LPS inhibits endothelin-1-mediated eNOS translocation to the cell membrane in sinusoidal endothelial cells.
OBJECTIVE
The objectives of this study were to develop a model for studying endothelin-1-mediated eNOS regulation in cultured sinusoidal endothelial cells and determine the effect of endothelin-1 and endotoxin (LPS) on eNOS localization.


METHODS
Changes in caveolin-1, calmodulin, and eNOS expression were determined by western blot and densitometric analysis. Endothelin receptor expression and localization and the intracellular localization of eNOS and caveolin-1 were assessed by confocal microscopy.


RESULTS
Sinusoidal endothelial cells expressed caveolin-1 and calmodulin, and expression was altered in cultured and passaged cells. eNOS expression decreased significantly in 24-h cultured cells, with expression dropping below the level of detection in passaged cells. Both endothelin A and endothelin B receptors were expressed on the cell surface after 24 h in culture. In 24-h cultured cells, caveolin-1 was localized in the perinuclear region and cell membrane, while eNOS was predominantly localized in the perinuclear region, where it co-localized with caveolin-1. Endothelin-1 stimulated eNOS translocation to the cell membrane. Pretreatment with LPS markedly inhibited the endothelin-1-mediated eNOS translocation.


CONCLUSIONS
These studies demonstrate an LPS-mediated uncoupling of endothelin receptor activation and eNOS translocation. This functional uncoupling may, in part, account for the hyperconstrictive effects of endothelin-1 during inflammatory conditions.
Merkel
Sandra M
SM
Department of Biology, University of North Carolina at Charlotte, Charlotte, North Carolina, USA.
Kamoun
Walid
W
Karaa
Amel
A
Korneszczuk
Katarzyna
K
Schrum
Laura W
LW
Clemens
Mark G
MG
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D019332:Endothelin-1 / Q000378:metabolism* / Q000502:physiology; D004730:Endothelium, Vascular / Q000166:cytology*; D007249:Inflammation; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008102:Liver Circulation; D008297:Male; D019001:Nitric Oxide Synthase / Q000032:analysis / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D014661:Vasoconstriction
D019332:Endothelin-1; D008070:Lipopolysaccharides; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat