RESUMEN
Airway inflammation and remodeling are major features of chronic obstructive pulmonary disease (COPD), whereas pulmonary hypertension is a common comorbidity associated with a poor disease prognosis. Recent studies in animal models have indicated that increased arginase activity contributes to features of asthma, including allergen-induced airway eosinophilia and mucus hypersecretion. Although cigarette smoke and lipopolysaccharide (LPS), major risk factors for COPD, may increase arginase expression, the role of arginase in COPD is unknown. This study aimed to investigate the role of arginase in pulmonary inflammation and remodeling using an animal model of COPD. Guinea pigs were instilled intranasally with LPS or saline twice weekly for 12 weeks and pretreated by inhalation of the arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH) or vehicle. Repeated LPS exposure increased lung arginase activity, resulting in increased l-ornithine/l-arginine and l-ornithine/l-citrulline ratios. Both ratios were reversed by ABH. ABH inhibited the LPS-induced increases in pulmonary IL-8, neutrophils, and goblet cells as well as airway fibrosis. Remarkably, LPS-induced right ventricular hypertrophy, indicative of pulmonary hypertension, was prevented by ABH. Strong correlations were found between arginase activity and inflammation, airway remodeling, and right ventricular hypertrophy. Increased arginase activity contributes to pulmonary inflammation, airway remodeling, and right ventricular hypertrophy in a guinea pig model of COPD, indicating therapeutic potential for arginase inhibitors in this disease.
Asunto(s)
Remodelación de las Vías Aéreas (Respiratorias) , Arginasa/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/enzimología , Animales , Arginasa/antagonistas & inhibidores , Fibrosis , Cobayas , Hipertensión Pulmonar/enzimología , Hipertensión Pulmonar/inmunología , Hipertensión Pulmonar/patología , Hipertrofia Ventricular Derecha/enzimología , Hipertrofia Ventricular Derecha/inmunología , Hipertrofia Ventricular Derecha/patología , Interleucina-8/metabolismo , Lipopolisacáridos/farmacología , Pulmón/irrigación sanguínea , Pulmón/enzimología , Pulmón/patología , Mucina 5AC/metabolismo , Neutrófilos/patología , Neumonía/enzimología , Neumonía/inmunología , Neumonía/patología , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Enfermedad Pulmonar Obstructiva Crónica/patología , Enfermedad Pulmonar Obstructiva Crónica/fisiopatologíaRESUMEN
Airway remodelling and emphysema are major structural abnormalities in chronic obstructive pulmonary disease (COPD). In addition, pulmonary vascular remodelling may occur and contribute to pulmonary hypertension, a comorbidity of COPD. Increased cholinergic activity in COPD contributes to airflow limitation and, possibly, to inflammation and airway remodelling. This study aimed to investigate the role of acetylcholine in pulmonary inflammation and remodelling using an animal model of COPD. To this aim, guinea pigs were instilled intranasally with lipopolysaccharide (LPS) twice weekly for 12 weeks and were treated, by inhalation, with the long-acting muscarinic receptor antagonist tiotropium. Repeated LPS exposure induced airway and parenchymal neutrophilia, and increased goblet cell numbers, lung hydroxyproline content, airway wall collagen and airspace size. Furthermore, LPS increased the number of muscularised microvessels in the adventitia of cartilaginous airways. Tiotropium abrogated the LPS-induced increase in neutrophils, goblet cells, collagen deposition and muscularised microvessels, but had no effect on emphysema. In conclusion, tiotropium inhibits remodelling of the airways as well as pulmonary inflammation in a guinea pig model of COPD, suggesting that endogenous acetylcholine plays a major role in the pathogenesis of this disease.
Asunto(s)
Remodelación de las Vías Aéreas (Respiratorias)/efectos de los fármacos , Antagonistas Colinérgicos/farmacología , Neumonía/tratamiento farmacológico , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Derivados de Escopolamina/farmacología , Acetilcolina/fisiología , Remodelación de las Vías Aéreas (Respiratorias)/inmunología , Animales , Animales no Consanguíneos , Modelos Animales de Enfermedad , Enfisema/tratamiento farmacológico , Enfisema/inmunología , Células Caliciformes/efectos de los fármacos , Células Caliciformes/inmunología , Células Caliciformes/metabolismo , Cobayas , Lipopolisacáridos/toxicidad , Pulmón/efectos de los fármacos , Pulmón/inmunología , Masculino , Mucina 5AC/metabolismo , Antagonistas Muscarínicos/farmacología , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Neumonía/inmunología , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Fibrosis Pulmonar/tratamiento farmacológico , Fibrosis Pulmonar/inmunología , Bromuro de TiotropioRESUMEN
Airway remodelling, characterised by increased airway smooth muscle (ASM) mass, subepithelial fibrosis, goblet cell hyperplasia and mucus gland hypertrophy, is a feature of chronic asthma. Increased arginase activity could contribute to these features via increased formation of polyamines and l-proline downstream of the arginase product l-ornithine, and via reduced nitric oxide synthesis. Using the specific arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH), we studied the role of arginase in airway remodelling using a guinea pig model of chronic asthma. Ovalbumin-sensitised guinea pigs were treated with ABH or PBS via inhalation before each of 12 weekly allergen or saline challenges, and indices of arginase activity, and airway remodelling, inflammation and responsiveness were studied 24 h after the final challenge. Pulmonary arginase activity of repeatedly allergen-challenged guinea pigs was increased. Allergen challenge also increased ASM mass and maximal contraction of denuded tracheal rings, which were prevented by ABH. ABH also attenuated allergen-induced pulmonary hydroxyproline (fibrosis) and putrescine, mucus gland hypertrophy, goblet cell hyperplasia, airway eosinophilia and interleukin-13, whereas an increased l-ornithine/l-citrulline ratio in the lung was normalised. Moreover, allergen-induced hyperresponsiveness of perfused tracheae was fully abrogated by ABH. These findings demonstrate that arginase is prominently involved in allergen-induced airway remodelling, inflammation and hyperresponsiveness in chronic asthma.
