Chloride channels in the small intestinal cell line IEC-18.
Small intestinal crypt cells play a critical role in modulating Cl- secretion during digestion. The types of Cl- channels mediating Cl- secretion in the small intestine was investigated using the intestinal epithelial cell line, IEC-18, which was derived from rat small intestine crypt cells. In initial radioisotope efflux studies, exposure to forskolin, ionomycin or a decrease in extracellular osmolarity significantly increased 36Cl efflux as compared to control cells. Whole cell patch clamp techniques were subsequently used to examine in more detail the swelling-, Ca2+-, and cAMP-activated Cl- conductance. Decreasing the extracellular osmolarity from 290 to 200 mOsm activated a large outwardly rectifying Cl- current that was voltage-independent and had an anion selectivity of I- > Cl-. Increasing cytosolic Ca2+ by ionomycin activated whole cell Cl- currents, which were also outwardly rectifying but were voltage-dependent. The increase in intracellular Ca2+ levels with ionomycin was confirmed with fura-2 loaded IEC-18 cells. A third type of whole cell Cl- current was observed after increases in intracellular cAMP induced by forskolin. These cAMP-activated Cl- currents have properties consistent with cystic fibrosis transmembrane regulator (CFTR) Cl- channels, as the currents were blocked by glibenclamide or NPPB but insensitive to DIDS. In addition, the current-voltage relationship was linear and had an anion selectivity of Cl- > I-. Confocal immunofluorescence studies and Western blots with two different anti-CFTR antibodies confirmed the expression of CFTR. These results suggest that small intestinal crypt cells express multiple types of Cl- channels, which may all contribute to net Cl- secretion.
Basavappa
Srisaila
S
Digestive Diseases Unit, Department of Medicine, University of Rochester School of Medicine Rochester, New York 14642, USA. srisaila_basavappa@urmc.rochester.edu
Vulapalli
Sreesatya Raju
SR
Zhang
Hui
H
Yule
David
D
Coon
Steven
S
Sundaram
Uma
U
D000818:Animals; D020013:Calcium Signaling / Q000502:physiology; D002460:Cell Line; D018118:Chloride Channels / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000037:antagonists & inhibitors / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism*; D045604:Extracellular Fluid / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D016257:Fura-2; D005905:Glyburide / Q000494:pharmacology; D007421:Intestine, Small / Q000378:metabolism*; D015759:Ionomycin / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009579:Nitrobenzoates / Q000494:pharmacology; D009994:Osmolar Concentration; D018408:Patch-Clamp Techniques; D011868:Radioisotopes; D051381:Rats
D018118:Chloride Channels; D002712:Chlorides; D009579:Nitrobenzoates; D011868:Radioisotopes; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; D015759:Ionomycin; D000242:Cyclic AMP; D005905:Glyburide; D016257:Fura-2