Direct demonstration of aquaporin-2 water channel recycling in
stably transfected llc-pk1 epithelial cells.
Katsura, Toshiya, Dennis A. Ausiello, and Dennis Brown.
Renal Unit, Massachusetts General Hospital and Departments of
Medicine and Pathology, Harvard Medical School, Boston, Massachusetts
02114
APStracts 3:0010F, 1996.
Vasopressin dependent membrane insertion of aquaporin-2 (AQP2) in
collecting duct principal cells has been demonstrated in vivo and in
vitro. However, the hypothesis that the AQP2 molecule recycles
between intracellular vesicles and the plasma membrane in response to
hormonal stimulation and withdrawal remains to be demonstrated
directly. In the present study, we examined AQP2 recycling between
intracellular vesicles and the plasma membrane in the absence of de
novo protein synthesis using LLC-PK1 cells transfected with an AQP2
-c-myc construct. Cells were treated with cycloheximide for 30 min
prior to vasopressin stimulation and all subsequent treatments were
performed in the continued presence of cycloheximide. Complete
inhibition of AQP2 biosynthesis by cycloheximide was verified by
immunoprecipitation. Immunofluorescence revealed that AQP2 was
located on intracellular vesicles in non-stimulated cells, but was
relocated to the plasma membrane after vasopressin treatment even in
the presence of cycloheximide. After vasopressin washout AQP2 was
retrieved to intracellular vesicles, and was relocated to the plasma
membrane after restimulation with forskolin. Subsequent forskolin
washout resulted in AQP2 endocytosis, and a second stimulation with
forskolin resulted in relocation to the plasma membrane. These data,
obtained in the absence of de novo protein synthesis, clearly
indicate that AQP2 can be recycled multiple times between
intracellular vesicles and the plasma membrane.
Received 6 September 1995; accepted in final form 18 December
1995.
APS Manuscript Number F295-5.
Article publication pending Am. J. Physiol. (Renal Fluid Electrolyte
Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 22 January 96