Protease‐activated receptor 2 activates airway apical membrane chloride permeability and increases ciliary beating

Abstract

Mucociliary clearance, driven by the engine of ciliary beating, is the primary physical airway defense against inhaled pathogens and irritants. A better understanding of the regulation of ciliary beating and mucociliary transport is necessary for identifying new receptor targets to stimulate improved clearance in airway diseases, such as cystic fibrosis and chronic rhinosinusitis. In this study, we examined the protease-activated receptor (PAR)-2, a GPCR previously shown to regulate airway cell cytokine and mucus secretion, and transepithelial Cl current. PAR-2 is activated by proteases secreted by airway neutrophils and pathogens. We cultured various airway cell lines, primary human and mouse sinonasal cells, and human bronchial cells at air-liquid interface and examined them using molecular biology, biochemistry, and live-cell imaging. We found that PAR-2 is expressed basolaterally, where it stimulates both intracellular Ca release and Ca influx, which activates low-level nitric oxide production, increases apical membrane Cl permeability ∼3-5-fold, and increases ciliary beating ∼20-50%. No molecular or functional evidence of PAR-4 was observed. These data suggest a novel and previously overlooked role of PAR-2 in airway physiology, adding to our understanding of the role of this receptor in airway Ca signaling and innate immunity.-McMahon, D. B., Workman, A. D., Kohanski, M. A., Carey, R. M., Freund, J. R., Hariri, B. M., Chen, B., Doghramji, L. J., Adappa, N. D., Palmer, J. N., Kennedy, D. W., Lee, R. J. Protease-activated receptor 2 activates airway apical membrane chloride permeability and increases ciliary beating.

Inverse regulation of secretion and inflammation in human airway gland serous cells by neuropeptides upregulated in allergy and asthma

“…Isolation of primary serous acinar cells, immunofluorescence, and live cell imaging of acinar cell volume, pHi (SNARF-5F), and Cl -(SPQ) was carried out as described (10-12, 47, 48). ASL height and pH measurements and ELISAs were carried out as previously reported (92,129,(151)(152)(153)(154)(155)(156)(157). Bacterial growth assays were carried out as previously described (158,159).…”

“…We hypothesized that Gi-coupled NPY receptors might blunt the magnitude of VIP-evoked cAMP increases, thus reducing Cland HCO3efflux through CFTR. We measured cellular Clpermeability using 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ), a dye quenched by Clbut not by NO3 - (48,92). Substitution of extracellular Clfor NO3results in electroneutral influx of NO3and efflux of Cl -, causing a decrease in intracellular [Cl -] ([Cl -]i) and increase in intracellular SPQ fluorescence.…”

Polarization of protease-activated receptor 2 (PAR-2) signaling is altered during airway epithelial remodeling and deciliation

“…A red calcium indicator was used to reduce effects of autofluorescence of the fungal media during acute addition. We and others previously showed that PAR-2 is linked to calcium in airway epithelial cells (8). It is important to note that arrestin binding and G-protein signaling are not mutually exclusive, and our goal was not to quantify the relative contributions of these two pathways (e.g., agonist biasing) in this study.…”

Bitter taste receptors stimulate phagocytosis in human macrophages through calcium, nitric oxide, and cyclic-GMP signaling

“…Unless indicated, all reagents and solutions were as described [7,8,67,68]. Full details of materials, reagents, and solution compositions are in the Supplementary Material.…”

“…Bitter agonists used and known cognate T2Rs are in Supplementary Table 1. Immunofluorescence (IF) microscopy was carried out as described [7,8,67,68], with more details in the Supplementary Material.…”