gastric fundus smooth muscles from telokin-/- mice and their wild-type littermates to KCl or CCh, and 8-Bromo-cGMP, SNP, or nitrergic neurotransmission, respectively. We compared the relaxation responses and telokin phosphorylation of gastric fundus smooth muscles from wild-type mice and W/W V mice which lack ICC-IM, to 8-Bromo-cGMP, SNP, or nitrergic neurotransmission. We found that telokin S13 is basally phosphorylated and that 8-Bromo-cGMP and SNP increased basal telokin phosphorylation. In muscles pre-contracted with KCl or CCh, 8-Bromo-cGMP and SNP had no effect on CPI-17 or MYPT1 phosphorylation, but increased telokin phosphorylation and reduced MLC phosphorylation. In telokin-/- gastric fundus smooth muscles, basal tone and constitutive MLC S19 phosphorylation were increased. Pre-contracted telokin-/- gastric fundus smooth muscles have increased contractile responses to KCl, CCh, or cholinergic neurotransmission and reduced relaxation to 8-Bromo-cGMP, SNP, and nitrergic neurotransmission. However, basal telokin phosphorylation was not increased when muscles were stimulated with lower concentrations of SNP or when the muscles were stimulated by nitrergic neurotransmission. SNP, but not nitrergic neurotransmission, increased telokin Ser13 phosphorylation in both wild-type and W/W V gastric fundus smooth muscles. Our findings indicate that telokin may play a role in attenuating constitutive MLC phosphorylation and provide an additional mechanism

  12. Histone deacetylase 8 regulates cortactin deacetylation and contraction in smooth muscle tissues

    PubMed Central

    Li, Jia; Chen, Shu; Cleary, Rachel A.; Wang, Ruping; Gannon, Olivia J.; Seto, Edward

    2014-01-01

    Histone deacetylases (HDACs) are a family of enzymes that mediate nucleosomal histone deacetylation and gene expression. Some members of the HDAC family have also been implicated in nonhistone protein deacetylation, which modulates cell-cycle control, differentiation, and cell migration. However, the role of HDACs in smooth muscle contraction is largely unknown. Here, HDAC8 was localized both in the cytoplasm and the nucleus of mouse and human smooth muscle cells. Knockdown of HDAC8 by lentivirus-encoding HDAC8 shRNA inhibited force development in response to acetylcholine. Treatment of smooth muscle tissues with HDAC8 inhibitor XXIV (OSU-HDAC-44) induced relaxation of precontracted smooth muscle tissues. In addition, cortactin is an actin-regulatory protein that undergoes deacetylation during migration of NIH 3T3 cells. In this study, acetylcholine stimulation induced cortactin deacetylation in mouse and human smooth muscle tissues, as evidenced by immunoblot analysis using antibody against acetylated lysine. Knockdown of HDAC8 by RNAi or treatment with the inhibitor attenuated cortactin deacetylation and actin polymerization without affecting myosin activation. Furthermore, expression of a charge-neutralizing cortactin mutant inhibited contraction and actin dynamics during contractile activation. These results suggest a novel mechanism for the regulation of smooth muscle contraction. In response to contractile stimulation, HDAC8 may mediate cortactin deacetylation, which subsequently promotes actin filament polymerization and smooth muscle contraction. PMID:24920679

  13. Calcium signaling in smooth muscle.

    PubMed

    Hill-Eubanks, David C; Werner, Matthias E; Heppner, Thomas J; Nelson, Mark T

    2011-09-01

    Changes in intracellular Ca(2+) are central to the function of smooth muscle, which lines the walls of all hollow organs. These changes take a variety of forms, from sustained, cell-wide increases to temporally varying, localized changes. The nature of the Ca(2+) signal is a reflection of the source of Ca(2+) (extracellular or intracellular) and the molecular entity responsible for generating it. Depending on the specific channel involved and the detection technology employed, extracellular Ca(2+) entry may be detected optically as graded elevations in intracellular Ca(2+), junctional Ca(2+) transients, Ca(2+) flashes, or Ca(2+) sparklets, whereas release of Ca(2+) from intracellular stores may manifest as Ca(2+) sparks, Ca(2+) puffs, or Ca(2+) waves. These diverse Ca(2+) signals collectively regulate a variety of functions. Some functions, such as contractility, are unique to smooth muscle; others are common to other excitable cells (e.g., modulation of membrane potential) and nonexcitable cells (e.g., regulation of gene expression).

  14. Autonomic Modification of Intestinal Smooth Muscle Contractility

    ERIC Educational Resources Information Center

    Montgomery, Laura E. A.; Tansey, Etain A.; Johnson, Chris D.; Roe, Sean M.; Quinn, Joe G.

    2016-01-01

    Intestinal smooth muscle contracts rhythmically in the absence of nerve and hormonal stimulation because of the activity of pacemaker cells between and within the muscle layers. This means that the autonomic nervous system modifies rather than initiates intestinal contractions. The practical described here gives students an opportunity to observe…

  15. Cross-bridge elasticity in single smooth muscle cells

    PubMed Central

    1983-01-01

    In smooth muscle, a cross-bridge mechanism is believed to be responsible for active force generation and fiber shortening. In the present studies, the viscoelastic and kinetic properties of the cross- bridge were probed by eliciting tension transients in response to small, rapid, step length changes (delta L = 0.3-1.0% Lcell in 2 ms). Tension transients were obtained in a single smooth muscle cell isolated from the toad (Bufo marinus) stomach muscularis, which was tied between a force transducer and a displacement device. To record the transients, which were of extremely small magnitude (0.1 microN), a high-frequency (400 Hz), ultrasensitive force transducer (18 mV/microN) was designed and built. The transients obtained during maximal force generation (Fmax = 2.26 microN) were characterized by a linear elastic response (Emax = 1.26 X 10(4) mN/mm2) coincident with the length step, which was followed by a biphasic tension recovery made up of two exponentials (tau fast = 5-20 ms, tau slow = 50-300 ms). During the development of force upon activation, transients were elicited. The relationship between stiffness and force was linear, which suggests that the transients originate within the cross-bridge and reflect the cross-bridge's viscoelastic and kinetic properties. The observed fiber elasticity suggests that the smooth muscle cross-bridge is considerably more compliant than in fast striated muscle. A thermodynamic model is presented that allows for an analysis of the factors contributing to the increased compliance of the smooth muscle cross-bridge. PMID:6413640

  16. Smooth Muscle-Mediated Connective Tissue Remodeling in Pulmonary Hypertension

    NASA Astrophysics Data System (ADS)

    Mecham, Robert P.; Whitehouse, Loren A.; Wrenn, David S.; Parks, William C.; Griffin, Gail L.; Senior, Robert M.; Crouch, Edmond C.; Stenmark, Kurt R.; Voelkel, Norbert F.

    1987-07-01

    Abnormal accumulation of connective tissue in blood vessels contributes to alterations in vascular physiology associated with disease states such as hypertension and atherosclerosis. Elastin synthesis was studied in blood vessels from newborn calves with severe pulmonary hypertension induced by alveolar hypoxia in order to investigate the cellular stimuli that elicit changes in pulmonary arterial connective tissue production. A two- to fourfold increase in elastin production was observed in pulmonary artery tissue and medial smooth muscle cells from hypertensive calves. This stimulation of elastin production was accompanied by a corresponding increase in elastin messenger RNA consistent with regulation at the transcriptional level. Conditioned serum harvested from cultures of pulmonary artery smooth muscle cells isolated from hypertensive animals contained one or more low molecular weight elastogenic factors that stimulated the production of elastin in both fibroblasts and smooth muscle cells and altered the chemotactic responsiveness of fibroblasts to elastin peptides. These results suggest that connective tissue changes in the pulmonary vasculature in response to pulmonary hypertension are orchestrated by the medial smooth muscle cell through the generation of specific differentiation factors that alter both the secretory phenotype and responsive properties of surrounding cells.

  17. Sex, stress and sleep apnoea: Decreased susceptibility to upper airway muscle dysfunction following intermittent hypoxia in females.

    PubMed

    O'Halloran, Ken D; Lewis, Philip; McDonald, Fiona

    2017-11-01

    Obstructive sleep apnoea syndrome (OSAS) is a devastating respiratory control disorder more common in men than women. The reasons for the sex difference in prevalence are multifactorial, but are partly attributable to protective effects of oestrogen. Indeed, OSAS prevalence increases in post-menopausal women. OSAS is characterized by repeated occlusions of the pharyngeal airway during sleep. Dysfunction of the upper airway muscles controlling airway calibre and collapsibility is implicated in the pathophysiology of OSAS, and sex differences in the neuro-mechanical control of upper airway patency are described. It is widely recognized that chronic intermittent hypoxia (CIH), a cardinal feature of OSAS due to recurrent apnoea, drives many of the morbid consequences characteristic of the disorder. In rodents, exposure to CIH-related redox stress causes upper airway muscle weakness and fatigue, associated with mitochondrial dysfunction. Of interest, in adults, there is female resilience to CIH-induced muscle dysfunction. Conversely, exposure to CIH in early life, results in upper airway muscle weakness equivalent between the two sexes at 3 and 6 weeks of age. Ovariectomy exacerbates the deleterious effects of exposure to CIH in adult female upper airway muscle, an effect partially restored by oestrogen replacement therapy. Intriguingly, female advantage intrinsic to upper airway muscle exists with evidence of substantially greater loss of performance in male muscle during acute exposure to severe hypoxic stress. Sex differences in upper airway muscle physiology may have relevance to human OSAS. The oestrogen-oestrogen receptor α axis represents a potential therapeutic target in OSAS, particularly in post-menopausal women. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Endocrine regulation of airway contractility is overlooked.

    PubMed

    Bossé, Ynuk

    2014-08-01

    Asthma is a prevalent respiratory disorder triggered by a variety of inhaled environmental factors, such as allergens, viruses, and pollutants. Asthma is characterized by an elevated activation of the smooth muscle surrounding the airways, as well as a propensity of the airways to narrow excessively in response to a spasmogen (i.e. contractile agonist), a feature called airway hyperresponsiveness. The level of airway smooth muscle (ASM) activation is putatively controlled by mediators released in its vicinity. In asthma, many mediators that affect ASM contractility originate from inflammatory cells that are mobilized into the airways, such as eosinophils. However, mounting evidence indicates that mediators released by remote organs can also influence the level of activation of ASM, as well as its level of responsiveness to spasmogens and relaxant agonists. These remote mediators are transported through circulating blood to act either directly on ASM or indirectly via the nervous system by tuning the level of cholinergic activation of ASM. Indeed, mediators generated from diverse organs, including the adrenals, pancreas, adipose tissue, gonads, heart, intestines, and stomach, affect the contractility of ASM. Together, these results suggest that, apart from a paracrine mode of regulation, ASM is subjected to an endocrine mode of regulation. The results also imply that defects in organs other than the lungs can contribute to asthma symptoms and severity. In this review, I suggest that the endocrine mode of regulation of ASM contractility is overlooked. © 2014 Society for Endocrinology.

  19. Increased IGF-IEc expression and mechano-growth factor production in intestinal muscle of fibrostenotic Crohn's disease and smooth muscle hypertrophy

    PubMed Central

    Li, Chao; Vu, Kent; Hazelgrove, Krystina

    2015-01-01

    The igf1 gene is alternatively spliced as IGF-IEa and IGF-IEc variants in humans. In fibrostenotic Crohn's disease, the fibrogenic cytokine TGF-β1 induces IGF-IEa expression and IGF-I production in intestinal smooth muscle and results in muscle hyperplasia and collagen I production that contribute to stricture formation. Mechano-growth factor (MGF) derived from IGF-IEc induces skeletal and cardiac muscle hypertrophy following stress. We hypothesized that increased IGF-IEc expression and MGF production mediated smooth muscle hypertrophy also characteristic of fibrostenotic Crohn's disease. IGF-IEc transcripts and MGF protein were increased in muscle cells isolated from fibrostenotic intestine under regulation by endogenous TGF-β1. Erk5 and MEF2C were phosphorylated in vivo in fibrostenotic muscle; both were phosphorylated and colocalized to nucleus in response to synthetic MGF in vitro. Smooth muscle-specific protein expression of α-smooth muscle actin, γ-smooth muscle actin, and smoothelin was increased in affected intestine. Erk5 inhibition or MEF2C siRNA blocked smooth muscle-specific gene expression and hypertrophy induced by synthetic MGF. Conditioned media of cultured fibrostenotic muscle induced muscle hypertrophy that was inhibited by immunoneutralization of endogenous MGF or pro-IGF-IEc. The results indicate that TGF-β1-dependent IGF-IEc expression and MGF production in patients with fibrostenotic Crohn's disease regulates smooth muscle cell hypertrophy a critical factor that contributes to intestinal stricture formation. PMID:26428636

  20. Increased IGF-IEc expression and mechano-growth factor production in intestinal muscle of fibrostenotic Crohn's disease and smooth muscle hypertrophy.

    PubMed

    Li, Chao; Vu, Kent; Hazelgrove, Krystina; Kuemmerle, John F

    2015-12-01

    The igf1 gene is alternatively spliced as IGF-IEa and IGF-IEc variants in humans. In fibrostenotic Crohn's disease, the fibrogenic cytokine TGF-β1 induces IGF-IEa expression and IGF-I production in intestinal smooth muscle and results in muscle hyperplasia and collagen I production that contribute to stricture formation. Mechano-growth factor (MGF) derived from IGF-IEc induces skeletal and cardiac muscle hypertrophy following stress. We hypothesized that increased IGF-IEc expression and MGF production mediated smooth muscle hypertrophy also characteristic of fibrostenotic Crohn's disease. IGF-IEc transcripts and MGF protein were increased in muscle cells isolated from fibrostenotic intestine under regulation by endogenous TGF-β1. Erk5 and MEF2C were phosphorylated in vivo in fibrostenotic muscle; both were phosphorylated and colocalized to nucleus in response to synthetic MGF in vitro. Smooth muscle-specific protein expression of α-smooth muscle actin, γ-smooth muscle actin, and smoothelin was increased in affected intestine. Erk5 inhibition or MEF2C siRNA blocked smooth muscle-specific gene expression and hypertrophy induced by synthetic MGF. Conditioned media of cultured fibrostenotic muscle induced muscle hypertrophy that was inhibited by immunoneutralization of endogenous MGF or pro-IGF-IEc. The results indicate that TGF-β1-dependent IGF-IEc expression and MGF production in patients with fibrostenotic Crohn's disease regulates smooth muscle cell hypertrophy a critical factor that contributes to intestinal stricture formation. Copyright © 2015 the American Physiological Society.

  1. Notch Signaling in Vascular Smooth Muscle Cells

    PubMed Central

    Baeten, J.T.; Lilly, B.

    2018-01-01

    The Notch signaling pathway is a highly conserved pathway involved in cell fate determination in embryonic development and also functions in the regulation of physiological processes in several systems. It plays an especially important role in vascular development and physiology by influencing angiogenesis, vessel patterning, arterial/venous specification, and vascular smooth muscle biology. Aberrant or dysregulated Notch signaling is the cause of or a contributing factor to many vascular disorders, including inherited vascular diseases, such as cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, associated with degeneration of the smooth muscle layer in cerebral arteries. Like most signaling pathways, the Notch signaling axis is influenced by complex interactions with mediators of other signaling pathways. This complexity is also compounded by different members of the Notch family having both overlapping and unique functions. Thus, it is vital to fully understand the roles and interactions of each Notch family member in order to effectively and specifically target their exact contributions to vascular disease. In this chapter, we will review the Notch signaling pathway in vascular smooth muscle cells as it relates to vascular development and human disease. PMID:28212801

  2. Electrodynamic smooth muscle sphincter: development and biomechanical evaluation of a novel porcine artificial smooth muscle sphincter in a new in vitro stoma simulator.

    PubMed

    Schrag, H J; Karwath, D; Grub, C; Fragoza Padilla, F; Noack, T; Hopt, U T

    2005-07-01

    Many authors have suggested that the activity of the enteric inhibitory nerves is important in regulating normal gastrointestinal motility and inducing smooth muscle relaxation. Hitherto, no experimental or clinical models exist that transfer these physiological aspects to creating an autologous artificial sphincter for the treatment of major incontinence. Therefore, this study was performed to determine the contractile and relaxant capacity of gastrointestinal muscle types and to investigate the efficiency of a novel smooth muscle sphincter, based on the non-adrenergic, non-cholinergic (NANC) receptive relaxation under electrical field stimulation (EFS). For the first step, the isometric tension from isolated circular porcine fundus and colon muscle strips was recorded during pharmacological stimulation (TTX, L-NNA and atropine) and EFS. As a result, a continent electrodynamic smooth muscle sphincter (ESMS) was created by wrapping a fundus muscle flap around an isolated segment of porcine distal colon. The EFS of the free nerve fibers of the flap was realized using a circular platinum wire electrode. Parameters such as threshold of continence, intra/preluminal pressure and fluid passage were analyzed in a newly designed in vitro stoma simulator. Electrical field stimulation produced a maximal and voltage-dependent fundus relaxation to --12.4 mN/mm(2) (frequency of 40 Hz, pulse duration, train duration and voltage of 5 ms, 1 s and 60 mA respectively), which were abolished by N-nitro-L -arginine (L-NNA; 10(-4) M) in a dose-dependent manner, confirming that relaxant responses were mediated by NANC nerves. The results of eight ESMS showed that circular electrical stimulation of the muscle flap caused muscle relaxation with a concomitant and effective reduction in the occlusion pressure. The NANC-induced relaxation mechanism of porcine fundus preparations could be transferred to an efficient smooth muscle sphincter with a high threshold of continence and electrically

  3. Allergic inflammation induces a persistent mechanistic switch in thromboxane-mediated airway constriction in the mouse

    PubMed Central

    Cyphert, Jaime M.; Allen, Irving C.; Church, Rachel J.; Latour, Anne M.; Snouwaert, John N.; Coffman, Thomas M.

    2012-01-01

    Actions of thromboxane (TXA2) to alter airway resistance were first identified over 25 years ago. However, the mechanism underlying this physiological response has remained largely undefined. Here we address this question using a novel panel of mice in which expression of the thromboxane receptor (TP) has been genetically manipulated. We show that the response of the airways to TXA2 is complex: it depends on expression of other G protein-coupled receptors but also on the physiological context of the signal. In the healthy airway, TXA2-mediated airway constriction depends on expression of TP receptors by smooth muscle cells. In contrast, in the inflamed lung, the direct actions of TXA2 on smooth muscle cell TP receptors no longer contribute to bronchoconstriction. Instead, in allergic lung disease, TXA2-mediated airway constriction depends on neuronal TP receptors. Furthermore, this mechanistic switch persists long after resolution of pulmonary inflammation. Our findings demonstrate the powerful ability of lung inflammation to modify pathways leading to airway constriction, resulting in persistent changes in mechanisms of airway reactivity to key bronchoconstrictors. Such alterations are likely to shape the pathogenesis of asthmatic lung disease. PMID:21984570

  4. Potential roles for BMP and Pax genes in the development of iris smooth muscle.

    PubMed

    Jensen, Abbie M

    2005-02-01

    The embryonic optic cup generates four types of tissue: neural retina, pigmented epithelium, ciliary epithelium, and iris smooth muscle. Remarkably little attention has focused on the development of the iris smooth muscle since Lewis ([1903] J. Am. Anat. 2:405-416) described its origins from the anterior rim of the optic cup neuroepithelium. As an initial step toward understanding iris smooth muscle development, I first determined the spatial and temporal pattern of the development of the iris smooth muscle in the chick by using the HNK1 antibody, which labels developing iris smooth muscle. HNK1 labeling shows that iris smooth muscle development is correlated in time and space with the development of the ciliary epithelial folds. Second, because neural crest is the only other neural tissue that has been shown to generate smooth muscle (Le Lievre and Le Douarin [1975] J. Embryo. Exp. Morphol. 34:125-154), I sought to determine whether iris smooth muscle development shares similarities with neural crest development. Two members of the BMP superfamily, BMP4 and BMP7, which may regulate neural crest development, are highly expressed by cells at the site of iris smooth muscle generation. Third, because humans and mice that are heterozygous for Pax6 mutations have no irides (Hill et al. [1991] Nature 354:522-525; Hanson et al. [1994] Nat. Genet. 6:168-173), I determined the expression of Pax6. I also examined the expression of Pax3 in the developing anterior optic cup. The developing iris smooth muscle coexpresses Pax6 and Pax3. I suggest that some of the eye defects caused by mutations in Pax6, BMP4, and BMP7 may be due to abnormal iris smooth muscle. Copyright 2004 Wiley-Liss, Inc.

  5. CysLT1 receptor-induced human airway smooth muscle cells proliferation requires ROS generation, EGF receptor transactivation and ERK1/2 phosphorylation

    PubMed Central

    Ravasi, Saula; Citro, Simona; Viviani, Barbara; Capra, Valérie; Rovati, G Enrico

    2006-01-01

    Background Cysteine-containing leukotrienes (cysteinyl-LTs) are pivotal inflammatory mediators that play important roles in the pathophysiology of asthma, allergic rhinitis, and other inflammatory conditions. In particular, cysteinyl-LTs exert a variety of effects with relevance to the aetiology of asthma such as smooth muscle contraction, eosinophil recruitment, increased microvascular permeability, enhanced mucus secretion and decreased mucus transport and, finally, airway smooth muscle cells (ASMC) proliferation. We used human ASMC (HASMC) to identify the signal transduction pathway(s) of the leukotriene D4 (LTD4)-induced DNA synthesis. Methods Proliferation of primary HASMC was measured by [3H]thymidine incorporation. Phosphorylation of EGF receptor (EGF-R) and ERK1/2 was assessed with a polyclonal anti-EGF-R or anti-phosphoERKl/2 monoclonal antibody. A Ras pull-down assay kit was used to evaluate Ras activation. The production of reactive oxygen species (ROS) was estimated by measuring dichlorodihydrofluorescein (DCF) oxidation. Results We demonstrate that in HASMC LTD4-stimulated thymidine incorporation and potentiation of EGF-induced mitogenic signaling mostly depends upon EGF-R transactivation through the stimulation of CysLT1-R. Accordingly, we found that LTD4 stimulation was able to trigger the increase of Ras-GTP and, in turn, to activate ERK1/2. We show here that EGF-R transactivation was sensitive to pertussis toxin (PTX) and phosphoinositide 3-kinase (PI3K) inhibitors and that it occurred independently from Src activity, despite the observation of a strong impairment of LTD4-induced DNA synthesis following Src inhibition. More interestingly, CysLT1-R stimulation increased the production of ROS and N-acetylcysteine (NAC) abolished LTD4-induced EGF-R phosphorylation and thymidine incorporation. Conclusion Collectively, our data demonstrate that in HASMC LTD4 stimulation of a Gi/o coupled CysLT1-R triggers the transactivation of the EGF-R through the

  6. Control of upper airway muscle activity in younger versus older men during sleep onset

    PubMed Central

    Fogel, Robert B; White, David P; Pierce, Robert J; Malhotra, Atul; Edwards, Jill K; Dunai, Judy; Kleverlaan, Darci; Trinder, John

    2003-01-01

    Pharyngeal dilator muscles are clearly important in the pathophysiology of obstructive sleep apnoea syndrome (OSA). We have previously shown that the activity of both the genioglossus (GGEMG) and tensor palatini (TPEMG) are decreased at sleep onset, and that this decrement in muscle activity is greater in the apnoea patient than in healthy controls. We have also previously shown this decrement to be greater in older men when compared with younger ones. In order to explore the mechanisms responsible for this decrement in muscle activity nasal continuous positive airway pressure (CPAP) was applied to reduce negative pressure mediated muscle activation. We then investigated the effect of sleep onset (transition from predominantly α to predominantly θ EEG activity) on ventilation, upper airway muscle activation and upper airway resistance (UAR) in middle-aged and younger healthy men. We found that both GGEMG and TPEMG were reduced by the application of nasal CPAP during wakefulness, but that CPAP did not alter the decrement in activity in either muscle seen in the first two breaths following an α to θ transition. However, CPAP prevented both the rise in UAR at sleep onset that occurred on the control night, and the recruitment in GGEMG seen in the third to fifth breaths following the α to θ transition. Further, GGEMG was higher in the middle-aged men than in the younger men during wakefulness and was decreased more in the middle-aged men with the application of nasal CPAP. No differences were seen in TPEMG between the two age groups. These data suggest that the initial sleep onset reduction in upper airway muscle activity is due to loss of a ‘wakefulness’ stimulus, rather than to loss of responsiveness to negative pressure. In addition, it suggests that in older men, higher wakeful muscle activity is due to an anatomically more collapsible upper airway with more negative pressure driven muscle activation. Sleep onset per se does not appear to have a greater

  7. [Relaxant effects of protopine on smooth muscles].

    PubMed

    Huang, Y H; Zhang, Z Z; Jiang, J X

    1991-01-01

    The relaxant effects of protopine (Pro) on smooth muscles were studied by recording isotonic contraction and radioimmunoassay. Pro relaxed the contraction of rabbit thoracic aorta, mesenteric artery, portal vein and guinea pig ileum and taenia colon induced by high K+ (70 mmol.L-1). Pro also inhibited the contraction of rabbit thoracic aorta, mesenteric artery, portal vein induced by NE (0.3 mumol.L-1) and guinea pig taenia colon induced by BaCl2 (1 mmol.L-1). Pro inhibited the intracellular Ca2+ release, but did not inhibit Ca2+ influx induced by NE. These results suggested that the smooth muscle relaxant mechanism of action of Pro may be the inhibition of intracellular Ca2+ release.

  8. A 310-bp minimal promoter mediates smooth muscle cell-specific expression of telokin.

    PubMed

    Smith, A F; Bigsby, R M; Word, R A; Herring, B P

    1998-05-01

    A cell-specific promoter located in an intron of the smooth muscle myosin light chain kinase gene directs transcription of telokin exclusively in smooth muscle cells. Transgenic mice were generated in which a 310-bp rabbit telokin promoter fragment, extending from -163 to +147, was used to drive expression of simian virus 40 large T antigen. Smooth muscle-specific expression of the T-antigen transgene paralleled that of the endogenous telokin gene in all smooth muscle tissues except uterus. The 310-bp promoter fragment resulted in very low levels of transgene expression in uterus; in contrast, a transgene driven by a 2.4-kb fragment (-2250 to +147) resulted in high levels of transgene expression in uterine smooth muscle. Telokin expression levels correlate with the estrogen status of human myometrial tissues, suggesting that deletion of an estrogen response element (ERE) may account for the low levels of transgene expression driven by the 310-bp rabbit telokin promoter in uterine smooth muscle. Experiments in A10 smooth muscle cells directly showed that reporter gene expression driven by the 2.4-kb, but not 310-bp, promoter fragment could be stimulated two- to threefold by estrogen. This stimulation was mediated through an ERE located between -1447 and -1474. Addition of the ERE to the 310-bp fragment restored estrogen responsiveness in A10 cells. These data demonstrate that in addition to a minimal 310-bp proximal promoter at least one distal cis-acting regulatory element is required for telokin expression in uterine smooth muscle. The distal element may include an ERE between -1447 and -1474.

  9. A Novel Orally Available Asthma Drug Candidate That Reduces Smooth Muscle Constriction and Inflammation by Targeting GABAA Receptors in the Lung.

    PubMed

    Forkuo, Gloria S; Nieman, Amanda N; Kodali, Revathi; Zahn, Nicolas M; Li, Guanguan; Rashid Roni, M S; Stephen, Michael Rajesh; Harris, Ted W; Jahan, Rajwana; Guthrie, Margaret L; Yu, Olivia B; Fisher, Janet L; Yocum, Gene T; Emala, Charles W; Steeber, Douglas A; Stafford, Douglas C; Cook, James M; Arnold, Leggy A

    2018-05-07

    We describe lead compound MIDD0301 for the oral treatment of asthma based on previously developed positive allosteric α 5 β 3 γ 2 selective GABA A receptor (GABA A R) ligands. MIDD0301 relaxed airway smooth muscle at single micromolar concentrations as demonstrated with ex vivo guinea pig tracheal rings. MIDD0301 also attenuated airway hyperresponsiveness (AHR) in an ovalbumin murine model of asthma by oral administration. Reduced numbers of eosinophils and macrophages were observed in mouse bronchoalveolar lavage fluid without changing mucous metaplasia. Importantly, lung cytokine expression of IL-17A, IL-4, and TNF-α were reduced for MIDD0301-treated mice without changing antiinflammatory cytokine IL-10 levels. Automated patch clamp confirmed amplification of GABA induced current mediated by α 1-3,5 β 3 γ 2 GABA A Rs in the presence of MIDD0301. Pharmacodynamically, transmembrane currents of ex vivo CD4 + T cells from asthmatic mice were potentiated by MIDD0301 in the presence of GABA. The number of CD4 + T cells observed in the lung of MIDD0301-treated mice were reduced by an oral treatment of 20 mg/kg b.i.d. for 5 days. A half-life of almost 14 h was demonstrated by pharmacokinetic studies (PK) with no adverse CNS effects when treated mice were subjected to sensorimotor studies using the rotarod. PK studies also confirmed very low brain distribution. In conclusion, MIDD0301 represents a safe and improved oral asthma drug candidate that relaxes airway smooth muscle and attenuates inflammation in the lung leading to a reduction of AHR at a dosage lower than earlier reported GABA A R ligands.

  10. YFa and analogs: Investigation of opioid receptors in smooth muscle contraction

    PubMed Central

    Kumar, Krishan; Goyal, Ritika; Mudgal, Annu; Mohan, Anita; Pasha, Santosh

    2011-01-01

    AIM: To study the pharmacological profile and inhibition of smooth muscle contraction by YFa and its analogs in conjunction with their receptor selectivity. METHODS: The effects of YFa and its analogs (D-Ala2) YFa, Y (D-Ala2) GFMKKKFMRF amide and Des-Phe-YGGFMKKKFMR amide in guinea pig ileum (GPI) and mouse vas deferens (MVD) motility were studied using an isolated tissue organ bath system, and morphine and DynA (1-13) served as controls. Acetylcholine was used for muscle stimulation. The observations were validated by specific antagonist pretreatment experiments using naloxonazine, naltrindole and norbinaltorphimine norBNI. RESULTS: YFa did not demonstrate significant inhibition of GPI muscle contraction as compared with morphine (15% vs 62%, P = 0.0002), but moderate inhibition of MVD muscle contraction, indicating the role of κ opioid receptors in the contraction. A moderate inhibition of GPI muscles by (Des-Phe) YFa revealed the role of anti-opiate receptors in the smooth muscle contraction. (D-Ala-2) YFa showed significant inhibition of smooth muscle contraction, indicating the involvement of mainly δ receptors in MVD contraction. These results were supported by specific antagonist pretreatment assays. CONCLUSION: YFa revealed its side-effect-free analgesic properties with regard to arrest of gastrointestinal transit. The study provides evidences for the involvement of κ and anti-opioid receptors in smooth muscle contraction. PMID:22110284

  11. YFa and analogs: investigation of opioid receptors in smooth muscle contraction.

    PubMed

    Kumar, Krishan; Goyal, Ritika; Mudgal, Annu; Mohan, Anita; Pasha, Santosh

    2011-10-28

    To study the pharmacological profile and inhibition of smooth muscle contraction by YFa and its analogs in conjunction with their receptor selectivity. The effects of YFa and its analogs (D-Ala2) YFa, Y (D-Ala2) GFMKKKFMRF amide and Des-Phe-YGGFMKKKFMR amide in guinea pig ileum (GPI) and mouse vas deferens (MVD) motility were studied using an isolated tissue organ bath system, and morphine and DynA (1-13) served as controls. Acetylcholine was used for muscle stimulation. The observations were validated by specific antagonist pretreatment experiments using naloxonazine, naltrindole and norbinaltorphimine norBNI. YFa did not demonstrate significant inhibition of GPI muscle contraction as compared with morphine (15% vs 62%, P = 0.0002), but moderate inhibition of MVD muscle contraction, indicating the role of κ opioid receptors in the contraction. A moderate inhibition of GPI muscles by (Des-Phe) YFa revealed the role of anti-opiate receptors in the smooth muscle contraction. (D-Ala-2) YFa showed significant inhibition of smooth muscle contraction, indicating the involvement of mainly δ receptors in MVD contraction. These results were supported by specific antagonist pretreatment assays. YFa revealed its side-effect-free analgesic properties with regard to arrest of gastrointestinal transit. The study provides evidences for the involvement of κ and anti-opioid receptors in smooth muscle contraction.

  12. EMMPRIN (CD147) Expression in Smooth Muscle Tumors of the Uterus.

    PubMed

    Kefeli, Mehmet; Yildiz, Levent; Gun, Seda; Ozen, Fatma Z; Karagoz, Filiz

    2016-01-01

    Smooth muscle tumors of the uterus are the most common mesenchymal tumors of the gynecologic tract. The vast majority of these are benign leiomyomas that present no diagnostic difficulty. Because some benign smooth muscle tumors may degenerate and uncommon variants exist, the diagnosis can be challenging in some cases. The goal of this research was to investigate EMMPRIN expression in leiomyomas, leiomyoma variants, and leiomyosarcomas (LMS) to determine whether it has a potential role in differential diagnosis. EMMPRIN expression was investigated with immunohistochemistry in 103 uterine smooth muscle tumors, which included 19 usual leiomyomas, 52 leiomyoma variants, and 32 LMS. They were evaluated on the basis of staining extent, intensity, and also their combined score, and the groups were compared. EMMPRIN expression was present in 3 of 19 (15.7%) usual leiomyomas, 23 of 52 (44.3%) leiomyoma variants, and 28 of 32 (87.5%) LMS. There were statistically significant differences in staining extent and intensity, and also for their combined scores, between the LMS and benign groups. Although uterine smooth muscle tumors are usually diagnosed easily with conventional diagnostic criteria, the differentiation of LMS from some variants of leiomyoma can be challenging based soley on morphology. EMMPRIN may be a valuable immunohistochemical marker for differentiating LMS from benign smooth muscle tumors in problematic cases.

  13. Airway-parenchymal interdependence

    PubMed Central

    Paré, Peter D; Mitzner, Wayne

    2015-01-01

    In this manuscript we discuss the interaction of the lung parenchyma and the airways as well as the physiological and pathophysiological significance of this interaction. These two components of the respiratory organ can be thought of as two independent elastic structures but in fact the mechanical properties of one influence the behavior of the other. Traditionally the interaction has focused on the effects of the lung on the airways but there is good evidence that the opposite is also true, i.e., that the mechanical properties of the airways influence the elastic properties of the parenchyma. The interplay between components of the respiratory system including the airways, parenchyma and vasculature is often referred to as “interdependence.” This interdependence transmits the elastic recoil of the lung to create an effective pressure that dilates the airways as transpulmonary pressure and lung volume increase. By using a continuum mechanics analysis of the lung parenchyma, it is possible to predict the effective pressure between the airways and parenchyma, and these predictions can be empirically evaluated. Normal airway caliber is maintained by this pressure in the adventitial interstitium of the airway, and it counteracts airway compression during forced expiration as well as the ability of airway smooth muscle to narrow airways. Interdependence has physiological and pathophysiological significance. Weakening of the forces of interdependence contributes to airway dysfunction and gas exchange impairment in acute and chronic airway diseases including asthma and emphysema. PMID:23723029

  14. Congenital smooth muscle hamartoma of the palpebral conjunctiva.

    PubMed

    Mora, L Evelyn; Rodríguez-Reyes, Abelardo A; Vera, Ana M; Rubio, Rosa Isela; Mayorquín-Ruiz, Mariana; Salcedo, Guillermo

    2012-01-01

    Smooth muscle hamartoma is defined as a disorganized focus or an overgrowth of mature smooth muscle, generally with low capacity of autonomous growth and benign behavior. The implicated tissues are mature and proliferate in a disorganized fashion. A healthy 5-day-old Mexican boy was referred to the authors' hospital in México city for evaluation of a "cystic" lesion of the right eye that had been noted since birth. The pregnancy and delivery were unremarkable. On physical examination, there was a reddish-pink soft lesion with a tender "cystic" appearance, which was probably emerging from the upper eyelid conjunctiva, which measured 2.7 cm in its widest diameter and transilluminated. Ultrasound imaging revealed an anterior "cystic" lesion with normally formed phakic eye. An excisional biopsy was performed, and the lesion was dissected from the upper tarsal subconjunctival space. Subsequent histologic and immunohistochemical findings were consistent with the diagnosis of congenital smooth muscle hamartoma (CSMH) of the tarsal conjunctiva. The authors' research revealed that only one case of CSMH localized in the conjunctiva (Roper GJ, Smith MS, Lueder GT. Congenital smooth muscle hamartoma of the conjunctival fornix. Am J Ophthalmol. 1999;128:643-4) has been reported to date in the literature. To the best of the authors' knowledge, this current case would be the second case reported of CSMH in this anatomic location. Therefore, the authors' recommendation is to include CSMH in the differential diagnosis of a cystic mass that presents in the fornix and palpebral conjunctiva.

  15. Control of resting membrane potential by delayed rectifier potassium currents in ferret airway smooth muscle cells.

    PubMed Central

    Fleischmann, B K; Washabau, R J; Kotlikoff, M I

    1993-01-01

    1. In order to determine the physiological role of specific potassium currents in airway smooth muscle, potassium currents were measured in freshly dissociated ferret trachealis cells using the nystatin-permeabilized, whole-cell method, at 35 degrees C. 2. The magnitude of the outward currents was markedly increased as bath temperature was increased from 22 to 35 degrees C. This increase was primarily due to the increase in maximum potassium conductance (gK,max), although there was also a small leftward shift in the relationship between gK and voltage at higher temperatures. The maximum conductance and the kinetics of current activation and inactivation were also temperature dependent. At 35 degrees C, gating of the current was steeply voltage dependent between -40 and 0 mV. Current activation was well fitted by fourth-order kinetics; the mean time constants of activation (30 mV clamp step) were 1.09 +/- 0.17 and 1.96 +/- 0.27 ms at 35 and 22 degrees C, respectively. 3. Outward currents using the nystatin method were qualitatively similar to delayed rectifier currents recorded in dialysed cells with high calcium buffering capacity solutions. 4-Aminopyridine (4-AP; 2 mM), a specific blocker of delayed rectifier potassium channels in this tissue, inhibited over 80% of the outward current evoked by voltage-clamp steps to between -10 and +20 mV (n = 6). Less than 5% of the outward current was blocked over the same voltage range by charybdotoxin (100 nM; n = 15), a specific antagonist of large-conductance, calcium-activated potassium channels in this tissue. 4. The degree to which delayed rectifier and calcium-activated potassium conductances control resting membrane potential was examined in current-clamp experiments. The resting membrane potential of current clamped cells was -33.6 +/- 1.0 mV (n = 62). Application of 4-AP (2 mM) resulted in a 14.4 +/- 1.0 mV depolarization (n = 8) and an increase in input resistance. Charybdotoxin (100 nM) had no effect on resting

  16. Hyaluronan mediates airway hyperresponsiveness in oxidative lung injury

    PubMed Central

    Lazrak, Ahmed; Creighton, Judy; Yu, Zhihong; Komarova, Svetlana; Doran, Stephen F.; Aggarwal, Saurabh; Emala, Charles W.; Stober, Vandy P.; Trempus, Carol S.; Garantziotis, Stavros

    2015-01-01

    Chlorine (Cl2) inhalation induces severe oxidative lung injury and airway hyperresponsiveness (AHR) that lead to asthmalike symptoms. When inhaled, Cl2 reacts with epithelial lining fluid, forming by-products that damage hyaluronan, a constituent of the extracellular matrix, causing the release of low-molecular-weight fragments (L-HA, <300 kDa), which initiate a series of proinflammatory events. Cl2 (400 ppm, 30 min) exposure to mice caused an increase of L-HA and its binding partner, inter-α-trypsin-inhibitor (IαI), in the bronchoalveolar lavage fluid. Airway resistance following methacholine challenge was increased 24 h post-Cl2 exposure. Intratracheal administration of high-molecular-weight hyaluronan (H-HA) or an antibody against IαI post-Cl2 exposure decreased AHR. Exposure of human airway smooth muscle (HASM) cells to Cl2 (100 ppm, 10 min) or incubation with Cl2-exposed H-HA (which fragments it to L-HA) increased membrane potential depolarization, intracellular Ca2+, and RhoA activation. Inhibition of RhoA, chelation of intracellular Ca2+, blockade of cation channels, as well as postexposure addition of H-HA, reversed membrane depolarization in HASM cells. We propose a paradigm in which oxidative lung injury generates reactive species and L-HA that activates RhoA and Ca2+ channels of airway smooth muscle cells, increasing their contractility and thus causing AHR. PMID:25747964

  17. Evidence Supports Tradition: The in Vitro Effects of Roman Chamomile on Smooth Muscles.

    PubMed

    Sándor, Zsolt; Mottaghipisheh, Javad; Veres, Katalin; Hohmann, Judit; Bencsik, Tímea; Horváth, Attila; Kelemen, Dezső; Papp, Róbert; Barthó, Loránd; Csupor, Dezső

    2018-01-01

    The dried flowers of Chamaemelum nobile (L.) All. have been used in traditional medicine for different conditions related to the spasm of the gastrointestinal system. However, there have been no experimental studies to support the smooth muscle relaxant effect of this plant. The aim of our research was to assess the effects of the hydroethanolic extract of Roman chamomile, its fractions, four of its flavonoids (apigenin, luteolin, hispidulin, and eupafolin), and its essential oil on smooth muscles. The phytochemical compositions of the extract and its fractions were characterized and quantified by HPLC-DAD, the essential oil was characterized by GC and GC-MS. Neuronally mediated and smooth muscle effects were tested in isolated organ bath experiments on guinea pig, rat, and human smooth muscle preparations. The crude herbal extract induced an immediate, moderate, and transient contraction of guinea pig ileum via the activation of cholinergic neurons of the gut wall. Purinoceptor and serotonin receptor antagonists did not influence this effect. The more sustained relaxant effect of the extract, measured after pre-contraction of the preparations, was remarkable and was not affected by an adrenergic beta receptor antagonist. The smooth muscle-relaxant activity was found to be associated with the flavonoid content of the fractions. The essential oil showed only the relaxant effect, but no contracting activity. The smooth muscle-relaxant effect was also detected on rat gastrointestinal tissues, as well as on strip preparations of human small intestine. These results suggest that Roman chamomile extract has a direct and prolonged smooth muscle-relaxant effect on guinea pig ileum which is related to its flavonoid content. In some preparations, a transient stimulation of enteric cholinergic motoneurons was also detected. The essential oil also had a remarkable smooth muscle relaxant effect in this setting. Similar relaxant effects were also detected on other visceral

  18. Tachykinin receptor expression and function in human esophageal smooth muscle.

    PubMed

    Kovac, Jason R; Chrones, Tom; Preiksaitis, Harold G; Sims, Stephen M

    2006-08-01

    Tachykinins are present in enteric nerves of the gastrointestinal tract and cause contraction of esophageal smooth muscle; however, the mechanisms involved are not understood. Our aim was to characterize tachykinin signaling in human esophageal smooth muscle. We investigated functional effects of tachykinins on human esophageal smooth muscle using tension recordings and isolated cells, receptor expression with reverse transcription (RT)-polymerase chain reaction (PCR) and immunoblotting, intracellular Ca2+ responses using fluorescent indicator dyes, and membrane currents with patch-clamp electrophysiology. The mammalian tachykinins [substance P and neurokinin (NK) A and NKB] elicited concentration-dependent contractions of human esophageal smooth muscle. These responses were not affected by muscarinic receptor or neuronal blockade indicating a direct effect on smooth muscle cells (SMCs). Immunofluorescence and RT-PCR identified tachykinin receptors (NK1, NK2, and NK3) on SMCs. Contraction was mediated through a combination of Ca2+ release from intracellular stores and influx through L-type Ca2+ channels. NK2 receptor blockade inhibited the largest proportion of tachykinin-evoked responses. NKA evoked a nonselective cation current (I(NSC)) with properties similar to that elicited by muscarinic stimulation. The following paradigm is suggested: tachykinin receptor binding to SMCs releases Ca2+ from stores along with activation of I(NSC), which in turn results in membrane depolarization, L-type Ca2+ channel opening, rise of Ca2+ concentration, and contraction. These studies reveal new aspects of tachykinin signaling in human esophageal SMCs. Excitatory tachykinin pathways may represent targets for pharmacological intervention in disorders of esophageal dysmotility.

