Apical and basolateral expression of Aquaporin-1 in transfected MDCK and LLC-PK cells and functional evaluation of their transcellular osmotic water permeabilities |
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Authors: | P M T Deen Søren Nielsen René J M Bindels Carel H van Os |
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Institution: | (1) 162, Department of Cell Physiology, University of Nijmegen, PO Box 9101, NL-6500 HB Nijmegen, The Netherlands, NL;(2) Department of Cell Biology, University of Aarhus, DK-8000 Aarhus C, Denmark, DK |
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Abstract: | Aquaporin-1 is present in the apical and basolateral membranes in proximal tubules and descending limbs of Henlé’s loop.
In order to be able to study the routing of Aquaporin-1 and the regulation of Aquaporin-1-mediated transcellular water flow,
we stably transfected LLC-PK1 and MDCK-HRS cell lines with an Aquaporin-1 expression construct. LLC-PK1 clone 7 and MDCK clone K integrated two and one copies, respectively, which was reflected in the amount of Aquaporin-1 mRNA expressed in both clones. The Aquaporin-1 protein levels, however, were similar. In both clones, immuno-electronmicroscopy
showed extensive labelling of Aquaporin-1 on the basolateral plasma membrane, endosomal vesicles and the apical plasma membrane,
including the microvilli. To measure transcellular water permeation, a simple method was applied using phenol-red as a cell-impermeant
marker of concentration. In contrast to the native cell lines, both clones revealed a high transcellular osmotic water permeability,
which could not be influenced by forskolin add/3-isobutyl-1-methylxanthine (IBMX) or the phorbol ester 12-O-tetradecanoyl 13-acetate (TPA). After glutaraldehyde fixation, it was inhibitable by HgCl2. These results indicate that targeting of Aquaporin-1 to the apical and basolateral plasma membrane is independent of cell
type and show for the first time that water flow through a cultured epithelium can be blocked by mercurial compounds.
Received: 9 October 1996 / Received after revision: 3 January 1997 / Accepted: 8 January 1997 |
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Keywords: | Water channel Transfection Kidney Transcellular osmotic transport Aquaporin-1 Protein routing |
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