Tracheal Smooth Muscle Tissues Research Articles

Rosuvastatin relaxes rat thoracic aorta, pulmonary artery, and trachea via nitric oxide, prostanoids, and potassium channels

Purpose: This study aimed to determine the functional effects and mechanisms of the action of rosuvastatin on vascular and tracheal smooth muscle tissues. 
 Materials and Methods: Vascular and tracheal rings (2-3 mm) isolated from the thoracic aortas, pulmonary arteries, and tracheas of Wistar Albino male rats (250-300 g) were placed in chambers in the isolated tissue bath system. As the resting tension, 1 g was selected. Vascular rings contracted with 10-6 M phenylephrine after a 90-minute equilibration period. Tracheal rings contracted with 10-5 M acetylcholine. After the contraction was steady, rosuvastatin (10-8-10-4 M) was cumulatively applied to the vascular and tracheal rings. The defined experimental methodology was repeated following the incubation of selective inhibitors of signaling pathways and K+ channel blockers to ascertain rosuvastatin's functional effect mechanisms. 
 Results: In the precontracted rat vascular and tracheal rings, rosuvastatin induced concentration-dependent relaxation. The maximal relaxation level in vessel samples was 96%. On the other hand, the maximal relaxation level in tracheal samples was found to be 75%. The vasorelaxant effects of rosuvastatin were dramatically attenuated by endothelium removal, L-NAME treatment, and indomethacin incubation (up to 27%). With the incubation of tetraethylammonium, glyburide, 4-Aminopyridine, and anandamide, rosuvastatin-mediated vascular smooth muscle relaxation levels were significantly decreased (up to 38%). Moreover, With the incubation of tetraethylammonium, glyburide, and 4-Aminopyridine rosuvastatin-mediated tracheal smooth muscle relaxation levels were significantly decreased (up to 30%). 
 Conclusion: Rosuvastatin has a noticeable relaxing effect on the vascular and tracheal smooth muscles. The vasorelaxant effect of rosuvastatin involves intact endothelium, nitric oxide, prostanoids, and K+ channels (BKCa, KV, and KATP channels). Furthermore, nitric oxide, prostanoids, BKCa channels, KV channels, and KATP channels play a role in rosuvastatin-induced tracheal smooth muscle relaxation

Relaxant Effect of Urginea maritima on Tracheal Smooth Muscle Mediated by the Effect on Beta-2 Adrenergic, Muscarinic Receptors and Calcium and Potassium Channels.

Urginea maritima (U. maritima) showed anti-inflammatory, antioxidant, antibacterial, diuretic, vasodilatation, and wound-healing effects on fungal infections, cardiac disorders, digestive disorders, rheumatoid disease, and respiratory disorders such as bronchitis, bronchial nosocomial infections, and severe cough. To examine the bronchodilatory effect of U. maritima, the relaxant effect of its extract on rat tracheal smooth muscle (TSM) and its possible mechanism was examined in this study. Male Wistar rats' TSM were divided into eight groups (n = 8 in each group). Four of these groups were TSM tissues, contracted with KCl (60 mM) incubated with atropine, glibenclamide, and indomethacin and nonincubated TSM, while the other four groups were TSM tissues contracted with methacholine (10 μM) for 5 min, incubated with propranolol, chlorpheniramine, and diltiazem and nonincubated TSM. Cumulative concentrations of U. maritima extract (12.5, 25, 50, 100, 20, and 400 μg/ml) were then added to organ bath every 5 min. Theophylline (0.2, 0.4, 0.6, and 0.8 mM) as positive control and saline (1 ml) as negative control were also examined in nonincubated tissues. A concentration-dependent relaxant effect of U. maritima on nonincubated TSM contracted with KCl (60 mM) or methacholine (10 μM) (p < 0.01 and p < 0.001) was observed. The relaxant effects of U. maritima extract in the incubated tissues with glibenclamide, propranolol, diltiazem, atropine, and chlorpheniramine were significantly lower than those in the nonincubated tissues (p < 0.05 to p < 0.001). EC50 values of U. maritima extract in the incubated TSM with glibenclamide, propranolol, diltiazem, and atropine were significantly higher than those in the nonincubated tissues (p < 0.05 for diltiazem-incubated tissues and p < 0.001 for other cases). U. maritima extract displayed considerable relaxant effect on TSM comparable to the effect of theophylline. Beta-2 adrenoceptor stimulation and muscarinic receptor inhibition as well as potassium opening and calcium channels blocking effects are the possible mechanisms for the relaxant effects of the plant.

Na+/Ca2+ Exchanger 1 in Airway Smooth Muscle of Allergic Inflammation Mouse Model.

Cytosolic free Ca2+ ([Ca2+]cyt) is essential for airway contraction, secretion and remodeling. [Ca2+]cyt homeostasis is controlled by several critical molecules, one of which is the Na+/Ca2+ exchanger 1 (NCX1) in the plasma membrane. Since little is currently known about NCX1 in the airway smooth muscle and its involvement in airway diseases, the present study was designed to investigate the expression and function of NCX1 in normal airway smooth muscle and its relevance to airway inflammation. Western blot analysis, tracheal smooth muscle contraction, and [Ca2+]cyt measurements were performed in mouse tracheal smooth muscle tissues and primary airway smooth muscle cell cultures. Additional studies were performed in a mouse model of allergic airway inflammation. Our data showed that NCX1 proteins were expressed in the human bronchial smooth muscle cells (HBSMCs), murine airway and whole lung. Carbachol raised [Ca2+]cyt in mouse tracheal smooth muscle cells and induced murine tracheal contraction, all of which were significantly attenuated by KB-R7943, a selective NCX inhibitor. Removal of extracellular Na+ increased [Ca2+]cyt in HBSMCs and mouse tracheal SMCs, which was dependent on extracellular Ca2+ and sensitive to KB-R7943. TNF-α treatment of HBSMCs significantly upregulated mRNA and protein expression of NCX1 and enhanced NCX activity. Finally, KB-R7943 abolished the airway hyperresponsiveness to methacholine in an ovalbumin-induced mouse model of allergic airway inflammation. Together, these findings indicate that NCX1 in airway smooth muscle may play an important role in the development of airway hyperresponsiveness, and downregulation or inhibition of NCX1 may serve as a potential therapeutic approach for asthma.

Rho kinase collaborates with p21-activated kinase to regulate actin polymerization and contraction in airway smooth muscle.

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. We found that ROCK regulates airway smooth muscle contraction by mediating activation of p21-activated kinase (Pak), a serine-threonine kinase, to promote actin polymerization. Pak catalyses paxillin phosphorylation on Ser273 and coupling of the GIT1-βPIX-Pak signalling module to paxillin, which activates the guanine nucleotide exchange factor (GEF) activity of βPIX towards Cdc42. Cdc42 is required for the activation of neuronal Wiskott-Aldrich syndrome protein (N-WASp), which transmits signals from Cdc42 to the Arp2/3 complex for the nucleation of actin filaments. Our 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.

Phosphodiesterase 9 (PDE9) regulates bovine tracheal smooth muscle relaxation.

The present study was designed to clarify phosphodiesterase 9 (PDE9) expression in bovine tracheal smooth muscle tissue, and to elucidate that PDE9 may contribute to the regulation of airway relaxation. PDE9 mRNA expression was detected in bovine tracheal smooth muscle. Sodium nitroprusside (an NO donor) and BAY 73-6691 (a selective PDE9 inhibitor) reduced high K+- and carbachol-induced contraction. BAY 73-6691 relaxed tracheal tissue on the same level with vardenafil (a selective PDE5 inhibitor). These results support our hypothesis that PDE9 plays functional role in the tracheal smooth muscle relaxation. PDE9 inhibitors are expected to be a novel target of the add-on treatment of airway hyperresponsiveness.

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

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. RhoA inactivation selectively inhibited phosphorylation of the NM myosin regulatory light chain (RLC), NM myosin filament assembly and contraction, although it did not inhibit SM RLC phosphorylation. We conclude that the assembly and activation of NM myosin II is regulated during contractile stimulation of airway SM tissues by RhoA-mediated NM myosin RLC phosphorylation and by NM myosin heavy chain Ser1943 phosphorylation. NM myosin II actomyosin cross-bridge cycling regulates the assembly of membrane adhesome complexes that mediate the cytoskeletal processes required for tension generation. NM myosin II plays a critical role in airway SM contraction that is independent and distinct from the function of SM myosin.

Viscoelastic Properties of Human Tracheal Tissues.

