An investigation of [3H]bumetanide uptake in a cultured renal cell line (MDCK).
The inhibition of ouabain-insensitive 86Rb+ (K+) flux in a cultured renal cell line (MDCK) by a series of loop diuretics, including bumetanide, (the 'cotransport' flux component) has been determined in order to define the concentration range over which [3H]bumetanide would be expected to bind to the membrane transporter involved. Half-maximal inhibition by bumetanide was observed at 0.26 +/- 0.12 (S.D.) microM. The time and concentration dependence of [3H]bumetanide uptake in intact MDCK cells has been determined in experimental conditions, which were as far as possible, identical to inhibition of K+ flux. Total cellular uptake of [3H]bumetanide (0-1 microM) may be separated into a linear component and a component displaying sigmoidal saturation kinetics with a concentration giving half-maximal uptake of 0.33 +/- 0.17 (S.D.) microM, maximal uptake of 1.17 +/- 0.47 pmol/10(6) cells, and a Hill coefficient of 1.59 +/- 0.28. There is also evidence for a second component of [3H]bumetanide uptake of lower affinity (less than 5 microM). Competition of [3H]bumetanide uptake by a series of loop diuretics at varying concentrations gives an order of potency identical to that observed for inhibition of the ouabain-insensitive 86Rb+ influx. The magnitude of the saturable component of [3H]bumetanide uptake is correlated with the magnitude of the diuretic-sensitive 86Rb+ influx in MDCK cells and in a variety of other cultured cells. The relationship between the diuretic-sensitive transport, the saturable component of [3H]bumetanide uptake and the cellular location of bumetanide uptake is discussed.
D000818:Animals; D001667:Binding, Competitive; D002034:Bumetanide / Q000378:metabolism*; D002460:Cell Line; D004232:Diuretics / Q000378:metabolism* / Q000494:pharmacology; D004285:Dogs; D005665:Furosemide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D009994:Osmolar Concentration; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000037:antagonists & inhibitors / Q000378:metabolism; D011868:Radioisotopes; D012413:Rubidium / Q000378:metabolism; D013449:Sulfonamides / Q000378:metabolism; D014018:Tissue Distribution; D014316:Tritium
D004232:Diuretics; D011868:Radioisotopes; D013449:Sulfonamides; D002034:Bumetanide; D014316:Tritium; D010042:Ouabain; D005665:Furosemide; C015451:piretanide; D012413:Rubidium; D011188:Potassium