Length oscillation induces force potentiation in infant guinea pig airway smooth muscle

Deep inspiration counteracts bronchospasm in normal subjects but triggers further bronchoconstriction in hyperresponsive airways. Although the exact mechanisms for this contrary response by normal and hyperresponsive airways are unclear, it has been suggested that the phenomenon is related to changes in force-generating ability of airway smooth muscle after mechanical oscillation. It is known that healthy immature airways of both humans and animals exhibit hyperresponsiveness. We hypothesize that the profile of active force generation after mechanical oscillation changes with maturation and that this change contributes to the expression of airway hyperresponsiveness in juveniles. We examined the effect of an acute sinusoidal length oscillation on the force-generating ability of tracheal smooth muscle from 1 wk, 3 wk, and 2- to 3-mo-old guinea pigs. We found that the length oscillation produced 15-20% initial reduction in active force equally in all age groups. This was followed by a force recovery profile that displayed striking maturation-specific features. Unique to tracheal strips from 1-wk-old animals, active force potentiated beyond the maximal force generated before oscillation. We also found that actin polymerization was required in force recovery and that prostanoids contributed to the maturation-specific force potentiation in immature airway smooth muscle. Our results suggest a potentiated mechanosensitive contractile property of hyperresponsive airway smooth muscle. This can account for further bronchoconstriction triggered by deep inspiration in hyperresponsive airways.     

Decreased airway narrowing and smooth muscle contraction in hyperresponsive pigs.

Increased smooth muscle contractility or reduced smooth muscle mechanical loads could account for the excessive airway narrowing and hyperresponsiveness seen in asthma. These mechanisms were investigated by using an allergen-induced porcine model of airway hyperresponsiveness. Airway narrowing to electric field stimulation was measured in isolated bronchial segments, over a range of transmural pressures (0-20 cmH(2)O). Contractile responses to ACh were measured in bronchial segments and in isolated tracheal smooth muscle strips isolated from control and test (ovalbumin sensitized and challenged) pigs. Test airways narrowed less than controls (P < 0.0001). Test pigs showed reduced contractility to ACh, both in isolated bronchi (P < 0.01) and smooth muscle strips (P < 0.01). Thus isolated airways from pigs exhibiting airway hyperresponsiveness in vivo are hyporesponsive in vitro. The decreased narrowing in bronchi from hyperresponsive pigs may be related to decreased smooth muscle contractility. These data suggest that mechanisms external to the airway wall may be important to the hyperresponsive nature of sensitized lungs.     

O3-induced mucosa-linked airway muscle hyperresponsiveness in the guinea pig.

We investigated the effects of ozone exposure (3.0 ppm, 2 h) on the responsiveness of guinea pig airway muscle in vitro from animals developing bronchial hyperreactivity. Muscarinic reactivity in vivo was determined by measuring specific airway resistance (sRaw) in response to increasing concentrations of aerosolized acetylcholine (ACh) administered before and 30 min after exposure. Immediately after reactivity testing, multiple tracheal rings from ozone- and air-exposed animals were prepared and the contractile responses to increasing concentrations of substance P, ACh, or KCl were assessed in the presence of 10 microM indomethacin with or without 1 microM phosphoramidon, an inhibitor of neutral endopeptidase. Isometric force generation in vitro was measured on stimulation by cumulative concentrations of the agonists, and force generation (in g/cm2) was calculated after determination of muscle cross-sectional area. The smooth muscle of mucosa-intact airways from guinea pigs with ozone-induced bronchial hyper-reactivity proved to be hyperresponsive in vitro to substance P and ACh but not to KCl. Pretreatment with phosphoramidon abolished the increase in substance P responsiveness but had no effect on muscarinic hyperresponsiveness after ozone exposure. Furthermore, substance P responsiveness was not augmented in ozone-exposed airways in which the mucosa had been removed before testing in vitro. Likewise, muscarinic hyperresponsiveness was not present in ozone-exposed airways without mucosa. Our data indicate that airway smooth muscle responsiveness is increased in guinea pigs with ozone-induced bronchial hyperreactivity and suggest that this hyperresponsiveness may be linked to non-cyclooxygenase mucosa-derived factors.     

