RESUMEN
In the present study, we show therapeutic amelioration of established ovalbumin (OVA)-induced allergic airway disease following house dust mite (HDM) peptide therapy. Mice were sensitized and challenged with OVA and HDM protein extract (Dermatophagoides species) to induce dual allergen sensitization and allergic airway disease. Treatment of allergic mice with peptides derived from the major allergen Der p 1 suppressed OVA-induced airway hyperresponsiveness, tissue eosinophilia, and goblet cell hyperplasia upon rechallenge with allergen. Peptide treatment also suppressed OVA-specific T-cell proliferation. Resolution of airway pathophysiology was associated with a reduction in recruitment, proliferation, and effector function of T(H)2 cells and decreased interleukin (IL)-17⺠T cells. Furthermore, peptide immunotherapy induced the regulatory cytokine IL-10 and increased the proportion of Fox p3⺠cells among those expressing IL-10. Tolerance to OVA was not associated with increased IL-35. In conclusion, our results provide in vivo evidence for the creation of a tolerogenic environment following HDM peptide immunotherapy, leading to the therapeutic amelioration of established OVA-induced allergic airway disease.
Asunto(s)
Alérgenos/inmunología , Antígenos Dermatofagoides/inmunología , Proteínas de Artrópodos/inmunología , Cisteína Endopeptidasas/inmunología , Interleucinas/biosíntesis , Ovalbúmina/efectos adversos , Hipersensibilidad Respiratoria/inmunología , Hipersensibilidad Respiratoria/metabolismo , Animales , Antígenos Dermatofagoides/administración & dosificación , Proteínas de Artrópodos/administración & dosificación , Cisteína Endopeptidasas/administración & dosificación , Desensibilización Inmunológica , Modelos Animales de Enfermedad , Epítopos de Linfocito T/inmunología , Femenino , Factores de Transcripción Forkhead/metabolismo , Interleucina-10/metabolismo , Interleucina-17/metabolismo , Interleucina-5/biosíntesis , Activación de Linfocitos/inmunología , Subgrupos Linfocitarios/inmunología , Subgrupos Linfocitarios/metabolismo , Ratones , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/inmunología , Neumonía/inmunología , Neumonía/metabolismo , Neumonía/patología , Hipersensibilidad Respiratoria/terapiaRESUMEN
BACKGROUND: The syndrome of allergic asthma features reversible bronchoconstriction, airway inflammation and hyperresponsiveness as well as airway remodelling, including goblet cell hyperplasia. Managing severe asthma is still a clinical challenge. Numerous studies report that furosemide, an inhibitor of Na(+)-K(+)-Cl(-) cotransporter (NKCC) reduces airway hyperresponsiveness (AHR) in asthmatic patients. However, the mechanism by which furosemide exerts anti-asthmatic action remains unclear. OBJECTIVE: This study sought to investigate the cellular profile of NKCC1 expression in the lung and examine the effects of furosemide on several outcome measurements in a mouse model of allergic asthma. METHODS: Mice were sensitized and challenged with ovalbumin (OVA). Before challenge, the OVA-sensitized mice were treated with furosemide (4.0 mg/kg/day, via daily intraperitoneal injection for 5 days). Outcome measurements in naïve, OVA-exposure, furosemide-treated naïve and furosemide-treated OVA-exposed mice included the slope of the relationship between inhaled methacholine (MCh) concentration and respiratory system resistance (Slope·R(RS)), bronchoalveolar lavage (BAL) cell counts and immunohistochemical and immunoblotting assays of lung tissues. RESULTS: NKCC1 immunoreactivity was observed in airway epithelial cells (AECs) and alveolar type II (ATII) cells of the control mice. OVA exposure enhanced the expression of NKCC1 in AECs and ATII cells, and increased the infiltration of NKCC1-expressing T lymphocytes in the lung. NKCC1 immunoreactivity was not detected in the airway smooth muscle (ASM) cells. Furosemide treatment reduced the Slope·R(RS) in both naïve and OVA-exposed mice by about 50%. Furosemide treatment also increased T lymphocyte infiltration to the lung in OVA-exposed mice by approximately 53%, but had no effect on pulmonary goblet cell hyperplasia. CONCLUSIONS AND CLINICAL RELEVANCE: Furosemide decreases basal airway responsiveness, thereby reducing the extent of allergen-induced AHR. However, the same treatment also increases T lymphocytes infiltration in the course of allergic asthma. Further studies are necessary to address the usefulness of furosemide in the clinical treatment of asthma.
Asunto(s)
Antiasmáticos/uso terapéutico , Asma/tratamiento farmacológico , Hiperreactividad Bronquial/tratamiento farmacológico , Furosemida/uso terapéutico , Hipersensibilidad/tratamiento farmacológico , Linfocitos T/inmunología , Alérgenos/inmunología , Animales , Antiasmáticos/farmacología , Asma/inmunología , Hiperreactividad Bronquial/etiología , Líquido del Lavado Bronquioalveolar/inmunología , Modelos Animales de Enfermedad , Furosemida/farmacología , Células Caliciformes/efectos de los fármacos , Células Caliciformes/patología , Hipersensibilidad/inmunología , Pulmón/inmunología , Pulmón/metabolismo , Cloruro de Metacolina/inmunología , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Miembro 2 de la Familia de Transportadores de Soluto 12 , Linfocitos T/efectos de los fármacos , Resultado del TratamientoRESUMEN
Asthma is functionally characterized by increased airway sensitivity and reactivity. Multiple mechanisms are believed to underlie these functional disorders, including impairment of airway wall barrier function. One proposed mechanism of impaired barrier function is through the direct consequence of proteolytic properties of inhaled allergens, including house dust mite (HDM). Here, we have observed the direct effects of HDM on airway barrier function and response to nebulized or intravenous methacholine. HDM naïve BALB/c mice were anesthetized, exposed to intranasal or intratracheal HDM (15 or 100 µg), and allowed to recover for 30 min or 2 h before methacholine challenge. A separate group of mice was exposed to intratracheal poly-L-lysine (PLL; 100 µg) for a duration of 30 min. This group served as a positive control for the presence of impaired barrier function and airway hypersensitivity. Negative control mice received saline challenges. Outcomes included assessment of lung mechanics in response to nebulized or intravenous methacholine as well as clearance of intratracheally instilled technetium-labeled ((99m)Tc) DTPA to evaluate airway epithelial barrier function. We found that PLL produced a leftward shift in the dose-response curve following nebulized but not intravenous methacholine challenge. This was associated with a significantly faster clearance of (99m)Tc-DTPA, indicating impairment in airway barrier function. However, HDM exposure did not produce changes in these outcomes when compared with saline-exposed mice. These findings suggest that direct impact on airway barrier function does not appear to be a mechanism by which HDM produces altered airway sensitivity in airway disease.
Asunto(s)
Dermatophagoides pteronyssinus/inmunología , Cloruro de Metacolina/administración & dosificación , Hipersensibilidad Respiratoria/etiología , Administración por Inhalación , Alérgenos/administración & dosificación , Animales , Antígenos Dermatofagoides/administración & dosificación , Modelos Animales de Enfermedad , Femenino , Humanos , Pulmón/inmunología , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica de Transmisión , Modelos Biológicos , Radiofármacos , Hipersensibilidad Respiratoria/diagnóstico por imagen , Hipersensibilidad Respiratoria/inmunología , Hipersensibilidad Respiratoria/fisiopatología , Mecánica Respiratoria , Pentetato de Tecnecio Tc 99m , Tomografía Computarizada de Emisión de Fotón ÚnicoRESUMEN
BACKGROUND: Asthma is a disease characterized by variable and reversible airway obstruction and is associated with airway inflammation, airway remodelling (including goblet cell hyperplasia, increased collagen deposition and increased smooth muscle mass) and increased airway responsiveness. It is believed that airway inflammation plays a critical role in the development of airway remodelling, with IL-13 and TGF-beta1 pathways being strongly associated with the disease progression. Mouse models of asthma are capable of recapitulating some components of asthma and have been used to look at both IL-13 and TGF-beta1 pathways, which use STAT6 and SMAD2 signalling molecules, respectively. OBJECTIVES: Using brief and chronic models of allergen exposure, we utilized BALB/c and C57Bl/6 to explore the hypothesis that observed differences in responses to allergen between these mouse strains will involve fundamental differences in IL-13 and TGF-beta1 responses. METHODS: The following outcome measurements were performed: airway physiology, bronchoalveolar lavage cell counts/cytokine analysis, histology, immunoblots and gene expression assays. RESULTS: We demonstrate in BALB/c mice an IL-13-dependent phosphorylation of STAT6, nuclear localized in inflammatory cells, which is associated with indices of airway remodelling and development of airway dysfunction. In BALB/c mice, phosphorylation of SMAD2 is delayed relative to STAT6 activation and also involves an IL-13-dependent mechanism. In contrast, despite an allergen-induced increase in IL-4, IL-13 and eosinophils, C57Bl/6 demonstrates a reduced and distinct pattern of phosphorylated STAT6, no SMAD2 phosphorylation changes and fail to develop indices of remodelling or changes in airway function. CONCLUSION: The activation of signalling pathways and nuclear translocation of signalling molecules downstream of IL-13 and TGF-beta1 further support the central role of these molecules in the pathology and dysfunction in animal models of asthma. Activation of signalling pathways downstream from IL-13 and TGF-beta1 may be more relevant in disease progression than elevations in airway inflammation alone.
