Nucleotide-Mediated Mucin Secretion from Differentiated Human Bronchial Epithelial Cells

Most current cell-based models for examining the regulation of mucin secretion demonstrate low signal-to-noise ratios, making experimental manipulation and data interpretation difficult. Using adenosine triphosphate (ATP) as a mucin secretagogue, we have developed a model of agonist-induced mucin secretion in differentiated human bronchial epithelial cells. Mucin secretory signals were estimated using enzyme-linked lectin assay, and typical signals of 300–400% of baseline were observed in response to a 30-min exposure to ATP (100 μM). ATP and uridine triphosphate equipotently stimulated mucin secretion consistent with mediation via P2Y2 receptor activation. Suramin and AR-C118925XX, a competitive P2Y2 receptor antagonist, inhibited adenosine 5′-o-(3-thiotriphosphate) (ATP-γS)–induced mucin secretion. A selective Gq G–protein antagonist (GP-ANT)–2A completely abrogated ATP-γS–induced mucin secretion. Pertussis toxin and the Gi/o-specific, GP-ANT-2, had no effect. The phospholipase C inhibitor, D609, and the protein kinase C inhibitor, calphostin C, substantially inhibited ATP-γS–induced mucin secretion. Phorbol myristate acetate also stimulated mucin secretion in a calphostin C–sensitive manner. ATP-γS–induced mucin secretion was inhibited by the Ca2+ chelator, 1,2-bis(o-aminophenoxy) ethane-N,N,N′,N′-tetra-acetic acid tetra (acetoxymethyl) ester. Ionomycin and thapsigargin both stimulated mucin secretion. Our data are broadly consistent with known G-protein–coupling and downstream signaling events associated with the P2Y2 receptor. The exceptional signal-to-noise ratios obtained using this model have permitted clear evaluation of the involvement of these mechanisms in agonist-induced mucin secretion from differentiated human bronchial epithelial cells.