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-subunit of Na-K-ATPase is incorporated into plasma membranes of mouse IMCD3 cells in response to hypertonicity
1The Water and Salt Research Center, Department of Cell Biology, Institute of Anatomy, and 2Institute of Medical Biochemistry, University of Aarhus, Aarhus, Denmark; and 3Division of Renal Diseases and Hypertension, Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado
Submitted 4 May 2004 ; accepted in final form 29 November 2004
Hypertonicity mediated by chloride upregulates the expression of the 
-subunit of Na-K-ATPase in cultured cells derived from the murine inner medullary collecting duct (IMCD3; Capasso JM, Rivard CJ, Enomoto LM, and Berl T. Proc Natl Acad Sci USA 100: 6428–6433, 2003). The purpose of this study was to examine the cellular locations and the time course of -subunit expression after long-term adaptation and acute hypertonic challenges induced with different salts. Cells were analyzed by confocal immunofluorescence and immunoelectron microscopy with antibodies against the COOH terminus of the Na-K-ATPase -subunit or the b splice variant. Cells grown in 300 mosmol/kgH2O showed no immunoreactivity for the -subunit, whereas cells adapted to 600 or 900 mosmol/kgH2O demonstrated distinct reactivity located at the plasma membrane of all cells. IMCD3 cell cultures acutely challenged to 550 mosmol/kgH2O with sodium chloride or choline chloride showed incorporation of into plasma membrane 12 h after osmotic challenge and distinct membrane staining in 
40% of the cells 48 h after osmotic shock. In contrast, challenging the IMCD3 cells to 550 mosmol/kgH2O by addition of sodium acetate did not result in expression of the -subunit in the membranes of surviving cells after 48 h. The present results demonstrate that the Na-K-ATPase -subunit becomes incorporated into the basolateral membrane of IMCD3 cells after both acute hyperosmotic challenge and hyperosmotic adaptation. We conclude that the -subunit has an important role in the function of Na-K-ATPase to sustain the cellular cation balance over the plasma membrane in a hypertonic environment.
inner medullary collecting duct cells; cell culture; osmotic challenge; sodium pump subunits; immunocytochemical localization
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