ascorbic-acid and diphenyleneiodonium

The role of reactive oxygen species (ROS) in ABA-induced increase in hydraulic conductivity was hypothesized to be dependent on an increase in aquaporin water channel (AQP) abundance. Single ABA application or its combination with ROS manipulators (ROS scavenger ascorbic acid and NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI)) were studied on detached roots of barley plants. We measured the osmotically driven flow rate of xylem sap and calculated root hydraulic conductivity. In parallel, immunolocalization of ABA and HvPIP2;2 AQPs was performed with corresponding specific antibodies. ABA treatment increased the flow rate of xylem, root hydraulic conductivity and immunostaining for ABA and HvPIP2;2, while the addition of antioxidants prevented the effects of this hormone. The obtained results confirmed the involvement of ROS in ABA effect on hydraulic conductivity, in particular, the importance of H

Topics: Abscisic Acid; Antioxidants; Aquaporins; Ascorbic Acid; Enzyme Inhibitors; Hordeum; NADPH Oxidases; Onium Compounds; Osmosis; Plant Proteins; Plant Roots; Reactive Oxygen Species; Xylem

In hulless barley, H 2 S mediated increases in H 2 O 2 induced by putrescine, and their interaction enhanced tolerance to UV-B by maintaining redox homeostasis and promoting the accumulation of UV-absorbing compounds. This study investigated the possible relationship between putrescence (Put), hydrogen sulfide (H2S) and hydrogen peroxide (H2O2) as well as the underlying mechanism of their interaction in reducing UV-B induced damage. UV-B radiation increased electrolyte leakage (EL) and the levels of malondialdehyde (MDA) and UV-absorbing compounds but reduced antioxidant enzyme activities and glutathione (GSH) and ascorbic acid (AsA) contents. Exogenous application of Put, H2S or H2O2 reduced some of the above-mentioned negative effects, but were enhanced by the addition of Put, H2S and H2O2 inhibitors. Moreover, the protective effect of Put against UV-B radiation-induced damage to hulless barley was diminished by DL-propargylglycine (PAG, a H2S biosynthesis inhibitor), hydroxylamine (HT, a H2S scavenger), diphenylene iodonium (DPI, a PM-NADPH oxidase inhibitor) and dimethylthiourea (DMTU, a ROS scavenger), and the effect of Put on H2O2 accumulation was abolished by HT. Taken together, as the downstream component of the Put signaling pathway, H2S mediated H2O2 accumulation, and H2O2 induced the accumulation of UV-absorbing compounds and maintained redox homeostasis under UV-B stress, thereby increasing the tolerance of hulless barley seedlings to UV-B stress.

Topics: Alkynes; Antioxidants; Ascorbic Acid; Enzyme Inhibitors; Glutathione; Glycine; Homeostasis; Hordeum; Hydrogen Peroxide; Hydrogen Sulfide; Hydroxylamine; NADPH Oxidases; Onium Compounds; Oxidation-Reduction; Oxidative Stress; Plant Proteins; Protective Agents; Putrescine; Seedlings; Signal Transduction; Thiourea; Ultraviolet Rays

The default growth pattern of primary roots of land plants is directed by gravity. However, roots possess the ability to sense and respond directionally to other chemical and physical stimuli, separately and in combination. Therefore, these root tropic responses must be antagonistic to gravitropism. The role of reactive oxygen species (ROS) in gravitropism of maize and Arabidopsis (Arabidopsis thaliana) roots has been previously described. However, which cellular signals underlie the integration of the different environmental stimuli, which lead to an appropriate root tropic response, is currently unknown. In gravity-responding roots, we observed, by applying the ROS-sensitive fluorescent dye dihydrorhodamine-123 and confocal microscopy, a transient asymmetric ROS distribution, higher at the concave side of the root. The asymmetry, detected at the distal elongation zone, was built in the first 2 h of the gravitropic response and dissipated after another 2 h. In contrast, hydrotropically responding roots show no transient asymmetric distribution of ROS Decreasing ROS levels by applying the antioxidant ascorbate, or the ROS-generation inhibitor diphenylene iodonium attenuated gravitropism while enhancing hydrotropism. Arabidopsis mutants deficient in Ascorbate Peroxidase 1 showed attenuated hydrotropic root bending. Mutants of the root-expressed NADPH oxidase RBOH C, but not rbohD, showed enhanced hydrotropism and less ROS in their roots apices (tested in tissue extracts with Amplex Red). Finally, hydrostimulation prior to gravistimulation attenuated the gravistimulated asymmetric ROS and auxin signals that are required for gravity-directed curvature. We suggest that ROS, presumably H