The physiological performance of trachea is highly dependent on its mechanical behavior, and therefore, the mechanical properties of its components. Mechanical characterization of trachea is key to succeed in new treatments such as tissue engineering, which requires the utilization of scaffolds which are mechanically compatible with the native human trachea. In this study, after isolating human trachea samples from brain-dead cases and proper storage, we assessed the viscoelastic properties of tracheal cartilage, smooth muscle, and connective tissue based on stress relaxation tests (at 5% and 10% strains for cartilage and 20%, 30%, and 40% for smooth muscle and connective tissue). After investigation of viscoelastic linearity, constitutive models including Prony series for linear viscoelasticity and quasi-linear viscoelastic, modified superposition, and Schapery models for nonlinear viscoelasticity were fitted to the experimental data to find the best model for each tissue. We also investigated the effect of age on the viscoelastic behavior of tracheal tissues. Based on the results, all three tissues exhibited a (nonsignificant) decrease in relaxation rate with increasing the strain, indicating viscoelastic nonlinearity which was most evident for cartilage and with the least effect for connective tissue. The three-term Prony model was selected for describing the linear viscoelasticity. Among different models, the modified superposition model was best able to capture the relaxation behavior of the three tracheal components. We observed a general (but not significant) stiffening of tracheal cartilage and connective tissue with aging. No change in the stress relaxation percentage with aging was observed. The results of this study may be useful in the design and fabrication of tracheal tissue engineering scaffolds.

Mechanical Characterization and Constitutive Modeling of Human Trachea: Age and Gender Dependency.

Tracheal disorders can usually reduce the free lumen diameter or wall stiffness, and hence limit airflow. Trachea tissue engineering seems a promising treatment for such disorders. The required mechanical compatibility of the prepared scaffold with native trachea necessitates investigation of the mechanical behavior of the human trachea. This study aimed at mechanical characterization of human tracheas and comparing the results based on age and gender. After isolating 30 human tracheas, samples of tracheal cartilage, smooth muscle, and connective tissue were subjected to uniaxial tension to obtain force-displacement curves and calculate stress-stretch data. Among several models, the Yeoh and Mooney-Rivlin hyperelastic functions were best able to describe hyperelastic behavior of all three tracheal components. The mean value of the elastic modulus of human tracheal cartilage was calculated to be 16.92 ± 8.76 MPa. An overall tracheal stiffening with age was observed, with the most considerable difference in the case of cartilage. Consistently, we noticed some histological alterations in cartilage and connective tissue with aging, which may play a role in age-related tracheal stiffening. No considerable effect of gender on the mechanical behavior of tracheal components was observed. The results of this study can be applied in the design and fabrication of trachea tissue engineering scaffolds.

Vinculin Phosphorylation at Tyr1065 Regulates Vinculin Conformation and Tension Development in Airway Smooth Muscle Tissues

Vinculin localizes to membrane adhesion junctions in smooth muscle tissues, where its head domain binds to talin and its tail domain binds to filamentous actin, thus linking actin filaments to the extracellular matrix. Vinculin can assume a closed conformation, in which the head and tail domains bind to each other and mask the binding sites for actin and talin, and an open activated conformation that exposes the binding sites for talin and actin. Acetylcholine stimulation of tracheal smooth muscle tissues induces the recruitment of vinculin to the cell membrane and its interaction with talin and actin, which is required for active tension development. Vinculin phosphorylation at Tyr(1065) on its C terminus increases concurrently with tension development in tracheal smooth muscle tissues. In the present study, the role of vinculin phosphorylation at Tyr(1065) in regulating the conformation and function of vinculin during airway smooth muscle contraction was evaluated. Vinculin constructs with point mutations at Tyr(1065) (vinculin Y1065F and vinculin Y1065E) and vinculin conformation-sensitive FRET probes were expressed in smooth muscle tissues to determine how Tyr(1065) phosphorylation affects smooth muscle contraction and the conformation and cellular functions of vinculin. The results show that vinculin phosphorylation at tyrosine 1065 is required for normal tension generation in airway smooth muscle during contractile stimulation and that Tyr(1065) phosphorylation regulates the conformation and scaffolding activity of the vinculin molecule. We conclude that the phosphorylation of vinculin at tyrosine 1065 provides a mechanism for regulating the function of vinculin in airway smooth muscle in response to contractile stimulation.

Protective effect of high-dose montelukast on salbutamol-induced homologous desensitisation in airway smooth muscle

Montelukast (MK) is a potent cysteinyl-leukotriene receptor antagonist that causes dose-related improvements in chronic asthma. We sought to determine whether MK was able to prevent salbutamol-induced tolerance in airway smooth muscle. Homologous β2-adrenoceptor desensitisation models were established in guinea-pigs and in human bronchial smooth muscle cells (BSMC) by chronic salbutamol administration. Characterisation tools included measurement of the response of tracheal smooth muscle tissues to salbutamol, analysis of gene expression and receptor trafficking, evaluation of intracellular cAMP levels and phosphodiesterase (PDE) activity in human bronchial smooth muscle cells. Salbutamol-induced β2-adrenoceptor desensitisation was characterised by β2-agonist hyporesponsiveness (−30%, p < 0.001) in desensitised tracheal smooth muscle, as compared to controls. MK, given intraperitoneally at 5 mg/kg/day for 6 consecutive days, completely restored tissue responsiveness to salbutamol. Prolonged salbutamol treatment significantly decreased cAMP synthesis, induced a complete removal of the β2-adrenoceptor from plasma membrane with a parallel increase in the cytosol and increased PDE4D5 gene transcription and PDE activity in human bronchial smooth muscle cells. In homologously desensitised BSMC, MK 30 μM for 24 h was able to prevent salbutamol subsensitivity and such an effect was associated with inhibition of salbutamol-induced PDE4 activity and restoration of membrane β2-adrenoceptor expression and function. These findings suggest the presence of a favourable interaction between MK and β2-adrenoceptor agonists that might improve the therapeutic index of bronchodilators in patients with chronic respiratory diseases.

Increased mechanical strain imposed on murine lungs during ventilation in vivo depresses airway responsiveness and activation of protein kinase Akt

Continuous positive airway pressure (CPAP) administered to tracheostomized rabbits and ferrets for 4 days or 2 wk suppresses bronchial reactivity in vivo and suppresses airway reactivity in lobes and tracheal segments isolated from these animals. In vitro studies of canine tracheal smooth muscle tissues indicate that mechanical loading suppresses the activation of the growth regulatory kinase, Akt, and that Akt is a negative regulator of smooth muscle differentiation. The transduction of mechanical signals in the tracheal tissues in vitro is mediated by integrin-associated adhesion complexes. To determine whether airway responsiveness and Akt activation are modulated by mechanical loads applied for short time periods to the airways of living animals in vivo, mice were mechanically ventilated for 2 h with high (5 cmH2O) or low (0-1 cmH2O) positive end-expiratory pressure (PEEP) and then ventilated at low PEEP for 30 min. Ventilation of mice with PEEP in vivo for 2 h depressed airway responsiveness to methacholine measured in vivo subsequent to the PEEP treatment. Airway narrowing in vitro in intraparenchymal airways in isolated lung slices and contractile responses of isolated tracheal segments in vitro were suppressed for at least 6 h subsequent to the in vivo exposure to PEEP. Tracheal segments isolated from high PEEP-treated mice exhibited significantly lower levels of Akt activation than tracheae from low PEEP-treated mice. The results indicate that mechanical loads imposed in vivo result in physiological and biochemical changes in the airway tissues after a relatively short 2-h period of in vivo loading.

Mechanical stimuli and IL-13 interact at integrin adhesion complexes to regulate expression of smooth muscle myosin heavy chain in airway smooth muscle tissue

Airway smooth muscle phenotype may be modulated in response to external stimuli under physiological and pathophysiological conditions. The effect of mechanical forces on airway smooth muscle phenotype were evaluated in vitro by suspending weights of 0.5 or 1 g from the ends of canine tracheal smooth muscle tissues, incubating the weighted tissues for 6 h, and then measuring the expression of the phenotypic marker protein, smooth muscle myosin heavy chain (SmMHC). Incubation of the tissues at a high load significantly increased expression of SmMHC compared with incubation at low load. Incubation of the tissues at a high load also decreased activation of PKB/Akt, as indicated by its phosphorylation at Ser 473. Inhibition of Akt or phosphatidylinositol-3,4,5 triphosphate-kinase increased SmMHC expression in tissues at low load but did not affect SmMHC expression at high load. IL-13 induced a significant increase in Akt activation and suppressed the expression of SmMHC protein at both low and high loads. The role of integrin signaling in mechanotransduction was evaluated by expressing a PINCH (LIM1-2) fragment in the muscle tissues that prevents the membrane localization of the integrin-binding IPP complex (ILK/PINCH/α-parvin), and also by expressing an inactive integrin-linked kinase mutant (ILK S343A) that inhibits endogenous ILK activity. Both mutants inhibited Akt activation and increased expression of SmMHC protein at low load but had no effect at high load. These results suggest that mechanical stress and IL-13 both act through an integrin-mediated signaling pathway to oppositely regulate the expression of phenotypic marker proteins in intact airway smooth muscle tissues. The stimulatory effects of mechanical stress on contractile protein expression oppose the suppression of contractile protein expression mediated by IL-13; thus the imposition of mechanical strain may inhibit changes in airway smooth muscle phenotype induced by inflammatory mediators.

