Bumetanide Hyperpolarizes Madin-Darby Canine Kidney Cells and Enhances Cellular Gentamicin Uptake by Elevating Cytosolic Ca2+ Thus Facilitating Intermediate Conductance Ca2+-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 similar to 60 %. This enhancement was suppressed by La3+, a non-selective cation channel (NSCC) blocker, and by K+ channel blockers Ba2+ 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 CFTRinh-172. Bumetanide and furosemide hyperpolarized MDCK cells by similar to 14 mV, increased whole-cell I/V slope conductance; the bumetanide-induced net current I/V showed a reversal potential (V (r)) similar to-80 mV. Bumetanide-induced hyperpolarization and I/V change was suppressed by Ba2+ or clotrimazole, and absent in elevated [Ca2+](i), but was not affected by apamin, 4-AP, TEA, glipizide, DPC, NFA, or CFTRinh-172. Bumetanide and furosemide stimulated a surge of Fluo-4-indicated cytosolic Ca2+. Ba2+ and clotrimazole alone depolarized cells by similar to 18 mV and reduced I/V slope with a net current V (r) near -85 mV, and reduced GTTR uptake by similar to 20 %. La3+ alone hyperpolarized the cells by similar to-14 mV, reduced the I/V slope with a net current V (r) near -10 mV, and inhibited GTTR uptake by similar to 50 %. In the presence of La3+, 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 Ca2+ and thus facilitating activation of the intermediate conductance Ca2+-activated K+ channels.