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.
Kwok
Willson
W
Department of Biology, University of North Carolina at Charlotte, Charlotte, North Carolina 28223, USA.
Clemens
Mark G
MG
D000818:Animals; D051242:Caveolin 1 / Q000096:biosynthesis / Q000235:genetics*; D019332:Endothelin-1 / Q000494:pharmacology / Q000502:physiology*; D004731:Endotoxins / Q000494:pharmacology*; D004789:Enzyme Activation; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008099:Liver / Q000098:blood supply / Q000201:enzymology*; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010766:Phosphorylation
D051242:Caveolin 1; D019332:Endothelin-1; D004731:Endotoxins; D008070:Lipopolysaccharides; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse