2002 Global Researcher Conference Proceeding
April 26 - 28, 2002
| Conference: | 2002 Global Researcher Conference |
|---|---|
| Title: | Protein Kinase C involvement in aquaporin-2 endocytosis in cell culture |
| Authors: | Nejsum, Lene Niemann; Promeneur, Dominique; Nielsen, Soren |
| Institutions: | The Water and Salt Research Center, University of Aarhus |
Aquaporin-2 (AQP2) is the predominant vasopressin regulated water channel of the kidney collecting duct and is involved in both acute and long term regulation of body water balance. In the acute regulation, vasopressin binds to the basolateral V2-recepter leading to an increase in cAMP levels and Protein Kinase A (PKA) phosphorylation of AQP2 (Ser256), leading to a translocation of AQP2 to the apical plasma membrane instantly increasing osmotic water permeability. On the removal of vasopressin, AQP2 is endocytosed instantly decreasing osmotic water permeability.
We aimed to examine the acute regulation of AQP2 endocytosis in AQP2-transfected Madin-Darby canine kidney (MDCK-C7) cells. MDCK-C7 cells were transiently transfected with wildtype AQP2 (WT-AQP2) and AQP2 mutated in the PKA sensitive site to mimic phosphorylated (AQP2-S256D) and non phosphorylated (AQP2-S256A) AQP2. Stimulation of WT-AQP2 with the cAMP elevating agent forskolin induced translocation of WT-AQP2 to the plasmamembrane. Following forskolin stimulation, the cells were stimulated with the Protein Kinase C (PKC) activator phorbol 12-myristate 13-acetate (TPA), which induced retrieval of WT-AQP2 from the plasma membrane to intracellular vesicles despite the remaining high levels of forskolin. Also AQP2-S256D was endocytoced when stimulated with TPA indicating the endocytosis to be independent of PKA. As a control, AQP2-S256A remained intracellular at stimulation with forskolin.
The use of TPA indicates that the stimulation of AQP2 endocytosis was mediated via PKC, independently of PKA. The data suggest that PKC is involved in a the regulation of AQP2 endocytosis between the membrane and intracellular vesicles in MDCK-C7 cells.