Asunto(s)
Alérgenos/inmunología , Asma/inmunología , Asma/fisiopatología , Modelos Animales de Enfermedad , Factor de Transcripción STAT6/metabolismo , Proteína Smad2/metabolismo , Alérgenos/farmacología , Animales , Asma/metabolismo , Hiperreactividad Bronquial/inmunología , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , Femenino , Humanos , Interleucina-13/biosíntesis , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Ovalbúmina/inmunología , Ovalbúmina/farmacología , Especificidad de la Especie , Factor de Crecimiento Transformador beta1/biosíntesisRESUMEN
Despite the effectiveness of corticosteroids at resolving airway inflammation, they are only moderately effective at attenuating airway hyperresponsiveness (AHR). The extent to which corticosteroids are able to reverse or inhibit the development of sustained AHR is not known. The present study aimed to determine whether budesonide can resolve and or prevent the development of sustained AHR in mice. Mice were chronically exposed to allergen and treated with budesonide either: 1) briefly during the final weeks of exposure to allergen; 2) prolonged concurrently throughout exposure to allergen; or 3) delayed following final exposure to allergen. AHR was assessed 24 h (brief treatment) or 4 weeks (prolonged concurrent and delayed treatments) following final exposure to allergen. Brief budesonide intervention significantly attenuated the inflammation-associated AHR assessed immediately following final exposure to allergen. Similarly, prolonged concurrent budesonide treatment prevented the development of sustained AHR. Delayed budesonide intervention, however, did not resolve sustained AHR. In conclusion, the early introduction and, importantly, the persistence of corticosteroid treatment prevented the development of sustained airway hyperresponsiveness; however, the inability of corticosteroids to reverse established airway dysfunction indicates a limitation in their use for the complete, long-term management of airway hyperresponsiveness.
Asunto(s)
Budesonida/uso terapéutico , Hipersensibilidad Respiratoria/tratamiento farmacológico , Corticoesteroides/farmacología , Alérgenos/química , Animales , Broncodilatadores/uso terapéutico , Colágeno/química , Modelos Animales de Enfermedad , Femenino , Inflamación , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Factores de Tiempo , Resultado del TratamientoRESUMEN
Asthma is characterised by an excessive airway narrowing in response to a variety of stimuli, called airway hyperresponsiveness (AHR). Previous comparisons between mouse strains have shown that increased velocity of airway narrowing correlates with baseline airway responsiveness. These data prompted the investigation into models of induced AHR to see whether airway narrowing dynamics correlated with in vivo responsiveness. In an attempt to reproduce some of the features of asthma, BALB/c mice were sensitised and subjected to either brief or chronic periods of allergen exposure. Brief exposure involved two challenges with intranasal chicken egg ovalbumin (OVA(in)). Chronic exposure involved six 2-day periods of OVA(in) challenges, each separated by 12 days. Control mice received intranasal saline challenges. Outcomes included videomicrometry of lung slices (magnitude and velocity of airway narrowing), in vivo respiratory physiology measurements and histological staining with morphometric analysis. Neither brief nor chronic allergen exposure resulted in greater airway narrowing and increased velocity compared with saline controls. Structural changes in the airway, such as goblet cell hyperplasia, subepithelial fibrosis and increased contractile tissue, were detected in mice chronically challenged with allergen. In conclusion, increased responsiveness to methacholine following allergen challenge may not be due to an intrinsic change to the smooth muscle per se, but rather to other changes in the lung, which ultimately manifest as an increase in respiratory resistance.