Topics: Antioxidants; Arabidopsis; Ascorbic Acid; Gravitropism; Isoenzymes; Microscopy, Confocal; Mutation; NADPH Oxidases; Onium Compounds; Plant Roots; Reactive Oxygen Species; Tropism

Canonical neutrophil antimicrobial effector mechanisms, such as degranulation, production of reactive oxygen species, and release of neutrophil extracellular traps (NETs), can result in severe pathology. Activation of neutrophils through immune complexes (ICs) plays a central role in the pathogenesis of many autoimmune inflammatory diseases. In this study, we report that immobilized ICs (iICs), which are hallmarks of several autoimmune diseases, induce the release of NETs from primary human neutrophils. The iIC-induced NET formation was found to require production of reactive oxygen species by NADPH oxidase and myeloperoxidase and to be mediated by FcγRIIIb. Blocking of the β2 integrin macrophage-1 Ag but not lymphocyte function-associated Ag-1 abolished iIC-induced NET formation. This suggests that FcγRIIIb signals in association with macrophage-1 Ag. As intracellular signaling pathways involved in iIC-induced NET formation we identified the tyrosine kinase Src/Syk pathway, which downstream regulates the PI3K/Akt, p38 MAPK, and ERK1/2 pathways. To our knowledge, the present study shows for the first time that iICs induce NET formation. Thus, we conclude that NETs contribute to pathology in autoimmune inflammatory disorders associated with surface-bound ICs.

Topics: Aminopyrine; Antigen-Antibody Complex; Antioxidants; Ascorbic Acid; Autoimmune Diseases; Butadienes; CD11a Antigen; CD18 Antigens; Cell Degranulation; Cells, Cultured; Extracellular Signal-Regulated MAP Kinases; GPI-Linked Proteins; Humans; Imidazoles; Inflammation; Intracellular Signaling Peptides and Proteins; Lymphocyte Function-Associated Antigen-1; Macrophage-1 Antigen; Mesalamine; Neutrophil Activation; Neutrophils; Nitriles; Onium Compounds; p38 Mitogen-Activated Protein Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-akt; Pyridines; Pyrimidines; Reactive Oxygen Species; Receptors, IgG; src-Family Kinases; Syk Kinase

Ethephon, an ethylene releasing compound, promoted leaf senescence, H2O2 elevation, and senescence-associated gene expression in sweet potato. It also affected the glutathione and ascorbate levels, which in turn perturbed H2O2 homeostasis. The decrease of reduced glutathione and the accumulation of dehydroascorbate correlated with leaf senescence and H2O2 elevation at 72h in ethephon-treated leaves. Exogenous application of reduced glutathione caused quicker and significant increase of its intracellular level and resulted in the attenuation of leaf senescence and H2O2 elevation. A small H2O2 peak produced within the first 4h after ethephon application was also eliminated by reduced glutathione. Diphenyleneiodonium (DPI), an NADPH oxidase inhibitor, delayed leaf senescence and H2O2 elevation at 72h, and its influence was effective only within the first 4h after ethephon treatment. Ethephon-induced senescence-associated gene expression was repressed by DPI and reduced glutathione at 72h in pretreated leaves. Leaves treated with l-buthionine sulfoximine, an endogenous glutathione synthetase inhibitor, did enhance senescence-associated gene expression, and the activation was strongly repressed by reduced glutathione. In conclusion, ethephon-mediated leaf senescence, H2O2 elevation and senescence-associated gene expression are all alleviated by reduced glutathione and NADPH oxidase inhibitor DPI. The speed and the amount of intracellular reduced glutathione accumulation influence its effectiveness of protection against ethephon-mediated effects. Reactive oxygen species generated from NADPH oxidase likely serves as an oxidative stress signal and participates in ethephon signaling. The possible roles of NADPH oxidase and reduced glutathione in the regulation of oxidative stress signal in ethephon are discussed.