  19. High concentrations of landiolol, a beta(1)-adrenoceptor antagonist, stimulate smooth muscle contraction of the rat trachea through the Rho-kinase pathway.

    PubMed

    Shibata, Osamu; Nishioka, Kenji; Yamaguchi, Masakazu; Makita, Tetsuji; Sumikawa, Koji

    2008-01-01

    Gradually progressing contraction of airway smooth muscle is suggested to be due to the Rho-kinase signaling pathway. In our preliminary study in rat tracheas, landiolol, a beta(1)-adrenoceptor antagonist, at high doses caused gradually progressing contraction, and this contraction reached a plateau after 20 min. Therefore, this study was carried out to clarify whether landiolol could stimulate the Rho-kinase pathway or the phosphatidylinositol (PI) response in the rat trachea. Seventy-eight male Wistar rats weighing 250-350 g were used for the experiments. Their tracheas were cut into 3-mm-wide ring segments or 1-mm-wide slices. Measurements of isometric tension and [(3)H] inositol monophosphate (IP(1)) production were conducted, using these tracheal rings or slices. Data values are expressed as means +/- SD, and statistical significance (P < 0.05) was determined using analysis of variance (ANOVA). Landiolol (700 microM)-induced contraction was completely inhibited by fasudil at 30 microM, while the landiolol-induced contraction was not inhibited by 4-diphenylacetoxy-N-methyl-piperidine methobromide (4-DAMP), ketanserin, or nicardipine. Landiolol did not stimulate IP(1) production. These results suggest that high concentrations of landiolol could cause airway smooth muscle contraction through the Rho-kinase pathway, but not through the PI response coupled with muscarinic M(3) receptors, 5-HT receptors or the activation of L-type Ca(2+) channels.

  20. The comparative effects of aminoglycoside antibiotics and muscle relaxants on electrical field stimulation response in rat bladder smooth muscle.

    PubMed

    Min, Chang Ho; Min, Young Sil; Lee, Sang Joon; Sohn, Uy Dong

    2016-06-01

    It has been reported that several aminoglycoside antibiotics have a potential of prolonging the action of non-depolarizing muscle relaxants by drug interactions acting pre-synaptically to inhibit acetylcholine release, but antibiotics itself also have a strong effect on relaxing the smooth muscle. In this study, four antibiotics of aminoglycosides such as gentamicin, streptomycin, kanamycin and neomycin were compared with skeletal muscle relaxants baclofen, tubocurarine, pancuronium and succinylcholine, and a smooth muscle relaxant, papaverine. The muscle strips isolated from the rat bladder were stimulated with pulse trains of 40 V in amplitude and 10 s in duration, with pulse duration of 1 ms at the frequency of 1-8 Hz, at 1, 2, 4, 6, 8 Hz respectively. To test the effect of four antibiotics on bladder smooth muscle relaxation, each of them was treated cumulatively from 1 μM to 0.1 mM with an interval of 5 min. Among the four antibiotics, gentamicin and neomycin inhibited the EFS response. The skeletal muscle relaxants (baclofen, tubocurarine, pancuronium and succinylcholine) and inhibitory neurotransmitters (GABA and glycine) did not show any significant effect. However, papaverine, had a significant effect in the relaxation of the smooth muscle. It was suggested that the aminoglycoside antibiotics have inhibitory effect on the bladder smooth muscle.

  1. A biomechanical model of agonist-initiated contraction in the asthmatic airway.

    PubMed

    Brook, B S; Peel, S E; Hall, I P; Politi, A Z; Sneyd, J; Bai, Y; Sanderson, M J; Jensen, O E

    2010-01-31

    This paper presents a modelling framework in which the local stress environment of airway smooth muscle (ASM) cells may be predicted and cellular responses to local stress may be investigated. We consider an elastic axisymmetric model of a layer of connective tissue and circumferential ASM fibres embedded in parenchymal tissue and model the active contractile force generated by ASM via a stress acting along the fibres. A constitutive law is proposed that accounts for active and passive material properties as well as the proportion of muscle to connective tissue. The model predicts significantly different contractile responses depending on the proportion of muscle to connective tissue in the remodelled airway. We find that radial and hoop-stress distributions in remodelled muscle layers are highly heterogenous with distinct regions of compression and tension. Such patterns of stress are likely to have important implications, from a mechano-transduction perspective, on contractility, short-term cytoskeletal adaptation and long-term airway remodelling in asthma. Copyright 2009 Elsevier B.V. All rights reserved.

  2. Desipramine improves upper airway collapsibility and reduces OSA severity in patients with minimal muscle compensation

    PubMed Central

    Taranto-Montemurro, Luigi; Sands, Scott A.; Edwards, Bradley A.; Azarbarzin, Ali; Marques, Melania; de Melo, Camila; Eckert, Danny J.; White, David P.; Wellman, Andrew

    2017-01-01

    We recently demonstrated that desipramine reduces the sleep-related loss of upper airway dilator muscle activity and reduces pharyngeal collapsibility in healthy humans without obstructive sleep apnoea (OSA). The aim of the present physiological study was to determine the effects of desipramine on upper airway collapsibility and apnoea–hypopnea index (AHI) in OSA patients. A placebo-controlled, double-blind, randomised crossover trial in 14 OSA patients was performed. Participants received treatment or placebo in randomised order before sleep. Pharyngeal collapsibility (critical collapsing pressure of the upper airway (Pcrit)) and ventilation under both passive (V′0,passive) and active (V′0,active) upper airway muscle conditions were evaluated with continuous positive airway pressure (CPAP) manipulation. AHI was quantified off CPAP. Desipramine reduced active Pcrit (median (interquartile range) −5.2 (4.3) cmH2O on desipramine versus −1.9 (2.7) cmH2O on placebo; p=0.049) but not passive Pcrit (−2.2 (3.4) versus −0.7 (2.1) cmH2O; p=0.135). A greater reduction in AHI occurred in those with minimal muscle compensation (defined as V′0,active−V′0, passive) on placebo (r=0.71, p=0.009). The reduction in AHI was driven by the improvement in muscle compensation (r=0.72, p=0.009). In OSA patients, noradrenergic stimulation with desipramine improves pharyngeal collapsibility and may be an effective treatment in patients with minimal upper airway muscle compensation. PMID:27799387

  3. Expression of protease activated receptor-2 (PAR-2) in central airways of smokers and non-smokers

    PubMed Central

    Miotto, D; Hollenberg, M; Bunnett, N; Papi, A; Braccioni, F; Boschetto, P; Rea, F; Zuin, A; Geppetti, P; Saetta, M; Maestrelli, P; Fabbri, L; Mapp, C

    2002-01-01

    Background: Protease activated receptor-2 (PAR-2) is a transmembrane G protein coupled receptor preferentially activated by trypsin and tryptase. The protease activated receptors play an important role in most components of injury responses including cell proliferation, migration, matrix remodelling, and inflammation. Cigarette smoking causes an inflammatory process in the central airways, peripheral airways, lung parenchyma, and adventitia of pulmonary arteries. Methods: To quantify the expression of PAR-2 in the central airways of smokers and non-smokers, surgical specimens obtained from 30 subjects undergoing lung resection for localised pulmonary lesions (24 with a history of cigarette smoking and six non-smoking control subjects) were examined. Central airways were immunostained with an antiserum specific for PAR-2 and PAR-2 expression was quantified using light microscopy and image analysis. Results: PAR-2 expression was found in bronchial smooth muscle, epithelium, glands, and in the endothelium and smooth muscle of bronchial vessels. PAR-2 expression was similar in the central airways of smokers and non-smokers. When smokers were divided according to the presence of symptoms of chronic bronchitis and chronic airflow limitation, PAR-2 expression was increased in smooth muscle (median 3.8 (interquartile range 2.9–5.8) and 1.4 (1.07–3.4) respectively); glands (33.3 (18.2–43.8) and 16.2 (11.5–22.2), respectively); and bronchial vessels (54.2 (48.7–56.8) and 40.0 (36–40.4), respectively) of smokers with symptoms of chronic bronchitis with normal lung function compared with smokers with chronic airflow limitation (COPD), but the increase was statistically significant (p<0.005) only for bronchial vessels. Conclusions: PAR-2 is present in bronchial smooth muscle, glands, and bronchial vessels of both smokers and non-smokers. An increased expression of PAR-2 was found in bronchial vessels of patients with bronchitis compared with those with COPD. PMID

  4. Cigarette smoke and α,β-unsaturated aldehydes elicit VEGF release through the p38 MAPK pathway in human airway smooth muscle cells and lung fibroblasts

    PubMed Central

    Volpi, Giorgia; Facchinetti, Fabrizio; Moretto, Nadia; Civelli, Maurizio; Patacchini, Riccardo

    2011-01-01

    BACKGROUND AND PURPOSE Vascular endothelial growth factor (VEGF) is an angiogenic factor known to be elevated in the sputum of asymptomatic smokers as well as smokers with bronchitis type of chronic obstructive pulmonary disease. The aim of this study was to investigate whether acute exposure to cigarette smoke extract altered VEGF production in lung parenchymal cells. EXPERIMENTAL APPROACH We exposed human airway smooth muscle cells (ASMC), normal human lung fibroblasts (NHLF) and small airways epithelial cells (SAEC) to aqueous cigarette smoke extract (CSE) in order to investigate the effect of cigarette smoke on VEGF expression and release. KEY RESULTS Vascular endothelial growth factor release was elevated by sub-toxic concentrations of CSE in both ASMC and NHLF, but not in SAEC. CSE-evoked VEGF release was mimicked by its component acrolein at concentrations (10–100 µM) found in CSE, and prevented by the antioxidant and α,β-unsaturated aldehyde scavenger, N-acetylcysteine (NAC). Both CSE and acrolein (30 µM) induced VEGF mRNA expression in ASMC cultures, suggesting an effect at transcriptional level. Crotonaldehyde and 4-hydroxy-2-nonenal, an endogenous α,β-unsaturated aldehyde, stimulated VEGF release, as did H2O2. CSE-evoked VEGF release was accompanied by rapid and lasting phosphorylation of p38 MAPK (mitogen-activated protein kinase), which was abolished by NAC and mimicked by acrolein. Both CSE- and acrolein-evoked VEGF release were blocked by selective inhibition of p38 MAPK signalling. CONCLUSIONS AND IMPLICATIONS α,β-Unsaturated aldehydes and possibly reactive oxygen species contained in cigarette smoke stimulate VEGF expression and release from pulmonary cells through p38 MAPK signalling. PMID:21306579

  5. Smooth muscle contraction: mechanochemical formulation for homogeneous finite strains.

    PubMed

    Stålhand, J; Klarbring, A; Holzapfel, G A

    2008-01-01

    Chemical kinetics of smooth muscle contraction affect mechanical properties of organs that function under finite strains. In an effort to gain further insight into organ physiology, we formulate a mechanochemical finite strain model by considering the interaction between mechanical and biochemical components of cell function during activation. We propose a new constitutive framework and use a mechanochemical device that consists of two parallel elements: (i) spring for the cell stiffness; (ii) contractile element for the sarcomere. We use a multiplicative decomposition of cell elongation into filament contraction and cross-bridge deformation, and suggest that the free energy be a function of stretches, four variables (free unphosphorylated myosin, phosphorylated cross-bridges, phosphorylated and dephosphorylated cross-bridges attached to actin), chemical state variable driven by Ca2+-concentration, and temperature. The derived constitutive laws are thermodynamically consistent. Assuming isothermal conditions, we specialize the mechanical phase such that we recover the linear model of Yang et al. [2003a. The myogenic response in isolated rat cerebrovascular arteries: smooth muscle cell. Med. Eng. Phys. 25, 691-709]. The chemical phase is also specialized so that the linearized chemical evolution law leads to the four-state model of Hai and Murphy [1988. Cross-bridge phosphorylation and regulation of latch state in smooth muscle. Am. J. Physiol. 254, C99-C106]. One numerical example shows typical mechanochemical effects and the efficiency of the proposed approach. We discuss related parameter identification, and illustrate the dependence of muscle contraction (Ca2+-concentration) on active stress and related stretch. Mechanochemical models of this kind serve the mathematical basis for analyzing coupled processes such as the dependency of tissue properties on the chemical kinetics of smooth muscle.

  6. Reflex regulation of airway sympathetic nerves in guinea-pigs

    PubMed Central

    Oh, Eun Joo; Mazzone, Stuart B; Canning, Brendan J; Weinreich, Daniel

    2006-01-01

    Sympathetic nerves innervate the airways of most species but their reflex regulation has been essentially unstudied. Here we demonstrate sympathetic nerve-mediated reflex relaxation of airway smooth muscle measured in situ in the guinea-pig trachea. Retrograde tracing, immunohistochemistry and electrophysiological analysis identified a population of substance P-containing capsaicin-sensitive spinal afferent neurones in the upper thoracic (T1–T4) dorsal root ganglia (DRG) that innervate the airways and lung. After bilateral vagotomy, atropine pretreatment and precontraction of the trachealis with histamine, nebulized capsaicin (10–60 μm) evoked a 63 ± 7% reversal of the histamine-induced contraction of the trachealis. Either the β-adrenoceptor antagonist propranolol (2 μm, administered directly to the trachea) or bilateral sympathetic nerve denervation of the trachea essentially abolished these reflexes (10 ± 9% and 6 ± 4% relaxations, respectively), suggesting that they were mediated primarily, if not exclusively, by sympathetic adrenergic nerve activation. Cutting the upper thoracic dorsal roots carrying the central processes of airway spinal afferents also markedly blocked the relaxations (9 ± 5% relaxation). Comparable inhibitory effects were observed following intravenous pretreatment with neurokinin receptor antagonists (3 ± 7% relaxations). These reflexes were not accompanied by consistent changes in heart rate or blood pressure. By contrast, stimulating the rostral cut ends of the cervical vagus nerves also evoked a sympathetic adrenergic nerve-mediated relaxation that were accompanied by marked alterations in blood pressure. The results indicate that the capsaicin-induced reflex-mediated relaxation of airway smooth muscle following vagotomy is mediated by sequential activation of tachykinin-containing spinal afferent and sympathetic efferent nerves innervating airways. This sympathetic nerve-mediated response may serve to oppose airway

  7. Vitamin D inhibits growth of human airway smooth muscle cells through growth factor-induced phosphorylation of retinoblastoma protein and checkpoint kinase 1

    PubMed Central

    Damera, G; Fogle, HW; Lim, P; Goncharova, EA; Zhao, H; Banerjee, A; Tliba, O; Krymskaya, VP; Panettieri, RA

    2009-01-01

    Background and purpose: Airway remodelling in asthma is manifested, in part, as increased airway smooth muscle (ASM) mass, reflecting myocyte proliferation. We hypothesized that calcitriol, a secosteroidal vitamin D receptor (VDR) modulator, would inhibit growth factor-induced myocyte proliferation. Experimental approach: Human ASM cell cultures were derived from bronchial samples taken during surgery. ASM cells were treated with platelet-derived growth factor (PDGF) (10 ng·mL−1) for 24 h in the presence of calcitriol, dexamethasone or a checkpoint kinase 1 (Chk1) inhibitor (SB218078). The effects of calcitriol on PDGF-mediated cell proliferation were assessed by thymidine incorporation assay, propidium iodide-based cell cycle analysis, caspase-3 assay and immunoblotting for specific cell cycle modulators. Key results: Calcitriol, but not dexamethasone, inhibited PDGF-induced ASM DNA synthesis concentration dependently (IC50= 520 ± 52 nM). These effects were associated with VDR-mediated expression of cytochrome CYP24A1 with no effects on ASM apoptosis. Calcitriol substantially inhibited (P < 0.01) PDGF-stimulated cell growth in ASM derived from both normal (59 ± 8%) and asthmatic subjects (57 ± 9%). Calcitriol inhibited PDGF-induced phosphorylation of retinoblastoma protein (Rb) and Chk1, with no effects on PDGF-mediated activation of extracellular signal-regulated kinases 1/2, PI3-kinase and S6 kinase, or expression of p21Waf/Cip-1, p27Kip1, cyclin D and E2F-1. Consistent with these observations, SB218078 also inhibited (IC50= 450 ± 100 pM) PDGF-induced cell cycle progression. Conclusions and implications: Calcitriol decreased PDGF-induced ASM cell growth by inhibiting Rb and Chk1 phosphorylation. This Research Paper is the subject of a Commentary in this issue by Clifford and Knox (pp. 1426–1428). To view this article visit http://www3.interscience.wiley.com/journal/121548564/issueyear?year=2009 PMID:19814732

  8. Recruitment of β-Catenin to N-Cadherin Is Necessary for Smooth Muscle Contraction*

    PubMed Central

    Wang, Tao; Wang, Ruping; Cleary, Rachel A.; Gannon, Olivia J.; Tang, Dale D.

    2015-01-01

    β-Catenin is a key component that connects transmembrane cadherin with the actin cytoskeleton at the cell-cell interface. However, the role of the β-catenin/cadherin interaction in smooth muscle has not been well characterized. Here stimulation with acetylcholine promoted the recruitment of β-catenin to N-cadherin in smooth muscle cells/tissues. Knockdown of β-catenin by lentivirus-mediated shRNA attenuated smooth muscle contraction. Nevertheless, myosin light chain phosphorylation at Ser-19 and actin polymerization in response to contractile activation were not reduced by β-catenin knockdown. In addition, the expression of the β-catenin armadillo domain disrupted the recruitment of β-catenin to N-cadherin. Force development, but not myosin light chain phosphorylation and actin polymerization, was reduced by the expression of the β-catenin armadillo domain. Furthermore, actin polymerization and microtubules have been implicated in intracellular trafficking. In this study, the treatment with the inhibitor latrunculin A diminished the interaction of β-catenin with N-cadherin in smooth muscle. In contrast, the exposure of smooth muscle to the microtubule depolymerizer nocodazole did not affect the protein-protein interaction. Together, these findings suggest that smooth muscle contraction is mediated by the recruitment of β-catenin to N-cadherin, which may facilitate intercellular mechanotransduction. The association of β-catenin with N-cadherin is regulated by actin polymerization during contractile activation. PMID:25713069

  9. Aspergillus fumigatus Infection-Induced Neutrophil Recruitment and Location in the Conducting Airway of Immunocompetent, Neutropenic, and Immunosuppressed Mice.

    PubMed

    Shevchenko, Marina A; Bogorodskiy, Andrey O; Troyanova, Natalia I; Servuli, Ekaterina A; Bolkhovitina, Elena L; Büldt, Georg; Fahlke, Christoph; Gordeliy, Valentin I; Gensch, Thomas; Borshchevskiy, Valentin I; Sapozhnikov, Alexander M

    2018-01-01

    Susceptibility to fungal infection is commonly associated with impaired neutrophil responses. To study the mechanisms underlying this association, we investigated neutrophil recruitment to the conducting airway wall after Aspergillus fumigatus conidium inhalation in mouse models of drug-induced immunosuppression and antibody-mediated neutrophil depletion (neutropenia) by performing three-dimensional confocal laser-scanning microscopy of whole-mount primary bronchus specimens. Actin staining enabled visualization of the epithelial and smooth muscle layers that mark the airway wall. Gr-1 + or Ly6G + neutrophils located between the epithelium and smooth muscles were considered airway wall neutrophils. The number of airway wall neutrophils for immunocompetent, immunosuppressed, and neutropenic mice before and 6 h after A. fumigatus infection were analyzed and compared. Our results show that the number of conducting airway wall neutrophils in immunocompetent mice significantly increased upon inflammation, while a dramatic reduction in this number was observed following immunosuppression and neutropenia. Interestingly, a slight increase in the infiltration of neutrophils into the airway wall was detected as a result of infection, even in immunosuppressed and neutropenic mice. Taken together, these data indicate that neutrophils are present in intact conducting airway walls and the number elevates upon A. fumigatus infection. Conducting airway wall neutrophils are affected by both neutropenia and immunosuppression.

  10. Aspergillus fumigatus Infection-Induced Neutrophil Recruitment and Location in the Conducting Airway of Immunocompetent, Neutropenic, and Immunosuppressed Mice

    PubMed Central

    Bogorodskiy, Andrey O.; Troyanova, Natalia I.; Servuli, Ekaterina A.; Bolkhovitina, Elena L.; Büldt, Georg; Fahlke, Christoph; Gordeliy, Valentin I.; Gensch, Thomas; Sapozhnikov, Alexander M.

    2018-01-01

    Susceptibility to fungal infection is commonly associated with impaired neutrophil responses. To study the mechanisms underlying this association, we investigated neutrophil recruitment to the conducting airway wall after Aspergillus fumigatus conidium inhalation in mouse models of drug-induced immunosuppression and antibody-mediated neutrophil depletion (neutropenia) by performing three-dimensional confocal laser-scanning microscopy of whole-mount primary bronchus specimens. Actin staining enabled visualization of the epithelial and smooth muscle layers that mark the airway wall. Gr-1+ or Ly6G+ neutrophils located between the epithelium and smooth muscles were considered airway wall neutrophils. The number of airway wall neutrophils for immunocompetent, immunosuppressed, and neutropenic mice before and 6 h after A. fumigatus infection were analyzed and compared. Our results show that the number of conducting airway wall neutrophils in immunocompetent mice significantly increased upon inflammation, while a dramatic reduction in this number was observed following immunosuppression and neutropenia. Interestingly, a slight increase in the infiltration of neutrophils into the airway wall was detected as a result of infection, even in immunosuppressed and neutropenic mice. Taken together, these data indicate that neutrophils are present in intact conducting airway walls and the number elevates upon A. fumigatus infection. Conducting airway wall neutrophils are affected by both neutropenia and immunosuppression. PMID:29577051

  11. Smooth Muscle-Like Cells Generated from Human Mesenchymal Stromal Cells Display Marker Gene Expression and Electrophysiological Competence Comparable to Bladder Smooth Muscle Cells.

    PubMed

    Brun, Juliane; Lutz, Katrin A; Neumayer, Katharina M H; Klein, Gerd; Seeger, Tanja; Uynuk-Ool, Tatiana; Wörgötter, Katharina; Schmid, Sandra; Kraushaar, Udo; Guenther, Elke; Rolauffs, Bernd; Aicher, Wilhelm K; Hart, Melanie L

    2015-01-01

    The use of mesenchymal stromal cells (MSCs) differentiated toward a smooth muscle cell (SMC) phenotype may provide an alternative for investigators interested in regenerating urinary tract organs such as the bladder where autologous smooth muscle cells cannot be used or are unavailable. In this study we measured the effects of good manufacturing practice (GMP)-compliant expansion followed by myogenic differentiation of human MSCs on the expression of a range of contractile (from early to late) myogenic markers in relation to the electrophysiological parameters to assess the functional role of the differentiated MSCs and found that differentiation of MSCs associated with electrophysiological competence comparable to bladder SMCs. Within 1-2 weeks of myogenic differentiation, differentiating MSCs significantly expressed alpha smooth muscle actin (αSMA; ACTA2), transgelin (TAGLN), calponin (CNN1), and smooth muscle myosin heavy chain (SM-MHC; MYH11) according to qRT-PCR and/or immunofluorescence and Western blot. Voltage-gated Na+ current levels also increased within the same time period following myogenic differentiation. In contrast to undifferentiated MSCs, differentiated MSCs and bladder SMCs exhibited elevated cytosolic Ca2+ transients in response to K+-induced depolarization and contracted in response to K+ indicating functional maturation of differentiated MSCs. Depolarization was suppressed by Cd2+, an inhibitor of voltage-gated Ca2+-channels. The expression of Na+-channels was pharmacologically identified as the Nav1.4 subtype, while the K+ and Ca2+ ion channels were identified by gene expression of KCNMA1, CACNA1C and CACNA1H which encode for the large conductance Ca2+-activated K+ channel BKCa channels, Cav1.2 L-type Ca2+ channels and Cav3.2 T-type Ca2+ channels, respectively. This protocol may be used to differentiate adult MSCs into smooth muscle-like cells with an intermediate-to-late SMC contractile phenotype exhibiting voltage-gated ion channel

  12. Neurogenic vasoreactive response of human internal thoracic artery smooth muscle.

    PubMed

    Canver, C C; Cooler, S D; Saban, R

    1997-09-01

    The interaction between primary afferent neurons containing neuropeptides and the vascular smooth muscle is incompletely understood. To explore the function of perivascular afferent neurons and to determine whether they produce local effects on vascular smooth muscle cells, we investigated the effects of acute capsaicin and substance P administration in vitro on human internal thoracic arteries (ITA). Vessels were obtained from patients undergoing coronary bypass or from multiorgan transplant donors. Fourteen ITA segments (5 mm wide) were suspended as rings between two stainless-steel stirrups in water-jacketed (37 degrees C) tissue baths under 2.5 to 3 g of basal tension. The tissue baths contained 10 mL physiological salt solution (PSS) of the following composition (mM): NaCl, 119; KCl, 4.7; NaH2PO4, 1.0; MgCl2, 0.5; CaCl2, 2.5; NaHCO3, 25; and glucose, 11; aerated continuously with 95% O2 and 5% CO2. Peptidase inhibitors (phosphoramidon and captopril) were added to PSS to decrease peptide degradation. Mechanical responses were measured isometrically and recorded on a polygraph via isotonic force transducers. Vessels were preconstricted with submaximal concentrations of norepinephrine. After the tension had stabilized, substance P or capsaicin was added cumulatively to the tissue bath. At the end of the experiments, the viability of ITA was verified by its responses to endothelial-dependent (acetylcholine) and endothelial-independent (sodium nitroprusside) vasodilators. In the endothelium-intact ITA segments, substance P produced relaxation of ITA smooth muscle while it induced slight contraction when the ITA was devoid of its endothelium (P = 0.0585). The addition of capsaicin to human ITA primarily produced contractile effects on the developed smooth muscle force. The capsaicin-induced contraction of the ITA smooth muscle was independent of endothelial cell integrity, although contraction was greater in the endothelium-intact ITA segments (P = 0.0165). The

  13. Segregation of striated and smooth muscle lineages by a Notch-dependent regulatory network

    PubMed Central

    2014-01-01

    Background Lineage segregation from multipotent epithelia is a central theme in development and in adult stem cell plasticity. Previously, we demonstrated that striated and smooth muscle cells share a common progenitor within their epithelium of origin, the lateral domain of the somite-derived dermomyotome. However, what controls the segregation of these muscle subtypes remains unknown. We use this in vivo bifurcation of fates as an experimental model to uncover the underlying mechanisms of lineage diversification from bipotent progenitors. Results Using the strength of spatio-temporally controlled gene missexpression in avian embryos, we report that Notch harbors distinct pro-smooth muscle activities depending on the duration of the signal; short periods prevent striated muscle development and extended periods, through Snail1, promote cell emigration from the dermomyotome towards a smooth muscle fate. Furthermore, we define a Muscle Regulatory Network, consisting of Id2, Id3, FoxC2 and Snail1, which acts in concert to promote smooth muscle by antagonizing the pro-myogenic activities of Myf5 and Pax7, which induce striated muscle fate. Notch and BMP closely regulate the network and reciprocally reinforce each other’s signal. In turn, components of the network strengthen Notch signaling, while Pax7 silences this signaling. These feedbacks augment the robustness and flexibility of the network regulating muscle subtype segregation. Conclusions Our results demarcate the details of the Muscle Regulatory Network, underlying the segregation of muscle sublineages from the lateral dermomyotome, and exhibit how factors within the network promote the smooth muscle at the expense of the striated muscle fate. This network acts as an exemplar demonstrating how lineage segregation occurs within epithelial primordia by integrating inputs from competing factors. PMID:25015411

  14. Effects of maternal folic acid supplementation on airway remodeling and allergic airway disease development.

    PubMed

    İscan, Burcin; Tuzun, Funda; Eroglu Filibeli, Berna; Cilekar Micili, Serap; Ergur, Bekir Ugur; Duman, Nuray; Ozkan, Hasan; Kumral, Abdullah

    2018-03-27

    Maternal folic acid supplementation has been recommended prior to and during the first trimester of pregnancy to reduce the risk of infant neural tube defects. However, an uncertain relationship between folic acid supplementation during pregnancy and development of childhood asthma exists. Recent data show a methyl donor-rich diet could increase the risk of developing allergic airway disease through DNA methylation and aberrant gene transcription. This study evaluated the effect of folic acid supplementation during pregnancy on airway remodeling and allergic airway disease vulnerability in a mouse asthma model. BALB/c mice were divided into four groups according to gestational folic acid supplementation and postnatal ovalbumin (OVA) exposure: Group 1 (whole pregnancy folic acid supplementation + OVA-exposed group), Group 2 (first gestational week folic acid supplementation + OVA-exposed group), Group 3 (no folic acid supplementation + OVA-exposed group), and Group 4 (control group). Offspring were sacrificed on day 45 for immunohistological and ultrastructural tests. In OVA challenged groups, folic acid supplementation led to a thicker epithelial and subepithelial smooth muscle layer than in the unsupplemented group. Moreover, folic acid supplementation during whole pregnancy (Group 1) was associated with a thicker epithelial and subepithelial smooth muscle layer than folic acid supplementation during the first week of pregnancy (Group 2), suggesting a duration-response relationship. Electron microscopic imaging revealed that structural changes including the loss of epithelial integrity, thickening of basement membrane, and subepithelial fibrosis were more prominent in the folic acid supplementation groups. This study suggested that maternal folic acid supplementation during pregnancy affects airway remodeling and increases the allergic responses induced by ovalbumin challenge in offspring. In addition, the effect size increased as the duration and

  15. Neostigmine but not sugammadex impairs upper airway dilator muscle activity and breathing

    PubMed Central

    Eikermann, M.; Zaremba, S.; Malhotra, A.; Jordan, A. S.; Rosow, C.; Chamberlin, N. L.

    2008-01-01

    Background Cholinesterase inhibitor-based reversal agents, given in the absence of neuromuscular block, evoke a partial upper airway obstruction by decreasing skeletal upper airway muscle function. Sugammadex reverses neuromuscular block by encapsulating rocuronium. However, its effects on upper airway integrity and breathing are unknown. Methods Fifty-one adult male rats were anaesthetized with isoflurane, tracheostomized, and a femoral artery and vein were cannulated. First, we compared the efficacy of sugammadex 15 mg kg−1 and neostigmine 0.06 mg kg−1 to reverse respiratory effects of rocuronium-induced partial paralysis [train-of-four ratio (T4/T1)=0.5]. Subsequently, we compared the safety of sugammadex and neostigmine given after recovery of the T4/T1 to 1, by measuring phasic genioglossus activity and breathing. Results During partial paralysis (T4/T1=0.5), time to recovery of minute volume to baseline values was 10.9 (2), 75.8 (18), and 153 (54) s with sugammadex, neostigmine, and placebo, respectively (sugammadex was significantly faster than neostigmine and placebo, P<0.05). Recovery of T4/T1 was also faster for sugammadex than neostigmine and placebo. Neostigmine administration after complete recovery of T4/T1 decreased upper airway dilator muscle activity to 64 (30)% of baseline and decreased tidal volume (P<0.05 for both variables), whereas sugammadex had no effect on either variable. Conclusions In contrast to neostigmine, which significantly impairs upper airway dilator muscle activity when given after recovery from neuromuscular block, a reversal dose of sugammadex given under the same conditions does not affect genioglossus muscle activity and normal breathing. Human studies will be required to evaluate the clinical relevance of our findings. PMID:18559352

  16. Sorcin modulation of Ca2+ sparks in rat vascular smooth muscle cells

    PubMed Central

    Rueda, Angélica; Song, Ming; Toro, Ligia; Stefani, Enrico; Valdivia, Héctor H

    2006-01-01

    Spontaneous, local Ca2+ release events or Ca2+ sparks by ryanodine receptors (RyRs) are important determinants of vascular tone and arteriolar resistance, but the mechanisms that modulate their properties in smooth muscle are poorly understood. Sorcin, a Ca2+-binding protein that associates with cardiac RyRs and quickly stops Ca2+ release in the heart, provides a potential mechanism to modulate Ca2+ sparks in vascular smooth muscle, but little is known about the functional role of sorcin in this tissue. In this work, we characterized the expression and intracellular location of sorcin in aorta and cerebral artery and gained mechanistic insights into its functional role as a modulator of Ca2+ sparks. Sorcin is present in endothelial and smooth muscle cells, as assessed by immunocytochemical and Western blot analyses. Smooth muscle sorcin translocates from cytosolic to membranous compartments in a Ca2+-dependent manner and associates with RyRs, as shown by coimmunoprecipitation and immunostaining experiments. Ca2+ sparks recorded in saponin-permeabilized vascular myocytes have increased frequency, duration and spatial spread but reduced amplitude with respect to Ca2+ sparks in intact cells, suggesting that permeabilization disrupts the normal organization of RyRs and releases diffusible substances that control Ca2+ spark properties. Perfusion of 2 μm sorcin onto permeabilized myocytes reduced the amplitude, duration and spatial spread of Ca2+ sparks, demonstrating that sorcin effectively regulates Ca2+ signalling in vascular smooth muscle. Together with a dense distribution in the perimeter of the cell along a pool of RyRs, these properties make sorcin a viable candidate to modulate vascular tone in smooth muscle. PMID:16931553

  17. Loss of Notch2 and Notch3 in vascular smooth muscle causes patent ductus arteriosus.

    PubMed

    Baeten, Jeremy T; Jackson, Ashley R; McHugh, Kirk M; Lilly, Brenda

    2015-12-01

    The overlapping roles of the predominant Notch receptors in vascular smooth muscle cells, Notch2 and Notch3, have not been clearly defined in vivo. In this study, we use a smooth muscle-specific deletion of Notch2 together with a global Notch3 deletion to produce mice with combinations of mutant and wild-type Notch2/3 alleles in vascular smooth muscle cells. Mice with complete loss of Notch3 and smooth muscle-expressed Notch2 display late embryonic lethality and subcutaneous hemorrhage. Mice without smooth muscle-Notch2 and only one wild-type copy of Notch3 die within one day of birth and present with vascular defects, most notably patent ductus arteriosus (DA) and aortic dilation. These defects were associated with decreased expression of contractile markers in both the DA and aorta. These results demonstrate that Notch2 and Notch3 have overlapping roles in promoting development of vascular smooth muscle cells, and together contribute to functional closure of the DA. © 2015 Wiley Periodicals, Inc.

  18. Voltage dependent potassium channel remodeling in murine intestinal smooth muscle hypertrophy induced by partial obstruction.

    PubMed

    Liu, Dong-Hai; Huang, Xu; Guo, Xin; Meng, Xiang-Min; Wu, Yi-Song; Lu, Hong-Li; Zhang, Chun-Mei; Kim, Young-chul; Xu, Wen-Xie

    2014-01-01

    Partial obstruction of the small intestine causes obvious hypertrophy of smooth muscle cells and motility disorder in the bowel proximate to the obstruction. To identify electric remodeling of hypertrophic smooth muscles in partially obstructed murine small intestine, the patch-clamp and intracellular microelectrode recording methods were used to identify the possible electric remodeling and Western blot, immunofluorescence and immunoprecipitation were utilized to examine the channel protein expression and phosphorylation level changes in this research. After 14 days of obstruction, partial obstruction caused obvious smooth muscle hypertrophy in the proximally located intestine. The slow waves of intestinal smooth muscles in the dilated region were significantly suppressed, their amplitude and frequency were reduced, whilst the resting membrane potentials were depolarized compared with normal and sham animals. The current density of voltage dependent potassium channel (KV) was significantly decreased in the hypertrophic smooth muscle cells and the voltage sensitivity of KV activation was altered. The sensitivity of KV currents (IKV) to TEA, a nonselective potassium channel blocker, increased significantly, but the sensitivity of IKv to 4-AP, a KV blocker, stays the same. The protein levels of KV4.3 and KV2.2 were up-regulated in the hypertrophic smooth muscle cell membrane. The serine and threonine phosphorylation levels of KV4.3 and KV2.2 were significantly increased in the hypertrophic smooth muscle cells. Thus this study represents the first identification of KV channel remodeling in murine small intestinal smooth muscle hypertrophy induced by partial obstruction. The enhanced phosphorylations of KV4.3 and KV2.2 may be involved in this process.

  19. Patent ductus arteriosus in mice with smooth muscle-specific Jag1 deletion

    PubMed Central

    Feng, Xuesong; Krebs, Luke T.; Gridley, Thomas

    2010-01-01

    The ductus arteriosus is an arterial vessel that shunts blood flow away from the lungs during fetal life, but normally occludes after birth to establish the adult circulation pattern. Failure of the ductus arteriosus to close after birth is termed patent ductus arteriosus and is one of the most common congenital heart defects. Mice with smooth muscle cell-specific deletion of Jag1, which encodes a Notch ligand, die postnatally from patent ductus arteriosus. These mice exhibit defects in contractile smooth muscle cell differentiation in the vascular wall of the ductus arteriosus and adjacent descending aorta. These defects arise through an inability to propagate the JAG1-Notch signal via lateral induction throughout the width of the vascular wall. Both heterotypic endothelial smooth muscle cell interactions and homotypic vascular smooth muscle cell interactions are required for normal patterning and differentiation of the ductus arteriosus and adjacent descending aorta. This new model for a common congenital heart defect provides novel insights into the genetic programs that underlie ductus arteriosus development and closure. PMID:21068062

  20. Abrogation of Airway Hyperresponsiveness but not Inflammation by Rho kinase Insufficiency

    PubMed Central

    Kasahara, David I.; Ninin, Fernanda M.C.; Wurmbrand, Allison P.; Liao, James K.; Shore, Stephanie A.

    2015-01-01

    Background Major features of allergic asthma include airway hyperresponsiveness (AHR), eosinophilic inflammation, and goblet cell metaplasia. Rho kinase (ROCK) is a serine/threonine protein kinase that regulates the actin cytoskeleton. By doing so, it can modulate airway smooth muscle cell contraction and leukocyte migration and proliferation. This study was designed to determine the contributions of the two ROCK isoforms, ROCK1 and ROCK2, to AHR, inflammation and goblet cell metaplasia in a mast-cell dependent model of allergic airways disease. Methods and Results Repeated intranasal challenges with OVA caused AHR, eosinophilic inflammation, and goblet cell hyperplasia in wildtype (WT) mice. OVA-induced AHR was partially or completely abrogated in mice haploinsufficient for ROCK2 (ROCK2+/−) or ROCK1 (ROCK1+/−), respectively. In contrast, there was no effect of ROCK insufficiency on allergic airways inflammation, although both ROCK1 and ROCK2 insufficiency attenuated mast cell degranulation. Goblet cell hyperplasia, as indicated by PAS staining, was not different in ROCK1+/− versus WT mice. However, in ROCK2+/− mice, goblet cell hyperplasia was reduced in medium but not large airways. Maximal acetylcholine-induced force generation was reduced in tracheal rings from ROCK1+/− and ROCK2+/− versus WT mice. The ROCK inhibitor, fasudil, also reduced airway responsiveness in OVA-challenged mice, without affecting inflammatory responses. Conclusion In a mast cell model of allergic airways disease, ROCK1 and ROCK2 both contribute to AHR, likely through direct effects on smooth muscle cell and effects on mast-cell degranulation. In addition, ROCK2 but not ROCK1 plays a role in allergen-induced goblet cell hyperplasia. PMID:25323425

  1. Smitin, a novel smooth muscle titin-like protein, interacts with myosin filaments in vivo and in vitro.

    PubMed

    Kim, Kyoungtae; Keller, Thomas C S

    2002-01-07

    Smooth muscle cells use an actin-myosin II-based contractile apparatus to produce force for a variety of physiological functions, including blood pressure regulation and gut peristalsis. The organization of the smooth muscle contractile apparatus resembles that of striated skeletal and cardiac muscle, but remains much more poorly understood. We have found that avian vascular and visceral smooth muscles contain a novel, megadalton protein, smitin, that is similar to striated muscle titin in molecular morphology, localization in a contractile apparatus, and ability to interact with myosin filaments. Smitin, like titin, is a long fibrous molecule with a globular domain on one end. Specific reactivities of an anti-smitin polyclonal antibody and an anti-titin monoclonal antibody suggest that smitin and titin are distinct proteins rather than differentially spliced isoforms encoded by the same gene. Smitin immunofluorescently colocalizes with myosin in chicken gizzard smooth muscle, and interacts with two configurations of smooth muscle myosin filaments in vitro. In physiological ionic strength conditions, smitin and smooth muscle myosin coassemble into irregular aggregates containing large sidepolar myosin filaments. In low ionic strength conditions, smitin and smooth muscle myosin form highly ordered structures containing linear and polygonal end-to-end and side-by-side arrays of small bipolar myosin filaments. We have used immunogold localization and sucrose density gradient cosedimentation analyses to confirm association of smitin with both the sidepolar and bipolar smooth muscle myosin filaments. These findings suggest that the titin-like protein smitin may play a central role in organizing myosin filaments in the contractile apparatus and perhaps in other structures in smooth muscle cells.

