title: 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.
authors.affiliation: Department of Biological Sciences and Center for Biomedical Engineering and Science, University of North Carolina at Charlotte, Charlotte, North Carolina.
authors.forename: Willson
authors.initials: W
authors.lastname: Kwok
authors.forename: Mark G
authors.initials: MG
authors.lastname: Clemens
mesh_terms: D000199:Actins / Q000378:metabolism; D000577:Amides / Q000737:chemistry; D000818:Animals; D051242:Caveolin 1 / Q000378:metabolism; D002478:Cells, Cultured; D051338:Cofilin 1 / Q000737:chemistry; D042783:Endothelial Cells / Q000166:cytology*; D019332:Endothelin-1 / Q000378:metabolism*; D004789:Enzyme Activation; D007249:Inflammation; D008070:Lipopolysaccharides; D008099:Liver / Q000098:blood supply / Q000378:metabolism*; D008297:Male; D008833:Microcirculation; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011725:Pyridines / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D018805:Sepsis; D012694:Serine / Q000378:metabolism; D054460:rho-Associated Kinases / Q000378:metabolism*
chemical_list: D000199:Actins; D000577:Amides; D051242:Caveolin 1; D051338:Cofilin 1; D019332:Endothelin-1; D008070:Lipopolysaccharides; D011725:Pyridines; C108830:Y 27632; D009569:Nitric Oxide; D012694:Serine; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; C580818:ROCK2 protein, rat; D054460:rho-Associated Kinases