Protein kinase C-α mediates hypertonicity-stimulated increase in urea transporter phosphorylation in the inner medullary collecting duct.
The UT-A1 urea transporter plays a critical role in the production of concentrated urine. Both vasopressin and hypertonicity increase urea permeability in rat terminal inner medullary collecting ducts (IMCD). Each agonist independently increases UT-A1 phosphorylation and apical plasma membrane accumulation. Vasopressin activates PKA and phosphorylates UT-A1 at serines 486 and 499. Hypertonicity stimulates urea permeability through protein kinase C (PKC) and intracellular calcium. To determine whether the hypertonic stimulation of urea permeability results from a PKC-mediated phosphorylation of UT-A1, rat IMCDs were metabolically labeled with [(32)P]. Hypertonicity stimulated UT-A1 phosphorylation, and this increase was blocked by preincubation with a PKC inhibitor. IMCDs were biotinylated to assess plasma membrane UT-A1. Hypertonicity increased biotinylated UT-A1, and this increase was blocked by preincubation with a PKC inhibitor. When PKC was directly activated using a phorbol ester, total UT-A1 phosphorylation increased, but phosphorylation at serine 486 was not increased, indicating that PKC did not phosphorylate UT-A1 at the same residue as PKA. Since PKC-α is a calcium-dependent PKC isoform and PKC-α knockout mice have a urine-concentrating defect, it suggested that PKC-α may mediate the response to hypertonicity. Consistent with this hypothesis, hypertonicity increased phospho-PKC-α in rat IMCDs. Finally, PKC-α knockout mice were used to determine whether hypertonicity could stimulate UT-A1 phosphorylation in the absence of PKC-α. Hypertonicity significantly increased UT-A1 phosphorylation in wild-type mice but not in PKC-α knockout mice. We conclude that PKC-α mediates the hypertonicity-stimulated increase in UT-A1 phosphorylation in the IMCD.
Emory University School of Medicine, Department of Medicine, Renal Division, 1639 Pierce Dr., Atlanta, GA 30322, USA. janet.klein@emory.edu
Janet D
JD
Klein
Christopher F
CF
Martin
Kimilia J
KJ
Kent
Jeff M
JM
Sands
D000818:Animals; D053119:Benzophenanthridines / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005950:Glucose Solution, Hypertonic / Q000494:pharmacology*; D007685:Kidney Tubules, Collecting / Q000187:drug effects / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D023421:Models, Animal; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014667:Vasopressins / Q000494:pharmacology; D000096982:Urea Transporters
D053119:Benzophenanthridines; D017868:Cyclic AMP-Dependent Protein Kinases; D004791:Enzyme Inhibitors; D005950:Glucose Solution, Hypertonic; D026901:Membrane Transport Proteins; D011493:Protein Kinase C; D014667:Vasopressins; D000096982:Urea Transporters; C016299:chelerythrine