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ATP is an efficacious secretagogue for mucin and chloride in the epithelial cell line HT29-Cl.16E. Mucin release has been measured as [³H]glucosamine-labeled product in extracellular medium and as single-cell membrane capacitance increases indicative of exocytosis-related increases in membrane area. The calcium-activated chloride channel blocker niflumic acid, also reported to modulate secretion, was used to probe for divergence in the purinergic signaling of mucin exocytosis and channel activation. With the use of whole cell patch clamping, ATP stimulated a transient capacitance increase of 15 ± 4%. Inclusion of niflumic acid significantly reduced the ATP-stimulated capacitance change to 3 ± 1%, although normalized peak currents were not significantly different. Ratiometric imaging was used to assess intracellular calcium (Ca[sup 2+, sub i]) dynamics during stimulation. In the presence of niflumic acid, the ATP-stimulated peak change in Ca[sup 2+, sub i] was unaffected, but the initial response and overall time to Ca[sup 2+, sub i] peak were significantly affected. Excluding external calcium before ATP stimulation or including the capacitative calcium entry blocker LACl[sub 3] during stimulation muted the initial calcium transient similar to that observed with niflumic acid and significantly reduced peak capacitance change, suggesting that a substantial portion of the ATP-stimulated mucin exocytosis in HT29Cl.16E depends on a rapid, brief calcium influx through the plasma membrane. Niflumic acid interferes with this influx independent of a chloride channel blockade effect. capacitative calcium entry; membrane capacitance; chloride channels; fura 2. [ABSTRACT FROM AUTHOR]

Assesses whether interleukin (IL)-13 could induce a change in goblet density (GCD) through a direct interaction with human airway epithelium. Effect on human bronchial epithelial cells (HBECS) that is sensitive to PD98059 but not AG1478.; Induction of mucus hypersectretary phenotype; Involvement of the MAP kinase kinase (MEK) pathway; No role of tyrosine kinase in the IL-13 driven increase.

Cultured primary epithelial cells are used to examine inflammation in cystic fibrosis (CF). We describe a new human model system using cultured nasal brushings. Nasal brushings were obtained from 16 F508del homozygous patients and 11 healthy controls. Cells were resuspended in airway epithelial growth medium and seeded onto collagen-coated flasks and membranes for use in patch-clamp, ion transport, and mediator release assays. Viable cultures were obtained with a 75% success rate from subjects with CF and 100% from control subjects. Amiloride-sensitive epithelial Na channel current of similar size was present in both cell types while forskolin-activated CF transmembrane conductance regulator current was lacking in CF cells. In Ussing chambers, cells from CF patients responded to UTP but not to forskolin. Spontaneous and cytomix-stimulated IL-8 release was similar (stimulated 29,448 ± 9,025 pg/ml; control 16,336 ± 3,308 pg/ml CF; means ± SE). Thus nasal epithelial cells from patients with CF can be grown from nasal brushings and used in electrophysiological and mediator release studies in CF research. [ABSTRACT FROM AUTHOR]

Background and Purpose: The epithelial sodium channel (ENaC) regulates airway mucosal hydration and mucus clearance. The lack of such regulation in cystic fibrosis patients leads to desiccation of the airway lumen, resulting in mucostasis that establishes the environment for infections. Osmotic agents and negative ENaC regulators can be used to restore mucosal hydration. We aimed to assess whether: (i) osmotically driven fluid flux into the rat lung could be quantified in vivo by magnetic resonance imaging (MRI); and (ii) the MRI signals could be modulated through the regulation of ENaC function.Experimental Approach: Lung images from spontaneously breathing rats were acquired following intra-tracheal (i.t.) administration of physiological or hypertonic saline (HS). Compounds known to modulate the ENaC function were given i.t. prior to saline. Volumes of fluid signals were quantified on the images.Key Results: A tonicity-dependent increase in lung fluid was demonstrated following HS administration. Pretreatment with the ENaC blockers, amiloride or 552-02, resulted in an enhancement of HS-induced lung fluid signals, which were detectable for up to 4 h, consistent with a role for ENaC in fluid clearance. Aprotinin, a serine protease inhibitor that attenuates ENaC function, likewise enhanced the HS-induced increase in lung fluid signal, while alpha(1)-anti-trypsin was without significant effect.Conclusions and Implications: Proton MRI provides a non-invasive technique for studying modulators of lung fluid hydration in rat lung in vivo. The pharmacological sensitivity of MRI-detected fluid signals is consistent with ENaC-mediated fluid reabsorption after HS. This target-related readout may be used to characterize new ENaC modulators. [ABSTRACT FROM AUTHOR]

Background and purpose:The epithelial sodium channel (ENaC) is a key regulator of airway mucosal hydration and mucus clearance. Negative regulation of airway ENaC function is predicted to be of clinical benefit in the cystic fibrosis lung. The aim of this study was to develop a small animal model to enable the direct assessment of airway ENaC function in vivo.Experimental approach:Tracheal potential difference (TPD) was utilized as a measure of airway epithelial ion transport in the guinea-pig. ENaC activity in the trachea was established with a dose–response assessment to a panel of well-characterized direct and indirect pharmacological modulators of ENaC function, delivered by intra-tracheal (i.t.) instillation.Key results:The TPD in anaesthetized guinea-pigs was attenuated by the direct ENaC blockers: amiloride, benzamil and CF552 with ED50 values of 16, 14 and 0.2 μg kg−1 (i.t.), respectively. 5-(N-Ethyl-N-isopropyl) amiloride, a structurally related compound but devoid of activity on ENaC, was without effect on the TPD. Intra-tracheal dosing of the Kunitz-type serine protease inhibitors aprotinin and placental bikunin, which have previously been demonstrated to inhibit proteolytic activation of ENaC, likewise potently attenuated TPD in guinea-pigs, whereas α1-antitrypsin and soya bean trypsin inhibitor were without effect.Conclusions and implications:The pharmacological sensitivity of the TPD to amiloride analogues and also to serine protease inhibitors are both consistent with that of ENaC activity in the guinea-pig trachea. The guinea-pig TPD therefore represents a suitable in vivo model of human airway epithelial ion transport.British Journal of Pharmacology (2008) 155, 1025–1033; doi:10.1038/bjp.2008.363; published online 22 September 2008 [ABSTRACT FROM AUTHOR]

Presents a study of the airway inflammation induced by acute cigarette smoke (CS) exposure to rats. Dose dependent changes in lung neutrophils and mucin secretion in airways; Reliance on BAL for marking lung tissue leucocyte changes; Elevation of tissue neutrophils due to dexamethasone.