Premium
The apical membrane is the rate‐determining barrier for vasopressin‐regulated trans‐epithelial urea transport in MDCK‐UTA1 cells
Author(s) -
Fröhlich Otto,
Yang Yuan,
Klein Janet D.,
Sands Jeff M.
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a906-b
Subject(s) - urea , chemistry , apical membrane , epithelial polarity , forskolin , phloretin , biophysics , biochemistry , membrane , biology , in vitro
In order to study the vasopressin regulation of urea transport in the inner medullary collecting duct (IMCD), we have developed the MDCK‐UTA1 cell culture model which permits measurements of trans‐epithelial tracer‐urea fluxes. This requires one to confirm that the regulation of urea transport in the MDCK‐UTA1 cells occurs, as in the IMCD, in the apical membrane. We permeabilized either the apical or basolateral side of the epithelial monolayer using amphotericin. Basolateral amphotericin had no effect on trans‐epithelial tracer urea flux in non‐stimulated cells (basal flux: 2 nmol cm −2 min −1 ). In contrast, apical amphotericin greatly increased urea flux to about 20 nmol cm −2 min −1 . This indicates that the apical membrane is the side with the limiting urea permeability. In the presence of forskolin, flux is stimulated approximately 10‐fold by activating the urea transporter UT‐A1. Again, basolateral amphotericin had no effect on the urea flux whereas apical amphotericin occasionally increased it a bit further. The apical urea permeabilities induced by amphotericin and forskolin were similar in magnitude. To confirm that the basolateral membrane indeed had the high urea permeability, we performed basolateral urea influx experiments. We measured influx rates of 10–20 nmol cm −2 min −1 , with near‐complete equilibration of basolateral and intracellular urea concentrations in 1–2 min. There was no significant stimulation of basolateral fluxes by forskolin. As MDCK cells do not appear to express native urea transporter, it is not clear which transport pathways might underlie the high basolateral urea permeability. Supported by NIH.