nitrogen-dioxide and linsidomine

Surfactant protein D (SP-D) is an important effector of innate immunity. We have previously shown that SP-D accumulates at sites of acute bacterial infection and neutrophil infiltration, a setting associated with the release of reactive species such as peroxynitrite. Incubation of native SP-D or trimeric SP-D lectin domains (NCRDs) with peroxynitrite resulted in nitration and nondisulfide cross-linking. Modifications were blocked by peroxynitrite scavengers or pH inactivation of peroxynitrite, and mass spectroscopy confirmed nitration of conserved tyrosine residues within the C-terminal neck and lectin domains. Mutant NCRDs lacking one or more of the tyrosines allowed us to demonstrate preferential nitration of Tyr314 and the formation of Tyr228-dependent cross-links. Although there was no effect of peroxynitrite or tyrosine mutations on lectin activity, incubation of SP-D dodecamers or murine lavage with peroxynitrite decreased the SP-D-dependent aggregation of lipopolysaccharide-coated beads, supporting our hypothesis that defective aggregation results from abnormal cross-linking. We also observed nitration, cross-linking of SP-D, and a significant decrease in SP-D-dependent aggregating activity in the lavage of mice acutely exposed to nitrogen dioxide. Thus, modification of SP-D by reactive oxygen-nitrogen species could contribute to alterations in the structure and function of SP-D at sites of inflammation in vivo.

Topics: Amino Acid Sequence; Animals; Bronchoalveolar Lavage Fluid; Humans; Mice; Molsidomine; Nitrogen Dioxide; Peroxynitrous Acid; Protein Structure, Quaternary; Protein Structure, Tertiary; Pulmonary Surfactant-Associated Protein D; Rats; Recombinant Proteins; Tandem Mass Spectrometry; Tyrosine

In blood, peroxynitrite (ONOO- ) and CO2 react to form NO2* and CO3* through the short-lived adduct ONOOCO2-, leading to protein-bound tyrosine nitration. ONOO(-) -modified LDL is atherogenic. Oxidatively modified LDL generally appears in the vessel wall surrounded by antioxidants. Human serum albumin (HSA) is one of them, partly associated to LDL as a LDL-albumin complex (LAC). The purpose was to study the effect of a mild nitration on LAC and whether albumin may interfere with LDL nitration. To do so, SIN-1 was used as ONOO- generator in the presence or absence of 25 mM HCO3-. The human serum albumin (HSA)-bound tyrosine nitration rate was found to be 4.4 x 10(-3) mol/mol in the presence of HCO3-. HSA decreased the LAC-tyrosine nitration rate from 2.5 x 10(-3) to 0.6 x 10(-3) mol/mol. It was concluded that HSA impaired the apoB-bound tyrosine nitration. These findings raise the question of the patho-physiological significance of these nitrations and their interactions which may potentially prevent both atheromatous plaque formation and endothelium dysfunction in particular and appear to be beneficial in terms of atherogenic risk.

Topics: Antioxidants; Atherosclerosis; Humans; Lipoproteins, LDL; Molsidomine; Nitric Oxide Donors; Nitrogen Dioxide; Oxidation-Reduction; Peroxynitrous Acid; Serum Albumin; Tyrosine

In mammalian cells DNA damage activates a checkpoint that halts progression through S phase. To determine the ability of nitrating agents to induce S-phase arrest, mouse C10 cells synchronized in S phase were treated with nitrogen dioxide (NO(2)) or SIN-1, a generator of reactive nitrogen species (RNS). SIN-1 or NO(2) induced S-phase arrest in a dose- and time-dependent manner. As for the positive controls adozelesin and cisplatin, arrest was accompanied by phosphorylation of ATM kinase; dephosphorylation of pRB; decreases in RF-C, cyclin D1, Cdc25A, and Cdc6; and increases in p21. Comet assays indicated that RNS induce minimal DNA damage. Moreover, in a cell-free replication system, nuclei from cells treated with RNS were able to support control levels of DNA synthesis when incubated in cytosolic extracts from untreated cells, whereas nuclei from cells treated with cisplatin were not. Induction of phosphatase activity may represent one mechanism of RNS-induced arrest, for the PP1/PP2A phosphatase inhibitor okadaic acid inhibited dephosphorylation of pRB; prevented decreases in the levels of RF-C, cyclin D1, Cdc6, and Cdc25A; and bypassed arrest by SIN-1 or NO(2), but not cisplatin or adozelesin. Our studies suggest that RNS may induce S-phase arrest through mechanisms that differ from those elicited by classical DNA-damaging agents.

