Forskolin stimulates phosphorylation and membrane accumulation of UT-A3.
UT-A1 is regulated by vasopressin and is localized to the apical membrane and intracellular compartment of inner medullary collecting duct (IMCD) cells. UT-A3 is also expressed in the IMCD and is regulated by forskolin in heterologous systems. The goal of the present study is to investigate mechanisms by which vasopressin regulates UT-A3 in rat IMCD. In fresh suspensions of rat IMCD, forskolin increases the phosphorylation of UT-A3, similar to UT-A1. Biotinylation studies indicate that UT-A3 is located in the plasma membrane. Forskolin treatment increases the abundance of UT-A3 in the plasma membrane similar to UT-A1. However, these two transporters do not form a complex through a protein-protein interaction, suggesting that transporter function is unique to each protein. While immunohistochemistry localized UT-A3 to the basal and lateral membranes, a majority of the staining was cytosolic. Immunohistochemistry of vasopressin-treated rat kidney sections also localized UT-A3 primarily to the cytosol with basal and lateral membrane staining but also showed some apical membrane staining in some IMCD cells. This suggests that under normal conditions, UT-A3 functions as the basolateral transporter but in a high cAMP environment, the transporter may move from the cytosol to all plasma membranes to increase urea flux in the IMCD. In summary, this study confirms that UT-A3 is located in the inner medullary tip where it is expressed in the basolateral membrane, shows that UT-A3 is a phosphoprotein in rat IMCD that can be trafficked to the plasma membrane independent of UT-A1, and suggests that vasopressin may induce UT-A3 expression in the apical plasma membrane of IMCD.
Renal Division, Emory University School of Medicine, Atlanta, GA 30322, USA. mabloun@emory.edu
Mitsi A
MA
Blount
Janet D
JD
Klein
Christopher F
CF
Martin
Dmitry
D
Tchapyjnikov
Jeff M
JM
Sands
D000818:Animals; D050034:Antidiuretic Agents / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology*; D007679:Kidney Medulla / Q000166:cytology / Q000378:metabolism*; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D014667:Vasopressins / Q000494:pharmacology; D000096982:Urea Transporters
D050034:Antidiuretic Agents; D005576:Colforsin; D026901:Membrane Transport Proteins; D014667:Vasopressins; D000096982:Urea Transporters