Transgenic overexpression of beta(2)-adrenergic receptors in airway epithelial cells decreases bronchoconstriction

Airway epithelial cells express beta(2)-adrenergic receptors (beta(2)-ARs), but their role in regulating airway responsiveness is unclear. With the Clara cell secretory protein (CCSP) promoter, we targeted expression of beta(2)-ARs to airway epithelium of transgenic (CCSP-beta(2)-AR) mice, thereby mimicking agonist activation of receptors only in these cells. In situ hybridization confirmed that transgene expression was confined to airway epithelium, and autoradiography showed that beta(2)-AR density in CCSP-beta(2)-AR mice was approximately twofold that of nontransgenic (NTG) mice. Airway responsiveness measured by whole body plethysmography showed that the methacholine dose required to increase enhanced pause to 200% of baseline (ED(200)) was greater for CCSP-beta(2)-AR than for NTG mice (345 +/- 34 vs. 157 +/- 14 mg/ml; P < 0.01). CCSP-beta(2)-AR mice were also less responsive to ozone (0.75 ppm for 4 h) because enhanced pause in NTG mice acutely increased to 77% over baseline (P < 0.05) but remained unchanged in the CCSP-beta(2)-AR mice. Although both groups were hyperreactive to methacholine 6 h after ozone exposure, the ED(200) for ozone-exposed CCSP-beta(2)-AR mice was equivalent to that for unexposed NTG mice. These findings show that epithelial cell beta(2)-ARs regulate airway responsiveness in vivo and that the bronchodilating effect of beta-agonists results from activation of receptors on both epithelial and smooth muscle cells.     

Targeted transgenic expression of beta(2)-adrenergic receptors to type II cells increases alveolar fluid clearance

Clearance of edema fluid from the alveolar space can be enhanced by endogenous and exogenous beta-agonists. To selectively delineate the effects of alveolar type II (ATII) cell beta(2)-adrenergic receptors (beta(2)-ARs) on alveolar fluid clearance (AFC), we generated transgenic (TG) mice that overexpressed the human beta(2)-AR under control of the rat surfactant protein C promoter. In situ hybridization showed that transgene expression was consistent with the distribution of ATII cells. TG mice expressed 4.8-fold greater beta(2)-ARs than nontransgenic (NTG) mice (939 +/- 113 vs. 194 +/- 18 fmol/mg protein; P < 0.001). Basal AFC in TG mice was approximately 40% greater than that in untreated NTG mice (15 +/- 1.4 vs. 10.9 +/- 0.6%; P < 0.005) and approached that of NTG mice treated with the beta-agonist formoterol (19.8 +/- 2.2%; P = not significant). Adrenalectomy decreased basal AFC in TG mice to 9.7 +/- 0.5% but had no effect on NTG mice (11.5 +/- 1.0%). Na(+)-K(+)-ATPase alpha(1)-isoform expression was unchanged, whereas alpha(2)-isoform expression was approximately 80% greater in the TG mice. These findings show that beta(2)-AR overexpression can be an effective means to increase AFC in the absence of exogenous agonists and that AFC can be stimulated by activation of beta(2)-ARs specifically expressed on ATII cells.     

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.     

Endogenous Gs-coupled receptors in smooth muscle exhibit differential susceptibility to GRK2/3-mediated desensitization

Although G protein-coupled receptor (GPCR) kinases (GRKs) have been shown to mediate desensitization of numerous GPCRs in studies using cellular expression systems, their function under physiological conditions is less well understood. In the current study, we employed various strategies to assess the effect of inhibiting endogenous GRK2/3 on signaling and function of endogenously expressed G s-coupled receptors in human airway smooth muscle (ASM) cells. GRK2/3 inhibition by expression of a Gbetagamma sequestrant, a GRK2/3 dominant-negative mutant, or siRNA-mediated knockdown increased intracellular cAMP accumulation mediated via beta-agonist stimulation of the beta-2-adrenergic receptor (beta 2AR). Conversely, neither 5'-( N-ethylcarboxamido)-adenosine (NECA; activating the A2b adenosine receptor) nor prostaglandin E2 (PGE 2; activating EP2 or EP4 receptors)-stimulated cAMP was significantly increased by GRK2/3 inhibition. Selective knockdown using siRNA suggested the majority of PGE 2-stimulated cAMP in ASM was mediated by the EP2 receptor. Although a minor role for EP3 receptors in influencing PGE 2-mediated cAMP was determined, the GRK2/3-resistant nature of EP2 receptor signaling in ASM was confirmed using the EP2-selective agonist butaprost. Somewhat surprisingly, GRK2/3 inhibition did not augment the inhibitory effect of the beta-agonist on mitogen-stimulated increases in ASM growth. These findings demonstrate that with respect to G s-coupled receptors in ASM, GRK2/3 selectively attenuates beta 2AR signaling, yet relief of GRK2/3-dependent beta 2AR desensitization does not influence at least one important physiological function of the receptor.     

Cytokines regulate beta-2-adrenergic receptor responsiveness in airway smooth muscle via multiple PKA- and EP2 receptor-dependent mechanisms

Beta2AR desensitization in airway smooth muscle (ASM) mediated by airway inflammation has been proposed to contribute to asthma pathogenesis and diminished efficacy of beta-agonist therapy. Mechanistic insight into this phenomenon is largely conceptual and lacks direct empirical evidence. Here, we employ molecular and genetic strategies to reveal mechanisms mediating cytokine effects on ASM beta2AR responsiveness. Ectopic expression of inhibitory peptide (PKI-GFP) or a mutant regulatory subunit of PKA (RevAB-GFP) effectively inhibited intracellular PKA activity in cultured human ASM cells and enhanced beta2AR responsiveness by mitigating both agonist-specific (beta-agonist-mediated) desensitization and cytokine (IL-1beta and TNF-alpha)-induced heterologous desensitization via actions on multiple targets. In the absence of cytokine treatment, PKA inhibition increased beta2AR-mediated signaling by increasing both beta2AR-G protein coupling and intrinsic adenylyl cyclase activity. PKI-GFP and RevAB-GFP expression also conferred resistance to cytokine-promoted beta2AR-G protein uncoupling and disrupted feed-forward mechanisms of PKA activation by attenuating the induction of COX-2 and PGE2. Cytokine treatment of tracheal ring preparations from wild-type mice resulted in a profound loss of beta-agonist-mediated relaxation of methacholine-contracted rings, whereas rings from EP2 receptor knockout mice were largely resistant to cytokine-mediated beta2AR desensitization. These findings identify EP2 receptor- and PKA-dependent mechanisms as the principal effectors of cytokine-mediated beta2AR desensitization in ASM.     

Effect of 1,1-dimethylphenyl 1,4-piperazinium on mouse tracheal smooth muscle responsiveness

Bronchial hyperresponsiveness is one of the main features of asthma. A nicotinic receptor agonist, 1,1-dimethylphenyl 1,4-piperazinium (DMPP), has been shown to have an inhibitory effect on airway response to methacholine in an in vivo model of asthma. The aims of this study were to 1) verify whether nicotinic acetylcholine receptors (nAChR) were present on mouse tracheal smooth muscle, 2) verify whether bronchoprotection observed in mice was due to a direct effect on airway smooth muscle, and 3) compare the effects of nicotinic agonists to that of salbutamol. Alpha3-, alpha4-, and alpha7-nAChR subunits were detected by immunofluorescence on tracheal tissues from normal BALB/c mice. The effect of DMPP on tracheal responsiveness was verified by an isometric method. Tracheas were isolated from normal mice, placed in organ baths, and contracted with a single dose of methacholine. Cumulative doses of DMPP or salbutamol were added to the baths. Results show that mouse tracheal smooth muscle is positive for alpha4- and alpha7-nAChR subunits and that the epithelium is positive for alpha3-, alpha4-, and alpha7-subunits. DMPP induced a greater dose-dependent relaxation of tracheal smooth muscles precontracted with methacholine than with salbutamol. These results suggest that the smooth muscle-relaxing effect of DMPP could have some interest in the treatment of obstructive pulmonary diseases.     

Enhanced airway reactivity and inflammation in A2A adenosine receptor-deficient allergic mice

A(2A) adenosine receptor (A(2A)AR) has potent anti-inflammatory properties, which may be important in the regulation of airway reactivity and inflammation. Inflammatory cells that possess A(2A)AR also produce nitrosative stress, which is associated with pathophysiology of asthma, so we hypothesized that A(2A)AR deficiency may lead to increased airway reactivity and inflammation through nitrosative stress. Thus the present study was carried out to investigate the role of A(2A)AR on airway reactivity, inflammation, NF-kappaB signaling, and nitrosative stress in A(2A)AR knockout (KO) and wild-type (WT) mice using our murine model of asthma. Animals were sensitized intraperitoneally on days 1 and 6 with 200 microg of ragweed, followed by aerosolized challenges with 0.5% ragweed on days 11, 12, and 13, twice a day. On day 14, airway reactivity to methacholine was assessed as enhanced pause (Penh) using whole body plethysmography followed by bronchoalveolar lavage (BAL) and lung collection for various analyses. Allergen challenge caused a significant decrease in expression of A(2A)AR in A(2A) WT sensitized mice, with A(2A)AR expression being undetected in A(2A) KO sensitized group leading to decreased lung cAMP levels in both groups. A(2A)AR deletion/downregulation led to an increase in Penh to methacholine and influx of total cells, eosinophils, lymphocytes, and neutrophils in BAL with highest values in A(2A) KO sensitized group. A(2A) KO sensitized group further had increased NF-kappaB expression and nitrosative stress compared with WT sensitized group. These data suggest that A(2A)AR deficiency leads to airway inflammation and airway hyperresponsiveness, possibly via involvement of nitrosative stress in this model of asthma.     

Inhibition of human airway smooth muscle cell proliferation by beta 2-adrenergic receptors and cAMP is PKA independent: evidence for EPAC involvement

Mechanisms by which beta-adrenergic receptor (beta AR) agonists inhibit proliferation of human airway smooth muscle (HASM) cells were investigated because of their potential relevance to smooth muscle hyperplasia in asthma. We hypothesized that beta AR agonists would inhibit mitogenesis in HASM cells via the beta 2AR, an increase in cAMP, and PKA activation. HASM cells were treated for 24 h with various agents and then analyzed for [3H]thymidine incorporation as a measure of cell proliferation. EGF stimulated proliferation by approximately 10-fold. The nonselective beta AR agonist isoproterenol and the beta 2AR-selective agonists albuterol and salmeterol inhibited EGF-stimulated proliferation by more than 50%, with half-maximal effects at 4.8 nM, 110 nM, and 6.7 nM, respectively. A beta 2AR-selective antagonist inhibited the isoproterenol effect with 100-fold greater potency than a beta 1AR-selective antagonist, confirming beta 2AR involvement in the inhibition of proliferation. The cAMP-elevating agents PGE2 and forskolin decreased EGF-induced proliferation, suggesting cAMP as the mediator. beta 2AR agonists and forskolin also inhibited proliferation stimulated by lysophosphatidic acid (LPA) as well as the synergistic proliferation stimulated by LPA+EGF. Importantly, PKA-selective cAMP analogs did not inhibit proliferation at concentrations that maximally activated PKA (10-100 microM), whereas a cAMP analog selective for the exchange protein directly activated by cAMP (EPAC), 8-(4-chlorophenylthio)-2'-O-methyl-cAMP, maximally inhibited proliferation at a concentration that did not activate PKA (10 microM). These data show that beta 2AR agonists and other cAMP-elevating agents decrease proliferation in HASM cells via a PKA-independent mechanism, and they provide pharmacological evidence for involvement of EPAC or an EPAC-like cAMP effector protein instead.     

Computational assessment of airway wall stiffness in vivo in allergically inflamed mouse models of asthma.

Allergic inflammation is known to cause airway hyperresponsiveness in mice. However, it is not known whether inflammation affects the stiffness of the airway wall, which would alter the load against which the circumscribing smooth muscle shortens when activated. Accordingly, we measured the time course of airway resistance immediately following intravenous methacholine injection in acutely and chronically allergically inflamed mice. We estimated the effective stiffness of the airway wall in these animals by fitting to the airway resistance profiles a computational model of a dynamically narrowing airway embedded in elastic parenchyma. Effective airway wall stiffness was estimated from the model fit and was found not to change from control in either the acute or chronic inflammatory groups. However, the acutely inflamed mice were hyperresponsive compared with controls, which we interpret as reflecting increased delivery of methacholine to the airway smooth muscle through a leaky pulmonary endothelium. These results support the notion that acutely inflamed BALB/c mice represent an animal model of functionally normal airway smooth muscle in a transiently abnormal lung.     

CD38-deficient mice have reduced airway hyperresponsiveness following IL-13 challenge

The transmembrane glycoprotein CD38 in airway smooth muscle is the source of cyclic-ADP ribose, an intracellular calcium-releasing molecule, and is subject to regulatory effects of cytokines such as interleukin (IL)-13, a cytokine implicated in asthma. We investigated the role of CD38 in airway hyperresponsiveness using a mouse model of IL-13-induced airway disease. Wild-type (WT) and CD38-deficient (CD38KO) mice were intranasally challenged with 5 microg of IL-13 three times on alternate days under isoflurane anesthesia. Lung resistance (R(L)) in response to inhaled methacholine was measured 24 h after the last challenge in pentobarbital-anesthetized, tracheostomized, and mechanically ventilated mice. Bronchoalveolar cytokines, bronchoalveolar and parenchymal inflammation, and smooth muscle contractility and relaxation using tracheal segments were also evaluated. Changes in methacholine-induced R(L) were significantly greater in the WT than in the CD38KO mice following intranasal IL-13 challenges. Airway reactivity after IL-13 exposure, as measured by the slope of the methacholine dose-response curve, was significantly higher in the WT than in the CD38KO mice. The rate of isometric force generation in tracheal segments (e.g., smooth muscle reactivity) was greater in the WT than in the CD38KO mice following incubation with IL-13. IL-13 treatment reduced isoproterenol-induced relaxations to similar magnitudes in tracheal segments obtained from WT and CD38KO mice. Both WT and CD38KO mice developed significant bronchoalveolar and parenchymal inflammation after IL-13 challenges compared with na?ve controls. The results indicate that CD38 contributes to airway hyperresponsiveness in lungs exposed to IL-13 at least partly by increasing airway smooth muscle reactivity to contractile agonists.     

Soluble guanylyl cyclase expression is reduced in allergic asthma

Soluble guanylyl cyclase (sGC) is an enzyme highly expressed in the lung that generates cGMP contributing to airway smooth muscle relaxation. To determine whether the bronchoconstriction observed in asthma is accompanied by changes in sGC expression, we used a well-established murine model of allergic asthma. Histological and biochemical analyses confirmed the presence of inflammation in the lungs of mice sensitized and challenged with ovalbumin (OVA). Moreover, mice sensitized and challenged with OVA exhibited airway hyperreactivity to methacholine inhalation. Steady-state mRNA levels for all sGC subunits (alpha1, alpha2, and beta1) were reduced in the lungs of mice with allergic asthma by 60-80%, as estimated by real-time PCR. These changes in mRNA were paralleled by changes at the protein level: alpha1, alpha2, and beta1 expression was reduced by 50-80% as determined by Western blotting. Reduced alpha1 and beta1 expression in bronchial smooth muscle cells was demonstrated by immunohistochemistry. To study if sGC inhibition mimics the airway hyperreactivity seen in asthma, we treated na?ve mice with a selective sGC inhibitor. Indeed, in mice receiving ODQ the methacholine dose response was shifted to the left. We conclude that sGC expression is reduced in experimental asthma contributing to the observed airway hyperreactivity.     

Interleukin-1beta-induced airway hyperresponsiveness enhances substance P in intrinsic neurons of ferret airway

Interleukin (IL)-1beta causes airway inflammation, enhances airway smooth muscle responsiveness, and alters neurotransmitter expression in sensory, sympathetic, and myenteric neurons. This study examines the role of intrinsic airway neurons in airway hyperresponsiveness (AHR) induced by IL-1beta. Ferrets were instilled intratracheally with IL-1beta (0.3 microg/0.3 ml) or saline (0.3 ml) once daily for 5 days. Tracheal smooth muscle contractility in vitro and substance P (SP) expression in tracheal neurons were assessed. Tracheal smooth muscle reactivity to acetylcholine (ACh) and methacholine (MCh) and smooth muscle contractions to electric field stimulation (EFS) both increased after IL-1beta. The IL-1beta-induced AHR was maintained in tracheal segments cultured for 24 h, a procedure that depletes SP from sensory nerves while maintaining viability of intrinsic airway neurons. Pretreatment with CP-99994, an antagonist of neurokinin 1 receptor, attenuated the IL-1beta-induced hyperreactivity to ACh and MCh and to EFS in cultured tracheal segments. SP-containing neurons in longitudinal trunk, SP innervation of superficial muscular plexus neurons, and SP nerve fiber density in tracheal smooth muscle all increased after treatment with IL-1beta. These results show that IL-1beta-enhanced cholinergic airway smooth muscle contractile responses are mediated by the actions of SP released from intrinsic airway neurons.     

