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1 Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
2 Novartis Respiratory Research Center, Horsham, United Kingdom
3 Institut National de la Sante et de la Recherche Medicale 94-04, Universite de Nantes, Nantes, France
4 Physiology and Biophysics, Case Western Reserve University, Cleveland, OH, USA
* To whom correspondence should be addressed. E-mail: cbertra{at}pitt.edu.
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 media, 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. Using 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 (Ca2+i) dynamics during stimulation. In the presence of niflumic acid, the ATP-stimulated peak change in Ca2+i was unaffected, but the initial response and overall time to Ca2+i 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.
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