Asunto(s)
Remodelación de las Vías Aéreas (Respiratorias)/fisiología , Arginasa/fisiología , Asma/enzimología , Asma/fisiopatología , Remodelación de las Vías Aéreas (Respiratorias)/efectos de los fármacos , Alérgenos/efectos adversos , Aminocaproatos/uso terapéutico , Animales , Antiasmáticos/uso terapéutico , Arginasa/antagonistas & inhibidores , Compuestos de Boro/uso terapéutico , Hiperreactividad Bronquial/tratamiento farmacológico , Enfermedad Crónica , Citrulina/análisis , Eosinofilia/tratamiento farmacológico , Glándulas Exocrinas/efectos de los fármacos , Células Caliciformes/efectos de los fármacos , Cobayas , Interleucina-13/análisis , Pulmón/química , Masculino , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Músculo Liso/efectos de los fármacos , Ornitina/análisis , Ovalbúmina/efectos adversos , Fibrosis Pulmonar/tratamiento farmacológico , Tráquea/efectos de los fármacos , Tráquea/fisiopatologíaRESUMEN
BACKGROUND: Clinical information on 24-h spirometric efficacy of combining tiotropium and salmeterol compared to single-agent therapy is lacking in patients with COPD. METHODS: A randomized, double-blind, four-way crossover study of 6-week treatment periods comparing combination therapy of tiotropium 18 microg plus qd or bid salmeterol 50 microg versus single-agent therapy. Serial 24-h spirometry (FEV(1), FVC), effects on dyspnea (TDI focal score) and rescue salbutamol use were evaluated in 95 patients. RESULTS: Tiotropium plus qd salmeterol was superior to tiotropium or salmeterol alone in average FEV(1) (0-24h) by 72 mL and 97 mL (p<0.0001), respectively. Compared to this qd regimen, combination therapy including bid salmeterol provided comparable daytime (0-12h: 12 mL, p=0.38) bronchodilator effects, but significantly more bronchodilation during the night-time (12-24h: 73 mL, p<0.0001). Clinically relevant improvements in TDI focal score were achieved with bronchodilator combinations including salmeterol qd or bid (2.56 and 2.71; p<0.005 versus components). Symptom benefit of combination therapies was also reflected in less need for reliever medication. All treatments were well tolerated. CONCLUSION: Compared to single-agent therapy, combination therapy of tiotropium plus salmeterol in COPD provided clinically meaningful improvements in airflow obstruction and dyspnea as well as a reduction in reliever medication.
Asunto(s)
Obstrucción de las Vías Aéreas/tratamiento farmacológico , Albuterol/análogos & derivados , Broncodilatadores/administración & dosificación , Disnea/tratamiento farmacológico , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Derivados de Escopolamina/administración & dosificación , Adulto , Obstrucción de las Vías Aéreas/fisiopatología , Albuterol/administración & dosificación , Estudios Cruzados , Método Doble Ciego , Esquema de Medicación , Quimioterapia Combinada , Disnea/fisiopatología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Xinafoato de Salmeterol , Espirometría , Bromuro de Tiotropio , Resultado del TratamientoRESUMEN
Peroxynitrite has been shown to be crucially involved in airway hyperresponsiveness (AHR) after the late asthmatic reaction (LAR). Peroxynitrite production may result from simultaneous synthesis of nitric oxide (NO) and superoxide by inducible NO-synthase (iNOS) at low L-arginine concentrations. L-arginine availability to iNOS is regulated by its cellular uptake, which can be inhibited by eosinophil-derived polycations and by arginase, which competes with iNOS for the common substrate. Using a guinea pig model of allergic asthma, we investigated whether aberrant L-arginine homeostasis could underlie peroxynitrite-mediated AHR after the LAR. After the LAR, arginase activity in the airways and eosinophil peroxidase release from bronchoalveolar lavage cells were increased. These changes were associated with a 2.0-fold AHR to methacholine as measured in isolated perfused tracheal preparations. AHR was reduced by exogenous L-arginine administration. Moreover, both the arginase inhibitor N(omega)-hydroxy-nor-L-arginine (nor-NOHA) and the polycation antagonist heparin normalised airway responsiveness. These effects were reversed by the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME), indicating that both agents reduced AHR by restoring bronchodilating NO production. In conclusion, in allergen-challenged guinea pigs, the AHR after the LAR is caused by arginase- and polycation-induced attenuation of L-arginine availability to iNOS, which may switch the enzyme to simultaneous production of superoxide and NO, and, consequently, peroxynitrite.
Asunto(s)
Arginina/deficiencia , Asma/metabolismo , Hiperreactividad Bronquial/metabolismo , Animales , Arginasa/metabolismo , Asma/patología , Hiperreactividad Bronquial/etiología , Modelos Animales de Enfermedad , Cobayas , NG-Nitroarginina Metil Éster/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Ovalbúmina/metabolismo , Perfusión , Ácido Peroxinitroso/metabolismo , Poliaminas , Polielectrolitos , Tráquea/metabolismoRESUMEN
Airway hyperresponsiveness (AHR) is a hallmark clinical symptom of asthma. At least two components of AHR have been identified: 1) baseline AHR, which is persistent and presumably caused by airway remodelling due to chronic recurrent airway inflammation; and 2) acute and variable AHR, which is associated with an episodic increase in airway inflammation due to environmental factors such as allergen exposure. Despite intensive research, the mechanisms underlying acute and chronic AHR are poorly understood. Owing to the complex variety of interactive processes that may be involved, in vitro model systems and animal models are indispensable to the unravelling of these mechanisms at the cellular and molecular level. The present paper focuses on a number of translational studies addressing the emerging central role of the airway smooth muscle cell, as a multicompetent cell involved in acute airway constriction as well as structural changes in the airways, in the pathophysiology of airway hyperresponsiveness.
Asunto(s)
Asma/diagnóstico , Asma/fisiopatología , Hiperreactividad Bronquial/patología , Enfermedad Aguda , Alérgenos/química , Animales , Modelos Animales de Enfermedad , Ambiente , Humanos , Inflamación , Modelos Biológicos , Miocitos del Músculo Liso/metabolismo , Fenotipo , Trastornos Respiratorios/inmunología , Trastornos Respiratorios/fisiopatología , Sistema Respiratorio/inmunología , Sistema Respiratorio/fisiopatología , Transducción de SeñalRESUMEN
Chronic inflammation in asthma and chronic obstructive pulmonary disease drives pathological structural remodelling of the airways. Using tiotropium bromide, acetylcholine was recently identified as playing a major regulatory role in airway smooth muscle remodelling in a guinea pig model of ongoing allergic asthma. The aim of the present study was to investigate other aspects of airway remodelling and to compare the effectiveness of tiotropium to the glucocorticosteroid budesonide. Ovalbumin-sensitised guinea pigs were challenged for 12 weeks with aerosolised ovalbumin. The ovalbumin induced airway smooth muscle thickening, hypercontractility of tracheal smooth muscle, increased pulmonary contractile protein (smooth-muscle myosin) abundance, mucous gland hypertrophy, an increase in mucin 5 subtypes A and C (MUC5AC)-positive goblet cell numbers and eosinophilia. It was reported previously that treatment with tiotropium inhibits airway smooth muscle thickening and contractile protein expression, and prevents tracheal hypercontractility. This study demonstrates that tiotropium also fully prevented allergen-induced mucous gland hypertrophy, and partially reduced the increase in MUC5AC-positive goblet cell numbers and eosinophil infiltration. Treatment with budesonide also prevented airway smooth muscle thickening, contractile protein expression, tracheal hypercontractility and mucous gland hypertrophy, and partially reduced MUC5AC-positive goblet cell numbers and eosinophilia. This study demonstrates that tiotropium and budesonide are similarly effective in inhibiting several aspects of airway remodelling, providing further evidence that the beneficial effects of tiotropium bromide might exceed those of bronchodilation.
Asunto(s)
Alérgenos/química , Budesonida/uso terapéutico , Hipersensibilidad/tratamiento farmacológico , Derivados de Escopolamina/uso terapéutico , Corticoesteroides/metabolismo , Animales , Broncodilatadores/farmacología , Budesonida/química , Antagonistas Colinérgicos/farmacología , Eosinofilia , Matriz Extracelular/metabolismo , Glucocorticoides/química , Cobayas , Humanos , Inflamación , Masculino , Músculo Liso/metabolismo , Ovalbúmina/química , Bromuro de Tiotropio , Tráquea/patologíaRESUMEN
BACKGROUND AND PURPOSE: We investigated expression of cannabinoid receptors and the effects of the endogenous cannabinoid virodhamine and the synthetic agonist CP55,940 on cAMP accumulation and interleukin-8 (IL-8) release in human bronchial epithelial cells. EXPERIMENTAL APPROACH: Human bronchial epithelial (16HBE14o(-)) cells were used. Total mRNA was isolated and cannabinoid receptor mRNAs were detected by RT-PCR. Expression of CB(1) and CB(2) receptor proteins was detected with Western blotting using receptor-specific antibodies. cAMP accumulation was measured by competitive radioligand binding assay. IL-8 release was measured by ELISA. KEY RESULTS: CB(1) and CB(2) receptor mRNAs and proteins were found. Both agonists concentration-dependently decreased forskolin-induced cAMP accumulation. This effect was inhibited by the CB(2) receptor antagonist SR144528, and was sensitive to Pertussis toxin (PTX), suggesting the involvement of CB(2) receptors and G(i/o)-proteins. Cell pretreatment with PTX unmasked a stimulatory component, which was blocked by the CB(1) receptor antagonist SR141716A. CB(2) receptor-mediated inhibition of cAMP production by virodhamine and CP55,940 was paralleled by inhibition of tumor necrosis factor-alpha (TNF-alpha) induced IL-8 release. This inhibition was insensitive to SR141716A. In the absence of agonist, SR144528 by itself reduced TNF-alpha induced IL-8 release. CONCLUSIONS AND IMPLICATIONS: Our results show for the first time that 16HBE14o(-) cells respond to virodhamine and CP55,940. CB(1) and CB(2) receptor subtypes mediated activation and inhibition of adenylyl cyclase, respectively. Stimulation of the dominant CB(2) receptor signalling pathway diminished cAMP accumulation and TNF-alpha-induced IL-8 release. These observations may imply that cannabinoids exert anti-inflammatory properties in airways by modulating cytokine release.