Asunto(s)
Alérgenos/efectos adversos , Asma/fisiopatología , Hiperreactividad Bronquial/fisiopatología , Pruebas de Provocación Bronquial/métodos , Resistencia de las Vías Respiratorias/efectos de los fármacos , Resistencia de las Vías Respiratorias/fisiología , Animales , Asma/inducido químicamente , Hiperreactividad Bronquial/inducido químicamente , Broncoconstrictores , Modelos Animales de Enfermedad , Procesamiento de Imagen Asistido por Computador , Cloruro de Metacolina , Ratones , Ratones Endogámicos BALB C , Microscopía por Video , Músculo Liso/fisiopatología , OvalbúminaRESUMEN
The role of inflammatory effector cells in the pathogenesis of airway allergy has been the subject of much investigation. However, whether systemic factors are involved in the development of local responses in both upper and lower airways has not been fully clarified. The present study was performed to investigate aspects of the pathogenesis of isolated allergic rhinitis in a murine model sensitized to ovalbumin (OVA). Both upper- and lower-airway physiological responsiveness and inflammatory changes were assessed, as well as bone marrow progenitor responses, by culture and immunohistological methods. Significant nasal symptoms and hyper-responsiveness appeared after intranasal OVA challenge (P < 0.0001 and P < 0.01, respectively), accompanied with significant nasal mucosal changes in CD4+ cells (P < 0.001), interleukin (IL)-4+ cells (P < 0.01), IL-5+ cells (P < 0.01), basophilic cells (P < 0.02) and eosinophils (P < 0.001), in the complete absence of hyper-responsiveness or inflammatory changes in the lower airway. In the bone marrow, there were significant increases in CD34+ cells, as well as in eosinophils and basophilic cells. In the presence in vitro of mouse recombinant IL-5, IL-3 or granulocyte-macrophage colony-stimulating factor (GM-CSF), the level of bone marrow eosinophil/basophil (Eo/Baso) colony-forming cells increased significantly in the OVA-sensitized group. We conclude that, in this murine model of allergic rhinitis, haemopoietic progenitors are upregulated, which is consistent with the involvement of bone marrow in the pathogenesis of nasal mucosal inflammation. Both local and systemic events, initiated in response to allergen provocation, may be required for the pathogenesis of allergic rhinitis. Understanding these events and their regulation could provide new therapeutic targets for rhinitis and asthma.
Asunto(s)
Alérgenos/inmunología , Ovalbúmina/inmunología , Rinitis/inmunología , Animales , Basófilos/inmunología , Células de la Médula Ósea/inmunología , Técnicas de Cultivo de Célula , Eosinófilos/inmunología , Femenino , Células Madre Hematopoyéticas/inmunología , Inmunoglobulina E/sangre , Recuento de Leucocitos , Masculino , Ratones , Ratones Endogámicos BALB C , Mucosa Nasal/inmunología , Mucosa Nasal/patología , Rinitis/patología , Azul de TripanoRESUMEN
We investigated the mechanisms underlying muscarinic excitation-contraction coupling in canine airway smooth muscle using organ bath, fura 2 fluorimetric, and patch-clamp techniques. Cyclopiazonic acid (CPA) augmented the responses to submaximal muscarinic stimulation in both tracheal (TSM) and bronchial smooth muscles (BSM), consistent with disruption of the barrier function of the sarcoplasmic reticulum. During maximal stimulation, however, CPA evoked substantial relaxation in TSM but not BSM. CPA reversal of carbachol tone persisted in the presence of tetraethylammoium or high KCl, suggesting that hyperpolarization is not involved; CPA relaxations were absent in tissues preconstricted with KCl alone or by permeabilization with beta-escin, ruling out a nonspecific effect on the contractile apparatus. Peak contractions were sensitive to inhibitors of tyrosine kinase (genistein) or Rho kinase (Y-27632). Sustained responses were dependent on Ca(2+) influx in TSM but not BSM; this influx was sensitive to Ni(2+) but not La(3+). In conclusion, there are several mechanisms underlying excitation-contraction coupling in airway smooth muscle, the relative importance of which varies depending on tissue and degree of stimulation.
Asunto(s)
Bronquios/fisiología , Contracción Muscular/fisiología , Músculo Liso/fisiología , Receptores Muscarínicos/fisiología , Tráquea/fisiología , Animales , Hiperreactividad Bronquial/fisiopatología , Calcio/metabolismo , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , ATPasas Transportadoras de Calcio/metabolismo , Carbacol/farmacología , Cloruros/metabolismo , Agonistas Colinérgicos/farmacología , Perros , Inhibidores Enzimáticos/farmacología , Genisteína/farmacología , Indoles/farmacología , Péptidos y Proteínas de Señalización Intracelular , Contracción Muscular/efectos de los fármacos , Relajación Muscular/efectos de los fármacos , Relajación Muscular/fisiología , Técnicas de Placa-Clamp , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Retículo Sarcoplasmático/enzimología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Quinasas Asociadas a rhoRESUMEN
Recently, we reported that freely moving Flinders sensitive line rats (FSL, selectively bred for their cholinergic hyperresponsiveness) are more susceptible to allergen-induced airway hyperresponsiveness than their control counterparts-Flinders resistant line (FRL) rats. In this study the two Flinders lines were compared for responsiveness of excised tracheal and primary bronchial smooth muscle in vitro. FSL tissues were slightly but significantly more sensitive to cholinergic stimulation than FRL tissues (slightly lower EC(50) value for carbachol) but the FRL tissues were more responsive, exhibiting larger amplitude of response. Surprisingly, previous exposure to allergen challenge was accompanied by reduced in vitro responses to spasmogens in both rat lines. We conclude that FSL and FRL airways do not differ greatly with respect to sensitivity to cholinergic stimulation in vitro and that inflamed airways show reduced in vitro responses to spasmogens. The discrepancy between the in vivo and in vitro findings suggests that responsiveness of airway smooth muscle involves regulation from both proximal and distal sites.
