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subunit of Na,K-ATPase is incorporated into the plasma membranes of mouse IMCD3 cells in response to hypertonicity
1 Department of Cell Biology, Institute of Anatomy, University of Aarhus, Water and Salt Research Center, Aarhus, Denmark
2 University of Aarhus, Institute of Medical Biochemistry, Aarhus, Denmark
3 Department of Medicine, University of Colorado Health Sciences Center, Division of Renal Diseases and Hypertension, Denver, CO, USA
* To whom correspondence should be addressed. E-mail: maunsbach{at}ana.au.dk.
Hypertonicity mediated by chloride up-regulates the expression of the 
subunit of Na,K-ATPase in cultured cells derived from the murine inner medullary collecting duct (IMCD3) (PNAS 100:6428, 2003). The purpose of this study was to examine the cellular locations and the time course of subunit expression following long-term adaptation and acute hypertonic challenges induced with different salts. Cells were analyzed by confocal immunofluorescence and immunoelectron microscopy with antibodies against the C-terminus of the Na,K-ATPase subunit or the b splice variant. Cells grown in 300 mOsm/kgH2O showed no immuno-reactivity for the subunit, while cells adapted to 600 or 900 mOsm/kgH2O demonstrated distinct
reactivity located at the plasma membrane of all cells. IMCD3 cell cultures acutely challenged to 550 mOsm/kgH2O with sodium chloride (NaCl) or choline chloride (ChoCl) showed incorporation of into plasma membrane after 12 h following osmotic challenge and distinct membrane staining in ~40% of the cells 48 h after osmotic shock. In contrast, challenging the IMCD3 cells to 550 mOsm/kgH2O by addition of
sodium acetate (NaAc) 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 following
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.
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