Topics: Ascorbic Acid; Buthionine Sulfoximine; Cellular Senescence; Chlorophyll; Ethylenes; Gene Expression Regulation, Plant; Glutathione; Hydrogen Peroxide; Ipomoea batatas; NADPH Oxidases; Onium Compounds; Organophosphorus Compounds; Plant Leaves

We investigated the signaling role of hydrogen peroxide (H(2)O(2)) in regulating the ascorbate (AsA) level after exogenous methanol (MeOH) application. The endogenous H(2)O(2) and AsA levels as well as the expression of related genes were monitored after MeOH treatment of cultures of Oncidium protocorm-like bodies (PLB). A high MeOH concentration was deleterious and caused irreversible consumption of endogenous AsA. However, a low MeOH concentration (50mM) triggered the synthesis of H(2)O(2) and was effective in enhancing the expression of AsA-biosynthetic genes of the Smirnoff-Wheeler and galacturonate (GalUA) pathways. The increased expression of these genes could be blocked by the addition of hydroxylamine, an inhibitor of alcohol oxidase (EC: 1.1.3.13), and diphenyleneiodonium chloride (DPI), an inhibitor of NADPH oxidase (EC: 1.6.3.1). Thus, the H(2)O(2) generated by MeOH application is a product of MeOH detoxification through alcohol oxidase and NADPH oxidase activation. In this chain, H(2)O(2) acts as a secondary messenger for the activation of AsA-related genes. Our results reveal the signaling function of H(2)O(2) and cellular AsA homeostasis in Oncidium orchids in response to MeOH stimulation. A mechanism for the MeOH effect on AsA production is suggested.

Topics: Alcohol Oxidoreductases; Ascorbic Acid; Gene Expression Regulation, Plant; Genes, Plant; Hexuronic Acids; Hydrogen Peroxide; Hydroxylamine; Methanol; NADPH Oxidases; Onium Compounds; Orchidaceae; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction

In the Japanese pear pathotype of Alternaria alternata, H2O2 is generated solely from penetration pegs and not from other portions of subcuticular hyphae within the pectin layers of host leaves. A close association between H2O2 generation and fungal aggressiveness is expected because the pegs are important for fungal penetration into the host epidermis. To determine the potential role of reactive oxygen species in microbial pathogenicity, we studied the inhibitory effects of the antioxidant reagent ascorbic acid and the NADPH oxidase inhibitor diphenylene iodonium on infection of the pathogen. In our study, we showed H2O2 generation to be inhibited by inoculation with the mixture of ascorbic acid or diphenylene iodonium and spores at the pegs in the spore-inoculated host leaves. The decrease of generation in the pegs resulted in penetration failure, indicating that H2O2 generation probably contributed to strengthening of the penetration and probably was recruited by NADPH oxidase.

Topics: Alternaria; Antioxidants; Ascorbic Acid; Host-Parasite Interactions; Hydrogen Peroxide; Hyphae; Onium Compounds; Plant Diseases; Plant Leaves; Plant Proteins; Pyrus; Reactive Oxygen Species

