RESUMEN
Glucagon is a hyperglycemic pancreatic hormone that has been shown to provide a beneficial effect against asthmatic bronchospasm. We investigated the role of this hormone on airway smooth muscle contraction and lung inflammation using both in vitro and in vivo approaches. The action of glucagon on mouse cholinergic tracheal contraction was studied in a conventional organ bath system, and its effect on airway obstruction was also investigated using the whole-body pletysmographic technique in mice. We also tested the effect of glucagon on lipopolysaccharide (LPS)-induced airway hyperreactivity (AHR) and inflammation. The expression of glucagon receptor (GcgR), CREB, phospho-CREB, nitric oxide synthase (NOS)-3, pNOS-3 and cyclooxygenase (COX)-1 was evaluated by western blot, while prostaglandin E2 (PGE2) and tumour necrosis factor-α were quantified by enzyme-linked immunoassay and ELISA respectively. Glucagon partially inhibited carbachol-induced tracheal contraction in a mechanism clearly sensitive to des-His1-[Glu9]-glucagon amide, a GcgR antagonist. Remarkably, GcgR was more expressed in the lung and trachea with intact epithelium than in the epithelium-denuded trachea. In addition, the glucagon-mediated impairment of carbachol-induced contraction was prevented by either removing epithelial cells or blocking NOS (L-NAME), COX (indomethacin) or COX-1 (SC-560). In contrast, inhibitors of either heme oxygenase or COX-2 were inactive. Intranasal instillation of glucagon inhibited methacholine-induced airway obstruction by a mechanism sensitive to pretreatment with L-NAME, indomethacin and SC-560. Glucagon induced CREB and NOS-3 phosphorylation and increased PGE2 levels in the lung tissue without altering COX-1 expression. Glucagon also inhibited LPS-induced AHR and bronchoalveolar inflammation. These findings suggest that glucagon possesses airway-relaxing properties that are mediated by epithelium-NOS-3-NO- and COX-1-PGE2-dependent mechanisms.
Asunto(s)
Broncodilatadores/farmacología , Neuronas Colinérgicas/efectos de los fármacos , Dinoprostona/metabolismo , Glucagón/farmacología , Músculo Liso/efectos de los fármacos , Óxido Nítrico/metabolismo , Tráquea/efectos de los fármacos , Administración Intranasal , Resistencia de las Vías Respiratorias/efectos de los fármacos , Animales , Asma/tratamiento farmacológico , Asma/inmunología , Asma/metabolismo , Hiperreactividad Bronquial/tratamiento farmacológico , Hiperreactividad Bronquial/inmunología , Hiperreactividad Bronquial/metabolismo , Broncodilatadores/administración & dosificación , Broncodilatadores/uso terapéutico , Neuronas Colinérgicas/inmunología , Neuronas Colinérgicas/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Glucagón/administración & dosificación , Glucagón/uso terapéutico , Técnicas In Vitro , Masculino , Ratones Endogámicos A , Relajación Muscular/efectos de los fármacos , Músculo Liso/inmunología , Músculo Liso/inervación , Músculo Liso/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Tráquea/inmunología , Tráquea/inervación , Tráquea/metabolismoRESUMEN
Previous studies have described the antispasmodic effect of mangiferin, a natural glucoside xanthone (2-C-ß-Dgluco-pyranosyl-1,3,6,7-tetrahydroxyxanthone) that is present in mango trees and other plants, but its mechanism of action remains unknown. The aim of this study was to examine the potential contribution of the nitric oxide-cyclic GMP pathway to the antispasmodic effect of mangiferin on isolated tracheal rings preparations. The functional effect of mangiferin on allergic and non-allergic contraction of guinea pig tracheal rings was assessed in conventional organ baths. Cultured tracheal rings were exposed to mangiferin or vehicle, and nitric oxide synthase (NOS) 3 and cyclic GMP (cGMP) levels were quantified using western blotting and enzyme immunoassays, respectively. Mangiferin (0.1-10 µM) inhibited tracheal contractions induced by distinct stimuli, such as allergen, histamine, 5-hydroxytryptamine or carbachol, in a concentration-dependent manner. Mangiferin also caused marked relaxation of tracheal rings that were precontracted by carbachol, suggesting that it has both anti-contraction and relaxant properties that are prevented by removing the epithelium. The effect of mangiferin was inhibited by the nitric oxide synthase inhibitor, Nω-nitro-L-arginine methyl ester (L-NAME) (100 µM), and the soluble guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10 µM), but not the adenylate cyclase inhibitor, 9-(tetrahydro-2-furyl)adenine (SQ22536) (100 µM). The antispasmodic effect of mangiferin was also sensitive to K⺠channel blockers, such as tetraethylammonium (TEA), glibenclamide and apamin. Furthermore, mangiferin inhibited Ca²âº-induced contractions in K⺠(60 mM)-depolarised tracheal rings preparations. In addition, mangiferin increased NOS3 protein levels and cGMP intracellular levels in cultured tracheal rings. Finally, mangiferin-induced increase in cGMP levels was abrogated by co-incubation with either ODQ or L-NAME. These data suggest that the antispasmodic effect of mangiferin is mediated by epithelium-nitric oxide- and cGMP-dependent mechanisms.
