Taurine Antagonizes The Ototoxicity Of Gentamycin And Modulates Calcium Homeostasis Of The Isolated Outer Hair Cells And Spiral Ganglion Cells

Ototoxicity is a well-known side effect of aminoglycoside antibiotics (AmAn), such as gentamycin. It has been reported that the ototoxicity of gentamycin will be enhanced with the concurrent use of loop diuretics, such as furosemide. Evidences have shown that free radicals are involved in the ototoxicity, and anti-oxidative therapy may have a protection against it. Taurine (2-enthansulfonic acid) is known to be an anti-oxidant and a membrane stabilizer with many physiological roles. It has been shown that taurine could attenuate the renal tubule damage by reducing gentamycin accumulation in the kidney. However, the role of taurine in the inner ear remains unclear. To date, few literatures can be found on whether taurine attenuates the ototoxicity of gentamycin.It has been reported that gentamycin inhibits calcium influx in isolated cochlear outer hair cells(OHC) and inhibits the increase of intracellular calcium ([Ca²⁺]i)at high K⁺ induced depolarization, in which the blockade or inactivation of voltage-gated calcium channels (VGCC) may be involved. This effect was also thought to be one of the mechanisms of acute ototoxicity of gentamycin. However, little is known aboutthe effect of gentamycin on the calcium mobilization of isolated spiral ganglion cells(SGC) and the underlying mechanism.Taurine has been reported to play a role in calcium modulation, which is thought to be involved in its cyto-protective effect. Data have shown that taurine antagonizes calcium overload in neurons and cardiomyocytes. On the other hand, it was shown that sole application of taurine induced a transient increase in [Ca2+]i in isolated cardiomyocytes and neurons. However, little is known about the effect of taurine on the calcium modulation of isolated OHC or SGC under physiological or ototoxic conditions.The present study was designed to explore the effect of taurine on the ototoxicity of gentamycin, and on the calcium modulation of isolated OHC and SGC, and the possible underlying mechanisms. Guinea pigs were used as subjects in this study. Auditory brainstem response(ABR),cochlear NO and MDA levels, and changes in cochlear morphology were detected for studying the effect of taurine on the ototoxicity of gentamycin and the underlying antioxidative mechanism in it. Furthermore, we explored the role of taurine on the ototoxicity induced by concurrent use of gentamycin and furosemide, and the involvement of NO and iNOS in the mechanism. Finally, the calcium modulatory effect of taurine on OHC and SGC was detected by confocal observation via applying the calcium indicator fluo-3 under physiological and otototoxic condition.1. Taurine attenuates gentamycin-induced ototoxicity in guinea pigsThe study was designed to explore the protective effect of taurine on gentamycin(GM)-induced ototoxicity in guinea pigs. Pigmented guinea pigs were randomly divided into three groups: GM group (GM 80mg/kg, i.m.),GM+Tau group(GM 80mg/kg, i.m., Tau 400 mg/kg p.o.)and control group(saline 0.5ml, i.m.).All drugs were administered once a day for a consecutive 14d. Before and Id after treatment, the duration of post-rotatory nystagmus (PRN), the thresholds ofauditory brainstem response (ABR) and the input-output function of click were measured respectively. The threshold shifts were calculated afterwards.Results: (1) In intra-groups and intra-individuals, there was no significant difference in the duration of post-rotatory nystagmus before and after drug administration. (2)The mean threshold shifts of ABR 14d post-treatment were 1.0-1.5 dB in control, 10.3dB of click and 6.0-20.0dB of tone in GM group with the most prominent at 6,8kHz stimuli. For GM+Tau group, the mean threshold shifts were 2.5dB after click, 1.5-3.0dB after short tone with the values of click and 6^ 8kHz significantly lower than those in the GM group (p<0.05), but not when compared with the control group (p>0.05). (3) The input-output functions of click 14d post-treatment were similar between GM+Tau and control group with no significant difference in amplitudes of wave III after different sound intensity. For GM group, all the amplitudes at different sound intensity were lower than those of the other two groups. (4) The cochlear surface preparation and SEM showed that the damage in GM group was severe but very slight in GM+Tau