Intracellularly generated reactive oxygen species (ROS) are thought to modulate redox sensitive signaling pathways and thus regulate cell physiology including proliferation and differentiation. However, the role of ROS in neuronal differentiation of embryonic pluripotent cells is unknown. For this reason, the modification of retinoic acid (RA) induced neuronal differentiation of mouse embryonal carcinoma cells P19 by selected ROS scavengers and flavoprotein inhibitor was evaluated.. Intracellular ROS was evaluated by flowcytometry. Cellular redox status was evaluated based on total levels of reduced thiol groups in cells. The activity of the RA responsive element (RARE) was evaluated by luciferase reporter assay. The RA-induced neuronal differentiation was determined based on changes in the expression of protein markers characteristic for undifferentiated (Oct-4) and neuron-like cell differentiated cells (N-cadherin and III-beta tubulin).. RA increased the intracellular ROS production that was accompanied by a decrease in thiol groups in cells. The ROS scavengers and flavoprotein inhibitor reduced RA-induced ROS production, RA-induced activity of RARE, and it decreased the RA-induced expression of N-cadherin and III-beta tubulin.. Our data outline a role of ROS as important molecules in the transduction of an intracellular signal during the neuronal differentiation of ES cells.

Topics: Acetophenones; Acetylcysteine; Ascorbic Acid; Blotting, Western; Cell Differentiation; Embryonal Carcinoma Stem Cells; Flow Cytometry; Free Radical Scavengers; Genes, Reporter; Glutathione; Humans; Neurons; Onium Compounds; Oxidation-Reduction; Reactive Oxygen Species; Sulfhydryl Compounds; Tretinoin; Vitamins

Previous in vitro studies in our laboratory have shown that mancozeb (MZ) and maneb (MB), both widely used EBDC fungicides, are equipotent neurotoxicants that produce cell loss in mesencephalic dopaminergic and GABAergic cells after an acute 24h exposure. Mitochondrial uncoupling and inhibition were associated with fungicide exposure. Inhibition of mitochondrial respiration is known to increase free radical production. Here the mechanism(s) of neuronal damage associated with MZ exposure was further explored by determining the role that reactive oxygen species (ROS) played in toxicity. Damage to mesencephalic dopamine and GABA cell populations were significantly attenuated when carried out in the presence of ascorbate or SOD, indicative of a free radical-mediated contribution to toxicity. ROS generation monitored by hydrogen peroxide (H(2)O(2)) production using Amplex Red increased in a dose-dependent manner in response to MZ. Inhibition of intracellular catalase with aminotriazole had little effect on H(2)O(2) generation, whereas exogenously added catalase significantly reduced H(2)O(2) production, demonstrating a large extracellular contribution to ROS generation. Conversely, cells preloaded with the ROS indicator dye DCF showed significant MZ-induced ROS production, demonstrating an increase in intracellular ROS. Both the organic backbone of MZ as well as its associated Mn ion, but not Zn ion, were responsible and required for H(2)O(2) generation. The functionally diverse NADPH oxidase inhibitors, diphenylene iodonium chloride, apocynin, and 4-(2-aminoethyl)benzene-sulfonyl fluoride hydrochloride significantly attenuated H(2)O(2) production by MZ. In growth medium lacking cells, MZ produced little H(2)O(2), but enhanced H(2)O(2) generation when added with xanthine plus xanthine oxidase whereas, in cultured cells, allopurinol partially attenuated H(2)O(2) production by MZ. Minocycline, an inhibitor of microglial activation, modestly reduced H(2)O(2) formation in mesencephalic cells. In contrast, neuronal-enriched cultures or cultures treated with MAC-1-SAP to kill microglia, did not show an attenuation of ROS production. These findings demonstrate that Mn-containing EBDC fungicides such as MZ and MB can produce robust ROS generation that likely occurs via redox cycling with extracellular and intracellular oxidases. The findings further show that microglia may contribute to but are not required for ROS production by MZ.

