Susan J. Gunst

Airway smooth muscle dynamics: a common pathway of airway obstruction in asthma

Excessive airway obstruction is the cause of symptoms and abnormal lung function in asthma. As airway smooth muscle (ASM) is the effecter controlling airway calibre, it is suspected that dysfunction of ASM contributes to the pathophysiology of asthma. However, the precise role of ASM in the series of events leading to asthmatic symptoms is not clear. It is not certain whether, in asthma, there is a change in the intrinsic properties of ASM, a change in the structure and mechanical properties of the noncontractile components of the airway wall, or a change in the interdependence of the airway wall with the surrounding lung parenchyma. All these potential changes could result from acute or chronic airway inflammation and associated tissue repair and remodelling. Anti-inflammatory therapy, however, does not “cure” asthma, and airway hyperresponsiveness can persist in asthmatics, even in the absence of airway inflammation. This is perhaps because the therapy does not directly address a fundamental abnormality of asthma, that of exaggerated airway narrowing due to excessive shortening of ASM. In the present study, a central role for airway smooth muscle in the pathogenesis of airway hyperresponsiveness in asthma is explored.

Actin polymerization stimulated by contractile activation regulates force development in canine tracheal smooth muscle.

1 The role of actin polymerization in the regulation of smooth muscle contractility was investigated in canine trachealis muscle strips. The effect of contractile activation on the content of monomeric globular (G)‐actin was estimated by the method of DNase I inhibition. The G‐actin content was 30 % lower in extracts of muscle strips activated with 10−4 M acetylcholine (ACh) than in extracts from unstimulated muscle strips. The decrease in G‐actin in response to contractile stimulation was prevented by latrunculin‐A, an agent that prevents actin polymerization by binding to G‐actin monomers. 2 The inhibition of actin polymerization by latrunculin‐A markedly depressed force development in response to ACh but had no effect on ACh‐induced myosin light chain (MLC) phosphorylation. Latrunculin also suppressed the length sensitivity of force during ACh‐induced isometric contractions. The actin‐capping agent cytochalasin‐D also markedly inhibited force and caused only a slight decrease in MLC phosphorylation. Cytochalasin‐D also inhibited force in α‐toxin‐permeabilized muscle strips that were activated either by Ca2+ or by ACh at constant pCa. No disorganization of smooth muscle cell ultrastructure was detected by electron microscopy or by immunofluorescence microscopy of muscles treated with either agent. 3 The results suggest that the polymerization of actin is stimulated by the contractile activation of tracheal smooth muscle and that this actin polymerization contributes directly to force development. In addition, actin filament remodelling contributes to the length sensitivity of tracheal smooth muscle contractility.

Actin cytoskeletal dynamics in smooth muscle: a new paradigm for the regulation of smooth muscle contraction

A growing body of data supports a view of the actin cytoskeleton of smooth muscle cells as a dynamic structure that plays an integral role in regulating the development of mechanical tension and the material properties of smooth muscle tissues. The increase in the proportion of filamentous actin that occurs in response to the stimulation of smooth muscle cells and the essential role of stimulus-induced actin polymerization and cytoskeletal dynamics in the generation of mechanical tension has been convincingly documented in many smooth muscle tissues and cells using a wide variety of experimental approaches. Most of the evidence suggests that the functional role of actin polymerization during contraction is distinct and separately regulated from the actomyosin cross-bridge cycling process. The molecular basis for the regulation of actin polymerization and its physiological roles may vary in diverse types of smooth muscle cells and tissues. However, current evidence supports a model for smooth muscle contraction in which contractile stimulation initiates the assembly of cytoskeletal/extracellular matrix adhesion complex proteins at the membrane, and proteins within this complex orchestrate the polymerization and organization of a submembranous network of actin filaments. This cytoskeletal network may serve to strengthen the membrane for the transmission of force generated by the contractile apparatus to the extracellular matrix, and to enable the adaptation of smooth muscle cells to mechanical stresses. Better understanding of the physiological function of these dynamic cytoskeletal processes in smooth muscle may provide important insights into the physiological regulation of smooth muscle tissues.

