• Sandra M Merkel, Walid Kamoun, Amel Karaa, Katarzyna Korneszczuk, Laura W Schrum, and Mark G Clemens.
• Department of Biology, University of North Carolina at Charlotte, Charlotte, North Carolina, USA.
• Microcirculation. 2005 Jul 1;12(5):433-42.

ObjectiveThe 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.MethodsChanges 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.ResultsSinusoidal 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.ConclusionsThese 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.

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