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Am J Physiol Cell Physiol 286: C247-C255, 2004. First published October 1, 2003; doi:10.1152/ajpcell.00593.2002
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PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON

Niflumic acid inhibits ATP-stimulated exocytosis in a mucin-secreting epithelial cell line

C. A. Bertrand,1 H. Danahay,2 C. T. Poll,2 C. Laboisse,3 U. Hopfer,4 and R. J. Bridges1

1Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261; 2Novartis Respiratory Research Center, Horsham RH125AB, United Kingdom; 3Institut National de la Santé et de la Recherche Médicale 94-04, Université de Nantes, F-44035 Nantes, France, and 4Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106

Submitted 19 December 2002 ; accepted in final form 15 September 2003

ATP is an efficacious secretagogue for mucin and chloride in the epithelial cell line HT29-Cl.16E. Mucin release has been measured as [3H]glucosamine-labeled product in extracellular medium and as single-cell membrane capacitance increases indicative of exocytosis-related increases in membrane area. The calcium-activated chloride channel blocker niflumic acid, also reported to modulate secretion, was used to probe for divergence in the purinergic signaling of mucin exocytosis and channel activation. With the use of whole cell patch clamping, ATP stimulated a transient capacitance increase of 15 ± 4%. Inclusion of niflumic acid significantly reduced the ATP-stimulated capacitance change to 3 ± 1%, although normalized peak currents were not significantly different. Ratiometric imaging was used to assess intracellular calcium () dynamics during stimulation. In the presence of niflumic acid, the ATP-stimulated peak change in was unaffected, but the initial response and overall time to peak were significantly affected. Excluding external calcium before ATP stimulation or including the capacitative calcium entry blocker LaCl3 during stimulation muted the initial calcium transient similar to that observed with niflumic acid and significantly reduced peak capacitance change, suggesting that a substantial portion of the ATP-stimulated mucin exocytosis in HT29-Cl.16E depends on a rapid, brief calcium influx through the plasma membrane. Niflumic acid interferes with this influx independent of a chloride channel blockade effect.

capacitative calcium entry; membrane capacitance; chloride channels; fura 2


Address for reprint requests and other correspondence: C. A. Bertrand, Univ. of Pittsburgh School of Medicine, S307 BST 3500 Terrace St., Pittsburgh, PA 15261 (E-mail: cbertra{at}pitt.edu).




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