Analysis of perilymphatic glutamate by in vivo microdialysis study

本文应用微透析技术对豚鼠耳蜗外淋巴谷氨酸进行了研究。(1)研究了高K~(2+)诱发豚鼠耳蜗去极化过程中外淋巴谷氨酸释放及其钙离子依赖性的时程变化。灌流10min可见谷氨酸升高的两个高峰。在无Ca~(2+)和Gt~(2+)存在的情况下，第一峰减弱，第二峰抑制最小。(2)本实验观察了闭塞小脑前下动脉(AICA)时豚鼠耳蜗外淋巴谷氨酸和耳蜗血流量的动态变化。本研究结果表明，耳蜗谷氨酸调节系统依赖于耳蜗血流量。(3)外淋巴液中谷氨酸含量变化的时程观察表明，卡那霉素和乙氰酸对内耳毛细胞有损伤作用。豚鼠皮下注射卡那霉素负荷量800 mg/kg，3小时后静脉注射40 mg/kg乙氰酸，在注射乙氰酸约2小时后明显释放谷氨酸。本研究结果为谷氨酸在氨基糖苷类药物中毒中的加重作用提供了进一步的证据。采用Northern印迹法分析了卡那霉素(KM)对大鼠耳蜗谷氨酸转运体(GLAST)基因表达的影响。KM(600 mg/kg/d)每日1次，连续给药20d后，GLAST mRNA的表达逐渐增加，在第20天达到高峰。KM治疗结束后12d，GLAST mRNA的表达仍维持在较高水平，但在2个月内降至正常水平。目前的研究结果表明，在KM给药过程中，细胞外高浓度的谷氨酸由坍塌的毛细胞释放，诱导了GLAST mRNA的表达。

We applied microdialysis technique to investigate the perilymphatic glutamate.(1) The time course of changes in perilymphatic glutamate release and their Ca^<2+>-dependency were studied in the guinea pig cochlea during high K^+-evoked depolarization. Two peaks of glutamate increase were found in response to perfusion for 10 min. In the absence of Ca^<2+> the first peak was diminished, whereas the inhibition of the second peak was minimal.(2) The dynamic changes in perilymphatic glutamate and cochlear blood flow were studied in the guinea pig cochlea during occulusion of anterior inferior cerbellum artery (AICA). The result of this study indicate that the glutamate regulating system in the cochlea is dependnt on cochlear blood flow.(3) The time course of changes in perilymphatic glutamate were observed during the application of kanamycin and ethacrynic acid, which are known to damage the hair cells in the inner ear. In guinea pigs receiving a loading dose of 800 mg/kg of kanamycin subcutaneously, followed three hours later by an intravenous injection of 40 mg/kg of ethacrynic acid, a marked glutamate release was clearly found about 2 hours after the injection of ethacrynic acid. The present findings provideadditional evidence that glutamate acts as an aggravating factor in aminoglycoside-inducedototoxicity.Kanamycin (KM) -induced changes in the gene expression for glutamate-aspartatetransporter (GLAST) in the rat cochlea were analyzed by Northern blotting. With theadministration of KM (600 mg/kg/day) once daily for 20 days, the expression of GLAST mRNAgradually increased and reached a peak on day 20. Although the expression of GLAST mRNA remained at a high level at 12 days after the completion of the KM treatment, it then fellto the normal level within 2 months. The present findings suggest that during the KMadministration, high concentrations of extracellular glutamate released by collapsing haircells induced GLAST mRNA expression.