Regulation of Rat Renal Inner Medullary Function

在过去十年中，我们和其他国家在理解长期 尿素转运蛋白的调节。下一个供资期的总体目标是调查 加压素增加UT-A1的顶端质膜积累，以确定加压素- 刺激增加UT-A1积累的信号通路，并研究UT-A1的回收、周转和 UT-A1的降解。尿素和水的渗透率经常一起变化，但也有情况下， 它们在IMCD中相互独立地调节。我们提出的研究将提供见解 一个有趣的生物学问题：加压素如何通过一个单一的受体，V2- 受体，在单个肾单位节段中独立于水渗透性调节尿素渗透性， 终端IMCD，事实上，可能独立地在同一细胞？假设一加压素 快速增加UT-A1在顶端质膜中的积累。具体目标1。确定是否 加压素通过V2-加压素增加UT-A1在顶端质膜中的积聚 受体的基本原理：我们的初步数据显示，急性加压素给药增加了 UT-A1在细胞膜上的表达。具体目标2。确定环AMP途径调节UT-A1 积累在质膜上。基本原理：我们的初步数据显示，激活Epac， 一种新的cAMP依赖性但PKA非依赖性途径，增加了IMCD血浆中UT-A1的蓄积 膜，但不改变AQP 2。这些数据表明，加压素的能力，独立 调节尿素和水的结果，至少部分来自不同cAMP依赖性信号的激活 IMCD细胞中的通路。假设2.加压素调节UT-A1的恢复和/或降解。 具体目标3。确定UT-A1在顶端质膜和细胞膜中的功能半衰期。 负责UT-A1检索和/或降级的系统。理由：我们在UT-A1中的初步数据- 结果表明：1）UT-A1是一种泛素化蛋白，存在于大鼠延髓和UT-A1-MDCK细胞中; 和2）用蛋白酶体抑制剂预处理产生尿素通量（功能）的显著增加，UT-A1 蛋白质丰度和生物素化UT-A1的大小。这些数据表明，泛素化可能 调节UT-A1从质膜的回收并靶向其在蛋白酶体中降解。

During the past decade, we and others have made tremendous progress in understanding the long-term regulation of urea transport proteins. The overall goals of the next funding period are to investigate whether vasopressin increases the apical plasma membrane accumulation of UT-A1, to determine the vasopressin- stimulated signaling pathway(s) that increase UT-A1 accumulation, and to study the retrieval, turnover, and degradation of UT-A1. Urea and water permeabilities often change together, but there are situations when they are regulated independently of one another in the IMCD. Our proposed studies will provide insights into an intriguing biological question: how does vasopressin, acting through a single receptor, the V2- receptor, regulate urea permeability independently of water permeability in a single nephron segment, the terminal IMCD and, indeed, probably independently within the same cells? HYPOTHESIS I. Vasopressin rapidly increases UT-A1 accumulation in the apical plasma membrane. Specific Aim 1. Determine whether vasopressin increases UT-A1 accumulation in the apical plasma membrane through the V2-vasopressin receptor. Rationale: our preliminary data show that acute vasopressin administration increases the amount of UT-A1 in the plasma membrane. Specific Aim 2. Determine which cyclic AMP pathway regulates UT-A1 accumulation in the plasma membrane. Rationale: our Preliminary Data show that activation of Epac, a novel cAMP-dependent but PKA-independent pathway, increases UT-A1 accumulation in the IMCD plasma membrane, but does not change AQP2. These data suggest that vasopressin's ability to independently regulate urea and water results, at least in part, from activation of different cAMP-dependent signaling pathways in IMCD cells. HYPOTHESIS II. Vasopressin regulates UT-A1 retrieval and/or degradation. Specific Aim 3. Determine the functional half-life of UT-A1 in the apical plasma membrane and the cellular systems responsible for UT-A1 retrieval and/or degradation. Rationale: our Preliminary Data in UT-A1- MDCK cells show that: 1) UT-A1 is a ubiquitinated protein in rat inner medulla and in UT-A1-MDCK cells; and 2) pre-treatment with a proteasome inhibitor produces a marked increase in urea flux (function), UT-A1 protein abundance, and the sizes of biotinylated UT-A1. These data suggest that ubiquitination may regulate retrieval of UT-A1 from the plasma membrane and target it for degradation in the proteasome.