Activation of Vinculin Induced by Cholinergic Stimulation Regulates Contraction of Tracheal Smooth Muscle Tissue

Vinculin localizes to membrane adhesion junctions where it links actin filaments to the extracellular matrix by binding to the integrin-binding protein talin at its head domain (Vh) and to actin filaments at its tail domain (Vt). Vinculin can assume an inactive (closed) conformation in which Vh and Vt bind to each other, masking the binding sites for actin and talin, and an active (open) conformation in which the binding sites for talin and actin are exposed. We hypothesized that the contractile activation of smooth muscle tissues might regulate the activation of vinculin and thereby contribute to the regulation of contractile tension. Stimulation of tracheal smooth muscle tissues with acetylcholine (ACh) induced the recruitment of vinculin to cell membrane and its interaction with talin and increased the phosphorylation of membrane-localized vinculin at the C-terminal Tyr-1065. Expression of recombinant vinculin head domain peptide (Vh) in smooth muscle tissues, but not the talin-binding deficient mutant head domain, VhA50I, inhibited the ACh-induced recruitment of endogenous vinculin to the membrane and the interaction of vinculin with talin and also inhibited vinculin phosphorylation. Expression of Vh peptide also inhibited ACh-induced smooth muscle contraction and inhibited ACh-induced actin polymerization; however, it did not affect myosin light chain phosphorylation, which is necessary for cross-bridge cycling. Inactivation of RhoA inhibited vinculin activation in response to ACh. We conclude that ACh stimulation regulates vinculin activation in tracheal smooth muscle via RhoA and that vinculin activation contributes to the regulation of active tension by facilitating connections between actin filaments and talin-integrin adhesion complexes and by mediating the initiation of actin polymerization.

Effect of off-axis cell orientation on mechanical properties in smooth muscle tissue

The cell alignment in a smooth muscle tissue plays a significant role in determining its mechanical proper-ties. The off-axis cell orientation "θ” not only effects the shortening strain but also modifies the shear stress relationship significantly. Both experiments and finite element analysis were carried out on a tracheal smooth muscle strip to study how the cell alignment in smooth muscle affects the shear stiffness and shear stresses as well as deformation. A simple model for shear stiffness is derived using the data from experiments. Shear stiffness results obtained from the model indicate that the muscle shear stiff-ness values increase non-linearly with strain and with higher off-axis alignment of cells. Results of deforma-tion and shear stresses obtained from finite element analsysis indicate that the maximum shear stress values of tracheal smooth muscle tissue at 45% of strain are 2.5 times the corresponding values at 20% of strain for all three off-axis cell orientation values θ = 15?, 30? and 45?.

Rosiglitazone reverses salbutamol-induced β2-adrenoceptor tolerance in airway smooth muscle

β₂-Adrenoceptor agonists are important therapeutic agents in the treatment of asthma and chronic obstructive pulmonary disease. The regular use of these drugs has been associated with proasthmatic-like changes that limit their efficacy and increase the risk of severe adverse reactions. We investigated whether the peroxisome-proliferator-activated receptor (PPAR)γ agonist rosiglitazone modulated salbutamol-induced β₂-adrenoceptor desensitization in vivo and in vitro. An in vivo model of homologous β₂-adrenoceptor desensitization, established in guinea-pigs by administering salbutamol continuously, was used to study the ability of rosiglitazone to prevent β₂-adrenoceptor tolerance. In vitro experiments on human bronchial smooth muscle cells were performed to increase the clinical relevance of the study. In tracheal smooth muscle tissues from desensitized animals, we observed a decrease in the protective effect of salbutamol on carbachol-induced contraction, a hyperresponsiveness to cholinergic stimuli, a modest underexpression of β₂-adrenoceptor gene and a marked decrease in β-adrenoceptor number, relative to control values. Treatment with rosiglitazone preserved salbutamol relaxant activity, mitigated carbachol hyperresponsiveness and partially restored β₂-adrenoceptor binding sites in tracheal tissues from homologously desensitized animals. The highly selective PPARγ agonist, GW1929, reproduced the effect of rosiglitazone, in vivo. In vitro β₂-adrenoceptor desensitization decreased salbutamol-mediated cAMP production, without affecting forskolin responses and β₂-adrenoceptor expression. Rosiglitazone and 15-deoxy-Δ¹²(,)¹⁴-prostaglandin J₂ restored salbutamol sensitivity in homologously desensitized cells. These data suggest a potential pharmacodynamic interaction between PPARγ agonists and salbutamol on airway smooth muscle responsiveness, supporting the therapeutic potential of this combination in chronic airway disease.

The effects of the small GTPase RhoA on the muscarinic contraction of airway smooth muscle result from its role in regulating actin polymerization

The small GTPase RhoA increases the Ca(2+) sensitivity of smooth muscle contraction and myosin light chain (MLC) phosphorylation by inhibiting the activity of MLC phosphatase. RhoA is also a known regulator of cytoskeletal dynamics and actin polymerization in many cell types. In airway smooth muscle (ASM), contractile stimulation induces MLC phosphorylation and actin polymerization, which are both required for active tension generation. The objective of this study was to evaluate the primary mechanism by which RhoA regulates active tension generation in intact ASM during stimulation with acetylcholine (ACh). RhoA activity was inhibited in canine tracheal smooth muscle tissues by expressing the inactive RhoA mutant, RhoA T19N, in the intact tissues or by treating them with the cell-permeant RhoA inhibitor, exoenzyme C3 transferase. RhoA inactivation reduced ACh-induced contractile force by approximately 60% and completely inhibited ACh-induced actin polymerization but inhibited ACh-induced MLC phosphorylation by only approximately 20%. Inactivation of MLC phosphatase with calyculin A reversed the reduction in MLC phosphorylation caused by RhoA inactivation, but calyculin A did not reverse the depression of active tension and actin polymerization caused by RhoA inactivation. The MLC kinase inhibitor, ML-7, inhibited ACh-induced MLC phosphorylation by approximately 80% and depressed active force by approximately 70% but did not affect ACh-induced actin polymerization, demonstrating that ACh-stimulated actin polymerization occurs independently of MLC phosphorylation. We conclude that the RhoA-mediated regulation of ACh-induced contractile tension in ASM results from its role in mediating actin polymerization rather than from effects on MLC phosphatase or MLC phosphorylation.

Cigarette smoke and lipopolysaccharide induce a proliferative airway smooth muscle phenotype

BackgroundA major feature of chronic obstructive pulmonary disease (COPD) is airway remodelling, which includes an increased airway smooth muscle (ASM) mass. The mechanisms underlying ASM remodelling in COPD are currently unknown. We hypothesized that cigarette smoke (CS) and/or lipopolysaccharide (LPS), a major constituent of CS, organic dust and gram-negative bacteria, that may be involved in recurrent airway infections and exacerbations in COPD patients, would induce phenotype changes of ASM.MethodsTo this aim, using cultured bovine tracheal smooth muscle (BTSM) cells and tissue, we investigated the direct effects of CS extract (CSE) and LPS on ASM proliferation and contractility.ResultsBoth CSE and LPS induced a profound and concentration-dependent increase in DNA synthesis in BTSM cells. CSE and LPS also induced a significant increase in BTSM cell number, which was associated with increased cyclin D1 expression and dependent on activation of ERK 1/2 and p38 MAP kinase. Consistent with a shift to a more proliferative phenotype, prolonged treatment of BTSM strips with CSE or LPS significantly decreased maximal methacholine- and KCl-induced contraction.ConclusionsDirect exposure of ASM to CSE or LPS causes the induction of a proliferative, hypocontractile ASM phenotype, which may be involved in airway remodelling in COPD.