  2. Notch signal reception is required in vascular smooth muscle cells for ductus arteriosus closure

    PubMed Central

    Krebs, Luke T.; Norton, Christine R.; Gridley, Thomas

    2017-01-01

    Summary The ductus arteriosus is an arterial vessel that shunts blood flow away from the lungs during fetal life, but normally occludes after birth to establish the adult circulation pattern. Failure of the ductus arteriosus to close after birth is termed patent ductus arteriosus, and is one of the most common congenital heart defects. Our previous work demonstrated that vascular smooth muscle cell expression of the Jag1 gene, which encodes a ligand for Notch family receptors, is essential for postnatal closure of the ductus arteriosus in mice. However, it was not known what cell population was responsible for receiving the Jag1-mediated signal. Here we show, using smooth muscle cell-specific deletion of the Rbpj gene, which encodes a transcription factor that mediates all canonical Notch signaling, that Notch signal reception in the vascular smooth muscle cell compartment is required for ductus arteriosus closure. These data indicate that homotypic vascular smooth muscle cell interactions are required for proper contractile smooth muscle cell differentiation and postnatal closure of the ductus arteriosus in mice. PMID:26742650

  3. Bidirectional regulation of human colonic smooth muscle contractility by tachykinin NK(2) receptors.

    PubMed

    Nakamura, Akihiro; Tanaka, Takahiro; Imanishi, Akio; Kawamoto, Makiko; Toyoda, Masao; Mizojiri, Gaku; Tsukimi, Yasuhiro

    2011-01-01

    In this study, we attempted to clarify the mechanism of tachykinin-induced motor response in isolated smooth muscle preparations of the human colon. Fresh specimens of normal colon were obtained from patients suffering from colonic cancer. Using mucosa-free smooth muscle strips, smooth muscle tension with circular direction was monitored isometrically. Substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) produced marked contraction. All of these contractions were inhibited by saredutant, a selective NK(2)-R antagonist, but not by CP122721, a selective NK(1)-R antagonist or talnetant, a selective NK(3)-R antagonist. βAla(8)-NKA(4-10) induced concentration-dependent contraction similar to NKA, but Sar(9)-Met(11)-SP and Met-Phe(7)-NKB did not cause marked contraction. Colonic contraction induced by βAla(8)-NKA(4-10) was completely blocked by saredutant, but not by atropine. Tetrodotoxin or N(G)-nitro-L-arginine methyl ester pretreatment significantly enhanced βAla(8)-NKA(4-10)-induced contraction. Immunohistochemical analysis showed that the NK(2)-R was expressed on the smooth muscle layers and myenteric plexus where it was also co-expressed with neuronal nitric oxide synthase in the myenteric plexus. These results suggest that the NK(2)-R is a major contributor to tachykinin-induced smooth muscle contraction in human colon and that the NK(2)-R-mediated response consists of an excitatory component via direct action on the smooth muscle and an inhibitory component possibly via nitric oxide neurons.

  4. Role of the adapter protein Abi1 in actin-associated signaling and smooth muscle contraction.

    PubMed

    Wang, Tao; Cleary, Rachel A; Wang, Ruping; Tang, Dale D

    2013-07-12

    Actin filament polymerization plays a critical role in the regulation of smooth muscle contraction. However, our knowledge regarding modulation of the actin cytoskeleton in smooth muscle just begins to accumulate. In this study, stimulation with acetylcholine (ACh) induced an increase in the association of the adapter protein c-Abl interactor 1 (Abi1) with neuronal Wiskott-Aldrich syndrome protein (N-WASP) (an actin-regulatory protein) in smooth muscle cells/tissues. Furthermore, contractile stimulation activated N-WASP in live smooth muscle cells as evidenced by changes in fluorescence resonance energy transfer efficiency of an N-WASP sensor. Abi1 knockdown by lentivirus-mediated RNAi inhibited N-WASP activation, actin polymerization, and contraction in smooth muscle. However, Abi1 silencing did not affect myosin regulatory light chain phosphorylation at Ser-19 in smooth muscle. In addition, c-Abl tyrosine kinase and Crk-associated substrate (CAS) have been shown to regulate smooth muscle contraction. The interaction of Abi1 with c-Abl and CAS has not been investigated. Here, contractile activation induced formation of a multiprotein complex including c-Abl, CAS, and Abi1. Knockdown of c-Abl and CAS attenuated the activation of Abi1 during contractile activation. More importantly, Abi1 knockdown inhibited c-Abl phosphorylation at Tyr-412 and the interaction of c-Abl with CAS. These results suggest that Abi1 is an important component of the cellular process that regulates N-WASP activation, actin dynamics, and contraction in smooth muscle. Abi1 is activated by the c-Abl-CAS pathway, and Abi1 reciprocally controls the activation of its upstream regulator c-Abl.

  5. Role of the Adapter Protein Abi1 in Actin-associated Signaling and Smooth Muscle Contraction*

    PubMed Central

    Wang, Tao; Cleary, Rachel A.; Wang, Ruping; Tang, Dale D.

    2013-01-01

    Actin filament polymerization plays a critical role in the regulation of smooth muscle contraction. However, our knowledge regarding modulation of the actin cytoskeleton in smooth muscle just begins to accumulate. In this study, stimulation with acetylcholine (ACh) induced an increase in the association of the adapter protein c-Abl interactor 1 (Abi1) with neuronal Wiskott-Aldrich syndrome protein (N-WASP) (an actin-regulatory protein) in smooth muscle cells/tissues. Furthermore, contractile stimulation activated N-WASP in live smooth muscle cells as evidenced by changes in fluorescence resonance energy transfer efficiency of an N-WASP sensor. Abi1 knockdown by lentivirus-mediated RNAi inhibited N-WASP activation, actin polymerization, and contraction in smooth muscle. However, Abi1 silencing did not affect myosin regulatory light chain phosphorylation at Ser-19 in smooth muscle. In addition, c-Abl tyrosine kinase and Crk-associated substrate (CAS) have been shown to regulate smooth muscle contraction. The interaction of Abi1 with c-Abl and CAS has not been investigated. Here, contractile activation induced formation of a multiprotein complex including c-Abl, CAS, and Abi1. Knockdown of c-Abl and CAS attenuated the activation of Abi1 during contractile activation. More importantly, Abi1 knockdown inhibited c-Abl phosphorylation at Tyr-412 and the interaction of c-Abl with CAS. These results suggest that Abi1 is an important component of the cellular process that regulates N-WASP activation, actin dynamics, and contraction in smooth muscle. Abi1 is activated by the c-Abl-CAS pathway, and Abi1 reciprocally controls the activation of its upstream regulator c-Abl. PMID:23740246

  6. Airway structural alterations selectively associated with severe asthma.

    PubMed

    Benayoun, Laurent; Druilhe, Anne; Dombret, Marie-Christine; Aubier, Michel; Pretolani, Marina

    2003-05-15

    To identify airway pathologic abnormalities selectively associated with severe asthma, we examined 10 control subjects, 10 patients with intermittent asthma, 15 patients with mild-to-moderate persistent asthma, 15 patients with severe persistent asthma, and 10 patients with chronic obstructive pulmonary disease. Bronchial biopsies were assessed for epithelial integrity; subepithelial basement membrane (SBM) thickness; collagen type III deposition; eosinophil, neutrophil, and fibroblast numbers; mucous gland and airway smooth muscle (ASM) areas; SBM-ASM distance; ASM hypertrophy (increased cell size); and the expression of the contractile proteins alpha-actin, smooth muscle myosin heavy-chain isoforms, myosin light-chain kinase, and the phosphorylated form of the regulatory light chain of myosin. Neither mucosal eosinophilia nor neutrophilia, epithelial damage, or SBM thickness reflected asthma severity. In contrast, higher numbers of fibroblasts (p < 0.001), an increase in collagen type III deposition (p < 0.020), larger mucous gland (p < 0.040) and ASM (p < 0.001) areas, augmented ASM cell size (p < 0.001), and myosin light-chain kinase expression (p < 0.005) distinguished patients with severe persistent asthma from patients with milder disease or with chronic obstructive pulmonary disease. Stepwise multivariate regression analysis established that fibroblast numbers and ASM cell size were negatively associated with prebronchodilator and postbronchodilator FEV1 values in patients with asthma. We conclude that fibroblast accumulation and ASM hypertrophy in proximal airways are selective determinants of severe persistent asthma.

  7. Inhibitory effects of botulinum toxin on pyloric and antral smooth muscle.

    PubMed

    James, Arlene N; Ryan, James P; Parkman, Henry P

    2003-08-01

    Botulinum toxin injection into the pylorus is reported to improve gastric emptying in gastroparesis. Classically, botulinum toxin inhibits ACh release from cholinergic nerves in skeletal muscle. The aim of this study was to determine the effects of botulinum toxin on pyloric smooth muscle. Guinea pig pyloric muscle strips were studied in vitro. Botulinum toxin type A was added; electric field stimulation (EFS) was performed every 30 min for 6 h. ACh (100 microM)-induced contractile responses were determined before and after 6 h. Botulinum toxin caused a concentration-dependent decrease of pyloric contractions to EFS. At a low concentration (2 U/ml), botulinum toxin decreased pyloric contractions to EFS by 43 +/- 9% without affecting ACh-induced contractions. At higher concentrations (10 U/ml), botulinum toxin decreased pyloric contraction to EFS by 75 +/- 7% and decreased ACh-induced contraction by 79 +/- 9%. In conclusion, botulinum toxin inhibits pyloric smooth muscle contractility. At a low concentration, botulinum toxin decreases EFS-induced contractile responses without affecting ACh-induced contractions suggesting inhibition of ACh release from cholinergic nerves. At higher concentrations, botulinum toxin directly inhibits smooth muscle contractility as evidenced by the decreased contractile response to ACh.

  8. Neurokinin subtype receptors mediating substance P contraction in immature rabbit airways.

    PubMed

    Kazem, E; John, C; Tanaka, D T

    1996-01-01

    Two-week-old rabbit tracheal smooth muscle (TSM) and bronchial smooth muscle (BSM) segments were placed in organ baths, and isometric contractions to substance P (SP) were obtained. In the presence of phosphoramidon (PHOS), a neutral endopeptidase inhibitor, BSM segments were significantly more reactive and sensitive to SP than TSM segments. Neither neostigmine (NEO) nor atropine (ATR) eliminated these regional differences. Airway contractile responses to: 1) Senktide (NK-3 agonist); 2) neurokinin A (NKA, a NK-2 agonist); and 3) Septide (a highly selective NK-1 agonist) were separately obtained. In the presence of PHOS and NEO, Senktide was virtually inactive in both BSM and TSM. In the presence of PHOS, NEO, and ATR, NKA was equipotent in all airway segments; in contrast, the Septide response was significantly more reactive in BSM than in TSM segments. After inhibition of NK-1 activity with GR 82334, a competitive NK-1 receptor antagonist, the regional differences in SP reactivity were greatly diminished. This latter indication of a NK-1 contribution was confirmed using Septide-mediated inactivation of NK-1 receptors whereby the regional differences in airway sensitivity to SP were eliminated. These findings indicate that both endogenous neutral endopeptidase activity as well as NK-1 and NK-2 receptor influences may modulate the contractile responses to SP in immature rabbit airways.

  9. Fragmented esophageal smooth muscle contraction segments on high resolution manometry: a marker of esophageal hypomotility.

    PubMed

    Porter, R F; Kumar, N; Drapekin, J E; Gyawali, C P

    2012-08-01

    Esophageal peristalsis consists of a chain of contracting striated and smooth muscle segments on high resolution manometry (HRM). We compared smooth muscle contraction segments in symptomatic subjects with reflux disease to healthy controls. High resolution manometry Clouse plots were analyzed in 110 subjects with reflux disease (50 ± 1.4 years, 51.5% women) and 15 controls (27 ± 2.1 years, 60.0% women). Using the 30 mmHg isobaric contour tool, sequences were designated fragmented if either smooth muscle contraction segment was absent or if the two smooth muscle segments were separated by a pressure trough, and failed if both smooth muscle contraction segments were absent. The discriminative value of contraction segment analysis was assessed. A total of 1115 swallows were analyzed (reflux group: 965, controls: 150). Reflux subjects had lower peak and averaged contraction amplitudes compared with controls (P < 0.0001 for all comparisons). Fragmented sequences followed 18.4% wet swallows in the reflux group, compared with 7.5% in controls (P < 0.0001), and were seen more frequently than failed sequences (7.9% and 2.5%, respectively). Using a threshold of 30% in individual subjects, a composite of failed and/or fragmented sequences was effective in segregating reflux subjects from control subjects (P = 0.04). Evaluation of smooth muscle contraction segments adds value to HRM analysis. Specifically, fragmented smooth muscle contraction segments may be a marker of esophageal hypomotility. © 2012 Blackwell Publishing Ltd.

  10. [The effects of leptin on apoptosis of airway smooth muscle cells via the PI3K/Akt signaling pathway].

    PubMed

    Liu, Wen-jing; Zhu, Shu-yang; Chen, Yu-ling; Wu, Xia; Ni, Wen-jing; Chen, Yun-feng; Zhao, Ling

    2012-12-01

    To observe the effects of leptin on the expression of Akt, Pho-Akt, Bcl-2, Bax, caspase-3 and the apoptosis of airway smooth muscle cells (ASMCs), and to explore the possible mechanisms. ASMCs were derived from rat airway tissue and cultured in vitro. The cells were randomly divided into 5 groups including a control group, leptin at concentrations of 50, 100, 200 µg/L groups (group Lep50, Lep100, Lep200), and PI3K specific antagonist with Lep200 group. Then the cells of different groups were incubated for 24 h. An apoptosis detection kit was used for annexin V and PI staining. The expression of Akt, phosphorylation Akt, Bcl-2, Bax, caspase-3 were measured by Western blot. The apoptosis rates of ASMCs in group Lep50, Lep100 and Lep200 were (3.97 ± 0.39)%, (1.88 ± 0.72)% and (0.77 ± 0.11)%, respectively, all significantly lower than that in the control group (7.38 ± 0.49)% (F = 89.57, P < 0.05). Furthermore, the concentration of leptin was negatively related to the apoptosis rate (r = -0.711, P < 0.05). The apoptosis rates of PI3K specific antagonist with Lep200 group (3.29 ± 0.36)% was higher than that of group Lep200 (0.77 ± 0.11)% (F = 89.57, P < 0.01). After the intervention of leptin, the expression of Bcl-2 was upregulated and positively correlated with leptin concentration (r = 0.939, P < 0.05); Bax was downregulated and negatively related to the leptin concentration (r = -0.908, P < 0.05); while the Bcl-2/Bax ratio was raised after leptin treatment (F = 20.56, P < 0.05). Leptin inhibited the activation of caspase-3 in the negative way. (r = -0.961, P < 0.05). The results also showed that leptin significantly increased phosphorylation of Akt that positively related to leptin concentration (r = 0.958, P < 0.05). Compared with group Lep200, the expression of Pho-Akt and Bcl-2 in PI3K specific antagonist with Lep200 group were downregulated (F = 32.93, 19.48, respectively, P < 0.05), while the expression of Bax and caspase-3 was increased (F = 10.10, 29

  11. Circulating smooth muscle progenitor cells in atherosclerosis and plaque rupture: current perspective and methods of analysis.

    PubMed

    Bentzon, Jacob F; Falk, Erling

    2010-01-01

    Smooth muscle cells play a critical role in the development of atherosclerosis and its clinical complications. They were long thought to derive entirely from preexisting smooth muscle cells in the arterial wall, but this understanding has been challenged by the claim that circulating bone marrow-derived smooth muscle progenitor cells are an important source of plaque smooth muscle cells in human and experimental atherosclerosis. This theory is today accepted by many cardiovascular researchers and authors of contemporary review articles. Recently, however, we and others have refuted the existence of bone marrow-derived smooth muscle cells in animal models of atherosclerosis and other arterial diseases based on new experiments with high-resolution microscopy and improved techniques for smooth muscle cell identification and tracking. These studies have also pointed to a number of methodological deficiencies in some of the seminal papers in the field. For those unaccustomed with the methods used in this research area, it must be difficult to decide what to believe and why to do so. In this review, we summarize current knowledge about the origin of smooth muscle cells in atherosclerosis and direct the reader's attention to the methodological challenges that have contributed to the confusion in the field. 2009 Elsevier Inc. All rights reserved.

  12. Abnormalities in the contractile properties of colonic smooth muscle in idiopathic slow transit constipation.

    PubMed

    Slater, B J; Varma, J S; Gillespie, J I

    1997-02-01

    The underlying pathophysiology of idiopathic slow transit constipation (ISTC) remains unclear. At present, there is little evidence to implicate a smooth muscle myopathy in the aetiology of this condition. This study compared the effect of cisapride on the cholinergic response of colonic muscle strips from patients with this condition with that of control tissue. Isometric tension production was recorded from circular smooth muscle strips taken from five patients undergoing colectomy for ISTC in response to cumulative concentrations of carbachol (100 nmol/1-100 mumol/l) alone and in the presence of cisapride 400 nmol/l. Similar dose-response activity was obtained for a control group consisting of six patients undergoing resection for colorectal carcinoma. In the absence of cisapride, smooth muscle from patients with carcinoma exhibited a significantly lower sensitivity to cholinergic stimulation (agonist concentration required to produce half-maximal activation (EC50) 4.83 mumol/l) than that from patients with ISTC (EC50 1.63 mumol/l, P = 0.036), and also a greater maximal frequency of the oscillatory activity associated with the increase in isometric tension (0.070 versus 0.049 Hz, P = 0.035). Cisapride had no effect on the sensitivity to carbachol of the carcinoma tissue but brought about a significant reduction in the sensitivity of smooth muscle from patients with ISTC (EC50 3.24 mumol/l, P = 0.043). These findings indicate that colonic smooth muscle from patients with ISTC is hypersensitive to cholinergic stimulation and suggest the existence of a smooth muscle myopathy in this condition.

  13. Airway reactivity in chronic obstructive pulmonary disease. Failure of in vivo methacholine responsiveness to correlate with cholinergic, adrenergic, or nonadrenergic responses in vitro.

    PubMed

    Taylor, S M; Paré, P D; Armour, C L; Hogg, J C; Schellenberg, R R

    1985-07-01

    This study aimed to determine whether in vivo airways hyperreactivity was manifested by either enhanced bronchial smooth muscle responses to contractile stimuli or by deficient responses to relaxant stimuli in vitro. Quantitative responses to nebulized methacholine were obtained in 12 human subjects prior to pulmonary resection. The provocative concentration of methacholine producing a 20% reduction in FEV1 (PC20) was calculated, and these values were compared with in vitro responses of bronchial smooth muscle strips from the surgical specimens. Both contractile cholinergic responses and relaxant nonadrenergic noncholinergic dose-response data were obtained for the in vitro bronchial specimens by electrical field stimulation. In addition, cumulative dose responses were obtained to exogenously added methacholine, the beta-adrenergic agonist salbutamol, and the adenylate cyclase activator forskolin. Despite a wide range of PC20 values, the in vivo airway responsiveness did not correlate with any of the in vitro responses examined, suggesting that airway reactivity is not due solely to the responsiveness of smooth muscle to contractile agonists nor to a localized deficiency in the nonadrenergic inhibitory system, beta-adrenergic inhibition, or abnormal cyclic-AMP-mediated pathways of relaxation.

  14. Cordyceps sinensis inhibits airway remodeling in rats with chronic obstructive pulmonary disease

    PubMed Central

    Yang, Lei; Jiao, Xingai; Wu, Jinxiang; Zhao, Jiping; Liu, Tian; Xu, Jianfeng; Ma, Xiaohui; Cao, Liuzao; Liu, Lin; Liu, Yahui; Chi, Jingyu; Zou, Minfang; Li, Shuo; Xu, Jiawei; Dong, Liang

    2018-01-01

    Cordyceps sinensis is a traditional Chinese herbal medicine that has been used for centuries in Asia as a tonic to soothe the lung for the treatment of respiratory diseases. The aim of the present study was to determine the effects of C. sinensis on airway remodeling in chronic obstructive pulmonary disease (COPD) and investigate the underlying molecular mechanisms. Rats with COPD were orally administered C. sinensis at low, moderate or high doses (2.5, 5 or 7.5 g/kg/day, respectively) for 12 weeks. Airway tissue histopathology, lung inflammation and airway remodeling were evaluated. C. sinensis treatment significantly ameliorated airway wall thickening, involving collagen deposition, airway wall fibrosis, smooth muscle hypertrophy and epithelial hyperplasia in model rats with COPD. Additionally, C. sinensis administration in rats with COPD reduced inflammatory cell accumulation and decreased inflammatory cytokine production, including tumor necrosis factor-α, interleukin-8 and transforming growth factor (TGF)-β1 in bronchoalveolar lavage fluid. Meanwhile, the increased levels of α-smooth muscle actin and collagen I in the COPD group were also markedly decreased by C. sinensis treatment. Furthermore, compared with untreated rats with COPD, C. sinensis reduced the expression level of phosphorylated (p)-Smad2, p-Smad3, TGF-β1 and its receptors, with the concomitant increased expression of Smad7 in the lungs of rats with COPD. These results indicated that treatment with C. sinensis may be a useful approach for COPD therapy. PMID:29456676

  15. Cordyceps sinensis inhibits airway remodeling in rats with chronic obstructive pulmonary disease.

    PubMed

    Yang, Lei; Jiao, Xingai; Wu, Jinxiang; Zhao, Jiping; Liu, Tian; Xu, Jianfeng; Ma, Xiaohui; Cao, Liuzao; Liu, Lin; Liu, Yahui; Chi, Jingyu; Zou, Minfang; Li, Shuo; Xu, Jiawei; Dong, Liang

    2018-03-01

    Cordyceps sinensis is a traditional Chinese herbal medicine that has been used for centuries in Asia as a tonic to soothe the lung for the treatment of respiratory diseases. The aim of the present study was to determine the effects of C. sinensi s on airway remodeling in chronic obstructive pulmonary disease (COPD) and investigate the underlying molecular mechanisms. Rats with COPD were orally administered C. sinensis at low, moderate or high doses (2.5, 5 or 7.5 g/kg/day, respectively) for 12 weeks. Airway tissue histopathology, lung inflammation and airway remodeling were evaluated. C. sinensis treatment significantly ameliorated airway wall thickening, involving collagen deposition, airway wall fibrosis, smooth muscle hypertrophy and epithelial hyperplasia in model rats with COPD. Additionally, C. sinensis administration in rats with COPD reduced inflammatory cell accumulation and decreased inflammatory cytokine production, including tumor necrosis factor-α, interleukin-8 and transforming growth factor (TGF)-β1 in bronchoalveolar lavage fluid. Meanwhile, the increased levels of α-smooth muscle actin and collagen I in the COPD group were also markedly decreased by C. sinensis treatment. Furthermore, compared with untreated rats with COPD, C. sinensis reduced the expression level of phosphorylated (p)-Smad2, p-Smad3, TGF-β1 and its receptors, with the concomitant increased expression of Smad7 in the lungs of rats with COPD. These results indicated that treatment with C. sinensis may be a useful approach for COPD therapy.

  16. Effects of nifedipine on anorectal smooth muscle in vitro.

    PubMed

    Cook, T A; Brading, A F; Mortensen, N J

    1999-06-01

    Glyceryl trinitrate reduces anal resting pressure and aids the healing of anal fissures. However, some patients develop tachyphylaxis and the fissure fails to heal, suggesting that other agents are needed. This study assesses the effects of nifedipine (a calcium channel antagonist) in modulating resting tone and agonist-induced contractions in human internal anal sphincter (IAS) and rectal circular muscle. Smooth muscle strips from the IAS and rectal circular muscle from ten patients undergoing surgical resection were mounted for isometric tension recording in a superfusion organ bath. The effects of noradrenaline and carbachol were assessed in the presence of various perfusates. LAS strips developed tone and spontaneous activity. Noradrenaline produced dose-dependent contractions. In calcium-free Krebs solution, tone and activity were abolished and no contractions were elicited in response to noradrenaline. Nifedipine also abolished tone and spontaneous activity, but contractions to noradrenaline were only slightly attenuated. In contrast, rectal smooth muscle strips developed spontaneous activity but no resting tone and contracted in response to carbachol. In calcium-free Krebs solution, the spontaneous activity and carbachol contractions were abolished. Addition of nifedipine to the perfusate abolished spontaneous activity and greatly reduced contractions. These data suggest that spontaneous activity and resting tone are dependent on extracellular calcium and flux across the cells. Agonist-induced contraction in the IAS is attributable mainly to the release of calcium from intracellular stores, whereas rectal circular smooth muscle depends principally on extracellular calcium entering the cell for contraction. The attenuation of contractions in both tissues and the abolition of resting tone in the IAS suggest that nifedipine may be useful in the management of patients with anorectal disorders.

  17. Bladder smooth muscle organ culture preparation maintains the contractile phenotype

    PubMed Central

    Wang, Tanchun; Kendig, Derek M.; Chang, Shaohua; Trappanese, Danielle M.; Chacko, Samuel

    2012-01-01

    Smooth muscle cells, when subjected to culture, modulate from a contractile to a secretory phenotype. This has hampered the use of cell culture for molecular techniques to study the regulation of smooth muscle biology. The goal of this study was to develop a new organ culture model of bladder smooth muscle (BSM) that would maintain the contractile phenotype and aid in the study of BSM biology. Our results showed that strips of BSM subjected to up to 9 days of organ culture maintained their contractile phenotype, including the ability to achieve near-control levels of force with a temporal profile similar to that of noncultured tissues. The technical aspects of our organ culture preparation that were responsible, in part, for the maintenance of the contractile phenotype were a slight longitudinal stretch during culture and subjection of the strips to daily contraction-relaxation. The tissues contained viable cells throughout the cross section of the strips. There was an increase in extracellular collagenous matrix, resulting in a leftward shift in the passive length-tension relationship. There were no significant changes in the content of smooth muscle-specific α-actin, calponin, h-caldesmon, total myosin heavy chain, protein kinase G, Rho kinase-I, or the ratio of SM1 to SM2 myosin isoforms. Moreover the organ cultured tissues maintained functional voltage-gated calcium channels and large-conductance calcium-activated potassium channels. Therefore, we propose that this novel BSM organ culture model maintains the contractile phenotype and will be a valuable tool for the use in cellular/molecular biology studies of bladder myocytes. PMID:22896042

  18. Apigenin inhibits TGF-β1-induced proliferation and migration of airway smooth muscle cells.

    PubMed

    Li, Li-Hua; Lu, Bin; Wu, Hong-Ke; Zhang, Hao; Yao, Fei-Fei

    2015-01-01

    It is well known that the proliferation and migration of ASM cells (ASMCs) plays an important role in the pathogenesis of airway remodeling in asthma. Previous studies reported that apigenin can inhibit airway remodeling in a mouse asthma model. However, its effects on the proliferation and migration of ASMCs in asthma remain unknown. Therefore, the aim of our present study was to investigate the effects of apigenin on ASMC proliferation and migration, and explore the possible molecular mechanism. We found that apigenin inhibited transforming growth factor-β1 (TGF-β1)-induced ASMC proliferation. The cell cycle was blocked at G1/S-interphase by apigenin. It also suppressed TGF-β1-induced ASMCs migration. Furthermore, apigenin inhibited TGF-β1-induced Smad 2 and Smad 3 phosphorylation in ASMCs. Taken together, these results suggested that apigenin inhibited the proliferation and migration of TGF-β1-stimulated ASMCs by inhibiting Smad signaling pathway. These data might provide useful information for treating asthma and show that apigenin has potential for attenuating airway remodeling.

  19. The regulation of smooth muscle contractility by zipper-interacting protein kinase.

    PubMed

    Ihara, Eikichi; MacDonald, Justin A

    2007-01-01

    Smooth muscle contractility is mainly regulated by phosphorylation of the 20 kDa myosin light chains (LC20), a process that is controlled by the opposing activities of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). Recently, intensive research has revealed that various protein kinase networks including Rho-kinase, integrin-linked kinase, zipper-interacting protein kinase (ZIPK), and protein kinase C (PKC) are involved in the regulation of LC20 phosphorylation and have important roles in modulating smooth muscle contractile responses to Ca2+ (i.e., Ca2+ sensitization and Ca2+ desensitization). Here, we review the general background and structure of ZIPK and summarize our current understanding of its involvement in a number of cell processes including cell death (apoptosis), cell motility, and smooth muscle contraction. ZIPK has been found to induce the diphosphorylation of LC20 at Ser-19 and Thr-18 in a Ca2+-independent manner and to regulate MLCP activity directly through its phosphorylation of the myosin-targeting subunit of MLCP or indirectly through its phosphorylation of the PKC-potentiated inhibitory protein of MLCP. Future investigations of ZIPK function in smooth muscle will undoubtably focus on determining the mechanisms that regulate its cellular activity, including the identification of upstream signaling pathways, the characterization of autoinhibitory domains and regulatory phosphorylation sites, and the development of specific inhibitor compounds.

  20. Endogenous cannabinoid receptor agonists inhibit neurogenic inflammations in guinea pig airways.

    PubMed

    Yoshihara, Shigemi; Morimoto, Hiroshi; Ohori, Makoto; Yamada, Yumi; Abe, Toshio; Arisaka, Osamu

    2005-09-01

    Although neurogenic inflammation via the activation of C fibers in the airway must have an important role in the pathogenesis of asthma, their regulatory mechanism remains uncertain. The pharmacological profiles of endogenous cannabinoid receptor agonists on the activation of C fibers in airway tissues were investigated and the mechanisms how cannabinoids regulate airway inflammatory reactions were clarified. The effects of endogenous cannabinoid receptor agonists on electrical field stimulation-induced bronchial smooth muscle contraction, capsaicin-induced bronchoconstriction and capsaicin-induced substance P release in guinea pig airway tissues were investigated. The influences of cannabinoid receptor antagonists and K+ channel blockers to the effects of cannabinoid receptor agonists on these respiratory reactions were examined. Both endogenous cannabinoid receptor agonists, anandamide and palmitoylethanolamide, inhibited electrical field stimulation-induced guinea pig bronchial smooth muscle contraction, but not neurokinin A-induced contraction. A cannabinoid CB2 antagonist, SR 144528, reduced the inhibitory effect of endogenous agonists, but not a cannabinoid CB1 antagonist, SR 141716A. Inhibitory effects of agonists were also reduced by the pretreatment of large conductance Ca2+ -activated K+ channel (maxi-K+ channel) blockers, iberiotoxin and charybdotoxin, but not by other K+ channel blockers, dendrotoxin or glibenclamide. Anandamide and palmitoylethanolamide blocked the capsaicin-induced release of substance P-like immunoreactivity from guinea pig airway tissues. Additionally, intravenous injection of palmitoylethanolamide dose-dependently inhibited capsaicin-induced guinea pig bronchoconstriction, but not neurokinin A-induced reaction. However, anandamide did not reduce capsaicin-induced guinea pig bronchoconstriction. These findings suggest that endogenous cannabinoid receptor agonists inhibit the activation of C fibers via cannabinoid CB2 receptors and

  1. Azithromycin ameliorates airway remodeling via inhibiting airway epithelium apoptosis.

    PubMed

    Liu, Yuanqi; Pu, Yue; Li, Diandian; Zhou, Liming; Wan, Lihong

    2017-02-01

    Azithromycin can benefit treating allergic airway inflammation and remodeling. In the present study, we hypothesized that azithromycin alleviated airway epithelium injury through inhibiting airway epithelium apoptosis via down regulation of caspase-3 and Bax/Bcl2 ratio in vivo and in vitro. Ovalbumin induced rat asthma model and TGF-β1-induced BEAS-2B cell apoptosis model were established, respectively. In vivo experiments, airway epithelium was stained with hematoxylin and eosin (HE) and periodic acid-Schiff (PAS) to histologically evaluate the airway inflammation and remodeling. Airway epithelium apoptotic index (AI) was further analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), while expression of apoptosis related gene (Bax, Bcl2, Caspase-3) in lungs were measured by qRT-PCR and western blotting, respectively. In vitro experiments, apoptosis were evaluated by Flow cytometry (FCM) and TUNEL. Above apoptosis related gene were also measured by qRT-PCR and western blotting. Compared with the OVA group, azithromycin significantly reduced the inflammation score, peribronchial smooth muscle layer thickness, epithelial thickening and goblet cell metaplasia (P<0.05), and effectively suppressed AI of airway epithelium (P<0.05). Moreover, the increasing mRNA and protein expressions of Caspase-3 and Bax/Bcl-2 ratio in lung tissue were all significantly decreased in azithromycin-treated rats (P<0.05). In vitro, azithromycin significantly suppressed TGF-β1-induced BEAS-2B cells apoptosis (P<0.05) and reversed TGF-β1 elevated Caspase-3 mRNA level and Bax/Bcl-2 ratio (P<0.05). Azithromycin is an attractive treatment option for reducing airway epithelial cell apoptosis by improving the imbalance of Bax/Bcl-2 ratio and inhibiting Caspase-3 level in airway epithelium. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Generalised smooth-muscle disease with defective muscarinic-receptor function.

    PubMed

    Bannister, R; Hoyes, A D

    1981-03-28

    A patient with widespread smooth-muscle disease presented with chronic intestinal pseudo-obstruction but had in addition defects of the bladder, pupils, sweating, and cardiovascular function. There was no evidence of a primary neural lesion, and minor changes in the muscle did not resemble those of a myopathy. In each organ affected muscarinic cholinergic function was at fault, but instead of supersensitivity to cholinergic drugs, which occurs in postganglionic autonomic neuropathies, there was a lack of response to cholinergic drugs and anticholinesterases. It was therefore concluded that the patient had a new type of defect of muscarinic-receptor function. The cause was unknown, but it may have been an autoimmune disease resembling myasthenia, in which there is a postjunctional defect of muscarinic receptors. In similar cases binding of muscarinic agonists and antagonists should be tested. When antibodies to purified human muscarinic receptors become available different patterns of smooth-muscle defect may be identifiable, enabling the lesion to be defined more precisely.

  3. Smooth muscle architecture within cell-dense vascular tissues influences functional contractility.

    PubMed

    Win, Zaw; Vrla, Geoffrey D; Steucke, Kerianne E; Sevcik, Emily N; Hald, Eric S; Alford, Patrick W

    2014-12-01

    The role of vascular smooth muscle architecture in the function of healthy and dysfunctional vessels is poorly understood. We aimed at determining the relationship between vascular smooth muscle architecture and contractile output using engineered vascular tissues. We utilized microcontact printing and a microfluidic cell seeding technique to provide three different initial seeding conditions, with the aim of influencing the cellular architecture within the tissue. Cells seeded in each condition formed confluent and aligned tissues but within the tissues, the cellular architecture varied. Tissues with a more elongated cellular architecture had significantly elevated basal stress and produced more contractile stress in response to endothelin-1 stimulation. We also found a correlation between the contractile phenotype marker expression and the cellular architecture, contrary to our previous findings in non-confluent tissues. Taken with previous results, these data suggest that within cell-dense vascular tissues, smooth muscle contractility is strongly influenced by cell and tissue architectures.

  4. Animal model for angiotensin II effects in the internal anal sphincter smooth muscle: mechanism of action.

    PubMed

    Fan, Ya-Ping; Puri, Rajinder N; Rattan, Satish

    2002-03-01

    Effect of ANG II was investigated in in vitro smooth muscle strips and in isolated smooth muscle cells (SMC). Among different species, rat internal and sphincter (IAS) smooth muscle showed significant and reproducible contraction that remained unmodified by different neurohumoral inhibitors. The AT(1) antagonist losartan but not AT(2) antagonist PD-123319 antagonized ANG II-induced contraction of the IAS smooth muscle and SMC. ANG II-induced contraction of rat IAS smooth muscle and SMC was attenuated by tyrosine kinase inhibitors genistein and tyrphostin, protein kinase C (PKC) inhibitor H-7, Ca(2+) channel blocker nicardipine, Rho kinase inhibitor Y-27632 or p(44/42) mitogen-activating protein kinase (MAPK(44/42)) inhibitor PD-98059. Combinations of nicardipine and H-7, Y-27632, and PD-98059 caused further attenuation of the ANG II effects. Western blot analyses revealed the presence of both AT(1) and AT(2) receptors. We conclude that ANG II causes contraction of rat IAS smooth muscle by the activation of AT(1) receptors at the SMC and involves multiple intracellular pathways, influx of Ca(2+), and activation of PKC, Rho kinase, and MAPK(44/42).

  5. Design of a muscle cell-specific expression vector utilising human vascular smooth muscle alpha-actin regulatory elements.

    PubMed

    Keogh, M C; Chen, D; Schmitt, J F; Dennehy, U; Kakkar, V V; Lemoine, N R

    1999-04-01

    The facility to direct tissue-specific expression of therapeutic gene constructs is desirable for many gene therapy applications. We describe the creation of a muscle-selective expression vector which supports transcription in vascular smooth muscle, cardiac muscle and skeletal muscle, while it is essentially silent in other cell types such as endothelial cells, hepatocytes and fibroblasts. Specific transcriptional regulatory elements have been identified in the human vascular smooth muscle cell (VSMC) alpha-actin gene, and used to create an expression vector which directs the expression of genes in cis to muscle cells. The vector contains an enhancer element we have identified in the 5' flanking region of the human VSMC alpha-actin gene involved in mediating VSMC expression. Heterologous pairing experiments have shown that the enhancer does not interact with the basal transcription complex recruited at the minimal SV40 early promoter. Such a vector has direct application in the modulation of VSMC proliferation associated with intimal hyperplasia/restenosis.

  6. Effects of tachykinins on uterine smooth muscle.

    PubMed

    Patak, E N; Pennefather, J N; Story, M E

    2000-11-01

    1. Sensory nerves supplying the mammalian uterus have been shown to contain substance P (SP) and neurokinin (NK)A. This review presents some of the advances that have led to a greater understanding of the effects of tachykinins on uterine smooth muscle. 2. The cell-surface peptidase neprilysin (EC.3 24.11, endopeptidase 24.11, enkephalinase, CALLA, CD10) has been shown to play a major role in regulating the actions of tachykinins on both rat and human myometrium. Because this peptidase is known to be regulated by steroids and pregnancy, its effects may be of physiological relevance. 3. Tachykinins produce contractions of isolated myometrial preparations from non-pregnant rats and mice. The NK2 receptor mediates these effects in rat uterus, while the NK1 receptor may mediate these effects in the mouse uterus. 4. The effects of tachykinins have been examined on myometrial preparations obtained at Caesarean section from near-term pregnant women. In the presence of the peptidase inhibitors (thiorphan, captopril and bestatin), the mammalian tachykinins SP, NKA and NKB produced concentration-dependent uterine contractions. 5. The order of agonist potency NKA > SP = NKB suggested that NK2 receptors mediate uterine contractions in the human. This was confirmed using the stable analogues [Sar9,Met(O2)11]SP, [Lys5MeLeu9Nle10]NKA(4-10) and [N-MePhe7]NKB, which are NK1, NK2 and NK3 receptor selective, respectively. Only [Lys5MeLeu9Nle10]NKA(4-10) produced concentration-related contractions of human uterine smooth muscle. 6. The experimental findings described in the present review, taken together with results published previously in the literature, indicate that tachykinin peptides may play a physiological or pathophysiological role in regulating uterine smooth muscle activity. However, more extensive research will be required to confirm such a role for these peptides.

  7. HOCl causes airway substance P hyperresponsiveness and neutral endopeptidase hypoactivity.

    PubMed

    Murlas, C G; Murphy, T P; Lang, Z

    1990-06-01

    We investigated whether exposure of guinea pig tracheal tissue to hypochlorous acid (HOCl) or hydrogen peroxide (H2O2) by perfusion through the airway lumen affected the responsiveness of airway muscle to ACh, KCl, or substance P in the presence or absence of 1 microM phosphoramidon, an inhibitor of neutral endopeptidase (NEP). Pairs of tracheal segments were immersed in a Krebs solution (pH 7.40 at 37 degrees C) and connected to perfusion circuits so that the lumen of one segment of each pair could be perfused with Krebs solution while the other was perfused for the same time (10 min) with either 0.1 microM HOCl or 10 mM H2O2. Segments after perfusion were cut into rings of similar size and placed in muscle chambers so that airway muscle force generation in vitro could be measured on stimulation by cumulative agonist doses. In addition, cell homogenates were made from other, similarly perfused tracheal segments to assess NEP activity using reverse-phase, high-pressure liquid chromatography (HPLC). We found that smooth muscle of mucosa-intact guinea pig airways perfused with HOCl, but not H2O2, was hyperresponsive to substance P but not to ACh or KCl. HOCl-perfused rings were not different from Krebs solution-exposed rings pretreated with phosphoramidon. There was no increase in substance P responsiveness of HOCl-exposed airways in which the mucosa had been removed before testing in vitro. The substance P hyperresponsiveness of HOCl-exposed, mucosa-intact airways was associated with decreased NEP activity.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. Detection of Leishmania spp. and associated inflammation in ocular-associated smooth and striated muscles in dogs with patent leishmaniosis.

    PubMed

    Naranjo, Carolina; Fondevila, Dolors; Leiva, Marta; Roura, Xavier; Peña, Teresa

    2010-05-01

    Canine leishmaniosis is a disease characterized by the wide distribution of the parasite throughout the tissues of the host. The purpose of this study was to describe the presence of Leishmania spp. and associated inflammation in ocular-associated muscles of dogs with patent leishmaniosis. Smooth muscles (iris dilator muscle, iris sphincter muscle, ciliary muscle, Müller muscle, smooth muscle of the periorbita and smooth muscle of the nictitating membrane) and striated muscles (orbicularis oculi muscle, obliquus dorsalis muscle and dorsal rectus muscle) were evaluated. Routine staining with hematoxylin and eosin and immunohistochemistry to detect Leishmania spp. were performed on tissue sections. Granulomatous inflammation was seen surrounding muscular fibers and was composed mainly of macrophages with scattered lymphocytes and plasma cells. This infiltrate could be seen in 52/473 (10.99%) samples of smooth muscle and 36/142 (25.35%) samples of striated muscle. Parasites were detected in 43/473 (9.09%) samples of smooth muscle and in 28/142 (19.71%) samples of striated muscle. To the authors' knowledge, this is the first report assessing the presence of Leishmania spp. and associated infiltrate in intraocular, extraocular and adnexal smooth and striated muscles. The inflammation present in those muscles could contribute to clinical signs already described, such as blepharitis, uveitis, and orbital cellulitis.

  9. The magnetic field of gastrointestinal smooth muscle activity

    NASA Astrophysics Data System (ADS)

    Bradshaw, Alan; Ladipo, Jk; Richards, William; Wikswo, John

    1997-11-01

    The gastrointestinal (GI) tract controls the absorption and transport of ingested materials. Its function is determined largely by the electrical activity of the smooth muscle that lines the GI tract. GI electrical activity consists of an omnipresent slowly oscillating wave known as the basic electrical rhythm (BER) that modulates a higher-frequency spiking activity associated with muscle contraction. The BER has been shown to be a reliable indicator of intestinal viability, and thus, recording of smooth muscle activity may have clinical value. The BER is difficult to measure with cutaneous electrodes because layers of low-conductivity fat between the GI tract and the abdominal surface attenuate the potential. On the other hand, the magnetic field associated with GI electrical activity is mostly unaffected by intervening fat layers. We recorded the magnetic fields from GI activity in 12 volunteers using a multichannel Superconducting QUantum Interference Device (SQUID) magnetometer. Characteristics typical of gastric and intestinal BER were apparent in the data. Channels near the epigastrium recorded gastric BER, and channels in intestinal areas recorded small bowel BER. These results suggest that a single multichannel SQUID magnetometer is able to measure gastrointestinal electrical activity from multiple locations around the abdomen simultaneously.

