内耳毛细胞钙通道性质和类型的重新评估

Ion channels are essential for mechano-electrical transduction in the inner ear ,wherein Ca2+ and calcium channels play a critical role.In order to get insight into the crucial role of Ca2+ and calcium channels in the auditory physiology and pathophysiology, this program aims at detecting calcium channels and calcium stores in the various types of hair cells within different location of the inner ear, using specific fluorescent indicators undrer the observation of laser scanning confocal microscopy. Evidences for the presence of L-type calcium channels in outer hair cells the cochlea and vestibule of guinea pigs were provided by dual fluorescent labeling with DMBODIPY-DHP, a specific indicator for the L-type calcium channel, and RH414, a dye for staining the cellular membrane. The densities of the L-type calcium channels were 1.16, 1.69 and 2.03 （P<0.05，n=11）for the forth, third and second turns of the cochlea，respectively, indicating that the densities had a tendency to increase gradually from low frequency areas to high frequency areas. These characteristics might reflect the mechanism for frequency encoding in the auditory system.This is the first report about the relationship between calcium channels in hair cells and the place theory of frequency encoding in mammalia. Sequential addition of 30 nmol/L thapsigargin and 10mmol/L caffeine induced a rise in [Ca2+]i from 1.0 at rest to 1.64, and then to 2.45. The defference of changes in [Ca2+]i evoked by these two agents was statistically significant tested with pairwise comparisons. The results revealed that Ca2+ was released from two distinct calcium stores in the hair cell, the inositol trisphosphate- and ryanodine-sensitive Ca2+ stores.The latter stores in the hair cell were reported for the first time. Guinea pigs were exposed to 4kHz noise at 110 dB SPL for 4h. Auditory brainstem response was used to monitor the auditory function and revealed a threthold shift of 32.77dB SPL after the noise exposure.The average ratios of fluo-3 and fura-red measured at 2h after the noise exposure were 1.74 ±0.23 and 1.16±0.12（mean±S, n＝11, p<0.001）in outer hair cells of the experimental group and the control group, respectively, indicating that the hearing loss after the noise exposure was associated with a rise in [Ca2+]i. The result suggests that the calcium overload in outer hair cells might be one of the pathogenetic factors for acoustic trauma. In addition to the physiological and pathophysiological relevance mentioned above, this program may open a new idea and provide a rationale for the study of channelopathy in otology and the application of calcium antagolists to deseases of the inner ear.

用膜片钳记录和激光扫描共聚焦显微镜两项技术，测定豚鼠、鸡和牛蛙毛细胞下通道的生物物理和药理学特性，观察特异荧光探针标记的L和N型钙通道的荧光图像。综合三项指标鉴定内耳毛细胞钙通道的类型和属性，重新评估毛细胞钙通道属性的传统观点，进一步阐明位听觉神经机制，为探讨Ca2+和钙拮抗剂在耳科病理学和药理学上的间谍奠定新的理论基础。