Aminoglycoside uptake and clearance in mammalian cells

说明(申请人提供)：像庆大霉素一样的氨基糖苷类抗生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，导致耳聋和前庭缺陷。氨基糖苷类化合物的内吞作用不够迅速，不足以解释它们迅速出现在细胞质中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类药物进入的快速、非内吞摄取机制。氨基糖苷类还可以通过转导通道和顶端内吞作用迅速进入毛细胞。毛细胞顶端浸泡在内淋巴中。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是如何从血管系统进入内淋巴的？TRPV通道TRPV4表达于血管纹内皮细胞和边缘细胞的管腔膜上。TRPV4可使氨基糖苷类药物从血液进入内皮细胞，并使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摄取和边缘细胞清除氨基糖苷类药物的模型，并验证TRPV4通道是双向氨基糖苷类允许的。我们将确定STATE外植体中的边缘细胞是否清除氨基糖苷类化合物，以及这一机制是否依赖于TRPV4。最后，我们将进行体内庆大霉素注射实验，并确定工业内皮细胞是否摄取庆大霉素，允许药物通过血管纹，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.