The small GTPase RhoA Stimulates the Contraction of Airway Smooth Muscle (ASM) by Regulating Actin Polymerization rather than Myosin Light Chain (MLC) Phosphorylation

Activation of RhoA has been shown to inhibit MLC phosphatase activity, thus increasing MLC phosphorylation and force in some smooth muscle. In ASM, actin polymerization is required for active contraction in addition to MLC phosphorylation. RhoA can regulate cytoskeletal processes and actin polymerization. We evaluated whether RhoA regulated the contraction of ASM primarily through its effects on MLC phosphorylation or actin polymerization. RhoA activity was inhibited in canine tracheal smooth muscle tissues by expressing the inactive RhoA mutant, RhoA T19N, or by treating tissues with the cell permeant RhoA inhibitor, C3 exoenzyme. RhoA inactivation inhibited ACh‐induced contractile force by 60–70%, inhibited ACh‐induced actin polymerization, and also caused a small (10–20%) decrease in MLC phosphorylation. Tissues were treated with the phosphatase inhibitor, calyculin A, by inactivate MLC phosphatase. Calyculin A induced MLC phosphorylation and contraction in the absence of ACh and potentiated MLC phosphorylation induced by ACh, but it did not affect ACh stimulated contraction or actin polymerization. However, inhibition of MLC phosphatase by calyculin A did not prevent the depression of force caused by the RhoA inactivation. This suggests RhoA activation regulates ASM contraction primarily through its effects on actin dynamics.Supported by HL29289, HL074099, ALA, and NIH T32 Grant GM12453‐03

HSP20 phosphorylation and airway smooth muscle relaxation

HSP20 (HSPB6) is a small heat shock protein expressed in smooth muscles that is hypothesized to inhibit contraction when phosphorylated by cAMP-dependent protein kinase. To investigate this hypothesis in airway smooth muscle (ASM) we showed that HSP20 was constitutively expressed as well as being inducible in cultured hASM cells by treatment with 1 μM isoproterenol or 10 μM salmeterol. In contrast, a mixture of proinflammatory mediators (interleukin-1β, tumor necrosis factor α, and interferon γ) inhibited expression of HSP20 by about 50% in 48 hours. To determine whether phosphorylation of HSP20 is sufficient to induce relaxation, canine tracheal smooth muscle was treated with a cell permeant phosphopeptide that mimics the phosphorylation of HSP20. The HSP20 phosphopeptide antagonized carbachol-induced contraction by 60% with no change in myosin light chain phosphorylation. Recombinant full length HSP20 inhibited skeletal actin binding to smooth muscle myosin subfragment 1 (S1), and recombinant cell permeant TAT-HSP20 S16D mutant reduced F-actin filaments in cultured hASM cells. Carbachol stimulation of canine tracheal smooth muscle tissue caused redistribution of HSP20 from large macromolecular complexes (200-500 kDa) to smaller complexes (<60 kDa). The results are consistent with HSP20 expression and macromolecular structure being dynamically regulated in airway smooth muscle. HSP20 is upregulated by beta agonists and downregulated by proinflammatory cytokines. HSP20 is phosphorylated in vivo in a cAMP-dependent manner and the phosphorylated form promotes airway smooth muscle relaxation, possibly through depolymerization of F-actin as well as inhibition of myosin binding to actin.

Actin Depolymerization Factor/Cofilin Activation Regulates Actin Polymerization and Tension Development in Canine Tracheal Smooth Muscle

The contractile activation of airway smooth muscle tissues stimulates actin polymerization, and the inhibition of actin polymerization inhibits tension development. Actin-depolymerizing factor (ADF) and cofilin are members of a family of actin-binding proteins that mediate the severing of F-actin when activated by dephosphorylation at serine 3. The role of ADF/cofilin activation in the regulation of actin dynamics and tension development during the contractile activation of smooth muscle was evaluated in intact canine tracheal smooth muscle tissues. Two-dimensional gel electrophoresis revealed that ADF and cofilin exist in similar proportions in the muscle tissues, and that approximately 40% of the total ADF/cofilin in unstimulated tissues is phosphorylated. Phospho-ADF/cofilin decreased concurrently with tension development in response to stimulation with acetylcholine (ACh) or potassium depolarization indicating the activation of ADF/cofilin. Expression of an inactive phospho-cofilin mimetic (cofilin S3E) but not wild type cofilin in the smooth muscle tissues inhibited endogenous ADF/cofilin dephosphorylation and ACh-induced actin polymerization. Expression of cofilin S3E in the tissues depressed tension development in response to ACh, but it did not affect myosin light chain phosphorylation. The ACh-induced dephosphorylation of ADF/cofilin required the Ca2+-dependent activation of calcineurin (PP2B). The results indicate that the activation of ADF/cofilin is regulated by contractile stimulation in tracheal smooth muscle and that cofilin activation is required for actin polymerization and tension development in response to contractile stimulation.