Stimulation Of Airway Smooth Muscle Research Articles

Dynamic cytosolic Ca2+ and force responses to muscarinic stimulation in airway smooth muscle.

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

EP2 and EP4 receptor antagonists: Impact on cytokine production and β2 -adrenergic receptor desensitization in human airway smooth muscle.

Prostaglandin E2 (PGE2 ) is a key prostanoid known to have both proinflammatory and anti-inflammatory impact in the context of chronic respiratory diseases. We hypothesize that these opposing effects may be the result of different prostanoid E (EP) receptor-mediated signaling pathways. In this study, we focus on two of the four EP receptors, EP2 and EP4 , as they are known to induce cyclic adenosine monophosphate (cAMP)-dependent signaling pathways. Using primary human airway smooth muscle (ASM) cells, we first focussed on the PGE2 -induced production of two cAMP-dependent proinflammatory mediators: interleukin 6 (IL-6) and cyclo-oxygenase 2 production. We show that PGE2 -induced IL-6 protein secretion occurs via an EP2 -mediated pathway, in a manner independent of receptor-mediated effects on messenger RNA (mRNA) expression and temporal activation kinetics of the transcription factor cAMP response element binding. Moreover, stimulation of ASM with PGE2 did not establish a positive, receptor-mediated, feedback loop, as mRNA expression for EP2 and EP4 receptors were not upregulated and receptor antagonists were without effect. Our studies revealed that the EP2 , but not the EP4 , receptor is responsible for β2 -adrenergic desensitization induced by PGE2 . We demonstrate that PGE2 -induced heterologous receptor desensitization responsible for tachyphylaxis to short- (salbutamol) or long- (formoterol) β2 -agonists (measured by cAMP release) can be reversed by the EP2 receptor antagonist PF-04418948. Importantly, this study highlights that inhibiting the EP2 receptor restores β2 -adrenergic receptor function in vitro and offers an attractive novel therapeutic target for treating infectious exacerbations in people suffering from chronic respiratory diseases in the future.

Non‐muscle (NM) Myosin II Mediates the Formation of Adhesome Signaling Complexes in Response to Contractile Stimulation of Airway Smooth Muscle (ASM)

Non‐muscle (NM) Myosin II Mediates the Formation of Adhesome Signaling Complexes in Response to Contractile Stimulation of Airway Smooth Muscle (ASM)

Non‐muscle (NM) and Smooth Muscle (SM) Myosin II Activation are Regulated by Different Mechanisms During Contractile Stimulation of Airway Smooth Muscle (ASM)

Myosin II is the major motor protein in SM, and is expressed as both SM and NM isoforms. However, the function of non‐muscle myosin II in SM is not understood. We hypothesized that NM and SM myosin II may serve different functions during ASM contraction and be regulated by different mechanisms. We detected both NM myosin IIA and IIB heavy chain isoforms in canine ASM tissues. The 20 kD myosin light chain (MLC) is expressed in both NM and SM isoforms, but these isoforms cannot be distinguished immunologically. We used 2‐D gel electrophoresis to distinguish NM and SM MLC isoforms and analyze their phosphorylation in response to acetylcholine (ACh). NM MLC represented a relatively small proportion of the total cellular MLC (20–30%). Both SM and NM MLCs were phosphorylated in similar proportions in response to ACh stimulation (about 50%). When RhoA was inactivated by expressing the inactive RhoA mutant, RhoA T19N, in muscle tissues, NM MLC phosphorylation was substantially inhibited but there was little effect on SM MLC phosphorylation. The MLC kinase inhibitor, ML‐7, suppressed ACh induced SM myosin II LC phosphorylation, but had little or no effect on NM myosin II MLC phosphorylation. We conclude that MLC kinase primarily regulates the phosphorylation of SM MLC, whereas NM MLC phosphorylation depends on RhoA activation. This suggests SM and NM myosin II play distinct roles in the regulation of SM contraction.HL29289, HL074099.

Reverse mode Na+/Ca2+ exchange mediated by STIM1 contributes to Ca2+ influx in airway smooth muscle following agonist stimulation.