Asunto(s)
Ácidos Araquidónicos/farmacología , AMP Cíclico/metabolismo , Ciclohexanoles/farmacología , Células Epiteliales/efectos de los fármacos , Interleucina-8/metabolismo , Inhibidores de Adenilato Ciclasa , Adenilil Ciclasas/metabolismo , Analgésicos/farmacología , Western Blotting , Bronquios/citología , Bronquios/efectos de los fármacos , Bronquios/metabolismo , Canfanos/farmacología , Cannabinoides/farmacología , Línea Celular , Colforsina/farmacología , Relación Dosis-Respuesta a Droga , Antagonismo de Drogas , Células Epiteliales/citología , Células Epiteliales/metabolismo , Humanos , Inmunosupresores/farmacología , Toxina del Pertussis/farmacología , Piperidinas/farmacología , Pirazoles/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor Cannabinoide CB1/genética , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/genética , Receptor Cannabinoide CB2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rimonabant , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
BACKGROUND AND PURPOSE: Recently, the use of inhaled insulin formulations for the treatment of type I and type II diabetes has been approved in Europe and in the United States. For regular use, it is critical that airway function remains unimpaired in response to insulin exposure. EXPERIMENTAL APPROACH: We investigated the effects of insulin on airway smooth muscle (ASM) contraction and contractile prostaglandin (PG) production, using guinea-pig open-ring tracheal smooth muscle preparations. KEY RESULTS: It was found that insulin (1 nM-1 microM) induced a concentration-dependent contraction that was insensitive to epithelium removal. These sustained contractions were susceptible to inhibitors of cyclooxygenase (indomethacin, 3 microM), Rho-kinase (Y-27632, 1 microM) and p42/44 MAP kinase (PD-98059, 30 microM and U-0126, 3 microM), but not of PI-3-kinase (LY-294002,10 microM). In addition, insulin significantly increased PGF(2alpha)-production which was inhibited by indomethacin, but not Y-27632. Moreover, the FP-receptor antagonist AL-8810 (10 microM) and the EP(1)-receptor antagonist AH-6809 (10 microM) strongly reduced insulin-induced contractions, supporting a pivotal role for contractile prostaglandins. CONCLUSIONS AND IMPLICATIONS: Collectively, the results show that insulin induces guinea-pig ASM contraction presumably through the production of contractile prostaglandins, which in turn are dependent on Rho-kinase for their contractile effects. The data suggest that administration of insulin as an aerosol could result in some acute adverse effects on ASM function.
Asunto(s)
Insulina/fisiología , Músculo Liso/fisiología , Tráquea/fisiología , Animales , Cobayas , Técnicas In Vitro , Insulina/farmacología , Masculino , Contracción Muscular , Músculo Liso/metabolismo , Prostaglandinas/biosíntesis , Mucosa Respiratoria/fisiología , Tráquea/metabolismoRESUMEN
In both children and adults with persistent asthma, treatment with an inhaled corticosteroid (ICS) is recommended. Moreover, inhaled bronchodilating agents have a clear role to play. The minimum effective dose of an ICS in the individual patient can be determined either by starting with a low dosage of ICS and increasing the dosage gradually on the basis of the symptoms (the 'step-up' approach), or by starting with a high dosage and, if the results are good, decreasing it to the pointwhere adequate control is maintained (the 'step-down' approach). In a study of the step-up approach with the ICS fluticasone, with or without salmeterol as a long-acting beta2-agonist (LABA) in adult patients with asthma, the approach with salmeterol produced the best results, namely, good asthma control in 71% of the patients and total control in 41%. In a study involving both children and adults with asthma, good results were obtained from treatment with a relatively low maintenance dose of ICS (budesonide) combined with a LABA (formoterol), whereby patients were permitted to use additional inhalations of the combination ICS and LABA. How the different therapeutic concepts result in long-term control, what the side effects are in the long term, and whether, in addition to the clinical symptoms, laboratory findings are also important as a therapeutic criterion are all unknown.
Asunto(s)
Corticoesteroides/uso terapéutico , Antiasmáticos/uso terapéutico , Asma/tratamiento farmacológico , Broncodilatadores/uso terapéutico , Administración por Inhalación , Adolescente , Corticoesteroides/administración & dosificación , Corticoesteroides/efectos adversos , Adulto , Antiasmáticos/administración & dosificación , Antiasmáticos/efectos adversos , Broncodilatadores/administración & dosificación , Broncodilatadores/efectos adversos , Niño , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Masculino , Resultado del TratamientoRESUMEN
Salbutamol consists of a racemic mixture of R- and S-salbutamol. R-salbutamol (levalbuterol) is the active bronchodilating enantiomer, whereas S-salbutamol is thought to be pharmacologically inactive or to exert adverse effects. This study evaluated the bronchoprotective effects of inhalation of therapeutically relevant doses of the racemate and individual enantiomers in guinea pigs. It was found that basal airway reactivity to histamine was similarly reduced 30 min after inhalation of equivalent doses of RS- and R-salbutamol; this protective effect disappeared within 3 h. Inhalation of RS- and R-salbutamol 30 min before and 5.5 h after allergen challenge suppressed allergen-induced airway hyperreactivity to histamine after the early and late asthmatic reaction, completely inhibiting the early asthmatic reaction and tending to reduce the development of the late asthmatic reaction. At 5 h after allergen challenge, the inhibition of airway hyperreactivity was more pronounced in animals treated with R-salbutamol compared to racemate-treated animals. Both basal airway reactivity and allergen-induced hyperreactivity were not affected by S-salbutamol. Inflammatory cell infiltration was not affected by the racemate or the individual enantiomers. In conclusion, inhalation of therapeutically relevant doses of R- and RS-salbutamol effectively suppress allergen-induced airway reactivity after the early and late asthmatic reactions, the R-enantiomer being slightly more potent with respect to early airway reactivity than the racemate. No adverse effects were observed for the S-enantiomer.
