TY - JOUR
T1 - TRPV1 regulators mediate gentamicin penetration of cultured kidney cells
AU - Myrdal, Sigrid E.
AU - Steyger, Peter S.
N1 - Funding Information:
We are grateful to Katherine Johnson for assistance with the cell cultures, and Dr. David Ellison for the gift of MDCK cells, and consultation throughout this project. These studies were funded by NIDCD Grants 04555 and 06084, with equipment grants from the American Foundation for Alternatives to Animal Research, and the Northwest Health Foundation. Portions of this work were presented in abstract form at the 27th Annual Midwinter Meeting of the Association for Research in Otolaryngology, Daytona Beach, February 2004.
PY - 2005/6
Y1 - 2005/6
N2 - Transient receptor potential (TRP) receptors are, typically, calcium-permeant cation channels that transduce environmental stimuli. Both kidney epithelial and inner ear sensory cells express TRPV1, are mechanosensors and accumulate the aminoglycoside antibiotic gentamicin. Recently, we showed that Texas Red-conjugated gentamicin (GTTR) enters kidney cells via an endosome-independent pathway. Here, we used GTTR to investigate this non-endocytotic mechanism of gentamicin uptake. In serum-free buffers, GTTR penetrated MDCK cells within 30 s and uptake was modulated by extracellular, multivalent cations (Ca2+, La3+, Gd3+) or protons. We verified the La3+ modulation of GTTR uptake using immunocytochemical detection of unconjugated gentamicin. Membrane depolarization, induced by high extracellular K+ or valinomycin, also reduced GTTR uptake, suggesting electrophoretic permeation through ion channels. GTTR uptake was enhanced by the TRPV1 agonists, resiniferatoxin and anandamide, in Ca2+-free media. Competitive antagonists of the TRPV1 cation current, iodo-resiniferatoxin and SB366791, also enhanced GTTR uptake independently of Ca2+, reinforcing these antagonists' potential as latent agonists in specific situations. Ruthenium Red blocked GTTR uptake in the presence or absence of these TRPV1-agonists and antagonists. In addition, GTTR uptake was blocked by RTX in the presence of more physiological levels (2 mM) of Ca2+. Thus gentamicin enters cells via cation channels, and gentamicin uptake can be modulated by regulators of the TRPV1 channel.
AB - Transient receptor potential (TRP) receptors are, typically, calcium-permeant cation channels that transduce environmental stimuli. Both kidney epithelial and inner ear sensory cells express TRPV1, are mechanosensors and accumulate the aminoglycoside antibiotic gentamicin. Recently, we showed that Texas Red-conjugated gentamicin (GTTR) enters kidney cells via an endosome-independent pathway. Here, we used GTTR to investigate this non-endocytotic mechanism of gentamicin uptake. In serum-free buffers, GTTR penetrated MDCK cells within 30 s and uptake was modulated by extracellular, multivalent cations (Ca2+, La3+, Gd3+) or protons. We verified the La3+ modulation of GTTR uptake using immunocytochemical detection of unconjugated gentamicin. Membrane depolarization, induced by high extracellular K+ or valinomycin, also reduced GTTR uptake, suggesting electrophoretic permeation through ion channels. GTTR uptake was enhanced by the TRPV1 agonists, resiniferatoxin and anandamide, in Ca2+-free media. Competitive antagonists of the TRPV1 cation current, iodo-resiniferatoxin and SB366791, also enhanced GTTR uptake independently of Ca2+, reinforcing these antagonists' potential as latent agonists in specific situations. Ruthenium Red blocked GTTR uptake in the presence or absence of these TRPV1-agonists and antagonists. In addition, GTTR uptake was blocked by RTX in the presence of more physiological levels (2 mM) of Ca2+. Thus gentamicin enters cells via cation channels, and gentamicin uptake can be modulated by regulators of the TRPV1 channel.
KW - Aminoglycosides
KW - Cytoplasmic drug uptake
KW - Non-endocytotic uptake
KW - TRP channel
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U2 - 10.1016/j.heares.2005.02.005
DO - 10.1016/j.heares.2005.02.005
M3 - Article
C2 - 15925202
AN - SCOPUS:19544392023
SN - 0378-5955
VL - 204
SP - 170
EP - 182
JO - Hearing Research
JF - Hearing Research
IS - 1-2
ER -