2-phenyl-4-4-5-5-tetramethylimidazoline-1-oxyl-3-oxide and linsidomine

Intracellular proteins involved in oxidative stress and apoptosis are nitrated in diseased tissues but not in normal tissues; definitive evidence to support a causative link between a specific protein that is nitratively modified with tissue injury in a specific disease is limited, however. The aims of the present study were to determine whether thioredoxin (Trx), a novel antioxidant and antiapoptotic molecule, is susceptible to nitrative inactivation and to establish a causative link between Trx nitration and postischemic myocardial apoptosis.. In vitro exposure of human Trx-1 to 3-morpholinosydnonimine resulted in significant Trx-1 nitration and almost abolished Trx-1 activity. 3-morpholinosydnonimine-induced nitrative Trx-1 inactivation was completely blocked by MnTE-2-PyP(5+) (a superoxide dismutase mimetic) and markedly attenuated by PTIO (a nitric oxide scavenger). Administration of either reduced or oxidized Trx-1 in vivo attenuated myocardial ischemia/reperfusion injury (>50% reduction in apoptosis and infarct size, P<0.01). However, administration of nitrated Trx-1 failed to exert a cardioprotective effect. In cardiac tissues obtained from ischemic/reperfused heart, significant Trx-1 nitration was detected, Trx activity was markedly inhibited, Trx-1/ASK1 (apoptosis signal-regulating kinase-1) complex formation was abolished, and apoptosis signal-regulating kinase-1 activity was increased. Treatment with either FP15 (a peroxynitrite decomposition catalyst) or MnTE-2-PyP(5+) 10 minutes before reperfusion blocked nitrative Trx inactivation, attenuated apoptosis signal-regulating kinase-1 activation, and reduced postischemic myocardial apoptosis.. These results strongly suggest that nitrative inactivation of Trx plays a proapoptotic role under those pathological conditions in which production of reactive nitrogen species is increased and that antinitrating treatment may have therapeutic value in those diseases, such as myocardial ischemia/reperfusion, in which pathological apoptosis is increased.

Topics: Amino Acid Substitution; Animals; Apoptosis; Cardiotonic Agents; Cyclic N-Oxides; Free Radical Scavengers; Humans; Imidazoles; Male; MAP Kinase Kinase Kinase 5; MAP Kinase Signaling System; Metalloporphyrins; Mice; Molsidomine; Mutagenesis, Site-Directed; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; NADP; Oxidation-Reduction; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Peroxynitrous Acid; Thioredoxins

NO-Aspirin (NCX-4016) releases nitric oxide (NO) in biological systems through as yet unidentified mechanisms. In LLC-PK1 kidney epithelial cells, a 5-h pretreatment with glyceryl trinitrate (GTN, 0.1-1 microM) significantly attenuated the cyclic GMP response to a subsequent challenge with both NO-aspirin or GTN. Similarly, NO-aspirin (10-100 microM) was found to induce tolerance to its own cyclic GMP stimulatory action and to that of GTN. In contrast, cyclic GMP stimulation by the spontaneous NO donor SIN-1, which releases NO independently of enzymatic catalysis, remained unimpaired in cells pretreated with GTN or NO-aspirin. The observed cross-tolerance between NO-aspirin and GTN cells indicates that bioactivation pathways of organic nitrates, which have been shown to involve cytochrome P450, may also be responsible for NO release from NO-aspirin. Prolonged treatment with NO-aspirin causes down-regulation of the cellular cyclic GMP response, suggesting that tolerance may occur during therapy with NO-aspirin.

Topics: Animals; Aspirin; Cell Line; Cyclic GMP; Cyclic N-Oxides; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Epithelial Cells; Free Radical Scavengers; Imidazoles; Kidney; Molsidomine; Nitric Oxide; Nitric Oxide Donors; Nitroglycerin; Protein Binding; Swine

