Mechanical state of airway smooth muscle at very short lengths.

Although the shortening of smooth muscle at physiological lengths is dominated by an interaction between external forces (loads) and internal forces, at very short lengths, internal forces appear to dominate the mechanical behavior of the active tissue. We tested the hypothesis that, under conditions of extreme shortening and low external force, the mechanical behavior of isolated canine tracheal smooth muscle tissue can be understood as a structure in which the force borne and exerted by the cross bridge and myofilament array is opposed by radially disposed connective tissue in the presence of an incompressible fluid matrix (cellular and extracellular). Strips of electrically stimulated tracheal muscle were allowed to shorten maximally under very low afterload, and large longitudinal sinusoidal vibrations (34 Hz, 1 s in duration, and up to 50% of the muscle length before vibration) were applied to highly shortened (active) tissue strips to produce reversible cross-bridge detachment. During the vibration, peak muscle force fell exponentially with successive forced elongations. After the episode, the muscle either extended itself or exerted a force against the tension transducer, depending on external conditions. The magnitude of this effect was proportional to the prior muscle stiffness and the amplitude of the vibration, indicating a recoil of strained connective tissue elements no longer opposed by cross-bridge forces. This behavior suggests that mechanical behavior at short lengths is dominated by tissue forces within a tensegrity-like structure made up of connective tissue, other extracellular matrix components, and active contractile elements.     

Structure-function relationships in smooth muscle: The missing links

Smooth muscle cells have developed a contractile machinery that allows them to exert tension on the surrounding extracellular matrix over their entire length. This has been achieved by coupling obliquely organized contractile filaments to a more-or-less longitudinal framework of cytoskeletal elements. Earlier structural data suggested that the cytoskeleton was composed primarily of intermediate filaments and played only a passive role. More recent findings highlight the segregation of actin isotypes and of actin-associated proteins between the contractile and cytoskeletal domains and raise the possibility that the cytoskeleton performs a more active function. Current efforts focus on defining the relative contributions of myosin cross-bridge cycling and actin-associated protein interactions to the maintenance of tension in smooth muscle tissue.     

Neurokinin-neurotrophin interactions in airway smooth muscle

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 Ca(2+) concentration ([Ca(2+)](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, [Ca(2+)](i) responses to 1 μM ACh as well as BDNF (30 min) effects on [Ca(2+)](i) regulation were enhanced by prior SP exposure, largely via increased Ca(2+) 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-F(c); 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 [Ca(2+)](i) (P < 0.05). These results suggest a novel interaction between SP and BDNF in regulating agonist-induced [Ca(2+)](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.     

Plasticity in canine airway smooth muscle

The large volume changes of some hollow viscera require a greater length range for the smooth muscle of their walls than can be accommodated by a fixed array of sliding filaments. A possible explanation is that smooth muscles adapt to length changes by forming variable numbers of contractile units in series. To test for such plasticity we examined the muscle length dependence of shortening velocity and compliance, both of which will vary directly with the number of thick filaments in series. Dog tracheal smooth muscle was studied because its cells are arrayed in long, straight, parallel bundles that span the length of the preparation. In experiments where muscle length was changed, both compliance and velocity showed a strong dependence on muscle length, varying by 1.7-fold and 2.2-fold, respectively, over a threefold range of length. The variation in isometric force was substantially less, ranging from a 1.2- to 1.3-fold in two series of experiments where length was varied by twofold to an insignificant 4% variation in a third series where a threefold length range was studied. Tetanic force was below its steady level after both stretches and releases, and increased to a steady level with 5-6 tetani at 5 min intervals. These results suggest strongly that the number of contractile units in series varies directly with the adapted muscle length. Temporary force depression after a length change would occur if the change transiently moved the filaments from their optimum overlap. The relative length independence of the adapted force is explained by the reforming of the filament lattice to produce optimum force development, with commensurate changes of velocity and compliance.     

