Bumetanide Hyperpolarizes Madin–Darby Canine Kidney Cells and Enhances Cellular Gentamicin Uptake by Elevating Cytosolic Ca 2+ Thus Facilitating Intermediate Conductance Ca 2+ -Activated Potassium Channels

Loop diuretics such as bumetanide and furosemide enhance aminoglycoside ototoxicity when co-administered to patients and animal models. The underlying mechanism(s) is poorly understood. We investigated the effect of these diuretics on cellular uptake of aminoglycosides, using Texas Red-tagged gentamicin (GTTR), and intracellular/whole-cell recordings of Madin–Darby canine kidney (MDCK) cells. We found that bumetanide and furosemide dose-dependently enhanced cytoplasmic GTTR fluorescence by ~60 %. This enhancement was suppressed by La 3+ , a non-selective cation channel (NSCC) blocker, and by K + channel blockers Ba 2+ and clotrimazole, but not by tetraethylammonium (TEA), 4-aminopyridine (4-AP) or glipizide, nor by Cl − channel blockers diphenylamine-2-carboxylic acid (DPC), niflumic acid (NFA), and CFTR inh -172. Bumetanide and furosemide hyperpolarized MDCK cells by ~14 mV, increased whole-cell I/V slope conductance; the bumetanide-induced net current I / V showed a reversal potential ( V r ) ~−80 mV. Bumetanide-induced hyperpolarization and I / V change was suppressed by Ba 2+ or clotrimazole, and absent in elevated [Ca 2+ ] i , but was not affected by apamin, 4-AP, TEA, glipizide, DPC, NFA, or CFTR inh -172. Bumetanide and furosemide stimulated a surge of Fluo-4-indicated cytosolic Ca 2+ . Ba 2+ and clotrimazole alone depolarized cells by ~18 mV and reduced I / V slope with a net current V r near −85 mV, and reduced GTTR uptake by ~20 %. La 3+ alone hyperpolarized the cells by ~−14 mV, reduced the I / V slope with a net current V r near −10 mV, and inhibited GTTR uptake by ~50 %. In the presence of La 3+ , bumetanide-caused negligible change in potential or I / V . We conclude that NSCCs constitute a major cell entry pathway for cationic aminoglycosides; bumetanide enhances aminoglycoside uptake by hyperpolarizing cells that increases the cation influx driving force; and bumetanide-induced hyperpolarization is caused by elevating intracellular Ca 2+ and thus facilitating activation of the intermediate conductance Ca 2+ -activated K + channels.