Localization of intracellular compartments that exchange Na,K-ATPase molecules with the plasma membrane in a hormone-dependent manner.
OBJECTIVE
Dopamine is a major regulator of sodium reabsorption in proximal tubule epithelia. By binding to D1-receptors, dopamine induces endocytosis of plasma membrane Na,K-ATPase, resulting in a reduced capacity of the cells to transport sodium, thus contributing to natriuresis. We have previously demonstrated several aspects of the molecular mechanism by which dopamine induces Na,K-ATPase endocytosis; however, the location of intracellular compartments containing Na,K-ATPase molecules has not been identified.


METHODS
In this study, we used different approaches to determine the localization of Na,K-ATPase-containing intracellular compartments. By expression of fluorescent-tagged Na,K-ATPase molecules in opossum kidney cells, a cell culture model of proximal tubule epithelia, we used fluorescence microscopy to determine cellular distribution of the fluorescent molecules and the effects of dopamine on this distribution. By labelling cell surface Na,K-ATPase molecules from the cell exterior with either biotin or an epitope-tagged antibody, we determined the localization of the tagged Na,K-ATPase molecules after endocytosis induced by dopamine.


RESULTS
In cells expressing fluorescent-tagged Na,K-ATPase molecules, there were intracellular compartments containing Na,K-ATPase molecules. These compartments were in very close proximity to the plasma membrane. Upon treatment of the cells with dopamine, the fluorescence labelling of these compartments was increased. The labelling of these compartments was also observed when the endocytosis of biotin- or antibody-tagged plasma membrane Na,K-ATPase molecules was induced by dopamine.


CONCLUSIONS
The intracellular compartments containing Na,K-ATPase molecules are located just underneath the plasma membrane.
Efendiev
R
R
College of Pharmacy, University of Houston, 4800 Calhoun Road, Houston, TX 77204, USA. refendiev@uh.edu
Das-Panja
K
K
Cinelli
A R
AR
Bertorello
A M
AM
Pedemonte
C H
CH
D000470:Alkaloids / Q000494:pharmacology; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D053119:Benzophenanthridines / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D004298:Dopamine / Q000494:pharmacology*; D004705:Endocytosis / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D042541:Intracellular Space / Q000187:drug effects* / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods; D008985:Monensin / Q000494:pharmacology; D009893:Opossums; D010042:Ouabain / Q000378:metabolism / Q000494:pharmacology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D000077191:Wortmannin
D000470:Alkaloids; D000730:Androstadienes; D053119:Benzophenanthridines; D008164:Luminescent Proteins; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D010042:Ouabain; D008985:Monensin; C016299:chelerythrine; D011493:Protein Kinase C; D000254:Sodium-Potassium-Exchanging ATPase; D004298:Dopamine; D000077191:Wortmannin