Aldosterone and amiloride alter ENaC abundance in vascular endothelium.
The amiloride-sensitive epithelial sodium channel (ENaC) is usually found in the apical membrane of epithelial cells but has also recently been described in vascular endothelium. Because little is known about the regulation and cell surface density of ENaC, we studied the influence of aldosterone, spironolactone, and amiloride on its abundance in the plasma membrane of human endothelial cells. Three different methods were applied, single ENaC molecule detection in the plasma membrane, quantification by Western blotting, and cell surface imaging using atomic force microscopy. We found that aldosterone increases the surface expression of ENaC molecules by 36% and the total cellular amount by 91%. The aldosterone receptor antagonist spironolactone prevents these effects completely. Acute application of amiloride to aldosterone-pretreated cells led to a decline of intracellular ENaC by 84%. We conclude that, in vascular endothelium, aldosterone induces ENaC expression and insertion into the plasma membrane. Upon functional blocking with amiloride, the channel disappears from the cell surface and from intracellular pools, indicating either rapid degradation and/or membrane pinch-off. This opens new perspectives in the regulation of ENaC expressed in the vascular endothelium.
Kusche-Vihrog
Kristina
K
Institute of Animal Physiology, University of Muenster, Hindenburgplatz 55, 48143 Muenster, Germany. kusche@uni-muenster.de
Sobczak
Katja
K
Bangel
Nadine
N
Wilhelmi
Marianne
M
Nechyporuk-Zloy
Volodymyr
V
Schwab
Albrecht
A
Schillers
Hermann
H
Oberleithner
Hans
H
D000450:Aldosterone / Q000494:pharmacology*; D000584:Amiloride / Q000494:pharmacology*; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000502:physiology; D002478:Cells, Cultured; D004232:Diuretics / Q000494:pharmacology*; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D053503:Epithelial Sodium Channels / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D018625:Microscopy, Atomic Force; D013148:Spironolactone / Q000494:pharmacology; D014471:Umbilical Veins / Q000166:cytology
D004232:Diuretics; D053503:Epithelial Sodium Channels; D013148:Spironolactone; D000450:Aldosterone; D000584:Amiloride