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- Title
NO suppresses while peroxynitrite sustains NF-κB: a paradigm to rationalize cytoprotective and cytotoxic actions attributed to NO
- Authors
Hattori, Yoshiyuki; Kasai, Kikuo; Gross, Steven S.
- Abstract
Objective: NO has both cytoprotective and cytotoxic effects. A key cytoprotective action of NO is attributed to inhibition of nuclear factor-κB (NF-κB)-mediated gene expression; this potentially endows NO with ubiquitous anti-inflammatory activity. Since immunostimulant-induced iNOS gene expression is itself dependent on NF-κB, NO is expected to limit its own synthesis. On the other hand, many cytotoxic actions of NO have been attributed to the chemical reactivity of peroxynitrite (ONOO-) formed from NO by near diffusion-limited reaction with O2-. To assess whether ONOO- shares the ability of NO to inhibit NF-κB activation and consequent iNOS gene expression, we compared effects of NO donors (NOR3 and SNAP), an ONOO- donor (SIN-1), and pure ONOO- on LPS-induced responses in vascular smooth muscle cells (VSMC). Methods and results: NO donors, but not ONOO-, suppressed LPS-induced NF-κB activation and expression of a murine iNOS promoter/reporter construct. An NO donor also suppressed NF-κB activation when induced by IL-1β or TNFα. Northern blot and RT-PCR analyses showed that NO, but not ONOO- or 8-bromo-cGMP, decreases LPS-induced expression of iNOS mRNA. Electrophoretic mobility shift assays (EMSA) and immunocytochemical analyses confirmed that NO but not ONOO- inhibits nuclear translocation of NF-κB. Although ONOO- generation from SIN-1 did not inhibit NF-κB activation, conversion of SIN-1 to a pure NO donor (by addition of excess superoxide dismutase) resulted in potent inhibition of NF-κB activation. Dose–response analyses suggest that the inhibitory effect of NO on iNOS gene transcription results specifically from inhibition of NF-κB activation, and is mediated by a G-cyclase-independent mechanism that is unavailable to ONOO-. LPS stimulates IκB-α phosphorylation by inducing IκB kinase (IKK) activity, and NO, but not ONOO-, inhibits LPS-induced IκB-α phosphorylation and IKK activity. Conclusion: We demonstrate that only NO inhibits the activation of NF-κB and suppresses iNOS gene expression. This distinction provides a novel paradigm to rationalize cytoprotective and cytotoxic actions attributed to NO.
- Subjects
VASCULAR smooth muscle; BLOOD vessels; GENE expression; NITRIC oxide
- Publication
Cardiovascular Research, 2004, Vol 63, Issue 1, p31
- ISSN
0008-6363
- Publication type
Article
- DOI
10.1016/j.cardiores.2004.03.014