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LPS Inhibits Endothelin‐1‐Mediated eNOS Translocation to the Cell Membrane in Sinusoidal Endothelial Cells
Author(s) -
MERKEL SANDRA M.,
KAMOUN WALID,
KARAA AMEL,
KORNESZCZUK KATARZYNA,
SCHRUM LAURA W.,
CLEMENS MARK G.
Publication year - 2005
Publication title -
microcirculation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.793
H-Index - 83
eISSN - 1549-8719
pISSN - 1073-9688
DOI - 10.1080/10739680590960377
Subject(s) - enos , endothelin 1 , endothelin receptor , caveolin , microbiology and biotechnology , biology , receptor , medicine , endocrinology , nitric oxide , nitric oxide synthase , signal transduction , caveolae , biochemistry
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.