Modulation of activator protein 1/DNA binding activity by acoustic overstimulation in the guinea-pig cochlea.

Changes in gene expression are part of the homeostatic machinery with which cells respond to external stimuli or assaults. The activity of the early response transcriptional factor activator protein-1 (AP-1) can be modulated by a variety of environmental stimuli including those that alter the cellular oxidation/reduction status. This study investigates the activation of AP-1/DNA binding in the guinea-pig cochlea in response to acoustic overstimulation which produces reactive oxygen species. Electrophoretic mobility shift assays revealed that binding of AP-1 to its radiolabeled oligonucleotide probe markedly changed in nuclear extracts of inner ear tissues following intense noise exposure (4 kHz octave band, 115 dB, 5 h). AP-1/DNA binding increased in the organ of Corti and the lateral wall tissues immediately after the exposure, returning to near-baseline levels 5 h later. At 15 h after noise, a second peak of binding activity occurred in the organ of Corti whereas stria vascularis showed a lesser but more sustained activity. Binding in nuclear extracts from the spiral ganglion did not change. Incubation of nuclear extracts with antibodies against Fos/Jun family proteins prior to a supershift assay showed Fra-2 as a major component of the AP-1 complex immediately after the noise exposure. In the organ of Corti, Fra-2 immunoreactivity was localized to the middle turn, i.e. the region which is most affected by the 4-kHz octave band exposure. The results suggest the modulation of gene expression via the activation of AP-1 as a consequence of noise trauma but also demonstrate differential responses in cochlear tissues.

ATP and nitric oxide modulate intracellular calcium in isolated pillar cells of the guinea pig cochlea.

Supporting cells in the mammalian cochlea have recently received attention as potential targets of neurotransmitters, neuromodulators, and neurohumoral agents. Calcium homeostasis in Deiters' and Hensen's cells, for example, is regulated by ATP and nitric oxide. We studied the intracellular calcium concentration [Ca2+]i in isolated pillar cells of the guinea pig cochlea in response to extracellular ATP and nitric oxide using the fluorescent indicator fluo-3. [Ca2+]i increased rapidly and significantly throughout the pillar cell in response to a bolus of ATP or 2-methylthio ATP while alpha,beta-methylene ATP was ineffective. The response to ATP was inhibited by suramin and Cibacron Blue but not by pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid. This pharmacological profile is consistent with a [Ca2+]i increase largely mediated by P2Y receptors. In Ca2+-free medium supplemented with EGTA, the response to extracellularATP was reduced by 33%, suggesting a contribution of calcium influx to the overall effect. The ATP-induced increase of [Ca2+] was attenuated by NO donors (sodium nitroprusside or diethylamine NONOate), and this attenuation was reversed by KT5823, an antagonist to protein kinase G. The results indicate the involvement of purinergic mechanisms and the nitric oxide/cyclic GMP/protein kinase G pathway in the regulation of [Ca2+]i in cochlear pillar cells.

Salicylate attenuates gentamicin-induced ototoxicity.

Aminoglycosides, primarily gentamicin, are the most commonly used antibiotics worldwide despite their toxicity to the kidney and the inner ear. A preventive therapy against these side effects should combine safety and efficacy with low cost because aminoglycoside-induced deafness is most prevalent in developing countries. We have previously shown that aminoglycosides catalyze the formation of free radicals in an iron-dependent reaction and have delineated the structure of an iron-gentamicin complex. Here we demonstrate that 2-hydroxybenzoate (salicylate), which can act as an iron chelator and antioxidant, effectively protects against gentamicin-induced hearing loss in guinea pigs. Co-therapy with salicylate reduced a profound gentamicin-induced auditory threshold shift of more than 60 dB to less than 20 dB. Morphological assessment of the inner ear confirmed protection of auditory sensory cells. Salicylate altered neither serum levels of gentamicin nor its antibacterial efficacy. Because the required salicylate levels correspond to anti-inflammatory levels in humans, this treatment holds promise for clinical application.

Biochemistry and pharmacology of aminoglycoside-induced hearing loss.

Discovered in the 1940s, the aminoglycoside antibiotics were the long-sought remedy for tuberculosis and other serious bacterial infections. They are still one of the most commonly used antibiotics today, thanks to the combination of their high efficacy with low cost. This review begins by addressing some of the history and the acute side effects of aminoglycosides. It then details their chronic toxicity to the inner ear (ototoxicity). The review concludes with recent advances that have demonstrated free-radical reactions as the underlying mechanism and intervention with antioxidant/chelation therapy to prevent aminoglycoside ototoxicity.

Biochemistry and pharmacology of aminoglycoside-induced hearing loss.

Discovered in the 1940s, the aminoglycoside antibiotics were the long-sought remedy for tuberculosis and other serious bacterial infections. They are still one of the most commonly used antibiotics today, thanks to the combination of their high efficacy with low cost. This review begins by addressing some of the history and the acute side effects of aminoglycosides. It then details their chronic toxicity to the inner ear (ototoxicity). The review concludes with recent advances that have demonstrated free-radical reactions as the underlying mechanism and intervention with antioxidant/chelation therapy to prevent aminoglycoside ototoxicity.

