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.     

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.     

Resident phenotypically modulated vascular smooth muscle cells in healthy human arteries

Vascular interstitial cells (VICs) are non‐contractile cells with filopodia previously described in healthy blood vessels of rodents and their function remains unknown. The objective of this study was to identify VICs in human arteries and to ascertain their role. VICs were identified in the wall of human gastro‐omental arteries using transmission electron microscopy. Isolated VICs showed ability to form new and elongate existing filopodia and actively change body shape. Most importantly sprouting VICs were also observed in cell dispersal. RT‐PCR performed on separately collected contractile vascular smooth muscle cells (VSMCs) and VICs showed that both cell types expressed the gene for smooth muscle myosin heavy chain (SM‐MHC). Immunofluorescent labelling showed that both VSMCs and VICs had similar fluorescence for SM‐MHC and αSM‐actin, VICs, however, had significantly lower fluorescence for smoothelin, myosin light chain kinase, h‐calponin and SM22α. It was also found that VICs do not have cytoskeleton as rigid as in contractile VSMCs. VICs express number of VSMC‐specific proteins and display features of phenotypically modulated VSMCs with increased migratory abilities. VICs, therefore represent resident phenotypically modulated VSMCs that are present in human arteries under normal physiological conditions.     

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.     

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.     

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.

     

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.     

Ultrastructural basis of airway smooth muscle contraction

The sliding filament theory of contraction that was developed for striated muscle is generally believed to be also applicable to smooth muscle. However, the well-organized myofilament lattice (i.e., the sarcomeric structure) found in striated muscle has never been clearly delineated in smooth muscle. There is evidence that the myofilament lattice in some smooth muscles, such as airway smooth muscle, is malleable; it can be reshaped to fit a large range of cell dimensions while the maximal overlap between the contractile filaments is maintained. In this review, some early models of the structurally static contractile apparatus of smooth muscle are described. The focus of the review, however, is on the recent findings supporting a model of structurally dynamic contractile apparatus and cytoskeleton for airway smooth muscle. A list of unanswered questions regarding smooth muscle ultrastructure is also proposed in this review, in the hope that it will provide some guidance for future research.     

Transgenic overexpression of beta(2)-adrenergic receptors in airway smooth muscle alters myocyte function and ablates bronchial hyperreactivity.

beta(2)-Adrenergic receptors (beta(2)AR) act to relax airway smooth muscle and can serve to counteract hyperresponsiveness, although the effect may not be ablative even in the presence of exogenous agonist. Within this signaling cascade that ultimately transduces smooth muscle relaxation, a significant "spare receptor" pool has been hypothesized to be present in the airway. In order to modify the relationship between beta(2)AR and downstream effectors, transgenic mice (TG) were created overexpressing beta(2)AR approximately 75-fold in airway smooth muscle using a mouse smooth muscle alpha-actin promoter. While >90% of these receptors were expressed on the smooth muscle cell surface, the percentage of receptors able to form the agonist-promoted high affinity complex was less than that found with nontransgenic (NTG) cells (R(H) = 18 versus 36%). Nevertheless, beta(2)AR signaling was found to be enhanced. Intact airway smooth muscle cells from TG had basal cAMP levels that were greater than NTG cells. A marked increase in agonist-stimulated cAMP levels was found in the TG ( approximately 200% stimulation over basal) compared with NTG ( approximately 50% over basal) cells. Adenylyl cyclase studies gave similar results and also showed a 10-fold lower EC(50) for TG cells. Tracheal rings from TG mice that were precontracted with acetylcholine had an enhanced responsiveness (relaxation) to beta-agonist, with a 60-fold decrease in the ED(50), indicating that the enhanced signaling imposed by overexpression results in an increase in the coordinated function of the intact airway cells. In vivo studies showed a significantly blunted airway resistance response to the inhaled bronchoconstrictor methacholine in the TG mice. Indeed, with beta-agonist pretreatment, the TG mice displayed no response whatsoever to methacholine. These results are consistent with beta(2)AR being the limiting factor in the transduction system. Increases in the initial component of this transduction system (the beta(2)AR) are sufficient to markedly alter signaling and airway smooth muscle function to the extent that bronchial hyperresponsiveness is ablated, consistent with an anti-asthma phenotype.     

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.

     

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.     

Cyclic nucleotide-dependent protein kinases in airway smooth muscle

Because of the potential importance of cyclic nucleotide-dependent protein kinases in the regulation of airway smooth muscle tone, we have examined some of the characteristics of these enzymes in the soluble fraction of canine trachealis homogenates. In the absence of added cAMP, the heat-stable cAMP-dependent protein kinase inhibitor (PKI) abolished only a half of the 32P incorporation into mixed histones. The remaining activity appeared to be contributed by a cyclic nucleotide-independent enzyme. Phosphotransferase activity was enhanced 5-fold by 5 microM cAMP but only 70% of the cAMP-stimulated activity could be inhibited by PKI. The sensitivity of the cyclic nucleotide-dependent, PKI-resistant enzyme to cAMP, cGMP, and Mg2+ indicated that it was cGMP-dependent protein kinase. Because of the large amount of cyclic nucleotide-independent activity, and the ability of cAMP to activate cGMP-dependent protein kinase, the traditional "-cAMP/+cAMP" ratio did not provide an accurate assessment of the in vivo activation state of cAMP-dependent protein kinase. However, a modified assay was developed which allowed the precise measurement of cAMP-dependent, cGMP-dependent, and cyclic nucleotide-independent protein kinase activities. Using this new method, the cAMP-dependent protein kinase activity ratio of 0.239 in untreated trachealis strips was increased to 0.355 and 0.386 by prior exposure of the intact tissue to the smooth muscle relaxants isoproterenol and prostaglandin E2, respectively. The results of this study are consistent with the proposed role of cAMP-dependent protein kinase in the regulation of smooth muscle contractile function.