Topics: Acetophenones; Allopurinol; Amitrole; Animals; Antioxidants; Ascorbic Acid; Catalase; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fungicides, Industrial; Hydrogen Peroxide; Maneb; Mesencephalon; Microglia; Minocycline; Molecular Structure; NADPH Oxidases; Neurons; Onium Compounds; Oxidation-Reduction; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Structure-Activity Relationship; Sulfones; Superoxide Dismutase; Time Factors; Xanthine Oxidase; Zineb

The balance between antioxidants, such as ascorbate (ASC) and glutathione, and oxidative reactive oxygen species (ROS) is known to play a pivotal role in the response of plant cells to abiotic stress. Here cell cultures of Arabidopsis thaliana were investigated with regard to their response to elevated levels of cadmium. At concentrations <100 microM, Cd induces a rapid and concentration-dependent H(2)O(2) accumulation. This response could be inhibited by diphenylene iodonium (DPI, 20 microM). Reverse transcription-PCR analysis of three RBOH (respiratory burst oxidase homologues) genes showed an increased transcription of RBOHF after 15 min. No change in ASC concentration was observed during the first 3 h after Cd addition. In contrast, glutathione levels completely diminished within 1 h. This drop could be attributed to an increase in phytochelatin 4. At the plasma membrane, Cd further induced a significant decrease in dehydroascorbate (DHA) uptake activity (up to 90% inhibition after 4 h). This decrease is not present when cells are treated with LaCl(3) before exposure to CdCl(2). LaCl(3) is a typical inhibitor of Ca channels and prevents Cd uptake in these cells as well as the Cd-induced ROS production. Therefore, these results appear to indicate that Cd uptake is a prerequisite for the change in DHA transport activity. However, DPI did not prevent the drop in DHA uptake activity present in Cd-treated Arabidopsis cells, indicating that this response seems to be independent of the Cd-induced H(2)O(2) production.

Topics: Arabidopsis; Ascorbic Acid; Cadmium; Cadmium Chloride; Cell Membrane; Cells, Cultured; Dehydroascorbic Acid; Enzyme Inhibitors; Glutathione; Hydrogen Peroxide; Lanthanum; Onium Compounds; Phytochelatins

Reactive oxygen species (ROS) and deposition of cleaved products of amyloid precursor protein (APP) are thought to contribute to neuronal loss observed in Alzheimer's disease (AD). The relationship between these factors was studied in a neuroblastoma and microglia co-culture system. Overexpression of wild-type APP (APP-wt) or APP with three mutations typical of familial AD (APP-3m) in SH-SY5Y neuroblastoma cells did not directly alter their morphology, growth rate, cell cycle or H(2)O(2) sensitivity. In a co-culture of APP-wt neuroblastoma cells with microglia, microglial cells generated ROS and neuronal cells died. The cell death was more pronounced in APP-3m-expressing neurons. Neuroblastoma cell death was attenuated by ROS-scavengers and was dose-dependently inhibited by the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI). Macrophage cell lines behaved similarly to microglia in the co-culture model. However, a macrophage cell line deficient in the NADPH oxidase subunit, gp91phox, failed to kill neurons. These results suggest that APP-dependent microglia activation and subsequent ROS generation by the phagocyte NADPH oxidase play a crucial role in neuronal killing in a cellular model of AD.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Antioxidants; Ascorbic Acid; Blotting, Western; Cell Count; Cell Culture Techniques; Cell Death; Cell Line, Tumor; Enzyme Inhibitors; Humans; Macrophages; Microglia; Microscopy, Fluorescence; Mutation; NADPH Oxidases; Neurons; Onium Compounds; Protease Nexins; Reactive Oxygen Species; Receptors, Cell Surface; Transduction, Genetic; Vitamin E