  10. Upper airway muscles awake and asleep.

    PubMed

    Sériès, Frédéric

    2002-06-01

    Upper airway (UA) structures are involved in different respiratory and non-respiratory tasks. The coordination of agonist and antagonist UA dilators is responsible for their mechanical function and their ability to maintain UA patency throughout the respiratory cycle. The activity of these muscles is linked with central respiratory activity but also depends on UA pressure changes and is greatly influenced by sleep. UA muscles are involved in determining UA resistance and stability (i.e. closing pressure), and the effect of sleep on these variables may be accounted for by its effect on tonic and phasic skeletal muscle activities. The mechanical effects of UA dilator contraction also depend on their physiological properties (capacity to generate tension in vitro, activity of the anaerobic enzymatic pathway, histo-chemical characteristics that may differ between subjects who may or may not have sleep-related obstructive breathing disorders). These characteristics may represent an adaptive process to an increased resistive loading of these muscles. The apparent discrepancy between the occurrence of UA closure and an increased capacity to generate tension in sleep apnea patients may be due to a reduction in the effectiveness of UA muscle contraction in these patients; such an increase in tissue stiffness could be accounted for by peri-muscular tissue characteristics. Therefore, understanding of UA muscle physiological characteristics should take into account its capacity for force production and its mechanical coupling with other UA tissues. Important research goals for the future will be to integrate these issues with other physiological features of the disease, such as UA size and dimension, histological characteristics of UA tissues and the effect of sleep on muscle function. Such integration will better inform understanding of the role of pharyngeal UA muscles in the pathophysiology of the sleep apnea/hypopnea syndrome.

  11. Neurokinin-1 receptor mediates stress-exacerbated allergic airway inflammation and airway hyperresponsiveness in mice.

    PubMed

    Joachim, Ricarda A; Sagach, Viktoriya; Quarcoo, David; Dinh, Q Thai; Arck, Petra C; Klapp, Burghard F

    2004-01-01

    A wealth of clinical observation has suggested that stress and asthma morbidity are associated. We have previously established a mouse model of stress-exacerbated allergic airway inflammation, which reflects major clinical findings. The aim of the current study was to investigate the role of the neurokinin- (NK-)1 receptor in the mediation of stress effects in allergic airway inflammation. BALB/c mice were systemically sensitized with ovalbumin (OVA) on assay days 1, 14, and 21 and repeatedly challenged with OVA aerosol on days 26 and 27. Sound stress was applied to the animals for 24 hours, starting with the first airway challenge. Additionally, one group of stressed and one group of nonstressed mice received the highly specific NK-1 receptor antagonist RP 67580. Bronchoalveolar lavage fluid was obtained, and cell numbers and differentiation were determined. Airway hyperreactivity was measured in vitro by electrical field stimulation of tracheal smooth-muscle elements. Application of stress in sensitized and challenged animals resulted in a significant increase in leukocyte number in the bronchoalveolar lavage fluid. Furthermore, stressed animals showed enhanced airway reactivity. The increase of inflammatory cells and airway reactivity was blocked by treatment of animals with the NK-1 receptor antagonist. These data indicate that the NK-1 receptor plays an important role in mediating stress effects in allergen-induced airway inflammation.

  12. Brain-Derived Neurotrophic Factor in the Airways

    PubMed Central

    Prakash, Y.S.; Martin, Richard J.

    2014-01-01

    In addition to their well-known roles in the nervous system, there is increasing recognition that neurotrophins such as brain derived neurotrophic factor (BDNF) as well as their receptors are expressed in peripheral tissues including the lung, and can thus potentially contribute to both normal physiology and pathophysiology of several diseases. The relevance of this family of growth factors lies in emerging clinical data indicating altered neurotrophin levels and function in a range of diseases including neonatal and adult asthma, sinusitis, influenza, and lung cancer. The current review focuses on 1) the importance of BDNF expression and signaling mechanisms in early airway and lung development, critical to both normal neonatal lung function and also its disruption in prematurity and insults such as inflammation and infection; 2) how BDNF, potentially derived from airway nerves modulate neurogenic control of airway tone, a key aspect of airway reflexes as well as dysfunctional responses to allergic inflammation; 3) the emerging idea that local BDNF production by resident airway cells such as epithelium and airway smooth muscle can contribute to normal airway structure and function, and to airway hyperreactivity and remodeling in diseases such as asthma. Furthermore, given its pleiotropic effects in the airway, BDNF may be a novel and appealing therapeutic target. PMID:24560686

  13. Mini-titins in striated and smooth molluscan muscles: structure, location and immunological crossreactivity.

    PubMed

    Vibert, P; Edelstein, S M; Castellani, L; Elliott, B W

    1993-12-01

    Invertebrate mini-titins are members of a class of myosin-binding proteins belonging to the immunoglobulin superfamily that may have structural and/or regulatory properties. We have isolated mini-titins from three molluscan sources: the striated and smooth adductor muscles of the scallop, and the smooth catch muscles of the mussel. Electron microscopy reveals flexible rod-like molecules about 0.2 micron long and 30 A wide with a distinctive polarity. Antibodies to scallop mini-titin label the A-band and especially the A/I junction of scallop striated muscle myofibrils by indirect immunofluorescence and immuno-electron microscopy. This antibody crossreacts with mini-titins in scallop smooth and Mytilus catch muscles, as well as with proteins in striated muscles from Limulus, Lethocerus (asynchronous flight muscle), and crayfish. It labels the A/I junction (I-region in Lethocerus) in these striated muscles as well as in chicken skeletal muscle. Antibodies to the repetitive immunoglobulin-like regions and also to the kinase domain of nematode twitchin crossreact with scallop mini-titin and label the A-band of scallop myofibrils. Electron microscopy of single molecules shows that antibodies to twitchin kinase bind to scallop mini-titin near one end of the molecule, suggesting how the scallop structure might be aligned with the sequence of nematode twitchin.

  14. Allergic asthma is distinguished by sensitivity of allergen-specific CD4+ T cells and airway structural cells to type 2 inflammation.

    PubMed

    Cho, Josalyn L; Ling, Morris F; Adams, David C; Faustino, Lucas; Islam, Sabina A; Afshar, Roshi; Griffith, Jason W; Harris, Robert S; Ng, Aylwin; Radicioni, Giorgia; Ford, Amina A; Han, Andre K; Xavier, Ramnik; Kwok, William W; Boucher, Richard; Moon, James J; Hamilos, Daniel L; Kesimer, Mehmet; Suter, Melissa J; Medoff, Benjamin D; Luster, Andrew D

    2016-10-05

    Despite systemic sensitization, not all allergic individuals develop asthma symptoms upon airborne allergen exposure. Determination of the factors that lead to the asthma phenotype in allergic individuals could guide treatment and identify novel therapeutic targets. We used segmental allergen challenge of allergic asthmatics (AA) and allergic nonasthmatic controls (AC) to determine whether there are differences in the airway immune response or airway structural cells that could drive the development of asthma. Both groups developed prominent allergic airway inflammation in response to allergen. However, asthmatic subjects had markedly higher levels of innate type 2 receptors on allergen-specific CD4 + T cells recruited into the airway. There were also increased levels of type 2 cytokines, increased total mucin, and increased mucin MUC5AC in response to allergen in the airways of AA subjects. Furthermore, type 2 cytokine levels correlated with the mucin response in AA but not AC subjects, suggesting differences in the airway epithelial response to inflammation. Finally, AA subjects had increased airway smooth muscle mass at baseline measured in vivo using novel orientation-resolved optical coherence tomography. Our data demonstrate that the development of allergic asthma is dependent on the responsiveness of allergen-specific CD4 + T cells to innate type 2 mediators as well as increased sensitivity of airway epithelial cells and smooth muscle to type 2 inflammation. Copyright © 2016, American Association for the Advancement of Science.

  15. Cultured smooth muscle cells of the human vesical sphincter are more sensitive to histamine than are detrusor smooth muscle cells.

    PubMed

    Neuhaus, Jochen; Oberbach, Andreas; Schwalenberg, Thilo; Stolzenburg, Jens-Uwe

    2006-05-01

    To compare histamine receptor expression in cultured smooth muscle cells from the human detrusor and internal sphincter using receptor-specific agonists. Smooth muscle cells from the bladder dome and internal sphincter were cultured from 5 male patients undergoing cystectomy for bladder cancer therapy. Calcium transients in cells stimulated with carbachol, histamine, histamine receptor 1 (H1R)-specific heptanecarboxamide (HTMT), dimaprit (H2R), and R-(alpha)-methylhistamine (H3R) were measured by calcium imaging. Histamine receptor proteins were detected by Western blot analysis and immunocytochemistry. H1R, H2R, and H3R expression was found in tissue and cultured cells. Carbachol stimulated equal numbers of detrusor and sphincter cells (60% and 51%, respectively). Histamine stimulated significantly more cells than carbachol in detrusor (100%) and sphincter (99.34%) cells. Calcium responses to carbachol in detrusor and sphincter cells were comparable and did not differ from those to histamine in detrusor cells. However, histamine and specific agonists stimulated more sphincter cells than did carbachol (P <0.001), and the calcium increase was greater in sphincter cells than in detrusor cells. Single cell analysis revealed comparable H2R responses in detrusor and sphincter cells, but H1R and H3R-mediated calcium reactions were significantly greater in sphincter cells. Histamine very effectively induces calcium release in smooth muscle cells. In sphincter cells, histamine is even more effective than carbachol regarding the number of reacting cells and the intracellular calcium increase. Some of the variability in the outcome of antihistaminic interstitial cystitis therapies might be caused by the ineffectiveness of the chosen antihistaminic or unintentional weakening of sphincteric function.

  16. Effect of ONO-1078, a leukotriene antagonist, on capsaicin- and substance P-induced bronchoconstriction and airway microvascular leakage in guinea pigs.

    PubMed

    Wei, E Q; Liu, J W; Zhang, L F; Zhang, W P; Bian, R L

    1996-05-01

    To study the effect of 4-oxo-8-[p-(4-phenylbutyloxy) benzoylamino]-2-(tetrazol-5-yl)-4H-1-benzopyran hemihydrate (ONO-1078), a specific leukotriene antagonist, on capsaicin (Cap)-sensitive sensory nerve functions in the airways, and clarify the modulating roles of endogenous peptido-leukotrienes. Changes in intrapulmonary pressure (IPP), Evans blue extravasation in airways, and contraction of bronchial smooth muscles of guinea pigs induced by Cap, substance P (SP) and leukotriene C4 (LTC4) were observed. Cap (0.05 mg.kg-1, i.v.), SP (1 microgram.kg-1, i.v.) and LTC4 (0.5 microgram.kg-1, i.v.) enhanced IPP, and Evans blue extravasation in bronchi and intrapulmonary airways. ONO-1078 0.03 mg.kg-1, i.v. completely blocked the responses to LTC4, attenuated those to Cap, but had no effect to SP. In isolated bronchial smooth muscles, ONO-1078 (1 mumol.L-1) inhibited the contractile response to Cap, but not to SP. ONO-1078 partly inhibits Cap-sensitive sensory nerve actions in airways, but has no direct effect on SP, a sensory neuropeptide.

  17. Evidence of direct smooth muscle relaxant effects of the fibrate gemfibrozil.

    PubMed

    Phelps, Laura E; Peuler, Jacob D

    2010-01-01

    Fibrates are commonly employed to treat abnormal lipid metabolism via their unique ability to stimulate peroxisome proliferator-activated receptor alpha (PPARalpha). Interestingly, they also decrease systemic arterial pressure, despite recent evidence that PPAR alpha may contribute to expression of renin and related hypertension. Yet, mechanisms responsible for their potential antihypertensive activity remain unresolved. Rapid decreases in arterial pressure following bolus intravenous injections of bezafibrate strongly suggest they may relax arterial smooth muscle directly. But since bezafibrate is highly susceptible to photodegradation in aqueous media, it has never been critically tested for this possibility in vitro with isolated arterial smooth muscle preparations. Accordingly, we tested gemfibrozil which is resistant to photodegradation. We examined it over a therapeutically-relevant range (50-400 microM) for both acute and delayed relaxant effects on contractions of the isolated rat tail artery; contractions induced by either depolarizing its smooth muscle cell membranes with high potassium or stimulating its membrane-bound receptors with norepinephrine and arginine-vasopressin. We also examined these same gemfibrozil levels for effects on spontaneously-occurring phasic rhythmic contractile activity, typically not seen in arteries under in vitro conditions but commonly exhibited by smooth muscle of uterus, duodenum and bladder. We found that gemfibrozil significantly relaxed all induced forms of contraction in the rat tail artery, acutely at the higher test levels and after a delay of a few hours at the lower test levels. The highest test level of gemfibrozil (400 microM) also completely abolished spontaneously-occurring contractile activity of the isolated uterus and duodenum and markedly suppressed it in the bladder. This is the first evidence that a fibrate drug can directly relax smooth muscle contractions, either induced by various contractile agents or

  18. Smooth muscle-like tissue constructs with circumferentially oriented cells formed by the cell fiber technology.

    PubMed

    Hsiao, Amy Y; Okitsu, Teru; Onoe, Hiroaki; Kiyosawa, Mahiro; Teramae, Hiroki; Iwanaga, Shintaroh; Kazama, Tomohiko; Matsumoto, Taro; Takeuchi, Shoji

    2015-01-01

    The proper functioning of many organs and tissues containing smooth muscles greatly depends on the intricate organization of the smooth muscle cells oriented in appropriate directions. Consequently controlling the cellular orientation in three-dimensional (3D) cellular constructs is an important issue in engineering tissues of smooth muscles. However, the ability to precisely control the cellular orientation at the microscale cannot be achieved by various commonly used 3D tissue engineering building blocks such as spheroids. This paper presents the formation of coiled spring-shaped 3D cellular constructs containing circumferentially oriented smooth muscle-like cells differentiated from dedifferentiated fat (DFAT) cells. By using the cell fiber technology, DFAT cells suspended in a mixture of extracellular proteins possessing an optimized stiffness were encapsulated in the core region of alginate shell microfibers and uniformly aligned to the longitudinal direction. Upon differentiation induction to the smooth muscle lineage, DFAT cell fibers self-assembled to coiled spring structures where the cells became circumferentially oriented. By changing the initial core-shell microfiber diameter, we demonstrated that the spring pitch and diameter could be controlled. 21 days after differentiation induction, the cell fibers contained high percentages of ASMA-positive and calponin-positive cells. Our technology to create these smooth muscle-like spring constructs enabled precise control of cellular alignment and orientation in 3D. These constructs can further serve as tissue engineering building blocks for larger organs and cellular implants used in clinical treatments.

  19. Smooth Muscle-Like Tissue Constructs with Circumferentially Oriented Cells Formed by the Cell Fiber Technology

    PubMed Central

    Hsiao, Amy Y.; Okitsu, Teru; Onoe, Hiroaki; Kiyosawa, Mahiro; Teramae, Hiroki; Iwanaga, Shintaroh; Kazama, Tomohiko; Matsumoto, Taro; Takeuchi, Shoji

    2015-01-01

    The proper functioning of many organs and tissues containing smooth muscles greatly depends on the intricate organization of the smooth muscle cells oriented in appropriate directions. Consequently controlling the cellular orientation in three-dimensional (3D) cellular constructs is an important issue in engineering tissues of smooth muscles. However, the ability to precisely control the cellular orientation at the microscale cannot be achieved by various commonly used 3D tissue engineering building blocks such as spheroids. This paper presents the formation of coiled spring-shaped 3D cellular constructs containing circumferentially oriented smooth muscle-like cells differentiated from dedifferentiated fat (DFAT) cells. By using the cell fiber technology, DFAT cells suspended in a mixture of extracellular proteins possessing an optimized stiffness were encapsulated in the core region of alginate shell microfibers and uniformly aligned to the longitudinal direction. Upon differentiation induction to the smooth muscle lineage, DFAT cell fibers self-assembled to coiled spring structures where the cells became circumferentially oriented. By changing the initial core-shell microfiber diameter, we demonstrated that the spring pitch and diameter could be controlled. 21 days after differentiation induction, the cell fibers contained high percentages of ASMA-positive and calponin-positive cells. Our technology to create these smooth muscle-like spring constructs enabled precise control of cellular alignment and orientation in 3D. These constructs can further serve as tissue engineering building blocks for larger organs and cellular implants used in clinical treatments. PMID:25734774

  20. Effect of 3-substituted 1,4-benzodiazepin-2-ones on bradykinin-induced smooth muscle contraction.

    PubMed

    Virych, P A; Shelyuk, O V; Kabanova, T A; Khalimova, E I; Martynyuk, V S; Pavlovsky, V I; Andronati, S A

    2017-01-01

    Biochemical properties of 3-substituted 1,4-benzodiazepine determined by the characteristics of their chemical structure. Influence of 3-substituted 1,4-benzodiazepin-2-ones on maximal normalized rate and amplitudes of isometric smooth muscle contraction in rats was investigated. Compounds MX-1775 and MX-1828 demonstrated the similar inhibition effect on bradykinin-induced contraction of smooth muscle like competitive inhibitor des-arg9-bradykinin-acetate to bradykinin B2-receptors. MX-1626 demonstrated unidirectional changes of maximal normalized rate and force of smooth muscle that proportionally depended on bradykinin concentration in the range 10-10-10-6 M. MX-1828 has statistically significant decrease of normalized rate of smooth muscle contraction for bradykinin concentrations 10-10 and 10-9 M by 20.7 and 8.6%, respectively, but for agonist concentration 10-6 M, this parameter increased by 10.7% and amplitude was reduced by 29.5%. Compounds MX-2011, MX-1785 and MX-2004 showed no natural effect on bradykinin-induced smooth muscle contraction. Compounds MX-1775, MX-1828, MX-1626 were selected for further research of their influence on kinin-kallikrein system and pain perception.

  1. Enhanced elastin synthesis and maturation in human vascular smooth muscle tissue derived from induced-pluripotent stem cells.

    PubMed

    Eoh, Joon H; Shen, Nian; Burke, Jacqueline A; Hinderer, Svenja; Xia, Zhiyong; Schenke-Layland, Katja; Gerecht, Sharon

    2017-04-01

    Obtaining vascular smooth muscle tissue with mature, functional elastic fibers is a key obstacle in tissue-engineered blood vessels. Poor elastin secretion and organization leads to a loss of specialization in contractile smooth muscle cells, resulting in over proliferation and graft failure. In this study, human induced-pluripotent stem cells (hiPSCs) were differentiated into early smooth muscle cells, seeded onto a hybrid poly(ethylene glycol) dimethacrylate/poly (l-lactide) (PEGdma-PLA) scaffold and cultured in a bioreactor while exposed to pulsatile flow, towards maturation into contractile smooth muscle tissue. We evaluated the effects of pulsatile flow on cellular organization as well as elastin expression and assembly in the engineered tissue compared to a static control through immunohistochemistry, gene expression and functionality assays. We show that culturing under pulsatile flow resulted in organized and functional hiPSC derived smooth muscle tissue. Immunohistochemistry analysis revealed hiPSC-smooth muscle tissue with robust, well-organized cells and elastic fibers and the supporting microfibril proteins necessary for elastic fiber assembly. Through qRT-PCR analysis, we found significantly increased expression of elastin, fibronectin, and collagen I, indicating the synthesis of necessary extracellular matrix components. Functionality assays revealed that hiPSC-smooth muscle tissue cultured in the bioreactor had an increased calcium signaling and contraction in response to a cholinergic agonist, significantly higher mature elastin content and improved mechanical properties in comparison to the static control. The findings presented here detail an effective approach to engineering elastic human vascular smooth muscle tissue with the functionality necessary for tissue engineering and regenerative medicine applications. Obtaining robust, mature elastic fibers is a key obstacle in tissue-engineered blood vessels. Human induced-pluripotent stem cells have

  2. MURC deficiency in smooth muscle attenuates pulmonary hypertension.

    PubMed

    Nakanishi, Naohiko; Ogata, Takehiro; Naito, Daisuke; Miyagawa, Kotaro; Taniguchi, Takuya; Hamaoka, Tetsuro; Maruyama, Naoki; Kasahara, Takeru; Nishi, Masahiro; Matoba, Satoaki; Ueyama, Tomomi

    2016-08-22

    Emerging evidence suggests that caveolin-1 (Cav1) is associated with pulmonary arterial hypertension. MURC (also called Cavin-4) is a member of the cavin family, which regulates caveolar formation and functions together with caveolins. Here, we show that hypoxia increased Murc mRNA expression in the mouse lung, and that Murc-null mice exhibited attenuation of hypoxia-induced pulmonary hypertension (PH) accompanied by reduced ROCK activity in the lung. Conditional knockout mice lacking Murc in smooth muscle also resist hypoxia-induced PH. MURC regulates the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) through Rho/ROCK signalling. Cav1 suppresses RhoA activity in PASMCs, which is reversed by MURC. MURC binds to Cav1 and inhibits the association of Cav1 with the active form of Gα13, resulting in the facilitated association of the active form of Gα13 with p115RhoGEF. These results reveal that MURC has a function in the development of PH through modulating Rho/ROCK signalling.

  3. Temporal and spatial dynamics underlying capacitative calcium entry in human colonic smooth muscle.

    PubMed

    Kovac, Jason R; Chrones, Tom; Sims, Stephen M

    2008-01-01

    Following smooth muscle excitation and contraction, depletion of intracellular Ca(2+) stores activates capacitative Ca(2+) entry (CCE) to replenish stores and sustain cytoplasmic Ca(2+) (Ca(2+)(i)) elevations. The objectives of the present study were to characterize CCE and the Ca(2+)(i) dynamics underlying human colonic smooth muscle contraction by using tension recordings, fluorescent Ca(2+)-indicator dyes, and patch-clamp electrophysiology. The neurotransmitter acetylcholine (ACh) contracted tissue strips and, in freshly isolated colonic smooth muscle cells (SMCs), caused elevation of Ca(2+)(i) as well as activation of nonselective cation currents. To deplete Ca(2+)(i) stores, the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitors thapsigargin and cyclopiazonic acid were added to a Ca(2+)-free bathing solution. Under these conditions, addition of extracellular Ca(2+) (3 mM) elicited increased tension that was inhibited by the cation channel blockers SKF-96365 (10 microM) and lanthanum (100 microM), suggestive of CCE. In a separate series of experiments on isolated SMCs, SERCA inhibition generated a gradual and sustained inward current. When combined with high-speed Ca(2+)-imaging techniques, the CCE-evoked rise of Ca(2+)(i) was associated with inward currents carrying Ca(2+) that were inhibited by SKF-96365. Regional specializations in Ca(2+) influx and handling during CCE were observed. Distinct "hotspot" regions of Ca(2+) rise and plateau were evident in 70% of cells, a feature not previously recognized in smooth muscle. We propose that store-operated Ca(2+) entry occurs in hotspots contributing to localized Ca(2+) elevations in human colonic smooth muscle.

  4. Catecholamines release mediators in the opossum oesophageal circular smooth muscle.

    PubMed Central

    Daniel, E E; Jager, L P; Jury, J

    1987-01-01

    1. Effects of catecholamines applied exogenously to the circular smooth muscle layer of the body of the oesophagus of the opossum (Didelphis marsupialis) were studied, simultaneously measuring changes in the membrane potential, the membrane conductance and the contractility of the muscle, using the double sucrose-gap technique. 2. Superfusion of the smooth muscle with Krebs solution at 27 degrees C containing dopamine (10(-6)-10(-4) M) dose-dependently caused a hyperpolarization of the smooth muscle cells and an increased membrane resistance followed after gradual repolarization by oscillations of the membrane potential, often accompanied by muscle action potentials. During the hyperpolarization, the tendency for the membrane potential to sag during prolonged application of hyperpolarizing currents was reduced and the 'off' depolarization following such currents was increased. This muscle did not develop active tension prior to treatment; it therefore did not relax during the hyperpolarizations, but contracted following the depolarized phase of oscillations. 3. The non-adrenergic, non-cholinergic nerve-mediated inhibitory junction potential (i.j.p.) showed a small reduction in amplitude during superfusion with dopamine, explicable as a result of the drug-induced hyperpolarization. The 'off' response following the i.j.p., decreased transiently when the membrane potential was hyperpolarized to its maximum value. Then it increased to values larger than control as the membrane repolarized. Vasoactive intestinal polypeptide (VIP, 10(-6) M) produced a similar response but hyperpolarizations were smaller. 4. Of the tested catecholamines, isoprenaline, phenylephrine, butylated hydroxytoluene-920 (BHT-920) and clonidine were ineffective whereas the potency order for other catecholamines was dopamine greater than noradrenaline greater than or equal to adrenaline greater than DOPA. The catecholamine-induced responses were not affected by alpha- or beta

  5. Inhibition of RhoA/Rho kinase pathway and smooth muscle contraction by hydrogen sulfide.

    PubMed

    Nalli, Ancy D; Wang, Hongxia; Bhattacharya, Sayak; Blakeney, Bryan A; Murthy, Karnam S

    2017-10-01

    Hydrogen sulfide (H 2 S) plays an important role in smooth muscle relaxation. Here, we investigated the expression of enzymes in H 2 S synthesis and the mechanism regulating colonic smooth muscle function by H 2 S. Expression of cystathionine-γ-lyase (CSE), but not cystathionine-β-synthase (CBS), was identified in the colonic smooth muscle of rabbit, mouse, and human. Carbachol (CCh)-induced contraction in rabbit muscle strips and isolated muscle cells was inhibited by l-cysteine (substrate of CSE) and NaHS (an exogenous H 2 S donor) in a concentration-dependent fashion. H 2 S induced S-sulfhydration of RhoA that was associated with inhibition of RhoA activity. CCh-induced Rho kinase activity also was inhibited by l-cysteine and NaHS in a concentration-dependent fashion. Inhibition of CCh-induced contraction by l-cysteine was blocked by the CSE inhibitor, dl-propargylglycine (DL-PPG) in dispersed muscle cells. Inhibition of CCh-induced Rho kinase activity by l-cysteine was blocked by CSE siRNA in cultured cells and DL-PPG in dispersed muscle cells. Stimulation of Rho kinase activity and muscle contraction in response to CCh was also inhibited by l-cysteine or NaHS in colonic muscle cells from mouse and human. Collectively, our studies identified the expression of CSE in colonic smooth muscle and determined that sulfhydration of RhoA by H 2 S leads to inhibition of RhoA and Rho kinase activities and muscle contraction. The mechanism identified may provide novel therapeutic approaches to mitigate gastrointestinal motility disorders. © 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.

  6. [Forskolin inhibits spontaneous contraction of gastric antral smooth muscle in rats].

    PubMed

    Jiang, Jing-Zhi; Sun, Qian; Xu, Dong-Yuan; Zhang, Mo-Han; Piao, Li-Hua; Cai, Ying-Lan; Jin, Zheng

    2013-04-25

    The aim of the present study was to investigate the effects of cyclic adenosine monophosphate (cAMP) on rat gastric antral circular smooth muscle function. Forskolin, a direct activator of adenylyl cyclase (AC), was used to observe the influences of cAMP. Multi-channel physiological recorder was used to record spontaneous contraction activity of gastric antral circular muscle from Wistar rats. And ELISA method was used to detect the change of cAMP production in perfusate. The results showed that forskolin concentration-dependently suppressed the amplitude and frequency of the spontaneous contraction of the gastric antral muscle, and lowered the baseline of contraction movement significantly. Forskolin concentration-dependently increased the production of cAMP in the perfusate, which showed a significant negative correlation with the contraction amplitude of gastric antral ring muscle. The inhibitory effect of forskolin on spontaneous contraction activity of rat gastric antral circular muscle could be blocked by cAMP-dependent protein kinase (PKA) inhibitor H-89. These results suggest forskolin increases cAMP production and then activates PKA pathway, resulting in the inhibition of the spontaneous contraction activity of rat gastric antral circular smooth muscle.

  7. K+-induced alterations in airway muscle responsiveness to electrical field stimulation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Murlas, C.; Ehring, G.; Suszkiw, J.

    1986-07-01

    We investigated possible pre- and postsynaptic effects of K+-induced depolarization on ferret tracheal smooth muscle (TSM) responsiveness to cholinergic stimulation. To assess electromechanical activity, cell membrane potential (Em) and tension (Tm) were simultaneously recorded in buffer containing 6, 12, 18, or 24 mM K+ before and after electrical field stimulation (EFS) or exogenous acetylcholine (ACh). In 6 mM K+, Em was -58.1 +/- 1.0 mV (mean +/- SE). In 12 mM K+, Em was depolarized to -52.3 +/- 0.9 mV, basal Tm did not change, and both excitatory junctional potentials and contractile responses to EFS at short stimulus duration weremore » larger than in 6 mM K+. No such potentiation occurred at a higher K+, although resting Em and Tm increased progressively above 12 mM K+. The sensitivity of ferret TSM to exogenous ACh appeared unaffected by K+. To determine whether the hyperresponsiveness in 12 mM K+ was due, in part, to augmented ACh release from intramural airway nerves, experiments were done using TSM preparations incubated with (3H)choline to measure (3H)ACh release at rest and during EFS. Although resting (3H)ACh release increased progressively in higher K+, release evoked by EFS was maximal in 12 mM K+ and declined in higher concentrations. We conclude that small elevations in the extracellular K+ concentration augment responsiveness of the airways, by increasing the release of ACh both at rest and during EFS from intramural cholinergic nerve terminals. Larger increases in K+ appear to be inhibitory, possibly due to voltage-dependent effects that occur both pre- and postsynaptically.« less

  8. Crosstalk between beta-2-adrenoceptor and muscarinic acetylcholine receptors in the airway.

    PubMed

    Pera, Tonio; Penn, Raymond B

    2014-06-01

    The M3 and M2 muscarinic acetylcholine receptors (mAChRs) and beta-2-adrenoceptors (β2ARs) are important regulators of airway cell function, and drugs targeting these receptors are among the first line drugs in the treatment of the obstructive lung diseases asthma and chronic obstructive lung disease (COPD). Cross-regulation or crosstalk between mAChRs and β2ARs in airway smooth muscle (ASM) helps determine the contractile state of the muscle, thus airway diameter and resistance to airflow. In this review we will detail mAChR and β2AR-signaling and crosstalk, focusing on events in the ASM cell but also addressing the function of these receptors in other cell types that impact airway physiology. We conclude by discussing how recent advances in GPCR pharmacology offer a unique opportunity to fine tune mAChR and β2AR signaling and their crosstalk, and thereby produce superior therapeutics for obstructive lung and other diseases. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Titanium Dioxide Modulation of the Contractibility of Visceral Smooth Muscles In Vivo

    NASA Astrophysics Data System (ADS)

    Tsymbalyuk, Olga V.; Naumenko, Anna M.; Rohovtsov, Oleksandr O.; Skoryk, Mykola A.; Voiteshenko, Ivan S.; Skryshevsky, Valeriy A.; Davydovska, Tamara L.

    2017-02-01

    Electronic scanning microscopy was used in the work to obtain the image and to identify the sizes of titanium dioxide (TiO2) nanoparticles 21 ± 5 nm. The qualitative and quantitative elemental analysis of the preparations of the caecum, antrum, myometrium, kidneys, and lungs of the rats, burdened with titanium dioxide, was also performed. It was established using the tenzometric method in the isometric mode that the accumulation of titanium dioxide in smooth muscles of the caecum resulted in the considerable, compared to the control, increase in the frequency of their spontaneous contractions, the decrease in the duration of the contraction-relaxation cycle, and the decrease in the indices of muscle functioning efficiency (the index of contractions in Montevideo units (MU) and the index of contractions in Alexandria units (AU)). In the same experimental conditions, there was not the increase, but the decrease in the frequency of spontaneous contractions, the duration of the contraction-relaxation cycle, and the increase in MU and AU indices in the smooth muscles of myometrium (in the group of rats, burdened with TiO2 for 30 days). It was also determined that TiO2 modulates both the mechanisms of the input of extracellular Ca2+ ions and the mechanisms of decreasing the concentration of these cations in smooth muscle cells of the caecum during the generation of the high potassium contraction. In these conditions, there is a considerable increase in the normalized maximal velocity of the contraction phase and the relaxation phase. It was demonstrated in the work that titanium dioxide also changes the cholinergic excitation in these muscles. The impact of titanium dioxide in the group of rats, burdened with TiO2, was accompanied with a considerable impairment of the kinetics of forming the tonic component of the oxytocin-induced contraction of the smooth muscles of myometrium.

  10. Optimisation of isolation of richly pure and homogeneous primary human colonic smooth muscle cells.

    PubMed

    Tattoli, I; Corleto, V D; Taffuri, M; Campanini, N; Rindi, G; Caprilli, R; Delle Fave, G; Severi, C

    2004-11-01

    Inherent properties of gastrointestinal smooth muscle can be assessed using isolated cell suspensions. Currently available isolation techniques, based on short 2-h enzymatic digestion, however, present the disadvantage of low cellular yield with brief viability. These features are an important limiting factor especially in studies in humans in which tissue may not be available daily and mixing of samples is not recommended. To optimise the isolation procedure of cells from human colon to obtain a richly pure primary smooth muscle cell preparation. Slices of circular muscle layer, obtained from surgical specimens of human colon, were incubated overnight in Dulbecco's modified eagle's medium supplemented with antibiotics, foetal bovine serum, an ATP-regenerating system and collagenase. On the following day, digested muscle strips were suspended in HEPES buffer, and spontaneously dissociated smooth muscle cells were harvested and used either immediately or maintained in suspension for up to 72 h. Cell yield, purity, viability, contractile responses, associated intracellular calcium signals and RNA and protein extraction were evaluated and compared to cell suspensions obtained with the current short digestion protocol. The overnight isolation protocol offers the advantage of obtaining a pure, homogeneous, long-life viable cell suspension that maintains a fully differentiated smooth muscle phenotype unchanged for at least 72 h and that allows multiple functional/biochemical studies and efficient RNA extraction from a single human specimen.

  11. Paradoxical behavior of neuromedin U in isolated smooth muscle cells and intact tissue.

    PubMed

    Brighton, Paul J; Wise, Alan; Dass, Narinder B; Willars, Gary B

    2008-04-01

    Neuromedin U (NmU) is a neuropeptide showing high levels of structural conservation across different species. Since its discovery in 1985, NmU has been implicated in numerous physiological roles, including smooth muscle contraction, energy homeostasis, stress, intestinal ion transport, pronociception, and circadian rhythm. Two G-protein-coupled receptors have been identified for NmU and cloned from humans, rats, and mice. Recombinantly expressed NmU receptors couple to both Galpha(q/11) and Galpha(i) G-proteins, and NmU binds essentially irreversibly, preventing signaling to repetitive applications of NmU. However, it is unclear whether these properties reflect those of endogenously expressed NmU receptors or how these properties influence the functional consequences of NmU receptor signaling. Here, we have explored the signaling by rat NmU receptors expressed endogenously in cultured rat colonic smooth muscle cells and explore the functional consequence of this signaling by investigating the NmU-mediated contraction of ex vivo rat colonic smooth muscle preparations. We demonstrate that endogenous rat NmU receptors couple to both Galpha(q/11) and Galpha(i) G-proteins. Furthermore, we show complex patterns of Ca(2+) signaling, including oscillations, and provide evidence of essentially irreversible binding of NmU to smooth muscle cells. Challenge of either circular or longitudinal rat isolated colonic smooth muscle preparations with NmU resulted in robust contractions. Stimulation was direct, and paradoxically, repetitive applications of NmU mediated repetitive contractions with no evidence of desensitization, highlighting a major discrepancy in the behavior of NmU in single cells and in intact tissues. The reason for this discrepancy is presently unknown.

  12. Acid-Sensing Ion Channel 1a Contributes to Airway Hyperreactivity in Mice

    PubMed Central

    Reznikov, Leah R.; Meyerholz, David K.; Adam, Ryan J.; Abou Alaiwa, Mahmoud; Jaffer, Omar; Michalski, Andrew S.; Powers, Linda S.; Price, Margaret P.; Stoltz, David A.; Welsh, Michael J.

    2016-01-01

    Neurons innervating the airways contribute to airway hyperreactivity (AHR), a hallmark feature of asthma. Several observations suggested that acid-sensing ion channels (ASICs), neuronal cation channels activated by protons, might contribute to AHR. For example, ASICs are found in vagal sensory neurons that innervate airways, and asthmatic airways can become acidic. Moreover, airway acidification activates ASIC currents and depolarizes neurons innervating airways. We found ASIC1a protein in vagal ganglia neurons, but not airway epithelium or smooth muscle. We induced AHR by sensitizing mice to ovalbumin and found that ASIC1a-/- mice failed to exhibit AHR despite a robust inflammatory response. Loss of ASIC1a also decreased bronchoalveolar lavage fluid levels of substance P, a sensory neuropeptide secreted from vagal sensory neurons that contributes to AHR. These findings suggest that ASIC1a is an important mediator of AHR and raise the possibility that inhibiting ASIC channels might be beneficial in asthma. PMID:27820848

  13. Fibronectin promotes differentiation of neural crest progenitors endowed with smooth muscle cell potential

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Costa-Silva, Bruno; Programa de Pos-graduacao em Neurociencias, Centro de Ciencias Biologicas, Universidade Federal de Santa Catarina, Campus Universitario - Trindade, 88040-900, Florianopolis, S.C.; Coelho da Costa, Meline

    The neural crest (NC) is a model system used to investigate multipotency during vertebrate development. Environmental factors control NC cell fate decisions. Despite the well-known influence of extracellular matrix molecules in NC cell migration, the issue of whether they also influence NC cell differentiation has not been addressed at the single cell level. By analyzing mass and clonal cultures of mouse cephalic and quail trunk NC cells, we show for the first time that fibronectin (FN) promotes differentiation into the smooth muscle cell phenotype without affecting differentiation into glia, neurons, and melanocytes. Time course analysis indicated that the FN-induced effectmore » was not related to massive cell death or proliferation of smooth muscle cells. Finally, by comparing clonal cultures of quail trunk NC cells grown on FN and collagen type IV (CLIV), we found that FN strongly increased both NC cell survival and the proportion of unipotent and oligopotent NC progenitors endowed with smooth muscle potential. In contrast, melanocytic progenitors were prominent in clonogenic NC cells grown on CLIV. Taken together, these results show that FN promotes NC cell differentiation along the smooth muscle lineage, and therefore plays an important role in fate decisions of NC progenitor cells.« less

  14. beta. -adrenergic relaxation of smooth muscle: differences between cells and tissues

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Scheid, C.R.

    1987-09-01

    The present studies were carried out in an attempt to resolve the controversy about the Na/sup +/ dependence of ..beta..-adrenergic relaxation in smooth muscle. Previous studies on isolated smooth muscle cells from the toad stomach had suggested that at least some of the actions of ..beta..-adrenergic agents, including a stimulatory effect on /sup 45/Ca efflux, were dependent on the presence of a normal transmembrane Na/sup +/ gradient. Studies by other investigators using tissues derived from mammalian sources had suggested that the relaxing effect of ..beta..-adrenergic agents was Na/sup +/ independent. Uncertainty remained as to whether these discrepancies reflected differences betweenmore » cells and tissues or differences between species. Thus, in the present studies, the authors utilized both tissues and cells from the same source, the stomach muscle of the toad Bufo marinus, and assessed the Na/sup +/ dependence of ..beta..-adrenergic relaxation. They found that elimination of a normal Na/sup +/ gradient abolished ..beta..-adrenergic relaxation of isolated cells. In tissues, however, similar manipulations had no effect on relaxation. The reasons for this discrepancy are unclear but do not appear to be attributable to changes in smooth muscle function following enzymatic dispersion. Thus the controversy concerning the mechanisms of ..beta..-adrenergic relaxation may reflect inherent differences between tissues and cells.« less

  15. Smooth muscle neurokinin-2 receptors mediate contraction in human saphenous veins.

    PubMed

    Mechiche, Hakima; Grassin-Delyle, Stanislas; Pinto, Francisco M; Buenestado, Amparo; Candenas, Luz; Devillier, Philippe

    2011-05-01

    Substance P (SP) and neurokinin A (NKA) are members of the tachykinin peptides family. SP causes endothelial-dependant relaxation but the contractile response to tachykinins in human vessels remains unknown. The objective was to assess the expression and the contractile effects of tachykinins and their receptors in human saphenous veins (SV). Tachykinin expression was assessed with RT-PCR, tachykinin receptors expression with RT-PCR and immunohistochemistry, and functional studies were performed in organ bath. Transcripts of all tachykinin and tachykinin receptor genes were found in SV. NK(1)-receptors were localized in both endothelial and smooth muscle layers of undistended SV, whereas they were only found in smooth muscle layers of varicose SV. The expression of NK(2)- and NK(3)-receptors was limited to the smooth muscle in both preparations. NKA induced concentration-dependent contractions in about half the varicose SV. Maximum effect reached 27.6±5.5% of 90 mM KCl and the pD(2) value was 7.3±0.2. NKA also induced the contraction of undistended veins from bypass and did not cause the relaxation of these vessels after precontraction. The NK(2)-receptor antagonist SR48968 abolished the contraction induced by NKA, and a rapid desensitization of the NK(2)-receptor was observed. In varicose SV, the agonists specific to NK(1)- or NK(3)-receptors did not cause either contraction or relaxation. The stimulation of smooth muscle NK(2)-receptors can induce the contraction of human SV. As SV is richly innervated, tachykinins may participate in the regulation of the tone in this portion of the low pressure vascular system. Copyright © 2011 Elsevier Ltd. All rights reserved.