O3-induced airway hyperresponsiveness to noncholinergic system and other stimuli.

The effect of O3 exposure (3 ppm, 1 h) on the in vivo and in vitro airway responsiveness, as well as the changes in cell contents in bronchoalveolar lavage (BAL) fluid, were evaluated 16-18 h after O3 exposure in sensitized and nonsensitized male guinea pigs. The sensitization procedure was performed through repeated inhalation of ovalbumin for 3 wk. Increase in pulmonary insufflation pressure produced by the excitatory nonadrenergic noncholinergic (eNANC) system, histamine, and antigen were assessed in in vivo conditions, whereas airway responsiveness to histamine and substance P was evaluated in in vitro conditions by use of tracheal chains with or without epithelium and lung parenchymal strips. We found that O3 exposure 1) increased the neutrophil content in BAL fluids in both sensitized and nonsensitized guinea pigs, 2) caused hyperresponsiveness to eNANC stimulation in nonsensitized guinea pigs (although combination of sensitization and O3 exposure paradoxically abolished the hyperresponsiveness to eNANC stimulation), 3) increased the in vivo bronchoconstrictor responses to histamine and antigen, 4) caused hyperresponsiveness to substance P in nonsensitized tracheae with or without epithelium and in sensitized tracheae with epithelium, 5) did not modify the responsiveness to histamine in tracheae with or without epithelium (and in addition, epithelium removal caused hyperresponsiveness to histamine even in those tracheae exposed to O3), and 6) produced hyperresponsiveness to histamine in lung parenchymal strips either from sensitized or nonsensitized guinea pigs.     

Repeated antigen challenge induces airway hyperresponsiveness with tissue eosinophilia in guinea pigs

To test the hypothesis that the development of airway hyperresponsiveness (AHR) lasting greater than or equal to 3 days after the last antigenic exposure required repeated mediator release, we compared dose-response changes in lung resistance (RL) to acetylcholine (ACh) in animals sensitized with 1% ovalbumin (OA), 4% Bordatella pertussis aerosol and subsequently challenged with 0.5% OA aerosol twice weekly for 4-6 wk vs. animals receiving saline aerosol instead of OA. Despite antihistamine pretreatment, each OA challenge produced cyanosis and inspiratory indrawing. Blood gas analysis in six guinea pigs revealed an immediate fall in arterial PO2 (PaO2) from 104.3 +/- 4.9 to 35.4 +/- 2.2 Torr after a 1-min exposure to aerosolized OA. ACh dose-response measurements of RL 3 days after the last OA challenge demonstrated a leftward shift and an increased magnitude of response. These differences were less marked at 7 days, and by 14 days after the last OA challenge, ACh dose-response curves were not different from those of control guinea pigs. Sensitization without repeated antigen challenge did not cause hyperresponsiveness. Morphometric analysis showed significantly increased numbers of eosinophils in the epithelium of airways in hyperresponsive guinea pigs, without neutrophil infiltration or alterations in epithelium and airway wall areas. We conclude that repeated antigenic challenge, but not sensitization alone, causes prolonged AHR in guinea pigs, which is associated with tissue eosinophilia.     