BackgroundAgonist stimulation of airway smooth muscle (ASM) results in IP3 mediated Ca2+ release from the sarcoplasmic reticulum followed by the activation of store operated and receptor operated non-selective cation channels. Activation of these non-selective channels also results in a Na+ influx. This localised increase in Na+ levels can potentially switch the Na+/Ca2+ exchanger into reverse mode and so result in a further influx of Ca2+. The aim of this study was to characterise the expression and physiological function of the Na+/Ca2+ exchanger in cultured human bronchial smooth muscle cells and determine its contribution to agonist induced Ca2+ influx into these cells.MethodsThe expression profile of NCX (which encodes the Na+/Ca2+ exchanger) homologues in cultured human bronchial smooth muscle cells was determined by reverse transcriptase PCR. The functional activity of reverse mode NCX was investigated using a combination of whole cell patch clamp, intracellular Ca2+ measurements and porcine airway contractile analyses. KB-R7943 (an antagonist for reverse mode NCX) and target specific siRNA were utilised as tools to inhibit NCX function.ResultsNCX1 protein was detected in cultured human bronchial smooth muscle cells (HBSMC) cells and NCX1.3 was the only mRNA transcript variant detected. A combination of intracellular Na+ loading and addition of extracellular Ca2+ induced an outwardly rectifying current which was augmented following stimulation with histamine. This outwardly rectifying current was inhibited by 10 μM KB-R7943 (an antagonist of reverse mode NCX1) and was reduced in cells incubated with siRNA against NCX1. Interestingly, this outwardly rectifying current was also inhibited following knockdown of STIM1, suggesting for the first time a link between store operated cation entry and NCX1 activation. In addition, 10 μM KB-R7943 inhibited agonist induced changes in cytosolic Ca2+ and induced relaxation of porcine peripheral airways.ConclusionsTaken together, these data demonstrate a potentially important role for NCX1 in control of Ca2+ homeostasis and link store depletion via STIM1 directly with NCX activation.

Potent bronchodilation and reduced stiffness by relaxant stimuli under dynamic conditions

Airway relaxation in response to isoprenaline, sodium nitroprusside (SNP) and electrical field stimulation (EFS) was compared under static and dynamic conditions. The capacity of relaxants to reduce airway stiffness and, thus, potentially contribute to bronchodilation was also investigated. Relaxation responses were recorded in fluid filled bronchial segments from pigs under static conditions and during volume oscillations simulating tidal and twice tidal manoeuvres. Bronchodilation was assessed from the reduction in carbachol-induced lumen pressure, at isovolume points in pressure cycles produced by volume oscillation, and stiffness was assessed from cycle amplitudes. Under static conditions, all three inhibitory stimuli produced partial relaxation of the carbachol-induced contraction. Volume oscillation alone also reduced the contraction in an amplitude-dependent manner. However, maximum relaxation was observed when isoprenaline or SNP were combined with volume oscillation, virtually abolishing contraction at the highest drug concentrations. The proportional effects of isoprenaline and EFS were not different under static or oscillating conditions, whereas relaxation to SNP was slightly greater in oscillating airways. All three inhibitory stimuli also strongly reduced carbachol-induced airway stiffening. The current authors conclude that bronchoconstriction is strongly suppressed by combining the inhibitory stimulation of airway smooth muscle with cyclical mechanical strains. The capacity of airway smooth muscle relaxants to also reduce stiffness may further contribute to bronchodilation.

The force is with α‐actinin: dynamic regulation of the extracellular matrix‐cytoskeletal connection in airway smooth muscle

The force is with α‐actinin: dynamic regulation of the extracellular matrix‐cytoskeletal connection in airway smooth muscle

IL-17A augments TNF-α–induced IL-6 expression in airway smooth muscle by enhancing mRNA stability

IL-17A is implicated in the regulation of inflammation and is found in increased amounts in the asthmatic airway. Human airway smooth muscle (ASM) cells synthesize cell adhesion molecules and cytokines in response to inflammatory mediators. In this study, we examined whether IL-17A modulated the synthetic function of ASM cells. Primary ASM cultures were treated with IL-17A alone and in combination with the proinflammatory cytokines TNF-alpha and IL-1beta. Intercellular adhesion molecule 1 expression, GM-CSF, and IL-6 secretion were measured by ELISA. Examination of transcriptional regulation was performed via transient transfection of promoter constructs, whereas mRNA stability was assessed by actinomycin D chase and quantitative real-time PCR. Airway smooth muscle did not secrete IL-17A after stimulation of ASM with TNF-alpha and IL-1beta. Furthermore, IL-17A (0.1-10 ng/mL) had no effect on TNF-alpha-induced and IL-1beta-induced intercellular adhesion molecule 1 expression or GM-CSF secretion. However, IL-17A (10 ng/mL) significantly augmented TNF-alpha-induced IL-6 secretion 12-fold (TNF-alpha, 2.3 +/- 0.4 ng/mL; IL-17A and TNF-alpha, 27.5 +/- 4.8 ng/mL; P <.05) while having no effect on IL-1beta-induced IL-6. Although IL-17A had no effect on nuclear factor kappaB-mediated transcriptional regulation of IL-6 gene expression induced by TNF-alpha, IL-17A significantly augmented TNF-alpha-induced IL-6 mRNA stability. Collectively, these results demonstrate that IL-17A amplifies the synthetic function of ASM cells, acting via a posttranscriptional pathway, rather than transcriptional mechanisms, to augment TNF-alpha-induced secretion of IL-6 from ASM cells.

