Regulation of UT-A1-mediated transepithelial urea flux in MDCK cells.
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 approximately 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 approximately 5 nmol.cm(-2).min(-1). 8-Bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP; 300 microM) 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 microM, 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.
Fröhlich
Otto
O
Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322, USA. otto.froehlich@emory.edu
Klein
Janet D
JD
Smith
Pauline M
PM
Sands
Jeff M
JM
Gunn
Robert B
RB
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D065092:Antidiuretic Hormone Receptor Antagonists; D001552:Benzazepines / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004285:Dogs; D004357:Drug Synergism; D004789:Enzyme Activation; D007546:Isoquinolines / Q000494:pharmacology; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000502:physiology*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D013449:Sulfonamides / Q000494:pharmacology; D014162:Transfection; D014508:Urea / Q000378:metabolism*; D014667:Vasopressins / Q000494:pharmacology; D000096982:Urea Transporters
D015056:1-Methyl-3-isobutylxanthine; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000262:Adenylyl Cyclases; D065092:Antidiuretic Hormone Receptor Antagonists; D001552:Benzazepines; D005576:Colforsin; D000242:Cyclic AMP; D007546:Isoquinolines; D026901:Membrane Transport Proteins; D010726:Phosphodiesterase Inhibitors; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; D014508:Urea; D014667:Vasopressins; D000096982:Urea Transporters; C076251:mozavaptan; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide