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 ~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 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 Ba²⁺ or clotrimazole, and absent in elevated Ca²⁺]_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 Ca²⁺. Ba²⁺ 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 %. La³⁺ 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 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.