Vascular endothelial growth factor-induced secretion of fibronectin is ERK dependent.

In severe asthma, cytokines and growth factors contribute to the proliferation of smooth muscle cells and blood vessels, and to the increased extracellular matrix deposition that constitutes the process of airway remodeling. Vascular endothelial growth factor (VEGF), which regulates vascular permeability and angiogenesis, also modulates the function of nonendothelial cell types. In this study, we demonstrate that VEGF induces fibronectin secretion by human airway smooth muscle (ASM) cells. In addition, stimulation of ASM with VEGF activates ERK, but not p38MAPK, and fibronectin secretion is ERK dependent. Both ERK activation and fibronectin secretion appear to be mediated through the VEGF receptor flt-1, as evidenced by the effects of the flt-1-specific ligand placenta growth factor. Finally, we demonstrate that ASM cells constitutively secrete VEGF, which is increased in response to PDGF, transforming growth factor-beta, IL-1beta, and PGE(2). We conclude that ASM-derived VEGF, through modulation of the extracellular matrix, may play an important role in airway remodeling seen in asthma.

Mitogenic signaling pathways in airway smooth muscle.

Increased airway smooth muscle mass has been demonstrated in patients with asthma, bronchopulmonary dysplasia and most recently, cystic fibrosis. These observations emphasize the need for further knowledge of the events involved in airway smooth muscle mitogenesis and hypertrophy. Workers in the field have developed cell culture systems involving tracheal and bronchial myocytes from different species. An emergent body of literature indicates that mutual signal transduction pathways control airway smooth muscle cell cycle entry across species lines. This article reviews what is known about mitogen-activated signal transduction in airway myocytes. The extracellular signal regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI 3-kinase) pathways appear to be key positive regulators of airway smooth muscle mitogenesis; recent studies have also demonstrated specific roles for reactive oxygen and the JAK/STAT pathway. It is also possible that growth factor stimulation of airway smooth muscle concurrently elicits signaling through negative regulatory intermediates such as p38 mitogen-activated protein (MAP) kinase and protein kinase C (PKC) delta, conceivably as a defense against extreme growth.

Mitogen-activated signaling and cell cycle regulation in airway smooth muscle.

Increased airway smooth muscle mass has been demonstrated in patients with bronchopulmonary dysplasia and asthma. These data highlight the need for a precise understanding of the events involved in airway smooth muscle mitogenesis. To that end, investigators have developed cell culture systems adopting tracheal and bronchial myocytes from different species. A growing body of literature suggests that common signal transduction pathways regulate airway smooth muscle cell cycle entry across species lines. This review summarizes what is known about mitogen-activated signal transduction in airway smooth muscle cells. The extracellular signal regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI 3-kinase) pathways appear to be major positive regulators of airway smooth muscle proliferation. It is also conceivable that growth factor stimulation of airway smooth muscle simultaneously elicits signaling through negative regulatory pathways such as the p38 mitogen-activated protein (MAP) kinase pathway, perhaps as a safeguard against excessive growth.