Airway responsiveness and prostaglandin generation in scorbutic guinea pigs

Airway responsiveness to histamine aerosol and lung prostaglandin generation were investigated in normal, partially vitamin C deficient and scorbutic guinea pigs. The ascorbic acid content of the lung expressed as microgram/100 mg wet weight lung parenchyma decreased from 22.1 +/- 1.8 (mean +/- SE) in the control group to 9.0 +/- 1.4 and 1.8 +/- 0.4 in tissues from partially ascorbic acid deficient and scorbutic animals, respectively. Guinea pigs on low and ascorbic acid deficient diets developed significant airway hyperresponsiveness to histamine aerosol after 3 and 4 weeks. Indomethacin (30 mg/Kg, i.p.) further increased the airway hyperresponsiveness in scorbutic animals but was without effect in control animals. Prostaglandin generation from different parts of the lung was significantly changed by the diets. However, airway hyperresponsiveness was not directly attributable to altered prostanoid generation. Scorbutic conditions did not alter the electrophysiological characteristics of airway smooth muscle namely, resting membrane potential and electrogenic sodium pump activity. In summary, ascorbic acid deficiency causes airway hyperresponsiveness to histamine in guinea pigs. This alteration seems not to be related to an altered prostaglandin generation by the lung or to the electrophysiological properties of airway smooth muscle.     

Different ontogeny of rate of force generation and shortening velocity in guinea pig trachealis.

Juveniles of many species, including humans, display greater airway responsiveness than do adults. This may involve changes in airway smooth muscle function. In the present work we studied force production and shortening velocity in trachealis from 1-wk-old (1 wk), 3-wk-old (3 wk), and 3-mo-old (adult) guinea pigs. Strips were electrically stimulated (60 Hz, 18 V) at their optimal length (l(o)) to obtain maximum active stress (P(o)) and rate of stress generation. Then, force-velocity curves were elicited at 2.5 s from the onset of the stimulus. By applying a recently developed modification of Hill's equation for airway smooth muscle, the maximum shortening velocity at zero load (V(o)) and the value alpha. gamma/beta, an index of internal resistance to shortening (Rsi), were calculated (alpha, beta, and gamma are the constants of the equation). P(o) increased little with maturation, whereas the rate of stress generation increased significantly (0.40 +/- 0.03, 0.45 +/- 0.03, 0. 51 +/- 0.03 P(o)/s for 1 wk, 3 wk, and adult animals). V(o) slightly increased early with maturation to decrease significantly later (1. 79 +/- 0.67, 2.45 +/- 0.92, and 0.55 +/- 0.09 l(o)/s for 1 wk, 3 wk, and adult animals), whereas the Rsi showed an opposite trend (14.98 +/- 5.19, 8.99 +/- 3.01, and 32.07 +/- 5.54 mN. mm(-2). l(o)(-1). s for 1 wk, 3 wk, and adult animals). This early increase of force generation in combination with late increase of Rsi may explain the changes of V(o) with age. An elevated V(o) may contribute to the incidence of airway hyperresponsiveness in healthy juveniles.     

A maturational model for the study of airway smooth muscle adaptation to mechanical oscillation

It has been shown that mechanical stretches imposed on airway smooth muscle (ASM) by deep inspiration reduce the subsequent contractile response of the ASM. This passive maneuver of lengthening and retraction of the muscle is beneficial in normal subjects to counteract bronchospasm. However, it is detrimental to hyperresponsive airways because it triggers further bronchoconstriction. Although the exact mechanisms for this contrary response by normal and hyperresponsive airways are unclear, it has been suggested that the phenomenon is related to changes in ASM adaptability to mechanical oscillation. Healthy immature airways of both human and animal exhibit hyperresponsiveness, but whether the adaptative properties of hyperresponsive airway differ from normal is still unknown. In this article, we review the phenomenon of ASM adaptation to mechanical oscillation and its relevance and implication to airway hyperresponsiveness. We demonstrate that the age-specific expression of ASM adaptation is prominent using an established maturational animal model developed in our laboratory. Our data on immature ASM showed potentiated contractile force shortly after a length oscillation compared with the maximum force generated before oscillation. Several potential mechanisms such as myogenic response, changes in actin polymerization, or changes in the quantity of the cytoskeletal regulatory proteins plectin and vimentin, which may underlie this age-specific force potentiation, are discussed. We suggest a working model of the structure of smooth muscle associated with force transmission, which may help to elucidate the mechanisms responsible for the age-specific expression of smooth muscle adaptation. It is important to study the maturational profile of ASM adaptation as it could contribute to juvenile hyperresponsiveness.     

