LPS inhibits endothelin-1-induced endothelial NOS activation in hepatic sinusoidal cells through a negative feedback involving caveolin-1.

During endotoxemia, liver microcirculation disruption is characterized by a hypersensitivity to the constrictor effects of endothelin 1 (ET-1). The shift of ET-1-mediated effects toward vasoconstriction may result from depressed ET-1-mediated vasodilation through decreased ET-1-induced nitric oxide (NO) production. We have previously shown that lipopolysaccharide (LPS) pretreatment abrogates ET-1-induced endothelial nitric oxide synthase (eNOS) translocation, but its effects on eNOS activation are yet to be determined. Our aim was to assess the effects of LPS on ET-1-mediated eNOS activation in hepatic sinusoidal endothelial cells (SECs) and to investigate the molecular mechanisms involved. SECs were treated with LPS (100 ng/mL) for 6 hours followed by 30 minutes ET-1 (10 nmol/L) stimulation. LPS significantly inhibited ET-1-mediated eNOS activation. This inhibition was associated with upregulation of Caveolin-1 (CAV-1) and a shift in ET-1-mediated eNOS phosphorylation from an activation (Ser1177) to an inhibition (Thr495). LPS treatment has been shown to induce ET-1 expression and secretion from endothelial cells. We therefore investigated the role of endogenous ET-1 in the inhibition of ET-1 activation of eNOS after LPS. Antagonizing ET-1 effects and blocking its activation in LPS pretreated SECs decreased the LPS-induced overexpression of CAV-1 as well as the inhibition of ET-1-induced NOS activity. Furthermore, 6 hours of ET-1 treatment exerted the same effects on eNOS activity, phosphorylation, and CAV-1 expression as LPS treatment. In conclusion, LPS-induced suppression of ET-1-mediated eNOS activation is ET-1 dependent and suggest a pivotal role of CAV-1 in eNOS induction inhibition under stress.

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.