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

• Published in: American Journal of Physiology: Cell Physiology 2006-10, Vol.291 (4), p.C600-C606
• Main Authors: Frohlich, Otto, Klein, Janet D, Smith, Pauline M, Sands, Jeff M, Gunn, Robert B
• Format: Article
• Language: English
• Subjects: 1-Methyl-3-isobutylxanthine - pharmacology; 8-Bromo Cyclic Adenosine Monophosphate - pharmacology; Adenylyl Cyclases - metabolism; Animals; Antidiuretic Hormone Receptor Antagonists; Benzazepines - pharmacology; Biological Transport - drug effects; Biological Transport - physiology; Cell Line; Cellular biology; Colforsin - pharmacology; Cyclic AMP - metabolism; Cyclin-dependent kinases; Dogs; Drug Synergism; Enzyme Activation; Isoquinolines - pharmacology; Kidney - cytology; Kidney - metabolism; Kidneys; Membrane Transport Proteins - genetics; Membrane Transport Proteins - physiology; Phosphodiesterase Inhibitors - pharmacology; Protein Kinase Inhibitors - pharmacology; Studies; Sulfonamides - pharmacology; Transfection; Urea - metabolism; Urea Transporters; Urine; Vasopressins - pharmacology
• ISSN: 0363-6143; 1522-1563
• DOI: 10.1152/ajpcell.00413.2005

1 Department of Physiology and 2 Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia Submitted 15 August 2005 ; accepted in final form 13 April 2006 Transepithelial [ 14 C]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 V 2 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