Aldosterone induces early activation and late accumulation of Na-K-ATPase at surface of A6 cells.
In A6 cells cultured on filters, aldosterone (10(-6) M) induces an early increase in the initial rate of ouabain binding to intact monolayers (in K-free buffer) that parallels an early increase in Na transport. This effect is independent of apical Na influx and precedes an increase in the synthesis of Na-K-adenosinetriphosphatase (ATPase) subunits. In the present study we observed in addition a late aldosterone-induced increase in the rate of ouabain binding (2.5 times from 3 to 120 h aldosterone). The role of Na-K-ATPase accumulation and/or translocation to the cell surface was tested by Western blotting, saturation ouabain binding, and cell-surface labeling using sulfosuccinimidobiotin or enzyme-mediated radioiodination. Only cell-surface beta 1-subunit was detected by blotting with streptavidin or autoradiography, because the alpha 1-subunit was not efficiently labeled. Three hours after hormone addition, none of the three parameters had increased significantly, whereas after 20 and 120 h similar increases (approximately 1.6 and 2.3 times, respectively) were detected by all three methods. In addition, it was shown that increasing intracellular Na with amphotericin B or a K-free preincubation also stimulated the rate of ouabain binding without increasing the surface labeling of beta 1-subunits. Taken together, these results suggest that a short aldosterone treatment or an increase in intracellular Na leads to an increase in the rate of ouabain binding that is due to an in situ activation of cell-surface Na-K-ATPase molecules. In contrast, the late increase in the rate of ouabain binding parallels an increase in the number of pumps.
Beron
J
J
Institute of Physiology, University of Zurich, Switzerland.
Verrey
F
F
D000450:Aldosterone / Q000494:pharmacology*; D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology*; D002999:Clone Cells; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004848:Epithelium / Q000187:drug effects / Q000201:enzymology; D007668:Kidney; D007700:Kinetics; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D010042:Ouabain / Q000378:metabolism; D026941:Sodium Channel Blockers; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D014982:Xenopus laevis
D046911:Macromolecular Substances; D008565:Membrane Proteins; D026941:Sodium Channel Blockers; D000450:Aldosterone; D010042:Ouabain; D000584:Amiloride; D000254:Sodium-Potassium-Exchanging ATPase