RESUMEN
Secretory granules (SGs) sequester significant calcium. Understanding roles for this calcium and potential mechanisms of release is hampered by the difficulty of measuring SG calcium directly in living cells. We adapted the Förster resonance energy transfer-based D1-endoplasmic reticulum (ER) probe to develop a unique probe (D1-SG) to measure calcium and pH in secretory granules. It significantly localizes to SGs and reports resting free Ca(2+) of 69 ± 15 µM and a pH of 5.8. Application of extracellular ATP to activate P2Y receptors resulted in a slow monotonic decrease in SG Ca(2+) temporally correlated with the occurrence of store-operated calcium entry (SOCE). Further investigation revealed a unique receptor-mediated mechanism of calcium release from SGs that involves SG store-operated Orai channels activated by their regulator stromal interaction molecule 1 (STIM1) on the ER. SG Ca(2+) release is completely antagonized by a SOCE antagonist, by switching to Ca(2+)-free medium, and by overexpression of a dominant-negative Orai1(E106A). Overexpression of the CRAC activation domain (CAD) of STIM1 resulted in a decrease of resting SG Ca(2+) by â¼75% and completely abolished the ATP-mediated release of Ca(2+) from SGs. Overexpression of a dominant-negative CAD construct(CAD-A376K) induced no significant changes in SG Ca(2+). Colocalization analysis suggests that, like the plasma membrane, SG membranes also possess Orai1 channels and that during SG Ca(2+) release, colocalization between SGs and STIM1 increases. We propose Orai channel opening on SG membranes as a potential mode of calcium release from SGs that may serve to raise local cytoplasmic calcium concentrations and aid in refilling intracellular calcium stores of the ER and exocytosis.
Asunto(s)
Canales de Calcio/fisiología , Calcio/metabolismo , Proteínas de la Membrana/fisiología , Proteínas de Neoplasias/fisiología , Vesículas Secretoras/metabolismo , Calcio/química , Señalización del Calcio/fisiología , Línea Celular Tumoral , Membrana Celular/metabolismo , Citoplasma/metabolismo , Retículo Endoplásmico/metabolismo , Exocitosis , Humanos , Concentración de Iones de Hidrógeno , Modelos Biológicos , Proteína ORAI1 , Fotoquímica/métodos , Molécula de Interacción Estromal 1RESUMEN
BACKGROUND: Exercise-induced bronchoconstriction (EIB) is a common cause of symptoms in a subgroup of asthmatic subjects. The pathobiology that makes this group of asthmatic subjects susceptible to bronchoconstriction after a brief period of exercise remains poorly understood. OBJECTIVE: We sought to determine whether there are differences in lower airway inflammation and production of cytokines and eicosanoids between asthmatic subjects with and without EIB. METHODS: Two distinct groups of asthmatic subjects based on a priori definitions were identified, one with moderate-to-severe EIB and the other without significant bronchoconstriction after exercise challenge. Both groups met the definition of asthma on the basis of bronchodilator response, bronchial hyperresponsiveness, or both. A comparative immunopathology study was conducted by using induced sputum to identify differences in lower airway inflammation and production of cytokines and eicosanoids. RESULTS: The groups had similar baseline lung function and bronchodilator response and did not have any asthma exacerbations within the prior year. The concentration of columnar epithelial cells was markedly higher in the group with EIB (1.4 x 10(5) vs 2.9 x 10(4) cells/mL, P=.01). The concentration of eosinophils was higher in the group with EIB (3.6 x 10(4) vs 4.9 x 10(3) cells/mL P=.04). Cysteinyl leukotrienes (CysLTs; 727.7 vs 151.9 pg/mL, P=.01) and the ratio of CysLTs to prostaglandin E(2) (1.85 vs 1.04, P=.002) in the airways were higher in the group with EIB. CONCLUSION: Injury to the airway epithelium, overexpression of CysLTs, relative under production of prostaglandin E(2), and greater airway eosinophilia are distinctive immunopathologic features of asthma with EIB.
