title: The gamma-subunit of Na-K-ATPase is incorporated into plasma membranes of mouse IMCD3 cells in response to hypertonicity.
abstract: Hypertonicity mediated by chloride upregulates the expression of the gamma-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 gamma-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 gamma-subunit or the gamma(b) splice variant. Cells grown in 300 mosmol/kgH(2)O showed no immunoreactivity for the gamma-subunit, whereas cells adapted to 600 or 900 mosmol/kgH(2)O demonstrated distinct reactivity located at the plasma membrane of all cells. IMCD3 cell cultures acutely challenged to 550 mosmol/kgH(2)O with sodium chloride or choline chloride showed incorporation of gamma into plasma membrane 12 h after osmotic challenge and distinct membrane staining in approximately 40% of the cells 48 h after osmotic shock. In contrast, challenging the IMCD3 cells to 550 mosmol/kgH(2)O by addition of sodium acetate did not result in expression of the gamma-subunit in the membranes of surviving cells after 48 h. The present results demonstrate that the Na-K-ATPase gamma-subunit becomes incorporated into the basolateral membrane of IMCD3 cells after both acute hyperosmotic challenge and hyperosmotic adaptation. We conclude that the gamma-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.
authors.affiliation: The Water and Salt Research Ctr., Dept. of Cell Biology, Institute of Anatomy, Univ. of Aarhus, DK-8000 Aarhus C, Denmark.
authors.forename: Kaarina
authors.initials: K
authors.lastname: Pihakaski-Maunsbach
authors.forename: Shigeki
authors.initials: S
authors.lastname: Tokonabe
authors.forename: Henrik
authors.initials: H
authors.lastname: Vorum
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Rivard
authors.forename: Juan M
authors.initials: JM
authors.lastname: Capasso
authors.forename: Tomas
authors.initials: T
authors.lastname: Berl
authors.forename: Arvid B
authors.initials: AB
authors.lastname: Maunsbach
mesh_terms: D000222:Adaptation, Physiological; D000818:Animals; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism*; D016764:Cell Polarity; D002478:Cells, Cultured; D006982:Hypertonic Solutions; D007150:Immunohistochemistry; D007679:Kidney Medulla / Q000166:cytology / Q000201:enzymology*; D051379:Mice; D009997:Osmotic Pressure; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D014882:Water-Electrolyte Balance / Q000502:physiology*
chemical_list: D006982:Hypertonic Solutions; C498816:Fxyd2 protein, mouse; D000254:Sodium-Potassium-Exchanging ATPase