Cyclic AMP Signaling Affects Urine Concentration through UT-A1

描述（由申请人提供）： 通过严格控制尿素转运蛋白功能来维持尿素浓度。最近的进展提供了对尿素转运蛋白长期调节的理解，包括：1）克隆两种尿素转运蛋白基因、其启动子和几种cDNA形式; 2）产生多克隆抗体;和3）尿素转运蛋白功能和丰度的生理学研究。我们已经表明，UT-A1尿素转运蛋白在大鼠内髓集合管（IMCD）悬浮液和Madin-Darby犬肾（MDCK）细胞系（UT-A1-MDCK），我们创建的，稳定表达UT-A1的磷酸化调节。我们还发现加压素以cAMP依赖性方式刺激UT-A1向大鼠IMCD膜的运输。我们的假设是，加压素介导的细胞内cAMP的增加刺激了一个特定的信号复合物的蛋白质，调节UT-A1的功能。加压素通过腺苷酸环化酶增加细胞内cAMP。随着时间的推移，这种刺激的性质是未知的。我们将鉴定UT-A1-MDCK细胞中的加压素敏感性腺苷酸环化酶，并监测cAMP随时间的合成。加压素敏感性磷酸二酯酶（PDE）位于IMCD中。我们将鉴定细胞中的PDE，并确定加压素是否增加PDE活性。加压素刺激UT-A1的磷酸化。用PKA抑制剂H-89处理减少磷酸化，表明PKA在UT-A1磷酸化中起作用。我们已经确定了UT-A1中的两个PKA位点。尚不清楚这些位点的磷酸化是否是加压素介导的。我们将研究加压素是否可以刺激UT-A1的PKA位点缺失结构的磷酸化。水通道蛋白2，IMCD中的另一种转运蛋白，在加压素处理后插入膜。插入已被证明是由一个cAMP信号复合物组成的AKAP，PKA，和PDE的调节。UT-A1在加压素刺激后也在膜中积累。我们将确定一个cAMP信号复合物，确定在这个复合物中的关键蛋白质，并监测由此产生的加压素介导的cAMP在UT-A1附近的质膜区室化。 公共卫生相关性：拟议的研究将产生新的信息，潜在的水稳态失调，发生在常见的临床疾病，如充血性心力衰竭，肝硬化和肾病综合征的机制。我们希望阐明尿素运动的机制，从而为治疗体内水平衡失调提供潜在的见解。

DESCRIPTION (provided by applicant): Urea concentration is maintained by tightly controlling urea transporter function. Recent advances have provided insight into the understanding of long-term regulation of urea transporters including: 1) cloning of two urea transporter genes, their promoters, and several cDNA is forms; 2) creation of polyclonal antibodies; and 3) physiologic studies of urea transporter function and abundance. We have shown that the UT-A1 urea transporter is regulated by phosphorylation in rat inner medullary collecting duct (IMCD) suspensions and in a Madin-Darby canine kidney (MDCK) cell line (UT-A1-MDCK), which we created, that stably expresses UT-A1. We have also seen that vasopressin stimulates UT-A1 trafficking to the membrane of rat IMCD in a cAMP-dependent manner. Our hypothesis is that the vasopressin-mediated increase in intracellular cAMP stimulates a specific signaling complex of proteins that regulate UT-A1 function. Vasopressin increases intracellular cAMP via adenylyl cyclases. The nature of this stimulation over time is unknown. We will identify vasopressin-sensitive adenylyl cyclases in UT-A1-MDCK cells and monitor the synthesis of cAMP over time. Vasopressin-sensitive phosphodiesterases (PDEs) are located in the IMCD. We will identify the PDE(s) in the cells and determine if PDE activity is increased by vasopressin. Vasopressin stimulates phosphorylation of UT-A1. Treatment with the PKA inhibitor H-89 reduced phosphorylation indicating that PKA plays a role in UT-A1 phosphorylation. We have identified two PKA sites in UT-A1. It is unknown if phosphorylation at these sites is vasopressin-mediated. We will investigate whether vasopressin can stimulate phosphorylation with PKA-site deletion constructs of UT-A1.Aquaporin-2, another transporter in the IMCD, is inserted into the membrane after vasopressin treatment. Insertion has been shown to be regulated by a cAMP-signaling complex composed of an AKAP, PKA, and a PDE. UT-A1 also accumulates in the membrane after vasopressin stimulation. We will identify a cAMP signaling complex, identify key proteins in this complex, and monitor the resulting vasopressin-mediated compartmentalization of cAMP at the plasma membrane near UT-A1. PUBLIC HEALTH RELEVANCE: The proposed studies will yield new information on the mechanisms underlying the disregulation of water homeostasis that occurs in common clinical disorders, such as congestive heart failure, cirrhosis, and nephrotic syndrome. We hope to clarify the mechanisms of urea movement to potentially provide insight into the treatment of disregulation of the body's water homeostasis.