AN INVESTIGATION OF [3H]BUMETANIDE UPTAKE IN A CULTURED RENAL CELL LINE (MDCK)

¹ Department of Physiology and Pharmacology, University of St Andrews, St Andrews, Fife KY16 9TS
² Department of Physiological Sciences, University of Newcastle-upon-Tyne, The Medical School, Framlington Place, Newcastle-upon-Tyne NE2 4HH

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.) µM. 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 µM) 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.) µM, maximal uptake of 1·17±0·47 pmol/10⁶ 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 ( 5 µM). 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.