Excitation-contraction coupling in airway smooth muscle

Excitation-contraction (EC) coupling in striated muscles is mediated by the cardiac or skeletal muscle isoform of voltage-dependent L-type Ca(2+) channel (Ca(v)1.2 and Ca(v)1.1, respectively) that senses a depolarization of the cell membrane, and in response, activates its corresponding isoform of intracellular Ca(2+) release channel/ryanodine receptor (RyR) to release stored Ca(2+), thereby initiating muscle contraction. Specifically, in cardiac muscle following cell membrane depolarization, Ca(v)1.2 activates cardiac RyR (RyR2) through an influx of extracellular Ca(2+). In contrast, in skeletal muscle, Ca(v)1.1 activates skeletal muscle RyR (RyR1) through a direct physical coupling that negates the need for extracellular Ca(2+). Since airway smooth muscle (ASM) expresses Ca(v)1.2 and all three RyR isoforms, we examined whether a cardiac muscle type of EC coupling also mediates contraction in this tissue. We found that the sustained contractions of rat ASM preparations induced by depolarization with KCl were indeed partially reversed ( approximately 40%) by 200 mum ryanodine, thus indicating a functional coupling of L-type channels and RyRs in ASM. However, KCl still caused transient ASM contractions and stored Ca(2+) release in cultured ASM cells without extracellular Ca(2+). Further analyses of rat ASM indicated that this tissue expresses as many as four L-type channel isoforms, including Ca(v)1.1. Moreover, Ca(v)1.1 and RyR1 in rat ASM cells have a similar distribution near the cell membrane in rat ASM cells and thus may be directly coupled as in skeletal muscle. Collectively, our data implicate that EC-coupling mechanisms in striated muscles may also broadly transduce diverse smooth muscle functions.     

Human airway smooth muscle in culture

We describe a method for culturing human airway smooth muscle. Cells were enzymatically and mechanically dispersed from strips of smooth muscle harvested from surgically removed lobar bronchi, and were seeded on to dishes containing Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. After 14-21 days confluent monolayers of cells formed, which were subcultured and identified as smooth muscle by positive immunocytochemical staining for actin and myosin. The retention of functional plasmalemmal receptors and of intracellular signal transduction pathways in cell culture was demonstrated in 45Ca-labelled monolayers by the stimulation of efflux of intracellularly stored 45Ca in response to extracellularly applied 10 microM carbachol or 10 microM histamine. Human airway smooth muscle in cell culture provides a novel preparation for investigating the physiology and pathophysiology of the human airways.     

TGF-beta potentiates airway smooth muscle responsiveness to bradykinin

The molecular mechanisms by which bradykinin induces excessive airway obstruction in asthmatics remain unknown. Transforming growth factor (TGF)-beta has been involved in regulating airway inflammation and remodeling in asthma, although it is unknown whether TGF-beta can modulate bradykinin-associated bronchial hyperresponsiveness. To test whether TGF-beta directly modulates airway smooth muscle (ASM) responsiveness to bradykinin, isolated murine tracheal rings were used to assess whether TGF-beta alters ASM contractile responsiveness to bradykinin. Interestingly, we found TGF-beta-treated murine rings (12.5 ng/ml, 18 h) exhibited increased expression of bradykinin 2 (B(2)) receptors and became hyperreactive to bradykinin, as shown by increases in maximal contractile responses and receptor distribution. We investigated the effect of TGF-beta on bradykinin-evoked calcium signals since calcium is a key molecule regulating ASM excitation-contraction coupling. We reported that TGF-beta, in a dose- (0.5-10 ng/ml) and time- (2-24 h) dependent manner, increased mRNA and protein expression of the B(2) receptor in cultured human ASM cells. Maximal B(2) receptor protein expression that colocalized with CD44, a marker of membrane cell surface, occurred after 18 h of TGF-beta treatment and was further confirmed using fluorescence microscopy. TGF-beta (2.5 ng/ml, 18 h) also increased bradykinin-induced intracellular calcium mobilization in fura-2-loaded ASM cells. TGF-beta-mediated enhancement of calcium mobilization was not attenuated with indomethacin, a cyclooxygenase inhibitor. These data demonstrate for the first time that TGF-beta may play a role in mediating airway hyperresponsiveness to bradykinin seen in asthmatics by enhancing ASM contractile responsiveness to bradykinin, possibly as a result of increased B(2) receptor expression and signaling.     