group. (5) The NO and MDA levels in the GM cochleae were all greater that those in GM+Tau and saline group (p<0.05 ) .Conclusion: Taurine has a potential protective role against gentamycin-induced ototoxicity possibly by its anti-oxidative effect.2. Taurine attenuates the combined ototoxicity of gentamycin and furosemide via down-regulating iNOS expressionIn our initial study, we found that the ototoxicity of gentamicin sulfate(GM) was potentiated when followed by an intravenous administration of furosemide (FM). The mechanism for this augmentation is unclear. The present study was designed to elucidate whether the changes in nitric oxide (NO) production and the inducible nitric oxide synthase (iNOS) expression are responsible for this enhanced ototoxicity and whether taurine (Tau), an antioxidant, provides protection against this interaction via attenuating iNOS expression. Guinea pigs received a single administration of GM100 mg/kg i.v. followed by FM 90 mg/kg i.v. (GM+FM);GM100 mg/kg i.v. alone;FM 90mg/kg i.v. alone;Tau (400mg/kg per day, i.p. for 5 days) before GM 100 mg/kg i.v. and FM 90 mg/kg i.v. (Tau+GM+FM), respectively. Animals received a single injection of saline served as controls.Results: There was a rapid and profound hearing loss in the GM+FM animals 3 days post-treatment, with the nitrite levels (Griess method) significantly increased and iNOS expression up-regulated in the cochleae, compared with GM, FM alone and saline control. In contrast, animals received Tau before GM+FM did not show evident ABR elevations, with nitrite concentration and the iNOS expression much lower than GM+FM animals.Conclusion: These findings provide new evidence that the enhanced ototoxicity induced by a combined single dose of GM+FM is caused, partially at least, by the increase in NO production and iNOS expression in the cochleae. Pre-treatment with taurine provides protection against this enhanced ototoxicity possibly via down-regulating the iNOS expression.3. Taurine modulates calcium influx through L-type voltage gated calcium channels in isolated cochlear outer hair cells in guinea pigs.Taurine has been proposed to play a role in calcium modulation. To explore the effect of taurine on intracellular calcium homeostasis of isolated cochlear outer hair cells and on the gentamycin -induced inhibition of calcium influx evoked by high K+ depolarization, we employed fluo-3 imaging of intracellular calcium ([Ca2+];) via confocal laser scanning microscopy to measure real-time changes of [Ca2+]i.Results: We found that the sole application of taurine (5,10,20mM) induced a transient [Ca2+]i increase in a concentration-dependent manner, which was inhibited either by the application of an L-type calcium-channel blocker nifedipine or a calcium-free medium. Pre-incubation with lmM gentamycin induced inhibition of [Ca2+]i elevation evoked by high K+. Short-term (lOmin) exposure with a high level of taurine (20mM) prevented this inhibition.Conclusion: These results indicated that taurine at a high concentration was able to promote calcium influx through L-type calcium channels in isolated outer hair cells and antagonize gentamycin-induced inhibition of calcium elevation evoked by high K+ by its calcium homeostatic effect.4. Taurine modulates calcium influx through L-type voltage gated calcium channels in isolated cochlear spiral ganglion cells in guinea pigs.Taurine has been proposed to play a role in calcium modulation. To explore the effect of taurine on intracellular calcium homeostasis of isolated cochlear spiral ganglion cells (SGC) and on the gentamycin -induced inhibition of calcium influx evoked by high K+ depolarization, we employed fluo-3 imaging of intracellular calcium ([Ca2+]j) via confocal laser scanning microscopy to measure real-time changes of [Ca2+];.Results: We found that the sole application of taurine (20mM) induced a transient [Ca +]i increase in SGC, which was inhibited either by the application of an L-type calcium-channel blocker nifedipine or a calcium-free medium. Pre-incubation with lmM gentamycin induced inhibition of [Ca2+]i elevation evoked by high K+. Short-term (lOmin) exposure with taurine (20mM) prevented this inhibition.Conclusion: These results indicated that taurine was able to promote calcium influx through L-type calcium channels in isolated spiral ganglion cells and antagonize gentamycin-induced inhibition of calcium elevation evoked by high K+ by its calcium homeostatic effect.