Deficiency of RAMP1 Attenuates Antigen-Induced Airway Hyperresponsiveness in Mice

Asthma is a chronic inflammatory disease affecting the lung, characterized by breathing difficulty during an attack following exposure to an environmental trigger. Calcitonin gene-related peptide (CGRP) is a neuropeptide that may have a pathological role in asthma. The CGRP receptor is comprised of two components, which include the G-protein coupled receptor, calcitonin receptor-like receptor (CLR), and receptor activity-modifying protein 1 (RAMP1). RAMPs, including RAMP1, mediate ligand specificity in addition to aiding in the localization of receptors to the cell surface. Since there has been some controversy regarding the effect of CGRP on asthma, we sought to determine the effect of CGRP signaling ablation in an animal model of asthma. Using gene-targeting techniques, we generated mice deficient for RAMP1 by excising exon 3. After determining that these mice are viable and overtly normal, we sensitized the animals to ovalbumin prior to assessing airway resistance and inflammation after methacholine challenge. We found that mice lacking RAMP1 had reduced airway resistance and inflammation compared to wildtype animals. Additionally, we found that a 50% reduction of CLR, the G-protein receptor component of the CGRP receptor, also ameliorated airway resistance and inflammation in this model of allergic asthma. Interestingly, the loss of CLR from the smooth muscle cells did not alter the airway resistance, indicating that CGRP does not act directly on the smooth muscle cells to drive airway hyperresponsiveness. Together, these data indicate that signaling through RAMP1 and CLR plays a role in mediating asthma pathology. Since RAMP1 and CLR interact to form a receptor for CGRP, our data indicate that aberrant CGRP signaling, perhaps on lung endothelial and inflammatory cells, contributes to asthma pathophysiology. Finally, since RAMP-receptor interfaces are pharmacologically tractable, it may be possible to develop compounds targeting the RAMP1/CLR interface to assist in the treatment of asthma.     

An endothelin receptor antagonist,SB-217242, inhibits airway hyperresponsiveness in allergic mice

Within the airways, endothelin-1 (ET-1) can exert a range of prominent effects, including airway smooth muscle contraction, bronchial obstruction, airway wall edema, and airway remodeling. ET-1 also possesses proinflammatory properties and contributes to the late-phase response in allergic airways. However, there is no direct evidence for the contribution of endogenous ET-1 to airway hyperresponsiveness in allergic airways. Allergic inflammation induced in mice by sensitization and challenge with the house dust mite allergen Der P1 was associated with elevated levels of ET-1 within the lung, increased numbers of eosinophils within bronchoalveolar lavage fluid and tissue sections, and development of airway hyperresponsiveness to methacholine (P < 0.05, n = 6 mice per group). Treatment of allergic mice with an endothelin receptor antagonist, SB-217242 (30 mg x kg(-1) x day(-1)), during allergen challenge markedly inhibited airway eosinophilia (bronchoalveolar lavage fluid and tissue) and development of airway hyperresponsiveness. These findings provide direct evidence for a mediator role for ET-1 in development of airway hyperresponsiveness and airway eosinophilia in Der P1-sensitized mice after antigen challenge.     

Exaggerated airway narrowing in mice treated with intratracheal cationic protein.

Airway hyperresponsiveness in mice with allergic airway inflammation can be attributed entirely to exaggerated closure of peripheral airways (Wagers S, Lundblad LK, Ekman M, Irvin CG, and Bates JHT. J Appl Physiol 96: 2019-2027, 2004). However, clinical asthma can be characterized by hyperresponsiveness of the central airways as well as the lung periphery. We, therefore, sought to establish a complementary model of hyperresponsiveness in the mouse due to excessive narrowing of the airways. We treated mice with a tracheal instillation of the cationic protein poly-l-lysine (PLL), hypothesizing that this would reduce the barrier function of the epithelium and thereby render the underlying airway smooth muscle more accessible to aerosolized methacholine. The PLL-treated animals were hypersensitive to methacholine: they exhibited an exaggerated response to submaximal doses but had a maximal response that was similar to controls. With the aid of a computational model of the mouse lung, we conclude that the methacholine responsiveness of PLL-treated mice is fundamentally different in nature to the hyperresponsiveness that we found previously in mice with allergically inflamed lungs.     

