Superoxide fluxes limit nitric oxide-induced signaling.
Independently, superoxide (O2-) and nitric oxide (NO) are biologically important signaling molecules. When co-generated, these radicals react rapidly to form powerful oxidizing and nitrating intermediates. Although this reaction was once thought to be solely cytotoxic, herein we demonstrate using MCF7, macrophage, and endothelial cells that when nanomolar levels of NO and O2- were produced concomitantly, the effective NO concentration was established by the relative fluxes of these two radicals. Differential regulation of sGC, pERK, HIF-1alpha, and p53 were used as biological dosimeters for NO concentration. Introduction of intracellular- or extracellular-generated O2- during NO generation resulted in a concomitant increase in oxidative intermediates with a decrease in steady-state NO concentrations and a proportional reduction in the levels of sGC, ERK, HIF-1alpha, and p53 regulation. NO responses were restored by addition of SOD. The intermediates formed from the reactions of NO with O2- were non-toxic, did not form 3-nitrotyrosine, nor did they elicit any signal transduction responses. H2O2 in bolus or generated from the dismutation of O2- by SOD, was cytotoxic at high concentrations and activated p53 independent of NO. This effect was completely inhibited by catalase, suppressed by NO, and exacerbated by intracellular catalase inhibition. We conclude that the reaction of O2- with NO is an important regulatory mechanism, which modulates signaling pathways by limiting steady-state levels of NO and preventing H2O2 formation from O2-.
Thomas
Douglas D
DD
Tumor Biology Section, Radiation Biology Branch, National Institutes of Health, Bethesda, MD 20892, USA. thomasdo@mail.nih.gov
Ridnour
Lisa A
LA
Espey
Michael Graham
MG
Donzelli
Sonia
S
Ambs
Stefan
S
Hussain
S Perwez
SP
Harris
Curtis C
CC
DeGraff
William
W
Roberts
David D
DD
Mitchell
James B
JB
Wink
David A
DA
D000818:Animals; D002417:Cattle; D002460:Cell Line; D018920:Coculture Techniques; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D016877:Oxidants / Q000378:metabolism; D010084:Oxidation-Reduction; D015290:Second Messenger Systems / Q000502:physiology*; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D016877:Oxidants; D016159:Tumor Suppressor Protein p53; D013481:Superoxides; D009569:Nitric Oxide; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase