Reconstitution of a regulated transepithelial water pathway in epithelial cells transfected with aquaporin-2 and an aquaporin-1/2 hybrid containing the AQP2-C-terminus

TORIANO, ROXANA; FORD, PAULA; RIVAROLA, VALERIA; TAMARAPPOO, B.K; VERKMAN, ALAN S; PARISI, MARIO

JOURNAL OF MEMBRANE BIOLOGY

Springer-Verlang

Lugar: New York; Año: 1998 vol. 161 p. 141 - 149

0022-2631

Transepithelial water permeability was measured in LLC-PK1 cells stably transfected with aquaporins (AQPs): AQP1, AQP2, and a chimera of AQP1 and AQP2 containing 41 amino acids of the C-terminus of AQP2. Transepithelial water fluxes (Jw) were not previously reported in cells transfected with aquaporins. Jw were now recorded each minute using a specially developed experimental device. A significant increase in Posm after 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 in 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 increases in 14C-Mannitol permeability. HgCl2 inhibited the hydrosmotic response to FK plus VP in AQP2 transfected epithelia. Results were comparable to those observed, in parallel experiments, in a native ADH-sensitive water channel containing epithelial barrier (the toad urinary bladder). Electron microscopy showed confluent LLC-PK1 cells with microvilli at the mucosal border. The presence of spherical or elongated intracellular vacuoles was observed in AQP2 transfected cells, specially after FK plus VP stimulus and under an osmotic gradient. These results demonstrate regulated transepithelial water permeability in epithelial cells transfected with AQP2.