papa-nonoate and linsidomine

Peroxynitrite, formed by nitric oxide (NO) and superoxide, can alter protein function by nitrating amino acids such as tyrosine, cysteine, trytophan, or methionine. Inducible nitric oxide synthase (Type 2 NOS or iNOS) converts arginine to citrulline, releasing NO. We hypothesized that peroxynitrite could function as a negative feedback modulator of NO production by nitration of iNOS. Confluent cultures of the murine lung epithelial cell line, LA-4 were stimulated with cytokines to express iNOS, peroxynitrite was added, and the flasks sealed. After 3 h, NO in the headspace above the culture was sampled. Peroxynitrite caused a concentration-dependent decrease in NO. Similar results were obtained when 3-morpholinosydnonimine (SIN-1), a peroxynitrite generator, was added to the flasks. PAPA-NONOate, the NO generator, did not affect the headspace NO. Nitration of the iNOS was confirmed by detection of 3-nitrotyrosine by Western blotting. These data suggest a mechanism for inhibition of NO synthesis at inflammatory sites where iNOS, NO, and superoxide would be expected.

Topics: Animals; Base Sequence; Cell Line; DNA Primers; Enzyme Inhibitors; Epithelial Cells; Gene Expression; Hydrazines; Lung; Mice; Molsidomine; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxidants; RNA, Messenger; Tyrosine

Peroxynitrite, formed by the reaction between nitric oxide (NO) and superoxide, has been implicated in the pathogenesis of numerous disease processes. Several studies have shown that peroxynitrite-induced protein nitration may compromise enzyme and protein function. We hypothesized that peroxynitrite may regulate cytokine function during inflammation. To test this hypothesis, the neutrophil and monocyte chemotactic responses of macrophage inflammatory protein-1alpha (MIP-1alpha) incubated with and without peroxynitrite were evaluated. Peroxynitrite attenuated neutrophil chemotactic activity (NCA) and monocyte chemotactic activity (MCA) by MIP-1alpha in a dose-dependent manner (P < .05). The inhibitory effects were not significant on NCA and MCA induced by leukotriene B4 or complement-activated serum incubated with peroxynitrite. The reducing agents deferoxamine, dithiothreitol, and exogenous L-tyrosine abrogated the NCA and MCA inhibition by peroxynitrite. Papa-NONOate, an NO donor, or a combination of xanthine and xanthine oxidase to generate superoxide, did not show an inhibitory effect on NCA and MCA induced by MIP-1alpha. In contrast, 3-morpholinosydnonimine (SIN-1), a peroxynitrite generator, elicited a concentration-dependent reduction in NCA and MCA induced by MIP-1alpha. Consistent with its capacity to reduce NCA and MCA, peroxynitrite treatment reduced MIP-1alpha binding to neutrophils and monocytes. Nitrotyrosine was detected in the MIP-1alpha incubated with peroxynitrite. These findings are consistent with nitration of tyrosine by peroxynitrite with subsequent inhibition of MIP-1alpha binding to neutrophils and monocytes and a reduction in NCA and MCA. These data demonstrate that peroxynitrite modulates the inflammatory cell migration by MIP-1alpha, and they suggest that oxidants may play an important role in the regulation of MIP-1alpha-induced inflammatory cell chemotaxis.

Topics: Chemokine CCL3; Chemokine CCL4; Chemotaxis, Leukocyte; Complement System Proteins; Deferoxamine; Humans; Hydrazines; In Vitro Techniques; Leukotriene B4; Macrophage Inflammatory Proteins; Molsidomine; Monocytes; Neutrophils; Nitrates; Nitric Oxide; Nitric Oxide Donors; Oxidants