Do inflammatory mediators influence the contribution of airway smooth muscle contraction to airway hyperresponsiveness in asthma?

It is now accepted that a host of cytokines, chemokines, growth factors, and other inflammatory mediators contributes to the development of nonspecific airway hyperresponsiveness in asthma. Yet, relatively little is known about how inflammatory mediators might promote airway structural remodeling or about the molecular mechanisms by which they might exaggerate smooth muscle shortening as observed in asthmatic airways. Taking a deep inspiration, which provides relief of bronchodilation in normal subjects, is less effective in asthmatic subjects, and some have speculated that this deficiency stems directly from an abnormality of airway smooth muscle and results in airway hyperresponsiveness to constrictor agonists. Here, we consider some of the mechanisms by which inflammatory mediators might acutely or chronically induce changes in the contractile apparatus that in turn might contribute to hyperresponsive airways in asthma.     

Inhibition of antigen-induced airway hyperresponsiveness, but not acute hypoxia nor airway eosinophilia, by an antagonist of platelet-activating factor

The role of platelet-activating factor (PAF) in Ag-induced airway hyperresponsiveness was evaluated in a guinea pig model using the PAF antagonist SDZ 64-412. Repeated OVA challenge by aerosol (twice weekly x 4 wk) of previously sensitized guinea pigs produced striking airway hyperresponsiveness as determined by pulmonary resistance changes to increasing doses of inhaled acetylcholine given 3 days after the last OVA challenge. Each OVA challenge produced significant hypoxia that was unaffected by oral pretreatment with 20 mg/kg SDZ 64-412, 2 h before each challenge (pO2 = 35 +/- 2 mm Hg for OVA alone vs 40 +/- 6 mm Hg for SDZ and OVA groups, respectively). SDZ 64-412 pretreatment abolished the airway hyperresponsiveness resulting from repeated Ag challenge. Morphometric analysis revealed that SDZ 64-412 treatment had no effect on the increased numbers of eosinophils that infiltrated the airways of OVA-challenged guinea pigs. These results suggest that PAF may be a primary mediator of airway hyperresponsiveness, but not acute bronchoconstriction, induced by repeated Ag challenge. This activity of PAF appears independent of eosinophil recruitment to airways.     

Bronchial mucosal permeability.

The tracheobronchial epithelium has well-developed tight junctions which on a morphologic basis should be markedly resistant to penetration by protein molecules. Despite this, antigen inhalation in monkeys allergic to Ascaris suum results in the rapid onset of pulmonary physiologic changes. Recent studies in man and animals have shown that a substantial number of mast cells exist in the bronchial lumen and epithelium. We suggest that antigen-antibody interaction initially occurs on these superficial mast cells leading to mediator release and the stimulation of airway irritant receptors. Antigen challenge also results in increased epithelial permeability to protein in the Ascaris-allergic monkey, and from studies on guinea pigs we suggest that this is due to alterations in the tight junctions. Antigen challenge in the monkey also produces increased permeability to labeled histamine and hyperresponsiveness to low concentrations of histamine. We suggest that the apparent airway hyperreactivity to inhaled histamine seen after inhalation of ozone, and NO2, or after upper respiratory infections could be due to damage to epithelial tight junctions. The resultant increase in mucosal permeability would result in an increased amount of histamine reaching airway smooth muscle for a given inhaled concentration.     