  16. Contribution of Rho-kinase to membrane excitability of murine colonic smooth muscle.

    PubMed

    Bayguinov, O; Dwyer, L; Kim, H; Marklew, A; Sanders, K M; Koh, S D

    2011-06-01

    The Rho-kinase pathway regulates agonist-induced contractions in several smooth muscles, including the intestine, urinary bladder and uterus, via dynamic changes in the Ca(2+) sensitivity of the contractile apparatus. However, there is evidence that Rho-kinase also modulates other cellular effectors such as ion channels. We examined the regulation of colonic smooth muscle excitability by Rho-kinase using conventional microelectrode recording, isometric force measurements and patch-clamp techniques. The Rho-kinase inhibitors, Y-27632 and H-1152, decreased nerve-evoked on- and off-contractions elicited at a range of frequencies and durations. The Rho-kinase inhibitors decreased the spontaneous contractions and the responses to carbachol and substance P independently of neuronal inputs, suggesting Y-27632 acts directly on smooth muscle. The Rho-kinase inhibitors significantly reduced the depolarization in response to carbachol, an effect that cannot be due to regulation of Ca(2+) sensitization. Patch-clamp experiments showed that Rho-kinase inhibitors reduce GTPγS-activated non-selective cation currents. The Rho-kinase inhibitors decreased contractions evoked by nerve stimulation, carbachol and substance P. These effects were not solely due to inhibition of the Ca(2+) sensitization pathway, as the Rho-kinase inhibitors also inhibited the non-selective cation conductances activated by excitatory transmitters. Thus, Rho-kinase may regulate smooth muscle excitability mechanisms by regulating non-selective cation channels as well as changing the Ca(2+) sensitivity of the contractile apparatus. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

  17. The effect of sleep onset on upper airway muscle activity in patients with sleep apnoea versus controls

    PubMed Central

    Fogel, Robert B; Trinder, John; White, David P; Malhotra, Atul; Raneri, Jill; Schory, Karen; Kleverlaan, Darci; Pierce, Robert J

    2005-01-01

    Pharyngeal dilator muscles are important in the pathophysiology of obstructive sleep apnoea syndrome (OSA). We have previously shown that during wakefulness, the activity of both the genioglossus (GGEMG) and tensor palatini (TPEMG) is greater in patients with OSA compared with controls. Further, EMG activity decreases at sleep onset, and the decrement is greater in apnoea patients than in healthy controls. In addition, it is known that the prevalence of OSA is greater in middle-aged compared with younger men. Thus, we had two goals in this study. First we compared upper airway muscle activity between young and middle-aged healthy men compared with men with OSA. We also explored the mechanisms responsible for the decrement in muscle activity at sleep onset in these groups. We investigated muscle activity, ventilation , and upper airway resistance (UAR) during wakefulness and sleep onset (transition from α to θ EEG activity) in all three groups. Measurements were obtained during basal breathing (BB) and nasal continuous positive airway pressure (CPAP) was applied to reduce negative pressure-mediated muscle activation). We found that during wakefulness there was a gradation of GGEMG and UAR (younger < older < OSA) and that muscle activity was reduced by the application of nasal CPAP (to a greater degree in the OSA patients). Although CPAP eliminated differences in UAR during wakefulness and sleep, GGEMG remained greater in the OSA patients. During sleep onset, a greater initial fall in GGEMG was seen in the OSA patients followed by subsequent muscle recruitment in the third to fifth breaths following the α to θ transition. On the CPAP night, and GGEMG still fell further in the OSA patients compared with control subjects. CPAP prevented the rise in UAR at sleep onset along with the associated recruitment in GGEMG. Differences in TPEMG among the groups were not significant. These data suggest that the middle-aged men had upper airway function midway between that of

  18. Mechanics of smooth muscle in isolated single microvessels.

    PubMed

    Gore, R W; Davis, M J

    1984-01-01

    In vivo studies on frog mesenteric arterioles (4) indicate that segmental differences in the response of microvessels to physical and chemical stimuli can be explained simply in terms of the length-tension characteristics of vascular smooth muscle at different points along the vascular tree. Studies on single, isolated arterioles in vitro were initiated to examine more closely the validity of this explanation for regional response differences. This paper reports some of the results. First-, second-, and third-order arterioles (18-60 micron i.d.) were dissected from hamster cheek pouches. The vessels were cannulated with a modified Burg microperfusion system, and their mechanical properties studied using the methods described by Duling and Gore. Vessels were activated in four stages with K+ and norepinephrine. During activation, transmural pressures were adjusted to minimize vascular smooth-muscle shortening. Active pressure-diameter curves were recorded while adjusting transmural pressure through the range 5 to 400 cm H20 in 5-25 cm steps. Vessel dimensions were measured with a videomicrometer. Passive curves were obtained after equilibration overnight in Ca2+-free medium. The vessels were then fixed and prepared for histologic sectioning, and measurements of vessel-wall composition were made. The Laplace relationship was used to construct length-tension diagrams, and the histologic data were used to normalize the dimensional data to smooth-muscle lengths. Maximum active tension of second-order arterioles (1,170 dynes/cm) was two times previous values reported by Gore et al. This was due presumably to refinements in techniques and dissection procedures. Maximum active stress averaged 3.9 X 10(+6) dynes/cm2 for second-order arterioles. This number is identical to data obtained from hog carotid strips by Dillon et al.

  19. MURC deficiency in smooth muscle attenuates pulmonary hypertension

    PubMed Central

    Nakanishi, Naohiko; Ogata, Takehiro; Naito, Daisuke; Miyagawa, Kotaro; Taniguchi, Takuya; Hamaoka, Tetsuro; Maruyama, Naoki; Kasahara, Takeru; Nishi, Masahiro; Matoba, Satoaki; Ueyama, Tomomi

    2016-01-01

    Emerging evidence suggests that caveolin-1 (Cav1) is associated with pulmonary arterial hypertension. MURC (also called Cavin-4) is a member of the cavin family, which regulates caveolar formation and functions together with caveolins. Here, we show that hypoxia increased Murc mRNA expression in the mouse lung, and that Murc-null mice exhibited attenuation of hypoxia-induced pulmonary hypertension (PH) accompanied by reduced ROCK activity in the lung. Conditional knockout mice lacking Murc in smooth muscle also resist hypoxia-induced PH. MURC regulates the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) through Rho/ROCK signalling. Cav1 suppresses RhoA activity in PASMCs, which is reversed by MURC. MURC binds to Cav1 and inhibits the association of Cav1 with the active form of Gα13, resulting in the facilitated association of the active form of Gα13 with p115RhoGEF. These results reveal that MURC has a function in the development of PH through modulating Rho/ROCK signalling. PMID:27546070

  20. Traction in smooth muscle cells varies with cell spreading

    NASA Technical Reports Server (NTRS)

    Tolic-Norrelykke, Iva Marija; Wang, Ning

    2005-01-01

    Changes in cell shape regulate cell growth, differentiation, and apoptosis. It has been suggested that the regulation of cell function by the cell shape is a result of the tension in the cytoskeleton and the distortion of the cell. Here we explore the association between cell-generated mechanical forces and the cell morphology. We hypothesized that the cell contractile force is associated with the degree of cell spreading, in particular with the cell length. We measured traction fields of single human airway smooth muscle cells plated on a polyacrylamide gel, in which fluorescent microbeads were embedded to serve as markers of gel deformation. The traction exerted by the cells at the cell-substrate interface was determined from the measured deformation of the gel. The traction was measured before and after treatment with the contractile agonist histamine, or the relaxing agonist isoproterenol. The relative increase in traction induced by histamine was negatively correlated with the baseline traction. On the contrary, the relative decrease in traction due to isoproterenol was independent of the baseline traction, but it was associated with cell shape: traction decreased more in elongated than in round cells. Maximum cell width, mean cell width, and projected area of the cell were the parameters most tightly coupled to both baseline and histamine-induced traction in this study. Wide and well-spread cells exerted larger traction than slim cells. These results suggest that cell contractility is controlled by cell spreading.

  1. Hydroxyapatite and Calcified Elastin Induce Osteoblast-like Differentiation in Rat Aortic Smooth Muscle Cells

    PubMed Central

    Lei, Yang; Sinha, Aditi; Nosoudi, Nasim; Grover, Ankit; Vyavahare, Naren

    2014-01-01

    Vascular calcification can be categorized into two different types. Intimal calcification related to atherosclerosis and elastin-specific medial arterial calcification (MAC). Osteoblast-like differentiation of vascular smooth muscle cells (VSMCs) has been shown in both types; however, how this relates to initiation of vascular calcification is unclear. We hypothesize that the initial deposition of hydroxyapatite-like mineral in MAC occurs on degraded elastin first and that causes osteogenic transformation of VSMCs. To test this, rat aortic smooth muscle cells (RASMCs) were cultured on hydroxyapatite crystals and calcified aortic elastin. Using RT-PCR and specific protein assays, we demonstrate that RASMCs lose their smooth muscle lineage markers like alpha smooth muscle actin (SMA) and myosin heavy chain (MHC) and undergo chondrogenic/osteogenic transformation. This is indicated by an increase in the expression of typical chondrogenic proteins such as aggrecan, collagen type II alpha 1(Col2a1) and bone proteins such as runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP) and osteocalcin (OCN). Furthermore, when calcified conditions are removed, cells return to their original phenotype. Our data supports the hypothesis that elastin degradation and calcification precedes VSMCs' osteoblast-like differentiation. PMID:24447384

  2. Pharmacological and molecular characterization of muscarinic receptor subtypes in human esophageal smooth muscle.

    PubMed

    Preiksaitis, H G; Krysiak, P S; Chrones, T; Rajgopal, V; Laurier, L G

    2000-12-01

    Esophageal peristalsis is dependent on activation of muscarinic receptors, but little is known about the roles of specific receptor subtypes in the human esophagus. We examined muscarinic receptor expression and function in human esophageal smooth muscle obtained from patients undergoing resection for cancer. [(3)H]Quinuclidinyl benzylate (QNB)-specific binding was similar in longitudinal muscle (B(max) = 106 +/- 22 fmol/mg of protein, K(d) = 68 +/- 9 pM) and circular muscle (B(max) = 81 +/- 16 fmol/mg of protein, K(d) = 79 +/- 15 pM). Subtype-selective antagonists inhibited [(3)H]QNB similarly in muscle from both layers. Further analysis of antagonist inhibition of [(3)H]QNB binding showed a major site (60-70%) with antagonist affinity profile consistent with the M2 subtype and a second site that could not be classified. Reverse transcription-polymerase chain reaction and immunoblotting demonstrated the presence of all five known muscarinic receptor subtypes, and immunocytochemistry on acutely isolated smooth muscle cells confirmed the expression of each subtype on the muscle cells. Subtype-selective antagonists had similar inhibitory effects on carbachol-evoked contractions in longitudinal muscle and circular muscle strips with pA(2) values of 9.5 +/- 0.1 and 9.6 +/- 0.2 for 4-diphenylacetoxy-N-methylpiperidine methiodide, 7.1 +/- 0.1 and 7.0 +/- 0.2 for pirenzepine, and 6.2 +/- 0.2 and 6.4 +/- 0.2 for methoctramine, respectively. We conclude that human esophageal smooth muscle expresses muscarinic receptor subtypes M1 through M5. The antagonist sensitivity profile for muscle contraction is consistent with activation of the M3 subtype.

  3. Structural limits on force production and shortening of smooth muscle.

    PubMed

    Siegman, Marion J; Davidheiser, Sandra; Mooers, Susan U; Butler, Thomas M

    2013-02-01

    This study determined the factors that limit force production and shortening in two smooth muscles having very different relationships between active and passive force as a function of muscle length. The rat anococcygeus muscle develops active force over the range of lengths 0.2-2.0× the optimum length for force production (Lo). Passive tension due to extension of the resting muscle occurs only at lengths exceeding Lo. In contrast, the rabbit taenia coli develops force in the range of lengths 0.4-1.1 Lo, and passive force which is detectable at 0.56 Lo, increases to ~0.45 maximum active force at Lo, and increases sharply with further extension. The anococcygeus muscle can shorten to 0.2 Lo and the taenia coli to 0.4 Lo. Dynamic stiffness and energy usage at short muscle lengths suggest that the limit of shortening in the taenia coli, in contrast to the anococcygeus muscle, is not due to a failure of cross bridge interaction. Phosphorylation of the regulatory myosin light chains in intact muscles decreased to a small extent at short lengths compared to the decrease in force production. The differences in force production and the extent of shortening in the two muscles was maintained even when, following permeabilization, the myosin light chains were irreversibly phosphorylated with ATPγS, indicating that differences in activation played little, if any role. Ultrastructural studies on resting and activated muscles show that the taenia coli, which is rich in connective tissue (unlike the anococcygeus muscle) undergoes marked cellular twisting and contractile filament misalignment at short lengths with compression of the extracellular matrix. As a result, force is not transmitted in the longitudinal axis of the muscle, but is dissipated against an internal load provided by the compressed extracellular matrix. These observations on two very different normal smooth muscles reveal how differences in the relative contribution of active and passive structural elements

  4. The effects of inhaled corticosteroids on intrinsic responsiveness and histology of airways from infant monkeys exposed to house dust mite allergen and ozone

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Joad, Jesse P.; Kott, Kayleen S.; Bric, John M.

    2008-01-15

    Inhaled corticosteroids (ICS) are recommended to treat infants with asthma, some with intermittent asthma. We previously showed that exposing infant monkeys to allergen/ozone resulted in asthma-like characteristics of their airways. We evaluated the effects of ICS on histology and intrinsic responsiveness of allergen/ozone-exposed and normal infant primate airways. Infant monkeys were exposed by inhalation to (1) filtered air and saline, (2) house dust mite allergen (HDMA) + ozone and saline, (3) filtered air and ICS (budesonide) or (4) HDMA + ozone and ICS. Allergen/ozone exposures started at 1 month and ICS at 3 months of age. At 6 months ofmore » age, methacholine-induced changes in luminal area of airways in proximal and distal lung slices were determined using videomicrometry, followed by histology of the same slices. Proximal airway responsiveness was increased by allergen/ozone and by ICS. Eosinophil profiles were increased by allergen/ozone in both proximal and distal airways, an effect that was decreased by ICS in distal airways. In both allergen/ozone- and air-exposed monkeys, ICS increased the number of alveolar attachments in distal airways, decreased mucin in proximal airways and decreased epithelial volume in both airways. ICS increased smooth muscle in air-exposed animals while decreasing it in allergen/ozone-exposed animals in both airways. In proximal airways, there was a small but significant positive correlation between smooth muscle and airway responsiveness, as well as between alveolar attachments and responsiveness. ICS change morphology and function in normal airways as well as allergen/ozone-exposed airways, suggesting that they should be reserved for infants with active symptoms.« less

  5. Cholesterol is necessary both for the toxic effect of Abeta peptides on vascular smooth muscle cells and for Abeta binding to vascular smooth muscle cell membranes.

    PubMed

    Subasinghe, Supundi; Unabia, Sharon; Barrow, Colin J; Mok, Su San; Aguilar, Marie-Isabel; Small, David H

    2003-02-01

    Accumulation of beta amyloid (Abeta) in the brain is central to the pathogenesis of Alzheimer's disease. Abeta can bind to membrane lipids and this binding may have detrimental effects on cell function. In this study, surface plasmon resonance technology was used to study Abeta binding to membranes. Abeta peptides bound to synthetic lipid mixtures and to an intact plasma membrane preparation isolated from vascular smooth muscle cells. Abeta peptides were also toxic to vascular smooth muscle cells. There was a good correlation between the toxic effect of Abeta peptides and their membrane binding. 'Ageing' the Abeta peptides by incubation for 5 days increased the proportion of oligomeric species, and also increased toxicity and the amount of binding to lipids. The toxicities of various Abeta analogs correlated with their lipid binding. Significantly, binding was influenced by the concentration of cholesterol in the lipid mixture. Reduction of cholesterol in vascular smooth muscle cells not only reduced the binding of Abeta to purified plasma membrane preparations but also reduced Abeta toxicity. The results support the view that Abeta toxicity is a direct consequence of binding to lipids in the membrane. Reduction of membrane cholesterol using cholesterol-lowering drugs may be of therapeutic benefit because it reduces Abeta-membrane binding.

  6. Electrical properties of purinergic transmission in smooth muscle of the guinea-pig prostate.

    PubMed

    Lam, Michelle; Mitsui, Retsu; Hashitani, Hikaru

    2016-01-01

    Prostatic smooth muscle develops spontaneous myogenic tone which is modulated by autonomic neuromuscular transmission. This study aimed to investigate the role of purinergic transmission in regulating electrical activity of prostate smooth muscle and whether its contribution may be altered with age. Intracellular recordings were simultaneously made with isometric tension recordings in smooth muscle preparations of the guinea-pig prostate. Immunostaining for P2X1 receptors on whole mount preparations was also performed. In prostate preparations which generated spontaneous slow waves, electrical field stimulation (EFS)-evoked excitatory junction potentials (EJPs) which were abolished by guanethidine (10 μM), α-β-methylene ATP (10 μM) or pyridoxal phosphate-6-azophenyl-2,4-disulfonic acid (PPADS, 10 μM) but not phentolamine (1 μM). Consistently, immunostaining revealed the expression of P2X1 receptors on prostatic smooth muscle. EJPs themselves did not cause contractions, but EJPs could sum to trigger a slow wave and associated contraction. Yohimbine (1 μM) and 3,7-dimethyl-1-propargylxanthine (DMPX, 10 μM) but not propranolol (1 μM) potentiated EJPs. Although properties of EJPs were not different between young and aging guinea-pig prostates, ectoATPase inhibitor ARL 67156 (100 μM) augmented EJP amplitudes by 64.2 ± 29.6% in aging animals, compared to 22.1 ± 19.9% in young animals. These results suggest that ATP released from sympathetic nerves acts on P2X1 purinoceptors located on prostate smooth muscle to evoke EJPs, while pre-junctional α2-adrenergic and adenosine A2 receptors may play a role in preventing excessive transmitter release. Age-related up-regulation of enzymatic ATP breakdown may be a compensatory mechanism for the enhanced purinergic transmission which would cause hypercontractility arising from increased ATP release in older animals. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Augmented vascular smooth muscle cell stiffness and adhesion when hypertension is superimposed on aging.

    PubMed

    Sehgel, Nancy L; Sun, Zhe; Hong, Zhongkui; Hunter, William C; Hill, Michael A; Vatner, Dorothy E; Vatner, Stephen F; Meininger, Gerald A

    2015-02-01

    Hypertension and aging are both recognized to increase aortic stiffness, but their interactions are not completely understood. Most previous studies have attributed increased aortic stiffness to changes in extracellular matrix proteins that alter the mechanical properties of the vascular wall. Alternatively, we hypothesized that a significant component of increased vascular stiffness in hypertension is due to changes in the mechanical and adhesive properties of vascular smooth muscle cells, and that aging would augment the contribution from vascular smooth muscle cells when compared with the extracellular matrix. Accordingly, we studied aortic stiffness in young (16-week-old) and old (64-week-old) spontaneously hypertensive rats and Wistar-Kyoto wild-type controls. Systolic and pulse pressures were significantly increased in young spontaneously hypertensive rats when compared with young Wistar-Kyoto rats, and these continued to rise in old spontaneously hypertensive rats when compared with age-matched controls. Excised aortic ring segments exhibited significantly greater elastic moduli in both young and old spontaneously hypertensive rats versus Wistar-Kyoto rats. were isolated from the thoracic aorta, and stiffness and adhesion to fibronectin were measured by atomic force microscopy. Hypertension increased both vascular smooth muscle cell stiffness and vascular smooth muscle cell adhesion, and these increases were both augmented with aging. By contrast, hypertension did not affect histological measures of aortic collagen and elastin, which were predominantly changed by aging. These findings support the concept that stiffness and adhesive properties of vascular smooth muscle cells are novel mechanisms contributing to the increased aortic stiffness occurring with hypertension superimposed on aging. © 2014 American Heart Association, Inc.

  8. Role of SM22 in the differential regulation of phasic vs. tonic smooth muscle

    PubMed Central

    Ali, Mehboob

    2015-01-01

    Preliminary proteomics studies between tonic vs. phasic smooth muscles identified three distinct protein spots identified to be those of transgelin (SM22). The latter was found to be distinctly downregulated in the internal anal sphincter (IAS) vs. rectal smooth muscle (RSM) SMC. The major focus of the present studies was to examine the differential molecular control mechanisms by SM22 in the functionality of truly tonic smooth muscle of the IAS vs. the adjoining phasic smooth muscle of the RSM. We monitored SMC lengths before and after incubation with pFLAG-SM22 (for SM22 overexpression), and SM22 small-interfering RNA. pFLAG-SM22 caused concentration-dependent and significantly greater relaxation in the IAS vs. the RSM SMCs. Conversely, temporary silencing of SM22 caused contraction in both types of the SMCs. Further studies revealed a significant reverse relationship between the levels of SM22 phosphorylation and the amount of SM22-actin binding in the IAS and RSM SMC. Data showed higher phospho-SM22 levels and decreased SM22-actin binding in the IAS, and reverse to be the case in the RSM SMCs. Experiments determining the mechanism for SM22 phosphorylation in these smooth muscles revealed that Y-27632 (Rho kinase inhibitor) but not Gö-6850 (protein kinase C inhibitor) caused concentration-dependent decreased phosphorylation of SM22. We speculate that SM22 plays an important role in the regulation of basal tone via Rho kinase-induced phosphorylation of SM22. PMID:25617350

  9. Voltage-dependent inward currents in smooth muscle cells of skeletal muscle arterioles

    PubMed Central

    Shirokov, Roman E.

    2018-01-01

    Voltage-dependent inward currents responsible for the depolarizing phase of action potentials were characterized in smooth muscle cells of 4th order arterioles in mouse skeletal muscle. Currents through L-type Ca2+ channels were expected to be dominant; however, action potentials were not eliminated in nominally Ca2+-free bathing solution or by addition of L-type Ca2+ channel blocker nifedipine (10 μM). Instead, Na+ channel blocker tetrodotoxin (TTX, 1 μM) reduced the maximal velocity of the upstroke at low, but not at normal (2 mM), Ca2+ in the bath. The magnitude of TTX-sensitive currents recorded with 140 mM Na+ was about 20 pA/pF. TTX-sensitive currents decreased five-fold when Ca2+ increased from 2 to 10 mM. The currents reduced three-fold in the presence of 10 mM caffeine, but remained unaltered by 1 mM of isobutylmethylxanthine (IBMX). In addition to L-type Ca2+ currents (15 pA/pF in 20 mM Ca2+), we also found Ca2+ currents that are resistant to 10 μM nifedipine (5 pA/pF in 20 mM Ca2+). Based on their biophysical properties, these Ca2+ currents are likely to be through voltage-gated T-type Ca2+ channels. Our results suggest that Na+ and at least two types (T- and L-) of Ca2+ voltage-gated channels contribute to depolarization of smooth muscle cells in skeletal muscle arterioles. Voltage-gated Na+ channels appear to be under a tight control by Ca2+ signaling. PMID:29694371

  10. Divergent modulation of Rho‐kinase and Ca2+ influx pathways by Src family kinases and focal adhesion kinase in airway smooth muscle

    PubMed Central

    Shaifta, Yasin; Irechukwu, Nneka; Prieto‐Lloret, Jesus; MacKay, Charles E; Marchon, Keisha A; Ward, Jeremy P T

    2015-01-01

    Background and Purpose The importance of tyrosine kinases in airway smooth muscle (ASM) contraction is not fully understood. The aim of this study was to investigate the role of Src‐family kinases (SrcFK) and focal adhesion kinase (FAK) in GPCR‐mediated ASM contraction and associated signalling events. Experimental Approach Contraction was recorded in intact or α‐toxin permeabilized rat bronchioles. Phosphorylation of SrcFK, FAK, myosin light‐chain‐20 (MLC20) and myosin phosphatase targeting subunit‐1 (MYPT‐1) was evaluated in cultured human ASM cells (hASMC). [Ca2+]i was evaluated in Fura‐2 loaded hASMC. Responses to carbachol (CCh) and bradykinin (BK) and the contribution of SrcFK and FAK to these responses were determined. Key Results Contractile responses in intact bronchioles were inhibited by antagonists of SrcFK, FAK and Rho‐kinase, while after α‐toxin permeabilization, they were sensitive to inhibition of SrcFK and Rho‐kinase, but not FAK. CCh and BK increased phosphorylation of MYPT‐1 and MLC20 and auto‐phosphorylation of SrcFK and FAK. MYPT‐1 phosphorylation was sensitive to inhibition of Rho‐kinase and SrcFK, but not FAK. Contraction induced by SR Ca2+ depletion and equivalent [Ca2+]i responses in hASMC were sensitive to inhibition of both SrcFK and FAK, while depolarization‐induced contraction was sensitive to FAK inhibition only. SrcFK auto‐phosphorylation was partially FAK‐dependent, while FAK auto‐phosphorylation was SrcFK‐independent. Conclusions and Implications SrcFK mediates Ca2+‐sensitization in ASM, while SrcFK and FAK together and individually influence multiple Ca2+ influx pathways. Tyrosine phosphorylation is therefore a key upstream signalling event in ASM contraction and may be a viable target for modulating ASM tone in respiratory disease. PMID:26294392

  11. Nociceptin effects in the airways.

    PubMed

    Peiser, C; Undem, B J; Fischer, A

    2000-07-01

    The opioid-like heptadecapeptide nociceptin (NC) has the following effects in the airways (investigated in isolated tracheae and bronchi from guinea pig or rat): the electric field stimulation (EFS)-induces release of acetylcholine (ACh), the tachykinin substance P (SP) and calcitonin gene-related peptide (CGRP) is reduced after pretreatment with NC, and EFS-induced tachykinergic nonadrenergic-noncholinergic (NANC) bronchoconstriction is inhibited by NC. Both the NC-mediated inhibition of neurotransmission and of smooth muscle contraction occurred in a concentration-dependent manner. Because these effects were naloxone-insensitive, were blocked by the NC receptor antagonist [F/G]NC(1-13)NH(2), and could be mimicked by the NC analogs, NCNH(2) and NC(1-13)NH(2), it is thought that they are distinct from the classic opioid receptors. That these pharmacological actions of NC are of relevance for airway physiology is highly probable given the presence of NC-immunoreactivity in the nerve fibers of the airways and of opioid-like receptor (ORL-1) transcripts in the jugular ganglia, from where the tachykinin-containing afferents arise.

  12. Effects of Gingko biloba extract (EGb 761) on vascular smooth muscle cell calcification induced by β-glycerophosphate.

    PubMed

    Li, En-Gang; Tian, Jun; Xu, Zhong-Hua

    2016-01-01

    To investigate the effects of Gingko biloba extract (EGb 761) on calcification induced by β-glycerophosphate in rat aortic vascular smooth muscle cells. Rat aortic vascular smooth muscle cells were cultured with various concentrations of EGb 761 and β-glycerophosphate for 7 days. Calcium content in the cells, alkaline phosphatase activity, cell protein content, NF-κB activation, and reactive oxygen species production were assayed, respectively. The calcium depositions of vascular smooth muscle cells of the β-glycerophosphate group were significantly higher than those of the control group (p < 0.01), and were inhibited by EGb 761 in a concentration-dependent manner (p < 0.05). Data showed β-glycerophosphate induced the enhanced expression of alkaline phosphatase, up-regulated the NF-κB activity and increased reactive oxygen species production of vascular smooth muscle cells while these decreased when administrated with EGb 761(p < 0.05). EGb 761 significantly reduced deposition of calcium induced by β-glycerophosphate in rat aortic vascular smooth muscle cells. It not only reduced the deposition of calcium, but also inhibited osteogenic transdifferentiation, which may be associated with decreasing expression of alkaline phosphatase, down-regulating the NF-κB activity, and reducing reactive oxygen species production of vascular smooth muscle cells, and may have the potential to serve as a role for vascular calcification in clinical situations.

  13. Endothelin ETA receptor expression in human cerebrovascular smooth muscle cells.

    PubMed

    Yu, J C; Pickard, J D; Davenport, A P

    1995-11-01

    1. Endothelin (ET) has been implicated in cerebrovasospasm for example, following subarachnoid haemorrhage, and blocking the interaction of ET with its receptors on cerebral vessels, may be of therapeutic benefit. The aim of our study was to characterize endothelin receptor sub-types on medial smooth muscle cells of human cerebral vessels. Cultures of vascular smooth muscle cells were explanted from human cerebral resistance vessels and characterized as human brain smooth muscle cells (HBSMCs). 2. Over a 48 h incubation period, HBSMC cultures secreted comparable levels of immunoreactive (IR) big endothelin-1 (big ET-1) and IR endothelin (ET): 12.7 +/- 10.3 and 8.3 +/- 5.6 pmol/10(6) cells, respectively (mean +/- s.e. mean from three different individuals), into the culture medium. 3. Total RNA was extracted from cultures of human brain smooth muscle cells. Reverse-transcriptase polymerase chain reaction (RI-PCR) assays and subsequent product separation by agarose gel electrophoresis revealed single bands corresponding to the expected product sizes encoding cDNA for ETA (299 base pairs) and ETB (428 base pairs) (n = 3 different cultures). 4. Autoradiography demonstrated the presence of specific binding sites for [125I]-ET-1 which labels all ET receptors, and [125I]-PD151242, an ETA subtype-selective antagonist which exclusively labels ETA receptors, but no specific-binding was detected using ETB subtype-selective [125I]-BQ3020 (n = 3 different cultures, in duplicate). 5. In saturation binding assays, [123I]-ET-1 bound with high affinity: KD = 0.8 +/- 0.1 nM and Bmax = 690 +/- 108 fmol mg-1. A one-site fit was preferred and Hill slopes were close to unity over the concentration range (10(-12) to 10(-8) M). [125I]-PD151242 also bound with similar affinity: KD = 0.4 +/- 0.1 nM and Bmax = 388 +/- 68 fmol mg-1 (mean +/- s.e. mean, n = 3 different cultures). Again, a one-site fit was preferred and Hill slopes were close to unity over the concentration range. Unlabelled PD

  14. KCl cotransport regulation and protein kinase G in cultured vascular smooth muscle cells.

    PubMed

    Adragna, N C; Zhang, J; Di Fulvio, M; Lincoln, T M; Lauf, P K

    2002-05-15

    K-Cl cotransport is activated by vasodilators in erythrocytes and vascular smooth muscle cells and its regulation involves putative kinase/phosphatase cascades. N-ethylmaleimide (NEM) activates the system presumably by inhibiting a protein kinase. Nitrovasodilators relax smooth muscle via cGMP-dependent activation of protein kinase G (PKG), a regulator of membrane channels and transporters. We investigated whether PKG regulates K-Cl cotransport activity or mRNA expression in normal, PKG-deficient-vector-only-transfected (PKG-) and PKG-catalytic-domain-transfected (PKG+) rat aortic smooth muscle cells. K-Cl cotransport was calculated as the Cl-dependent Rb influx, and mRNA was determined by semiquantitative RT-PCR. Baseline K-Cl cotransport was higher in PKG+ than in PKG- cells (p <0.01). At 0.5 mM, NEM stimulated K-Cl cotransport by 5-fold in PKG- but not in PKG+ cells. However, NEM was more potent although less effective to activate K-Cl cotransport in normal (passage 1-3) and PKG+ than in PKG- cells. In PKG- cells, [(dihydroindenyl) oxy] alkanoic acid (300 mM) but not furosemide (1 mM) inhibited K-Cl cotransport. Furthermore, no difference in K-Cl cotransport mRNA expression was observed between these cells. In conclusion, this study shows that manipulation of PKG expression in vascular smooth muscle cells affects K-Cl cotransport activity and its activation by NEM.

  15. Laboratory practical to study the differential innervation pathways of urinary tract smooth muscle.

    PubMed

    Rembetski, Benjamin E; Cobine, Caroline A; Drumm, Bernard T

    2018-06-01

    In the mammalian lower urinary tract, there is a reciprocal relationship between the contractile state of the bladder and urethra. As the bladder fills with urine, it remains relaxed to accommodate increases in volume, while the urethra remains contracted to prevent leakage of urine from the bladder to the exterior. Disruptions to the normal contractile state of the bladder and urethra can lead to abnormal micturition patterns and urinary incontinence. While both the bladder and urethra are smooth-muscle organs, they are differentially contracted by input from cholinergic and sympathetic nerves, respectively. The laboratory practical described here provides an experiential approach to understanding the anatomy of the lower urinary tract. Several key factors in urinary tract physiology are outlined, e.g., the bladder is contracted by activation of the parasympathetic pathway via cholinergic stimulation on muscarinic receptors, whereas the urethra is contracted by activation of the sympathetic pathway via adrenergic stimulation on α 1 -adrenoceptors. This is achieved by measuring the force generated by bladder and urethra smooth muscle to demonstrate that acetylcholine contracts the smooth muscle of the bladder, whereas adrenergic agonists contract the urethral smooth muscle. An inhibition of these effects is also demonstrated by application of the muscarinic receptor antagonist atropine and the α 1 -adrenergic receptor blocker phentolamine. A list of suggested techniques and exam questions to evaluate student understanding on this topic is also provided.

  16. Value of counting positive PHH3 cells in the diagnosis of uterine smooth muscle tumors

    PubMed Central

    Pang, Shu-Jie; Li, Cheng-Cheng; Shen, Yan; Liu, Yian-Zhu; Shi, Yi-Quan; Liu, Yi-Xin

    2015-01-01

    The diagnosis of uterine smooth muscle tumors including leiomyosarcomas (LMS), smooth muscle tumors of uncertain malignant potential (STUMP), bizarre (atypical) leiomyoma (BLM), mitotically active leiomyoma (MAL) and leiomyoma (LM) depends on a combination of microscopic features, such as mitoses, cytologic atypia, and coagulative tumor cell necrosis. However, a small number of these tumors still pose difficult diagnostic challenges. The assessment of accurate mitotic figures (MF) is one of the major parameters in the proper classification of uterine smooth muscle tumors. This assessment can be hampered by the presence of increased number of apoptotic bodies or pyknotic nuclei, which frequently mimic mitoses. Phospho-histone H3 (PHH3) is a recently described immunomarker specific for cells undergoing mitoses. In our study, we collected 132 cases of uterine smooth muscle tumors, including 26 LMSs, 16 STUMPs, 30 BLMs, 30 MALs and 30 LMs. We used mitosis specific marker PHH3 to count mitotic indexes (MI) of uterine smooth muscle tumors and compared with the mitotic indexes of hematoxylin and eosin (H&E). There is a positive correlation with the number of mitotic figures in H&E-stained sections and PHH3-stained sections (r=0.944, P<0.05). The ratio of PHH3-MI to H&E-MI has no statistically significant difference in each group except for LMs (P>0.05). The counting value of PHH3 in LMSs have significantly higher than STUMPs, BLMs, MALs and LMs (P<0.001) and the counting value of PHH3 is 1.5±0.5 times of the number of mitotic indexes in H&E. To conclude, our results show that counting PHH3 is a useful index in the diagnosis of uterine smooth muscle tumors and it can provide a more accurate index instead of the time-honored mitotic figure counts at a certain ratio. PMID:26191133

  17. Kir2.1 encodes the inward rectifier potassium channel in rat arterial smooth muscle cells

    PubMed Central

    Bradley, Karri K; Jaggar, Jonathan H; Bonev, Adrian D; Heppner, Thomas J; Flynn, Elaine RM; Nelson, Mark T; Horowitz, Burton

    1999-01-01

    The molecular nature of the strong inward rectifier K+ channel in vascular smooth muscle was explored by using isolated cell RT-PCR, cDNA cloning and expression techniques.RT-PCR of RNA from single smooth muscle cells of rat cerebral (basilar), coronary and mesenteric arteries revealed transcripts for Kir2.1. Transcripts for Kir2.2 and Kir2.3 were not found.Quantitative PCR analysis revealed significant differences in transcript levels of Kir2.1 between the different vascular preparations (n = 3; P < 0.05). A two-fold difference was detected between Kir2.1 mRNA and β-actin mRNA in coronary arteries when compared with relative levels measured in mesenteric and basilar preparations.Kir2.1 was cloned from rat mesenteric vascular smooth muscle cells and expressed in Xenopus oocytes. Currents were strongly inwardly rectifying and selective for K+.The effect of extracellular Ba2+, Ca2+, Mg2+ and Cs2+ ions on cloned Kir2.1 channels expressed in Xenopus oocytes was examined. Ba2+ and Cs+ block were steeply voltage dependent, whereas block by external Ca2+ and Mg2+ exhibited little voltage dependence. The apparent half-block constants and voltage dependences for Ba2+, Cs+, Ca2+ and Mg2+ were very similar for inward rectifier K+ currents from native cells and cloned Kir2.1 channels expressed in oocytes.Molecular studies demonstrate that Kir2.1 is the only member of the Kir2 channel subfamily present in vascular arterial smooth muscle cells. Expression of cloned Kir2.1 in Xenopus oocytes resulted in inward rectifier K+ currents that strongly resemble those that are observed in native vascular arterial smooth muscle cells. We conclude that Kir2.1 encodes for inward rectifier K+ channels in arterial smooth muscle. PMID:10066894

  18. Differential Effect of Zoledronic Acid on Human Vascular Smooth Muscle Cells

    PubMed Central

    Albadawi, Hassan; Haurani, Mounir J.; Oklu, Rahmi; Trubiano, Jordan P.; Laub, Peter J.; Yoo, Hyung-Jin; Watkins, Michael T.

    2012-01-01

    Introduction The activation of human vascular smooth muscle cell proliferation, adhesion and migration is essential for intimal hyperplasia formation. These experiments were designed to test whether Zoledronic Acid (ZA) would modulate indices of human smooth muscle cell activation, exert differential effects on proliferating vs. quiescent cells and determine whether these effects were dependent on GTPase binding proteins prenylation. ZA was chosen for testing in these experiments because it is clinically used in humans with cancer, and has been shown to modulate rat smooth muscle cell proliferation and migration. Methods Human aortic smooth muscle cells (HASMC) were cultured under either proliferating or growth arrest (quiescent) conditions in the presence or absence of ZA for 48 hours, whereupon the effect of ZA on HASMC proliferation, cellular viability, metabolic activity and membrane integrity were compared. In addition, the effect of ZA on adhesion and migration were assessed in proliferating cells. The effect of increased concentration of ZA on the mevalonate pathway and genomic/cellular stress related poly ADP Ribose polymerase (PARP) enzyme activity were assessed using the relative prenylation of Rap-1A/B protein and the formation of poly ADP- ribosylated proteins (PAR) respectively. Results There was a dose dependent inhibition of cellular proliferation, adhesion and migration following ZA treatment. ZA treatment decreased indices of cellular viability and significantly increased membrane injury in proliferating vs. quiescent cells. This was correlated with the appearance of unprenylated Rap-1A protein and dose dependent down regulation of PARP activity. Conclusions These data suggest that ZA is effective in inhibiting HASMC proliferation, adhesion and migration which coincide with the appearance of unprenylated RAP-1A/B protein, thereby suggesting that the mevalonate pathway may play a role in the inhibition of HASMC activation. PMID:23164362

  19. Role of smooth muscle cells on endothelial cell cytosolic free calcium in porcine coronary arteries.

    PubMed

    Budel, S; Schuster, A; Stergiopoulos, N; Meister, J J; Bény, J L

    2001-09-01

    We tested the hypothesis that the cytosolic free calcium concentration in endothelial cells is under the influence of the smooth muscle cells in the coronary circulation. In the left descending branch of porcine coronary arteries, cytosolic free calcium concentration ([Ca(2+)](i)) was estimated by determining the fluorescence ratio of two calcium probes, fluo 4 and fura red, in smooth muscle and endothelial cells using confocal microscopy. Acetylcholine and potassium, which act directly on smooth muscle cells to increase [Ca(2+)](i), were found to indirectly elevate [Ca(2+)](i) in endothelial cells; in primary cultures of endothelial cells, neither stimulus affected [Ca(2+)](i), yet substance P increased the fluorescence ratio twofold. In response to acetylcholine and potassium, isometric tension developed by arterial strips with intact endothelium was attenuated by up to 22% (P < 0.05) compared with strips without endothelium. These findings suggest that stimuli that increase smooth muscle [Ca(2+)](i) can indirectly influence endothelial cell function in porcine coronary arteries. Such a pathway for negative feedback can moderate vasoconstriction and diminish the potential for vasospasm in the coronary circulation.

  20. Alcohol and Airways Function in Health and Disease

    PubMed Central

    Sisson, Joseph H.

    2007-01-01

    The volatility of alcohol promotes the movement of alcohol from the bronchial circulation across the airway epithelium and into the conducting airways of the lung. The exposure of the airways through this route likely accounts for many of the biologic effects of alcohol on lung airway functions. The impact of alcohol on lung airway functions is dependent on the concentration, duration and route of exposure. Brief exposure to mild concentrations of alcohol may enhance mucociliary clearance, stimulates bronchodilation and probably attenuates the airway inflammation and injury observed in asthma and COPD. Prolonged and heavy exposure to alcohol impairs mucociliary clearance, may complicate asthma management and likely worsens outcomes including lung function and mortality in COPD patients. Non-alcohol congeners and alcohol metabolites act as triggers for airway disease exacerbations especially in atopic asthmatics and in Asian populations who have a reduced capacity to metabolize alcohol. Research focused on the mechanisms of alcohol-mediated changes in airway functions has identified specific mechanisms that mediate alcohol effects within the lung airways. These include prominent roles for the second messengers calcium and nitric oxide, regulatory kinases including PKG and PKA, alcohol and acetaldehyde-metabolizing enzymes such as aldehyde dehydrogenase type 2 (ALDH2). The role alcohol may play in the pathobiology of airway mucus, bronchial blood flow, airway smooth muscle regulation and the interaction with other airway exposure agents, such as cigarette smoke, represent opportunities for future investigation. PMID:17764883

  1. Alcohol and airways function in health and disease.

    PubMed

    Sisson, Joseph H

    2007-08-01

    The volatility of alcohol promotes the movement of alcohol from the bronchial circulation across the airway epithelium and into the conducting airways of the lung. The exposure of the airways through this route likely accounts for many of the biologic effects of alcohol on lung airway functions. The effect of alcohol on lung airway functions is dependent on the concentration, duration, and route of exposure. Brief exposure to mild concentrations of alcohol may enhance mucociliary clearance, stimulates bronchodilation, and probably attenuates the airway inflammation and injury observed in asthma and chronic obstructive pulmonary disease (COPD). Prolonged and heavy exposure to alcohol impairs mucociliary clearance, may complicate asthma management, and likely worsens outcomes including lung function and mortality in COPD patients. Nonalcohol congeners and alcohol metabolites act as triggers for airway disease exacerbations especially in atopic asthmatics and in Asian populations who have a reduced capacity to metabolize alcohol. Research focused on the mechanisms of alcohol-mediated changes in airway functions has identified specific mechanisms that mediate alcohol effects within the lung airways. These include prominent roles for the second messengers calcium and nitric oxide, regulatory kinases including PKG and PKA, alcohol- and acetaldehyde-metabolizing enzymes such as aldehyde dehydrogenase 2. The role alcohol may play in the pathobiology of airway mucus, bronchial blood flow, airway smooth muscle regulation, and the interaction with other airway exposure agents, such as cigarette smoke, represents opportunities for future investigation.

  2. Crystal Structure of a Phosphorylated Light Chain Domain of Scallop Smooth-Muscle Myosin

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kumar, V.S.; Robinson, H.; O-Neall-Hennessey, E.

