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; VERKMAN, ALAN; PARISI, MARIO

Paris - Francia

Congreso; Molecular Phyiology of Water Transport; 1997

Constitutive and regulated membrane expression of aquaporin-2 water channels (AQP2) in stably transfected LLC-PK1 epithelial cells has been reported, as well as direct demonstration, in the same cellular system, of AQP2 water channel recycling. However permeability measurements were made, in these previous experiments, by following cell volume changes in response to an osmotic gradient The cells were grown on glass coverslips, avoiding the measurement of transcellular fluxes. Now the cells were grown on a permeable supporí (3 m m Nucleopore filters, Costar holders) and the net transepithelial water fluxes (Jw) were minute by minute recorded, employing an specially developed experimental device. Unidirectional 14C-mannitol fluxes were simultaneously run in some experiments and morpho-functional correlations were established from electron-microscopy studies. Transepithelial resistance was routinely controlled. The toad or frog urinary bladder, a well known epithelial barrier containing ADH sensitive water channels, was employed as a comparative model to test the reconstitution experiments. The obtained results indícate that the insertion of AQP2 into LLC-PK1 cells leads to the development of a functional, sensitive to vasopressin plus forskolin, transepithelial water pathway. Several points must be remarked: 1) The combined action of forskolin plus vasopressin induced an increase in the observed Jw both in the toad urinary bíadder and in the AQP2 transfected epithelium. This Jw appeared in the absence of a transepithelial osmotic gradient and was not paralleled by an increase in the 14C-mannitol permeability. These results can be straightforward interpretad as a transcellular water movement associated to an ionic transport and that must be further investigated. 2) HgCI2 inhibited both the non-osmotic effect of forskolin plus vasopressin as well as the osmotic pathway, either in the toad urinary bladder as in the AQP2 transfected cells It must be here remarked that, in all experiments, HgCI2 was added to both, the apical and the basolateral media. 3) The similarity between the responses to vasopressin plus forskolin in the AQP2 transfected cells and in the toad urinary bladder, as well as the similar effect of HgCI2 in both cases, allow us to consider that the functional reconstitution of the. vasopressin-regulated transepithelial water pathway was achieved.