Requirement for Reactive Oxygen Species in Serum-induced and Platelet-derived Growth Factor-induced Growth of Airway Smooth Muscle

Reactive oxygen species have been recently identified as important mediators of mitogenic signaling in a number of cell types. We therefore explored their role in mediating mitogenesis of airway smooth muscle. The antioxidants catalase, N-acetylcysteine, and probucol significantly reduced proliferation in primary cultures of rat tracheal smooth muscle stimulated with fetal bovine serum or platelet-derived growth factor, without affecting cell viability or inducing apoptosis. N-Acetylcysteine also significantly reduced serum-stimulated elevation of c-Fos but did not prevent the normal mitogen-induced increase in c-fos mRNA. Fractionation of ribosomes by sucrose density centrifugation and subsequent dot-blot Northern analysis revealed that antioxidants reduced incorporation of c-fos mRNA into the heaviest polyribosomes, suggesting redox regulation of c-fos mRNA translation. Serum treatment of monolayers produced a small but reproducibly significant rise in superoxide dismutase-inhibitable reduction of ferricytochrome c by myocyte monolayers. Serum-induced ferricytochrome c reduction, cellular proliferation, and c-Fos elevation were decreased by the flavoprotein-dependent enzyme inhibitor dipheyleneiodonium. Growth responses to fetal bovine serum and superoxide dismutase-inhibitable reduction of ferricytochrome c were not different between cultured tracheal myocytes from wild-type versus gp91 phagocyte oxidase null mice. These results suggest that mitogen stimulation of airway smooth muscle induces signal transduction of cell proliferation that is in part dependent on generation of partially reduced oxygen species, generated by an NADH or NADPH oxidoreductase that is different from the oxidase in phagocytic cells.

Cellular and molecular mechanisms regulating airway smooth muscle proliferation and cell adhesion molecule expression.

Asthma as well as bronchiolitis obliterans and chronic bronchitis are chronic lung diseases characterized by airflow obstruction and airway inflammation. Although asthma typically induces reversible airflow obstruction, in some patients airflow obstruction becomes irreversible. The irreversible airway remodeling that occurs in asthma has been attributed in part to increased smooth muscle mass. However, the precise mechanisms regulating increases in myocyte number in vivo remain unknown. Frequent stimulation of airway smooth muscle by contractile agonists, inflammatory mediators, and growth factors may contribute to stimulation of myocyte proliferation. The recognition of phosphatidylinositol 4,5-bisphosphate hydrolysis as a ubiquitous receptor-activated signaling mechanism has led to the discovery that phospholipids, in particular 3-PI, may play a critical role in signaling cell growth. Infiltrating inflammatory cells may indirectly stimulate myocyte growth and activate PI 3-kinase through secretion of agonists and growth factors. In addition, new evidence reveals that direct cell- cell interaction between immunocytes and airway smooth muscle may also modulate airway smooth muscle cell function. Studies have shown that activated T lymphocytes can adhere to cultured airway smooth muscle, and the functional consequence of this adherence is the upregulation of cell adhesion molecules and the stimulation of DNA synthesis in human airway smooth muscle cells. The identification of the critical regulatory sites that mediate airway smooth muscle cell proliferation or modulate cell adhesion molecule expression in these cells may improve our understanding of the mechanisms that regulate airway inflammation and possibly provide new therapeutic approaches to alter airway remodeling seen in patients with chronic airflow obstruction.

The Effects of Halothane Pretreatment on Manganese Influx Induced by Muscarinic Stimulation of Airway Smooth Muscle

The Effects of Halothane Pretreatment on Manganese Influx Induced by Muscarinic Stimulation of Airway Smooth Muscle

The Effects of Halothane Pretreatment on Manganese Influx Induced by Muscarinic Stimulation of Airway Smooth Muscle

We hypothesized that halothane inhibits contraction of canine airway smooth muscle in part by depleting sarcoplasmic reticulum (SR) calcium stores, which affects subsequent force and calcium influx. This hypothesis was tested by using the rate of quenching of fura-2 fluorescence by manganese (Mn2+) as an index of calcium influx. When added 10 min before submaximum muscarinic stimulation (with 0.3 microM acetylcholine [ACh]), halothane (0.60 +/- 0.04 mM [mean +/- SE]) reduced subsequent isometric force and intracellular calcium concentration ([Ca2+]i) measured 10 min after contraction (to 55%) +/- 5% and 69% +/- 4% of control, respectively). The Mn2+ influx measured concurrently was significantly increased by halothane (by 57% +/- 22%). Depletion of SR calcium stores by ACh prior to contraction also increased Mn2+ influx (by 46% +/- 6%) but did not affect developed force or increase [Ca2+]i in response to submaximum muscarinic stimulation. Halothane did not affect [Ca2+]i or Mn2+ influx when added prior to maximum stimulation with 100 microM ACh but significantly reduced developed force. These findings are consistent with the hypothesis that halothane-induced SR depletion prior to contraction stimulates subsequent calcium influx, but they further suggest that halothane-induced SR depletion itself does not contribute significantly to the reduction in contractility produced by halothane in the canine airway smooth muscle.