    2011-11-02

    We have determined the crystal structure of a phosphorylated smooth-muscle myosin light chain domain (LCD). This reconstituted LCD is of a sea scallop catch muscle myosin with its phosphorylatable regulatory light chain (RLC SmoA). In the crystal structure, Arg{sup 16}, an arginine residue that is present in this isoform but not in vertebrate smooth-muscle RLC, stabilizes the phosphorylation site. This arginine interacts with the carbonyl group of the phosphorylation-site serine in the unphosphorylated LCD (determined previously), and with the phosphate group when the serine is phosphorylated. However, the overall conformation of the LCD is essentially unchanged upon phosphorylation. This resultmore » provides additional evidence that phosphorylation of the RLC is unlikely to act as an on-switch in regulation of scallop catch muscle myosin.« less

  3. THE EFFECT OF SMOOTH MUSCLE ON THE INTERCELLULAR SPACES IN TOAD URINARY BLADDER

    PubMed Central

    DiBona, Donald R.; Civan, Mortimer M.

    1970-01-01

    Phase microscopy of toad urinary bladder has demonstrated that vasopressin can cause an enlargement of the epithelial intercellular spaces under conditions of no net transfer of water or sodium. The suggestion that this phenomenon is linked to the hormone's action as a smooth muscle relaxant has been tested and verified with the use of other agents effecting smooth muscle: atropine and adenine compounds (relaxants), K+ and acetylcholine (contractants). Furthermore, it was possible to reduce the size and number of intercellular spaces, relative to a control, while increasing the rate of osmotic water flow. A method for quantifying these results has been developed and shows that they are, indeed, significant. It is concluded, therefore, that the configuration of intercellular spaces is not a reliable index of water flow across this epithelium and that such a morphologic-physiologic relationship is tenuous in any epithelium supported by a submucosa rich in smooth muscle. PMID:4915450

  4. MicroRNA-20b-5p inhibits platelet-derived growth factor-induced proliferation of human fetal airway smooth muscle cells by targeting signal transducer and activator of transcription 3.

    PubMed

    Tang, Jin; Luo, Lingying

    2018-06-01

    Pediatric asthma is still a health threat to the pediatric population in recent years. The airway remodeling induced by abnormal airway smooth muscle (ASM) cell proliferation is an important cause of asthma. MicroRNAs (miRNAs) are important regulators of ASM cell proliferation. Numerous studies have reported that miR-20b-5p is a critical regulator for cell proliferation. However, whether miR-20b-5p is involved in regulating ASM cell proliferation remains unknown. In this study, we aimed to investigate the potential role of miR-20b-5p in regulating the proliferation of fetal ASM cell induced by platelet-derived growth factor (PDGF). Here, we showed that miR-20b-5p was significantly decreased in fetal ASM cells treated with PDGF. Biological experiments showed that the overexpression of miR-20b-5p inhibited the proliferation while miR-20b-5p inhibition markedly promoted the proliferation of fetal ASM cells. Bioinformatics analysis and luciferase reporter assay showed that miR-20b-5p directly targeted the 3'-UTR of signal transducer and activator of transcription 3 (STAT3). Further data showed that miR-20b-5p negatively regulated the expression of STAT3 in fetal ASM cells. Moreover, miR-20b-5p regulates the transcriptional activity of STAT3 in fetal ASM cells. Overexpression of STAT3 reversed the inhibitory effect of miR-20b-5p overexpression on fetal ASM cell proliferation while the knockdown of STAT3 abrogated the promoted effect of miR-20b-5p inhibition on fetal ASM cell proliferation. Overall, our results show that miR-20b-5p impedes PDGF-induced proliferation of fetal ASM cells through targeting STAT3. Our study suggests that miR-20b-5p may play an important role in airway remodeling during asthma and suggests that miR-20b-5p may serve as a potential therapeutic target for pediatric asthma. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  5. Arterial Smooth Muscle Mitochondria Amplify Hydrogen Peroxide Microdomains Functionally Coupled to L-Type Calcium Channels

    PubMed Central

    Chaplin, Nathan L.; Nieves-Cintrón, Madeline; Fresquez, Adriana M.; Navedo, Manuel F.; Amberg, Gregory C.

    2015-01-01

    Rationale Mitochondria are key integrators of convergent intracellular signaling pathways. Two important second messengers modulated by mitochondria are calcium and reactive oxygen species. To date, coherent mechanisms describing mitochondrial integration of calcium and oxidative signaling in arterial smooth muscle are incomplete. Objective To address and add clarity to this issue we tested the hypothesis that mitochondria regulate subplasmalemmal calcium and hydrogen peroxide microdomain signaling in cerebral arterial smooth muscle. Methods and Results Using an image-based approach we investigated the impact of mitochondrial regulation of L-type calcium channels on subcellular calcium and ROS signaling microdomains in isolated arterial smooth muscle cells. Our single cell observations were then related experimentally to intact arterial segments and to living animals. We found that subplasmalemmal mitochondrial amplification of hydrogen peroxide microdomain signaling stimulates L-type calcium channels and that this mechanism strongly impacts the functional capacity of the vasoconstrictor angiotensin II. Importantly, we also found that disrupting this mitochondrial amplification mechanism in vivo normalized arterial function and attenuated the hypertensive response to systemic endothelial dysfunction. Conclusions From these observations we conclude that mitochondrial amplification of subplasmalemmal calcium and hydrogen peroxide microdomain signaling is a fundamental mechanism regulating arterial smooth muscle function. As the principle components involved are fairly ubiquitous and positioning of mitochondria near the plasma membrane is not restricted to arterial smooth muscle, this mechanism could occur in many cell types and contribute to pathological elevations of intracellular calcium and increased oxidative stress associated with many diseases. PMID:26390880

  6. Intercellular ultrafast Ca2+ wave in vascular smooth muscle cells: numerical and experimental study

    NASA Astrophysics Data System (ADS)

    Quijano, J. C.; Raynaud, F.; Nguyen, D.; Piacentini, N.; Meister, J. J.

    2016-08-01

    Vascular smooth muscle cells exhibit intercellular Ca2+ waves in response to local mechanical or KCl stimulation. Recently, a new type of intercellular Ca2+ wave was observed in vitro in a linear arrangement of smooth muscle cells. The intercellular wave was denominated ultrafast Ca2+ wave and it was suggested to be the result of the interplay between membrane potential and Ca2+ dynamics which depended on influx of extracellular Ca2+, cell membrane depolarization and its intercel- lular propagation. In the present study we measured experimentally the conduction velocity of the membrane depolarization and performed simulations of the ultrafast Ca2+ wave along coupled smooth muscle cells. Numerical results reproduced a wide spectrum of experimental observations, including Ca2+ wave velocity, electrotonic membrane depolarization along the network, effects of inhibitors and independence of the Ca2+ wave speed on the intracellular stores. The numerical data also provided new physiological insights suggesting ranges of crucial model parameters that may be altered experimentally and that could significantly affect wave kinetics allowing the modulation of the wave characteristics experimentally. Numerical and experimental results supported the hypothesis that the propagation of membrane depolarization acts as an intercellular messenger mediating intercellular ultrafast Ca2+ waves in smooth muscle cells.

  7. Electrically Stimulated Adipose Stem Cells on Polypyrrole-Coated Scaffolds for Smooth Muscle Tissue Engineering.

    PubMed

    Björninen, Miina; Gilmore, Kerry; Pelto, Jani; Seppänen-Kaijansinkko, Riitta; Kellomäki, Minna; Miettinen, Susanna; Wallace, Gordon; Grijpma, Dirk; Haimi, Suvi

    2017-04-01

    We investigated the use of polypyrrole (PPy)-coated polymer scaffolds and electrical stimulation (ES) to differentiate adipose stem cells (ASCs) towards smooth muscle cells (SMCs). Since tissue engineering lacks robust and reusable 3D ES devices we developed a device that can deliver ES in a reliable, repeatable, and cost-efficient way in a 3D environment. Long pulse (1 ms) or short pulse (0.25 ms) biphasic electric current at a frequency of 10 Hz was applied to ASCs to study the effects of ES on ASC viability and differentiation towards SMCs on the PPy-coated scaffolds. PPy-coated scaffolds promoted proliferation and induced stronger calponin, myosin heavy chain (MHC) and smooth muscle actin (SMA) expression in ASCs compared to uncoated scaffolds. ES with 1 ms pulse width increased the number of viable cells by day 7 compared to controls and remained at similar levels to controls by day 14, whereas shorter pulses significantly decreased viability compared to the other groups. Both ES protocols supported smooth muscle expression markers. Our results indicate that electrical stimulation on PPy-coated scaffolds applied through the novel 3D ES device is a valid approach for vascular smooth muscle tissue engineering.

  8. Markers for human brain pericytes and smooth muscle cells.

    PubMed

    Smyth, Leon C D; Rustenhoven, Justin; Scotter, Emma L; Schweder, Patrick; Faull, Richard L M; Park, Thomas I H; Dragunow, Mike

    2018-06-07

    Brain pericytes and vascular smooth muscle cells (vSMCs) are a critical component of the neurovascular unit and are important in regulating cerebral blood flow and blood-brain barrier integrity. Identification of subtypes of mural cells in tissue and in vitro is important to any study of their function, therefore we identified distinct mural cell morphologies in neurologically normal post-mortem human brain. Further, the distribution of mural cell markers platelet-derived growth factor receptor-β (PDGFRβ), α-smooth muscle actin (αSMA), CD13, neural/glial antigen-2 (NG2), CD146 and desmin was examined. We determined that PDGFRβ, NG2, CD13, and CD146 were expressed in capillary-associated pericytes. NG2, and CD13 were also present on vSMCs in large vessels, however abundant CD146 and desmin staining was also detected in vSMCs on large vessels, co-labelling with αSMA. To determine whether cultures recapitulated observations from tissue, primary human brain pericytes derived from neurologically normal autopsies were analysed for the presence of pericyte markers by immunocytochemistry, western blotting and qPCR. The proteins observed in brain pericytes in tissue (PDGFRβ, αSMA, desmin, CD146, CD13, and NG2) were present in vitro, validating a panel of proteins that can be used to label brain pericytes and vSMCs in tissue and in vitro. Finally, we showed that the proteins CD146 and desmin that are expressed on large vessels in situ, are also selective markers of a smooth muscle cell phenotype in vitro. Copyright © 2018 Elsevier B.V. All rights reserved.

  9. Lysyl oxidase propeptide inhibits smooth muscle cell signaling and proliferation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hurtado, Paola A.; Vora, Siddharth; Sume, Siddika Selva

    2008-02-01

    Lysyl oxidase is required for the normal biosynthesis and maturation of collagen and elastin. It is expressed by vascular smooth muscle cells, and its increased expression has been previously found in atherosclerosis and in models of balloon angioplasty. The lysyl oxidase propeptide (LOX-PP) has more recently been found to have biological activity as a tumor suppressor, and it inhibits Erk1/2 Map kinase activation. We reasoned that LOX-PP may have functions in normal non-transformed cells. We, therefore, investigated its effects on smooth muscle cells, focusing on important biological processes mediated by Erk1/2-dependent signaling pathways including proliferation and matrix metalloproteinase-9 (MMP-9) expression.more » In addition, we investigated whether evidence for accumulation of LOX-PP could be found in vivo in a femoral artery injury model. Recombinant LOX-PP was expressed and purified, and was found to inhibit primary rat aorta smooth muscle cell proliferation and DNA synthesis by more than 50%. TNF-{alpha}-stimulated MMP-9 expression and Erk1/2 activation were both significantly inhibited by LOX-PP. Immunohistochemistry studies carried out with affinity purified anti-LOX-PP antibody showed that LOX-PP epitopes were expressed at elevated levels in vascular lesions of injured arteries. These novel data suggest that LOX-PP may provide a feedback control mechanism that serves to inhibit properties associated with the development of vascular pathology.« less

  10. Lidocaine effect on flotillin-2 distribution in detergent-resistant membranes of equine jejunal smooth muscle in vitro.

    PubMed

    Tappenbeck, Karen; Schmidt, Sonja; Feige, Karsten; Naim, Hassan Y; Huber, Korinna

    2014-05-01

    Lidocaine is the most commonly chosen prokinetic for treating postoperative ileus in horses, a motility disorder associated with ischaemia-reperfusion injury of intestinal tissues. Despite the frequent use of lidocaine, the mechanism underlying its prokinetic effects is still unclear. Previous studies suggested that lidocaine altered cell membrane characteristics of smooth muscle cells. Therefore, the present study aimed to elucidate effects of lidocaine administration on characteristics of detergent-resistant membranes in equine jejunal smooth muscle. Lidocaine administration caused significant redistribution of flotillin-2, a protein marker of detergent-resistant membranes, in fractions of sucrose-density-gradients obtained from ischaemia-reperfusion injured smooth muscle solubilised with Triton X-100. It was concluded that lidocaine induced disruption of detergent-resistant membranes which might affect ion channel activity and therefore enhance smooth muscle contractility. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Numerous eosinophilic globules (skeinoid fibers) in a duodenal stromal tumor: an exceptional case showing smooth muscle differentiation.

    PubMed

    Matsukuma, S; Doi, M; Suzuki, M; Ikegawa, K; Sato, K; Kuwabara, N

    1997-11-01

    A unique case of duodenal stromal tumor in a 51-year-old man is reported. The tumor histologically showed spindle cell proliferation and numerous eosinophilic globules. Most globules were composed of tangled 45 nm thick fibrils, which were ultrastructurally identical to 'skeinoid fibers'. The presence of glycogen granules in the tumor cells and the immunoreactivity for alpha-smooth muscle actin suggested smooth muscle differentiation. Focal ultrastructural findings also supported the smooth muscle nature of this tumor. There were no immunohistochemical and ultrastructural features indicating neural differentiation. In previous studies, the presence of such 'skeinoid fibers' was suggested to be a histological marker for neural differentiation in gastrointestinal stromal tumor. However, the findings in the present case suggest that numerous 'skeinoid fibers' can be identified in duodenal stromal tumor with smooth muscle differentiation, although this condition may be rare.

  12. Pharmacological and histological examinations of regional differences of guinea-pig lung: a role of pleural surface smooth muscle in lung strip contraction.

    PubMed Central

    Wong, W. S.; Bloomquist, S. L.; Bendele, A. M.; Fleisch, J. H.

    1992-01-01

    1. Parenchymal lung strip preparations have been widely used as an in vitro model of peripheral airway smooth muscle. The present study examined functional responses of 4 consecutive guinea-pig lung parenchymal strips isolated from the central region (segment 1) to the distal edge (segment 4) of the lower lung lobe. The middle two segments were designated as segments 2 and 3. 2. Lung segments 1 and 4 exhibited significantly greater contraction than the other 2 segments to KCl when responses were expressed as mg force per mg tissue weight. Contractile responses to bronchospastic agents including histamine, carbachol, endothelin-1, leukotrienes (LT) B4 and D4, and the thromboxane A2-mimetic U46619 demonstrated no significant difference in EC50 values among the 4 lung segments. 3. Contractile responses of segments 1 and 4 to antigen-challenge (ovalbumin), ionophore A23187 and substance P were significantly greater than the other 2 segments with respect to either sensitivity or maximum responsiveness. 4. U46619-induced contractions of the 4 lung segments were relaxed in similar manner by papaverine and theophylline up to 100%, salbutamol up to 80%, and sodium nitroprusside by only 20%. In contrast, sodium nitroprusside markedly reversed U46619-induced contraction of pulmonary arterial rings and bronchial rings. 5. Histological studies identified 2-4 layers of smooth muscle cells underlying the lung pleural surface. Mast cells were prominent in this area. Moreover, morphometric studies showed that segment 4 possessed the least amount of smooth muscle structures from bronchial/bronchiolar wall and vasculatures as compared to the other 3 segments, and a significant difference in this respect was evident between segment 1 and segment 4.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 1 Figure 6 PMID:1378341

  13. Transforming Growth Factor β1 Function in Airway Remodeling and Hyperresponsiveness. The Missing Link?

    PubMed

    Ojiaku, Christie A; Yoo, Edwin J; Panettieri, Reynold A

    2017-04-01

    The pathogenesis of asthma includes a complex interplay among airway inflammation, hyperresponsiveness, and remodeling. Current evidence suggests that airway structural cells, including bronchial smooth muscle cells, myofibroblasts, fibroblasts, and epithelial cells, mediate all three aspects of asthma pathogenesis. Although studies show a connection between airway remodeling and changes in bronchomotor tone, the relationship between the two remains unclear. Transforming growth factor β1 (TGF-β1), a growth factor elevated in the airway of patients with asthma, plays a role in airway remodeling and in the shortening of various airway structural cells. However, the role of TGF-β1 in mediating airway hyperresponsiveness remains unclear. In this review, we summarize the literature addressing the role of TGF-β1 in airway remodeling and shortening. Through our review, we aim to further elucidate the role of TGF-β1 in asthma pathogenesis and the link between airway remodeling and airway hyperresponsiveness in asthma and to define TGF-β1 as a potential therapeutic target for reducing asthma morbidity and mortality.

  14. Heterotrimeric G Stimulatory Protein α Subunit Is Required for Intestinal Smooth Muscle Contraction in Mice.

    PubMed

    Qin, Xiaoteng; Liu, Shangming; Lu, Qiulun; Zhang, Meng; Jiang, Xiuxin; Hu, Sanyuan; Li, Jingxin; Zhang, Cheng; Gao, Jiangang; Zhu, Min-Sheng; Feil, Robert; Li, Huashun; Chen, Min; Weinstein, Lee S; Zhang, Yun; Zhang, Wencheng

    2017-04-01

    The α subunit of the heterotrimeric G stimulatory protein (Gsa), encoded by the guanine nucleotide binding protein, α-stimulating gene (Gnas, in mice), is expressed ubiquitously and mediates receptor-stimulated production of cyclic adenosine monophosphate and activation of the protein kinase A signaling pathway. We investigated the roles of Gsa in vivo in smooth muscle cells of mice. We performed studies of mice with Cre recombinase-mediated disruption of Gnas in smooth muscle cells (Gsa SMKO and SM22-CreER T2 , induced in adult mice by tamoxifen). Intestinal tissues were collected for histologic, biochemical, molecular, cell biology, and physiology analyses. Intestinal function was assessed in mice using the whole-gut transit time test. We compared gene expression patterns of intestinal smooth muscle from mice with vs without disruption of Gnas. Biopsy specimens from ileum of patients with chronic intestinal pseudo-obstruction and age-matched control biopsies were analyzed by immunohistochemistry. Disruption of Gnas in smooth muscle of mice reduced intestinal motility and led to death within 4 weeks. Tamoxifen-induced disruption of Gnas in adult mice impaired contraction of intestinal smooth muscle and peristalsis. More than 80% of these died within 3 months of tamoxifen exposure, with features of intestinal pseudo-obstruction characterized by chronic intestinal dilation and dysmotility. Gsa deficiency reduced intestinal levels of cyclic adenosine monophosphate and transcriptional activity of the cyclic adenosine monophosphate response element binding protein 1 (CREB1); this resulted in decreased expression of the forkhead box F1 gene (Foxf1) and protein, and contractile proteins, such as myosin heavy chain 11; actin, α2, smooth muscle, aorta; calponin 1; and myosin light chain kinase. We found decreased levels of Gsa, FOXF1, CREB1, and phosphorylated CREB1 proteins in intestinal muscle layers of patients with chronic intestinal pseudo

  15. EP2 receptors mediate airway relaxation to substance P, ATP, and PGE2.

    PubMed

    Fortner, C N; Breyer, R M; Paul, R J

    2001-08-01

    Substance P (SP) and ATP evoke transient, epithelium-dependent relaxation of constricted mouse tracheal smooth muscle. Relaxation to either SP or ATP is blocked by indomethacin, but the specific eicosanoid(s) involved have not been definitively identified. SP and ATP are reported to release PGE2 from airway epithelium in other species, suggesting PGE2 as a likely mediator in epithelium-dependent airway relaxation. Using mice homozygous for a gene-targeted deletion of the EP2 receptor [EP2(-/-)], one of the PGE2 receptors, we tested the hypothesis that PGE2 is the primary mediator of relaxation to SP or ATP. Relaxation in response to SP or ATP was significantly reduced in tracheas from EP2(-/-) mice. There were no differences between EP2(-/-) and wild-type tracheas in their physical dimensions, contraction to ACh, or relaxation to isoproterenol, thus ruling out any general alterations of smooth muscle function. There were also no differences between EP2(-/-) and wild-type tracheas in basal or stimulated PGE2 production. Exogenous PGE2 produced significantly less relaxation in EP2(-/-) tracheas compared with the wild type. Taken together, this experimental evidence supports the following two conclusions: EP2 receptors are of primary importance in airway relaxation to PGE2 and relaxation to SP or ATP is mediated through PGE2 acting on EP2 receptors.

  16. Effects of One Resistance Exercise Session on Vascular Smooth Muscle of Hypertensive Rats

    PubMed Central

    da Silva, Tharciano Luiz Teixeira Braga; Mota, Marcelo Mendonça; Fontes, Milene Tavares; Araújo, João Eliakim dos Santos; Carvalho, Vitor Oliveira; Bonjardim, Leonardo Rigoldi; Santos, Márcio Roberto Viana

    2015-01-01

    Background Hypertension is a public health problem and increases the incidence of cardiovascular diseases. Objective To evaluate the effects of a resistance exercise session on the contractile and relaxing mechanisms of vascular smooth muscle in mesenteric arteries of NG-nitro L-arginine methyl ester (L-NAME)-induced hypertensive rats. Methods Wistar rats were divided into three groups: control (C), hypertensive (H), and exercised hypertensive (EH). Hypertension was induced by administration of 20 mg/kg of L-NAME for 7 days prior to experimental protocols. The resistance exercise protocol consisted of 10 sets of 10 repetitions and intensity of 40% of one repetition maximum. The reactivity of vascular smooth muscle was evaluated by concentration‑response curves to phenylephrine (PHEN), potassium chloride (KCl) and sodium nitroprusside (SNP). Results Rats treated with L-NAME showed an increase (p < 0.001) in systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial pressure (MAP) compared to the initial period of induction. No difference in PHEN sensitivity was observed between groups H and EH. Acute resistance exercise reduced (p < 0.001) the contractile response induced by KCl at concentrations of 40 and 60 mM in group EH. Greater (p < 0.01) smooth muscle sensitivity to NPS was observed in group EH as compared to group H. Conclusion One resistance exercise session reduces the contractile response induced by KCl in addition to increasing the sensitivity of smooth muscle to NO in mesenteric arteries of hypertensive rats. PMID:26107814

  17. Effects of one resistance exercise session on vascular smooth muscle of hypertensive rats.

    PubMed

    Silva, Tharciano Luiz Teixeira Braga da; Mota, Marcelo Mendonça; Fontes, Milene Tavares; Araújo, João Eliakim Dos Santos; Oliveira Carvalho, Vitor; Bonjardim, Leonardo Rigoldi; Santos, Márcio Roberto Viana

    2015-08-01

    Hypertension is a public health problem and increases the incidence of cardiovascular diseases. To evaluate the effects of a resistance exercise session on the contractile and relaxing mechanisms of vascular smooth muscle in mesenteric arteries of NG-nitro L-arginine methyl ester (L-NAME)-induced hypertensive rats. Wistar rats were divided into three groups: control (C), hypertensive (H), and exercised hypertensive (EH). Hypertension was induced by administration of 20 mg/kg of L-NAME for 7 days prior to experimental protocols. The resistance exercise protocol consisted of 10 sets of 10 repetitions and intensity of 40% of one repetition maximum. The reactivity of vascular smooth muscle was evaluated by concentration‑response curves to phenylephrine (PHEN), potassium chloride (KCl) and sodium nitroprusside (SNP). Rats treated with L-NAME showed an increase (p < 0.001) in systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial pressure (MAP) compared to the initial period of induction. No difference in PHEN sensitivity was observed between groups H and EH. Acute resistance exercise reduced (p < 0.001) the contractile response induced by KCl at concentrations of 40 and 60 mM in group EH. Greater (p < 0.01) smooth muscle sensitivity to NPS was observed in group EH as compared to group H. One resistance exercise session reduces the contractile response induced by KCl in addition to increasing the sensitivity of smooth muscle to NO in mesenteric arteries of hypertensive rats.

  18. The expression and crucial roles of BMP signaling in development of smooth muscle progenitor cells in the mouse embryonic gut.

    PubMed

    Torihashi, Shigeko; Hattori, Takako; Hasegawa, Hirotaka; Kurahashi, Masaaki; Ogaeri, Takunori; Fujimoto, Toyoshi

    2009-03-01

    Bone morphogenetic protein (BMP) signaling is essential for normal development of the gastrointestinal (GI) tract. BMPs also play multiple roles in vascular smooth muscle cells; however, the BMP signaling in the development of the GI musculature remains to be clarified. We investigated the expression of BMPs and their receptors in mouse embryonic GI tracts by immunohistochemistry and in situ hybridization. We demonstrated that BMP2, BMP receptor Ib and BMP receptor II were expressed in the smooth muscle progenitors from E12 to E13 for the first time. BMP signaling on smooth muscle differentiation was examined by implantation of agarose beads soaked with BMPs in the in vitro developmental model that is gut-like structures from mouse embryonic stem (ES) cells. BMP2 rather than BMP4 beads enhanced smooth muscle differentiation, and increased gut-like structures showing spontaneous contractions and expressing intensive alpha-smooth muscle actin immunoreactivity. This increase was confirmed by up-regulation of SM22 mRNA shown by real-time PCR. By addition of noggin beads or noggin to the medium at BMP2 bead implantation, the ratio of contractive gut-like structures decreased. Implantation of BMP2 beads at EB7 (EB--embryoid bodies) (corresponding to E12 or E13 of mouse embryo) showed the highest effects and up-regulation of transcription factors msx-1 after 24h. This increase was blocked by noggin, and msx-1 decreased to almost the control level after 60 h. BMP2 beads at EB7 increased platelet-derived growth factor-A (PDGF-A) in the differentiating smooth muscle cells. We have recently reported that PDGF-A is expressed in the developing inner circular smooth muscle and is crucial for the longitudinal smooth muscle differentiation. Taken together, BMP signaling was expressed for a short window in the smooth muscle progenitors and the signal, especially BMP2, plays an essential role in smooth muscle differentiation in cooperation with PDGF signaling.

  19. A Contractile Network of Interstitial Cells of Cajal in the Supratarsal Mueller's Smooth Muscle Fibers With Sparse Sympathetic Innervation

    PubMed Central

    Yuzuriha, Shunsuke; Matsuo, Kiyoshi; Ban, Ryokuya; Yano, Shiharu; Moriizumi, Tetsuji

    2012-01-01

    Background: We previously reported that the supratarsal Mueller's muscle is innervated by both sympathetic efferent fibers and trigeminal proprioceptive afferent fibers, which function as mechanoreceptors-inducing reflexive contractions of both the levator and frontalis muscles. Controversy still persists regarding the role of the mechanoreceptors in Mueller's muscle; therefore, we clinically and histologically investigated Mueller's muscle. Methods: We evaluated the role of phenylephrine administration into the upper fornix in contraction of Mueller's smooth muscle fibers and how intraoperative stretching of Mueller's muscle alters the degree of eyelid retraction in 20 patients with aponeurotic blepharoptosis. In addition, we stained Mueller's muscle in 7 cadavers with antibodies against α-smooth muscle actin, S100, tyrosine hydroxylase, c-kit, and connexin 43. Results: Maximal eyelid retraction occurred approximately 3.8 minutes after administration of phenylephrine and prolonged eyelid retraction for at least 20 minutes after administration. Intraoperative stretching of Mueller's muscle increased eyelid retraction due to its reflexive contraction. The tyrosine hydroxylase antibody sparsely stained postganglionic sympathetic nerve fibers, whereas the S100 and c-kit antibodies densely stained the interstitial cells of Cajal (ICCs) among Mueller's smooth muscle fibers. A connexin 43 antibody failed to stain Mueller's muscle. Conclusions: A contractile network of ICCs may mediate neurotransmission within Mueller's multiunit smooth muscle fibers that are sparsely innervated by postganglionic sympathetic fibers. Interstitial cells of Cajal may also serve as mechanoreceptors that reflexively contract Mueller's smooth muscle fibers, forming intimate associations with intramuscular trigeminal proprioceptive fibers to induce reflexive contraction of the levator and frontalis muscles. PMID:22359687

  20. Ex vivo pharmacology of surgical samples of the uterosacral ligament. Part I: Effects of carbachol and oxytocin on smooth muscle.

    PubMed

    Drews, Ulrich; Renz, Matthias; Busch, Christian; Reisenauer, Christl

    2012-11-01

    In a previous study we observed impaired smooth muscle in the uterosacral ligament (USL) of patients with pelvic organ prolapse. The aims of the study were to describe the method of the novel microperfusion system and to determine normal function and pharmacology of smooth muscle in the USL. Samples from the USL were obtained during hysterectomy for benign reasons. Small stretches of connective tissue were mounted in a perfusion chamber under the stereomicroscope. Isotonic contractions of smooth muscle were monitored by digital time-lapse video and quantified by image processing. Constant perfusion with carbachol elicited tonic and pulse stimulation with carbachol and oxytocin rhythmic contractions of smooth muscle in the ground reticulum. Under constant perfusion with relaxin the tonic contraction after carbachol was abolished. With the novel microperfusion system, isotonic contractions of smooth muscle in the USL can be recorded and quantified in the tissue microenvironment on the microscopic level. The USL smooth muscle is cholinergic, stimulated by oxytocin and modulated by relaxin. Copyright © 2012 Wiley Periodicals, Inc.

  1. Theophylline Represses IL-8 Secretion from Airway Smooth Muscle Cells Independently of Phosphodiesterase Inhibition. Novel Role as a Protein Phosphatase 2A Activator.

    PubMed

    Patel, Brijeshkumar S; Rahman, Md Mostafizur; Rumzhum, Nowshin N; Oliver, Brian G; Verrills, Nicole M; Ammit, Alaina J

    2016-06-01

    Theophylline is an old drug experiencing a renaissance owing to its beneficial antiinflammatory effects in chronic respiratory diseases, such as asthma and chronic obstructive pulmonary disease. Multiple modes of antiinflammatory action have been reported, including inhibition of the enzymes that degrade cAMP-phosphodiesterase (PDE). Using primary cultures of airway smooth muscle (ASM) cells, we recently revealed that PDE4 inhibitors can potentiate the antiinflammatory action of β2-agonists by augmenting cAMP-dependent expression of the phosphatase that deactivates mitogen-activated protein kinase (MAPK)-MAPK phosphatase (MKP)-1. Therefore, the aim of this study was to address whether theophylline repressed cytokine production in a similar, PDE-dependent, MKP-1-mediated manner. Notably, theophylline did not potentiate cAMP release from ASM cells treated with the long-acting β2-agonist formoterol. Moreover, theophylline (0.1-10 μM) did not increase formoterol-induced MKP-1 messenger RNA expression nor protein up-regulation, consistent with the lack of cAMP generation. However, theophylline (at 10 μM) was antiinflammatory and repressed secretion of the neutrophil chemoattractant cytokine IL-8, which is produced in response to TNF-α. Because theophylline's effects were independent of PDE4 inhibition or antiinflammatory MKP-1, we then wished to elucidate the novel mechanisms responsible. We investigated the impact of theophylline on protein phosphatase (PP) 2A, a master controller of multiple inflammatory signaling pathways, and show that theophylline increases TNF-α-induced PP2A activity in ASM cells. Confirmatory results were obtained in A549 lung epithelial cells. PP2A activators have beneficial effects in ex vivo and in vivo models of respiratory disease. Thus, our study is the first to link theophylline with PP2A activation as a novel mechanism to control respiratory inflammation.

  2. Differentiation of Human Adipose Derived Stem Cells into Smooth Muscle Cells Is Modulated by CaMKIIγ

    PubMed Central

    Aji, Kaisaier; Maimaijiang, Munila; Aimaiti, Abudusaimi; Rexiati, Mulati; Azhati, Baihetiya; Tusong, Hamulati

    2016-01-01

    The multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is known to participate in maintenance and switches of smooth muscle cell (SMC) phenotypes. However, which isoform of CaMKII is involved in differentiation of adult mesenchymal stem cells into contractile SMCs remains unclear. In the present study, we detected γ isoform of CaMKII in differentiation of human adipose derived stem cells (hASCs) into SMCs that resulted from treatment with TGF-β1 and BMP4 in combination for 7 days. The results showed that CaMKIIγ increased gradually during differentiation of hASCs as determined by real-time PCR and western blot analysis. The siRNA-mediated knockdown of CaMKIIγ decreased the protein levels and transcriptional levels of smooth muscle contractile markers (a-SMA, SM22a, calponin, and SM-MHC), while CaMKIIγ overexpression increases the transcriptional and protein levels of smooth muscle contractile markers. These results suggested that γ isoform of CaMKII plays a significant role in smooth muscle differentiation of hASCs. PMID:27493668

  3. [Urothelium-dependent modulation of urinary bladder smooth muscle contractions by menthol].

    PubMed

    Paduraru, O M; Filippov, I B; Boldyriev, O I; Vladymyrova, I A; Naĭd'onov, V H; Shuba, Ia M

    2011-01-01

    TRPM8 cold receptor/channel is considered amongst the variety of receptors that support and modulate sensory function of urothelium, although the information regarding this is still quite contradictory. Here we have studied the effects of nonspecific TRPM8 activator menthol on the contractions of the smooth muscle strips of the rat bladder with intact and removed urothelium, and assessed the expression in them of TRPM8 mRNA using semi-quantitative RT-PCR. Menthol (100 microM) decreased the basal tone and the amplitude of spontaneous contractions only in the strips with intact urothelium. Irrespective of the presence of urothelium it similarly inhibited (by approximately 45 %) the contractions evoked by high-potassium depolarization. Contractions induced by muscarinic agonist carbachol (1 microM) were inhibited by menthol much stronger (by approximately 63%) if the urothelium was present than without it (by approximately 12%). Expression of TRPM8 mRNA in urothelium was not detected, whilst in detrusor smooth muscle it was found very low. We conclude that modulation of contractile responses by menthol is most likely explained by its blocking action on voltage-gated calcium channels ofdetrusor smooth muscle cells (SMC) and by menthol-stimulated release from urothelium of some factor(s) with relaxant effects on SMCs. Stimulation of the secretion of these factors from urothelial cells most likely involves menthol-induced, TRPM8-independent mobilization of calcium.

  4. Epigenetic regulation of vascular smooth muscle cell function in atherosclerosis.

    PubMed

    Findeisen, Hannes M; Kahles, Florian K; Bruemmer, Dennis

    2013-04-01

    Epigenetics involve heritable and acquired changes in gene transcription that occur independently of the DNA sequence. Epigenetic mechanisms constitute a hierarchic upper-level of transcriptional control through complex modifications of chromosomal components and nuclear structures. These modifications include, for example, DNA methylation or post-translational modifications of core histones; they are mediated by various chromatin-modifying enzymes; and ultimately they define the accessibility of a transcriptional complex to its target DNA. Integrating epigenetic mechanisms into the pathophysiologic concept of complex and multifactorial diseases such as atherosclerosis may significantly enhance our understanding of related mechanisms and provide promising therapeutic approaches. Although still in its infancy, intriguing scientific progress has begun to elucidate the role of epigenetic mechanisms in vascular biology, particularly in the control of smooth muscle cell phenotypes. In this review, we will summarize epigenetic pathways in smooth muscle cells, focusing on mechanisms involved in the regulation of vascular remodeling.

  5. Epigenetic regulation of vascular smooth muscle cell function in atherosclerosis.

    PubMed

    Findeisen, Hannes M; Kahles, Florian K; Bruemmer, Dennis

    2013-05-01

    Epigenetics involve heritable and acquired changes in gene transcription that occur independently of the DNA sequence. Epigenetic mechanisms constitute a hierarchic upper-level of transcriptional control through complex modifications of chromosomal components and nuclear structures. These modifications include, for example, DNA methylation or post-translational modifications of core histones; they are mediated by various chromatin-modifying enzymes; and ultimately they define the accessibility of a transcriptional complex to its target DNA. Integrating epigenetic mechanisms into the pathophysiologic concept of complex and multifactorial diseases such as atherosclerosis may significantly enhance our understanding of related mechanisms and provide promising therapeutic approaches. Although still in its infancy, intriguing scientific progress has begun to elucidate the role of epigenetic mechanisms in vascular biology, particularly in the control of smooth muscle cell phenotypes. In this review, we will summarize epigenetic pathways in smooth muscle cells, focusing on mechanisms involved in the regulation of vascular remodeling.

  6. Immortalization of cat iris sphincter smooth muscle cells by SV40 virus: growth, morphological, biochemical and pharmacological characteristics.

    PubMed

    Ocklind, A; Yousufzai, S Y; Ghosh, S; Coca-Prados, M; St Jernschantz, J; Abdel-Latif, A A

    1995-11-01

    The purpose of this study was to establish immortalized cell cultures of cat iris sphincter smooth muscle cells for a model investigating ocular receptors and their signal transduction pathways. Cultured cat iris sphincter muscle cells were immortalized by viral transformation with SV40 virus and the morphological and immunocytochemical properties of the normal and immortalized cells were investigated. The transformed cell clone, SV-CISM-2, was further characterized biochemically and pharmacologically. The normal muscle cells showed characteristics of smooth muscle cells, as judged by their growth and the presence of smooth muscle alpha-actin and desmin. After seven passages the normal cells ceased to proliferate. In contrast, the immortalized cells retained their proliferative ability for more than 220 population doublings over 55 passages. The transformation phenotype in these cells was confirmed by their expression of the large T-antigen, the incorporation of viral DNA into cellular DNA, growth in agarose and in low-serum medium, and complete loss of contact inhibition. The immortalized cells expressed smooth muscle alpha-actin, desmin and MLC protein. Biochemical and pharmacological studies on the SV-CISM cells revealed the presence of several functional receptors including muscarinic cholinergic, beta-adrenergic, peptidergic (substance P and endothelin). Platelet-activating factor, and prostaglandin (PG). Muscarinic stimulation of these cells resulted in: (a) a dose-dependent increase in the release of arachidonic acid (AA) and (PGs) and enhancement in the production of inositol trisphosphate (IP3); and (b) a substantial increase in MLC phosphorylation (118%), an indicator of smooth muscle contractility. The stimulatory effects of carbachol on these responses were completely blocked by atropine, a muscarinic receptor antagonist. This study constitutes the first successful immortalization of iris sphincter smooth muscle cells. The SV-CISM-2 cells can serve as

  7. Ca2+-recruitment in tachykinin-induced contractions of gut smooth muscle from African clawed frog, Xenopus laevis and rainbow trout, Oncorhynchus mykiss.

    PubMed

    Johansson, Agot; Holmgren, Susanne

    2003-04-01

    Changes in intracellular Ca(2+) concentration control many essential cellular functions like the contraction of smooth muscle cells. The aim of this study was to investigate if the tachykinin substance P (SP) engages external Ca(2+)-sources, internal Ca(2+)-sources, or both in the contraction of the gastrointestinal smooth muscle of rainbow trout (Oncorhynchus mykiss) and the African clawed frog (Xenopus laevis). Strip preparations made of either longitudinal smooth muscle of proximal intestine or circular smooth muscle of cardiac stomach were mounted in organ baths and the tension was recorded via force transducers. Ca(2+)-free Ringer's solution containing the Ca(2+) chelating agent EGTA (2mM) abolished all spontaneous contractions. Exposure to SP in Ca(2+)-free solution decreased the response. Preparations were also treated with the Ca(2+)-ATPase inhibitor thapsigargin (10 microM) during 30 min. Thapsigargin reduced the effect of SP on intestinal longitudinal smooth muscle in rainbow trout and on stomach circular smooth muscle in the African clawed frog and to a less extent in the intestinal longitudinal smooth muscle. The results show that external Ca(2+) is of great importance, but is not the only source of Ca(2+) recruitment in SP-activation of gastrointestinal smooth muscle in rainbow trout and the African clawed frog.

  8. Piezo1 in Smooth Muscle Cells Is Involved in Hypertension-Dependent Arterial Remodeling.

    PubMed

    Retailleau, Kevin; Duprat, Fabrice; Arhatte, Malika; Ranade, Sanjeev Sumant; Peyronnet, Rémi; Martins, Joana Raquel; Jodar, Martine; Moro, Céline; Offermanns, Stefan; Feng, Yuanyi; Demolombe, Sophie; Patel, Amanda; Honoré, Eric

    2015-11-10

    The mechanically activated non-selective cation channel Piezo1 is a determinant of vascular architecture during early development. Piezo1-deficient embryos die at midgestation with disorganized blood vessels. However, the role of stretch-activated ion channels (SACs) in arterial smooth muscle cells in the adult remains unknown. Here, we show that Piezo1 is highly expressed in myocytes of small-diameter arteries and that smooth-muscle-specific Piezo1 deletion fully impairs SAC activity. While Piezo1 is dispensable for the arterial myogenic tone, it is involved in the structural remodeling of small arteries. Increased Piezo1 opening has a trophic effect on resistance arteries, influencing both diameter and wall thickness in hypertension. Piezo1 mediates a rise in cytosolic calcium and stimulates activity of transglutaminases, cross-linking enzymes required for the remodeling of small arteries. In conclusion, we have established the connection between an early mechanosensitive process, involving Piezo1 in smooth muscle cells, and a clinically relevant arterial remodeling. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Hypotension Due to Kir6.1 Gain‐of‐Function in Vascular Smooth Muscle

    PubMed Central

    Li, Anlong; Knutsen, Russell H.; Zhang, Haixia; Osei‐Owusu, Patrick; Moreno‐Dominguez, Alex; Harter, Theresa M.; Uchida, Keita; Remedi, Maria S.; Dietrich, Hans H.; Bernal‐Mizrachi, Carlos; Blumer, Kendall J.; Mecham, Robert P.; Koster, Joseph C.; Nichols, Colin G.

    2013-01-01

    Background KATP channels, assembled from pore‐forming (Kir6.1 or Kir6.2) and regulatory (SUR1 or SUR2) subunits, link metabolism to excitability. Loss of Kir6.2 results in hypoglycemia and hyperinsulinemia, whereas loss of Kir6.1 causes Prinzmetal angina–like symptoms in mice. Conversely, overactivity of Kir6.2 induces neonatal diabetes in mice and humans, but consequences of Kir6.1 overactivity are unknown. Methods and Results We generated transgenic mice expressing wild‐type (WT), ATP‐insensitive Kir6.1 [Gly343Asp] (GD), and ATP‐insensitive Kir6.1 [Gly343Asp,Gln53Arg] (GD‐QR) subunits, under Cre‐recombinase control. Expression was induced in smooth muscle cells by crossing with smooth muscle myosin heavy chain promoter–driven tamoxifen‐inducible Cre‐recombinase (SMMHC‐Cre‐ER) mice. Three weeks after tamoxifen induction, we assessed blood pressure in anesthetized and conscious animals, as well as contractility of mesenteric artery smooth muscle and KATP currents in isolated mesenteric artery myocytes. Both systolic and diastolic blood pressures were significantly reduced in GD and GD‐QR mice but normal in mice expressing the WT transgene and elevated in Kir6.1 knockout mice as well as in mice expressing dominant‐negative Kir6.1 [AAA] in smooth muscle. Contractile response of isolated GD‐QR mesenteric arteries was blunted relative to WT controls, but nitroprusside relaxation was unaffected. Basal KATP conductance and pinacidil‐activated conductance were elevated in GD but not in WT myocytes. Conclusions KATP overactivity in vascular muscle can lead directly to reduced vascular contractility and lower blood pressure. We predict that gain of vascular KATP function in humans would lead to a chronic vasodilatory phenotype, as indeed has recently been demonstrated in Cantu syndrome. PMID:23974906

  10. Serelaxin Elicits Bronchodilation and Enhances β-Adrenoceptor-Mediated Airway Relaxation

    PubMed Central

    Lam, Maggie; Royce, Simon G.; Donovan, Chantal; Jelinic, Maria; Parry, Laura J.; Samuel, Chrishan S.; Bourke, Jane E.

