Characterization of two distinct chloride channels in cultured dog pancreatic duct epithelial cells.
Cl- secretion by pancreatic duct epithelial cells (PDEC) regulates cellular HCO3- secretion, an important component of the exocrine pancreas. In cystic fibrosis, for example, impaired function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel results in decreased pancreatic secretion and secondary pancreatic insufficiency. Studies of ion transport by PDEC have been hindered by the lack of a practical in vitro model. We have successfully cultured nontransformed dog PDEC on Vitrogen-coated permeable membranes overlying a feeder layer of myofibroblasts and report the characterization of Cl- channels in these cells. Cl- conductance, assessed through efflux of 125I from PDEC, was stimulated by agents acting via adenosine 3',5'-cyclic monophosphate (cAMP) or cytosolic Ca2+. The Cl- conductances activated by cAMP and Ca2+ were distinct, since they were differentially inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and, to a lesser extent, by 5-nitro-2-(3-phenylpropylamino)benzoic acid and diphenylamine-2 carboxylate. Patch-clamp studies confirmed the presence of Cl- channels activated by cAMP and Ca2+, with differential inhibition by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. The presence of CFTR Cl- channels in PDEC was confirmed by immunoblotting. These cultured PDEC are an optimal model for studies of pancreatic duct secretion.
Nguyen
T D
TD
Department of Medicine, University of Washington, Seattle, Washington 98108, USA. tlnguyen@u.washington.edu
Koh
D S
DS
Moody
M W
MW
Fox
N R
NR
Savard
C E
CE
Kuver
R
R
Hille
B
B
Lee
S P
SP
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000502:physiology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002478:Cells, Cultured; D018118:Chloride Channels / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000037:antagonists & inhibitors / Q000502:physiology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004285:Dogs; D004847:Epithelial Cells; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D007455:Iodine / Q000378:metabolism; D009579:Nitrobenzoates / Q000494:pharmacology; D010183:Pancreatic Ducts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D062367:ortho-Aminobenzoates / Q000494:pharmacology
D002121:Calcium Channel Blockers; D018118:Chloride Channels; D009579:Nitrobenzoates; D062367:ortho-Aminobenzoates; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D000001:Calcimycin; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; C001209:fenamic acid; D007455:Iodine; D000242:Cyclic AMP; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002118:Calcium