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.