© 2004 by European Society of Cardiology
Copyright © 2004, European Society of Cardiology
NO suppresses while peroxynitrite sustains NF-
B: a paradigm to rationalize cytoprotective and cytotoxic actions attributed to NO
aDepartment of Endocrinology and Metabolism, Dokkyo University School of Medicine, Mibu, Tochigi, 321-0293, Japan
bDepartment of Pharmacology and the Program in Biochemistry and Structural Biology, Weill Medical College of Cornell University, New York, NY, USA
*Corresponding author. Tel./fax: +81-282-87-2150. Email address: yhattori{at}dokkyomed.ac.jp
Objective: NO has both cytoprotective and cytotoxic effects. A key cytoprotective action of NO is attributed to inhibition of nuclear factor-
B (NF-B)-mediated gene expression; this potentially endows NO with ubiquitous anti-inflammatory activity. Since immunostimulant-induced iNOS gene expression is itself dependent on NF-B, 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-B 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-B activation and expression of a murine iNOS promoter/reporter construct. An NO donor also suppressed NF-B activation when induced by IL-1β or TNF
. 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-B. Although ONOO– generation from SIN-1 did not inhibit NF-B activation, conversion of SIN-1 to a pure NO donor (by addition of excess superoxide dismutase) resulted in potent inhibition of NF-B activation. Dose–response analyses suggest that the inhibitory effect of NO on iNOS gene transcription results specifically from inhibition of NF-B activation, and is mediated by a G-cyclase-independent mechanism that is unavailable to ONOO–. LPS stimulates IB- phosphorylation by inducing IB kinase (IKK) activity, and NO, but not ONOO–, inhibits LPS-induced IB- phosphorylation and IKK activity. Conclusion: We demonstrate that only NO inhibits the activation of NF-B and suppresses iNOS gene expression. This distinction provides a novel paradigm to rationalize cytoprotective and cytotoxic actions attributed to NO.
KEYWORDS Nitric oxide; Peroxynitrite; Nuclear factor B; IB kinase; Vascular smooth muscle cells
Time for primary review 26 days
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