Aminoglycoside antibiotics impair calcium entry but not viability and motility in isolated cochlear outer hair cells

Abstract

Cochlear outer hair cells have been well established as primary targets of the ototoxic actions of aminoglycoside antibiotics. These cells, isolated from the guinea pig cochlea and maintained in short-term culture, were used as a model for evaluating the acute effects of gentamicin on cell viability, depolarization-induced transmembrane calcium flux, and depolarization-induced motile responses. On the basis of morphology and fluorochromasia, the presence of extracellular gentamicin as high as 5 mM did not affect the viability of the cells for up to 6 hr, the longest time tested. Viable cells showed binding of fluorescently tagged gentamicin to their base but excluded the drug from their cytoplasm. In response to [K + ]-depolarization, intracellular calcium levels (monitored with the fluorescent calcium-sensitive dye fluo-3) increased from a resting value of 218 ± 102 nM to 2,018 ± 1,077 nM concomitant with a cell shortening of 0.7% ± 1.3%. The depolarization-induced calcium increase was apparently caused by calcium entry into the cell as it was inhibited by the calcium-channel blocker methoxyverapamil and prevented in the absence of extracellular calcium. Both gentamicin and neomycin blocked the [K + ]-induced calcium increase at an IC 50 of 50 ΜM. Despite the inhibition of calcium entry the ability of the outer hair cells to shorten under [K + ]-depolarization was not impaired; in fact, cell shortening was even more pronounced in the absence of calcium influx (2.6% ± 1.4%). This argues effectively against the existence of a calcium-dependent actomyosin-mediated component in [K + ]-induced shape changes. The results suggest the existence of voltage-gated calcium channels in outer hair cells and that calcium influx through these channels is impaired by the aminoglycoside antibiotics neomycin and gentamicin. This action may be part of the acute ototoxic mechanism of these molecules. Furthermore, the results not only confirm the calcium independence of the depolarization-induced motility but also suggest that calcium influx into outer hair cells opposes cell shortening.