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 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.
Kemp
Philip A
PA
Novartis Respiratory Research Centre, Wimblehurst Road, Horsham, West Sussex RH12 5AB, UK.
Sugar
Rosemary A
RA
Jackson
Alan D
AD
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D001667:Binding, Competitive; D001980:Bronchi / Q000166:cytology / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002273:Carcinogens / Q000494:pharmacology; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002614:Chelating Agents / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D006801:Humans; D037102:Lectins / Q000378:metabolism; D009077:Mucins / Q000187:drug effects / Q000378:metabolism*; D009281:Naphthalenes / Q000378:metabolism; D037342:Pertussis Toxin / Q000494:pharmacology; D018048:Receptors, Purinergic P2 / Q000378:metabolism; D058473:Receptors, Purinergic P2Y2; D015398:Signal Transduction / Q000187:drug effects; D013498:Suramin / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014544:Uridine Triphosphate / Q000494:pharmacology
D002273:Carcinogens; D002614:Chelating Agents; D037102:Lectins; D009077:Mucins; D009281:Naphthalenes; C546385:P2RY2 protein, human; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; D013498:Suramin; D000255:Adenosine Triphosphate; D037342:Pertussis Toxin; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; C058819:calphostin C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D014544:Uridine Triphosphate