Asunto(s)
Resistencia de las Vías Respiratorias/genética , Alérgenos/farmacología , Hiperreactividad Bronquial/genética , Broncoconstricción/genética , Ratas Endogámicas/fisiología , Resistencia de las Vías Respiratorias/efectos de los fármacos , Alérgenos/toxicidad , Animales , Bronquios/efectos de los fármacos , Hiperreactividad Bronquial/inducido químicamente , Broncoconstricción/efectos de los fármacos , Broncoconstrictores/farmacología , Carbacol/farmacología , Colinérgicos/farmacología , Estimulación Eléctrica , Femenino , Músculo Liso/efectos de los fármacos , Músculo Liso/inervación , Ovalbúmina/farmacología , Ovalbúmina/toxicidad , Sistema Nervioso Parasimpático/fisiología , Cloruro de Potasio/farmacología , Ratas , Pruebas de Función Respiratoria , Tráquea/efectos de los fármacosRESUMEN
Increases in bone-marrow (BM) inflammatory cell progenitors are associated with allergen-induced airway hyperresponsiveness and inflammation in asthmatics and dogs. Here, for the first time, we compare the time course of airway hyperresponsiveness, inflammation, and marrow progenitor responses in a mouse model of airway allergen challenge. Sensitized BALB/c mice were studied at 2, 12, 24, 48, and 72 h after intranasal ovalbumin or saline challenges. Outcome measurements included airway responsiveness, airway inflammation as assessed via bronchoalveolar lavage (BAL) and lung tissue sections, and BM eosinophil colony-forming units (Eo-CFU) as enumerated using a semisolid culture assay with optimal concentrations of interleukin-5. We observed significant increases in BAL fluid eosinophils, neutrophils, lymphocytes, and macrophages by 2 h after the second of two intranasal allergen challenges (P < 0.05). Significant increases in airway responsiveness or BM Eo-CFU were observed at 24 h and persisted until 48 h after the second challenge (P < 0.05). Airway inflammation, including eosinophils, persisted until at least 72 h (P < 0.05). We observed that allergen-induced airway eosinophilia is accompanied by increases in BM eosinophil progenitors, indicating that in this model, increased eosinophil production involves an expansion of the relevant stem-cell population. These findings support the use of this model to explore the mechanisms of increased eosinopoiesis observed in human asthma.
Asunto(s)
Alérgenos/administración & dosificación , Hiperreactividad Bronquial/etiología , Eosinofilia/etiología , Animales , Asma/etiología , Asma/patología , Médula Ósea/efectos de los fármacos , Médula Ósea/patología , Hiperreactividad Bronquial/patología , Ensayo de Unidades Formadoras de Colonias , Modelos Animales de Enfermedad , Perros , Eosinofilia/patología , Eosinófilos/efectos de los fármacos , Eosinófilos/patología , Hematopoyesis/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/patología , Humanos , Interleucina-5/farmacología , Cloruro de Metacolina/farmacología , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/inmunologíaRESUMEN
Cromolyn and nedocromil are often used in the treatment of asthma. Recently, these agents have been shown to block Cl- currents and/or Ca2+ currents in a variety of cell preparations. Ca2+ and Cl- currents play central roles in excitation-contraction coupling in airway smooth muscle. This study therefore aimed to investigate the effects of these agents on membrane currents, elevations of [Ca2+] and contractions evoked by depolarization and/or acetylcholine in airway smooth muscle. Patch-clamp, fura-2 fluorimetric and muscle-bath techniques were used to monitor ion currents, [Ca2+] and contractions, respectively, in canine tracheal smooth muscle in the presence and absence of the chromones. Cromolyn and nedocromil eliminated voltage-dependent Ca2+ currents, leading to a reduction in depolarization-evoked K+ currents. Both chromones had little or no effect on either acetylcholine-evoked release of internal Ca2+ or the subsequent contraction; however, cromolyn (but not nedocromil) at high concentrations suppressed Ca2+-dependent Cl- currents triggered by acetylcholine. In conclusion, cromolyn and nedocromil abolished voltage-dependent Ca2+ currents and cromolyn also suppressed Ca2+-dependent Cl- currents in airway smooth muscle; neither chromone greatly altered either the release of internally sequestered Ca2+ or the resultant contractions. Further investigation is needed to determine whether the local concentrations obtained by inhaled chromones within the airway wall allow these cellular effects to occur in patients in vivo.