Caveolar localization dictates physiologic signaling of beta 2-adrenoceptors in neonatal cardiac myocytes

There is a growing body of evidence that G protein-coupled receptors function in the context of plasma membrane signaling compartments. These compartments may facilitate interaction between receptors and specific downstream signaling components while restricting access to other signaling molecules. We recently reported that beta(1)- and beta(2)-adrenergic receptors (AR) regulate the intrinsic contraction rate in neonatal mouse myocytes through distinct signaling pathways. By studying neonatal myocytes isolated from beta(1)AR and beta(2)AR knockout mice, we found that stimulation of the beta(1)AR leads to a protein kinase A-dependent increase in the contraction rate. In contrast, stimulation of the beta(2)AR has a biphasic effect on the contraction rate. The biphasic effect includes an initial protein kinase A-independent increase in the contraction rate followed by a sustained decrease in the contraction rate that can be blocked by pertussis toxin. Here we present evidence that caveolar localization is required for physiologic signaling by the beta(2)AR but not the beta(1)AR in neonatal cardiac myocytes. Evidence for beta(2)AR localization to caveolae includes co-localization by confocal imaging, co-immunoprecipitation of the beta(2)AR and caveolin 3, and co-migration of the beta(2)AR with a caveolin-3-enriched membrane fraction. The beta(2)AR-stimulated increase in the myocyte contraction rate is increased by approximately 2-fold and markedly prolonged by filipin, an agent that disrupts lipid rafts such as caveolae and significantly reduces co-immunoprecipitation of beta(2)AR and caveolin 3 and co-migration of beta(2)AR and caveolin-3 enriched membranes. In contrast, filipin has no effect on beta(1)AR signaling. These observations suggest that beta(2)ARs are normally restricted to caveolae in myocyte membranes and that this localization is essential for physiologic signaling of this receptor subtype.     

Disruption of ET-1 gene enhances pulmonary responses to methacholine via functional mechanism in knockout mice.

Endothelin (ET)-1 has been shown to have various pathophysiological roles in the lung. Recently, it has been reported that ET-1 and a gene encoding ET-1 (Edn1) might be involved in airway hyperresponsiveness, which is a major feature of bronchial asthma. Meanwhile, it remains unclear whether ET-1 might be involved in airway remodeling in vivo. In the present study, we hypothesized whether ET-1 might play a role in airway remodeling, leading to altered responsiveness. To test this hypothesis, we investigated airway function in vivo and airway wall structure in Edn1(+/-) heterozygous knockout mice, which genetically produce lower levels of ET-1, and Edn1(+/+) wild-type mice. In the physiological study, enhanced responses in lung elastance and resistance to methacholine administration were observed in Edn1(+/-) mice, whereas there was no difference in serotonin responsiveness. In the morphometric study, there were no differences in either lamina propria or airway smooth muscle thickness between Edn1(+/-) mice and Edn1(+/+) mice. These findings suggest that ET-1 gene disruption is involved in methacholine pulmonary hyperresponsiveness via functional mechanism, but not airway remodeling, in mice. The ET-1 knockout mice may provide appropriate models to study diseases related to ET-1 metabolism.     

Intrinsic and antigen-induced airway hyperresponsiveness are the result of diverse physiological mechanisms.

Airway hyperresponsiveness (AHR) is a defining feature of asthma. We have previously shown, in mice sensitized and challenged with antigen, that AHR is attributable to normal airway smooth muscle contraction with exaggerated airway closure. In the present study we sought to determine if the same was true for mice known to have intrinsic AHR, the genetic strain of mice, A/J. We found that A/J mice have AHR characterized by minimal increase in elastance following aerosolized methacholine challenge compared with mice (BALB/c) that have been antigen sensitized and challenged [concentration that evokes 50% change in elastance (PC(50)): 22.9 +/- 5.7 mg/ml for A/J vs. 3.3 +/- 0.4 mg/ml for antigen-challenged and -sensitized mice; P < 0.004]. Similar results were found when intravenous methacholine was used (PC(30) 0.22 +/- 0.08 mg/ml for A/J vs. 0.03 +/- 0.004 mg/ml for antigen-challenged and -sensitized mice). Computational model analysis revealed that the AHR in A/J mice is dominated by exaggerated airway smooth muscle contraction and that when the route of methacholine administration was changed to intravenous, central airway constriction dominates. Absorption atelectasis was used to provide evidence of the lack of airway closure in A/J mice. Bronchoconstriction during ventilation with 100% oxygen resulted in a mean 9.8% loss of visible lung area in A/J mice compared with 28% in antigen-sensitized and -challenged mice (P < 0.02). We conclude that the physiology of AHR depends on the mouse model used and the route of bronchial agonist administration.