    2016-01-01

    Treatment with β-adrenoceptor agonists does not fully overcome the symptoms associated with severe asthma. Serelaxin elicits potent uterine and vascular relaxation via its cognate receptor, RXFP1, and nitric oxide (NO) signaling, and is being clinically evaluated for the treatment of acute heart failure. However, its direct bronchodilator efficacy has yet to be explored. Tracheal rings were prepared from male Sprague-Dawley rats (250–350 g) and tricolor guinea pigs, and precision cut lung slices (PCLSs) containing intrapulmonary airways were prepared from rats only. Recombinant human serelaxin (rhRLX) alone and in combination with rosiglitazone (PPARγ agonist; recently described as a novel dilator) or β-adrenoceptor agonists (isoprenaline, salbutamol) were added either to pre-contracted airways, or before contraction with methacholine or endothelin-1. Regulation of rhRLX responses by epithelial removal, indomethacin (cyclooxygenase inhibitor), L-NAME (nitric oxide synthase inhibitor), SQ22536 (adenylate cyclase inhibitor) and ODQ (guanylate cyclase inhibitor) were also evaluated. Immunohistochemistry was used to localize RXFP1 to airway epithelium and smooth muscle. rhRLX elicited relaxation in rat trachea and PCLS, more slowly than rosiglitazone or isoprenaline, but potentiated relaxation to both these dilators. It markedly increased β-adrenoceptor agonist potency in guinea pig trachea. rhRLX, rosiglitazone, and isoprenaline pretreatment also inhibited the development of rat tracheal contraction. Bronchoprotection by rhRLX increased with longer pre-incubation time, and was partially reduced by epithelial removal, indomethacin and/or L-NAME. SQ22536 and ODQ also partially inhibited rhRLX-mediated relaxation in both intact and epithelial-denuded trachea. RXFP1 expression in the airways was at higher levels in epithelium than smooth muscle. In summary, rhRLX elicits large and small airway relaxation via epithelial-dependent and -independent mechanisms, likely

  11. IL-17A induces eotaxin-1/CC chemokine ligand 11 expression in human airway smooth muscle cells: role of MAPK (Erk1/2, JNK, and p38) pathways.

    PubMed

    Rahman, Muhammad Shahidur; Yamasaki, Akira; Yang, Jie; Shan, Lianyu; Halayko, Andrew J; Gounni, Abdelilah Soussi

    2006-09-15

    Recently, IL-17A has been shown to be expressed in higher levels in respiratory secretions from asthmatics and correlated with airway hyperresponsiveness. Although these studies raise the possibility that IL-17A may influence allergic disease, the mechanisms remain unknown. In this study, we investigated the molecular mechanisms involved in IL-17A-mediated CC chemokine (eotaxin-1/CCL11) production from human airway smooth muscle (ASM) cells. We found that incubation of human ASM cells with rIL-17A resulted in a significant increase of eotaxin-1/CCL11 release from ASM cells that was reduced by neutralizing anti-IL-17A mAb. Moreover, IL-17A significantly induced eotaxin-1/CCL11 release and mRNA expression, an effect that was abrogated with cycloheximide and actinomycin D treatment. Furthermore, transfection studies using a luciferase-driven reporter construct containing eotaxin-1/CCL11 proximal promoter showed that IL-17A induced eotaxin-1/CCL11 at the transcriptional level. IL-17A also enhanced significantly IL-1beta-mediated eotaxin-1/CCL11 mRNA, protein release, and promoter activity in ASM cells. Primary human ASM cells pretreated with inhibitors of MAPK p38, p42/p44 ERK, JNK, or JAK but not PI3K, showed a significant decrease in eotaxin-1/CCL11 release upon IL-17A treatment. In addition, IL-17A mediated rapid phosphorylation of MAPK (p38, JNK, and p42/44 ERK) and STAT-3 but not STAT-6 or STAT-5 in ASM cells. Taken together, our data provide the first evidence of IL-17A-induced eotaxin-1/CCL11 expression in ASM cells via MAPK (p38, p42/p44 ERK, JNK) signaling pathways. Our results raise the possibility that IL-17A may play a role in allergic asthma by inducing eotaxin-1/CCL11 production.

  12. K/sup +/-induced alterations in airway muscle responsiveness to electrical field stimulation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Murlas, C.; Ehring, G.; Suszkiw, J.

    1986-07-01

    The authors investigated possible pre- and postsynaptic effects of K/sup +/-induced depolarization on ferret tracheal smooth muscle (TSM) responsiveness to cholinergic stimulation. To assess electromechanical activity, cell membrane potential (E/sub m/) and tension (T/sub m/) were simultaneously recorded in buffer containing 6, 12, 18, or 24 mM K/sup +/ before and after electrical field stimulation (EFS) or exogenous acetylcholine (ACh). In 6 mM K/sup +/ E/sub m/ was -58.1 +/- 1.0 m V (mean +/- SE). In 12 mM K/sup +/, E/sub m/ was depolarized to -52.3 +/- 0.9 mV, basal T/sub m/ did not change, and both excitatory junctionalmore » potentials and contractile responses to EFS at short stimulus duration were larger than in 6 mM K/sup +/. No such potentiation occurred at a higher K/sup +/, although resting E/sub m/ and T/sub m/ increased progressively above 12 mM K/sup +/. The sensitivity of ferret TSM to exogenous ACh appeared unaffected by K/sup +/. To determine whether the hyperresponsiveness in 12 mM K/sup +/ was due, in part, to augmented ACh release from intramural airway nerves, experiments were done using TSM preparations incubated with (/sup 3/H)choline to measure (/sup 3/H)ACh release at rest and during EFS. Although resting (/sup 3/H)ACh release increased progressively in higher K/sup +/, release evoked by EFS was maximal in 12 mM K/sup +/ and declined in higher concentrations. They conclude that small elevations in the extracellular K/sup +/ concentration augment responsiveness of the airways, by increasing the release of ACh both at rest and during EFS from intramural cholinergic nerve terminals. Larger increases in K/sup +/ appear to be inhibitory, possibly due to voltage-dependent effects that occur both pre- and postsynaptically.« less

  13. Administration of SIN-1 induces guinea pig airway hyperresponsiveness through inactivation of airway neutral endopeptidase.

    PubMed

    Kanazawa, H; Hirata, K; Yoshikawa, J

    1999-12-01

    Peroxynitrite plays an important role in the pathogenesis of airway inflammation. We have already found that peroxynitrite may contribute to decreased beta(2)-adrenoceptor responses in airway smooth muscle. However, it is not known whether peroxynitrite can alter neutral endopeptidase (EC 3.4.24.11; NEP) activity in the airways. This study was designed to determine whether peroxynitrite induces airway hyperresponsiveness to substance P (SP) and endothelin-1 (ET-1) through the inactivation of airway NEP. We examined whether the administration of S-morpholinosydnonimine (SIN-1), a compound that releases peroxynitrite, increased bronchoconstrictor responses to SP and ET-1 in anesthetized guinea pigs. In addition, we assayed NEP activity in the airways of SIN-1-exposed guinea pigs. Though SIN-1 (10(-7) M) alone had no effect on pulmonary resistance, pretreatment with SIN-1 significantly enhanced SP- and ET-1-induced bronchoconstriction. Pretreatment with phosphoramidon, an NEP inhibitor, also enhanced SP- and ET-1-induced bronchoconstriction. However, simultaneous administration of phosphoramidon and SIN-1 had no additive effect on SP- and ET-1-induced bronchoconstriction. Peroxynitrite formation by SIN-1 was completely inhibited by N-acetylcysteine (NAC) and glutathione (GSH) in vitro, and pretreatment with NAC and GSH significantly reversed the potentiation by SIN-1 of SP-induced bronchoconstriction. In addition, the NEP activity of the trachea after SIN-1 exposure was significantly reduced compared to the level in control guinea pigs (solvent for SIN-1: 30.0+/-4.2 fmol.min(-1).mg tissue(-1); 10(-7) M SIN-1; 15.5+/-4.5 fmol.min(-1).mg tissue(-1), p<0.05). These findings suggest that peroxynitrite induces airway hyperresponsiveness to SP and ET-1 through the inactivation of airway NEP, and that peroxynitrite is an important mediator of the alterations in airway functions.

  14. LRP1 in brain vascular smooth muscle cells mediates local clearance of Alzheimer's amyloid-β.

    PubMed

    Kanekiyo, Takahisa; Liu, Chia-Chen; Shinohara, Mitsuru; Li, Jie; Bu, Guojun

    2012-11-14

    Impaired clearance of amyloid-β (Aβ) is a major pathogenic event for Alzheimer's disease (AD). Aβ depositions in brain parenchyma as senile plaques and along cerebrovasculature as cerebral amyloid angiopathy (CAA) are hallmarks of AD. A major pathway that mediates brain Aβ clearance is the cerebrovascular system where Aβ is eliminated through the blood-brain barrier (BBB) and/or degraded by cerebrovascular cells along the interstitial fluid drainage pathway. An Aβ clearance receptor, the low-density lipoprotein receptor-related protein 1 (LRP1), is abundantly expressed in cerebrovasculature, in particular in vascular smooth muscle cells. Previous studies have indicated a role of LRP1 in endothelial cells in transcytosing Aβ out of the brain across the BBB; however, whether this represents a significant pathway for brain Aβ clearance remains controversial. Here, we demonstrate that Aβ can be cleared locally in the cerebrovasculature by an LRP1-dependent endocytic pathway in smooth muscle cells. The uptake and degradation of both endogenous and exogenous Aβ were significantly reduced in LRP1-suppressed human brain vascular smooth muscle cells. Conditional deletion of Lrp1 in vascular smooth muscle cell in amyloid model APP/PS1 mice accelerated brain Aβ accumulation and exacerbated Aβ deposition as amyloid plaques and CAA without affecting Aβ production. Our results demonstrate that LRP1 is a major Aβ clearance receptor in cerebral vascular smooth muscle cell and a disturbance of this pathway contributes to Aβ accumulation. These studies establish critical functions of the cerebrovasculature system in Aβ metabolism and identify a new pathway involved in the pathogenesis of both AD and CAA.

  15. LRP1 in Brain Vascular Smooth Muscle Cells Mediates Local Clearance of Alzheimer's Amyloid-β

    PubMed Central

    Kanekiyo, Takahisa; Liu, Chia-Chen; Shinohara, Mitsuru; Li, Jie; Bu, Guojun

    2012-01-01

    Impaired clearance of amyloid-β (Aβ) is a major pathogenic event for Alzheimer’s disease (AD). Aβ depositions in brain parenchyma as senile plaques and along cerebrovasculature as cerebral amyloid angiopathy (CAA) are hallmarks of AD. A major pathway that mediates brain Aβ clearance is the cerebrovascular system where Aβ is eliminated through the blood-brain barrier (BBB) and/or degraded by cerebrovascular cells along the interstitial fluid drainage pathway. An Aβ clearance receptor, the low-density lipoprotein receptor-related protein 1 (LRP1), is abundantly expressed in cerebrovasculature, in particular in vascular smooth muscle cells. Previous studies have indicated a role of LRP1 in endothelial cells in transcytosing Aβ out of the brain across the BBB; however, whether this represents a significant pathway for brain Aβ clearance remains controversial. Here, we demonstrate that Aβ can be cleared locally in the cerebrovasculature by an LRP1-dependent endocytic pathway in smooth muscle cells. The uptake and degradation of both endogenous and exogenous Aβ were significantly reduced in LRP1-suppressed human brain vascular smooth muscle cells. Conditional deletion of Lrp1 in vascular smooth muscle cell in amyloid model APP/PS1 mice accelerated brain Aβ accumulation and exacerbated Aβ deposition as amyloid plaques and CAA without affecting Aβ production. Our results demonstrate that LRP1 is a major Aβ clearance receptor in cerebral vascular smooth muscle cell and a disturbance of this pathway contributes to Aβ accumulation. These studies establish critical functions of the cerebrovasculature system in Aβ metabolism and identify a new pathway involved in the pathogenesis of both AD and CAA. PMID:23152628

  16. Distinct function of estrogen receptor α in smooth muscle and fibroblast cells in prostate development.

    PubMed

    Vitkus, Spencer; Yeh, Chiuan-Ren; Lin, Hsiu-Hsia; Hsu, Iawen; Yu, Jiangzhou; Chen, Ming; Yeh, Shuyuan

    2013-01-01

    Estrogen signaling, through estrogen receptor (ER)α, has been shown to cause hypertrophy in the prostate. Our recent report has shown that epithelial ERα knockout (KO) will not affect the normal prostate development or homeostasis. However, it remains unclear whether ERα in different types of stromal cells has distinct roles in prostate development. This study proposed to elucidate how KO of ERα in the stromal smooth muscle or fibroblast cells may interrupt cross talk between prostate stromal and epithelial cells. Smooth muscle ERαKO (smERαKO) mice showed decreased glandular infolding with the proximal area exhibiting a significant decrease. Fibroblast ERαKO mouse prostates did not exhibit this phenotype but showed a decrease in the number of ductal tips. Additionally, the amount of collagen observed in the basement membrane was reduced in smERαKO prostates. Interestingly, these phenotypes were found to be mutually exclusive among smERαKO or fibroblast ERαKO mice. Compound KO of ERα in both fibroblast and smooth muscle showed combined phenotypes from each of the single KO. Further mechanistic studies showed that IGF-I and epidermal growth factor were down-regulated in prostate smooth muscle PS-1 cells lacking ERα. Together, our results indicate the distinct functions of fibroblast vs. smERα in prostate development.

  17. Regional Differences in Rat Vaginal Smooth Muscle Contractility and Morphology

    PubMed Central

    Skoczylas, Laura C.; Jallah, Zegbeh; Sugino, Yoshio; Stein, Suzan E.; Feola, Andrew; Yoshimura, Naoki

    2013-01-01

    The objective of this study was to define the regional differences in rat vaginal smooth muscle contractility and morphology. We evaluated circumferential segments from the proximal, middle, and distal rat vagina (n = 21) in vitro. Contractile responses to carbachol, phenylephrine, potassium chloride, and electrical field stimulation (EFS) were measured. Immunohistochemical analyses were also performed. The dose–response curves for carbachol- and phenylephrine-dependent contractions were different in the distal (P = .05, P = .04) compared to the proximal/middle regions. Adjusted for region-dependent changes in contractility, the distal vagina generated lower force in response to carbachol and higher force in response to phenylephrine. There was less force with increasing EFS frequency in the distal (P = .03), compared to the proximal/middle regions. Cholinergic versus adrenergic nerves were more frequent in the proximal region (P = .03). In summary, the results indicate that functional and morphological differences in smooth muscle and nerve fibers of the distal versus proximal/middle regions of the vagina exist. PMID:23298869

  18. [Wood smoke condensate had weak proliferation and strong necrotic effects on human airway smooth muscle cells].

    PubMed

    Pan, Yi-ling; Li, Bing; Ran, Pi-xin

    2013-08-01

    To investigate the effect of wood smoke condensate (WSC) on proliferation and necrosis of human airway smooth muscle cells (HASMCs). Primary cultured HASMCs between passage 2 and 8 were divided into 3 groups: a control group, a WSC group and a cigarette smoke condensate (CSC) group. The viability of cells was examined by the CCK8 assays. The ratio of cellular proliferative stage (S phase) and cell cycle index were examined by fluorescent-labeled thymidine analogue uptake assays and flow cytometry. The expression of cyclin D1 was detected by quantitative reverse transcriptase polymerase chain reaction (Q-PCR) and Western blot. Cell apoptosis and necrosis were observed by the annexin-V and PI staining. Statistical analysis was performed by using the One-way ANOVA and LSD-t test. Cell viability reached peak at WSC 1 mg/L[(126 ± 12)%] and at CSC 10 mg/L exposure level [(142 ± 11) %] respectively. While at WSC 10 mg/L and CSC 60 mg/L exposure levels, cell viability decreased significantly to 86% and 76%, respectively, as compared with that of the blank control group[(100 ± 0)%] (q = 3.63- 9.33, P < 0.05). In the WSC 1 mg/L group, the cell proliferation ratio and the expression of cyclin D1 protein were (124 ± 20)% and 1.31 ± 0.64, respectively, the differences being significant as compared with the blank control group [(100 ± 0)%, 1.0 ± 0.0] (q = 5.85, 5.91, P < 0.05), while the expression of cyclin D1 mRNA and the percentage of S+G2M phase were 1.18 ± 0.21 and (103 ± 4)%, respectively, not significantly different as compared to the control group [(100 ± 0)%, 1.0 ± 0.0], (q = 1.16, 2.05, P > 0.05). In the CSC 10 mg/L group, the above-mentioned values were (204 ± 45)%, 1.80 ± 0.25, (140 ± 6)%, 1.48 ± 0.2, respectively, which were significantly higher than those in the blank control group (q = 5.38-16.51, P < 0.05) and in the WSC group (q = 3.33-15.35, P < 0.05). However, when HASMCs were exposed to WSC 10 mg/L, the cell death ratio was (13.39 ± 0

  19. Corticosteroids inhibit sphingosine 1-phosphate-induced interleukin-6 secretion from human airway smooth muscle via mitogen-activated protein kinase phosphatase 1-mediated repression of mitogen and stress-activated protein kinase 1.

    PubMed

    Che, Wenchi; Parmentier, Johannes; Seidel, Petra; Manetsch, Melanie; Ramsay, Emma E; Alkhouri, Hatem; Ge, Qi; Armour, Carol L; Ammit, Alaina J

    2014-02-01

    Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that plays an important proinflammatory role in asthmatic airways. Corticosteroids are first-line antiinflammatories in asthma; however, their repressive effects on S1P-induced cytokine secretion have not been investigated. To address this, our in vitro study reveals the molecular mechanisms by which corticosteroids inhibit S1P-induced IL-6 expression in the pivotal immunomodulatory cell type, airway smooth muscle (ASM). We first uncover the cellular signaling pathways responsible: S1P activates a cyclic adenosine monophosphate/cAMP response-element-binding protein (CREB)/CRE-dependent pathway to induce IL-6 transcription, concomitant with stimulation of the mitogen-activated protein kinase (MAPK) superfamily and downstream mitogen and stress-activated protein kinase 1 (MSK1) and histone H3 phosphorylation. In this way, S1P stimulates parallel signaling pathways to induce IL-6 secretion via CRE-driven transcription of the IL-6 gene promoter in a relaxed chromatin environment achieved through histone H3 phosphorylation. Second, we investigated how corticosteroids mediate their repressive effects. The corticosteroid dexamethasone inhibits S1P-induced IL-6 protein secretion and mRNA expression, but CREB/CRE transrepression, inhibition of IL-6 mRNA stability, or subcellular relocation of MSK1 were not responsible for the repressive effects of dexamethasone. Rather, we show that dexamethasone rapidly induces up-regulation of the MAPK deactivator MAPK phosphatase 1 (MKP-1) and that MKP-1 blocks the MAPK-driven activation of MSK1 and phosphorylation of histone H3. This was confirmed by treatment with triptolide, an inhibitor of MKP-1 up-regulation, where repressive effects of corticosteroids were reversed. Our study reveals the molecular mechanism underlying the antiinflammatory capacity of corticosteroids to repress proinflammatory functions induced by the potent bioactive sphingolipid S1P in the lung.

  20. Continuum vs. spring network models of airway-parenchymal interdependence

    PubMed Central

    Ma, Baoshun

    2012-01-01

    The outward tethering forces exerted by the lung parenchyma on the airways embedded within it are potent modulators of the ability of the airway smooth muscle to shorten. Much of our understanding of these tethering forces is based on treating the parenchyma as an elastic continuum; yet, on a small enough scale, the lung parenchyma in two dimensions would seem to be more appropriately described as a discrete spring network. We therefore compared how the forces and displacements in the parenchyma surrounding a contracting airway are predicted to differ depending on whether the parenchyma is modeled as an elastic continuum or as a spring network. When the springs were arranged hexagonally to represent alveolar walls, the predicted parenchymal stresses and displacements propagated substantially farther away from the airway than when the springs were arranged in a triangular pattern or when the parenchyma was modeled as a continuum. Thus, to the extent that the parenchyma in vivo behaves as a hexagonal spring network, our results suggest that the range of interdependence forces due to airway contraction may have a greater influence than was previously thought. PMID:22500006

  1. Trinitrobenzenesulfonic Acid Colitis Induces Changes in the Contractile Response of Circular Smooth Muscle in the Distal Colon

    DTIC Science & Technology

    1996-03-27

    contractile response of circular smooth muscle in the rat distal colon" Name of Candidate: Jeanette M. Hosseini Doctor of Philosophy Degree 27 March 1996... muscle in the rat distal colon" beyond brief excerpts is with the pennission of the copyright owner, and will save and hold harmless the Unifonned...induces changes in the contractile response of circular smooth muscle 10 the rat colon. Jeanette Marie Hosseini, 1996 Dissertation directed by: Terez

  2. Mediation of acetylcholine and substance P induced contractions by myosin light chain phosphorylation in feline colonic smooth muscle.

    PubMed

    Washabau, Robert J; Holt, David E; Brockman, Daniel J

    2002-05-01

    To determine the role of myosin light chain phosphorylation in feline colonic smooth muscle contraction. Colonic tissue was obtained from eight 12- to 24-month-old cats. Colonic longitudinal smooth muscle strips were attached to isometric force transducers for measurements of isometric stress. Myosin light chain phosphorylation was determined by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Stress and phosphorylation were determined following stimulation with ACh or SP, in the absence or presence of a calmodulin antagonist (W-7; 0.1 to 1.0 mM), myosin light chain kinase inhibitor (ML-9; 1 to 10 microM), or extracellular calcium free solutions. Unstimulated longitudinal colonic smooth muscle contained low amounts (6.9+/-3.2%) of phosphorylated myosin light chain. Phosphorylation of the myosin light chains was dose and time dependent with maximal values of 58.5% at 30 seconds of stimulation with 100 microM Ach and 60.2% at 45 seconds of stimulation with 100 nM SP Active isometric stress development closely paralleled phosphorylation of the myosin light chains in ACh- or SP-stimulated muscle. W-7 and ML-9 dose dependently inhibited myosin light chain phosphorylation and isometric stress development associated with ACh or SP stimulation. Removal of extracellular calcium inhibited myosin light chain phosphorylation and isometric stress development in ACh-stimulated smooth muscle. Feline longitudinal colonic smooth muscle contraction is calcium-, calmodulin-, and myosin light chain kinase-dependent. Myosin light chain phosphorylation is necessary for the initiation of contraction in feline longitudinal colonic smooth muscle. These findings may prove useful in determining the biochemical and molecular defects that accompany feline colonic motility disorders.

  3. Effects of fenoterol on beta-adrenoceptor and muscarinic M2 receptor function in bovine tracheal smooth muscle.

    PubMed

    De Vries, B; Roffel, A F; Kooistra, J M; Meurs, H; Zaagsma, J

    2001-05-11

    Prolonged (18 h) incubation of isolated bovine tracheal smooth muscle with the beta2-adrenoceptor agonist fenoterol (10 microM) induced desensitization of isoprenaline-induced adenylyl cyclase activity in bovine tracheal smooth muscle membranes, characterized by a 25% decrease in maximal effect (Emax) (P < 0.05), while the sensitivity to the agonist (pEC50) was unchanged. The Emax value of isoprenaline-induced smooth muscle relaxation of submaximal methacholine-induced contractile tones was similarly reduced by about 25% (P < 0.001), while the pEC50 value was diminished by 1.0 log unit (P < 0.001). As determined by 30 microM gallamine-induced muscarinic M2 receptor antagonism and pertussis toxin-induced inactivation of G(i alpha), muscarinic M2 receptor-mediated functional antagonism did not play a role in isoprenaline-induced relaxation of bovine tracheal smooth muscle contracted by methacholine, both in control and in 18-h fenoterol-treated tissue. In line with these observations, we found no enhanced muscarinic M2 receptor-mediated inhibition of 1 microM forskolin-stimulated adenylyl cyclase activity after 18-h fenoterol treatment. These data indicate that 18-h fenoterol treatment of bovine tracheal smooth muscle induces beta2-adrenoceptor desensitization and reduced functional antagonism of methacholine-induced contraction by beta-adrenoceptor agonists, without a change of muscarinic M2 receptor function.

  4. Discoidin Domain Receptor-1 Deficiency Attenuates Atherosclerotic Calcification and Smooth Muscle Cell-Mediated Mineralization

    PubMed Central

    Ahmad, Pamela J.; Trcka, Daniel; Xue, Siming; Franco, Christopher; Speer, Mei Y.; Giachelli, Cecilia M.; Bendeck, Michelle P.

    2009-01-01

    Intimal calcification is a feature of advanced atherosclerotic disease that predicts a two- to eightfold increase in the risk of coronary events. Type I collagen promotes vascular smooth muscle cell-mediated calcification, although the mechanism by which this occurs is unknown. The discoidin domain receptor 1 (DDR1) is a collagen receptor that is emerging as a critical mediator of atherosclerosis. To determine whether DDR1 is involved in intimal calcification, we fed male Ddr1−/−;Ldlr−/− and Ddr1+/+;Ldlr−/− mice an atherogenic diet for 6, 12, or 24 weeks. DDR1 deficiency significantly reduced the calcium content of the aortic arch, and microcomputed tomography demonstrated a significant decrease in hydroxyapatite deposition after 24 weeks of atherogenic diet. Reduced calcification was correlated with decreases in macrophage accumulation and tumor necrosis factor α staining, suggesting that the reduction in calcification was in part due to decreased inflammation. The chondrogenic markers type II collagen, type X collagen, and Sox-9 were expressed within the mineralized foci. An in vitro assay performed with vascular smooth muscle cells revealed that DDR1 was required for cell-mediated calcification of the matrix, and Ddr1+/+ smooth muscle cells expressed more alkaline phosphatase activity, whereas Ddr1−/− smooth muscle cells expressed elevated levels of mRNA for nucleotide pyrophosphatase phosphodiesterase 1, an inhibitor of tissue mineralization. Taken together, our results demonstrate that DDR1 mediates an important mechanism for atherosclerotic calcification. PMID:19893047

  5. Discoidin domain receptor-1 deficiency attenuates atherosclerotic calcification and smooth muscle cell-mediated mineralization.

    PubMed

    Ahmad, Pamela J; Trcka, Daniel; Xue, Siming; Franco, Christopher; Speer, Mei Y; Giachelli, Cecilia M; Bendeck, Michelle P

    2009-12-01

    Intimal calcification is a feature of advanced atherosclerotic disease that predicts a two- to eightfold increase in the risk of coronary events. Type I collagen promotes vascular smooth muscle cell-mediated calcification, although the mechanism by which this occurs is unknown. The discoidin domain receptor 1 (DDR1) is a collagen receptor that is emerging as a critical mediator of atherosclerosis. To determine whether DDR1 is involved in intimal calcification, we fed male Ddr1(-/-);Ldlr(-/-) and Ddr1(+/+);Ldlr(-/-) mice an atherogenic diet for 6, 12, or 24 weeks. DDR1 deficiency significantly reduced the calcium content of the aortic arch, and microcomputed tomography demonstrated a significant decrease in hydroxyapatite deposition after 24 weeks of atherogenic diet. Reduced calcification was correlated with decreases in macrophage accumulation and tumor necrosis factor alpha staining, suggesting that the reduction in calcification was in part due to decreased inflammation. The chondrogenic markers type II collagen, type X collagen, and Sox-9 were expressed within the mineralized foci. An in vitro assay performed with vascular smooth muscle cells revealed that DDR1 was required for cell-mediated calcification of the matrix, and Ddr1(+/+) smooth muscle cells expressed more alkaline phosphatase activity, whereas Ddr1(-/-) smooth muscle cells expressed elevated levels of mRNA for nucleotide pyrophosphatase phosphodiesterase 1, an inhibitor of tissue mineralization. Taken together, our results demonstrate that DDR1 mediates an important mechanism for atherosclerotic calcification.

  6. Enkephalinase inhibitor potentiates substance P- and capsaicin-induced bronchial smooth muscle contractions in humans.

    PubMed

    Honda, I; Kohrogi, H; Yamaguchi, T; Ando, M; Araki, S

    1991-06-01

    To determine the roles of endogenously released tachykinins (substance P, neurokinins A and B) in human bronchial tissues, and to determine the roles of enkephalinase (neutral endopeptidase, E.C. 3.4.24.11) in regulating the effects of the tachykinins, we studied the effects of substance P and capsaicin, which releases tachykinins, on human bronchial smooth muscle contraction in the presence or absence of enkephalinase inhibitor phosphoramidon in vitro. Substance P alone caused human bronchial smooth muscle contraction at 10(-6) M or more. Phosphoramidon (10(-7) to 10(-5) M) potentiated the substance P-induced contraction in a dose-dependent fashion, and phosphoramidon shifted the dose-response curve to lower concentrations. Capsaicin (10(-5) or 10(-4) M) alone caused bronchial smooth muscle contraction in four tissues from nine patients. After the contraction by capsaicin reached a plateau, phosphoramidon (10(-5) M) increased and prolonged the contraction significantly. Furthermore, pretreatment of bronchial tissues with phosphoramidon (10(-5) M) potentiated capsaicin-induced contraction in all tissues from five patients. Phosphoramidon (10(-5) M) shifted the dose-response curve to capsaicin to lower concentrations more than 1 log unit. Captopril did not alter the contractile response to substance P, suggesting that angiotensin-converting enzyme does not regulate the contractile response to substance P in human bronchial smooth muscle in vitro. These results suggest that enkephalinase regulates the contractile effects of exogenous substance P and endogenous substances, probably tachykinins, released by capsaicin in the human bronchus.

  7. Myosin Va Plays a Role in Nitrergic Smooth Muscle Relaxation in Gastric Fundus and Corpora Cavernosa of Penis

    PubMed Central

    Carew, Josephine A.; Goyal, Raj K.; Sullivan, Maryrose P.

    2014-01-01

    The intracellular motor protein myosin Va is involved in nitrergic neurotransmission possibly by trafficking of neuronal nitric oxide synthase (nNOS) within the nerve terminals. In this study, we examined the role of myosin Va in the stomach and penis, proto-typical smooth muscle organs in which nitric oxide (NO) mediated relaxation is critical for function. We used confocal microscopy and co-immunoprecipitation of tissue from the gastric fundus (GF) and penile corpus cavernosum (CCP) to localize myosin Va with nNOS and demonstrate their molecular interaction. We utilized in vitro mechanical studies to test whether smooth muscle relaxations during nitrergic neuromuscular neurotransmission is altered in DBA (dilute, brown, non-agouti) mice which lack functional myosin Va. Myosin Va was localized in nNOS-positive nerve terminals and was co-immunoprecipitated with nNOS in both GF and CCP. In comparison to C57BL/6J wild type (WT) mice, electrical field stimulation (EFS) of precontracted smooth muscles of GF and CCP from DBA animals showed significant impairment of nitrergic relaxation. An NO donor, Sodium nitroprusside (SNP), caused comparable levels of relaxation in smooth muscles of WT and DBA mice. These normal postjunctional responses to SNP in DBA tissues suggest that impairment of smooth muscle relaxation resulted from inhibition of NO synthesis in prejunctional nerve terminals. Our results suggest that normal physiological processes of relaxation of gastric and cavernosal smooth muscles that facilitate food accommodation and penile erection, respectively, may be disrupted under conditions of myosin Va deficiency, resulting in complications like gastroparesis and erectile dysfunction. PMID:24516539

  8. Selectivity of ROCK inhibitors in the spontaneously tonic smooth muscle.

    PubMed

    Rattan, Satish; Patel, Chirag A

    2008-03-01

    The selectivity of different Rho kinase (ROCK) inhibitors in the spontaneously tonic smooth muscle has not been investigated. We examined this issue using Y-27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarbox anecarboxamide, 2HCl], H-1152 [(S)-(+)-(2-methyl-5-isoquinolinyl) sulfonylhomopiperazine, 2HCl], HA-1077 [(5 isoquinolinesulfonyl) homopiperazine, 2HCl], and ROCK inhibitor II [N-(4-pyridyl)-N'-(2,4,6-trichlorophenyl)urea]. We compared these inhibitors in the spontaneously tonic smooth muscle of the internal anal sphincter (IAS). ROCK, protein kinase C (PKC), and myosin light chain kinase (MLCK) activities were determined in the IAS, before and after different ROCK inhibitors. Y-27632 and H-1152 were approximately 30-fold more potent in the IAS (IC(50): 4.4 x 10(-7) and 7.9 x 10(-8) M, respectively) vs. the phasic rectal smooth muscle (RSM) (IC(50): 1.3 x 10(-5) and 2.5 x 10(-6) M, respectively). HA-1077 and ROCK inhibitor II were equipotent in the IAS vs. RSM. In the IAS, H-1152 was the most potent whereas ROCK inhibitor II is the least. Y-27632 and H-1152 caused concentration-dependent decrease in the IAS tone that correlates directly with the decreases in ROCK activity, without significant effect in the PKC and MLCK activities. This specifically selective correlation between ROCK activity and decrease in the IAS tone was absent in the case of HA-1077 and ROCK inhibitor II, which also inhibited PKC and MLCK. We conclude that the IAS tone is critically dependent on ROCK activity, and H-1152 and Y-27632 are the most selective and potent ROCK inhibitors in the IAS.

  9. Cutaneous Malignant Melanoma With Rhabdoid Morphology and Smooth Muscle Differentiation: A Challenging Histopathologic Diagnosis.

    PubMed

    Prieto-Torres, Lucía; Alegría-Landa, Victoria; Llanos, Concepción; Córdoba, Alicia; Kutzner, Heinz; Requena, Luis

    2017-05-01

    Divergent differentiation or metaplastic change is a rare feature exhibited occasionally in malignant melanoma (MM), which is characterized by the development of morphologically, immunochemically, and/or ultrastructurally nonmelanocytic cells within the tumor. Smooth muscle differentiation in MM is an exceedingly rare phenomenon reported only in a few cases in the literature. We report the case of a 69-year-old woman who presented with a pure dermal amelanotic MM with smooth muscle cell differentiation and an area of rhabdoid morphology, which made the accurate histopathologic diagnostic of MM challenging.

  10. Expression Profiling Identifies Klf15 as a Glucocorticoid Target That Regulates Airway Hyperresponsiveness

    PubMed Central

    Masuno, Kiriko; Haldar, Saptarsi M.; Jeyaraj, Darwin; Mailloux, Christina M.; Huang, Xiaozhu; Panettieri, Rey A.; Jain, Mukesh K.

    2011-01-01

    Glucocorticoids (GCs), which activate GC receptor (GR) signaling and thus modulate gene expression, are widely used to treat asthma. GCs exert their therapeutic effects in part through modulating airway smooth muscle (ASM) structure and function. However, the effects of genes that are regulated by GCs on airway function are not fully understood. We therefore used transcription profiling to study the effects of a potent GC, dexamethasone, on human ASM (HASM) gene expression at 4 and 24 hours. After 24 hours of dexamethasone treatment, nearly 7,500 genes had statistically distinguishable changes in expression; quantitative PCR validation of a 40-gene subset of putative GR-regulated genes in 6 HASM cell lines suggested that the early transcriptional targets of GR signaling are similar in independent HASM lines. Gene ontology analysis implicated GR targets in controlling multiple aspects of ASM function. One GR-regulated gene, the transcription factor, Kruppel-like factor 15 (Klf15), was already known to modulate vascular smooth and cardiac muscle function, but had no known role in the lung. We therefore analyzed the pulmonary phenotype of Klf15−/− mice after ovalbumin sensitization and challenge. We found diminished airway responses to acetylcholine in ovalbumin-challenged Klf15−/− mice without a significant change in the induction of asthmatic inflammation. In cultured cells, overexpression of Klf15 reduced proliferation of HASM cells, whereas apoptosis in Klf15−/− murine ASM cells was increased. Together, these results further characterize the GR-regulated gene network in ASM and establish a novel role for the GR target, Klf15, in modulating airway function. PMID:21257922

  11. [Effect of substance P on the potassium and calcium currents of colonic smooth muscle cells].

    PubMed

    Tang, Qincai; Luo, Hesheng; Quan, Xiaojing; Fan, Han; Yu, Guang

    2015-08-11

    To investigate the effect of substance P(SP) on the spontaneous contractile activity of smooth muscle cells,the large-conductance calcium-activated potassium channel currents (IBKCa) and the L-type calcium channel currents (ICaL) in rat smooth muscle cells of the proximal colon. A total of 24 healthy male Wista rats were used in this test. The change of smooth muscle strips spontaneous contraction of rat proximal colon after adding SP was recorded by a physiological signal stystem (RM6240). The IBKCa and ICaL were measured via the whole cell patch-clamp technique. The longitudinal muscle contraction was obviously increased concentration-dependently after adding different concentrations of SP (10(-7)-10(-6) mol/L), so as the circular muscle while adding SP(10(-8)-10(-6) mol/L) (all P<0.05). Compared with the control group, IBKCa was decreased after adding SP(10(-6) mol/L). Under the stimulating voltage of 60 mV, the IBKCa current density was (11.71±1.65) pA/pF, which was significantly lower compared with the control group (14.42±2.89) pA/pF (P<0.05). The ICaL) was apparently increased. Under the stimulating voltage of 0 mV, the ICaL) currents density was (-5.04±0.67) pA/pF, compared with the control group (-4.25±0.46) pA/pF, which was significantly increased (P<0.01). SP can promote the spontaneous contractile activity of colon smooth muscle of rats in vitro.And SP decrease IBKCa representatively while apparently increase ICaL). That is probably one of the mechanism SP regulate the gastrointestinal motility.

  12. Nitric oxide deficiency contributes to impairment of airway relaxation in cystic fibrosis mice.

    PubMed

    Mhanna, M J; Ferkol, T; Martin, R J; Dreshaj, I A; van Heeckeren, A M; Kelley, T J; Haxhiu, M A

    2001-05-01

    The pulmonary disease of cystic fibrosis (CF) is characterized by persistent airway obstruction, which has been attributed to chronic endobronchial infection and inflammation. The levels of exhaled nitric oxide (NO) are reduced in CF patients, which could contribute to bronchial obstruction through dysregulated constriction of airway smooth muscle. Because airway epithelium from CF mice has been shown to have reduced expression of inducible NO synthase, we examined airway responsiveness and relaxation in isolated tracheas of CF mice. Airway relaxation as measured by percent relaxation of precontracted tracheal segments to electrical field stimulation (EFS) and substance P, a nonadrenergic, noncholinergic substance, was significantly impaired in CF mice. The airway relaxation in response to prostaglandin E2 was similar in CF and non-CF animals. Treatment with the NO synthase inhibitor NG-nitro-L-arginine methylester reduced tracheal relaxation induced by EFS in wild-type animals but had virtually no effect in the CF mice. Conversely, exogenous NO and L-arginine, a NO substrate, reversed the relaxation defect in CF airway. We conclude that the relative absence of NO compromises airways relaxation in CF, and may contribute to the bronchial obstruction seen in the disease.

  13. The role of TRPP2 in agonist-induced gallbladder smooth muscle contraction.

    PubMed

    Zhong, Xingguo; Fu, Jie; Song, Kai; Xue, Nairui; Gong, Renhua; Sun, Dengqun; Luo, Huilai; He, Wenzhu; Pan, Xiang; Shen, Bing; Du, Juan

    2016-04-01

    TRPP2 channel protein belongs to the superfamily of transient receptor potential (TRP) channels and is widely expressed in various tissues, including smooth muscle in digestive gut. Accumulating evidence has demonstrated that TRPP2 can mediate Ca(2+) release from Ca(2+) stores. However, the functional role of TRPP2 in gallbladder smooth muscle contraction still remains unclear. In this study, we used Ca(2+) imaging and tension measurements to test agonist-induced intracellular Ca(2+) concentration increase and smooth muscle contraction of guinea pig gallbladder, respectively. When TRPP2 protein was knocked down in gallbladder muscle strips from guinea pig, carbachol (CCh)-evoked Ca(2+) release and extracellular Ca(2+) influx were reduced significantly, and gallbladder contractions induced by endothelin 1 and cholecystokinin were suppressed markedly as well. CCh-induced gallbladder contraction was markedly suppressed by pretreatment with U73122, which inhibits phospholipase C to terminate inositol 1,4,5-trisphosphate receptor (IP3) production, and 2-aminoethoxydiphenyl borate (2APB), which inhibits IP3 recepor (IP3R) to abolish IP3R-mediated Ca(2+) release. To confirm the role of Ca(2+) release in CCh-induced gallbladder contraction, we used thapsigargin (TG)-to deplete Ca(2+) stores via inhibiting sarco/endoplasmic reticulum Ca(2+)-ATPase and eliminate the role of store-operated Ca(2+) entry on the CCh-induced gallbladder contraction. Preincubation with 2 μmol L(-1) TG significantly decreased the CCh-induced gallbladder contraction. In addition, pretreatments with U73122, 2APB or TG abolished the difference of the CCh-induced gallbladder contraction between TRPP2 knockdown and control groups. We conclude that TRPP2 mediates Ca(2+) release from intracellular Ca(2+) stores, and has an essential role in agonist-induced gallbladder muscle contraction.

  14. Effects of lubiprostone on human uterine smooth muscle cells.

    PubMed

    Cuppoletti, John; Malinowska, Danuta H; Chakrabarti, Jayati; Ueno, Ryuji

    2008-06-01

    Lubiprostone, a bicyclic fatty acid derivative and member of a new class of compounds called prostones, locally activates ClC-2 Cl(-) channels without activation of prostaglandin receptors. The present study was specifically designed to test and compare lubiprostone and prostaglandin effects at the cellular level using human uterine smooth muscle cells. Effects on [Ca(2+)](i), membrane potential and [cAMP](i) in human uterine smooth muscle cells were measured. 10 nM lubiprostone significantly decreased [Ca(2+)](i) from 188 to 27 nM, which was unaffected by 100 nM SC-51322, a prostaglandin EP receptor antagonist. In contrast 10nM PGE(2) and PGE(1) both increased [Ca(2+)](i) 3-5-fold which was blocked by SC-51322. Similarly, lubiprostone and prostaglandins had opposite/different effects on membrane potential and [cAMP](i). Lubiprostone caused SC-51322-insensitive membrane hyperpolarization and no effect on [cAMP](i). PGE(2) and PGE(1) both caused SC-51322-sensitive membrane depolarization and increased [cAMP](i). Lubiprostone has fundamentally different cellular effects from prostaglandins that are not mediated by EP receptors.