Mechanical strain memory in airway smooth muscle

We investigated the effect of a singlerapid stretch on poststretch force and myosin phosphorylation in bovinetracheal smooth muscle. When unstimulated muscle strips were stretchedfrom suboptimal length to optimal length (L_o),poststretch steady-state force was not significantly different fromthat of unstretched control at L_o. However, whencarbachol-activated muscle strips were stretched from suboptimal lengthto L_o, poststretch force and myosin phosphorylation were lower than control and significantly correlated with initial length. When poststretch muscle strips were allowed to relax for 1 hand then activated by K⁺ depolarization, the developedforce remained significantly correlated with initial length. When thesame strain was applied in 23 increments to minimize peak stress,poststretch force and myosin phosphorylation increased significantly,approaching the levels expected at L_o. Furthermore,poststretch force development increased after each cycle of contractionand relaxation, approaching the control level after four cycles. Theseresults suggest that activated airway smooth muscle cells can retainrelatively precise memory of past strain when they are stretchedrapidly with high stress.

     

Inspiratory rhythm in airway smooth muscle tone

In anesthetized paralyzed open-chested cats ventilated with low tidal volumes at high frequency, we recorded phrenic nerve activity, transpulmonary pressure (TPP), and either the tension in an upper tracheal segment or the impulse activity in a pulmonary branch of the vagus nerve. The TPP and upper tracheal segment tension fluctuated with respiration, with peak pressure and tension paralleling phrenic nerve activity. Increased end-tidal CO2 or stimulation of the carotid chemoreceptors with sodium cyanide increased both TPP and tracheal segment tension during the increased activity of the phrenic nerve. Lowering end-tidal CO2 or hyperinflating the lungs to achieve neural apnea (lack of phrenic activity) caused a decrease in TPP and tracheal segment tension and abolished the inspiratory fluctuations. During neural apnea produced by lowering end-tidal CO2, lung inflation caused no further decrease in tracheal segment tension and TPP. Likewise, stimulation of the cervical sympathetics, which caused a reduction in TPP and tracheal segment tension during normal breathing, caused no further reduction in these parameters when the stimulation occurred during neural apnea. During neural apnea the tracheal segment tension and TPP were the same as those following the transection of the vagi or the administration of atropine (0.5 mg/kg). Numerous fibers in the pulmonary branch of the vagus nerve fired in synchrony with the phrenic nerve. Only these fibers had activity which paralleled changes in TPP and tracheal tension. We propose that the major excitatory input to airway smooth muscle arises from cholinergic nerves that fire during inspiration, which have preganglionic cell bodies in the ventral respiratory group in the region of the nucleus ambiguus and are driven by the same pattern generators that drive the phrenic and inspiratory intercostal motoneurons.     

Resting calcium influx in airway smooth muscle

Plasma membrane Ca2+ leak remains the most uncertain of the cellular Ca2+ regulation pathways. During passive Ca2+ influx in non-stimulated smooth muscle cells, basal activity of constitutive Ca2+ channels seems to be involved. In vascular smooth muscle, the 3 following Ca2+ entry pathways contribute to this phenomenon: (i) via voltage-dependent Ca2+ channels, (ii) receptor gated Ca2+ channels, and (iii) store operated Ca2+ channels, although, in airway smooth muscle it seems only 2 passive Ca2+ influx pathways are implicated, one sensitive to SKF 96365 (receptor gated Ca2+ channels) and the other to Ni2+ (store operated Ca2+ channels). Resting Ca2+ entry could provide a sufficient amount of Ca2+ and contribute to resting intracellular Ca2+ concentration ([Ca2+]i), maintenance of the resting membrane potential, myogenic tone, and sarcoplasmic reticulum-Ca2+ refilling. However, further research, especially in airway smooth muscle, is required to better explore the physiological role of this passive Ca2+ influx pathway as it could be involved in airway hyperresponsiveness.     

Relaxation of canine airway smooth muscle

Relaxation of airway smooth muscle is an inadequately understood yet critical process that, if impaired, may have significant implications for asthma. Here we explore why relaxation is an important process to consider, how it may determine airway hyperresponsiveness, and some of the factors that influence relaxation of the airway smooth muscle. These include mechanical and biochemical factors such as deep inspirations or large amplitude oscillation of the muscle, plastic properties of the muscle, the load the muscle experiences, calcium, phosphorylation of the myosin light chain, cytoskeletal proteins, and sensitization.     

Adaptation to chronic length change in explanted airway smooth muscle.

