Apical and basolateral expression of aquaporin-1 in transfected MDCK and LLC-PK cells and functional evaluation of their transcellular osmotic water permeabilities.
Aquaporin-1 is present in the apical and basolateral membranes in proximal tubules and descending limbs of Henlé's loop. In order ot 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.
Deen
P M
PM
Department of Cell Physiology, University of Nijmegen, The Netherlands.
Nielsen
S
S
Bindels
R J
RJ
van Os
C H
CH
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D051398:Aquaporin 1; D020346:Aquaporins; D015152:Blotting, Northern; D015139:Blotting, Southern; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D004285:Dogs; D015151:Immunoblotting; D007473:Ion Channels / Q000096:biosynthesis* / Q000235:genetics; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018374:LLC-PK1 Cells; D016253:Microscopy, Immunoelectron; D009994:Osmolar Concentration; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D013552:Swine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D020346:Aquaporins; D007473:Ion Channels; D010726:Phosphodiesterase Inhibitors; D012333:RNA, Messenger; D051398:Aquaporin 1; D013755:Tetradecanoylphorbol Acetate; D015056:1-Methyl-3-isobutylxanthine