Aminoglycoside uptake and clearance in mammalian cells

描述（由申请方提供）：氨基糖苷类（如庆大霉素）是用于治疗严重感染的抗生素。然而，这些药物也会杀死内耳感觉毛细胞，导致耳聋和前庭功能障碍。氨基糖苷类内吞进入细胞的速度不足以解释其在细胞质中的快速出现和观察到的细胞毒性。最近，本实验室描述了一种快速，非内吞摄取机制的氨基糖苷类药物进入，调节TRPV通道调节剂。氨基糖苷类还可以通过转导通道和顶端内吞作用快速进入毛细胞。毛细胞顶端浸于内淋巴液中。如果内淋巴途径是氨基糖苷类药物进入毛细胞的关键，那么氨基糖苷类药物如何从血管系统进入内淋巴？TRPV通道TRPV4在血管纹中的边缘细胞和内皮细胞的管腔膜处表达。TRPV4可使氨基糖苷类药物从血液进入内皮细胞，并从边缘细胞进入内淋巴。我们将以内耳内皮细胞和肾小管细胞为模型，研究内耳内皮细胞对氨基糖苷类药物的摄取和边缘细胞对氨基糖苷类药物的清除，并验证TRPV 4通道是双向的氨基糖苷类药物允许通道。我们将确定是否边缘细胞在移植物清除氨基糖苷类，如果这种机制是TRPV4依赖。最后，我们将进行体内庆大霉素注射实验，并确定血管内皮细胞是否摄取庆大霉素，允许药物通过血管纹，以及这个过程是否也依赖于TRPV 4通道。

DESCRIPTION (provided by applicant): Aminoglycosides like gentamicin are antibiotics used for treating serious infections. However, these drugs also kill inner ear sensory hair cells, causing deafness and vestibular deficits. Endocytosis of aminoglycosides into cells is insufficiently rapid to account for their swift appearance in the cytoplasm and observed cytotoxicity. Recently, this laboratory described a rapid, non-endocytotic uptake mechanism for aminoglycoside entry, modulated by TRPV channel regulators. Aminoglycosides can also rapidly enter hair cells through transduction channels and apical endocytosis. The hair cell apex is bathed in endolymph. If the endolymph pathway is critical for aminoglycosides to enter hair cells, how do aminoglycosides enter the endolymph from the vasculature? A TRPV channel, TRPV4, is expressed at the luminal membrane of marginal and endothelial cells in the stria vascularis. TRPV4 could allow aminoglycosides to enter endothelial cells from the blood, and exit marginal cells into endolymph. We will use aortal endothelial cells and kidney tubule cells as models of endothelial uptake and marginal cell clearance of aminoglycosides in the inner ear, and verify that TRPV4 channel is bi-directionally aminoglycoside-permissive. We will determine if marginal cells in strial explants clear aminoglycosides and if this mechanism is TRPV4-dependent. Finally, we will conduct in vivo gentamicin injection experiments and determine if strial endothelial cells uptake gentamicin, allowing the drug to pass through stria vascularis, and if this process is also dependent on TRPV4 channel.