It has been shown that airway smooth muscle in vitro is able to maintain active force over a large length range by adaptation in the absence of periodic stimulations at 4 degrees C (Wang L, Paré PD, and Seow CY. J Appl Physiol 90: 734-740, 2001). In this study, we show that such adaptation also takes place at body temperature and that long-term adaptation results in irreversible functional change in the muscle that could lead to airway hyperresponsiveness. Rabbit tracheal muscle explants were passively maintained at shortened and in situ length for 3 and 7-8 days in culture media; the length-tension relationship was then examined. The length associated with maximal force generation decreased by 10.5 +/- 3.8% (SE) after 3 days and 37.7 +/- 8.5% after 7 or 8 days of passive shortening. At day 3, the left shift in the length-tension curve due to adaptation at short lengths was reversible by readapting the muscle at a longer length. The shift was, however, not completely reversible after 7 days. The results suggest that long-term adaptation of airway smooth muscle could lead to increased muscle stiffness and force-generating ability at short lengths. Under in vivo condition, this could translate into resistance to stretch-induced relaxation and excessive airway narrowing.     

Plasticity in airway smooth muscle: an update

At a similar meeting 10 years ago, we proposed (i) that the long functional range of some smooth muscles is accommodated by plastic alterations that place more myofilaments in series at longer lengths, (ii) that this plasticity is facilitated by myosin filament evanescence, with filaments dissociating partially during relaxation and reforming upon activation, and (iii) that filament lengthening during the rise of activation would cause velocity to fall. Since that meeting, we have accumulated a substantial body of evidence to support these proposals, as follows: (i) muscles develop nearly the same force when adapted to a range of lengths that can vary by 3-fold; (ii) other physiological parameters including shortening velocity, maximum power, compliance, ATPase rate, and thick-filament mass increase by about 2/3 for a doubling of muscle length; (iii) thick-filament density increases substantially during the rise of activation; and (iv) velocity falls as force rises during the rise of tetanic force, and when correction is made for differences in activation, velocity and force vary exactly in inverse proportion. This review explains the rationale for the different experimental measurements and their interpretation.     

Stiffness changes in cultured airway smooth muscle cells

Airwaysmooth muscle (ASM) cells in culture stiffen when exposed tocontractile agonists. Such cell stiffening may reflect activation ofthe contractile apparatus as well as polymerization of cytoskeletalbiopolymers. Here we have assessed the relative contribution of thesemechanisms in cultured ASM cells stimulated with serotonin(5-hydroxytryptamine; 5-HT) in the presence or absence of drugs thatinhibit either myosin-based contraction or polymerization offilamentous (F) actin. Magnetic twisting cytometry was used to measurecell stiffness, and associated changes in structural organization ofactin cytoskeleton were evaluated by confocal microscopy. We found that5-HT increased cell stiffness in a dose-dependent fashion and alsoelicited rapid formation of F-actin as marked by increased intensity ofFITC-phalloidin staining in these cells. A calmodulin antagonist (W-7),a myosin light chain kinase inhibitor (ML-7) and a myosin ATPaseinhibitor (BDM) each ablated the stiffening response but not theF-actin polymerization induced by 5-HT. Agents that inhibited theformation of F-actin (cytochalasin D, latrunculin A, C3 exoenzyme, andY-27632) attenuated both baseline stiffness and the extent of cellstiffening in response to 5-HT. Together, these data suggest thatagonist-evoked stiffening of cultured ASM cells requires actinpolymerization as well as myosin activation and that neitheractin polymerization nor myosin activation by itself is sufficient toaccount for the cell stiffening response.

     

Emergence of airway smooth muscle functions related to structural malleability

The function of a complex system such as a smooth muscle cell is the result of the active interaction among molecules and molecular aggregates. Emergent macroscopic manifestations of these molecular interactions, such as the length-force relationship and its associated length adaptation, are well documented, but the molecular constituents and organization that give rise to these emergent muscle behaviors remain largely unknown. In this minireview, we describe emergent properties of airway smooth muscle that seem to have originated from inherent fragility of the cellular structures, which has been increasingly recognized as a unique and important smooth muscle attribute. We also describe molecular interactions (based on direct and indirect evidence) that may confer malleability on fragile structural elements that in turn may allow the muscle to adapt to large and frequent changes in cell dimensions. Understanding how smooth muscle works may hinge on how well we can relate molecular events to its emergent macroscopic functions.