Asunto(s)
Resistencia de las Vías Respiratorias/efectos de los fármacos , Cromolin Sódico/farmacología , Canales Iónicos/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Nedocromil/farmacología , Animales , Calcio/metabolismo , Canales de Calcio/efectos de los fármacos , Canales de Cloruro/efectos de los fármacos , Perros , Potenciales de la Membrana/efectos de los fármacos , Contracción Muscular/efectos de los fármacos , Técnicas de Placa-Clamp , Tráquea/efectos de los fármacosRESUMEN
To investigate the role of prostaglandin (PG) E2 in allergen-induced hyperresponsiveness, dogs inhaled either the allergen Ascaris suum or vehicle (Sham). Twenty-four hours after inhalation, some animals exposed to allergen demonstrated an increased responsiveness to acetylcholine challenge in vivo (Hyp-Resp), whereas others did not (Non-Resp). Strips of tracheal smooth muscle, either epithelium intact or epithelium denuded, were suspended on stimulating electrodes, and a concentration-response curve to carbachol (10(-9) to 10(-5) M) was generated. Tissues received electrical field stimulation, and organ bath fluid was collected to determine PGE2 content. With the epithelium present, all three groups contracted similarly to 10(-5) M carbachol, whereas epithelium-denuded tissues from animals that inhaled allergen contracted more than tissues from Sham dogs. In response to electrical field stimulation, Hyp-Resp tissues contracted less than Sham tissues in the presence of epithelium and more than Sham tissues in the absence of epithelium. PGE2 release in the muscle bath was greater in Non-Resp tissues than in Sham or Hyp-Resp tissues when the epithelium was present. Removal of the epithelium greatly inhibited PGE2 release. We conclude that tracheal smooth muscle is hyperresponsive in vitro after in vivo allergen exposure only when the modulatory effect of the epithelium, largely through PGE2 release, is removed.
Asunto(s)
Alérgenos/administración & dosificación , Asma/fisiopatología , Hiperreactividad Bronquial/inducido químicamente , Dinoprostona/metabolismo , Tráquea/metabolismo , Administración por Inhalación , Alérgenos/farmacología , Animales , Antígenos Helmínticos/administración & dosificación , Antígenos Helmínticos/farmacología , Ascaris suum , Carbacol/farmacología , Perros , Estimulación Eléctrica , Epitelio/inmunología , Epitelio/metabolismo , Femenino , Masculino , Contracción Muscular/efectos de los fármacos , Parasimpaticomiméticos/farmacología , Tráquea/inmunologíaRESUMEN
We investigated Ca2+ handling in airway smooth muscle (SM) using fura 2 fluorescence, ion currents, and contractions as indexes of intracellular Ca2+ concentration ([Ca2+]i). Carbachol evoked a transient elevation of [Ca2+]i, the magnitude of which was smaller and the rate of decay faster at 37 degrees C, indicating that some temperature-sensitive mechanism contributed to recovery. Removal of external Na+ had no effect on agonist-evoked Ca2+ transients or contractions or on spontaneous Ca(2+)-dependent K+ currents. Cyclopiazonic acid, a selective inhibitor of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, evoked a transient elevation of [Ca2+]i and contraction, markedly slowed recovery of the cholinergic Ca2+ transient, and depleted the SR. Sodium vanadate evoked a sustained elevation of [Ca2+]i and markedly slowed the decay of the cholinergic Ca2+ transient. We conclude that, in canine airway SM, 1) Na+/Ca2+ exchange makes at best only a minor contribution to Ca2+ homeostasis, 2) the SR Ca(2+)-ATPase compensates for spontaneous and agonist-triggered release of Ca2+, and 3) [Ca2+]i homeostasis involves some other extrusion pathway, likely the plasmalemmal Ca(2+)-ATPase.
Asunto(s)
Bronquios/metabolismo , ATPasas Transportadoras de Calcio/fisiología , Calcio/metabolismo , Proteínas Portadoras/fisiología , Membranas Intracelulares/metabolismo , Músculo Liso/metabolismo , Tráquea/metabolismo , Animales , Bronquios/efectos de los fármacos , Bronquios/fisiología , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Colinérgicos/farmacología , Perros , Electrofisiología , Inhibidores Enzimáticos/farmacología , Técnicas In Vitro , Indoles/farmacología , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Concentración Osmolar , Potasio/fisiología , Sodio/farmacología , Intercambiador de Sodio-Calcio , Temperatura , Tráquea/efectos de los fármacos , Tráquea/fisiología , Vanadatos/farmacologíaRESUMEN
We investigated allergen-induced airway hyperresponsiveness (AH) in bronchial tissues obtained from dogs that inhaled Ascaris suum leading to AH (RESP) in vivo or that exhibited no change (NON-RESP) as well as from dogs that inhaled saline (SHAM). RESP tissues were not hyperresponsive to KCl or to carbachol, whereas contractions to electrical field stimulation (EFS) were reduced. This reduction was reversed partially by indomethacin and completely by replacement of the bathing fluid. Radioimmunoassay revealed marked elevation of prostaglandin (PG) E2 generation in RESP tissues compared with SHAM and NON-RESP tissues. EFS-evoked contractions were often followed by a slowly developing secondary contraction in RESP tissues but not in SHAM or NON-RESP tissues. However, indomethacin unmasked such secondary contractions in many SHAM and NON-RESP tissues and markedly enhanced those in RESP tissues, whereas L-655,240 (thromboxane A2/PGD2 receptor antagonist) abolished such contractions in all groups. We were unable to detect thromboxane using radioimmunoassay. We conclude that allergen-induced AH involves altered generation of cyclooxygenase metabolites of arachidonic acid (particularly PGE2) as well as of a nonprostanoid inhibitory factor; as such, the responsiveness of the tissue in vitro is dependent on the relative levels of inhibitory and excitatory metabolites.
Asunto(s)
Alérgenos , Ácidos Araquidónicos/metabolismo , Ascaris suum , Bronquios/fisiología , Hiperreactividad Bronquial/fisiopatología , Dinoprostona/metabolismo , Músculo Liso/fisiología , Animales , Bronquios/efectos de los fármacos , Bronquios/inervación , Carbacol/farmacología , Perros , Estimulación Eléctrica , Femenino , Técnicas In Vitro , Indometacina/farmacología , Masculino , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Músculo Liso/efectos de los fármacos , Músculo Liso/inervación , Nifedipino/farmacología , Cloruro de Potasio/farmacologíaRESUMEN
Cyclopiazonic acid (selective blocker of the internal Ca+2 pump) evoked tonic contraction in canine bronchial smooth muscle (BSM) and tracheal smooth muscle. This contraction was biphasic, including an initial component that was relatively insensitive to blockade of Ca+2 influx (e.g., removal of external Ca+2; nifedipine; hyperpolarization using lemakalim) followed by a component that was sensitive to all such interventions. In BSM, but not in tracheal smooth muscle, electrical field stimulation (EFS) evoked relaxations that were not affected by interventions designed to prevent release of autacoids from nerve endings or the epithelium, Na+/Ca+2 exchange or Ca(+2)-ATPase activities (internal or plasmalemmal). EFS evoked little or no relaxant response in carbachol-precontracted BSM in the presence of propranolol. After Ca+2 was replaced with Sr+2, however, carbachol evoked comparable contraction after which EFS evoked non-neurogenic relaxations. We found that the EFS-evoked relaxations were abolished by TEA or high KCI, were reduced significantly by charydotoxin or quinine, were reduced partially by ouabain and were unaffected by removal of external K+, by apamin or by glybenclamide. In addition, the relaxations were reduced significantly by the free radical scavenger N-acetylcysteine, were mimicked by H2O2 but were unaffected by superoxide dismutase or catalase. These observations suggest that the cyclopiazonic acid-evoked contraction involves pharmacomechanical coupling mechanisms (i.e., Ca(+2)-release) initially, followed by electromechanical coupling (i.e., voltage-dependent Ca+2 influx). After depletion of the internal Ca+2 store (e.g., by cyclopiazonic acid or Sr+2), EFS is able to evoke in BSM (but not in tracheal smooth muscle) relaxations that seem to involve opening of K+ channels (including those of the large-conductance Ca(+2)-dependent type) by EFS-liberated free radicals.
