T84 cells: anion selectivity demonstrates expression of Cl- conductance affected in cystic fibrosis.
The T84 cell line possesses an adenosine 3',5'-cyclic monophosphate (cAMP)-activated Cl- conductance and expresses high levels of the cystic fibrosis (CF) gene product, implicating it as a good model for CF research. To evaluate whether T84 Cl- conductance properties are consistent with those described in CF target epithelial, we used transepithelial measurements (verified by selective permeabilization of the basal membrane) to determine the apparent anion selectivity properties of the apical and basolateral membranes of stimulated and unstimulated T84 cells. Unstimulated epithelial cells were almost electrically inert, having a low transepithelial voltage (Vt; -6 mV, apical surface negative), a small equivalent short-circuit current (Isc,(eq.) 2.2 microA/cm2), a very high transepithelial resistance (Rt; 2,500 omega.cm2), and poor anion permselectivity properties at both membrane surfaces (0.8 less than PX/PCl- less than 1.1), where X is NO3-, Br-, I-, or gluconate. When stimulated with forskolin (10(-6) M), Vt increased 8-fold, Isc(eq) increased 30-fold, Rt fell to one-third of unstimulated values, and the apical surface became highly anion selective, i.e., NO3- (1.4) greater than Br- (1.2) greater than Cl- (1.0) greater than I- (0.7) greater than gluconate (0.0), where numbers in parentheses are PX/PCl-. I- was less permeable than Cl- and probably directly inhibits the anion conductance, since Rt was substantially greater after I- substitution than after substitution with the impermeable anion gluconate. Bumetanide (10(-4) M) significantly attenuated the response of Vt to anion substitutions at the basal membrane surface, indicating that the effects of substitution were predominantly on the Na(+)-K(+)-2Cl- cotransporter.(ABSTRACT TRUNCATED AT 250 WORDS)
Division of Biomedical Sciences, University of California, Riverside 92521.
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D000584:Amiloride / Q000494:pharmacology; D000838:Anions; D001386:Azides / Q000494:pharmacology; D002034:Bumetanide / Q000494:pharmacology; D002712:Chlorides / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D003110:Colonic Neoplasms; D000242:Cyclic AMP / Q000502:physiology; D003550:Cystic Fibrosis / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004553:Electric Conductivity / Q000187:drug effects; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism; D019810:Sodium Azide; D014407:Tumor Cells, Cultured
D000838:Anions; D001386:Azides; C505032:CFTR protein, human; D002712:Chlorides; D008565:Membrane Proteins; D002034:Bumetanide; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D000584:Amiloride; D019810:Sodium Azide; D000242:Cyclic AMP; D011188:Potassium