title: 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.
abstract: 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 CFTRinh-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 CFTRinh-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.
authors.affiliation: Oregon Hearing Research Center, NRC04, Department of Otolaryngology, Oregon Health & Science University, Portland, OR 97239, USA.
authors.forename: Tian
authors.initials: T
authors.lastname: Wang
authors.forename: Yu-Qin
authors.initials: YQ
authors.lastname: Yang
authors.forename: Takatoshi
authors.initials: T
authors.lastname: Karasawa
authors.forename: Qi
authors.initials: Q
authors.lastname: Wang
authors.forename: Amanda
authors.initials: A
authors.lastname: Phillips
authors.forename: Bing-Cai
authors.initials: BC
authors.lastname: Guan
authors.forename: Ke-Tao
authors.initials: KT
authors.lastname: Ma
authors.forename: Meiyan
authors.initials: M
authors.lastname: Jiang
authors.forename: Ding-Hua
authors.initials: DH
authors.lastname: Xie
authors.forename: Peter S
authors.initials: PS
authors.lastname: Steyger
authors.forename: Zhi-Gen
authors.initials: ZG
authors.lastname: Jiang
mesh_terms: D000818:Animals; D002034:Bumetanide / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D016764:Cell Polarity / Q000187:drug effects*; D004232:Diuretics / Q000494:pharmacology*; D004285:Dogs; D005786:Gene Expression Regulation; D005839:Gentamicins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D061985:Madin Darby Canine Kidney Cells; D008564:Membrane Potentials / Q000187:drug effects; D018408:Patch-Clamp Techniques; D024681:Potassium Channels, Calcium-Activated / Q000378:metabolism*; D028021:Sodium-Potassium-Chloride Symporters / Q000235:genetics / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism; D050916:TRPV Cation Channels / Q000235:genetics / Q000378:metabolism; D014966:Xanthenes / Q000737:chemistry
chemical_list: D002121:Calcium Channel Blockers; D004232:Diuretics; D005839:Gentamicins; D024681:Potassium Channels, Calcium-Activated; D028021:Sodium-Potassium-Chloride Symporters; D050916:TRPV Cation Channels; D014966:Xanthenes; D002034:Bumetanide; C034657:Texas red; D000254:Sodium-Potassium-Exchanging ATPase; D002118:Calcium