Acute and chronic effects of aminoglycosides on cochlear hair cells.

The first detectable effect on the auditory system after a single high-dose injection of an aminoglycosidic antibiotic (AA) like gentamicin (GM) is the reversible blockade of medial efferent function, probably via blockade of calcium channels at the base of the outer hair cells (OHC). The kinetics of this effect are compatible with that of the molecule in perilymph. In the course of chronic treatment with lower doses, however, ototoxicity develops only after several days of treatment. Still GM can be observed inside the OHCs as soon as 24 hours after the first injection, and will be still present in some OHCs as long as 11 months after a chronic, nonototoxic 6-day treatment. In vitro, the short-term viability of isolated OHCs is not affected by exposure to AAs, but their transduction channels and their response to acetylcholine are reversibly blocked. However, developing organs of Corti in culture are highly and rapidly affected by exposure to AAs. Yet during direct intracochlear perilymphatic perfusion of GM, 2-mM solutions are not ototoxic, and with perfusion with a 20-mM solution ototoxicity develops only after several days of perfusion. From these various observations one can describe some aspects of the mechanisms of ototoxicity of AAs, from their access to perilymph and endolymph, to penetration in the hair cells, likely via endocytosis at their apical pole, and intracellular cytotoxic events.

Increasing intracellular free calcium induces circumferential contractions in isolated cochlear outer hair cells.

The relationship between intracellular free calcium and the motile responses of outer hair cells isolated from the guinea pig cochlea was examined. Calcium levels were modulated by the addition of the calcium ionophores ionomycin or A23187 to the incubation medium and monitored with the fluorescent calcium indicator fluo-3. In the presence of 1.25 mM external calcium, the application of either ionophore (10 microM) led to an increase in intracellular free calcium from 157 +/- 76 nM to 1200 +/- 500 nM within 30-60 sec. Concurrently, cells elongated by 1-2 microns, cell diameter decreased, and cell volume shrank by 269 +/- 220 microns 3 (5.0 +/- 4.1%). The reduction in diameter was most pronounced in the middle portion of the cell (4.4% +/- 4.2%), also evident in the apical region (3.1% +/- 4.8%) but not significant in the basal region near the nucleus. This response was observed in outer hair cells from basal and apical turns of the cochlea and was reversed when the cells were rinsed with calcium-free medium supplemented with 2 mM EGTA. Optical imaging of the cell membrane with the potentiometric dye 1-(3-sulfonatopropyl)-4-[beta] [2-(di-n-butylaminol)-6-naphthyl vinyl] pyridinium betaine during the elevation of intracellular calcium demonstrated features of contractility at the lateral cell membrane. A rise in intracellular calcium as well as the motile response was still observed after a 5-min exposure of the cells to a calcium-free solution (supplemented with 2 mM EGTA), indicating that the ionophore was also able to liberate calcium from intracellular sites. However, depletion of calcium stores through prolonged incubation of the cells in calcium-free medium (30-60 min) suppressed both the calcium signal and the cell response. The calmodulin inhibitors trifluoperazine and pimozide (30 microM) blocked the cell motility induced by ionomycin while they left the increase of intracellular calcium unaffected. These observations suggest that calcium-dependent circumferential contractions in outer hair cells are mediated by calmodulin. The application to the extracellular medium of putative neurotransmitters of the cochlear efferent system such as acetylcholine and GABA led to neither an increase in intracellular calcium nor a modification of cell shape. Therefore, these neurotransmitters may not be directly involved in calcium-induced contractions in outer hair cells. The circumferential contractions altered the stiffness of the plasma membrane and the turgor of the cell. Under normal conditions, changes in cell volume were inversely proportional to the osmotic pressure of the extracellular medium following van't Hoff's law.(ABSTRACT TRUNCATED AT 400 WORDS)

Dynamic changes following combined treatment with gentamicin and ethacrynic acid with and without acoustic stimulation. Cellular uptake and functional correlates.

Regional selectivity of gentamicin (GM) ototoxicity was studied in guinea pigs (GPs) using electrophysiological, morphological, autoradiographic and immunohistological observations following combined treatment with GM (150 mg/kg i.m.) and ethacrynic acid (EA) (30 mg/kg i.c. or i.v., 1.5 h after GM injection). The GPs were either continuously stimulated every 5 min with a series of 256 clicks (70 dB peSPL, 10/s) during 3 h for monitoring fast changes in VIII nerve compound action potential (CAP) after the EA injection, and thereafter kept in the animal quarters (background noise of 60 dB SPL) (group I), or similarly monitored for only 10 min after the EA injection and thereafter kept in a soundproof room (around 0 dB SPL) (group II). Whenever GM labelling was observed it was localized only in the sensory hair cells. From 3 h after EA injection, the GPs in group I presented threshold elevations in the high-frequency region, which progressed to 60-80 dB at all frequencies at and after 48 h. Parallel to the threshold pattern, GM uptake in outer hair cells (OHCs) was seen with an increasing concentration from apex toward base from 3 to 24 h, while after 48 h almost all OHCs were destroyed and inner hair cells (IHCs) were marked by GM. In group II no changes in CAP thresholds were observed until more than 24 h, although GM was detected in the hair cells from 6 h on. At this early stage, the distribution of GM lacked a clear pattern, particularly without a clear apex-base gradient, and GM deposits were found only around the basal body. However in both groups, in late stage (greater than 24 h), the base-apex gradient was more pronounced and GM was found throughout the cell body, with a marked concentration below the cuticular plate. These results suggest that GM may penetrate hair cells around the basal body and that activating the cells by sound potentiates both GM uptake and its intracellular toxicity.

Potassium-depolarization induces motility in isolated outer hair cells by an osmotic mechanism.

Outer hair cells in vitro contract in response to various stimuli: electrical stimulation, K+-depolarization, elevation of intracellular calcium or osmotic changes of the extracellular medium. The characteristics of motile responses induced by K+-depolarization, osmotic changes, and calcium injection were compared in this study in order to delineate the underlying mechanisms. Slow shape changes in outer hair cells were induced by changes of the osmolality or the K+/Na+-ratio of the bathing medium, or by intracellular injections of calcium. K+- and osmotically induced contractions of isolated outer hair cells had identical morphological features and the same rate (50-200 nm/s) and amplitude (up to greater than 10% of original length) of shortening. The shortening of the cells was linearly related to an increase in volume in both cases. In contrast, the active contraction induced by Ca2+/ATP exhibited a somewhat faster rate and no increase in volume. Furthermore, the K+-induced contractions in outer hair cells, unlike those reported in smooth muscle cells, were unaffected by the removal of external Ca2+ (i.e. medium without Ca2+/Mg2+ and supplemented with 1 mM EGTA) or the presence of D600, an inhibitor of the Ca2+ inward current. The results strongly suggest that K+ induces shape changes of outer hair cells via osmotic forces and that intracellular calcium mediates contractions by a different mechanism.