Mechanosensitive tyrosine phosphorylation of paxillin and focal adhesion kinase in tracheal smooth muscle

We investigated the role of the integrin-associated proteins focal adhesion kinase (FAK) and paxillin as mediators of mechanosensitive signal transduction in tracheal smooth muscle. In muscle strips contracted isometrically with ACh, we observed higher levels of tyrosine phosphorylation of FAK and paxillin at the optimal muscle length (Lo) than at shorter muscle lengths of 0.5 or 0.75 Lo. Paxillin phosphorylation was also length sensitive in muscles activated by K+ depolarization and adjusted rapidly to changes in muscle length imposed after contractile activation by either ACh or K+ depolarization. Ca2+ depletion did not affect the length sensitivity of paxillin and FAK phosphorylation in muscles activated with ACh, indicating that the mechanotransduction process can be mediated by a Ca2+-independent pathway. Since Ca2+-depleted muscles do not generate significant active tension, this suggests that the mechanotransduction mechanism is sensitive to muscle length rather than tension. We conclude that FAK and paxillin participate in an integrin-mediated mechanotransduction process in tracheal smooth muscle. We propose that this pathway may initiate alterations in smooth muscle cell structure and contractility via the remodeling of actin filaments and/or via the mechanosensitive regulation of signaling molecules involved in contractile protein activation.We investigated the role of the integrin-associated proteins focal adhesion kinase (FAK) and paxillin as mediators of mechanosensitive signal transduction in tracheal smooth muscle. In muscle strips contracted isometrically with ACh, we observed higher levels of tyrosine phosphorylation of FAK and paxillin at the optimal muscle length ( L o) than at shorter muscle lengths of 0.5 or 0.75 L o. Paxillin phosphorylation was also length sensitive in muscles activated by K+ depolarization and adjusted rapidly to changes in muscle length imposed after contractile activation by either ACh or K+depolarization. Ca2+ depletion did not affect the length sensitivity of paxillin and FAK phosphorylation in muscles activated with ACh, indicating that the mechanotransduction process can be mediated by a Ca2+-independent pathway. Since Ca2+-depleted muscles do not generate significant active tension, this suggests that the mechanotransduction mechanism is sensitive to muscle length rather than tension. We conclude that FAK and paxillin participate in an integrin-mediated mechanotransduction process in tracheal smooth muscle. We propose that this pathway may initiate alterations in smooth muscle cell structure and contractility via the remodeling of actin filaments and/or via the mechanosensitive regulation of signaling molecules involved in contractile protein activation.

Cytoskeletal remodeling of the airway smooth muscle cell: a mechanism for adaptation to mechanical forces in the lung

Airway smooth muscle is continuously subjected to mechanical forces caused by changes in lung volume during breathing. These mechanical oscillations have profound effects on airway smooth muscle contractility both in vivo and in vitro. Alterations in airway smooth muscle properties in response to mechanical forces may result from adaptive changes in the organization of the actin cytoskeleton. Recent advances suggest that in airway smooth muscle, two cytosolic signaling proteins that associate with focal adhesion complexes, focal adhesion kinase (FAK) and paxillin, are involved in transducing external mechanical signals. FAK and paxillin regulate changes in the organization of the actin cytoskeleton and the activation of contractile proteins. Actin is in a dynamic state in airway smooth muscle and undergoes polymerization and depolymerization during the contraction-relaxation cycle. The organization of the cytoskeletal proteins, vinculin, talin, and alpha-actinin, which mediate linkages between actin filaments and transmembrane integrins, is also regulated by contractile stimulation in airway smooth muscle. The fluidity of the cytoskeletal structure of the airway smooth muscle cell may be fundamental to its ability to adapt and respond to the mechanical forces imposed on it in the lung during breathing.

