Reflex regulation of airway smooth muscle tone.

Autonomic nerves in most mammalian species mediate both contractions and relaxations of airway smooth muscle. Cholinergic-parasympathetic nerves mediate contractions, whereas adrenergic-sympathetic and/or noncholinergic parasympathetic nerves mediate relaxations. Sympathetic-adrenergic innervation of human airway smooth muscle is sparse or nonexistent based on histological analyses and plays little or no role in regulating airway caliber. Rather, in humans and in many other species, postganglionic noncholinergic parasympathetic nerves provide the only relaxant innervation of airway smooth muscle. These noncholinergic nerves are anatomically and physiologically distinct from the postganglionic cholinergic parasympathetic nerves and differentially regulated by reflexes. Although bronchopulmonary vagal afferent nerves provide the primary afferent input regulating airway autonomic nerve activity, extrapulmonary afferent nerves, both vagal and nonvagal, can also reflexively regulate autonomic tone in airway smooth muscle. Reflexes result in either an enhanced activity in one or more of the autonomic efferent pathways, or a withdrawal of baseline cholinergic tone. These parallel excitatory and inhibitory afferent and efferent pathways add complexity to autonomic control of airway caliber. Dysfunction or dysregulation of these afferent and efferent nerves likely contributes to the pathogenesis of obstructive airways diseases and may account for the pulmonary symptoms associated with extrapulmonary disorders, including gastroesophageal reflux disease, cardiovascular disease, and rhinosinusitis.     

Pro-inflammatory mechanisms of muscarinic receptor stimulation in airway smooth muscle

Background

Acetylcholine, the primary parasympathetic neurotransmitter in the airways, plays an important role in bronchoconstriction and mucus production. Recently, it has been shown that acetylcholine, by acting on muscarinic receptors, is also involved in airway inflammation and remodelling. The mechanism(s) by which muscarinic receptors regulate inflammatory responses are, however, still unknown.

Methods

The present study was aimed at characterizing the effect of muscarinic receptor stimulation on cytokine secretion by human airway smooth muscle cells (hASMc) and to dissect the intracellular signalling mechanisms involved. hASMc expressing functional muscarinic M₂ and M₃ receptors were stimulated with the muscarinic receptor agonist methacholine, alone, and in combination with cigarette smoke extract (CSE), TNF-α, PDGF-AB or IL-1β.

Results

Muscarinic receptor stimulation induced modest IL-8 secretion by itself, yet augmented IL-8 secretion in combination with CSE, TNF-α or PDGF-AB, but not with IL-1β. Pretreatment with GF109203X, a protein kinase C (PKC) inhibitor, completely normalized the effect of methacholine on CSE-induced IL-8 secretion, whereas PMA, a PKC activator, mimicked the effects of methacholine, inducing IL-8 secretion and augmenting the effects of CSE. Similar inhibition was observed using inhibitors of IκB-kinase-2 (SC514) and MEK1/2 (U0126), both downstream effectors of PKC. Accordingly, western blot analysis revealed that methacholine augmented the degradation of IκBα and the phosphorylation of ERK1/2 in combination with CSE, but not with IL-1β in hASMc.

Conclusions

We conclude that muscarinic receptors facilitate CSE-induced IL-8 secretion by hASMc via PKC dependent activation of IκBα and ERK1/2. This mechanism could be of importance for COPD patients using anticholinergics.     

Effects of hypoxia on rat airway smooth muscle cell proliferation.

Although it is well known that hypoxemia induces pulmonary vasoconstriction and vascular remodeling, due to the proliferation of both vascular smooth muscle cells and fibroblasts, the effects of hypoxemia on airway smooth muscle cells are not well characterized. The present study was designed to assess the in vitro effects of hypoxia (1 or 3% O(2)) on rat airway smooth muscle cell growth and response to mitogens (PDGF and 5-HT). Cell growth was assessed by cell counting and cell cycle analysis. Compared with normoxia (21% O(2)), there was a 42.2% increase in the rate of proliferation of cells exposed to 3% O(2) (72 h, P = 0.006), as well as an enhanced response to PDGF (13.9% increase; P = 0.023) and to 5-HT (17.2% increase; P = 0.039). Exposure to 1% O(2) (72 h) decreased cell proliferation by 21.0% (P = 0.017) and reduced the increase in cell proliferation induced by PGDF and 5-HT by 16.2 and 15.7%, respectively (P = 0.019 and P = 0.011). A significant inhibition in hypoxia-induced cell proliferation was observed after the administration of bisindolylmaleimide GF-109203X (a specific PKC inhibitor) or downregulation of PKC with PMA. Pretreatment with GF-109203X decreased proliferation by 21.5% (P = 0.004) and PMA by 31.5% (P = 0.005). These results show that hypoxia induces airway smooth muscle cell proliferation, which is at least partially dependent on PKC activation. They suggest that hypoxia could contribute to airway remodeling in patients suffering from chronic, severe respiratory diseases.     