Asunto(s)
Albuterol/farmacología , Asma/tratamiento farmacológico , Asma/fisiopatología , Hiperreactividad Bronquial/tratamiento farmacológico , Albuterol/química , Alérgenos , Animales , Asma/inducido químicamente , Hiperreactividad Bronquial/inducido químicamente , Modelos Animales de Enfermedad , Cobayas , Isomerismo , Masculino , Cloruro de Sodio , Resultado del TratamientoRESUMEN
In the present study, we investigated the effect of fenoterol-induced constitutive beta(2)-adrenoceptor activity on muscarinic receptor agonist- and histamine-induced bovine tracheal smooth muscle contractions. Bovine tracheal smooth muscle strips were incubated with 10 microM fenoterol or vehicle for various periods of time (5, 30 min, 18 h) at 37 degrees C. After extensive washout (3 h, 37 degrees C), isometric contractions were measured to the full muscarinic receptor agonist methacholine, the partial muscarinic receptor agonist 4-(m-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium (McN-A-343) and histamine. Fenoterol treatment significantly reduced the sensitivity (pEC(50)) to methacholine in a time-dependent manner, without affecting maximal contraction (E(max)). Fenoterol treatment similarly reduced the pEC(50) of McN-A-343 and histamine; however, E(max) values were also reduced, to approximately 70% of control after 18-h treatment. The inverse agonist timolol, having no effect on control preparations, consistently restored the reduced pEC(50) and E(max) values of the contractile agonists. Remarkably, in the presence of timolol the pEC(50) values of McN-A-343 and histamine in fenoterol-treated airways were significantly enhanced compared to controls. In conclusion, fenoterol-induced constitutive beta(2)-adrenoceptor activity reduces muscarinic receptor agonist- and histamine-induced contractions of bovine tracheal smooth muscle, which can be reversed by the inverse agonist timolol. Moreover, after beta(2)-adrenoceptor agonist treatment, inverse agonism by beta-adrenoceptor antagonists may cause enhanced airway reactivity to contractile mediators.
Asunto(s)
Músculo Liso/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Agonistas de Receptores Adrenérgicos beta 2 , Animales , Bovinos , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Fenoterol/farmacología , Histamina/farmacología , Técnicas In Vitro , Cloruro de Metacolina/farmacología , Agonistas Muscarínicos/farmacología , Contracción Muscular , Timolol/farmacología , Tráquea/metabolismoRESUMEN
In a guinea pig model of allergic asthma, we investigated the effects of the selective phosphodiesterase inhibitors rolipram (phosphodiesterase 4-selective), Org 9935 (phosphodiesterase 3-selective) and Org 20241 (dual phosphodiesterase 4/phosphodiesterase 3-selective), administered by aerosol inhalation in approximately equipotent bronchodilatory doses, on allergen-induced early and late asthmatic reactions, airway hyperreactivity and airway inflammation. Using ovalbumin-sensitized non-challenged animals, different nebulizer concentrations of each inhibitor were tested for their protective effects against histamine-induced bronchoconstriction. Inhalation of 2.5 mM rolipram, 100 mM 4,5-dihydro-6-(5,6-dimethoxybenzo[b]thien-2-yl-5-methyl-3(2H)pyridazinone (Org 9935) and 10 and 100 mM N-hydroxy-4-(3,4-dimethoxyphenyl)-thiazole-2-carboximidamide HCl (Org 20241) provided a similar, 1.8-fold (P<0.01), 2.0-fold (P<0.05), and 1.8- and 1.9-fold (P<0.05) protection, respectively. The duration of these bronchoprotective effects were different, the rate of decline being faster with rolipram and the lower Org 20241 concentration than with Org 9935 and the higher concentration of Org 20241. All compounds strongly protected against the immediate allergen-induced bronchoconstriction and significantly (P<0.05) diminished the overall early asthmatic reaction from 0 to 6 h following allergen-provocation. The severity of the late asthmatic reaction was also significantly inhibited by rolipram (P<0.05) and Org 9935 (P<0.05). Allergen-induced airway hyperreactivity to inhaled histamine after the early reaction, at 6 h after ovalbumin challenge, was strongly reduced by rolipram (P<0.05) and completely prevented by the two other phosphodiesterase inhibitors; in addition, airway hyperreactivity after the late asthmatic reaction, at 24 h, was abolished in all treatment groups. Bronchoalveolar lavage performed at 24 h after allergen challenge revealed no inhibition of eosinophil infiltration in the rolipram-treated animals, whereas inhalation of Org 9935 and the higher-but not the lower-concentration of Org 20241 strongly reduced the influx of these cells. Eosinophil peroxidase activity in the lavage fluid tended to be diminished in all treatment groups but significance was not reached with the exception of the lower concentration of Org 20241. Infiltration of lymphocytes and macrophages was significantly inhibited by Org 9935 only (P<0.05 and P<0.01, respectively), whereas neutrophil influx was not significantly affected. The results indicate that inhalation of phosphodiesterase 3-, phosphodiesterase 4- and dual phosphodiesterase 3/phosphodiesterase 4-selective inhibitors afford protection against acute histamine- and allergen-induced bronchoconstriction and prevent the development of airway hyperreactivity both after the early and late asthmatic reaction predominantly through inhibition of phosphodiesterase 4; in contrast, for significant reduction of eosinophil infiltration, both phosphodiesterase 3 and phosphodiesterase 4 inhibition seems to be required.