To better understand the mechanism(s) underlying nitric oxide (. NO)-mediated toxicity, in the presence and absence of concomitant oxidant exposure, postmitotic terminally differentiated NT2N cells, which are incapable of producing. NO, were exposed to PAPA-NONOate (PAPA/NO) and 3-morpholinosydnonimine (SIN-1). Exposure to SIN-1, which generated peroxynitrite in the range of 25-750 nM/min, produced a concentration- and time-dependent delayed cell death. In contrast, a critical threshold concentration (>440 nM/min) was required for. NO to produce significant cell injury. Examination of cells by electron microscopy shows a largely necrotic injury after peroxynitrite exposure but mainly apoptotic-like morphology after. NO exposure. Cellular levels of reduced thiols correlated with cell death, and pretreatment with N-acetylcysteine (NAC) fully protected from cell death in either PAPA/NO or SIN-1 exposure. NAC given within the first 3 h posttreatment further delayed cell death and increased the intracellular thiol level in SIN-1 but not. NO-exposed cells. Cell injury from. NO was independent of cGMP, caspases, and superoxide or peroxynitrite formation. Overall, exposure of non-. NO-producing cells to. NO or peroxynitrite results in delayed cell death, which, although occurring by different mechanisms, appears to be mediated by the loss of intracellular redox balance.

Topics: Acetylcysteine; Animals; Cell Death; Cell Differentiation; Cell Line; Cell Survival; Cyclic GMP; Hydrazines; Molsidomine; Necrosis; Neurons; Nitrates; Nitric Oxide; Nitric Oxide Donors; Oxidants; Sulfhydryl Compounds

Hydrogen carbonate (bicarbonate, HCO(3)(-)) has been proposed to accelerate the decomposition of N(2)O(3) because N-nitrosation of morpholine via a nitric oxide/oxygen mixture ((*)NO/O(2)) was inhibited by the addition of HCO(3)(-) at pH 8.9 [Caulfield, J. L., Singh, S. P., Wishnok, J. S., Deen, W. M., and Tannenbaum, S. R. (1996) J. Biol. Chem. 271, 25859-25863]. In the study presented here, it is shown that carbon dioxide (CO(2)) is responsible for this kind of protective effect because of formation of amine carbamates, whereas an inhibitory function of HCO(3)(-) is excluded. N-Nitrosation of morpholine (1-10 mM) at pH 7.4-7.5 by the (*)NO-donor compounds PAPA NONOate and MAMA NONOate (0.5 mM each) was not affected by the presence of large amounts of HCO(3)(-) (up to 100 mM) in aerated aqueous solution. Similar results were obtained by replacing the (*)NO-donor compounds with authentic (*)NO (900 microM). In agreement with data from the study cited above, (*)NO/O(2)-mediated formation of N-nitrosomorpholine (NO-Mor) was indeed inhibited by about 45% in the presence of 50 mM HCO(3)(-) at pH 8.9. However, 500 MHz (13)C NMR analysis with (13)C-enriched bicarbonate revealed that significant amounts of morpholine carbamate are formed from reaction of equilibrated CO(2) with morpholine (1-100 mM) at pH 8.9, but only to a minor extent at pH 7. 5. The protective effect of morpholine carbamate formation is explained by a significantly reduced charge density at nitrogen. This view is supported by the results of density functional theory/natural population analysis, i.e., quantumchemical calculations for morpholine and morpholine carbamate. In agreement with its lower pK(a), another secondary amine, piperazine, had already produced significant amounts of piperazine carbamate at pH 7. 4 as shown by (13)C NMR spectrometry. Consequently, and in contrast to morpholine, N-nitrosation of piperazine (2 mM) by both (*)NO/O(2) (PAPA NONOate, 0.5 mM) and the (*)NO/O(2)(-)(*)-releasing compound SIN-1 (1 mM) was inhibited by about 66% in the presence of 200 mM HCO(3)(-).

Topics: Bicarbonates; Carbamates; Carbon Dioxide; Hydrazines; Molsidomine; Morpholines; Nitric Oxide; Nitrosation

