TY - JOUR
T1 - Bumetanide Hyperpolarizes Madin-Darby Canine Kidney Cells and Enhances Cellular Gentamicin Uptake by Elevating Cytosolic Ca2+ Thus Facilitating Intermediate Conductance Ca2+-Activated Potassium Channels
AU - Wang, Tian
AU - Yang, Yu qin
AU - Karasawa, Takatoshi
AU - Wang, Qi
AU - Phillips, Amanda
AU - Guan, Bing Cai
AU - Ma, Ke Tao
AU - Jiang, Meiyan
AU - Xie, Ding Hua
AU - Steyger, Peter S.
AU - Jiang, Zhi Gen
PY - 2013/4
Y1 - 2013/4
N2 - 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.
AB - 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.
KW - Aminoglycoside
KW - Chloride channels
KW - Cytosolic calcium
KW - Ion current
KW - Loop diuretics
KW - Membrane potential
KW - Ototoxicity
UR - http://www.scopus.com/inward/record.url?scp=84875218947&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84875218947&partnerID=8YFLogxK
U2 - 10.1007/s12013-012-9442-2
DO - 10.1007/s12013-012-9442-2
M3 - Article
C2 - 23109177
AN - SCOPUS:84875218947
SN - 1085-9195
VL - 65
SP - 381
EP - 398
JO - Cell Biochemistry and Biophysics
JF - Cell Biochemistry and Biophysics
IS - 3
ER -