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LPS inhibits endothelin‐1 (ET‐1)‐mediated endothelial nitric oxide synthase (eNOS) activation through the RhoA/Rho‐kinase (ROCK‐2) pathway in hepatic sinusoidal endothelial cells (SECs)
Author(s) -
Kwok Willson,
Lee SangHo,
Korneszczuk Katarzyna,
Culberson Cathy,
Gray David L.,
Clemens Mark G.
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1190.12
Subject(s) - enos , rhoa , rho associated protein kinase , rock1 , rho kinase inhibitor , nitric oxide , downregulation and upregulation , chemistry , nitric oxide synthase , medicine , endocrinology , microbiology and biotechnology , kinase , signal transduction , biology , biochemistry , gene
The complex interactions among ET‐1, its receptors, caveolin and the components of the caveolae in the regulation of nitric oxide (NO) production are important determinants of hepatic functions in sepsis and endotoxemia. However, the exact mechanism following stresses such as endotoxin is unknown. We hypothesize that endotoxin disrupts ET‐1 signaling via RhoA and its downstream effector ROCK‐2 in hepatic SECs. In SECs isolated from Sprague‐Dawley rat livers, Western blot analysis indicated that LPS increased RhoA and ROCK‐2 expressions. The inhibition of ROCK‐2 using a competitive inhibitor Y27632 increased eNOS activity (1.61 ± 0.12), as measured by the conversion of [3H]‐L‐Arginine to [3H]‐L‐citrulline. Dose and time response experiments indicated that preincubation of 10 μM Y27632 in SECs for 30 min was most effective in enhancing eNOS activity in control media. In LPS, eNOS activity was not significantly increased by the treatment of ET‐1 (1.35 ± 0.11) or Y27632 alone (1.35 ± 0.17). However, the combined treatment of Y27632 and ET‐1 rescued eNOS activity in LPS (1.49 ± 0.14) associated with an upregulation of eNOS‐Ser1177 phosphorylation. Images from immunofluorescence confocal microscopy indicated that Y27632 inhibition of ROCK‐2 did not alter ROCK‐2 localization. These results suggest that upregulation of RhoA/ROCK‐2 may contribute to the inhibition of eNOS following LPS. Supported by R01 DK38201‐19