Cyclic AMP Signaling Affects Urine Concentration through UT-A1

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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.