Furosemide-sensitive salt transport in the Madin-Darby canine kidney cell line. Evidence for the cotransport of Na+, K+, and Cl-.
Confluent monolayer cultures of the Madin-Darby canine kidney (MDCK) cell line have been shown to possess a furosemide and bumetanide-sensitive (Na+,K+)-cotransport system. We have studied the effect of anion substitutions on (Na+,K+)-cotransport. In Na+-depleted cells, bumetanide-sensitive uptake of 22Na+ or 86Rb+ exhibited an absolute requirement for extracellular Cl-. Chloride could be replaced in the buffers by Br-, but not by F-, I-, acetate, nitrate, thiocyanate, sulfate, or gluconate. The effect of Cl- was saturating, and Na+-stimulated 86RB+ uptake as well as K+-stimulated 22Na+ uptake was shown to be dependent on the square of the Cl- concentration. The concentration of Cl- which gave half-maximal stimulation of cation cotransport varied between 58 and 70 mM. There was a small degree of cooperativity between the binding affinities for Cl- and K+ at constant Na+ concentrations. Bumetanide-sensitive 36Cl- uptake could be demonstrated when extracellular Na+ and K+ were present simultaneously. Uptake through this system was unaffected by changes in the membrane potential or by the imposition of pH gradients. Together these data strongly suggest that the bumetanide-sensitive transport system in Madin-Darby canine kidney cells co-transports Na+, K+, and Cl- in a ratio of 1:1:2.
D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D002034:Bumetanide / Q000494:pharmacology; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism* / Q000494:pharmacology; D004285:Dogs; D005665:Furosemide / Q000494:pharmacology*; D007668:Kidney / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D011188:Potassium / Q000378:metabolism* / Q000494:pharmacology; D012413:Rubidium / Q000378:metabolism; D012492:Salts; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D002712:Chlorides; D012492:Salts; D002034:Bumetanide; D005665:Furosemide; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium; D011188:Potassium