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Published ahead of print on July 1, 2004, doi:10.1165/rcmb.2003-0211OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 446-455, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0211OC

Nucleotide-Mediated Mucin Secretion from Differentiated Human Bronchial Epithelial Cells

Philip A. Kemp, Rosemary A. Sugar and Alan D. Jackson

Novartis Institutes for Biomedical Research, Horsham, West Sussex, United Kingdom

Address correspondence to: Dr. Alan D. Jackson, Novartis Respiratory Research Centre, Wimblehurst Road, Horsham, West Sussex RH12 5AB, UK. E-mail alan.jackson{at}pharma.novartis.com

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-{gamma}S)–induced mucin secretion. A selective Gq G–protein antagonist (GP-ANT)–2A completely abrogated ATP-{gamma}S–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-{gamma}S–induced mucin secretion. Phorbol myristate acetate also stimulated mucin secretion in a calphostin C–sensitive manner. ATP-{gamma}S–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.

Abbreviations: alcian blue, AB • adenosine diphosphate, ADP • air–liquid interface, ALI • adenosine triphosphate, ATP • adenosine 5'-o-(3-thiotriphosphate), ATP-{gamma}S • 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetra-acetic acid tetra (acetoxymethyl) ester, BAPTA-AM • bronchial–epithelial basal medium, BEBM • bronchial–epithelial growth medium, BEGM • collagen 1, Col I • cholera toxin, CTX • Dulbecco's modified Eagle's medium, DMEM • agonist concentration needed to produce 50% of the maximal response, EC50 • enzyme-linked immunosorbent lectin assay, ELISLA • enzyme-linked lectin assay, ELLA • G-protein antagonist, GP-ANT • GP–coupled receptor, GPCR • sequence scrambled GP-ANT-2A, SGP-2A • human bronchial epithelial cell, HBEC • horseradish peroxidase, HRP • concentration producing 50% inhibition, IC50 • lactate dehydrogenase, LDH • 2-methylthioadenosine 5'-triphosphate, 2MeSATP • affinity constant defined by the log of the concentration of antagonist required to shift an agonist concentration response curve 2-fold to the right, pA2 • periodic acid–Schiff, PAS • phosphate-buffered saline, PBS • phosphatidylcholine-specific phospholipase C, PC-PLC • phosphatidylinositol-specific PLC, PI-PLC • protein kinase C, PKC • phorbol myristate acetate, PMA • phospholipase C, PLC • pertussis toxin, PTX • sodium dodecyl sulfate, SDS • uridine diphosphate, UDP • Ulex europaeus agglutinin type 1, UEA-1 • uridine triphosphate, UTP • wheat germ agglutinin, WGA • HRP-conjugated WGA, HRP-WGA




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