SIN-1 has been used, in vitro, to simultaneously generate nitric oxide (*NO) and superoxide (O*-2). However, the pharmacological activity of SIN-1 resembles that of a *NO donor. SIN-1 decays by a three-step mechanism. After initial isomerization to an open ring form, SIN-1A reduces oxygen by a one-electron transfer reaction to give O*-2 and the SIN-1 cation radical, which decomposes to form SIN-1C and *NO. Here we report that one-electron oxidizing agents, in addition to oxygen, can oxidize SIN-1A, resulting in the release of *NO without the concomitant formation of O*-2. We demonstrate that easily reducible nitroxides, such as the nitronyl and imino nitroxides, are able to oxidize SIN-1. Biological oxidizing agents such as ferricytochrome c also stimulate *NO production from SIN-1. In addition, decomposition of SIN-1 by human plasma or by the homogenate of rat liver, kidney, and heart tissues results in the formation of *NO. Our findings suggest that SIN-1 may react with heme proteins and other electron acceptors in biological systems to produce *NO. Thus, at the relatively low in vivo oxygen concentrations, SIN-1 is likely to behave more like an *NO donor than a peroxynitrite donor. The relevance of this reaction to myocardial protection afforded by SIN-1 in ischemia/reperfusion-induced injury is discussed.

Topics: Animals; Cyclic N-Oxides; Free Radical Scavengers; Hemeproteins; Humans; Imidazoles; Kidney; Liver; Molsidomine; Myocardium; Nitrates; Nitric Oxide Donors; Oxidants; Rats

In this work, we addressed the issue of whether exogenous NO and ONOO- (peroxynitrite) are able to alter growth, viability and/or differentiation of normal epithelial cells using cultured normal human keratinocytes as a model. 3-Morpholino-sydnonimine (SIN-1), a donor of both NO and O2(-)., leading to the production of ONOO-, dose-dependently inhibited growth of human keratinocytes without loss of viability. This inhibitory effect was lowered when SIN-1 was transformed into a pure NO donor by scavenging O2(-). with superoxide dismutase/catalase. Finally, scavenging NO release from SIN-1 with carboxy-1H-imidazol-1-yloxy,2-(4-carboxyp henyl)-4,5-dihydro-4,4,5,5 -tetramethyl-3-oxide (PTIO) resulted in a loss of the inhibitory effect of SIN-1. Together these findings suggest that both ONOO- and NO exert a growth inhibitory effect on human keratinocytes without cytotoxicity. Further support for this conclusion came from the treatment of human keratinocytes with the NO. donor propanamine 3-(2-hydroxy-2-nitroso-1-propyl hydrazino) or with authentic peroxynitrite. Moreover, only SIN-1 or peroxynitrite, and not NO, was able to trigger the expression of markers of terminal differentiation in human keratinocytes. From a physiological perspective, the ability of peroxynitrite, a known genotoxic and potentially carcinogenic agent, to direct proliferating keratinocytes towards terminal differentiation may be crucial to protect the genomic stability of this barrier epithelium.

Topics: Catalase; Cell Differentiation; Cell Division; Cyclic N-Oxides; Fluorescent Antibody Technique; Free Radical Scavengers; Humans; Hydrazines; Imidazoles; Keratinocytes; Molsidomine; Nitrates; Nitric Oxide; Superoxide Dismutase; Thymidine

In C6 glioma cells, the nitric oxide (NO) donor 3-morpholinosynonimine hydrochloride (SIN-1) (0.5 mM) produced a significant decrease in the stimulatory G-protein alpha subunit (G alpha(s)) levels. Northern hydridization did not detect any differences in G alpha(s) mRNA levels after SIN-1 treatment. Furthermore SIN-1 increased endogenous and cholera toxin-catalyzed ADP-ribosylation of G alpha(s). 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) (0.5mM), a NO scavenger, had no effect on endogenous or cholera toxin-catalyzed ADP-ribosylation of G alpha(s), but reversed the increase in endogenous and cholera toxin-catalyzed ADP-ribosylation of G alpha(s) induced by SIN-1. These results suggest that increasing ADP-ribosylation may be involved in SIN-1 mediated G alpha(s) down-regulation.

Topics: Adenosine Diphosphate Ribose; Animals; Blotting, Northern; Cholera Toxin; Cyclic N-Oxides; Down-Regulation; Enzyme Inhibitors; Glioma; GTP-Binding Proteins; Imidazoles; Molsidomine; Nitric Oxide; Rats; RNA, Messenger; Tumor Cells, Cultured