Bumetanide Hyperpolarizes Madin-Darby Canine Kidney Cells and Enhances Cellular Gentamicin Uptake by Elevating Cytosolic Ca2+ Thus Facilitating Intermediate Conductance Ca2+-Activated Potassium Channels

Tian Wang, Yu qin Yang, Takatoshi Karasawa, Qi Wang, Amanda Phillips, Bing Cai Guan, Ke Tao Ma, Meiyan Jiang, Ding Hua Xie, Peter S. Steyger, Zhi Gen Jiang

Research output: Contribution to journalArticle

8 Scopus citations

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 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 ~14 mV, increased whole-cell I/V slope conductance; the bumetanide-induced net current I/V showed a reversal potential (Vr) ~-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 ~18 mV and reduced I/V slope with a net current Vr 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 Vr 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.

Original languageEnglish (US)
Pages (from-to)381-398
Number of pages18
JournalCell Biochemistry and Biophysics
Volume65
Issue number3
DOIs
StatePublished - Jan 1 2013

Keywords

  • Aminoglycoside
  • Chloride channels
  • Cytosolic calcium
  • Ion current
  • Loop diuretics
  • Membrane potential
  • Ototoxicity

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology

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