Asunto(s)
Bronquios/fisiología , Relajación Muscular , Canales de Potasio/fisiología , Tráquea/fisiología , Animales , Bronquios/inervación , Calcio/fisiología , Perros , Estimulación Eléctrica , Radicales Libres , Técnicas In Vitro , Indoles/farmacología , ATPasa Intercambiadora de Sodio-Potasio/fisiología , Tráquea/inervaciónRESUMEN
The importance of the potent neutrophil chemoattractant leukotriene (LT)B4 in causing ozone-induced bronchoconstriction, airway inflammation, and airway hyperresponsiveness in dogs was studied using the LTB4-receptor antagonist SC-53228. Seven dogs from random sources were studied three times, at least 2 wk apart. On each occasion, acetylcholine (Ach) airway responsiveness was measured before and 1 h after ozone (3 ppm, 30 min) or dry air inhalation, followed by a bronchoalveolar lavage (BAL). On the first day, dogs were treated with SC-53228 (0.4 mg/kg intravenously) followed by a continuous intravenous infusion of 1.2 mg/kg/h before ozone inhalation. On the other two days, diluent was infused followed by ozone or dry air inhalation. Cell counts were measured in BAL and cell activation was measured by spontaneous and by phorbol myristate acetate-stimulated (PMA) (2.4 mumol/L) oxygen radical release, measured from washed BAL cells (4 x 10(6) cells) by lucigenin-enhanced chemiluminescence. Ozone inhalation caused bronchoconstriction and airway hyperresponsiveness. SC-53228 inhibited the ozone-induced airway hyperresponsiveness (p = 0.006), but not the bronchoconstriction. Spontaneous (p = 0.004) and PMA-stimulated (p = 0.04) lucigenin-enhanced chemiluminescence were increased after ozone inhalation. The ozone-induced increases in PMA-stimulated chemiluminescence were significantly attenuated by treatment with SC-53228 (p = 0.04). These results suggest that LTB4 is involved in the pathogenesis of ozone-induced airway hyperresponsiveness, possibly through activation of airway inflammatory cell.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Benzamidas/farmacología , Benzopiranos/farmacología , Hiperreactividad Bronquial/inducido químicamente , Bronquitis/inducido químicamente , Ozono/toxicidad , Receptores de Leucotrieno B4/antagonistas & inhibidores , Acetilcolina , Animales , Hiperreactividad Bronquial/fisiopatología , Pruebas de Provocación Bronquial/métodos , Bronquitis/fisiopatología , Líquido del Lavado Bronquioalveolar/citología , Broncoconstricción/efectos de los fármacos , Broncoconstricción/fisiología , Perros , Femenino , Mediciones Luminiscentes , Masculino , Distribución Aleatoria , Receptores de Leucotrieno B4/fisiología , Estallido Respiratorio/efectos de los fármacosRESUMEN
Both ozone and allergen inhalation increase the capacity to produce oxygen radicals by bronchoalveolar lavage cells in dogs. The purpose of these studies was to determine whether inhaled corticosteroids inhibits these increases in oxygen radical production from bronchoalveolar lavage cells. Six random source dogs were studied after dry air or ozone inhalation (3 ppm, 30 min). Seven random source dogs were studied after diluent or allergen inhalation. The dogs inhaled budesonide (2.74 mg/day) or lactose powder, twice daily for 7 days before ozone and allergen. 90 min after ozone or dry air, and 24 h after Ascaris suum or diluent a bronchoalveolar lavage was carried out. Spontaneous luminol-enhanced chemiluminescence was measured from bronchoalveolar lavage cells (4 x 10(6) cells) for 10 min, followed by a measurement of phorbol myristate acetate (PMA 2.4 micromol/l) stimulated chemiluminescence for 10 min. Both ozone and allergen inhalation caused an increase in PMA stimulated chemiluminescence (P<0.05). Budesonide pretreatment inhibited ozone-induced (P<0.008), but not allergen-induced PMA stimulated chemiluminescence (P>0.90). Both ozone and allergen inhalation caused an increase in the bronchoalveolar lavage neutrophils. Budesonide pretreatment significantly inhibited the ozone-induced (P=0.007), but not the ascaris-induced neutrophil influx (P=0.93). These results demonstrate that ozone, but not allergen, stimulated oxygen radical release and neutrophil influx are attenuated by inhaled corticosteroids. This suggests that luminol-enhanced chemiluminescence from bronchoalveolar lavage cells measures oxygen radicals derived from neutrophils, and that ozone-and allergen-induced bronchoalveolar lavage neutrophilia are caused by different mechanisms.