title: A highly potent agonist to protease-activated receptor-2 reveals apical activation of the airway epithelium resulting in Ca2+-regulated ion conductance.
abstract: The airway epithelium provides a barrier that separates inhaled air and its various particulates from the underlying tissues. It provides key physiological functions in both sensing the environment and initiating appropriate innate immune defenses to protect the lung. Protease-activated receptor-2 (PAR2) is expressed both apically and basolaterally throughout the airway epithelium. One consequence of basolateral PAR2 activation is the rapid, Ca(2+)-dependent ion flux that favors secretion in the normally absorptive airway epithelium. However, roles for apically expressed PAR2 activation have not been demonstrated, in part due to the lack of specific, high-potency PAR2 ligands. In the present study, we used the newly developed PAR2 ligand 2at-LIGRLO(PEG3-Pam)-NH2 in combination with well-differentiated, primary cultured airway epithelial cells from wild-type and PAR2 (-/-) mice to examine the physiological role of PAR2 in the conducting airway after apical activation. Using digital imaging microscopy of intracellular Ca(2+) concentration changes, we verified ligand potency on PAR2 in primary cultured airway cells. Examination of airway epithelial tissue in an Ussing chamber showed that apical activation of PAR2 by 2at-LIGRLO(PEG3-Pam)-NH2 resulted in a transient decrease in transepithelial resistance that was due to increased apical ion efflux. We determined pharmacologically that this increase in ion conductance was through Ca(2+)-activated Cl(-) and large-conductance K(+) channels that were blocked with a Ca(2+)-activated Cl(-) channel inhibitor and clotrimazole, respectively. Stimulation of Cl(-) efflux via PAR2 activation at the airway epithelial surface can increase airway surface liquid that would aid in clearing the airway of noxious inhaled agents.
authors.affiliation: Arizona Respiratory Center, University of Arizona, Tucson, Arizona; The BIO5 Collaborative Research Institute, University of Arizona, Tucson, Arizona.
authors.forename: Cara L
authors.initials: CL
authors.lastname: Sherwood
authors.affiliation: Arizona Respiratory Center, University of Arizona, Tucson, Arizona; Department of Pediatrics, Arizona Health Sciences Center, University of Arizona, Tucson, Arizona; The BIO5 Collaborative Research Institute, University of Arizona, Tucson, Arizona.
authors.forename: Michael O
authors.initials: MO
authors.lastname: Daines
authors.affiliation: Department of Pharmacology, Arizona Health Sciences Center, University of Arizona, Tucson, Arizona; and The BIO5 Collaborative Research Institute, University of Arizona, Tucson, Arizona.
authors.forename: Theodore J
authors.initials: TJ
authors.lastname: Price
authors.affiliation: The BIO5 Collaborative Research Institute, University of Arizona, Tucson, Arizona.
authors.forename: Josef
authors.initials: J
authors.lastname: Vagner
authors.affiliation: Arizona Respiratory Center, University of Arizona, Tucson, Arizona; Department of Physiology, Arizona Health Sciences Center, University of Arizona, Tucson, Arizona; The BIO5 Collaborative Research Institute, University of Arizona, Tucson, Arizona sboitano@email.arizona.edu.
authors.forename: Scott
authors.initials: S
authors.lastname: Boitano
mesh_terms: D000818:Animals; D018927:Anti-Asthmatic Agents / Q000494:pharmacology*; D020013:Calcium Signaling; D002478:Cells, Cultured; D018118:Chloride Channels / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D015640:Ion Channel Gating; D008564:Membrane Potentials / Q000187:drug effects; D008810:Mice, Inbred C57BL; D009952:Ornithine / Q000031:analogs & derivatives / Q000494:pharmacology; D010168:Palmitates / Q000494:pharmacology*; D024681:Potassium Channels, Calcium-Activated / Q000378:metabolism*; D044464:Receptor, PAR-2 / Q000819:agonists* / Q000378:metabolism; D020545:Respiratory Mucosa / Q000166:cytology; D014132:Trachea / Q000166:cytology
chemical_list: C000595466:2at-LIGRLO(PEG3-Pam)-NH2; D018927:Anti-Asthmatic Agents; D018118:Chloride Channels; D010168:Palmitates; D024681:Potassium Channels, Calcium-Activated; D044464:Receptor, PAR-2; D009952:Ornithine
keywords: allergic asthma; asthmagen; ion conductance; lipidated agonist; protease-activated receptor-2