  15. Smooth muscle membrane organization in the normal and dysfunctional human urinary bladder: a structural analysis.

    PubMed

    Burkhard, Fiona C; Monastyrskaya, Katia; Studer, Urs E; Draeger, Annette

    2005-01-01

    The decline in contractile properties is a characteristic feature of the dysfunctional bladder as a result of infravesical outlet obstruction. During clinical progression of the disease, smooth muscle cells undergo structural modifications. Since adaptations to constant changes in length require a high degree of structural organization within the sarcolemma, we have investigated the expression of several proteins, which are involved in smooth muscle membrane organization, in specimens derived from normal and dysfunctional organs. Specimen from patients with urodynamically normal/equivocal (n = 4), obstructed (n = 2), and acontractile (n = 2) bladders were analyzed relative to their structural features and sarcolemmal protein profile. Smooth muscle cells within the normal urinary bladder display a distinct sarcolemmal domain structure, characterized by firm actin-attachment sites, alternating with flexible "hinge" regions. In obstructed bladders, foci of cells displaying degenerative sarcolemmal changes alternate with areas of hypertrophic cells in which the membrane appears unaffected. In acontractile organs, the overall membrane structure remains intact, however annexin 6, a protein belonging to a family of Ca2+-dependent, "membrane-organizers," is downregulated. Degenerative changes in smooth muscle cells, which are chronically working against high resistance, are preferentially located within the actin-attachment sites. In acontractile bladders, the downregulation of annexin 6 might have a bearing on the fine-tuning of the plasma membrane during contraction/relaxation cycles. Copyright 2005 Wiley-Liss, Inc.

  16. Consequences of metabolic inhibition in smooth muscle isolated from guinea-pig stomach.

    PubMed

    Nakayama, S; Chihara, S; Clark, J F; Huang, S M; Horiuchi, T; Tomita, T

    1997-11-15

    1. In smooth muscle isolated from the guinea-pig stomach, cyanide (CN) and iodoacetic acid (IAA) were applied to block oxidative phosphorylation and glycolysis, respectively. Effects of IAA on generation of spontaneous mechanical and electrical activities were systematically investigated by comparing those of CN. Spontaneous activity ceased in 10-20 min during applications of 1 mM IAA. On the other hand, application of 1 mM CN also reduced the spontaneous activity, but never terminated it. In the presence of CN the negativity of the resting membrane potential was slightly reduced. 2. When spontaneous activity ceased with IAA, the resting membrane potential was not significantly affected. Also, before ceasing, the amplitude and duration of the spontaneous electrical activity were significantly reduced. The amplitude of the electrotonic potential was, however, not changed by IAA. Further, glibenclamide did not prevent the effects of IAA. These results suggest that, unlike cardiac muscle, activation of metabolism-dependent K+ channels in stomach smooth muscle does not seem to play a major role in reducing and terminating spontaneous activity during metabolic inhibition. 3. Carbachol-induced contraction transiently increased, and subsequently decreased gradually during application of IAA. 4. After 50 min application of IAA, when there was no spontaneous activity, the concentrations of phosphocreatine (PCr) and ATP measured with 31P nuclear magnetic resonance decreased to 60 and 80% of the control, respectively, while inorganic phosphate (Pi) concentration paradoxically fell to below detectable levels. During subsequent prolonged application of IAA, high-energy phosphates steadily decreased. On the other hand, after 50 min CN application, [PCr] and [ATP] decreased to approximately 30 and 80% of the control, respectively, while [Pi] increased by 2.6-fold. 5. In the presence of either CN or IAA, spontaneous mechanical and electrical activities were reduced or eliminated

  17. Smooth muscle myosin light chain kinase efficiently phosphorylates serine 15 of cardiac myosin regulatory light chain

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Josephson, Matthew P.; Sikkink, Laura A.; Penheiter, Alan R.

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer Cardiac myosin regulatory light chain (MYL2) is phosphorylated at S15. Black-Right-Pointing-Pointer Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase. Black-Right-Pointing-Pointer It is a widely believed that MYL2 is a poor substrate for smMLCK. Black-Right-Pointing-Pointer In fact, smMLCK efficiently and rapidly phosphorylates S15 in MYL2. Black-Right-Pointing-Pointer Phosphorylation kinetics measured by novel fluorescence method without radioactivity. -- Abstract: Specific phosphorylation of the human ventricular cardiac myosin regulatory light chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca{sup 2+} sensitivity of contraction in cardiac tissue. Smooth muscle myosin light chainmore » kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory light chain S15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (S20 in smooth muscle) but failed to phosphorylate the myosin regulatory light chain in rabbit skeletal myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicates similar Michaelis-Menten V{sub max} and K{sub M} for regulatory light chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially

  18. Extracellular Cl- regulates electrical slow waves and setting of smooth muscle membrane potential by interstitial cells of Cajal in mouse jejunum.

    PubMed

    Saravanaperumal, Siva Arumugam; Gibbons, Simon J; Malysz, John; Sha, Lei; Linden, David R; Szurszewski, Joseph H; Farrugia, Gianrico

    2018-01-01

    What is the central question of this study? The aim was to investigate the roles of extracellular chloride in electrical slow waves and resting membrane potential of mouse jejunal smooth muscle by replacing chloride with the impermeant anions gluconate and isethionate. What is the main finding and its importance? The main finding was that in smooth muscle cells, the resting Cl - conductance is low, whereas transmembrane Cl - movement in interstitial cells of Cajal (ICCs) is a major contributor to the shape of electrical slow waves. Furthermore, the data confirm that ICCs set the smooth muscle membrane potential and that altering Cl - homeostasis in ICCs can alter the smooth muscle membrane potential. Intracellular Cl - homeostasis is regulated by anion-permeable channels and transporters and contributes to excitability of many cell types, including smooth muscle and interstitial cells of Cajal (ICCs). Our aims were to investigate the effects on electrical activity in mouse jejunal muscle strips of replacing extracellular Cl - (Cl - o ) with the impermeant anions gluconate and isethionate. On reducing Cl - o , effects were observed on electrical slow waves, with small effects on smooth muscle membrane voltage (E m ). Restoration of Cl - hyperpolarized smooth muscle E m proportional to the change in Cl - o concentration. Replacement of 90% of Cl - o with gluconate reversibly abolished slow waves in five of nine preparations. Slow waves were maintained in isethionate. Gluconate and isethionate substitution had similar concentration-dependent effects on peak amplitude, frequency, width at half peak amplitude, rise time and decay time of residual slow waves. Gluconate reduced free ionized Ca 2+ in Krebs solutions to 0.13 mm. In Krebs solutions containing normal Cl - and 0.13 mm free Ca 2+ , slow wave frequency was lower, width at half peak amplitude was smaller, and decay time was faster. The transient hyperpolarization following restoration of Cl - o was not observed

  19. Hydrogen sulfide mediates hypoxia-induced relaxation of trout urinary bladder smooth muscle.

    PubMed

    Dombkowski, Ryan A; Doellman, Meredith M; Head, Sally K; Olson, Kenneth R

    2006-08-01

    Hydrogen sulfide (H2S) is a recently identified gasotransmitter that may mediate hypoxic responses in vascular smooth muscle. H2S also appears to be a signaling molecule in mammalian non-vascular smooth muscle, but its existence and function in non-mammalian non-vascular smooth muscle have not been examined. In the present study we examined H2S production and its physiological effects in urinary bladder from steelhead and rainbow trout (Oncorhynchus mykiss) and evaluated the relationship between H2S and hypoxia. H2S was produced by trout bladders, and its production was sensitive to inhibitors of cystathionine beta-synthase and cystathionine gamma-lyase. H2S produced a dose-dependent relaxation in unstimulated and carbachol pre-contracted bladders and inhibited spontaneous contractions. Bladders pre-contracted with 80 mmol l(-1) KCl were less sensitive to H2S than bladders contracted with either 80 mmol l(-1) KC2H3O2 (KAc) or carbachol, suggesting that some of the H2S effects are mediated through an ion channel. However, H2S relaxation of bladders was not affected by the potassium channel inhibitors, apamin, charybdotoxin, 4-aminopyridine, and glybenclamide, or by chloride channel/exchange inhibitors 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt, tamoxifen and glybenclamide, or by the presence or absence of extracellular HCO3-. Inhibitors of neuronal mechanisms, tetrodotoxin, strychnine and N-vanillylnonanamide were likewise ineffective. Hypoxia (aeration with N2) also relaxed bladders, was competitive with H2S for relaxation, and it was equally sensitive to KCl, and unaffected by neuronal blockade or the presence of extracellular HCO3-. Inhibitors of H2S synthesis also inhibited hypoxic relaxation. These experiments suggest that H2S is a phylogenetically ancient gasotransmitter in non-mammalian non-vascular smooth muscle and that it serves as an oxygen sensor/transducer, mediating the effects of hypoxia.

  20. Isolation of pulmonary artery smooth muscle cells from neonatal mice.

    PubMed

    Lee, Keng Jin; Czech, Lyubov; Waypa, Gregory B; Farrow, Kathryn N

    2013-10-19

    Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al. that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 μM in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

  1. Chitosan-based scaffolds for the support of smooth muscle constructs in intestinal tissue engineering

    PubMed Central

    Zakhem, Elie; Raghavan, Shreya; Gilmont, Robert R; Bitar, Khalil N

    2012-01-01

    Intestinal tissue engineering is an emerging field due to a growing demand for intestinal lengthening and replacement procedures secondary to massive resections of the bowel. Here, we demonstrate the potential use of a chitosan/collagen scaffold as a 3D matrix to support the bioengineered circular muscle constructs maintain their physiological functionality. We investigated the biocompatibility of chitosan by growing rabbit colonic circular smooth muscle cells (RCSMCs) on chitosan-coated plates. The cells maintained their spindle-like morphology and preserved their smooth muscle phenotypic markers. We manufactured tubular scaffolds with central openings composed of chitosan and collagen in a 1:1 ratio. Concentrically-aligned 3D circular muscle constructs were bioengineered using fibrin-based hydrogel seeded with RCSMCs. The constructs were placed around the scaffold for 2 weeks, after which they were taken off and tested for their physiological functionality. The muscle constructs contracted in response to Acetylcholine (Ach) and potassium chloride (KCl) and they relaxed in response to vasoactive intestinal peptide (VIP). These results demonstrate that chitosan is a biomaterial possibly suitable for intestinal tissue engineering applications. PMID:22483012

  2. [The effect of 18beta-glycyrrhetinic acid on gap junction among cerebral arteriolar smooth muscle cells in Wistar rat and spontaneously hypertensive rat].

    PubMed

    Chen, Xin-Yan; Si, Jun-Qiang; Li, Li; Zhao, Lei; Wei, Li-Li; Jiang, Xue-Wei; Ma, Ke-Tao

    2013-05-01

    This study compared Wistar rat with spontaneously hypertensive rat (SHR) on the electrophysiology and coupling force of the smooth muscle cells in the cerebral arteriolar segments and observe the influence of 18beta-glycyrrhetinic acid(18beta-GA) on the gap junctions between the arterial smooth muscle cells. The outer layer's connective tissue of the cerebral arteriolar segments was removed. Whole-cell patch clamp recordings were used to observe the 18beta-GA's impaction on the arteriolar segment membrane's input capacitance (C(input)), input conductance (G(input)) and input resistance (R(input)) of the smooth muscle cells. (1) The C(input) and G(input) of the SHR arteriolar segment smooth muscle cells was much higher than the Wistar rats, there was significant difference (P < 0.05). (2) 18beta-GA concentration-dependently reduced C(input) and G(input) (or increase R(input)) on smooth muscle cells in arteriolar segment. IC50 of 18beta-GA suppression's G(input) of the Wistar rat and SHR were 1.7 and 2.0 micromol/L respectively, there was not significant difference (P > 0.05). After application of 18beta-GA concentration > or = 100 micrmol/L, the C(input), G(input) and R(input) of the single smooth muscle cells was very close. Gap junctional coupling is enhanced in the SHR cerebral arterial smooth muscle cells. 18beta-GA concentration-dependent inhibits Wistar rat's and SHR cerebral arteriolar gap junctions between arterial smooth muscle cells. The inhibitory potency is similar between the two different rats. When 18beta-GA concentration is > or = 100 micromol/L, it can completely block gap junctions between arteriolar smooth muscle cells.

  3. Akt1/PKB upregulation leads to vascular smooth muscle cell hypertrophy and polyploidization

    PubMed Central

    Hixon, Mary L.; Muro-Cacho, Carlos; Wagner, Mark W.; Obejero-Paz, Carlos; Millie, Elise; Fujio, Yasushi; Kureishi, Yasuko; Hassold, Terry; Walsh, Kenneth; Gualberto, Antonio

    2000-01-01

    Vascular smooth muscle cells (VSMCs) at capacitance arteries of hypertensive individuals and animals undergo marked age- and blood pressure–dependent polyploidization and hypertrophy. We show here that VSMCs at capacitance arteries of rat models of hypertension display high levels of Akt1/PKB protein and activity. Gene transfer of Akt1 to VSMCs isolated from a normotensive rat strain was sufficient to abrogate the activity of the mitotic spindle cell–cycle checkpoint, promoting polyploidization and hypertrophy. Furthermore, the hypertrophic agent angiotensin II induced VSMC polyploidization in an Akt1-dependent manner. These results demonstrate that Akt1 regulates ploidy levels in VSMCs and contributes to vascular smooth muscle polyploidization and hypertrophy during hypertension. PMID:11032861

  4. Inhibition of Smooth Muscle Proliferation by Urea-Based Alkanoic Acids via Peroxisome Proliferator-Activated Receptor α–Dependent Repression of Cyclin D1

    PubMed Central

    Ng, Valerie Y.; Morisseau, Christophe; Falck, John R.; Hammock, Bruce D.; Kroetz, Deanna L.

    2007-01-01

    Objective Proliferation of smooth muscle cells is implicated in cardiovascular complications. Previously, a urea-based soluble epoxide hydrolase inhibitor was shown to attenuate smooth muscle cell proliferation. We examined the possibility that urea-based alkanoic acids activate the nuclear receptor peroxisome proliferator-activated receptor α (PPARα) and the role of PPARα in smooth muscle cell proliferation. Methods and Results Alkanoic acids transactivated PPARα, induced binding of PPARα to its response element, and significantly induced the expression of PPARα-responsive genes, showing their function as PPARα agonists. Furthermore, the alkanoic acids attenuated platelet-derived growth factor–induced smooth muscle cell proliferation via repression of cyclin D1 expression. Using small interfering RNA to decrease endogenous PPARα expression, it was determined that PPARα was partially involved in the cyclin D1 repression. The antiproliferative effects of alkanoic acids may also be attributed to their inhibitory effects on soluble epoxide hydrolase, because epoxyeicosatrienoic acids alone inhibited smooth muscle cell proliferation. Conclusions These results show that attenuation of smooth muscle cell proliferation by urea-based alkanoic acids is mediated, in part, by the activation of PPARα. These acids may be useful for designing therapeutics to treat diseases characterized by excessive smooth muscle cell proliferation. PMID:16917105

  5. Graded effects of unregulated smooth muscle myosin on intestinal architecture, intestinal motility and vascular function in zebrafish.

    PubMed

    Abrams, Joshua; Einhorn, Zev; Seiler, Christoph; Zong, Alan B; Sweeney, H Lee; Pack, Michael

    2016-05-01

    Smooth muscle contraction is controlled by the regulated activity of the myosin heavy chain ATPase (Myh11). Myh11 mutations have diverse effects in the cardiovascular, digestive and genitourinary systems in humans and animal models. We previously reported a recessive missense mutation, meltdown (mlt), which converts a highly conserved tryptophan to arginine (W512R) in the rigid relay loop of zebrafish Myh11. The mlt mutation disrupts myosin regulation and non-autonomously induces invasive expansion of the intestinal epithelium. Here, we report two newly identified missense mutations in the switch-1 (S237Y) and coil-coiled (L1287M) domains of Myh11 that fail to complement mlt Cell invasion was not detected in either homozygous mutant but could be induced by oxidative stress and activation of oncogenic signaling pathways. The smooth muscle defect imparted by the mlt and S237Y mutations also delayed intestinal transit, and altered vascular function, as measured by blood flow in the dorsal aorta. The cell-invasion phenotype induced by the three myh11 mutants correlated with the degree of myosin deregulation. These findings suggest that the vertebrate intestinal epithelium is tuned to the physical state of the surrounding stroma, which, in turn, governs its response to physiologic and pathologic stimuli. Genetic variants that alter the regulation of smooth muscle myosin might be risk factors for diseases affecting the intestine, vasculature, and other tissues that contain smooth muscle or contractile cells that express smooth muscle proteins, particularly in the setting of redox stress. © 2016. Published by The Company of Biologists Ltd.

  6. Transforming growth factor-β stimulates the expression of eotaxin/CC chemokine ligand 11 and its promoter activity through binding site for nuclear factor-κB in airway smooth muscle cells

    PubMed Central

    Matsukura, S.; Odaka, M.; Kurokawa, M.; Kuga, H.; Homma, T.; Takeuchi, H.; Notomi, K.; Kokubu, F.; Kawaguchi, M.; Schleimer, R. P.; Johnson, M. W.; Adachi, M.

    2013-01-01

    Summary Background Chemokines ligands of CCR3 including eotaxin/CC chemokine ligand 11 (CCL11) may contribute to the pathogenesis of asthma. These chemokines and a growth factor (TGF-β) may be involved in the process of airway remodelling. Objective We analysed the effects of TGF-β on the expression of CCR3 ligands in human airway smooth muscle (HASM) cells and investigated the mechanisms. Methods HASM cells were cultured and treated with TGF-β and Th2 cytokines IL-4 or IL-13. Expression of mRNA was analysed by real-time PCR. Secretion of CCL11 into the culture medium was analysed by ELISA. Transcriptional regulation of CCL11 was analysed by luciferase assay using CCL11 promoter-luciferase reporter plasmids. Results IL-4 or IL-13 significantly up-regulated the expression of mRNAs for CCL11 and CCL26. TGF-β alone did not increase the expression of chemokine mRNAs, but enhanced the induction of only CCL11 by IL-4 or IL-13 among CCR3 ligands. Activity of the CCL11 promoter was stimulated by IL-4, and this activity was enhanced by TGF-β. Activation by IL-4 or IL-4 plus TGF-β was lost by mutation of the binding site for signal transducers and activators of transcription-6 (STAT6) in the promoter. Cooperative activation by IL-4 and TGF-β was inhibited by mutation of the binding site for nuclear factor-κB (NF-κB) in the promoter. Pretreatment with an inhibitor of NF-κB and glucocorticoid fluticasone propionate significantly inhibited the expression of CCL11 mRNA induced by IL-4 plus TGF-β, indicating the importance of NF-κB in the cooperative activation of CCL11 transcription by TGF-β and IL-4. Conclusion These results indicate that Th2 cytokines and TGF-β may contribute to the pathogenesis of asthma by stimulating expression of CCL11. The transcription factors STAT6 and NF-κB may play pivotal roles in this process. PMID:20214667

  7. Transforming growth factor-β stimulates the expression of eotaxin/CC chemokine ligand 11 and its promoter activity through binding site for nuclear factor-κβ in airway smooth muscle cells.

    PubMed

    Matsukura, S; Odaka, M; Kurokawa, M; Kuga, H; Homma, T; Takeuchi, H; Notomi, K; Kokubu, F; Kawaguchi, M; Schleimer, R P; Johnson, M W; Adachi, M

    2010-05-01

    Chemokines ligands of CCR3 including eotaxin/CC chemokine ligand 11 (CCL11) may contribute to the pathogenesis of asthma. These chemokines and a growth factor (TGF-beta) may be involved in the process of airway remodelling. We analysed the effects of TGF-beta on the expression of CCR3 ligands in human airway smooth muscle (HASM) cells and investigated the mechanisms. HASM cells were cultured and treated with TGF-beta and Th2 cytokines IL-4 or IL-13. Expression of mRNA was analysed by real-time PCR. Secretion of CCL11 into the culture medium was analysed by ELISA. Transcriptional regulation of CCL11 was analysed by luciferase assay using CCL11 promoter-luciferase reporter plasmids. IL-4 or IL-13 significantly up-regulated the expression of mRNAs for CCL11 and CCL26. TGF-beta alone did not increase the expression of chemokine mRNAs, but enhanced the induction of only CCL11 by IL-4 or IL-13 among CCR3 ligands. Activity of the CCL11 promoter was stimulated by IL-4, and this activity was enhanced by TGF-beta. Activation by IL-4 or IL-4 plus TGF-beta was lost by mutation of the binding site for signal transducers and activators of transcription-6 (STAT6) in the promoter. Cooperative activation by IL-4 and TGF-beta was inhibited by mutation of the binding site for nuclear factor-kappaB (NF-kappaB) in the promoter. Pretreatment with an inhibitor of NF-kappaB and glucocorticoid fluticasone propionate significantly inhibited the expression of CCL11 mRNA induced by IL-4 plus TGF-beta, indicating the importance of NF-kappaB in the cooperative activation of CCL11 transcription by TGF-beta and IL-4. These results indicate that Th2 cytokines and TGF-beta may contribute to the pathogenesis of asthma by stimulating expression of CCL11. The transcription factors STAT6 and NF-kappaB may play pivotal roles in this process.

  8. Differential gene expression profiling of human adipose stem cells differentiating into smooth muscle-like cells by TGFβ1/BMP4

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Elçin, Ayşe Eser; Parmaksiz, Mahmut; Dogan, Arin

    Regenerative repair of the vascular system is challenging from the perspectives of translational medicine and tissue engineering. There are fundamental hurdles in front of creating bioartificial arteries, which involve recaputilation of the three-layered structure under laboratory settings. Obtaining and maintaining smooth muscle characteristics is an important limitation, as the transdifferentiated cells fail to display mature phenotype. This study aims to shed light on the smooth muscle differentiation of human adipose stem cells (hASCs). To this end, we first acquired hASCs from lipoaspirate samples. Upon characterization, the cells were induced to differentiate into smooth muscle (SM)-like cells using a variety ofmore » inducer combinations. Among all, TGFβ1/BMP4 combination had the highest differentiation efficiency, based on immunohistochemical analyses. hSM-like cell samples were compared to hASCs and to the positive control, human coronary artery-smooth muscle cells (hCA-SMCs) through gene transcription profiling. Microarray findings revealed the activation of gene groups that function in smooth muscle differentiation, signaling pathways, extracellular modeling and cell proliferation. Our results underline the effectiveness of the growth factors and suggest some potential variables for detecting the SM-like cell characteristics. Evidence in transcriptome level was used to evaluate the TGFβ1/BMP4 combination as a previously unexplored effector for the smooth muscle differentiation of adipose stem cells. - Highlights: • Human adipose stem cells (hASCs) were isolated, characterized and cultured. • Growth factor combinations were evaluated for their effectiveness in differentiation using IHC. • hASCs were differentiated into smooth muscle (SM)-like cells using TGF-β1 and BMP4 combination. • Microarray analysis was performed for hASCs, SM-like cells and coronary artery-SMCs. • Microarray data was used to perform hierarchical clustering and

  9. Influenza infection causes airway hyperresponsiveness by decreasing enkephalinase.

    PubMed

    Jacoby, D B; Tamaoki, J; Borson, D B; Nadel, J A

    1988-06-01

    Ferret tracheal segments were infected with human influenza virus A/Taiwan/86 (H1N1) in vitro. After 4 days, the smooth muscle contractile responses to acetylcholine and to substance P were measured. The response to substance P was markedly accentuated, with a threefold increase in force of contraction at a substance P concentration of 10(-5) M, the highest concentration tested. In contrast, the response to acetylcholine was not affected by viral infection. Histological examination of tissues revealed extensive epithelial desquamation. Activity of enkephalinase (neutral metallo-endopeptidase, EC.3.4.24.11), an enzyme that degrades substance P, was decreased by 50% in infected tissues. Inhibiting enkephalinase activity by pretreating with thiorphan (10(-5) M) increased the response to substance P to the same final level in both infected and control tissues. Inhibiting other substance P-degrading enzymes including kininase II (angiotensin-converting enzyme), serine proteases, and aminopeptidases did not affect the response to substance P. Inhibiting cyclooxygenase and lipoxygenase activity using indomethacin and BW 755c did not affect hyperresponsiveness to substance P. Pretreating tissues with antagonists of alpha-adrenoceptors, beta-adrenoceptors, and H1 histamine receptors (phentolamine 10(-5) M, propranolol 5 X 10(-6) M, and pyrilamine 10(-5) M, respectively) had no effect on substance P-induced contraction. These results demonstrate that infection of ferret airway tissues with influenza virus increases the contractile response of airway smooth muscle to substance P. This effect is caused by decreased enkephalinase activity in infected tissues.

  10. Smooth muscle cells of penis in the rat: noninvasive quantification with shear wave elastography.

    PubMed

    Zhang, Jia-Jie; Qiao, Xiao-Hui; Gao, Feng; Bai, Ming; Li, Fan; Du, Lian-Fang; Xing, Jin-Fang

    2015-01-01

    Smooth muscle cells (SMCs) of cavernosum play an important role in erection. It is of great significance to quantitatively analyze the level of SMCs in penis. In this study, we investigated the feasibility of shear wave elastography (SWE) on evaluating the level of SMCs in penis quantitatively. Twenty healthy male rats were selected. The SWE imaging of penis was carried out and then immunohistochemistry analysis of penis was performed to analyze the expression of alpha smooth muscle actin in penis. The measurement index of SWE examination was tissue stiffness (TS). The measurement index of immunohistochemistry analysis was positive area percentage of alpha smooth muscle actin (AP). Sixty sets of data of TS and AP were obtained. The results showed that TS was significantly correlated with AP and the correlation coefficient was -0.618 (p < 0.001). The result of TS had been plotted against the AP measurements. The relation between the two results has been fitted with quadric curve; the goodness-of-fit index was 0.364 (p < 0.001). The level of SMCs in penis was successfully quantified in vivo with SWE. SWE can be used clinically for evaluating the level of SMCs in penis quantitatively.

  11. Genetic control of indirect airway responsiveness in the rat.

    PubMed

    Pauwels, R A; Germonpré, P R; Kips, J C; Joos, G F

    1995-11-01

    Many of the airway responses to endogenous and exogenous stimuli are caused by indirect mechanisms such as the activation of neurons and/or inflammatory cells. In the present study we compare the bronchoconstrictor and the plasma protein extravasation response to adenosine and tachykinins in two highly inbred rat strains, F344 and BDE. BDE-rats have a bronchoconstrictor response to adenosine at lower doses. Challenge with the A3-adenosine receptor agonist APNEA demonstrates that the difference in airway responsiveness to adenosine between BDE- and F344-rats is probably related to a higher number of A3-receptors on the airway mast cells of BDE-rats. In contrast, F344-rats have a higher airway responsiveness to tachykinins than BDE-rats. Tachykinins cause bronchoconstriction in F344-rats mainly by an indirect mechanism, involving stimulation of NK1-receptors and mast cell activation. In BDE-rats they cause bronchoconstriction by a direct effect on airway smooth muscle via activation of NK2-receptors. Finally we also observed a difference between F344- and BDE-rats with regard to the mechanisms involved in the plasma protein extravasation in the airways caused by substance P or capsaicin. In F344-rats but not in BDE-rats mast cell activation and the release of 5-hydroxytryptamine is partly responsible for this plasma protein extravasation.

  12. Onset of diabetes modulates the airway smooth muscle reactivity of guinea pigs: role of epithelial mediators.

    PubMed

    Saidullah, Bano; Muralidhar, Kambadur; Fahim, Mohammad

    2014-01-01

    Diabetes induces lung dysfunction, leading to alteration in the pulmonary functions. Our aim was to investigate whether the early stage of diabetes alters the epithelium-dependent bronchial responses and whether nitric oxide (NO), KATP channels and cyclooxygenase (COX) pathways contribute in this effect. Guinea pigs were treated with a single injection of streptozotocin (180 mg/kg, i.p.) for induction of diabetes. Airway conductivity was assessed by inhaled histamine, using a non-invasive body plethysmography. The contractile responses of tracheal rings induced by acetylcholine (ACh) and relaxant responses of precontracted rings, induced by isoproterenol (IP) were compared in the presence and absence of the epithelium. Effects of N(ω)-Nitro-L-arginine methyl ester (L-NAME, a nitric oxide synthase inhibitor), glybenclamide (a KATP channel inhibitor) and indomethacin (a COX inhibitor) were also assessed in diabetic guinea pigs. Early stage diabetes did not alter the airway conductivity. ACh-induced bronchoconstriction in epithelium intact tracheal rings was not affected by the onset of diabetes, however a reduction in the increased ACh responses due to epithelium removal, to L-NAME or to indomethacin was observed. The relaxation response to IP was impaired in trachea from guinea pigs in which diabetes had just developed. Early diabetes significantly reduced the IP response to glybenclamide and to indomethacin. Our results demonstrate that the early stage of diabetes, modulate the bronchial reactivity to both ACh and IP by disrupting the NO, KATP channels and COX pathways, without affecting the airway conductivity in guinea pigs.

  13. Vascular smooth muscle cell phenotypic changes in patients with Marfan syndrome.

    PubMed

    Crosas-Molist, Eva; Meirelles, Thayna; López-Luque, Judit; Serra-Peinado, Carla; Selva, Javier; Caja, Laia; Gorbenko Del Blanco, Darya; Uriarte, Juan José; Bertran, Esther; Mendizábal, Yolanda; Hernández, Vanessa; García-Calero, Carolina; Busnadiego, Oscar; Condom, Enric; Toral, David; Castellà, Manel; Forteza, Alberto; Navajas, Daniel; Sarri, Elisabet; Rodríguez-Pascual, Fernando; Dietz, Harry C; Fabregat, Isabel; Egea, Gustavo

    2015-04-01

    Marfan's syndrome is characterized by the formation of ascending aortic aneurysms resulting from altered assembly of extracellular matrix microfibrils and chronic tissue growth factor (TGF)-β signaling. TGF-β is a potent regulator of the vascular smooth muscle cell (VSMC) phenotype. We hypothesized that as a result of the chronic TGF-β signaling, VSMC would alter their basal differentiation phenotype, which could facilitate the formation of aneurysms. This study explores whether Marfan's syndrome entails phenotypic alterations of VSMC and possible mechanisms at the subcellular level. Immunohistochemical and Western blotting analyses of dilated aortas from Marfan patients showed overexpression of contractile protein markers (α-smooth muscle actin, smoothelin, smooth muscle protein 22 alpha, and calponin-1) and collagen I in comparison with healthy aortas. VSMC explanted from Marfan aortic aneurysms showed increased in vitro expression of these phenotypic markers and also of myocardin, a transcription factor essential for VSMC-specific differentiation. These alterations were generally reduced after pharmacological inhibition of the TGF-β pathway. Marfan VSMC in culture showed more robust actin stress fibers and enhanced RhoA-GTP levels, which was accompanied by increased focal adhesion components and higher nuclear localization of myosin-related transcription factor A. Marfan VSMC and extracellular matrix measured by atomic force microscopy were both stiffer than their respective controls. In Marfan VSMC, both in tissue and in culture, there are variable TGF-β-dependent phenotypic changes affecting contractile proteins and collagen I, leading to greater cellular and extracellular matrix stiffness. Altogether, these alterations may contribute to the known aortic rigidity that precedes or accompanies Marfan's syndrome aneurysm formation. © 2015 American Heart Association, Inc.

  14. Relaxant effect of ghrelin on guinea pig isolated tracheal smooth muscle: role of epithelial NO and PGE2.

    PubMed

    Al-Ayed, Mohammed Saeed Zayed

    2018-06-01

    This study aimed at investigating the potential ghrelin relaxing effect on guinea pig isolated tracheal smooth muscle (TSM). Using an in vitro experimental approach, the physiological role of the airway epithelium on smooth muscle relaxation has been investigated by analyzing the dose-response curves for carbachol- or histamine-induced contractions on epithelium intact versus denuded tracheal tissue. The relaxant effect of ghrelin (5-200 μmol/L) then investigated on carbachol-contracted, non-sensitized, and ovalbumin (OVA)-sensitized guinea pig TSM with an intact or denuded epithelium. The isolated TSMs from identical guinea pigs were incubated in Krebs solution aerated with 95% O 2 and 5% CO 2 through an automated tissue organ bath system (n = 6 for each group). The ghrelin relaxation mechanism was assessed by adding L-NAME, indomethacin, and YIL-781 for GHS-R1 into the tissue chamber. The spasmogens carbachol and histamine have shown a significantly higher contracting effect on epithelium-denuded than in epithelium-intact TSM confirmed by the significantly higher mean pEC50 of both agonists on the epithelium-denuded trachea (p < 0.05). Ghrelin has shown a concentration-dependent relaxing effect on carbachol-contracted TSM (r = 0.96, p = 0.00). The effect was more evident in the intact non-sensitized than in epithelium-denuded or OVA-sensitized groups (p < 0.05). Preincubation with nitric oxide (NO) and prostaglandin E 2 (PGE 2 ) inhibitors has significantly reduced the ghrelin-induced relaxation on epithelium-intact TSM suggesting an epithelium-dependant mechanism. However, GHS-R1a antagonist has also succeeded to reduce ghrelin relaxant effect, which needs further clarification. Ghrelin proved to have a potential TSM relaxant effect possibly through epithelium-dependant mechanisms involving NO and PGE 2 .

  15. Telocytes in skeletal, cardiac and smooth muscle interstitium: morphological and functional aspects.

    PubMed

    Marini, Mirca; Rosa, Irene; Ibba-Manneschi, Lidia; Manetti, Mirko

    2018-04-25

    Telocytes (TCs) represent a new distinct type of cells found in the stromal compartment of many organs, including the skeletal, cardiac and smooth muscles. TCs are morphologically defined as interstitial cells with a small cellular body from which arise very long (up to hundreds of micrometers) and thin moniliform processes (named telopodes) featuring the alternation of slender segments (called podomers) and small dilated portions (called podoms) accommodating some organelles. Although these stromal cells are mainly characterized by their ultrastructural traits, in the last few years TCs have been increasingly studied for their immunophenotypes, microRNA profiles, and gene expression and proteomic signatures. By their long-distance spreading telopodes, TCs build a three-dimensional network throughout the whole stromal space and communicate with each other and neighboring cells through homocellular and heterocellular junctions, respectively. Moreover, increasing evidence suggests that TCs may exert paracrine functions being able to transfer genetic information and signaling molecules to other cells via the release of different types of extracellular vesicles. A close relationship between TCs and stem/progenitor cell niches has also been described in several organs. However, the specific functions of TCs located in the muscle interstitium remain to be unraveled. Here, we review the morphological and possible functional aspects of TCs in skeletal, cardiac and smooth muscle tissues. The potential involvement of TCs in muscle tissue pathological changes and future possibilities for targeting TCs as a novel promising therapeutic strategy to foster muscle tissue regeneration and repair are also discussed.

  16. Identification of a rhythmic firing pattern in the enteric nervous system that generates rhythmic electrical activity in smooth muscle.

    PubMed

    Spencer, Nick J; Hibberd, Timothy J; Travis, Lee; Wiklendt, Lukasz; Costa, Marcello; Hu, Hongzhen; Brookes, Simon J; Wattchow, David A; Dinning, Phil G; Keating, Damien J; Sorensen, Julian

    2018-05-28

    The enteric nervous system (ENS) contains millions of neurons essential for organization of motor behaviour of the intestine. It is well established the large intestine requires ENS activity to drive propulsive motor behaviours. However, the firing pattern of the ENS underlying propagating neurogenic contractions of the large intestine remains unknown. To identify this, we used high resolution neuronal imaging with electrophysiology from neighbouring smooth muscle. Myoelectric activity underlying propagating neurogenic contractions along murine large intestine (referred to as colonic migrating motor complexes, CMMCs) consisted of prolonged bursts of rhythmic depolarizations at a frequency of ∼2 Hz. Temporal coordination of this activity in the smooth muscle over large spatial fields (∼7mm, longitudinally) was dependent on the ENS. During quiescent periods between neurogenic contractions, recordings from large populations of enteric neurons, in mice of either sex, revealed ongoing activity. The onset of neurogenic contractions was characterized by the emergence of temporally synchronized activity across large populations of excitatory and inhibitory neurons. This neuronal firing pattern was rhythmic and temporally synchronized across large numbers of ganglia at ∼2 Hz. ENS activation preceded smooth muscle depolarization, indicating rhythmic depolarizations in smooth muscle were controlled by firing of enteric neurons. The cyclical emergence of temporally coordinated firing of large populations of enteric neurons represents a unique neural motor pattern outside the central nervous system. This is the first direct observation of rhythmic firing in the ENS underlying rhythmic electrical depolarizations in smooth muscle. The pattern of neuronal activity we identified underlies the generation of CMMCs. SIGNIFICANCE STATEMENT How the enteric nervous system (ENS) generates neurogenic contractions of smooth muscle in the gastrointestinal (GI) tract has been a long

  17. Ionotropic and metabotropic proton-sensing receptors involved in airway inflammation in allergic asthma.

    PubMed

    Aoki, Haruka; Mogi, Chihiro; Okajima, Fumikazu

    2014-01-01

    An acidic microenvironment has been shown to evoke a variety of airway responses, including cough, bronchoconstriction, airway hyperresponsiveness (AHR), infiltration of inflammatory cells in the lung, and stimulation of mucus hyperproduction. Except for the participation of transient receptor potential vanilloid-1 (TRPV1) and acid-sensing ion channels (ASICs) in severe acidic pH (of less than 6.0)-induced cough and bronchoconstriction through sensory neurons, the molecular mechanisms underlying extracellular acidic pH-induced actions in the airways have not been fully understood. Recent studies have revealed that ovarian cancer G protein-coupled receptor 1 (OGR1)-family G protein-coupled receptors, which sense pH of more than 6.0, are expressed in structural cells, such as airway smooth muscle cells and epithelial cells, and in inflammatory and immune cells, such as eosinophils and dendritic cells. They function in a variety of airway responses related to the pathophysiology of inflammatory diseases, including allergic asthma. In the present review, we discuss the roles of ionotropic TRPV1 and ASICs and metabotropic OGR1-family G protein-coupled receptors in the airway inflammation and AHR in asthma and respiratory diseases.

  18. Hippo signaling is required for Notch-dependent smooth muscle differentiation of neural crest.

    PubMed

    Manderfield, Lauren J; Aghajanian, Haig; Engleka, Kurt A; Lim, Lillian Y; Liu, Feiyan; Jain, Rajan; Li, Li; Olson, Eric N; Epstein, Jonathan A

    2015-09-01

    Notch signaling has well-defined roles in the assembly of arterial walls and in the development of the endothelium and smooth muscle of the vasculature. Hippo signaling regulates cellular growth in many tissues, and contributes to regulation of organ size, in addition to other functions. Here, we show that the Notch and Hippo pathways converge to regulate smooth muscle differentiation of the neural crest, which is crucial for normal development of the aortic arch arteries and cranial vasculature during embryonic development. Neural crest-specific deletion of the Hippo effectors Yap and Taz produces neural crest precursors that migrate normally, but fail to produce vascular smooth muscle, and Notch target genes such as Jagged1 fail to activate normally. We show that Yap is normally recruited to a tissue-specific Jagged1 enhancer by directly interacting with the Notch intracellular domain (NICD). The Yap-NICD complex is recruited to chromatin by the DNA-binding protein Rbp-J in a Tead-independent fashion. Thus, Hippo signaling can modulate Notch signaling outputs, and components of the Hippo and Notch pathways physically interact. Convergence of Hippo and Notch pathways by the mechanisms described here might be relevant for the function of these signaling cascades in many tissues and in diseases such as cancer. © 2015. Published by The Company of Biologists Ltd.

  19. Endothelial and Smooth Muscle Cell Ion Channels in Pulmonary Vasoconstriction and Vascular Remodeling

    PubMed Central

    Makino, Ayako; Firth, Amy L.; Yuan, Jason X.-J.

    2017-01-01

    The pulmonary circulation is a low resistance and low pressure system. Sustained pulmonary vasoconstriction and excessive vascular remodeling often occur under pathophysiological conditions such as in patients with pulmonary hypertension. Pulmonary vasoconstriction is a consequence of smooth muscle contraction. Many factors released from the endothelium contribute to regulating pulmonary vascular tone, while the extracellular matrix in the adventitia is the major determinant of vascular wall compliance. Pulmonary vascular remodeling is characterized by adventitial and medial hypertrophy due to fibroblast and smooth muscle cell proliferation, neointimal proliferation, intimal, and plexiform lesions that obliterate the lumen, muscularization of precapillary arterioles, and in situ thrombosis. A rise in cytosolic free Ca2+ concentration ([Ca2+]cyt) in pulmonary artery smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction, while increased release of mitogenic factors, upregulation (or downregulation) of ion channels and transporters, and abnormalities in intracellular signaling cascades are key to the remodeling of the pulmonary vasculature. Changes in the expression, function, and regulation of ion channels in PASMC and pulmonary arterial endothelial cells play an important role in the regulation of vascular tone and development of vascular remodeling. This article will focus on describing the ion channels and transporters that are involved in the regulation of pulmonary vascular function and structure and illustrating the potential pathogenic role of ion channels and transporters in the development of pulmonary vascular disease. PMID:23733654

  20. The inhibitory actions of prostaglandins on respiratory smooth muscle

    PubMed Central

    Main, I. H. M.

    1964-01-01

    Prostaglandin E1, in concentrations as low as 1 ng/ml., relaxed isolated tracheal muscle from cat, monkey, rabbit, guinea-pig and ferret. Tracheal muscle from the cat, monkey and rabbit did not exhibit inherent tone and the effect of prostaglandin E1 on these preparations was seen only after a sustained contraction had been produced by a previous dose of acetylcholine or of another agonist. Prostaglandins E2, E3 and F1α also relaxed isolated cat tracheal muscle which had been stimulated by acetylcholine: their activities relative to that of prostaglandin E1 were, respectively, 1.0, 0.2 and 0.002. In the anaesthetized cat prostaglandin E1 increased lung “resistance to inflation” (presumably comparable to bronchial resistance) and the heart rate. In the anaesthetized rabbit and guinea-pig, prostaglandin E1 antagonized the rise in resistance to inflation of the lungs obtained after vagal stimulation or after the intravenous injection of histamine; it sometimes lowered the resistance to inflation in these species. The possibility that prostaglandin may have a local physiological role in the control of bronchial smooth muscle tone is discussed. ImagesFig. 5Fig. 7 PMID:14211681