Regulation of the inner medullary collecting duct (IMCD) urea transporter by protein kinases

It is generally accepted that reabsorption of urea in the IMCD is regulated by vasopressin via a cAMP dependent pathway, a process which is important during urinary concentration. The objective of this study was to investigate the direct effect of protein kinase (PK) activation on the IMCD urea transporter UT1 by Xenopus oocyte expression studies. Whereas preincubation of oocytes expressing UT1 with PKC activators (phorbol 12-myristate 13-acetate or phorbol 12,13-dibutyrate) resulted in a minor decrease (20-25%) in passive urea uptake, pre-incubation with dibutyryl cyclic-AMP stimulated urea uptake 2 to 5-fold. This stimulation was dose and time dependent and was inhibited by the specific PKA inhibitor, Rp-cAMP. Immunocytochemistry revealed no alteration in the level of UT1 expression in the oocyte plasma membrane after incubation with cAMP agonists, suggesting that activation occurs by direct phosphorylation. UT1 consists of two homologous halves connected by a 72 amino acid residue segment. Expression of these halves, either individually or together (by coexpression), stimulated passive urea transport that was PKA independent, indicating that the phosphorylation site resides in the connecting segment between these halves. Site-directed mutagenesis of 3 potential PKA phosphorylation sites in this segment revealed that serine 499 is crucial for cAMP responsiveness of UT1. Taken together, our data indicate that cAMP stimulates urea permeability in the IMCD by phosphorylation of the urea transporter UT1.