Although several studies demonstrated that lead induced abnormal vascular responses in low level lead exposed animals, investigations of the direct effects of lead on blood vessels are limited. In this study we tested the hypothesis that lead was able to directly affect the contractile reactivities of vessels. Male Wistar rat aortae were removed and cultured in PMRI 1640 with 1 ppm lead acetate for 0.5, 6, 12, 24 and 48 h, and then their responses to norepinephrine bitartrate (NE) and serotonin (5-hydroxytryptamine, 5-HT) were examined. The contractile responses to 5-HT of lead exposed aortae were significantly increased when the aortae were cultured for 24 and 48 h. Denudation of endothelium was able to abolish the increased contractile response completely. Diphenyleneiodonium (DPI), an inhibitor of the NAD(P)H oxidase, could abolish the increased contractile response to 5-HT. However, Vitamin C (VC) enhanced the contractile response of both groups to higher dosages of 5-HT. The expression of 5-HT(2B) receptor was not significantly altered by incubation with 1 ppm lead for 24 h. These data suggest that exposure to low levels of lead can directly increase the contraction of aorta to 5-HT. This effect is endothelium dependent, which is not mediated by increased expression of the 5-HT 2B receptor. The increased contraction to 5-HT may be related to increased production of superoxide (O2*-) induced by lead exposure.

Topics: Animals; Antioxidants; Aorta, Thoracic; Ascorbic Acid; Blotting, Western; Cells, Cultured; Endothelium, Vascular; Enzyme Inhibitors; Lead; Male; Muscle Contraction; Muscle, Smooth, Vascular; NADPH Oxidases; Norepinephrine; Onium Compounds; Rats; Rats, Wistar; Reactive Oxygen Species; Receptor, Serotonin, 5-HT2B; Serotonin; Superoxides; Up-Regulation; Vasoconstrictor Agents

We previously found that human chymase cleaves big endothelins (ETs) at the Tyr(31)-Gly(32) bond and produces 31-amino acid ETs (1-31), without any further degradation products. In the present study, we investigated the effects of various antioxidants on the ET-1 (1-31)-induced change in intracellular signaling and proliferation of cultured rat aortic smooth muscle cells (RASMC). ET-1 (1-31) stimulated rapid and significant activation of the mitogen-activated protein (MAP) kinase family, i.e. extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun NH(2)-terminal kinase (JNK), and p38 MAPK, in RASMC to an extent similar to that of ET-1. All of the antioxidants examined, i.e. N-acetyl-L-cysteine (NAC), diphenyleneiodonium chloride (DPI), and L-(+)-ascorbic acid (ascorbic acid), inhibited both ET-1 (1-31)- and ET-1-induced JNK and p38 MAPK activation but not ERK1/2 activation. Electron paramagnetic resonance (EPR) spectroscopy measurements revealed that NAC, DPI, and ascorbic acid inhibited xanthine oxidase-induced superoxide (O(2)(.-)) generation in a cell-free system. ET-1 (1-31) in addition to ET-1 increased the generation of cellular reactive oxygen species (ROS) in RASMC. ET-1 (1-31)- and ET-1-induced cellular ROS generation was inhibited similarly by NAC, DPI, and ascorbic acid in RASMC. Gel-mobility shift analysis showed that ET-1 (1-31) and ET-1 caused an increase in activator protein-1 (AP-1)-DNA binding activity in RASMC that was inhibited by the above three antioxidants. ET-1 (1-31) increased [3H]thymidine incorporation into cells to an extent similar to that of ET-1. This ET-1 (1-31)-induced increase in [3H]thymidine incorporation was also inhibited by NAC and DPI, but not by ascorbic acid. These results suggest that antioxidants inhibit ET-1 (1-31)-induced RASMC proliferation by inhibiting ROS generation within the cells. The underlying mechanisms of the inhibition of cellular proliferation by antioxidants may be explained, in part, by the inhibition of JNK activation and the resultant inhibition of AP-1-DNA binding.

Topics: Acetylcysteine; Animals; Antioxidants; Ascorbic Acid; Cell Division; Cyclic N-Oxides; DNA; Dose-Response Relationship, Drug; Endothelin-1; Endothelins; Enzyme Activation; Enzyme Inhibitors; Humans; Imidazoles; JNK Mitogen-Activated Protein Kinases; Male; Mitogen-Activated Protein Kinases; Muscle, Smooth, Vascular; Onium Compounds; p38 Mitogen-Activated Protein Kinases; Peptide Fragments; Pyridines; Rats; Rats, Sprague-Dawley; Time Factors; Transcription Factor AP-1

