Aminoglycoside uptake and clearance in mammalian cells

说明（由申请人提供）：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。氨基糖苷进入细胞的内吞作用不够快，不足以解释它们在细胞质中迅速出现和观察到的细胞毒性。最近，该实验室描述了一种由TRPV通道调节剂调节的氨基糖苷进入的快速非内吞摄取机制。氨基糖苷也可通过转导通道和根尖内吞作用迅速进入毛细胞。毛细胞顶端浸没在内淋巴中。如果内淋巴通路对氨基糖苷进入毛细胞至关重要，那么氨基糖苷是如何从脉管系统进入内淋巴的呢？TRPV通道TRPV4在血管纹边缘细胞和内皮细胞的管腔膜上表达。TRPV4能使氨基糖苷从血液进入内皮细胞，并从边缘细胞进入内淋巴。我们将使用主动脉内皮细胞和肾小管细胞作为内耳内皮细胞对氨基糖苷摄取和边缘细胞清除的模型，并验证TRPV4通道是双向氨基糖苷允许的。我们将确定试验外植体的边缘细胞是否清除氨基糖苷，以及这种机制是否依赖于trpv4。最后，我们将进行体内庆大霉素注射实验，确定试验体内皮细胞是否摄取庆大霉素，使药物通过血管纹，以及这一过程是否也依赖于TRPV4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.