title: Acute regulation of mUT-A3 urea transporter expressed in a MDCK cell line.
abstract: Renal facilitative urea transporters play a vital role in the urinary concentrating mechanism. UT-A3 is a phloretin-sensitive urea transporter that in the mouse is expressed on the basolateral membrane of renal inner medullary collecting duct (IMCD) cells. In this study, we engineered a Madin-Darby canine kidney (MDCK) I cell line that stably expresses mouse UT-A3 (MDCK-mUT-A3). Immunoblotting using the UT-A-targeted antibody ML446 detected a approximately 40-kDa signal in MDCK-mUT-A3 protein that corresponds to mUT-A3. Using cultured epithelial monolayers, radioactive (14)C-urea flux experiments determined that basolateral urea transport was no different between MDCK-mUT-A3 and control MDCK-FLZ cells under basal conditions [not significant (NS), ANOVA]. However, exposure to arginine vasopressin (AVP) significantly stimulated basolateral urea flux in MDCK-mUT-A3 monolayers (P < 0.05, ANOVA), while it had no effect in control MDCK-FLZ monolayers (NS, ANOVA). The AVP-stimulated basolateral urea transport in MDCK-mUT-A3 was inhibited by 1,3 dimethyl urea (P < 0.05, ANOVA) or phloretin (P < 0.05, ANOVA), both known inhibitors of facilitative urea transporters. MDCK-mUT-A3 basolateral urea flux was also stimulated by increasing intracellular levels of cAMP, via forskolin (P < 0.05, ANOVA), or intracellular calcium, via ATP (P < 0.05, ANOVA). Finally, 1-h preincubation with a specific PKA inhibitor, H89, significantly inhibited the increase in urea transport produced by AVP (P < 0.05, ANOVA). In conclusion, we have produced the first renal cell line to stably express the mUT-A3 urea transporter. Our results indicate that mUT-A3 is acutely regulated by AVP, via a PKA-dependent pathway. These findings have important implications for the regulation of urea transport in the renal IMCD and the urinary concentrating mechanism.
authors.affiliation: Faculty of Life Sciences, 2nd Floor, Core Technology Facility, Univ. of Manchester, Grafton St., Manchester M13 9NT, UK. gavin.s.stewart@manchester.ac.uk
authors.forename: Gavin S
authors.initials: GS
authors.lastname: Stewart
authors.forename: Sarah L
authors.initials: SL
authors.lastname: King
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Potter
authors.forename: Craig P
authors.initials: CP
authors.lastname: Smith
mesh_terms: D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D000242:Cyclic AMP / Q000494:pharmacology; D004285:Dogs; D005786:Gene Expression Regulation / Q000187:drug effects; D015151:Immunoblotting; D007546:Isoquinolines / Q000494:pharmacology; D026901:Membrane Transport Proteins / Q000096:biosynthesis* / Q000187:drug effects / Q000502:physiology; D008782:Methylurea Compounds / Q000494:pharmacology; D051379:Mice; D009865:Oocytes / Q000187:drug effects; D010693:Phloretin / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D013449:Sulfonamides / Q000494:pharmacology; D014162:Transfection; D014508:Urea / Q000378:metabolism; D014668:Vasotocin / Q000037:antagonists & inhibitors / Q000494:pharmacology; D014982:Xenopus laevis; D000096982:Urea Transporters
chemical_list: D002118:Calcium; D000242:Cyclic AMP; D007546:Isoquinolines; D026901:Membrane Transport Proteins; D008782:Methylurea Compounds; D010693:Phloretin; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; D014508:Urea; D014668:Vasotocin; D000096982:Urea Transporters; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; C018276:1,3-dimethylurea