Topics: Animals; Ataxia Telangiectasia Mutated Proteins; cdc25 Phosphatases; Cell Cycle; Cell Cycle Proteins; Cell Line; Cells, Cultured; Cisplatin; Cyclin D1; DNA; DNA Damage; DNA-Binding Proteins; Dose-Response Relationship, Drug; In Vitro Techniques; Mice; Molsidomine; Nitrogen Dioxide; Nuclear Proteins; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Protein Serine-Threonine Kinases; Reactive Nitrogen Species; Replication Protein C; Retinoblastoma Protein; S Phase; Time Factors; Tumor Suppressor Proteins

This study demonstrated the direct formation of the nitrogen dioxide (*NO2) radical during the decomposition of 3-morpholinosydnonimine (SIN-1) in biological buffer 4-morpholinoethanosulfone acid solution. Consequently, at approximately pH 4, SIN-1 can be used successfully as a source of *NO2. This conclusion is drawn from a comparison of the reactions of cis-[Cr(C2O4)(L- L)(OH2)2]+, where L-L denotes pyridoxamine (Hpm) or histamine (hm), with the gaseous *NO2 radical obtained by two methods: from SIN-1 and from a simple redox reaction. These reactions were investigated using the stopped-flow technique. The measurements were carried out at temperatures ranging from 5 to 25 degrees C over a pH range from 6.52 to 9.11 for cis-[Cr(C2O4)(Hpm) (OH2)2]+ and from 6.03 to 8.15 for cis-[Cr(C2O4)(hm)(OH2)2] +. We also determined the thermodynamic activation parameter (E(a)) and the uptake mechanism for each of the coordination compounds studied.

Topics: Chromium; Chromium Compounds; Free Radicals; Histamine; Hydrogen-Ion Concentration; Kinetics; Molsidomine; Nitrogen Dioxide; Pyridoxamine; Spectrophotometry; Temperature; Thermodynamics

SIN-1 is frequently used in cell culture studies as an extracellularly operating generator of peroxynitrite. However, little is known about the nature of the reactive species produced intracellulary from SIN-1. SIN-1 can easily penetrate cells as exemplified for both L-929 mouse fibroblasts and bovine aortic endothelial cells (BAECs) by utilizing capillary zone electrophoresis. In L-929 cells, SIN-1 produced nitric oxide (*NO) as monitored by the fluorescent *NO scavenger FNOCT-1 and by means of a *NO electrode, as well as reactive nitrogenoxide species (RNOS, e.g. peroxynitrite, nitrogen dioxide, dinitrogen trioxide), as detected with the fluorescent indicator DAF-2. Laser scanning microscopy revealed that in L-929 cells SIN-1 -derived species initially oxidized the major fraction of the NAD(P)H within the cytosol and the nuclei, whereas the mitochondrial NAD(P)H level was somewhat increased. In marked contrast to this, in BAECs no evidence for *NO formation was found although the intracellular amount of SIN-1 was four-fold higher than in L-929 cells. In BAECs, the level of NAD(P)H was slightly decreased within the first 10 min after administration of SIN-1 in both the cytosol/nuclei and mitochondria. These observations reflect the capability of SIN-1 to generate intracellularly either almost exclusively RNOS as in BAECs, or RNOS and freely diffusing *NO as in L-929 cells. Nitric oxide as well as RNOS may decisively affect cellular metabolism as indicated by the alterations in the NAD(P)H level. Hence, care should be taken when applying SIN-1 as an exclusively peroxynitrite-generating compound in cell culture systems.

Topics: Animals; Cattle; Cell Line; Endothelial Cells; Fibroblasts; Free Radicals; Mice; Molecular Structure; Molsidomine; NADP; Nitric Oxide; Nitrogen Dioxide; Time Factors

Endogenous sources of reactive nitrogen species (RNS) act as second messengers in a variety of cell signaling events, whereas environmental sources of RNS like nitrogen dioxide (NO2) inhibit cell survival and growth through covalent modification of cellular macromolecules. To examine the effects of RNS on cell cycle progression, murine type II alveolar C10 cells arrested in G0 by serum deprivation were exposed to either NO2 or SIN-1, a generator of RNS, during cell cycle re-entry. In serum-stimulated cells, RNS did not prevent the immediate early gene response by AP-1, but rather blocked cyclin D1 gene expression, resulting cell cycle arrest at the boundary between G0 and G1. Dichlorofluorescin diacetate (DCF) fluorescence indicated that RNS induced sustained production of intracellular hydrogen peroxide (H2O2), which normally is produced only transiently in response to serum growth factors. Loading cells with catalase did not diminish the formation of 3-nitrotyrosine on the cell surface, but rather prevented enhanced DCF fluorescence and rescued cyclin D1 expression and S phase entry. These studies indicate environmental RNS interfere with cell cycle re-entry through an H2O2-dependent mechanism that influences expression of cyclin D1 and progression from G0 to the G1 phase of the cell cycle.

Topics: Animals; Catalase; Cell Cycle; Cells, Cultured; Cyclin D1; Epithelial Cells; Fluoresceins; Gene Expression Regulation; Hydrogen Peroxide; Mice; Molsidomine; Nitric Oxide Donors; Nitrogen Dioxide; Oxidants; Pulmonary Alveoli; Signal Transduction; Superoxide Dismutase; Transcription Factor AP-1; Tyrosine

Nitrogen dioxide (*NO2) is commonly known as an indoor and outdoor air pollutant. Inhalation of *NO2 is associated with epithelial cell injury, inflammation, and the aggravation of asthma. *NO2 can also be formed during inflammation, by the metabolism of nitric oxide. We describe a gas-phase exposure system for in vitro exposure of lung epithelial cells to *NO2. Immunofluorescence revealed 3-nitrotyrosine immunoreactivity of rat alveolar type II epithelial cells exposed to 5 parts per million of *NO2 for 4 h. Comparative analysis of log-phase and confluent cultures demonstrated that cell death occurred extensively in log-phase cells, whereas minimal death was observed in confluent cultures. Peroxynitrite (ONOO-) or the ONOO- generator 3-morpholinosydnonimine (SIN-1) caused similar amounts of death. Further, exposure of wounded cell cultures to *NO(2) or SIN-1 revealed that death was restricted to cells repopulating a wounded area. Cycloheximide or actinomycin D, inhibitors or protein and messenger RNA synthesis, respectively, significantly reduced terminal transferase reactivity, suggesting that a new protein(s) may be required for cell death. These results suggest that during restitution after pulmonary injury, epithelium may be sensitive to cell death by reactive nitrogen species.

Topics: Cell Count; Cell Death; Cells, Cultured; Culture Media, Conditioned; DNA, Single-Stranded; In Situ Nick-End Labeling; Lung; Molsidomine; Nitrates; Nitric Oxide Donors; Nitrites; Nitrogen Dioxide; Oxidative Stress; Respiratory Mucosa

Peroxynitrite is the product of the reaction between nitric oxide and superoxide. It is an oxidant which can also decompose to form the hydroxyl radical and nitrogen dioxide. In this report we show that a powerful oxidant with reactivity similar to that of the hydroxyl radical is formed from the generation of superoxide from xanthine oxidase and nitric oxide from S-nitroso-n-acetylpenicillamine (SNAP). Simultaneous generation of these two radicals by either xanthine oxidase/SNAP or the sydnonimine SIN-1 in the presence of low-density lipoprotein (LDL) results in the depletion of alpha-tocopherol and formation of its oxidised product alpha-tocopheroquinone. The mechanism of oxidation required both the formation of nitric oxide and superoxide. In contrast to the promotion of LDL oxidation by transition metals the oxidation of LDL by SIN-1 was not sensitive to the addition of exogenous lipid hydroperoxide.

Topics: Acetaldehyde; Catalase; Free Radicals; Humans; Hydroxides; Hydroxyl Radical; Lipoproteins, LDL; Molsidomine; Nitric Oxide; Nitrogen Dioxide; Oxidation-Reduction; Penicillamine; S-Nitroso-N-Acetylpenicillamine; Superoxide Dismutase; Superoxides; Thiobarbiturates; Vitamin E; Xanthine Oxidase