Journal
AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY
Volume 285, Issue 2, Pages C433-C445Publisher
AMER PHYSIOLOGICAL SOC
DOI: 10.1152/ajpcell.00509.2002
Keywords
Ussing chamber; short-circuit current; RT-PCR; immunoblot
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Funding
- NHLBI NIH HHS [HL-47122] Funding Source: Medline
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Pancreatic duct cells express a Ca2+-activated Cl- conductance (CaCC), upregulation of which may be beneficial to patients with cystic fibrosis. Here, we report that HPAF, a human pancreatic ductal adenocarcinoma cell line that expresses CaCC, develops into a high-resistance, anion-secreting epithelium. Mucosal ATP (50 muM) caused a fourfold increase in short-circuit current (I-sc), a hyperpolarization of transepithelial potential difference (from -4.9 +/- 0.73 to -8.5 +/- 0.84 mV), and a fall in resistance to less than one-half of resting values. The effects of ATP were inhibited by mucosal niflumic acid (100 muM), implicating an apical CaCC in the response. RT-PCR indicated expression of hClC-2, hClC-3, and hClC-5, but surprisingly not hCLCA-1 or hCLCA-2. K+ channel activity was necessary to maintain the ATP-stimulated I-sc. Using a pharmacological approach, we found evidence for two types of K+ channels in the mucosal and serosal membranes of HPAF cells, one activated by chlorzoxazone (500 muM) and sensitive to clotrimazole (30 muM), as well as one blocked by clofilium (100 muM) but not chromanol 293B (5 muM). RT-PCR indicated expression of the Ca2+-activated K+ channel KCNN4, as well as the acid-sensitive, four transmembrane domain, two pore K+ channel, KCNK5 (hTASK-2). Western blot analysis verified the expression of CLC channels, as well as KCNK5. We conclude that HPAF will be a useful model system for studying channels pertinent to anion secretion in human pancreatic duct cells.
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