Cytoskeletal remodeling in differentiated vascular smooth muscle is actin isoform dependent and stimulus dependent

Dynamic remodeling of the actin cytoskeleton plays an essential role in the migration and proliferation of vascular smooth muscle cells. It has been suggested that actin remodeling may also play an important functional role in nonmigrating, nonproliferating differentiated vascular smooth muscle (dVSM). In the present study, we show that contractile agonists increase the net polymerization of actin in dVSM, as measured by the differential ultracentrifugation of vascular smooth muscle tissue and the costaining of single freshly dissociated cells with fluorescent probes specific for globular and filamentous actin. Furthermore, induced alterations of the actin polymerization state, as well as actin decoy peptides, inhibit contractility in a stimulus-dependent manner. Latrunculin pretreatment or actin decoy peptides significantly inhibit contractility induced by a phorbol ester or an alpha-agonist, but these procedures have no effect on contractions induced by KCl. Aorta dVSM expresses alpha-smooth muscle actin, beta-actin, nonmuscle gamma-actin, and smooth muscle gamma-actin. The incorporation of isoform-specific cell-permeant synthetic actin decoy peptides, as well as isoform-specific probing of cell fractions and two-dimensional gels, demonstrates that actin remodeling during alpha-agonist contractions involves the remodeling of primarily gamma-actin and, to a lesser extent, beta-actin. Taken together, these results show that net isoform- and agonist-dependent increases in actin polymerization regulate vascular contractility.

Silencing of p21-activated kinase attenuates vimentin phosphorylation on Ser-56 and reorientation of the vimentin network during stimulation of smooth muscle cells by 5-hydroxytryptamine

Vimentin intermediate filaments undergo spatial reorganization in endothelial cells and fibroblasts in response to stimulation with platelet-derived growth factor and epidermal growth factor. In the present study, the vimentin network exhibited a curved filamentous structure in unstimulated smooth muscle cells. Vimentin filaments became straight and were arranged along the long axis of cells upon stimulation with 5-hydroxytryptamine (5-HT; serotonin). Stimulation of smooth muscle cells with 5-HT also induced phosphorylation of vimentin on Ser-56. Treatment of cells with small interfering RNA selectively down-regulated the expression of PAK1 (p21-activated kinase 1) without affecting the content of smooth muscle alpha-actin. The silencing of PAK1 inhibited the site-specific phosphorylation and spatial rearrangement of the vimentin network in response to stimulation with 5-HT. Neither the disruption of stress fibres by cytochalasin D nor the inhibition of protein tyrosine phosphorylation affects the spatial reorganization of vimentin intermediate filaments in response to stimulation with 5-HT. In addition, stimulation of smooth muscle cells with 5-HT increased the ratio of soluble to insoluble vimentin. PAK1 silencing attenuated increases in the ratio of soluble to insoluble vimentin upon stimulation with 5-HT. These results suggest that the PAK-mediated site-specific phosphorylation of vimentin may play a role in regulating the reorganization of vimentin intermediate filaments during stimulation of smooth muscle cells with 5-HT.

HALOTHANE, ENFLURANE, AND ISOFLURANE DEPRESS THE PERIPHERAL VAGAL MOTOR PATHWAY IN ISOLATED CANINE TRACHEAL SMOOTH MUSCLE

Volatile anesthetics are potent bronchodilators, but the site of action for the dilation is unclear. To determine the site of action of halothane, enflurane, and isoflurane on the peripheral vagal motor pathway, isolated strips of canine trachealis muscle were stimulated before and during exposure to halothane at 0.3, 1.0, 1.7, or 2.4 MAC, enflurane at 1 MAC, or isoflurane at 1 MAC. The sites and methods of stimulation were: 1) postsynaptic nicotinic cholinergic receptors in the intramural parasympathetic ganglia, with 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP); 2) postganglionic cholinergic nerve fibers, with electrical field stimulation (EFS); and 3) muscarinic cholinergic receptors of the smooth muscle, with acetylcholine (ACh). The concentration-response curve to DMPP was significantly shifted to the right by 0.3 MAC halothane, whereas 0.3 MAC halothane had no significant effect on the concentration-response curves to ACh and EFS. At concentrations greater than 1 MAC of halothane, enflurane, or isoflurane, concentration-response curves to all three stimuli were shifted significantly to the right; i.e., the contractile responses to ACh, EFS, and DMPP were reduced. At all concentrations of halothane the force of contraction was significantly more reduced during stimulation with DMPP than during stimulation with ACh, and at halothane concentrations greater than or equal to 1.7 MAC the response to EFS was significantly more reduced than that to ACh. We conclude that halothane, enflurane, and isoflurane attenuated airway constriction by several mechanisms, including 1) reduced excitability of the postsynaptic nicotinic receptors of the intramural parasympathetic ganglia and 2) an effect on the smooth muscle and/or on the muscarinic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of non-phosphorylatable paxillin mutants in canine tracheal smooth muscle inhibits tension development