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.     

Effects of cyclopiazonic acid on cytosolic calcium in bovine airway smooth muscle cells

In many cells, inhibition of sarcoplasmic reticulum (SR) Ca2+-ATPase activity induces a steady-state increase in cytosolic calcium concentration ([Ca2+]i) that is sustained by calcium influx. The goal was to characterize the response to inhibition of SR Ca2+-ATPase activity in bovine airway smooth muscle cells. Cells were dispersed from bovine trachealis and loaded with fura 2-AM (0.5 microM) for imaging of single cells. Cyclopiazonic acid (CPA; 5 microM) inhibited refilling of both caffeine- and carbachol-sensitive calcium stores. In the presence of extracellular calcium, CPA caused a transient increase in [Ca2+]i from 166 +/- 11 to 671 +/- 100 nM, and then [Ca2+]i decreased to a sustained level (CPA plateau; 236 +/- 19 nM) significantly above basal. The CPA plateau spontaneously declined toward basal levels after 10 min and was attenuated by discharging intracellular calcium stores. When CPA was applied during sustained stimulation with caffeine or carbachol, decreases in [Ca2+]i were observed. We concluded that the CPA plateau depended on the presence of SR calcium and that SR Ca2+-ATPase activity contributed to sustained increases in [Ca2+]i during stimulation with caffeine and, to a lesser extent, carbachol.     

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.     

Effects of decorin and biglycan on human airway smooth muscle cell adhesion

Growth on a decorin matrix results in decreased human airway smooth muscle cell (HASMC) number, by decreasing proliferation and increasing apoptosis. We questioned whether these effects were related to abnormal extracellular matrix (ECM)-cell adhesion. HASMCs were seeded on decorin, biglycan, collagen type I or plastic. Actin organization and focal adhesion formation were assessed by staining for filamentous (F) and globular (G) actin, and vinculin, respectively. Gene expression for focal adhesion proteins, ECM molecules and HASMC receptors was measured. Protein levels for fibronectin, α(2), α(5), α(v) and β(3) integrin subunits and, focal adhesion kinase (FAK) were assessed. F-actin filaments were prominent in cells seeded on collagen I and plastic, less apparent in cells cultured on biglycan and faint in cells on decorin. Vinculin clustering was decreased in cells seeded on decorin and biglycan, as was vinculin gene expression. Compared to cells on plastic, cells on decorin had an increase in fibronectin gene expression. Seeding on decorin caused an increase in α(2) integrin subunit and platelet-derived growth factor receptor A gene expression. There was also an increase in α(2) and α(v) integrin subunit protein. Finally, FAK protein levels in cells seeded on decorin or biglycan were decreased compared to cells seeded on plastic or collagen I. Cells grown on proteoglycan matrices demonstrate evidence of abnormalities during many of the key processes involved in normal cell adhesion. Upregulation of cell surface receptor proteins, such as α(2) integrin subunit, may represent a compensatory mechanism to overcome poor adhesion induced by growth on these matrices.     

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.     

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.     

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.     

Neuropeptide modulation of lymphatic smooth muscle tone in the canine forelimb

Neurokinin A and B are putative inflammatory mediators. We assessed their ability to alter prenodal lymphatic resistance. Intralymphatic neurokinin A (3.0 x 10(-6), 3.0 x 10(-5) and 3.0 x 10(-4) mol l(-1)) significantly constricted lymphatics at the two highest doses. Preliminary experiments suggested that neurokinin B might dilate lymphatics. To test this, lymphatic pressure was increased by norepinephrine (3.1 x 10(-6) mol l(-1)). Neurokinin B (2.7 x 10(-4) mol l(-1)) was then infused intralymphatically during norepinephrine infusion. Norepinephrine increased perfusion pressure from 5.6 +/- 0.6 mmHg to 12.1 +/- 1.4 mmHg. Subsequent infusion of neurokinin B significantly decreased lymphatic perfusion pressure from 11.9 +/- 1.3 mmHg to 9.9 +/- 1.1 mmHg. These data indicate that neurokinin A and B can alter lymphatic resistance and are consistent with the hypothesis that lymph vessel function may be subject to modulation by neurokinins.     

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.     

Effects of micropatterned curvature on the motility and mechanical properties of airway smooth muscle cells

Geometric features such as size and shape of the microenvironment are known to alter cell behaviors such as growth, differentiation, apoptosis, and migration. Little is known, however, about the effect of curvature on cell behaviors despite that many cells reside in curved space of tubular organs such as the bronchial airways. To address this question, we fabricated micropatterned strips that mimic airway walls with varying curvature. Then, we cultured airway smooth muscle cells (ASMCs) on these strips and investigated the cells’ motility and mechanical properties using time-lapse imaging microscopy and optical magnetic twisting cytometry (OMTC). We found that both motility and mechanical properties of the ASMCs were influenced by the curvature. In particular, when the curvature increased from 0 to 1/150 μm⁻¹, the velocity of cell migration first decreased (0–1/750 μm⁻¹), and then increased (1/750–1/150 μm⁻¹). In contrast, the cell stiffness increased and then decreased. Thus, at the intermediate curvature (1/750 μm⁻¹) the ASMCs were the least motile, but most stiff. The contractility instead decreased consistently as the curvature increased. The level of F-actin, and vinculin expression within the ASMCs appeared to correlate with the contractility and motility, respectively, in relation to the curvature. These results may provide valuable insights to understanding the heterogeneity of airway constrictions in asthma as well as the developing and functioning of other tubular organs and tissue engineering.     

Effects of vibrotactile stimulation on the control of muscle tone and movement facilitation in children with cerebral injury

Afferent signals from the muscle's proprioceptors play important role in the control of muscle tone and in the facilitation of movements. Peripheral afferent pathway enables the restoration of connections with supraspinal structures and so includes mechanism of synaptic inhibition in the performance of normal movement. Different sensory stimuli, as vibrotactile stimulation, excite muscle's proprioceptors which then send sensorimotor information via spinal cord. In this way afferent signals promote cortical control and modulation of movements. The goal of this study is to evaluate the effects of vibrotactile stimulation on the spasticity and motor performance in children with cerebral injury. Subjects included in this study were 13 children who were developing the classification of spastic cerebral palsy. For all children perinatal brain damage was documented by medical reports and neonatal brain ultrasound scan. At the mean age of 3 years and 6 months subject underwent the assessment of motor development by Gross Motor Function Measurement (GMFM-88). Gross Motor Classification System (GMFCS) has been used to classify functions of lower extremities. Therapeutic intervention was conducted once a week during 3 months. All subjects were stimulated with vibrotactile stimuli of 40Hz in duration of 20 minutes in order to reduce spasticity. After the ending of the treatment subjects underwent second assessment of motor performance and the classification of lower extremities functions. The results have shown that there was a significant improvement in motor performance, what has been seen in the facilitation of rotations, better postural trunk stability and head control and in greater selectivity of movements. Further randomized, control trial investigations with bigger sample and included spasm scale are needed to gain better insight in the role of vibrotactile stimulation in the facilitation of normal movements.     

Effects of age on muscarinic agonist-induced contraction and IP accumulation in airway smooth muscle.

The effects of age on carbachol-stimulated force development and [3H]inositol phosphate production was studied in tracheal rings from guinea pigs aged 1 month and 25 months of age. The pD2 for the contractile response to carbachol was significantly reduced in tracheal tissues from old animals as compared to that of the young tissues (6.49 +/- 0.04, 7.09 +/- 0.04, n = 12), respectively. In contrast, inositol phosphate formation was not altered with increasing age when stimulated by carbachol or NaF, a direct activator of G proteins. Carbachol-induced inositol phosphate accumulation was inhibited by treatment with 1 micrograms/ml pertussis toxin, suggesting that IP1 accumulation is coupled to a pertussis-toxin-sensitive protein. The pD2 values for contraction (7.09 +/- 0.09, 6.49 +/- 0.04) were significantly different from the pD2 values for IP1 accumulation (4.72 +/- 0.14, 5.10 +/- 0.18) in both young and old tissues, respectively. These data suggest that IP1 accumulation is not responsible for the decreased contractile ability in tracheal smooth muscle during aging.