Superoxide production and reactive oxygen species signaling by endothelial nitric-oxide synthase.
Reactive oxygen species can function as intracellular messengers, but linking these signaling events with specific enzymes has been difficult. Purified endothelial nitric-oxide synthase (eNOS) can generate superoxide (O(2)) under special conditions but is only known to participate in cell signaling through NO. Here we show that eNOS regulates tumor necrosis factor alpha (TNFalpha) through a mechanism dependent on the production of O(2) and completely independent of NO. Expression of eNOS in transfected U937 cells increased phorbol 12-myristate 13-acetate-induced TNFalpha promoter activity and TNFalpha production. N(omega)-Methyl-l-arginine, an inhibitor of eNOS that blocks NO production but not its NADPH oxidase activity, did not prevent TNFalpha up-regulation. Likewise, Gln(361)eNOS, a competent NADPH oxidase that lacks NOS activity, retained the ability to increase TNFalpha. Similar to the effect of eNOS, a O(2) donor dose-dependently increased TNFalpha production in differentiated U937 cells. In contrast, cotransfection of superoxide dismutase with eNOS prevented TNFalpha up-regulation, as did partial deletion of the eNOS NADPH binding site, a mutation associated with loss of O(2) production. Thus, eNOS may straddle a bifurcating pathway that can lead to the formation of either NO or O(2), interrelated but often opposing free radical messengers. This arrangement has possible implications for atherosclerosis and septic shock where endothelial dysfunction results from imbalances in NO and O(2) production.
Wang
W
W
Critical Care Medicine Department, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland 20892, USA.
Wang
S
S
Yan
L
L
Madara
P
P
Del Pilar Cintron
A
A
Wesley
R A
RA
Danner
R L
RL
D001665:Binding Sites; D005998:Glycine / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D009249:NADP / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D017382:Reactive Oxygen Species; D017384:Sequence Deletion; D015398:Signal Transduction; D013481:Superoxides / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D020298:U937 Cells
D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D013481:Superoxides; D009569:Nitric Oxide; D009249:NADP; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D005998:Glycine