Bronchial responses to substance P after antigen challenge in the guinea-pig: in vivo and in vitro studies

The effect of antigen challenge on the airway responses to substance P and on the epithelial neutral endopeptidase (NEP) activity was investigated in aerosol sensitized guinea-pigs. In vivo, bronchial responses to aerosolized substance P were similar to the responses observed in antigen-challenged guinea-pigs and in the control groups. In contrast, when the guinea-pigs were pretreated with the NEP inhibitor, phosphoramidon, a significant increase in the airway responses to substance P was observed after antigen challenge in vivo. However, in vitro, the contractile responses of the tracheal smooth muscle to substance P were similar between groups of guinea-pigs, in respect to the presence or absence of the epithelium and/or phosphoramidon. Histological studies showed an accumulation of eosinophils in the tracheal submucosa after antigen challenge and intact epithelial cells. These results show that in vivo bronchial hyperresponsiveness to substance P after antigen challenge in the guinea-pig is not associated with increased responses of the smooth muscle to exogenous SP in vitro. In addition, the results with phosphoramidon suggest that loss of NEP activity cannot account for the in vivo bronchial hyperresponsiveness to substance P presently observed.     

Smooth muscle molecular mechanics in airway hyperresponsiveness and asthma

Asthma is a respiratory disorder characterized by airway inflammation and hyperresponsiveness associated with reversible airway obstruction. The relative contributions of airway hyperresponsiveness and inflammation are still debated, but ultimately, airway narrowing mediated by airway smooth muscle contraction is the final pathway to asthma. Considerable effort has been devoted towards identifying the factors that lead to the airway smooth muscle hypercontractility observed in asthma, and this will be the focus of this review. Airway remodeling has been observed in severe and fatal asthma. However, it is unclear whether remodeling plays a protective role or worsens airway responsiveness. Smooth muscle plasticity is a mechanism likely implicated in asthma, whereby contractile filament rearrangements lead to maximal force production, independent of muscle length. Increased smooth muscle rate of shortening via altered signaling pathways or altered contractile protein expression has been demonstrated in asthma and in numerous models of airway hyperresponsiveness. Increased rate of shortening is implicated in counteracting the relaxing effect of tidal breathing and deep inspirations, thereby creating a contracted airway smooth muscle steady-state. Further studies are therefore required to understand the numerous mechanisms leading to the airway hyperresponsiveness observed in asthma as well as their multiple interactions.     

Epithelial modulation of allergen and drug effects in guinea pig airways.

The effect of egg albumin (EA) challenge on tracheal tube preparations from sensitized guinea pigs was studied with regard to EA permeability, histamine release and penetrability, and the contractile response of the preparation. We used a plethysmographic method that allowed simultaneous measurement of changes in smooth muscle tension and collection of samples for determination of mediators. Our results clearly show that epithelial damage potentiates the contractile response to histamine, potassium ions, and acetylcholine. Epithelial damage did not alter the maximal contractile response in preparations challenged with high antigen concentrations (EA, 1 mg/ml), but histamine release measured in the perfusate increased substantially. The permeability of the preparations to EA was greater when the epithelium was damaged. No increase in the permeability in response to the EA challenge was observed. The present study has demonstrated that guinea pig airway epithelium constitutes a barrier for both antigen and drugs. We also present a method for recording contractile responses from intact whole tracheal preparations, in which the epithelium can still act as a barrier, as is the case in vivo.     

Epithelium modulates the reactivity of ovalbumin-sensitized guinea-pig airway smooth muscle

Mechanical removal of the airway epithelium alters the in vitro reactivity of airway smooth muscle. The modulation of reactivity may involve the release of inhibitory and excitatory factors from epithelial cells. Guinea pigs sensitized with ovalbumin have been used as an animal model of airway hyperreactivity. We evaluated the influence of the epithelium on the reactivity of in vitro tracheal smooth muscle from control and ovalbumin-sensitized guinea pigs, and the extent to which the presence of the epithelium affects the contractile response to in vitro challenge with ovalbumin. In both control and ovalbumin-sensitized tissues, epithelium removal increased the sensitivity of the preparations to histamine, methacholine and isoproterenol to a similar extent, i.e., 2- to 2.5-fold. Epithelium removal resulted in an 8.1-fold increase in sensitivity to ovalbumin in sensitized tissues. The epithelium appears not only to modulate the reactivity of the tissues to bronchoactive agents, but it also influences the magnitude of the contractile response following antigen challenge.     

Effect of acetylcholinesterase activity on pathogenesis of airway hyperresponsiveness in guinea pigs.

To clarify the effect of acetylcholinesterase (AChE) on the pathogenesis of airway hyperresponsiveness, AChE activities in tracheal smooth muscle and lung tissue from congenitally bronchial-hypersensitive (BHS) and bronchial-hyposensitive (BHR) guinea pigs were compared. For this purpose, AChE activities were determined by measuring the rate of absorbance of tissue homogenate. Relative amounts of AChE mRNA were also evaluated by the RT-PCR method. In both tracheal smooth muscle and lung tissue from BHS, the AChE activity and the relative amount of AChE mRNA were less than those in BHR. These results suggest that the reduced AChE activity is at least a candidate for inducing airway hyperresponsiveness.     

Double-stranded RNA causes airway hyperreactivity and neuronal M2 muscarinic receptor dysfunction.

Viral infection causes dysfunction of inhibitory M2 muscarinic receptors (M2Rs) on parasympathetic nerves, leading to airway hyperreactivity. The mechanisms of M2R dysfunction are incompletely understood. Double-stranded RNA (dsRNA), a product of viral replication, promotes the expression of interferons. Interferon-gamma decreases M2R gene expression in cultured airway parasympathetic neurons. In this study, guinea pigs were treated with dsRNA (1 mg/kg ip) on 2 consecutive days. Twenty-four hours later, anesthetized guinea pigs had dysfunctional M2Rs and were hyperresponsive to electrical stimulation of the vagus nerves, in the absence of inflammation. DsRNA did not affect either cholinesterase or the function of postjunctional M3 muscarinic receptors on smooth muscle. M2Rs on the nerves supplying the heart were also dysfunctional, but M2Rs on the heart muscle itself functioned normally. Thus dsRNA causes increased bronchoconstriction and bradycardia via increased release of ACh from the vagus nerves because of loss of M2R function on parasympathetic nerves in the lungs and heart. Production of dsRNA may be a mechanism by which viruses cause dysfunction of neuronal M2Rs and airway hyperreactivity.     

Transgenic smooth muscle expression of the human CysLT1 receptor induces enhanced responsiveness of murine airways to leukotriene D4

Cysteinyl leukotrienes (CysLTs) exert potent proinflammatory actions and contribute to many of the symptoms of asthma. Using a model of allergic sensitization and airway challenge with Aspergillus fumigatus (Af), we have found that Th2-type inflammation and airway hyperresponsiveness (AHR) to methacholine (MCh) were associated with increased LTD(4) responsiveness in mice. To explore the importance of increased CysLT signaling in airway smooth muscle function, we generated transgenic mice that overexpress the human CysLT1 receptor (hCysLT(1)R) via the alpha-actin promoter. These receptors were expressed abundantly and induced intracellular calcium mobilization in airway smooth muscle cells from transgenic mice. Force generation in tracheal ring preparations ex vivo and airway reactivity in vivo in response to LTD(4) were greatly amplified in hCysLT(1)R-overexpressing mice, indicating that the enhanced signaling induces coordinated functional changes of the intact airway smooth muscle. The increase of AHR imposed by overexpression of the hCysLT(1)R was greater in transgenic BALB/c mice than in transgenic B6 x SJL mice. In addition, sensitization- and challenge-induced increases in airway responsiveness were significantly greater in transgenic mice than that of nontransgenic mice compared with their respective nonsensitized controls. The amplified AHR in sensitized transgenic mice was not due to an enhanced airway inflammation and was not associated with similar enhancement in MCh responsiveness. These results indicate that a selective hCysLT(1)R-induced contractile mechanism synergizes with allergic AHR. We speculate that hCysLT(1)R signaling contributes to a hypercontractile state of the airway smooth muscle.     

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.