bay-11-7082 and thiazolyl-blue

A defect in mitochondrial activity contributes to many diseases. We have shown that monolayers of the human colonic T84 epithelial cell line exposed to dinitrophenol (DNP, uncouples oxidative phosphorylation) and nonpathogenic Escherichia coli (E. coli) (strain HB101) display decreased barrier function. Here the impact of DNP on macrophage activity and the effect of TNF-alpha, DNP, and E. coli on epithelial permeability were assessed. DNP treatment of the human THP-1 macrophage cell line resulted in reduced ATP synthesis, and, although hyporesponsive to LPS, the metabolically stressed macrophages produced IL-1beta, IL-6, and TNF-alpha. Given the role of TNF-alpha in inflammatory bowel disease (IBD) and the association between increased permeability and IBD, recombinant TNF-alpha (10 ng/ml) was added to the DNP (0.1 mM) + E. coli (10(6) colony-forming units), and this resulted in a significantly greater loss of T84 epithelial barrier function than that elicited by DNP + E. coli. This increased epithelial permeability was not due to epithelial death, and the enhanced E. coli translocation was reduced by pharmacological inhibitors of NF-kappabeta signaling (pyrrolidine dithiocarbamate, NF-kappabeta essential modifier-binding peptide, BAY 11-7082, and the proteosome inhibitor, MG132). In contrast, the drop in transepithelial electrical resistance was unaffected by the inhibitors of NF-kappabeta. Thus, as an integrative model system, our findings support the induction of a positive feedback loop that can severely impair epithelial barrier function and, as such, could contribute to existing inflammation or trigger relapses in IBD. Thus metabolically stressed epithelia display increased permeability in the presence of viable nonpathogenic E. coli that is exaggerated by TNF-alpha released by activated immune cells, such as macrophages, that retain this ability even if they themselves are experiencing a degree of metabolic stress.

Topics: Adenosine Triphosphate; Blotting, Western; Caspases; Cell Death; Cell Line; Cells, Cultured; Dinitrophenols; Electric Impedance; Epithelium; Escherichia coli Infections; Feedback, Physiological; Humans; Interleukin-1beta; Interleukin-6; Leupeptins; Lipopolysaccharides; Macrophages; NF-kappa B; Nitriles; Permeability; Stress, Physiological; Sulfones; Tetrazolium Salts; Thiazoles; Tumor Necrosis Factor-alpha; Uncoupling Agents

Lipid peroxidation and its end-product, 4-hydroxyhexenal (HHE), are known to affect redox balance during aging, which causes various degenerative processes including vascular alterations from endothelial cell deterioration. To better understand the molecular action of HHE in the development of vascular abnormalities during the aging process, we investigated whether the upregulation of inducible endothelial nitric oxide synthase (iNOS) by HHE is mediated through nuclear factor kappaB (NF-kappaB) activation. Results indicate that HHE stimulates iNOS by the transcriptional regulation of NF-kappaB activation through cytosolic kappaB degradation inhibitors (IkappaB). Pretreatment with NF-kappaB inhibitors Bay 11-7082 and N-acetyl cysteine (NAC) suppressed the upregulation of iNOS by blunting IkappaB degradation and NF-kappaB binding activity. Because inflammatory stimuli induce iNOS to generate large amounts of nitric oxide (NO), intracellular NO levels in the presence of Bay 11-7082, NAC, and caffeic acid methyl ester were estimated. These inhibitors significantly suppressed the HHE-induced NO levels to a basal level. These findings strongly suggest that in endothelial cells, HHE induces iNOS gene expression through NF-kappaB activation, which can lead to vascular dysfunction by the activation of various proinflammatory genes.

Topics: Acetylcysteine; Aging; Aldehydes; Animals; Apoptosis; Blotting, Western; Cell Nucleus; Cell Survival; Coloring Agents; Culture Media, Serum-Free; Cytosol; Dose-Response Relationship, Drug; Enzyme Activation; Free Radicals; Gene Expression Regulation; Lipid Peroxidation; Luciferases; Male; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitriles; Oxidation-Reduction; Oxidative Stress; Prostate; Rats; Sulfones; Tetrazolium Salts; Thiazoles; Time Factors; Transcription, Genetic; Transfection; Up-Regulation