Cl- transport in an immortalized human epithelial cell line (NCM460) derived from the normal transverse colon.
Cells of a newly described, immortalized, epithelial, human transverse colonic cell line, NCM460, reach approximately 90% confluence on plastic and develop transepithelial resistances of 120-250 Omega . cm2 on porous substrates. Its utility as a model for the transverse human colon was validated by comparing second messenger-mediated Cl- transport, using the fluorescent probe 6-methoxy-quinolyl acetoethyl ester, in NCM460 cells and colonocytes isolated from human transverse crypts. Basal Cl- influx was increased (P < 0.01) by PGE1 (1 microM), forskolin (1 microM), 8-bromoadenosine 3'5'-cyclic monophosphate (100 microM), heat-stable Escherichia coli enterotoxin (STa; 1 microM), 8-bromoguanosine 3'5'-cyclic monophosphate (100 microM), histamine (1 microM), and phorbol 12,13-dibutyrate (1 microM) in both cell types. The Cl- channel blocker diphenylamine 2-carboxylic acid (50 microM) and the Na+-K+-2Cl- cotransport inhibitor furosemide (1 microM), but not the K+ channel blocker Ba2+ (3 mM), inhibited these Cl- permeabilities. These cells possess transcripts for cystic fibrosis transmembrane conductance regulator, Na+-K+-2Cl- cotransporter, STa receptor, and intestine-specific cGMP-dependent protein kinase II. Thus cAMP-, cGMP-, and Ca2+-dependent secretagogues act on NCM460 and primary colonocytes to stimulate Cl- transport. This validates the utility of NCM460 as a model for transverse colonic crypts and is the first demonstration of a colonic cell line whose origin is known.
Sahi
J
J
Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
Nataraja
S G
SG
Layden
T J
TJ
Goldstein
J L
JL
Moyer
M P
MP
Rao
M C
MC
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000527:Alprostadil / Q000494:pharmacology; D001427:Bacterial Toxins / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002461:Cell Line, Transformed; D002712:Chlorides / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D003106:Colon / Q000502:physiology*; D000242:Cyclic AMP / Q000502:physiology; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology / Q000502:physiology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000502:physiology*; D004768:Enterotoxins / Q000494:pharmacology; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D007413:Intestinal Mucosa / Q000502:physiology*; D008954:Models, Biological; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic
D001427:Bacterial Toxins; C505032:CFTR protein, human; D002712:Chlorides; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; C034151:heat stable toxin (E coli); D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D015124:8-Bromo Cyclic Adenosine Monophosphate; C016276:8-bromocyclic GMP; D015240:Phorbol 12,13-Dibutyrate; D006632:Histamine; D000242:Cyclic AMP; D000527:Alprostadil; D006152:Cyclic GMP