Asunto(s)
Antiinflamatorios no Esteroideos/administración & dosificación , Asma/tratamiento farmacológico , Broncodilatadores/administración & dosificación , Modelos Animales de Enfermedad , Inhibidores de Fosfodiesterasa/administración & dosificación , Administración por Inhalación , Alérgenos/efectos adversos , Animales , Asma/inmunología , Asma/fisiopatología , Hiperreactividad Bronquial/tratamiento farmacológico , Hiperreactividad Bronquial/fisiopatología , Broncoconstricción/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Cobayas , Histamina/farmacología , Inflamación/inmunología , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunologíaRESUMEN
Although airway inflammation and airway hyperreactivity are observed after allergen inhalation both in allergic humans and animals, little is known about the mechanisms by which inflammatory cells can contribute to allergen-induced airway hyperreactivity. To understand how inflammatory cell infiltration can contribute to airway hyperreactivity, the location of these cells within the airways may be crucial Using a guinea pig model of acute allergic asthma, we investigated the inflammatory cell infiltration in different airway compartments at 6 and 24 h (i.e. after the early and the late asthmatic reaction, respectively) after allergen or saline challenge in relation to changes in airway reactivity (AR) to histamine. At 6 h after allergen challenge, a threefold (p < 0.01) increase in the AR to histamine was observed. At 24 h after challenge, the AR to histamine was lower, but still significantly enhanced (1.6-fold, p < 0.05). Adventitial eosinophil and neutrophil numbers in both bronchi and bronchioli were significantly increased at 6 h post-allergen provocation as compared with saline (p < 0.01 for all), while there was a strong tendency to enhanced eosinophils in the bronchial submucosa at this time point (p = 0.08). At 24h after allergen challenge, the eosinophilic and neutrophilic cell infiltration was reduced. CD3+ T lymphocytes were increased in the adventitial compartment of the large airways (p < 0.05) and in the parenchyma (p < 0.05) at 24h post-allergen, while numbers of CD8+ cells did not differ from saline treatment at any time point post-provocation. The results indicate that, after allergen provocation, inflammatory cell numbers in the airways are mainly elevated in the adventitial compartment. The adventitial inflammation could be important for the development of allergen-induced airway hyperreactivity.
Asunto(s)
Asma/inmunología , Eosinófilos/inmunología , Pulmón/inmunología , Neutrófilos/inmunología , Linfocitos T/inmunología , Animales , Antígenos/inmunología , Modelos Animales de Enfermedad , Cobayas , Liberación de Histamina/inmunología , Masculino , Ovalbúmina/inmunología , Factores de TiempoRESUMEN
1. In the present study, the roles of nitric oxide (NO) and superoxide anions (O2(-)) in allergen-induced airway hyperreactivity (AHR) after the late asthmatic reaction (LAR) were investigated ex vivo, by examining the effects of the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) and superoxide dismutase (SOD) on the responsiveness to methacholine of isolated perfused guinea-pig tracheae from unchallenged (control) animals and from animals 24 h after ovalbumin challenge. 2. At 24 h after allergen challenge, the animals developed AHR in vivo, as indicated by a mean 2.63 +/- 0.54 fold (P < 0.05) increase in sensitivity to histamine inhalation. 3. Compared to unchallenged controls, tracheal preparations from the ovalbumin-challenged guinea-pigs displayed a significant 1.8 fold (P < 0.01) increase in the maximal response (E(max)) to methacholine, both after intraluminal (IL) and extraluminal (EL) administration of the agonist. No changes were observed in the sensitivity (pEC(50)) to the agonist. Consequently, the DeltapEC(50) (EL-IL), as a measure of epithelial integrity, was unchanged. 4. In the presence of L-NAME (100 microM, IL), tracheae from control guinea-pigs showed a 1.6 fold (P < 0.05) increase in the E(max) of IL methacholine. By contrast, the E(max) of IL methacholine was significantly decreased in the presence of 100 u ml(-1) EL SOD (54% of control, P < 0.01). 5. Remarkably, the increased responsiveness to IL methacholine at 24 h after allergen challenge was reversed by L-NAME to control (P < 0.01), and a similar effect was observed with SOD (P < 0.01). 6. The results indicate that both NO and O2(-) are involved in the tracheal hyperreactivity to methacholine after the LAR, possibly by promoting airway smooth muscle contraction through the formation of peroxynitrite.
