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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Regulation of UT-A1-mediated transepithelial urea flux in MDCK cells

Otto Fröhlich,¹ Janet D. Klein,² Pauline M. Smith,^1, Jeff M. Sands,^1,2 and Robert B. Gunn^1,

¹Department of Physiology and ²Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia

Submitted 15 August 2005 ; accepted in final form 13 April 2006

Transepithelial [¹⁴C]urea fluxes were measured across cultured Madin-Darby canine kidney (MDCK) cells permanently transfected to express the urea transport protein UT-A1. The urea fluxes were typically increased from a basal rate of 2 to 10 and 25 nmol·cm^–2·min^–1 in the presence of vasopressin and forskolin, respectively. Flux activation consisted of a rapid-onset component of small amplitude that leveled off within 10 min and at times even decreased again, followed by a delayed, strong increase over the next 30–40 min. Forskolin activated urea transport through activation of adenylyl cyclase; dideoxyforskolin was inactive. Vasopressin activated urea transport only from the basolateral side and was blocked by OPC-31260, indicating that its action was mediated by basolateral V₂ receptors. In the presence of the phosphodiesterase inhibitor IBMX, vasopressin activated as strongly as forskolin. By itself, IBMX caused a slow increase over 50 min to 5 nmol·cm^–2·min^–1. 8-Bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP; 300 µM) activated urea flux only when added basolaterally. IBMX augmented the activation by basolateral 8-BrcAMP. Urea flux activation by vasopressin and forskolin were only partially blocked by the protein kinase A inhibitor H-89. Even at concentrations >10 µM, urea flux after 60 min of stimulation was reduced by <50%. The rapid-onset component appeared unaffected by the presence of H-89. These data suggest that activation of transepithelial urea transport across MDCK-UT-A1 cells by forskolin and vasopressin involves cAMP as a second messenger and that it is mediated by one or more signaling pathways separate from and in addition to protein kinase A.

urea transporter; Madin-Darby canine kidney cells

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