Induction of NF-kappaB, IkappaB-alpha, and iNOS in rat gastric mucosa during endotoxemia.
Upregulation of inducible nitric oxide synthase (iNOS) contributes to the development of gastric injury during endotoxemia. The molecular mechanisms related to its induction are unknown. Because the transcription factor nuclear factor-kappa B (NF-kappaB) regulates inflammatory genes in response to endotoxemia, we hypothesized that its activity in gastric mucosa would increase while its inhibitor IkappaB-alpha would decrease commensurate with changes in iNOS expression. Rats were given intraperitoneal saline or lipopolysaccharide (LPS; 20 mg/kg) for 5, 15, or 30 min, or for 1, 3, or 5 h, and killed, and the gastric mucosa was prepared for determination of iNOS and IkappaB-alpha by Western immunoblotting, iNOS mRNA by quantitative real-time RT-PCR, and NF-kappaB by electrophoretic mobility-shift assay. LPS caused a significant increase in iNOS mRNA and protein immunoreactivity at 1, 3, and 5 h compared to controls. NF-kappaB-binding activity increased in the nuclear fraction of gastric mucosa at 1 h and steadily increased over time after LPS administration. The activated NF-kappaB consisted mainly of p50 with a lesser amount of p65 subunits as demonstrated by a supershift assay. IkappaB-alpha decreased in gastric cytosolic fractions over time, consistent with its degradation. These data suggest that during endotoxemia expression of the inflammatory mediator iNOS in the gastric mucosa may be upregulated by degradation of IkappaB-alpha and subsequent translocation of NF-kappaB into the nucleus and increased NF-kappaB activity.
Helmer
Kenneth S
KS
Department of Surgery, University of Texas Medical School, Houston, Texas 77030, USA.
Chang
Lily
L
Cui
Yan
Y
Mercer
David W
DW
D000818:Animals; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D019446:Endotoxemia / Q000378:metabolism*; D005260:Female; D005753:Gastric Mucosa / Q000187:drug effects / Q000378:metabolism*; D020672:I-kappa B Proteins / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D016328:NF-kappa B / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors
D020672:I-kappa B Proteins; D008070:Lipopolysaccharides; D016328:NF-kappa B; D004247:DNA; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat