Aldosterone does not alter apical cell-surface expression of epithelial Na+ channels in the amphibian cell line A6.
The steroid hormone aldosterone regulates reabsorptive Na+ transport across specific high resistance epithelia. The increase in Na+ transport induced by aldosterone is dependent on protein synthesis and is due, in part, to an increase in Na+ conductance of the apical membrane mediated by amiloride-sensitive Na+ channels. To examine whether an increment in the biochemical pool of Na+ channels expressed at the apical cell surface is a mechanism by which aldosterone increases apical membrane Na+ conductance, apical cell-surface proteins from the epithelial cell line A6 were specifically labeled by an enzyme-catalyzed radioiodination procedure following exposure of cells to aldosterone. Labeled Na+ channels were immunoprecipitated to quantify the biochemical pool of Na+ channels at the apical cell surface. The activation of Na+ transport across A6 cells by aldosterone was not accompanied by alterations in the biochemical pool of Na+ channels at the apical plasma membrane, despite a 3.7-4.2-fold increase in transepithelial Na+ transport. Similarly, no change in the distribution of immunoreactive protein was resolved by immunofluorescence microscopy. The oligomeric subunit composition of the channel remained unaltered, with one exception. A 75,000-Da polypeptide and a broad 70,000-Da polypeptide were observed in controls. Following addition of aldosterone, the 75,000-Da polypeptide was not resolved, and the 70,000-Da polypeptide was the major polypeptide found in this molecular mass region. Aldosterone did not alter rates of Na+ channel biosynthesis. These data suggest that neither changes in rates of Na+ channel biosynthesis nor changes in its apical cell-surface expression are required for activation of transepithelial Na+ transport by aldosterone. Post-translational modification of the Na+ channel, possibly the 75,000 or 70,000-Da polypeptide, may be one of the cellular events required for Na+ channel activation by aldosterone.
Kleyman
T R
TR
Department of Medicine, University of Pennsylvania, Philadelphia.
Coupaye-Gerard
B
B
Ernst
S A
SA
D000450:Aldosterone / Q000494:pharmacology*; D000818:Animals; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004848:Epithelium / Q000378:metabolism; D007668:Kidney; D007700:Kinetics; D046911:Macromolecular Substances; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D015222:Sodium Channels / Q000187:drug effects / Q000378:metabolism*; D013148:Spironolactone / Q000494:pharmacology; D013462:Sulfur Radioisotopes; D014982:Xenopus laevis
D046911:Macromolecular Substances; D008565:Membrane Proteins; D015222:Sodium Channels; D013462:Sulfur Radioisotopes; D013148:Spironolactone; D000450:Aldosterone; D008715:Methionine