Angiotensin II (Ang II) induces vascular smooth muscle cell (VSMC) hypertrophy, which results in several cardiovascular diseases. Ang II-induced cellular events have been mediated, in part, by reactive oxygen species (ROS) which also involve activation of mitogen-activated protein (MAP) kinases. Although it has been proposed that the therapeutic administration of antioxidants is useful for vascular diseases, the precise mechanisms which regulate ROS-sensitive signaling events have not been well characterized. Thus, we hypothesized that antioxidants may affect ROS-mediated MAP kinases activation induced by Ang II. The present findings showed that Ang II stimulated rapid and significant activation of ERK 1/2, JNK and p38 MAPK in cultured rat aortic smooth muscle cells (RASMC). Ang II-induced ERK 1/2 activation was not affected by all antioxidants examined, whereas JNK was sensitive to all antioxidants. In contrast, p38 MAPK activation was inhibited by DPI and ascorbic acid concentration-dependently, but by NAC only at high concentration. DETC and Trolox C had no effects on p38 MAPK activation by Ang II. We further examined the effects of antioxidants on Ang II-induced increases in oxygen consumption as an index of ROS generation in RASMC. DPI strongly inhibited Ang II-induced increases in oxygen consumption. DETC also inhibited Ang II-induced oxygen consumption, whereas ascorbic acid markedly augmented it. These findings suggest that the inhibitory effects of antioxidants on MAP kinases activation in VSMC are attributable, in part, to their modulating effects on ROS generation by Ang II in VSMC. Thus, inhibition of MAP kinases by antioxidants may imply their usefulness for relief of cardiovascular diseases.

Topics: Acetylcysteine; Angiotensin II; Animals; Antioxidants; Aorta, Thoracic; Ascorbic Acid; Cells, Cultured; Chelating Agents; Chromans; Ditiocarb; Dose-Response Relationship, Drug; Enzyme Inhibitors; Free Radical Scavengers; JNK Mitogen-Activated Protein Kinases; Male; MAP Kinase Kinase 4; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Muscle, Smooth, Vascular; Onium Compounds; Oxygen Consumption; p38 Mitogen-Activated Protein Kinases; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Vasoconstrictor Agents

In the previous paper [Kawano et al. (2000a) Plant Cell Physiol. 41: 1251], we demonstrated that addition of phenylethylamine (PEA) and benzylamine can induce an immediate and transient burst of active oxygen species (AOS) in tobacco suspension culture. Detected AOS include H2O2, superoxide anion and hydroxyl radicals. Use of several inhibitors suggested the presence of monoamine oxidase-like H2O2-generating activity in the cellular soluble fraction. It was also suggested that peroxidase(s) or copper amine oxidase(s) are involved in the extracellular superoxide production as a consequence of H2O2 production. Since more than 85% of the PEA-dependent AOS generating activity was localized in the extracellular space (extracellular fluid + cell wall), extracellularly secreted enzymes, probably peroxidases, may largely contribute to the oxidative burst induced by PEA. The PEA-induced AOS generation was also observed in the horseradish peroxidase (HRP) reaction mixture, supporting the hypothesis that peroxidases catalyze the oxidation of PEA leading to AOS generation. In addition to AOS production, we observed that PEA induced an increase in monodehydroascorbate radicals (MDA) in the cell suspension culture and in HRP reaction mixture using electron spin resonance spectroscopy and the newly invented MDA reductase-coupled method. Here we report that MDA production is an indicator of peroxidase-mediated generation of PEA radical species in tobacco suspension culture.

Topics: Amines; Ascorbic Acid; Calcium; Catalase; Clorgyline; Dehydroascorbic Acid; Dose-Response Relationship, Drug; Free Radicals; Hydrogen Peroxide; Luminol; Monoamine Oxidase Inhibitors; Nicotiana; Onium Compounds; Oxidation-Reduction; Pargyline; Phenethylamines; Plants, Toxic; Reactive Oxygen Species; Salicylamides; Superoxide Dismutase