ascorbic-acid and 2-phenyl-4-4-5-5-tetramethylimidazoline-1-oxyl-3-oxide

Various nitric oxide modulators (NO donors--SNP, GSNO, DEA NONOate and scavengers--PTIO, cPTIO) were tested to highlight the role of NO under Cd excess in various ontogenetic stages of chamomile (Matricaria chamomilla). Surprisingly, compared to Cd alone, SNP and PTIO elevated Cd uptake (confirmed also by PhenGreen staining) but depleted glutathione (partially ascorbic acid) and phytochelatins PC2 and PC3 in both older plants (cultured hydroponically) and seedlings (cultured in deionised water). Despite these anomalous impacts, fluorescence staining of NO and ROS confirmed predictable assumptions and revealed reciprocal changes (decrease in NO but increase in ROS after PTIO addition and the opposite after SNP application). Subsequent tests using alternative modulators and seedlings confirmed changes to NO and ROS after application of GSNO and DEA NONOate as mentioned above for SNP while cPTIO altered only NO level (depletion). On the contrary to SNP and PTIO, GSNO, DEA NONOate and cPTIO did not elevate Cd content and phytochelatins (PC2, PC3) were rather elevated. These data provide evidence that various NO modulators are useful in terms of NO and ROS manipulation but interactions with intact plants affect metal uptake and must therefore be used with caution. In this view, cPTIO and DEA NONOate revealed the less pronounced side impacts and are recommended as suitable NO scavenger/donor in plant physiological studies under Cd excess.

Topics: Antioxidants; Ascorbic Acid; Cadmium; Chamomile; Cyclic N-Oxides; Glutathione; Imidazoles; Microscopy, Confocal; Microscopy, Fluorescence; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Reactive Oxygen Species; S-Nitrosoglutathione; Seeds

The involvement of NO in O(2)(.-) generation, rootlet development and antioxidant defence were investigated in the adventitious root cultures of mountain ginseng. Treatments of NO producers (SNP, sodium nitroprusside; SNAP, S-nitroso-N-acetylpenicillamine; and sodium nitrite with ascorbic acid), and NO scavenger (PTIO, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl3-oxide) revealed that NO is involved in the induction of new rootlets. Severe decline in number of new rootlets compared to the control under PTIO treatment indicates that NO acts downstream of auxin action in the process. NO producers (SNP, SNAP and sodium nitrite with ascorbic acid) activated NADPH oxidase activity, resulting in greater O(2)(.-) generation and higher number of new rootlets in the adventitious root explants. Moreover, treatment of diphenyliodonium chloride, a NADPH oxidase inhibitor, individually or along with SNP, inhibited root growth, NADPH oxidase activity and O(2)(.-) anion generation. NO supply also enhanced the activities of antioxidant enzymes that are likely to be responsible for reducing H(2)O(2)levels and lipid peroxidation as well as modulation of ascorbate and non-protein thiol concentrations in the adventitious roots. Our results suggest that NO-induced generation of O(2) (.-) by activating NADPH oxidase activity is related to adventitious root formation in mountain ginseng.

Topics: Antioxidants; Ascorbic Acid; Cyclic N-Oxides; Hydrogen Peroxide; Imidazoles; Lipid Peroxidation; NADPH Oxidases; Nitric Oxide; Nitric Oxide Donors; Panax; Penicillamine; Plant Roots; Reactive Oxygen Species; S-Nitroso-N-Acetylpenicillamine; Superoxide Dismutase; Superoxides

Nitric oxide (NO) as a key signaling molecule has been involved in mediation of various biotic and abiotic stress-induced physiological responses in plants. In the present study, we investigated the effect of NO on Cassia tora L. plants exposed to aluminum (Al). Plants pre-treated for 12 h with 0.4 mM sodium nitroprusside (SNP), an NO donor, and subsequently exposed to 10 microM Al treatment for 24 h exhibited significantly greater root elongation as compared with the plants without SNP treatment. The NO-promoted root elongation was correlated with a decrease in Al accumulation in root apexes. Furthermore, oxidative stress associated with Al treatment increased lipid peroxidation and reactive oxygen species, and the activation of lipoxygenase and antioxidant enzymes was reduced by NO. Such effects were confirmed by the histochemical staining for the detection of peroxidation of lipids and loss of membrane integrity in roots. The ameliorating effect of NO was specific, because the NO scavenger cPTIO [2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylinidazoline-1-oxyl-3-oxide] completely reversed the effect of NO on root growth in the presence of Al. These results indicate that NO plays an important role in protecting the plant against Al-induced oxidative stress.