Asunto(s)
GMP Cíclico/metabolismo , Óxido Nítrico/metabolismo , Parasimpatolíticos/farmacología , Transducción de Señal/efectos de los fármacos , Tráquea/efectos de los fármacos , Xantonas/farmacología , Animales , Calcio/metabolismo , Guanilato Ciclasa/antagonistas & inhibidores , Guanilato Ciclasa/metabolismo , Cobayas , Masculino , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Bloqueadores de los Canales de Potasio/farmacología , Tráquea/fisiologíaRESUMEN
Inhalation of JMF2-1, an analog of lidocaine with reduced anesthetic activity, prevents airway contraction and lung inflammation in experimental asthma models. We sought to test if the JMF2-1 effects are a consequence of increased intracellular cAMP levels in asthma cell targets, such as smooth muscle cells and T cells. Functional effect of JMF2-1 on carbachol-induced contraction of intact or epithelial-denuded rat trachea was assessed in conventional organ baths. cAMP was quantified by radioimmunoassay in cultured guinea pig tracheal smooth muscle cells, as well as lymph node cells from BALB/c mice, exposed to JMF2-1. We found that JMF2-1 (0.1-1mM) concentration-dependently inhibited epithelium-intact tracheal ring contraction induced by carbachol challenge. The antispasmodic effect remained unaltered following epithelium removal or pretreatment with NG-nitro-L-arginine methyl ester (100µM), but it was clearly sensitive to 9-(tetrahydro-2-furyl) adenine (SQ22,536, 100µM), an adenylate cyclase inhibitor. JMF2-1 (300 and 600µM) also dose-dependently increased cAMP intracellular levels of both cultured airway smooth muscle cells and T lymphocytes. This effect was consistently abrogated by SQ22,536 and reproduced by forskolin in both systems. JMF2-1 induced apoptosis of anti-CD3 activated T cells in a mechanism sensitive to zIETD, indicating that JMF2-1 mediates caspase-8-dependent apoptosis. Furthermore, forskolin also inhibited anti-CD3 induced T cell proliferation and survival. Our results suggest that JMF2-1 inhibits respiratory smooth muscle contraction as well as T cell proliferation and survival through enhancement of intracellular cAMP levels. These findings may help to explain the anti-inflammatory and antispasmodic effects of JMF2-1 observed in previous studies.
Asunto(s)
Antiinflamatorios/farmacología , AMP Cíclico/metabolismo , Lidocaína/análogos & derivados , Parasimpatolíticos/farmacología , Adenilil Ciclasas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Asma/tratamiento farmacológico , Asma/metabolismo , Carbacol/farmacología , Caspasa 8/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Colforsina/farmacología , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Cobayas , Inflamación/prevención & control , Lidocaína/farmacología , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Contracción Muscular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/metabolismo , Ratas , Ratas Wistar , Sistema Respiratorio/efectos de los fármacos , Sistema Respiratorio/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Tráquea/efectos de los fármacos , Tráquea/metabolismoRESUMEN
Allergic asthma is a chronic inflammatory disease of the lung whose incidence and morbidity continues to rise in developed nations. Despite being a hallmark of asthma, the molecular mechanisms that determine airway hyperresponsiveness (AHR) are not completely established. Transcription factors of the NFAT family are involved in the regulation of several asthma-related genes. It has been shown that the absence of NFAT1 leads to an increased pleural eosinophilic allergic response accompanied by an increased production of Th2 cytokines, suggesting a role for NFAT1 in the regulation of allergic diseases. Herein, we analyze NFAT1-/- mice to address the role of NFAT1 in a model of allergic airway inflammation and its influence in AHR. NFAT1-/- mice submitted to airway inflammation display a significant exacerbation of several features of the allergic disease, including lung inflammation, eosinophilia, and serum IgE levels, which were concomitant with elevated Th2 cytokine production. However, in spite of the increased allergic phenotype, NFAT1-/- mice failed to express AHR after methacholine aerosol. Refractoriness of NFAT1-/- mice to methacholine was confirmed in naïve mice, suggesting that this refractoriness occurs in an intrinsic way, independent of the lung inflammation. In addition, NFAT1-/- mice exhibit increased AHR in response to serotonin inhalation, suggesting a specific role for NFAT1 in the methacholine pathway of bronchoconstriction. Taken together, these data add support to the interpretation that NFAT1 acts as a counterregulatory mechanism to suppress allergic inflammation. Moreover, our findings suggest a novel role for NFAT1 protein in airway responsiveness mediated by the cholinergic pathway.