In vivo airway reactivity: predictive value of morphological estimates of airway smooth muscle.

Airway responsiveness to methacholine and other bronchoconstrictors is highly variable within and among species. The aim of the experiments in this report was to evaluate the importance of the quantity of airway smooth muscle as a determinant of intra- and inter-species variability in airway responsiveness. To do this we established concentration-response curves to methacholine in a sample of normal guinea pigs as well as in rat, rabbit, and dog. After challenge we excised the lungs for the quantitation of smooth muscle by morphometry. Animals were anesthetized with pentobarbital and mechanically ventilated using a Harvard ventilator. Aerosols of methacholine were administered in progressively doubling concentrations from 0.0625 to 256 mg/mL for a period of 30 s for each concentration. The maximal response, determined from pulmonary resistance (RL), and the concentration of methacholine required to effect 50% of the maximal RL were determined. After provocation testing the lungs were removed and fixed with 10% Formalin. Midsagittal sections and parahilar sections were stained with hematoxylin-phloxine-saffron for microscopic examination of smooth muscle. The images of all airways in the sections were traced using a camera lucida side-arm attachment and digitized using commercial software. The area of the airway wall occupied by smooth muscle was determined and standardized for airway size by dividing it by the square of the epithelial basement membrane length. The variability in airway smooth muscle in the intraparenchymal airways was significantly greater between than within individual guinea pigs (n = 13). This was not true of extraparenchymal airways.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autocrine regulation of asthmatic airway inflammation: role of airway smooth muscle

Chronic airway inflammation is one of the main features of asthma. Release of mediators from infiltrating inflammatory cells in the airway mucosa has been proposed to contribute directly or indirectly to changes in airway structure and function. The airway smooth muscle, which has been regarded as a contractile component of the airways responding to various mediators and neurotransmitters, has recently been recognised as a rich source of pro-inflammatory cytokines, chemokines and growth factors. In this review, we discuss the role of airway smooth muscle cells in the regulation and perpetuation of airway inflammation that contribute to the pathogenesis of asthma.     

Autocrine regulation of asthmatic airway inflammation: role of airway smooth muscle

Chronic airway inflammation is one of the main features of asthma. Release of mediators from infiltrating inflammatory cells in the airway mucosa has been proposed to contribute directly or indirectly to changes in airway structure and function. The airway smooth muscle, which has been regarded as a contractile component of the airways responding to various mediators and neurotransmitters, has recently been recognised as a rich source of pro-inflammatory cytokines, chemokines and growth factors. In this review, we discuss the role of airway smooth muscle cells in the regulation and perpetuation of airway inflammation that contribute to the pathogenesis of asthma.     

Autonomic response characteristics of porcine airway smooth muscle in vivo

We studied the autonomic response characteristics of airways in 65 swine in vivo. Tracheal smooth muscle response was measured isometrically in situ; bronchial response was measured simultaneously as change in airway resistance and dynamic compliance. To determine the optimal resting length at which maximal tracheal contraction was obtained, length-tension studies were generated in four animals using maximal electrical stimulation of the vagus nerves determined from stimulus-response characteristics in eight other swine. Pharmacological studies were performed in 25 animals to determine the relative potency and intrinsic activity of agonists (acetylcholine greater than histamine much greater than norepinephrine) causing contraction of trachea and bronchial airways. In 13 swine, the effects of autonomic stimulation were studied by intravenous administration of dimethylphenylpiperazinium (DMPP) after muscarinic blockade with 1.5 mg/kg iv atropine. Tracheal contraction caused by topical application of 3.4 X 10(-4) mol histamine (13.4 +/- 1.54 g/cm) was 96 +/- 7.2% blocked by 25 micrograms/kg iv DMPP in adrenal-intact animals; minimal relaxation was demonstrated in adrenalectomized animals, indicating absence of substantial sympathetic innervation to porcine trachea. Nonadrenergic innervation was not demonstrated. After beta-adrenergic blockade, sympathetic stimulation caused alpha-adrenergic contraction in bronchial airways but not in trachea. These data define the unique response characteristics of the airways of swine and demonstrate their utility for acute experimental study of airway responses in vivo.     

