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.
Kwok
Willson
W
Department of Biology, University of North Carolina at Charlotte, Charlotte, North Carolina 28223, USA.
Lee
Sang Ho
SH
Culberson
Cathy
C
Korneszczuk
Katarzyna
K
Clemens
Mark G
MG
D000818:Animals; D000924:Anticholesteremic Agents / Q000494:pharmacology; D021941:Caveolae / Q000187:drug effects; D051242:Caveolin 1 / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002784:Cholesterol / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D019332:Endothelin-1 / Q000494:pharmacology*; D004731:Endotoxins / Q000494:pharmacology*; D005372:Filipin / Q000494:pharmacology; D008099:Liver / Q000166:cytology*; D008297:Male; D008954:Models, Biological; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D009685:Nuclear Envelope / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D047392:beta-Cyclodextrins / Q000494:pharmacology
D000924:Anticholesteremic Agents; C495537:Cav1 protein, rat; D051242:Caveolin 1; D019332:Endothelin-1; D004731:Endotoxins; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D009569:Nitric Oxide; D005372:Filipin; D002784:Cholesterol; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat