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

Abstract

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-β-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.