Heme oxygenase modulates oxidant-signaled airway smooth muscle contractility: role of bilirubin

Reactive oxygen species (ROS) increase the contractile response of airway smooth muscle (ASM). Heme oxygenase (HO) catabolizes heme to the powerful antioxidant bilirubin. Because HO is expressed in the airways, we investigated its effects on ASM contractility and ROS production in guinea pig trachea. HO expression was higher in the epithelium than in tracheal smooth muscle. Incubation of tracheal rings (TR) with the HO inhibitor tin protoporphyrin (SnPP IX) or the HO substrate hemin increased and decreased, respectively, ASM contractile response to carbamylcholine. The effect of hemin was reversed by SnPP and mimicked by the antioxidants superoxide dismutase (SOD) and catalase. Hemin significantly reduced the effect of carbamylcholine in rings treated with the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), compared with ODQ-treated rings without hemin incubation, suggesting that the CO-guanosine 3',5'-cyclic monophosphate pathway was not involved in the control of tracheal reactivity. SnPP and hemin increased and decreased ROS production by TR by 18 and 38%, respectively. Bilirubin (100 pM) significantly decreased TR contractility and ROS production. Hemin, bilirubin, and SOD/catalase decreased phosphorylation of the contractile protein myosin light chain, whereas SnPP significantly augmented it. These data suggest that modulation of the redox status by HO and, moreover, by bilirubin modulates ASM contractility by modulating levels of phosphorylated myosin light chain.     

In vivo recording of electrical activity of canine tracheal smooth muscle.

Electrical activity of the tracheal smooth muscle was studied using extracellular bipolar electrodes in 37 decerebrate, paralyzed, and mechanically ventilated dogs. A spontaneous oscillatory potential that consisted of a slow sinusoidal wave of 0.57 +/- 0.13 (SD) Hz mean frequency but lacked a fast spike component was recorded from 15 dogs. Lung collapse accomplished by bilateral pneumothoraxes evoked or augmented the slow potentials that were associated with an increase in tracheal muscle contraction in 26 dogs. This suggests that the inputs from the airway mechanoreceptors reflexly activate the tracheal smooth muscle cells. Bilateral vagal transection abolished both the spontaneous and the reflexly evoked slow waves and provided relaxation of the tracheal smooth muscle. Electrical stimulation of the distal nerve with a train pulse (0.5 ms, 1-30 Hz) evoked slow-wave oscillatory potentials accompanied by a contraction of the tracheal smooth muscle in all the experimental animals. Our observations in this in vivo study confirm that the electrical activity of tracheal smooth muscle consists of slow oscillatory potentials and that tracheal contraction is at least partly coupled to the slow-wave activity of the smooth muscle.     

Selected contribution: plasticity of airway smooth muscle stiffness and extensibility: role of length-adaptive mechanisms.

Airway smooth muscle exhibits the property of length adaptation, which enables it to optimize its contractility to the mechanical conditions under which it is activated. Length adaptation has been proposed to result from a dynamic modulation of contractile and cytoskeletal filament organization, in which the cell structure adapts to changes in cell shape at different muscle lengths. Changes in filament organization would be predicted to alter muscle stiffness and extensibility. We analyzed the effects of tracheal muscle length at the time of contractile activation on the stiffness and extensibility of the muscle during subsequent stretch over a constant range of muscle lengths. Muscle strips were significantly stiffer and less extensible after contractile activation at a short length than after activation at a long length, consistent with the prediction of a shorter, thicker array of the cytoskeletal filaments at a short muscle length. Stretch beyond the length of contractile activation resulted in a persistent reduction in stiffness, suggesting a stretch-induced structural rearrangement. Our results support a model in which the filament organization of airway smooth muscle cells is plastic and can be acutely remodeled to adapt to the changes in the external physical environment.     

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.     

Interleukin-9 influences chemokine release in airway smooth muscle: role of ERK

Interleukin (IL)-9 is a pleiotropic cytokine that has been proposed as a candidate gene for asthma. As IL-9 expression is correlated with airway hyperresponsiveness in animals, we examined the effects of IL-9 on cultured human airway smooth muscle (HASM) cells. IL-9 alone had no effect on IL-8 release, but at concentrations of > or =30 ng/ml, IL-9 significantly increased IL-8 release induced by TNF-alpha. IL-9 increased phosphorylation of extracellular signal-regulated protein kinase (ERK, p42 and p44) in a concentration- and time-dependent fashion, and U-0126 (10 micro M), which inhibits ERK phosphorylation, abolished the synergism between TNF-alpha and IL-9 on IL-8 release. IL-9 alone had no effect on eotaxin release into HASM cell supernatants but at concentrations of > or =10 ng/ml caused an approximately 50% increase in release of eotaxin evoked by IL-13 (10 ng/ml). U-0126 blocked the synergism between IL-9 and IL-13 on eotaxin release. IL-9 had no effect on cyclooxygenase-2 (COX-2) expression or PGE(2) release and did not augment the COX-2 expression that was induced by IL-1beta. Our results indicate that airway smooth muscle is a target for IL-9 and that IL-9 amplifies the potential for these cells to recruit eosinophils and neutrophils into the airways by a mechanism involving ERK.     

NO responsiveness in pulmonary artery and airway smooth muscle: the role of cGMP regulation

The purpose of this study was to assess intrinsic smooth muscle mechanisms contributing to greater nitric oxide (NO) responsiveness in pulmonary vascular vs. airway smooth muscle. Canine pulmonary artery smooth muscle (PASM) and tracheal smooth muscle (TSM) strips were used to perform concentration response studies to an NO donor, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO). PASM exhibited a greater NO responsiveness whether PASM and TSM were contracted with receptor agonists, phenylephrine and acetylcholine, respectively, or with KCl. The >10-fold difference in NO sensitivity in PASM was observed with both submaximal and maximal contractions. This difference in NO responsiveness was not due to differences in endothelial or epithelial barriers, since these were removed, nor was it due to the presence of cGMP-independent NO-mediated relaxation in either tissue. At equal concentrations of NO, the intracellular cGMP concentration ([cGMP]i) was also greater in PASM than in TSM. Phosphodiesterase (PDE) inhibition using isobutylmethylxanthine indicated that the greater [cGMP]i in PASM was not due to greater PDE activity in TSM. Expression of soluble guanylate cyclase (sGC) subunit mRNA (2 +/- 0.2 and 1.3 +/- 0.2 attomol/microg total RNA, respectively) and protein (47.4 +/- 2 and 27.8 +/- 3.9 ng/mg soluble homogenate protein, respectively) was greater in PASM than in TSM. sGCalpha1 and sGCbeta1 mRNA expression was equal in PASM but was significantly different in TSM, suggesting independent regulation of their expression. An intrinsic smooth muscle mechanism accounting for greater NO responsiveness in PASM vs. TSM is greater sGC activity.     

Historical perspective on airway smooth muscle: the saga of a frustrated cell.

Despite the lack of a clearly defined physiological function, airway smooth muscle receives substantial attention because of its involvement in the pathogenesis of asthma. Recent investigations have turned to the ways in which the muscle is influenced by its dynamic microenvironment. Ordinarily, airway smooth muscle presents little problem, even when maximally activated, because unending mechanical perturbations provided by spontaneous tidal breathing put airway smooth muscle in a perpetual state of "limbo," keeping its contractile machinery off balance and unable to achieve its force-generating potential. The dynamic microenvironment affects airway smooth muscle in at least two ways: by acute changes associated with disruption of myosin binding and by chronic changes associated with plastic restructuring of contractile and cytoskeletal filament organization. Plastic restructuring can occur when dynamic length changes occur between sequential contractile events or within a single contractile event. Impairment of these normal responses of airway smooth muscle to its dynamic environment may be implicated in airway hyperresponsiveness in asthma.     

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.     

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.     

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.     

Series-to-parallel transition in the filament lattice of airway smooth muscle.

Force-velocity curves measured at different times during tetani of sheep trachealis muscle were analyzed to assess whether velocity slowing could be explained by thick-filament lengthening. Such lengthening increases force by placing more cross bridges in parallel on longer filaments and decreases velocity by reducing the number of filaments spanning muscle length. From 2 s after the onset of stimulation, when force had achieved 42% of it final value, to 28 s, when force had been at its tetanic plateau for approximately 15 s, velocity decreases were exactly matched by force increases when force was adjusted for changes in activation, as assessed from the maximum power value in the force-velocity curves. A twofold change in velocity could be quantitatively explained by a series-to-parallel change in the filament lattice without any need to postulate a change in cross-bridge cycling rate.     

Bi-directional activation between human airway smooth muscle cells and T lymphocytes: role in induction of altered airway responsiveness

Because both T lymphocyte and airway smooth muscle (ASM) cell activation are events fundamentally implicated in the pathobiology of asthma, this study tested the hypothesis that cooperative intercellular signaling between activated T cells and ASM cells mediates proasthmatic changes in ASM responsiveness. Contrasting the lack of effect of resting human T cells, anti-CD3-activated T cells were found to adhere to the surface of naive human ASM cells, increase ASM CD25 cell surface expression, and induce increased constrictor responsiveness to acetylcholine and impaired relaxation responsiveness to isoproterenol in isolated rabbit ASM tissues. Comparably, exposure of resting T cells to ASM cells prestimulated with IgE immune complexes reciprocally elicited T cell adhesion to ASM cells and up-regulated T cell expression of CD25. Extended studies demonstrated that: 1) ASM cells express mRNAs and proteins for the cell adhesion molecules (CAMs)/costimulatory molecules, CD40, CD40L, CD80, CD86, ICAM-1 (CD54), and LFA-1 (CD11a/CD18); 2) apart from LFA-1, ASM cell surface expression of the latter molecules is up-regulated in the presence of activated T cells; and 3) pretreatment of ASM cells and tissues with mAbs directed either against CD11a or the combination of CD40 and CD86 completely abrogated both the activated T cell-induced changes in expression of the above CAMs/costimulatory molecules in ASM cells and altered ASM tissue responsiveness. Collectively, these observations identify the presence of bi-directional cross-talk between activated T cells and ASM cells that involves coligation of specific CAMs/costimulatory molecules, and this cooperative intercellular signaling mediates the induction of proasthmatic-like changes in ASM responsiveness.