Oxidative stress disrupts nitric oxide synthase activation in liver endothelial cells.
Oxidative stress may mediate vascular disruption associated with a loss of endothelial nitric oxide synthase (eNOS) activity and a hypersensitivity to the constrictor effects of endothelin-1 (ET-1). We hypothesize that this is due, in part, to uncoupling of ET(B) receptors from eNOS activation. Thus, we tested whether oxidative stress (OS) affects liver vascular relaxation by reducing basal and ET-1-induced NO production. Primary sinusoidal endothelial cell cultures were pretreated with H(2)O(2) (25 microM) for 1 or 6 h before a 10-min ET-1 stimulation. OS resulted in a significant basal and ET-1-induced decrease in NO production. Acute OS increased the monomeric form of the inhibitory protein caveolin-1 (1.2 +/- 0.05 vs 0.9 +/- 0.02, p < 0.01) and increased the eNOS-caveolin association as determined by coimmunoprecipitation (1.24 +/- 0.04 vs 0.97 +/- 0.04, p < 0.05). ET-1 stimulation further exacerbated these effects. Subacute OS inhibited ET-1-induced eNOS phosphorylation of serine 1177 (activation residue) (1 +/- 0.07 vs 1.6 +/- 0.04, p < 0.05) and dephosphorylation of the inhibitory residue threonine 495 (1.5 +/- 0.08 vs 0.7 +/- 0.02, p < 0.01). Additionally subacute OS resulted in dissociation of eNOS from ET(B) (0.8 +/- 0.09 vs 1.2 +/- 0.06, p < 0.05). Our findings indicate that acute and subacute oxidative stress result in the inhibition of induced nitric oxide synthase activity through distinct mechanisms dependent on caveolin-1 inhibition, ET(B) dissociation, and eNOS phosphorylation.
Karaa
Amel
A
Department of Biology, University of North Carolina at Charlotte, 9201 University City Boulevard, Charlotte, NC 28223, USA.
Kamoun
Walid S
WS
Clemens
Mark G
MG
D000818:Animals; D015153:Blotting, Western; D022461:Caveolins / Q000378:metabolism; D002470:Cell Survival; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000166:cytology; D019332:Endothelin-1 / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology*; D004789:Enzyme Activation; D005609:Free Radicals; D006861:Hydrogen Peroxide / Q000494:pharmacology; D047468:Immunoprecipitation; D008099:Liver / Q000166:cytology* / Q000378:metabolism; D008297:Male; D008954:Models, Biological; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D018384:Oxidative Stress; D010766:Phosphorylation; D012313:RNA / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013912:Threonine / Q000737:chemistry; D013997:Time Factors
D022461:Caveolins; D019332:Endothelin-1; D005609:Free Radicals; D013912:Threonine; D009569:Nitric Oxide; D012313:RNA; D006861:Hydrogen Peroxide; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III