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.
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.
Authors: Willson Kwok; Sang Ho Lee; Cathy Culberson; Katarzyna Korneszczuk; Mark G Clemens Journal: Am J Physiol Gastrointest Liver Physiol Date: 2009-11 Impact factor: 4.052
Authors: Victoria C Cogger; Gregory P McNerney; Tun Nyunt; Laurie D DeLeve; Peter McCourt; Bård Smedsrød; David G Le Couteur; Thomas R Huser Journal: J Struct Biol Date: 2010-06-04 Impact factor: 2.867