RECONSTITUTION OF A REGULATED TRANSEPITHELIAL WATER PATHWAY IN CELLS TRANSFECTED WITH AQP2 AND AN AQP1 AQP2 HYBRID CONTAINING THE AQP2-C TERMINUS/

Transepithelial water permeability was measured in LLC-PK1 cells stabl y transfected with aquaporins (AQPs): AQP1, AQP2, and a chimera of AQP 1 and AQP2 containing 41 amino acids of the C-terminus of AQP2. Transe pithelial water fluxes (Jw) were not previously reported in cells tran sfected with aquaporins. Jw were now recorded each minute using a spec ially developed experimental device. A significant increase in Posm af ter forskolin (FK) plus vasopressin (VP) was found in AQP2 transfected cells (39.9 +/- 8.2 vs. 12.5 +/- 3.3 cm . sec(-1). 10(-3)), but not i n cells transfected with AQP1 (15.3 +/- 3.6 vs. 13.4 +/- 3.6 cm . sec( -1). 10(-3)). In the case of the AQP1/2 cells (chimera) the FK plus VP induced Posm was smaller than in AQP2 cells but significantly higher than in mock cells at rest (18.1 +/- 4.8 vs. 6.7 +/- 1.0 cm . sec(-1). 10(-3)). The increases in Posm values were not paralleled by increase s in C-14-Mannitol permeability. HgCl2 inhibited the hydrosmotic respo nse to FK plus VP in AQP2 transfected epithelia. Results were comparab le to those observed, in parallel experiments, in a native ADH-sensiti ve water channel containing epithelial barrier (the toad urinary bladd er). Electron microscopy showed confluent LLC-PK1 cells with microvill i at the mucosal border. The presence of spherical or elongated intrac ellular vacuoles was observed in AQP2 transfected cells, specially aft er FK plus VP stimulus and under an osmotic gradient, These results de monstrate regulated transepithelial water permeability in epithelial c ells transfected with AQP2.