AQP5 is blocked by amiloride

Abstract

We have previously shown that amiloride inhibits transepithelial water flux in airway epithelia. Since similar effects were elicited with HgCl2, we speculated that amiloride directly blocked an aquaporin. To test this hypothesis, we measured the rate of cell shrinkage of untransfected MDCK cells, and those stably-expressing AQP1 or 5, or expressing AQP3 after 24 h exposure to a mildly hypertonic media. Following mucosal exposure to a hypertonic Ringer solution, MDCK cultures grown to confluency on permeable filtures shrunk by 8% (n = 6) after 30 s, as measured by XZ confocal microscopy. In contrast MDCKAQP1 or MDCKAQP3 cultures shrunk by 20% over the same period and MDCKAQP5 cultures shrunk by 35% (all n=6). A 10 min pretreatment with 200 μM amiloride abolished hypertonic Ringer-induced cell shrinkage in MDCK5 cultures (93% at 30 s; n=6) yet had no affect on cell shrinkage in the other cell lines. Addition of amiloride immediately (<10 s) before hypertonic Ringer addition did not block MDCKAQP5 cell shrinkage (69%, n = 6). Amiloride is known to be internalized over several minutes suggesting that it blocks AQP5 internally. To confirm that AQP5 is present in airway epithelia, we performed immunostaining with an AQP5 antibody and found that AQP5 is highly expressed at the apical membrane of bronchial epithelia. We conclude that amiloride blocks AQP5, which is abundantly expressed in bronchi. Supported by NHLBI RO1 HL074158