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 similar to 90% confluence on plastic and develop transepithelial resistances of 120-250 Omega . cm(2) on p orous substrates. Its utility as a model for the transverse human colo n was validated by comparing second messenger-mediated Cl- transport, using the fluorescent probe 6-methoxy-quinolyl acetoethyl ester, in NC M460 cells and colonocytes isolated from human transverse crypts. Basa l Cl- influx was increased (P < 0.01) by PGE(1) (1 mu M), forskolin (1 mu M), 8-bromoadenosine 3'5'-cyclic monophosphate (100 mu M), heat-st able Escherichia coli enterotoxin (STa; 1 mu M), 8-bromoguanosine 3'5' -cyclic monophosphate (100 mu M), histamine (1 mu M), and phorbol 12,1 3-dibutyrate (1 mu M) in both cell types. The Cl- channel blocker diph enylamine 2-carboxylic acid (50 mu M) and the Na+-K+-2Cl(-) cotranspor t inhibitor furosemide (1 mu M), but not the K+ channel blocker Ba2+ ( 3 mM), inhibited these Cl- permeabilities. These cells possess transcr ipts for cystic fibrosis transmembrane conductance regulator, Na+-K+-2 Cl(-) cotransporter, STa receptor, and intestine-specific cGMP-depende nt protein kinase II. Thus cAMP-, cGMP-, and Ca2+-dependent secretagog ues 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 or igin is known.