Characterization of Na+/K+/Cl- cotransport in cultured HT29 human colonic adenocarcinoma cells.
A Na+/K+/Cl- cotransport pathway has been examined in the HT29 human colonic adenocarcinoma cell line using 86Rb as the K congener. Ouabain-resistant bumetanide-sensitive (OR-BS) K+ influx in attached HT29 cells was 17.9 +/- 0.9 nmol/min per mg protein at 25 degrees C. The identity of this pathway as a Na+/K+/Cl- cotransporter has been deduced from the following findings: (a) OR-BS K+ influx ceased if the external Cl- (Cl-o) was replaced by NO3- or the external Na+ (Na+o) by choline; (b) neither OR-BS 24Na+ nor 36Cl- influx was detectable in the absence of external K+ (K+o); and (c) concomitant measurements of 86Rb+, 22Na+, and 36Cl- influx indicated that the stoichiometry of the cotransport system approached a ratio of 1N+:1K+:2Cl-. In addition, OR-BS K+ influx was exquisitely sensitive to cellular ATP levels. Depletion of the normal ATP content of 35-40 nmol/mg protein to 10-15 nmol/mg protein, a concentration at which the ouabain-sensitive K+ influx was unaffected, completely abolished K+ cotransport. OR-BS K+ influx was slightly reduced by the divalent cations Ca2+, Ba2+, Mg2+ and Mn2+. Although changes in cell volume, whether shrinking or swelling, did not influence OR-BS K+ influx, ouabain-sensitive K+ influx was activated by cell swelling. As in T84 cells, we found that the OR-BS K+ influx in HT29 cells was stimulated by exogenous cyclic AMP analogues and by augmented cyclic AMP content in response to vasoactive intestinal peptide, forskolin, norepinephrine and forskolin or prostaglandin E1.
Kim
H D
HD
Department of Pharmacology, School of Medicine, University of Missouri-Columbia 65212.
Tsai
Y S
YS
Franklin
C C
CC
Turner
J T
JT
D000230:Adenocarcinoma / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002413:Cations, Divalent / Q000494:pharmacology; D003110:Colonic Neoplasms / Q000378:metabolism*; D000242:Cyclic AMP / Q000502:physiology*; D005665:Furosemide / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D009994:Osmolar Concentration; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000502:physiology*; D028021:Sodium-Potassium-Chloride Symporters; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D002413:Cations, Divalent; D028021:Sodium-Potassium-Chloride Symporters; D010042:Ouabain; D005665:Furosemide; D000242:Cyclic AMP; D011188:Potassium