The adapter protein paxillin has been implicated in the regulation of cytoskeletal organization and cell motility. Paxillin undergoes tyrosine phosphorylation in response to the contractile stimulation of smooth muscle, and the depletion of paxillin by antisense inhibits smooth muscle contraction. In the present study, acetylcholine (ACh)‐stimulation of tracheal smooth muscle tissues increased paxillin phosphorylation at tyr‐31 and tyr‐118 by three‐ to fourfold. The role of tyr‐31 and tyr‐118 phosphorylation of paxillin in smooth muscle was evaluated by introducing plasmids encoding wild type paxillin or paxillin mutants F31, F118 or F31/118 (phenylalanine substitution at tyrosine sites 31, 118) into tracheal smooth muscle strips by reversible permeabilization, and incubating the tissues for 2 days. The expression of recombinant proteins was confirmed by immunoblot and immunofluorescence analysis. Expression of the paxillin mutants F31, F118 or F31/118 inhibited the contractile response to ACh stimulation but did not inhibit the increase in myosin light chain phosphorylation. The expression of wild type paxillin had no significant affect on force or myosin light chain phosphorylation. ACh stimulation reduced G‐actin/F‐actin ratio in tissues expressing wild type paxillin; whereas the agonist‐induced decrease in G‐actin/F‐actin was inhibited in strips expressing paxillin mutant F31/118. The paxillin mutant F31/118 showed a marked decrease in their interaction with the SH2/SH3 adaptor protein CrkII but not with vinculin or focal adhesion kinase. We conclude that paxillin phosphorylation at tyr‐31 and tyr‐118 regulates active tension development during contractile stimulation. Paxillin phosphorylation at these two sites may be important in regulating actin filament dynamics and organization during smooth muscle contraction.

Dynamic association between α-actinin and β-integrin regulates contraction of canine tracheal smooth muscle

The adhesion junctions of smooth muscle cells may be dynamically regulated during smooth muscle contraction, and this dynamic regulation may be important for the development of active tension. In the present study, the role of α‐actinin during smooth muscle contraction was evaluated in tracheal smooth muscle tissues and freshly dissociated cells. Stimulation with acetylcholine (ACh) increased the localization of α‐actinin at the membrane of freshly dissociated smooth muscle cells, and increased the amount of β1 integrin that coprecipitated with α‐actinin from muscle tissue homogenates. GFP‐α‐actinin fusion proteins were expressed in muscle tissues and visualized in live freshly dissociated cells. GFP‐α‐actinin translocated to the membrane within seconds of stimulation of the cells with ACh. Expression of the integrin‐binding rod domain of α‐actinin in smooth muscle tissues depressed active contraction in response to ACh. Expression of the α‐actinin rod domain also inhibited the translocation of endogenous α‐actinin to the membrane, and inhibited the association of endogenous α‐actinin with β1‐integrin in α‐actinin immunoprecipitates from tissue extracts. However, the expression of α‐actinin rod domain peptides did not inhibit increases in myosin light chain phosphorylation or actin polymerization in response to stimulation with ACh. Results suggest that contractile stimulation of smooth muscle causes the rapid recruitment of α‐actinin to β‐integrin complexes at the membrane, and that the recruitment of α‐actinin to integrin complexes is necessary for active tension development in smooth muscle.