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 prox imal tubules and descending limbs of Henle'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 bot h clones. The Aquaporin-1 protein levels, however, were similar. In bo th clones, immuno-electronmicroscopy showed extensive labelling of Aqu aporin-1 on the basolateral plasma membrane, endosomal vesicles and th e apical plasma membrane, including the microvilli. To measure transce llular water permeation, a simple method was applied using phenol-red as a cell-impermeant marker of concentration. In contrast to the nativ e cell lines, both clones revealed a high transcellular osmotic water permeability, which could not be influenced by forskolin add/3-isobuty l-1-methylxanthine (IBMX) or the phorbol ester 12-O-tetradecanoyl 13-a cetate (TPA). After glutaraldehyde fixation, it was inhibitable by HgC l2. 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 b e blocked by mercurial compounds.