s-nitro-n-acetylpenicillamine and Inflammation

Using the electromobility shift assay (EMSA) in the rat myoblast system, the activation of transcription factor NF-kappaB by reactive nitrogen species was evaluated. Two distinct patterns of activation were demonstrated. Whereas NO donor, SNAP, activated NF-kappaB in the classical pathway, which led to a transient response, NF-kappaB activation by peroxynitrite donor, SIN-1, was mediated by an alternative pathway, which has been demonstrated in previous works to involve tyrosine nitration of the NF-kappaB inhibitory protein I-kappaB alpha. This led to a constitutive non-transient activation of NF-kappaB and a prolonged inflammatory reaction. Lymphocytes exposed to mild intensity of cigarette smoke for 8 h, which activated NF-kappaB, exhibited a decrease in the fraction of apoptotic cells from 27% to 19% compared with lymphocytes exposed to atmospheric air, using the FACS Annexin V assay. This also has been shown in previous works to be mediated by peroxynitrite. Thus, mild exposure to cigarette smoke induces NF-kappaB activation, which can attenuate apoptosis in human lymphocytes and lead to prolonged inflammatory response. A possible proposed mechanism for induction of chronic inflammatory response may involve peroxynitrite-induced activation of NF-kappaB.

Topics: Animals; Cell Line; Cells, Cultured; Humans; Inflammation; Lymphocytes; Molsidomine; NF-kappa B; Nicotiana; Nitric Oxide Donors; Penicillamine; Peroxynitrous Acid; Rats; Smoke

Cyclooxygenase-1 and cyclooxygenase-2 mRNAs and proteins and prostaglandin E(2) production are evaluated in a rat model of inflammation in which Escherichia coli lipopolysaccharide is intraperitoneally injected or intravesically instilled into the bladder. While cyclooxygenase-1 mRNA and protein and cyclooxygenase-2 mRNA do not change in bladders treated with lipopolysaccharide, cyclooxygenase-2 protein is elevated in bladders from rats intravesically instilled with lipopolysaccharide or phosphate buffered saline (PBS) or intraperitoneally injected with lipopolysaccharide. Urinary prostaglandin E(2) levels and prostaglandin E(2) synthesis in bladder particulates are elevated by intravesical instillation and intraperitoneal injection of lipopolysaccharide. The nitric oxide donor, S-nitroso-N-acetyl-D,L-penicillamine, increases prostaglandin E(2) synthesis in bladders from lipopolysaccharide intravesically instilled and intraperitoneally injected rats. Lipopolysaccharide increases prostaglandin E(2) synthesis by increasing cyclooxygenase-2 protein levels in rat bladder and prostaglandin E(2) synthesis may be further elevated by increases in nitric oxide caused by an up-regulation of inducible nitric oxide synthase (iNOS).

Topics: Administration, Intravesical; Animals; Blotting, Western; Cyclooxygenase 1; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Dinoprostone; Female; Inflammation; Injections, Intraperitoneal; Isoenzymes; Lipopolysaccharides; Membrane Proteins; Niflumic Acid; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Oxidants; Penicillamine; Prostaglandin-Endoperoxide Synthases; Protamines; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Up-Regulation; Urinary Bladder

Extracellular-superoxide dismutase (EC-SOD) is bound to the vascular endothelial cell surface with an affinity for heparan sulfate proteoglycan. The binding of EC-SOD to the human umbilical vein endothelial cell (HUVEC) and bovine aortic endothelial cell surface proteoglycans was significantly decreased by the incubation with S-nitroso-N-acetyl-DL-penicillamine (SNAP) and +/- -N-[(E)-4-ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexene-1-yl]-3-pyridine carboxamide (NOR4), potent nitric oxide (NO) donors. NO derived from lipopolysaccharide-stimulated J774 A-1 cells also decreased the binding of EC-SOD to HUVEC, and this decrease was blocked by N(G)-nitro-L-arginine, a nitric oxide synthase inhibitor. SNAP and NOR4 also decreased the binding of EC-SOD to immobilized heparin. Furthermore, the decomposed derivatives of NO donors and sodium nitrite decreased the binding of EC-SOD. These observations suggest that excess NO produced in the inflammatory conditions decreases the binding of EC-SOD to the vascular endothelial cell surface, which results in a loss of the ability to protect the endothelial cell surface from oxidative stress.

Topics: Animals; Cattle; Cell Membrane; Cells, Cultured; Dose-Response Relationship, Drug; Endothelium, Vascular; Enzyme Inhibitors; Extracellular Matrix; Heparin; Humans; Inflammation; Lipopolysaccharides; Macrophages; Nitric Oxide; Nitric Oxide Donors; Nitrites; Oxidative Stress; Penicillamine; Protein Binding; Proteoglycans; Pyridines; Superoxide Dismutase; Umbilical Veins

Vascular endothelial cell injury plays an important role in the pathogenesis of inflammatory-mediated tissue injury. In the current study, we assessed injury in primary cultures of endothelial cells obtained from different sites within the same species, comparing rat dermal microvascular and rat lung microvascular endothelial cells. Dermal microvascular-derived endothelial cells were more sensitive to killing by PMA (phorbol myristate acetate)-activated human neutrophils than were endothelial cells derived from lung microvasculature. Lung endothelial cells stimulated with interferon-gamma plus lipopolysaccharide (IFNgamma + LPS) generated high levels of nitric oxide (*NO), while dermal endothelial cells stimulated with IFNgamma + LPS generated significantly lower levels of *NO. Under conditions of *NO generation (IFNgamma + LPS stimulation), or in the presence of the *NO donor, S-nitroso-N-acetyl penicillamine (SNAP), endothelial cell killing by PMA-activated neutrophils was reduced. Lung endothelial cells stimulated with PMA generated less superoxide (02*-) than dermal endothelial cells. Under conditions of *NO generation (IFNgamma + LPS stimulation), or in the presence of SNAP, O2*- release from endothelial cells was reduced. Endothelial cell-derived *NO appeared to play a significant role in attenuating the neutrophil-mediated killing. The differences in the ability of endothelial cells to generate *NO and 02*- underlies, at least in part, the differences in susceptibility of these cells to injury by activated neutrophils.

Topics: Animals; Catalase; Cells, Cultured; E-Selectin; Endothelium, Vascular; Fluorescent Dyes; Free Radical Scavengers; Humans; Inflammation; Intercellular Adhesion Molecule-1; Interferon-gamma; Lipopolysaccharides; Lung; Neutrophils; Nitric Oxide; Penicillamine; Rats; Rats, Long-Evans; Reactive Oxygen Species; Skin; Superoxides; Tumor Necrosis Factor-alpha