Asunto(s)
Alérgenos/inmunología , Asma/metabolismo , Hiperreactividad Bronquial/metabolismo , Óxido Nítrico/metabolismo , Superóxidos/metabolismo , Animales , Asma/inducido químicamente , Asma/inmunología , Asma/fisiopatología , Hiperreactividad Bronquial/inducido químicamente , Hiperreactividad Bronquial/inmunología , Hiperreactividad Bronquial/fisiopatología , Broncoconstrictores/farmacología , Cobayas , Histamina/farmacología , Cloruro de Metacolina/farmacología , NG-Nitroarginina Metil Éster/farmacología , Ovalbúmina/inmunología , Perfusión , Respiración/efectos de los fármacos , Superóxido Dismutasa/farmacología , Tráquea/efectos de los fármacos , Tráquea/metabolismo , Tráquea/fisiopatologíaRESUMEN
Carp were cannulated in the dorsal aorta, and after 2 days of recovery they were infused with 1) norepinephrine, 2) yohimbine (alpha(2)-antagonist) plus norepinephrine, 3) clonidine (alpha(2)-agonist), and 4) isoproterenol (nonselective beta-agonist). Norepinephrine lowered the plasma free fatty acid (FFA) level and raised the plasma glucose level for several hours. Norepinephrine in combination with the alpha(2)-antagonist yohimbine resulted in retardation of the FFA decrease, indicating the involvement of alpha(2)-adrenoceptors. Infusion with the partial alpha(2)-agonist clonidine had a smaller effect. Infusion with isoproterenol caused a marked increase of glucose levels, and unexpectedly a decline of plasma FFA levels, indicating a direct involvement of beta-adrenoceptors. Combination of isoproterenol with either atenolol (beta(1)-antagonist) or ICI-118,551 (beta(2)-antagonist) showed that both beta(1)- and beta(2)-adrenoceptors were involved in the glucose release by isoproterenol. Remarkably, the decline of FFA levels was augmented in the presence of ICI-118,551, whereas with atenolol present plasma FFA levels were increased by isoproterenol. Thus it is concluded that in carp both beta(1)- and beta(2)-adrenoceptors mediate glucose release, whereas lipolysis is controlled by inhibitory beta(1)-adrenoceptors and stimulatory beta(2)-adrenoceptors, as well as by inhibitory alpha(2)-adrenoceptors.
Asunto(s)
Glucemia/metabolismo , Carpas/sangre , Epinefrina/sangre , Ácidos Grasos no Esterificados/sangre , Norepinefrina/farmacología , Receptores Adrenérgicos beta/fisiología , Agonistas Adrenérgicos/sangre , Agonistas alfa-Adrenérgicos/sangre , Antagonistas Adrenérgicos alfa/farmacología , Agonistas Adrenérgicos beta/farmacología , Antagonistas Adrenérgicos beta/farmacología , Animales , Atenolol/farmacología , Clonidina/farmacología , Isoproterenol/farmacología , Ácido Láctico/sangre , Propanolaminas/farmacología , Yohimbina/farmacologíaRESUMEN
Prolonged (18 h) incubation of isolated bovine tracheal smooth muscle with the beta2-adrenoceptor agonist fenoterol (10 microM) induced desensitization of isoprenaline-induced adenylyl cyclase activity in bovine tracheal smooth muscle membranes, characterized by a 25% decrease in maximal effect (Emax) (P < 0.05), while the sensitivity to the agonist (pEC50) was unchanged. The Emax value of isoprenaline-induced smooth muscle relaxation of submaximal methacholine-induced contractile tones was similarly reduced by about 25% (P < 0.001), while the pEC50 value was diminished by 1.0 log unit (P < 0.001). As determined by 30 microM gallamine-induced muscarinic M2 receptor antagonism and pertussis toxin-induced inactivation of G(i alpha), muscarinic M2 receptor-mediated functional antagonism did not play a role in isoprenaline-induced relaxation of bovine tracheal smooth muscle contracted by methacholine, both in control and in 18-h fenoterol-treated tissue. In line with these observations, we found no enhanced muscarinic M2 receptor-mediated inhibition of 1 microM forskolin-stimulated adenylyl cyclase activity after 18-h fenoterol treatment. These data indicate that 18-h fenoterol treatment of bovine tracheal smooth muscle induces beta2-adrenoceptor desensitization and reduced functional antagonism of methacholine-induced contraction by beta-adrenoceptor agonists, without a change of muscarinic M2 receptor function.
Asunto(s)
Agonistas Adrenérgicos beta/farmacología , Fenoterol/farmacología , Músculo Liso/efectos de los fármacos , Receptores Adrenérgicos beta/efectos de los fármacos , Receptores Muscarínicos/efectos de los fármacos , Toxina de Adenilato Ciclasa , Adenilil Ciclasas/metabolismo , Animales , Bovinos , Interacciones Farmacológicas , Cloruro de Metacolina/farmacología , Agonistas Muscarínicos/farmacología , Contracción Muscular/efectos de los fármacos , Músculo Liso/enzimología , Toxina del Pertussis , Receptor Muscarínico M2 , Tráquea , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
According to the two state receptor model, the beta(2)-adrenergic receptor (beta(2)-AR) isomerizes between an inactive state and a constitutively active state, which couples to the stimulatory G-protein in the absence of agonist. In bovine tracheal smooth muscle (BTSM), we investigated the effect of short and long term beta(2)-AR activation by fenoterol on constitutive receptor activity. Preincubation of the BTSM strips for 5 min, 30 min and 18 h with 10 microM fenoterol, followed by extensive washout (3 h, 37 degrees C), caused a rapid and time-dependent inhibition of KCl-induced contraction, reaching 68+/-10, 51+/-6 and 46+/-4% of control, respectively, at 40 mM KCl (P:<0.05 all). At all time points, the EC(50) values to KCl were significantly reduced as well. Preincubation of BTSM with 0.1, 1.0 and 10 microM fenoterol during 18 h caused a concentration-dependent decrease of the 40 mM KCl response to 70+/-5, 47+/-12 and 43+/-9% of control, respectively (P:<0.05 all). The reduced KCl contractions were reversed in the presence of 1 microM timolol. Moreover, the sensitivity to KCl in the presence of timolol was enhanced after fenoterol incubation. Inverse agonism was also found for other beta-blockers, with a rank order of efficacy of pindolol >/=timolol=propranolol>alprenolol>/=sotalol>labetalol. At 25 mM KCl-induced tone, the contraction induced by cumulative timolol administration was competitively antagonized by the less efficacious inverse agonist labetalol, indicating that the fenoterol-induced effects cannot be explained by residual beta-agonist binding. In conclusion, fenoterol treatment of BTSM causes a time- and concentration-dependent development of constitutive beta(2)-AR activity, which can be reversed by various inverse agonists. The beta-agonist-induced changes could represent a novel regulation mechanism of beta(2)-AR activity.
