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的功能来维持尿素浓度。最近的进展为了解尿素转运蛋白的长期调控提供了洞察力，包括：1)两个尿素转运蛋白基因及其启动子和几种形式的cDNAs的克隆；2)多克隆抗体的产生；以及3)尿素转运蛋白功能和丰度的生理学研究。我们已经证明，UT-A1尿素转运蛋白在大鼠内髓集合管(IMCD)悬液和我们创建的稳定表达UT-A1的Madin-Darby犬肾(MDCK)细胞系(UT-A1-MDCK)中受磷酸化调控。我们还看到，加压素以cAMP依赖的方式刺激UT-A1转运到大鼠IMCD的膜上。我们的假设是，加压素介导的细胞内cAMP增加刺激了调节UT-A1功能的蛋白质的特定信号复合体。加压素通过腺酰环化酶增加细胞内cAMP。随着时间的推移，这种刺激的性质尚不清楚。我们将鉴定UT-A1-MDCK细胞中对加压素敏感的腺苷环化酶，并随时间监测cAMP的合成。加压素敏感的磷酸二酯酶(PDE)位于IMCD中。我们将鉴定细胞中的PDE(S)，并确定血管加压素是否增加PDE的活性。加压素刺激UT-A1的磷酸化。用PKA抑制剂H-89处理降低了磷酸化，表明PKA在UT-A1磷酸化中起作用。我们已经在UT-A1确定了两个PKA位点。目前尚不清楚这些位点的磷酸化是否由加压素介导。我们将研究加压素是否能刺激UT-A1的PKA位点缺失结构的磷酸化。在加压素治疗后，IMCD中的另一转运体Aquaporin-2被插入膜中。已证明插入受由AKAP、PKA和PDE组成的cAMP信号复合体的调控。UT-A1在后叶加压素刺激后也会在膜上积聚。我们将确定cAMP信号复合体，确定该复合体中的关键蛋白，并监测加压素介导的cAMP在UT-A1附近质膜上的区域化。 公共卫生相关性：拟议的研究将提供有关常见临床疾病(如充血性心力衰竭、肝硬变和肾病综合征)中水平衡失调的潜在机制的新信息。我们希望阐明尿素运动的机制，以潜在地为治疗体内水稳态失调提供洞察力。

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.