Abstract
Aminoglycosides (AG) are commonly prescribed antibiotics with potent bactericidal activities. One main side effect is permanent sensorineural hearing loss, induced by selective inner ear sensory hair cell death. Much work has focused on AG's initiating cell death processes, however, fewer studies exist defining mechanisms of AG uptake by hair cells. The current study investigated two proposed mechanisms of AG transport in mammalian hair cells: mechanotransducer (MET) channels and endocytosis. To study these two mechanisms, rat cochlear explants were cultured as whole organs in gentamicin-containing media. Two-photon imaging of Texas Red conjugated gentamicin (GTTR) uptake into live hair cells was rapid and selective. Hypocalcemia, which increases the open probability of MET channels, increased AG entry into hair cells. Three blockers of MET channels (curare, quinine, and amiloride) significantly reduced GTTR uptake, whereas the endocytosis inhibitor concanavalin A did not. Dynosore quenched the fluorescence of GTTR and could not be tested. Pharmacologic blockade of MET channels with curare or quinine, but not concanavalin A or dynosore, prevented hair cell loss when challenged with gentamicin for up to 96 hours. Taken together, data indicate that the patency of MET channels mediated AG entry into hair cells and its toxicity. Results suggest that limiting permeation of AGs through MET channel or preventing their entry into endolymph are potential therapeutic targets for preventing hair cell death and hearing loss.
Highlights
Inner ear hair cells are the mechanosensory cells essential for hearing function
The current study demonstrates that aminoglycoside entry via MET channels is primarily responsible for uptake leading to hair cell death
Hair cell toxicity caused by aminoglycosides The first step in assessing AG toxicity and entry mechanisms into sensory hair cells was to develop an in vitro preparation where toxicity could be reproducibly assessed, keeping in mind the limited time course that was available for imaging entry
Summary
As mammalian hair cells do not regenerate, damage or loss of hair cells leads to permanent hearing impairment. Up to 25% of patients treated with aminoglycosides develop irreversible sensorineural hearing loss [3,4]. Entry of aminoglycosides into hair cells is necessary to induce cell death [7]. Hair cell death is thought to be mediated by reactive oxygen species [8,9] and caspase activation [1,2,10,11], caspase-independent cell death can occur [12]. Extensive work has characterized the intracellular events occurring after aminoglycosides enter hair cells [3,8,13], yet studies examining the mechanism of aminoglycoside entry into hair cells are limited
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