Eosinophils and increased production of nitric oxide (NO) and superoxide, components of peroxynitrite, have been implicated in the pathogenesis of a number of allergic disorders including asthma. Peroxynitrite induced protein nitration may compromise enzyme and protein function. We hypothesized that peroxynitrite may modulate eosinophil migration by modulating chemotactic cytokines. To test this hypothesis, the eosinophil chemotactic responses of regulated on activation, normal T cell expressed and secreted (RANTES) and interleukin (IL)-5 incubated with and without peroxynitrite were evaluated. Peroxynitrite-attenuated RANTES and IL-5 induced eosinophil chemotactic activity (ECA) in a dose-dependent manner (P < 0.05) but did not attenuate leukotriene B4 or complement-activated serum ECA. The reducing agents deferoxamine and dithiothreitol reversed the ECA inhibition by peroxynitrite, and exogenous L-tyrosine abrogated the inhibition by peroxynitrite. PAPA-NONOate, a NO donor, or superoxide generated by lumazine or xanthine and xanthine oxidase, did not show an inhibitory effect on ECA. The peroxynitrite generator, 3-morpholinosydnonimine, caused a concentration-dependent inhibition of ECA. Peroxynitrite reduced RANTES and IL-5 binding to eosinophils and resulted in nitrotyrosine formation. These findings are consistent with nitration of tyrosine by peroxynitrite with subsequent inhibition of RANTES and IL-5 binding to eosinophils and suggest that peroxynitrite may play a role in regulation of eosinophil chemotaxis.

Topics: Chemokine CCL5; Chemotaxis; Deferoxamine; Dithiothreitol; Dose-Response Relationship, Drug; Eosinophils; Humans; Hydrazines; Interleukin-5; Leukotriene B4; Molsidomine; Nitrates; Nitric Oxide; Nitrogen; Pteridines; Reactive Oxygen Species; Superoxides; Tyrosine; Xanthine

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

Clinically, nitric oxide (NO*) is widely used as a pulmonary vaso- and bronchodilator agent. However, the precise molecular mechanisms by which NO. induces smooth muscle relaxation are not well established. It has been suggested that NO. relaxes airway smooth muscle (ASM) via a 3',5'-cyclic guanosine monophosphate (cGMP)-dependent pathway, and our previous work has shown that Ca2+-activated K+ (KCa) channels are susceptible to cGMP-dependent protein kinase (PKG)-dependent phosphorylation (A. Alioua, J. P. Huggins, and E. Rousseau. Am. J. Physiol. 1995;268:L1057-L1063). To assess whether KCa channels are also directly activated by NO. or one of its derivatives such as peroxynitrite, the activity of these channels was measured upon fusion of sarcolemmal vesicles derived from bovine tracheal smooth muscle cells into planar lipid bilayers (PLB). It was found that in the absence of adenosine triphosphate (ATP), cGMP, and cGMP-dependent protein kinase, NO* donors such as 1-propanamine-3-(2-hydroxy-2-nitroso-1-propylhydrazine) (PAPA NONOate) or 3-morpholinosydnonimine hydrochloride (SIN-1) in the presence of superoxide dismutase (SOD), added on either side of the bilayer, caused a concentration- dependent increase in the open probability (Po) of KCa channels without altering their unitary conductance. Release of NO*, which was measured by chemiluminescence analysis in parallel experiments, affected the gating behavior of KCa channels in the presence of SOD and ethyleneglycol-bis-(beta-aminoethyl ether)- N,N'-tetraacetic acid (EGTA) by reducing the mean closed times and increasing the number and duration of short open events. PAPA NONOate, a true NO. donor, had similar effects in the presence of ethylenediaminetetraacetic acid (EDTA), a heavy-metal chelator, and K-urate, a peroxynitrite scavenger. Addition of either 5 mM dithiothreitol (DTT) or 5 mM reduced glutathione (GSH), as well as 5 mM N-ethylmaleimide (NEM)-an alkylating agent-to the trans (intracellular) side of an experimental chamber slightly increased channel Po but prevented further channel activation by NO* donors. However, neither DTT nor GSH was able to reverse the effect of NO*. In contrast to SIN-1, DTT had no effect when added to the cis (extracellular) side of the chamber. This suggests that the effect of NO* is most likely due to a chemical modification (nitrothiosylation) of intracellular sulfhydryl group(s). Neither PAPA NONOate (NO*), nor SIN-1 had any effect on sarcolemmal Cl- channels rec

Topics: Animals; Bronchi; Calcium; Charybdotoxin; Cyclic GMP; Dithiothreitol; Ethylmaleimide; Glutathione; Humans; Hydrazines; Lipid Bilayers; Male; Molsidomine; Muscle, Smooth; Nitric Oxide; Potassium Channels; Rats; Rats, Sprague-Dawley