Asunto(s)
Asma Inducida por Ejercicio/inmunología , Asma Inducida por Ejercicio/patología , Pulmón/inmunología , Pulmón/patología , Adulto , Citocinas/análisis , Citocinas/inmunología , Eicosanoides/análisis , Eicosanoides/inmunología , Eosinófilos/inmunología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Esputo/química , Esputo/citología , Esputo/inmunologíaRESUMEN
RATIONALE: Exercise-induced bronchoconstriction (EIB) is a highly prevalent condition with unclear pathogenesis. Two competing theories of the pathogenesis of EIB differ regarding the inflammatory basis of this condition. OBJECTIVES: Our goals were to establish whether epithelial cell and mast cell activation with release of inflammatory mediators occurs during EIB and how histamine and cysteinyl leukotriene antagonists alter the airway events occurring during EIB. METHODS: Induced sputum was used to measure mast cell mediators and eicosanoids at baseline and 30 minutes after exercise challenge in 25 individuals with asthma with EIB. In a randomized, double-blind crossover study, the cysteinyl leukotriene antagonist montelukast and antihistamine loratadine or two matched placebos were administered for two doses before exercise challenge. MAIN RESULTS: The percentage of columnar epithelial cells in induced sputum at baseline was associated with the severity of EIB. After exercise challenge, histamine, tryptase, and cysteinyl leukotrienes significantly increased and prostaglandin E(2) and thromboxane B(2) significantly decreased in the airways, and there was an increase in columnar epithelial cells in the airways. The concentration of columnar epithelial cells was associated with the levels of histamine and cysteinyl leukotrienes in the airways. Treatment with montelukast and loratadine inhibited the release of cysteinyl leukotrienes and histamine into the airways, but did not inhibit the release of columnar epithelial cells into the airways. CONCLUSIONS: These data indicate that epithelial cells, mast cell mediators, and eicosanoids are released into the airways during EIB, supporting an inflammatory basis for EIB.
Asunto(s)
Asma/complicaciones , Asma/metabolismo , Bronquitis/etiología , Bronquitis/fisiopatología , Broncoconstricción , Ejercicio Físico , Mediadores de Inflamación/metabolismo , Acetatos/uso terapéutico , Adolescente , Adulto , Asma/tratamiento farmacológico , Asma/fisiopatología , Estudios Cruzados , Ciclopropanos , Método Doble Ciego , Femenino , Volumen Espiratorio Forzado , Antagonistas de los Receptores Histamínicos H1 no Sedantes/uso terapéutico , Humanos , Antagonistas de Leucotrieno/uso terapéutico , Loratadina/uso terapéutico , Pulmón/fisiopatología , Masculino , Persona de Mediana Edad , Quinolinas/uso terapéutico , Índice de Severidad de la Enfermedad , SulfurosRESUMEN
Pancreatic duct epithelial cells (PDEC) mediate the secretion of fluid and electrolytes and are exposed to refluxed bile. In nontransformed cultured dog PDEC, which express many ion transport pathways of PDEC, 1 mM taurodeoxycholic acid (TDCA) stimulated an (125)I(-) efflux inhibited by DIDS and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and a (86)Rb(+) efflux inhibited by charybdotoxin. Inhibition by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM suggests mediation via increased intracellular Ca(2+) concentration, whereas the absence of lactate dehydrogenase release excludes cellular toxicity. At 1 mM, TDCA stimulated a larger (125)I(-) efflux than glycodeoxycholate; two dihydroxy bile acids, taurochenodeoxycholate and TDCA, were similarly effective, whereas a trihydroxy bile acid, taurocholate, was ineffective. In Ussing chambers, 1 mM serosal or 2 mM luminal TDCA stimulated an I(sc) increase from confluent PDEC monolayers. TDCA also stimulated 1) a short-circuit current (I(sc)) increase from basolaterally permeabilized PDEC subject to a serosal-to-luminal Cl(-) gradient that was inhibited by BAPTA-AM, DIDS, and NPPB and 2) an I(sc) increase from apically permeabilized PDEC subject to a luminal-to-serosal K(+) gradient inhibited by BAPTA-AM and charybdotoxin. Along with the efflux studies, these findings suggest that TDCA interacts directly with PDEC to stimulate Ca(2+)-activated apical Cl(-) channels and basolateral K(+) channels. Monolayer transepithelial resistance was only minimally affected by 1 mM serosal and 2 mM luminal TDCA but decreased after exposure to higher TDCA concentrations (2 mM serosal and 4 mM luminal). A secretory role for bile acids should be considered in pancreatic diseases associated with bile reflux.