DIRECT DEMONSTRATION OF AQUAPORIN-2 WATER CHANNEL RECYCLING IN STABLYTRANSFECTED LLC-PK1 EPITHELIAL-CELLS

Vasopressin-dependent membrane insertion of aquaporin-2 (AQP-2) in col lecting duct principal cells has been demonstrated in vivo and in vitr o. However, the hypothesis that the AQP-2 molecule recycles between in tracellular vesicles and the plasma membrane in response to hormonal s timulation and withdrawal remains to he demonstrated directly. In the present study, we examined AQP-2 recycling between intracellular vesic les and the plasma membrane in the absence of de novo protein synthesi s using LLC-PK1 cells transfected with an AQP-2-c-myc construct. Cells were treated with cycloheximide for 30 min prior to vasopressin stimu lation, and all subsequent treatments were performed in the continued presence of cycloheximide. Complete inhibition of AQP-2 biosynthesis b y cycloheximide was verified by immunoprecipitation. Immunofluorescenc e revealed that AQP-2 was located on intracellular vesicles in stimula ted cells but was relocated to the plasma membrane after vasopressin t reatment, even in the presence of cycloheximide. After vasopressin was hout, AQP-2 was retrieved to intracellular vesicles and was relocated to;the plasma membrane after restimulation with forskolin. Subsequent forskolin washout resulted in AQP-2 endocytosis, and a second stimulat ion with forskolin resulted in relocation to the plasma membrane. Thes e data, obtained in the absence of de novo protein synthesis, clearly indicate that AQP-2 can be recycled multiple times between intracellul ar vesicles and the plasma membrane.