Asunto(s)
Agonistas Adrenérgicos beta/farmacología , Músculo Liso/efectos de los fármacos , Receptores Adrenérgicos beta 2/efectos de los fármacos , Tráquea/efectos de los fármacos , Antagonistas Adrenérgicos beta/farmacología , Animales , Bovinos , Relación Dosis-Respuesta a Droga , Fenoterol/farmacología , Técnicas In Vitro , Contracción Muscular/efectos de los fármacos , Músculo Liso/fisiología , Cloruro de Potasio/farmacología , Receptores Adrenérgicos beta 2/fisiología , Timolol/farmacología , Tráquea/fisiologíaRESUMEN
Cholinergic airway constriction is functionally antagonized by agonist-induced constitutive nitric oxide synthase (cNOS)-derived nitric oxide (NO). Since cNOS and arginase, which hydrolyzes L-arginine to L-ornithine and urea, use L-arginine as a common substrate, competition between both enzymes for the substrate could be involved in the regulation of cholinergic airway reactivity. Using a perfused guinea-pig tracheal tube preparation, we investigated the modulation of methacholine-induced airway constriction by the recently developed, potent and specific arginase inhibitor N(Omega)-hydroxy-nor-L-arginine (nor-NOHA). Intraluminal (IL) administration of nor-NOHA caused a concentration-dependent inhibition of the maximal effect (E(max)) in response to IL methacholine, which was maximal in the presence of 5 microM nor-NOHA (E(max)=31.2+/-1.6% of extraluminal (EL) 40 mM KCl-induced constriction versus 51.6+/-2.1% in controls, P<0.001). In addition, the pEC(50) (-log(10) EC(50)) was slightly but significantly reduced in the presence of 5 microM nor-NOHA. The inhibition of E(max) by 5 microM nor-NOHA was concentration-dependently reversed by the NOS inhibitor N(Omega)-nitro-L-arginine methyl ester (L-NAME), reaching an E(max) of 89.4+/-7.7% in the presence of 0.5 mM L-NAME (P<0.01). A similar E(max) in the presence of 0.5 mM L-NAME was obtained in control preparations (85.2+/-9.7%, n.s.). In the presence of excess of exogenously applied L-arginine (5 mM), 5 microM nor-NOHA was ineffective (E(max)=33.1+/-5.8 versus 31.1+/-7.5% in controls, n.s.). The results indicate that endogenous arginase activity potentiates methacholine-induced airway constriction by inhibition of NO production, presumably by competition with cNOS for the common substrate, L-arginine. This finding may represent an important novel regulation mechanism of airway reactivity.
Asunto(s)
Arginasa/metabolismo , Arginina/análogos & derivados , Broncoconstrictores/farmacología , Cloruro de Metacolina/farmacología , Óxido Nítrico/metabolismo , Tráquea/efectos de los fármacos , Animales , Arginasa/antagonistas & inhibidores , Arginina/farmacología , Broncoconstricción/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Cobayas , Técnicas In Vitro , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , Organismos Libres de Patógenos Específicos , Tráquea/metabolismo , Tráquea/fisiologíaRESUMEN
Histamine, released from activated mast cells, causes bronchoconstriction mediated by H(1) receptors, whereas beta(2)-agonists are widely used for the relief of bronchoconstriction. In this study, we examined the effects of the beta(2)-adrenoceptor agonist, fenoterol, on the expression of H(1) receptors at the mRNA and protein levels, and functional responses. Incubation of bovine tracheal smooth muscle with fenoterol (10(-7) M) for 2 h increased H(1) receptor mRNA (maximum approximately 190%). The number of H(1) receptors was increased after 12 and 18 h without any change in binding affinity. In the contraction experiments, the concentration-response curves for histamine-induced contraction were shifted significantly to the left after 18-h exposure to fenoterol, consistent with the increase in receptor number. The fenoterol-induced increase in H(1) receptor mRNA was concentration-dependent and was abolished by propranolol and ICI 118551, but not by CGP 20712A, indicating that fenoterol acts via beta(2)-adrenoceptors. These effects were mimicked by other cAMP-elevating agents forskolin and prostaglandin E(2), and by the stable cAMP analog 8-bromo-cAMP. Cycloheximide alone produced superinduction of H(1) receptor mRNA and augmented the fenoterol-induced increase in H(1) receptor mRNA. Fenoterol increased both the stability and the transcription rate of H(1) receptor mRNA. Pretreatment with dexamethasone did not prevent fenoterol-induced up-regulation of H(1) receptor mRNA. Thus, fenoterol increases the expression of airway smooth muscle H(1) receptors via activation of the cAMP system through increased gene transcription and mRNA stability. This mechanism may be involved in the adverse responses encountered with the clinical use of short-acting beta(2)-agonists.