Rho-kinase activation contributes to Lps-induced impairment of endothelial nitric oxide synthase activation by endothelin-1 in cultured hepatic sinusoidal endothelial cells.

The purpose of this study is to understand the role of rho-kinase (ROCK-2) in the regulation of liver microcirculation after inflammatory stress. Endothelin-1 (ET-1)-induced nitric oxide (NO) is essential in the regulation of blood flow in hepatic sinusoids. Lipopolysaccharide (LPS) inhibits this ET-1-induced NO production and disrupts liver microcirculation; however, the exact molecular mechanism is unknown. Liver sinusoidal endothelial cells were isolated, pretreated with 10 ng/mL LPS for 6 h, and treated with 10 µM Y27632 (ROCK-2 inhibitor) for 30 min and 10 nM ET-1 for 30 min. Lipopolysaccharide induced RhoA membrane translocation that was attenuated by methyl-ß-cyclodextrin (cholesterol sequester) or targeted mutation of caveolin-1. Lipopolysaccharide increased ROCK-2 expressions (+60%) and ROCK-2 activity (+36%). Endothelin-1 increased endothelial NO synthase (eNOS) activity (+70%), but LPS inhibited this ET-1-mediated eNOS response. Treatment with Y27632 restored ET-1-mediated eNOS activity (+61%) and stimulated NO production in the perinuclear region after LPS pretreatment. This treatment reduced cofilin-Ser3 phosphorylation (-73%), increased vasodilator-stimulated phosphoprotein-Ser239 phosphorylation (+88%), and stimulated globular actin/eNOS association. Lipopolysaccharide induces Rho/ROCKs signaling pathway to disrupt the ET-1-mediated eNOS activation in liver sinusoidal endothelial cells. Rho-kinase ROCK-2 inhibition restores ET-1-mediated NO production after the LPS pretreatment, in part, through an increase in actin depolymerization.

Targeted mutation of Cav-1 alleviates the effect of endotoxin in the inhibition of ET-1-mediated eNOS activation in the liver.

Dysfunction of hepatic microcirculation during inflammatory stress conditions is associated with overexpression of caveolin 1 (Cav-1) in sinusoidal endothelial cells. Because Cav-1 binds and inhibits eNOS, it was suggested that Cav-1 overexpression inhibits endothelin 1 (ET-1)-mediated eNOS activation after endotoxemia in the liver; however, a causal link between stress-mediated suppression of eNOS and Cav-1 overexpression has not been fully established. We hypothesize that genetic knockout of Cav-1 reverses the LPS-suppressed ET-1-mediated eNOS activation. In this report, liver sinusoidal endothelial cells (LSECs) from wild-type (WT) and Cav-1 knockout (KO) mice were isolated, pretreated with 100 ng/mL LPS for 6 h, and treated with 10 nmol ET-1 for 30 min. Data showed that LPS increased Cav-1 protein expression (+88%; P < 0.05) and inhibited ET-1-mediated eNOS activation and NO production in WT LSECs. Genetic deletion of Cav-1 increased basal eNOS activity (0.40 in KO vs. 0.15 fmol/min per well in WT; +262%; P < 0.05) and reversed LPS inhibition of ET-1-stimulated eNOS activity (+25.7%; P < 0.05) by increasing eNOS-Ser1177 (+40.3%; P < 0.05) and decreasing eNOS-threonine-495 (-8.8%; P < 0.05) phosphorylation. The reversal of LPS inhibition resulted in an increase in ET-1-induced eNOS translocation to the plasma membrane and an augmentation of NO production in the perinuclear region and plasma membrane of Cav-1 KO LSECs. These results showed that genetic knockout of Cav-1 increased basal eNOS activity and at least partially restored ET-1-mediated eNOS translocation and NO production in LSECs after LPS treatment. In conclusion, Cav-1 overexpression is a requirement for decreased eNOS activity in LSECs after endotoxemia.

Caveolin-1 mediates endotoxin inhibition of endothelin-1-induced endothelial nitric oxide synthase activity in liver sinusoidal endothelial cells.

Endothelin-1 (ET-1) plays a key role in the regulation of endothelial nitric oxide synthase (eNOS) activation in liver sinusoidal endothelial cells (LSECs). In the presence of endotoxin, an increase in caveolin-1 (Cav-1) expression impairs ET-1/eNOS signaling; however, the molecular mechanism is unknown. The objective of this study was to investigate the molecular mechanism of Cav-1 in the regulation of LPS suppression of ET-1-mediated eNOS activation in LSECs by examining the effect of caveolae disruption using methyl-beta-cyclodextrin (CD) and filipin. Treatment with 5 mM CD for 30 min increased eNOS activity (+255%, P < 0.05). A dose (0.25 microg/ml) of filipin for 30 min produced a similar effect (+111%, P < 0.05). CD induced the perinuclear localization of Cav-1 and eNOS and stimulated NO production in the same region. Readdition of 0.5 mM cholesterol to saturate CD reversed these effects. Both the combined treatment with CD and ET-1 (CD + ET-1) and with filipin and ET-1 stimulated eNOS activity; however, pretreatment with endotoxin (LPS) abrogated these effects. Following LPS pretreatment, CD + ET-1 failed to stimulate eNOS activity (+51%, P > 0.05), which contributed to the reduced levels of eNOS-Ser1177 phosphorylation and eNOS-Thr495 dephosphorylation, the LPS/CD-induced overexpression and translocation of Cav-1 in the perinuclear region, and the increased perinuclear colocalization of eNOS with Cav-1. These results supported the hypothesis that Cav-1 mediates the action of endotoxin in suppressing ET-1-mediated eNOS activation and demonstrated that the manipulation of caveolae produces significant effects on ET-1-mediated eNOS activity in LSECs.