Biochemistry and pharmacology of aminoglycoside-induced hearing loss

Discovered in the 1940s, the aminoglycoside antibiotics were the long-sought remedy for tuberculosis and other serious bacterial infections. They are still one of the most commonly used antibiotics today, thanks to the combination of their high efficacy with low cost. This review begins by addressing some of the history and the acute side effects of aminoglycosides. It then details their chronic toxicity to the inner ear (ototoxicity). The review concludes with recent advances that have demonstrated free-radical reactions as the underlying mechanism and intervention with antioxidant/chelation therapy to prevent aminoglycoside ototoxicity.

Are aminoglycoside antibiotics excitotoxic?

Guinea pigs received gentamicin to induce a profound hearing loss (61 dB auditory threshold shift at 18 kHz). Concomitant administration of maleic or tartaric acid dissolved in dimethyl sulfoxide (DMSO) significantly reduced the threshold shift to < 40 dB. The results have several important implications. First, they support the hypothesis of a free-radical mechanism of gentamicin toxicity since the protective compounds are metal chelators and scavengers. Second, they caution against these and similar chemicals, commonly found in drug preparations, as vehicles in tests of aminoglycoside toxicity. For example, a recent study by others describing attenuation of aminoglycoside ototoxicity by NMDA antagonists may have been influenced by the presence of maleate, tartrate and DMSO. Third, they suggest simple antioxidants as a potentially efficient and inexpensive clinical prophylaxis of aminoglycoside-induced hearing loss.

Mechanically induced calcium increases in isolated vestibular hair cells of the guinea pig.

Intracellular Ca2+([Ca2+]i) is elevated by depolarization or mechanical stimulation in some hair cell systems. It is not clear whether both these stimuli promote Ca2+ entry in mammalian vestibular hair cells. We monitored [Ca2+]i with the indicator fluo-3 in isolated type I vestibular hair cells of the guinea pig maintained in Hanks' balanced salt solution (HBSS). Mechanical stimulation by bolus application of HBSS led to an immediate rise of [Ca2+]i. The effect depended upon the presence of extracellular Ca2+([Ca2+]o) and no increase occurred in calcium-free HBSS supplemented with calcium-chelators. When the cells were depolarized by bolus application of KCl (final concentration, 100 mM KCl in modified HBSS), the increase in [Ca2+]i was similar to that elicited by HBSS. In the absence of [Ca2+]o, the application of KCI/HBSS led to a slow sustained increase in the fluorescence of the cells suggesting release of calcium from intracellular stores. Finally, treatment of cells with BAPTA prior to mechanical stimulation prevented the rise in [Ca2+]i indicating the need for intact stereociliary tip-links. The results are consistent with the hypothesis that mechanical stimulation elevates [Ca2+]i in isolated vestibular hair cells via calcium influx through mechanotransduction channels.

Glutathione protection against gentamicin ototoxicity depends on nutritional status.

This study demonstrates that gentamicin ototoxicity depends on dietary factors and correlates with tissue glutathione levels. After 15 days of gentamicin injections (100 mg/kg/day s.c.) guinea pigs on a regular protein diet (18.5% protein) had an average hearing loss of 9 dB at 3 kHz, 31 dB at 8 kHz and 42 dB at 18 kHz. Guinea pigs on a 7% protein diet showed an increased hearing loss of 52 dB at 3 kHz, 63 dB at 8 kHz and 74 dB at 18 kHz. Supplementing the low protein diet with either essential or sulfur-containing amino acids did not protect against gentamicin ototoxicity. Glutathione levels in the cochlear sensory epithelium were decreased in animals on a low protein diet and could be restored to normal by oral administration of glutathione monoethyl ester (1.2 g/kg/day) in combination with vitamin C (100 mg/kg/day). Glutathione supplementation significantly reduced the magnitude of hearing loss in the low protein diet group at all frequencies (43 dB reduction at 3 kHz, 27 dB reduction at 8 kHz and 21 dB reduction at 18 kHz). In animals on a full protein diet, dietary glutathione neither increased cochlear glutathione levels nor attenuated hearing loss. Serum gentamicin levels did not differ between animals on the various diets with or without glutathione supplement. These results suggest that gentamicin toxicity and detoxifying mechanisms are affected by the metabolic state of the animal and the glutathione content of the tissue. Thus, compounds that could potentially protect against gentamicin ototoxicity may be more correctly assessed in animal models of deficient nutritional states in which endogenous detoxifying mechanisms are compromised. This animal model might also be more realistically related to the clinical situation of a critically ill patient receiving gentamicin treatment.

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)

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.

Membrane perturbation by aminoglycosides as a simple screen of their toxicity.

A simple test to assess the toxicity of aminoglycosides was designed based on the interaction of these drugs with phosphatidylinositol bisphosphate. The fluorescent probe 1-anilino-8-naphthalene sulfonate was incorporated into liposomes composed of L-alpha-phosphatidylcholine and phosphatidylinositol bisphosphate (4:1 molar ratio), and changes in fluorescence induced by the aminoglycosides were measured. The magnitude of these changes correlated well with the intrinsic ototoxicity of the drugs previously established in cochlear perfusions: neomycin greater than gentamicin approximately equal to tobramycin greater than amikacin approximately equal to kanamycin approximately equal to netilmicin greater than neamine approximately equal to spectinomycin. Correlations were not accurate when other phospholipids were substituted for phosphatidylinositol bisphosphate. An alternative in vitro test, measurement of the zeta potential of liposomes, ranked the drugs according to their electrostatic charge but not their toxicity. These results suggest that: a simple screen for aminoglycoside toxicity can be established and perturbation of membranes containing phosphatidylinositol bisphosphate is a key step in aminoglycoside ototoxicity.