Airway hyperresponsiveness: mechanisms and relevance

Airway provocation tests such as the methacholine inhalation test or histamine inhalation test are commonly used for the quantitation of the airway hyperresponsiveness characteristic of asthma. The reduced provocation concentration of methacholine (PC20M) or histamine (PC20H) required to produce a 20% fall in FEV1 may be due to a number of underlying mechanisms, particularly airway mucosal inflammation. The characteristic eosinophilic mucosal inflammation of asthma apparently contributes to hyperresponsiveness through an enhancement of neuromuscular responses to methacholine and histamine in addition to increased responses to various stimuli through the release of inflammatory mediators. Methacholine airway hyperresponsiveness and airway mucosal inflammation usually are both present in asthma, but these findings do not always coexist. Patients with chronic cough and a normal PC20M may have eosinophilic airway inflammation; treatment with inhaled steroid can reduce the cough, sputum production and the number of eosinophils and mast cells in the sputum. Airway responses in provocation tests with histamine or methacholine seem to depend, at least in part, upon direct stimulation of airway smooth muscle and have been referred to as direct tests of airway responsiveness. In contrast, tests with stimuli such as allergen or adenosine, which elicit mediator release, have been referred to as indirect tests of airway hyperresponsiveness. There are complex interrelationships and an interdependence between methacholine airway hyperresponsiveness and airway inflammation; progress in the assessment of underlying mechanisms has been greatly enhanced by biochemical, immunologic and pathologic-type measurements in various types of specimens from the airways to directly assess the pathogenetic processes. Some features of asthma such as exercise and nocturnal symptoms correlate well with the level of methacholine airway hyperresponsiveness. Conversely, the occurrence of cough or the occurrence of asthmatic attacks are not predicted accurately by a measurement of methacholine airway hyperresponsiveness. The assessment of asthma severity seems to require the consideration of a number of types of parameters including the level of ongoing symptoms, need for inhaled B2-agonist, frequency and severity of acute attacks, level of treatment required to maintain control of asthma, baseline airflow rates and tests of variability of airflow such as serial measurements of variability of PEF or of PC20M. Measurement of the PC20M is a sensitive test of changes in airway responsiveness and can be useful in serial measurements. In some instances, airway reactions can be identified by serial measurements of PC20M but not by serial measurement of airway calibre such as the FEV1.

Halothane Changes the Relationships Between Lung Resistances and Lung Volume

The authors hypothesized that relaxation of airway smooth muscle by halothane lessens the dependence of airway resistance on lung volume, and that halothane alters the relationship between pulmonary resistance and lung volume by changing both the airway and tissue components of pulmonary resistance. The relationship among airway resistance, tissue resistance, and lung volume was examined in mongrel dogs before and during the administration of halothane, both in airways with reduced smooth muscle tone (after vagotomy) and during moderate increases in smooth muscle tone caused by vagus nerve stimulation (VNS). Resistance were measured at several levels of positive end-expiratory pressure (PEEP, 4-15 cmH2O) using an alveolar capsule technique. Before halothane administration, airway resistance increased at low PEEP; VNS accentuated this increase. Tissue resistance increased at low PEEP only during VNS. Halothane had no significant effect on any resistance before VNS. During VNS, halothane markedly blunted increases in airway resistance and tissue resistance as PEEP decreased. The authors conclude that during stimulation of airway smooth muscle in dogs, halothane attenuates increases in airway resistance and tissue resistance with reductions in lung volume in dogs. Thus, moderate changes in lung volume have little effect on these resistances during halothane anesthesia under these conditions.

Muscarinic regulation of membrane ion channels in airway smooth muscle cells

We have demonstrated that stimulation of airway smooth muscle by muscarinic agonists results in a coordinated modulation of two membrane ion channel proteins. Both channels are modulated in a similar way, although their effects on open-channel probability are opposite. The voltage dependence of channel activity is shifted to more positive potentials in the case of K Ca, and to more negative potentials in the case of the voltage-dependent calcium channels. Similarly, K Ca channel dwell-time kinetics are shifted to short open lifetimes, whereas the long open state is favored for the large-amplitude voltage-dependent calcium channel. Although little is known about the molecular coupling of calcium channels, muscarinic inhibition of K Ca channels is mediated through a pertussis toxin-sensitive guanine nucleotide binding protein.