Topics: Aluminum; Ascorbic Acid; Cassia; Catalase; Cyclic N-Oxides; Enzyme Activation; Histocytochemistry; Imidazoles; Lipid Peroxidation; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Oxidative Stress; Peroxidase; Plant Roots; Superoxide Dismutase

The quintessential nitrosating species produced during NO autoxidation is N(2)O(3). Nitrosation of amine, thiol, and hydroxyl residues can modulate critical cell functions. The biological mechanisms that control reactivity of nitrogen oxide species formed during autoxidation of nano- to micromolar levels of NO were examined using the synthetic donor NaEt(2)NN(O)NO (DEA/NO), human tumor cells, and 4,5-diaminofluorescein (DAF). Both the disappearance of NO and formation of nitrosated product from DAF in aerobic aqueous buffer followed second order processes; however, consumption of NO and nitrosation within intact cells were exponential. An optimal ratio of DEA/NO and 2-phenyl-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (PTIO) was used to form N(2)O(3) through the intermediacy of NO(2). This route was found to be most reflective of the nitrosative mechanism within intact cells and was distinct from the process that occurred during autoxidation of NO in aqueous media. Manipulation of the endogenous scavengers ascorbate and glutathione indicated that the location, affinity, and concentration of these substances were key determinants in dictating nitrosative susceptibility of molecular targets. Taken together, these findings suggest that the functional effects of nitrosation may be organized to occur within discrete domains or compartments. Nitrosative stress may develop when scavengers are depleted and this architecture becomes compromised. Although NO(2) was not a component of aqueous NO autoxidation, the results suggest that the intermediacy of this species may be a significant factor in the advent of either nitrosation or oxidation chemistry in biological systems.

Topics: Ascorbic Acid; Cyclic N-Oxides; Fluorescein; Glutathione; Humans; Imidazoles; Indicators and Reagents; Kinetics; Models, Chemical; Nitric Oxide; Nitrogen Oxides; Nitrosation; Oxygen; Protein Structure, Tertiary; Reactive Oxygen Species; Spectrometry, Fluorescence; Stress, Physiological; Time Factors; Tumor Cells, Cultured

Nitric oxide (NO*) derived from exogenous donors has been shown to increase the permeability of cultured intestinal epithelial monolayers, an effect that is augmented by mildly acidic conditions. Because interferon-gamma (IFN-gamma) also increases intestinal epithelial permeability, at least partly by an NO*-dependent mechanism, we sought to determine whether IFN-gamma-induced hyperpermeability is increased under acidic conditions.. Human intestinal epithelial (Caco-2BBe) cells were grown as monolayers on permeable supports in bicameral chambers. Permeability was assessed by measuring transepithelial electrical resistance (TER) or the transepithelial passage of fluorescein disulfonic acid. Inducible nitric oxide synthase (iNOS) messenger RNA expression was determined by northern blot analysis. Concentrations of nitrite and nitrate (NO2-/NO3-), stable oxidation products of NO*, were determined using the Greiss reaction. Cellular adenosine triphosphate (ATP) levels were determined using the luciferin/luciferase method.. Incubation of Caco-2BBe monolayers with INF-gamma (1000 units/mL) at an extracellular pH (pHo) of 7.4 increased permeability to fluorescein disulfonic acid and decreased TER. However, incubation of monolayers with IFN-gamma under mildly acidic conditions (i.e., pHo 7.0-6.6) accelerated the decrease in TER and augmented the increase in permeability induced by the cytokine. IFN-gamma-induced iNOS messenger RNA expression and NO2-/NO3- accumulation in medium were unaffected by acidic conditions. At pHo 7.4, incubation of Caco-2BBe monolayers with IFN-gamma (1000 units/mL) for 72 hrs had no effect on intracellular ATP content compared with monolayers simultaneously incubated under the same conditions but in the absence of the cytokine. However, when the cells were incubated for 72 hrs with the same concentration of IFN-gamma under mildly acidic conditions (i.e., pHo 7.0 or 6.6), ATP levels were significantly decreased. At pHo 7.0, IFN-gamma-induced increases in permeability were ameliorated by addition of the following agents: 2-phenyl-4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide (a NO* scavenger), N(G)-monomethyl-L-arginine (a iNOS inhibitor), dimethyl sulfoxide (a hydroxyl radical scavenger), and ascorbate (a peroxynitrous acid scavenger).. Mild acidosis augments IFN-gamma-induced intestinal epithelial hyperpermeability and ATP depletion, possibly by fostering the formation of peroxynitrous acid and/or hydroxyl radical.

Topics: Adenosine Triphosphate; Ascorbic Acid; Caco-2 Cells; Cell Membrane Permeability; Cyclic N-Oxides; Dimethyl Sulfoxide; Drug Evaluation, Preclinical; Electric Impedance; Free Radical Scavengers; Humans; Hydrogen-Ion Concentration; Imidazoles; Interferon-gamma; Intestinal Mucosa; Nitric Oxide; Nitric Oxide Synthase; Nitrous Acid; omega-N-Methylarginine; Peroxynitrous Acid; Time Factors