Nucleotide-mediated mucin secretion from differentiated human bronchial epithelial cells.

Abstract

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 microM). 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-gammaS)-induced mucin secretion. A selective Gq G-protein antagonist (GP-ANT)-2A completely abrogated ATP-gammaS-induced mucin secretion. Pertussis toxin and the G(i/o)-specific, GP-ANT-2, had no effect. The phospholipase C inhibitor, D609, and the protein kinase C inhibitor, calphostin C, substantially inhibited ATP-gammaS-induced mucin secretion. Phorbol myristate acetate also stimulated mucin secretion in a calphostin C-sensitive manner. ATP-gammaS-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.