ascorbic-acid and 2-2--(hydroxynitrosohydrazono)bis-ethanamine

S-nitrosothiols (SNO) release nitric oxide (NO) through interaction with ascorbic acid (AA). However, little is known about their combined effect in the vasculature. The aim of this study was to investigate the effect of AA on SNO-mediated NO release, proliferation, cell cycle progression, cell death, and oxidative stress in vascular cells.. Vascular smooth muscle cells and adventitial fibroblasts harvested from the aortae of Sprague-Dawley rats were treated with AA, ± S-nitrosoglutathione (GSNO), or ± diethylenetriamine NONOate (DETA/NO). NO release, proliferation, cell cycle progression, cell death, and oxidative stress were determined by the Griess reaction, [(3)H]-thymidine incorporation, flow cytometry, trypan blue exclusion, and 5-(and-6)chloromethyl-2',7'dichlorodihydrofluorescein staining, respectively.. AA increased NO release from GSNO 3-fold (P < .001). GSNO and DETA/NO significantly decreased proliferation, but AA abrogated this effect (P < .05). Mirroring the proliferation data, changes in cell cycle progression induced by GSNO and DETA/NO were reversed by the addition of AA. GSNO- and DETA/NO-mediated increases in oxidative stress were significantly decreased by the addition of AA (P < .001).. Despite causing increased NO release from GSNO, AA reduced the antiproliferative and cell cycle effects of GSNO and DETA/NO through the modulation of oxidative stress.

Topics: Animals; Antioxidants; Ascorbic Acid; Cell Cycle; Cell Proliferation; Cells, Cultured; Connective Tissue; Fibroblasts; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; S-Nitrosoglutathione

Previously, we have reported that the exposure of PC12 cells to the aluminum-maltolate complex (Al(maltol)(3)) results in decreased cell viability via the apoptotic cell death pathway. In this study, we have used several nitric oxide synthase (NOS) inhibitors and the NO generator diethylenetriamine NONOate (DETA NONOate) to examine whether or not intracellular nitric oxide (NO) generation is involved in the onset mechanism of Al(maltol)(3)-induced cell death. Cell viability was assessed by measuring lactate dehydrogenase (LDH) release and caspase-3 activity. Treatment of the cells with 150 microM Al(maltol)(3) for 48 h resulted in intracellular NO generation. Exposure of the cells to DETA NONOate also induced a marked decrease in cell viability. Pre-treatment of the cells with a general NOS inhibitor or with a selective inducible NOS (iNOS) inhibitor effectively prevented Al(maltol)(3)-induced cell death. However, a neuronal NOS (nNOS) inhibitor did not exhibit any protective effect against Al(maltol)(3)-induced cell death. In addition, ascorbic acid markedly inhibited Al(maltol)(3)- and DETA NONOate-induced cell death. Based on these results, we discussed the involvement of intracellular NO generation in the onset mechanisms of Al(maltol)(3)-induced cell death.

Topics: Aluminum; Animals; Antioxidants; Ascorbic Acid; Caspase 3; Caspases; Cell Death; Cell Nucleus; Cell Survival; Enzyme Inhibitors; L-Lactate Dehydrogenase; Neoplasm Proteins; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitroso Compounds; Organometallic Compounds; PC12 Cells; Pyrones; Rats; Thiourea

The use of exogenous nitric oxide (NO) has been shown to alter the regulation of other endothelially derived mediators of vascular tone, such as endothelin-1 (ET-1). However, the interaction between NO and ET-1 appears to be complex and remains incompletely understood. One of the major actions of NO is the activation of soluble guanylate cyclase (sGC) with the subsequent generation of cGMP. Therefore, we undertook this study to test the hypothesis that NO regulates ET-1 production via the activation of the sGC/cGMP pathway. The results obtained indicated that the exposure of primary cultures of 4-wk-old ovine pulmonary arterial endothelial cells (4-wk PAECs) to the long-acting NO donor DETA NONOate induced both a dose- and time-dependent decrease in secreted ET-1. This decrease in ET-1 secretion occurred in the absence of changes in endothelin-converting enzyme-1 or sGC expression but in conjunction with a decrease in prepro-ET-1 mRNA. The changes in ET-1 release were inversely proportional to the cellular cGMP content. Furthermore, the NO-independent activator of sGC, YC-1, or treatment with a cGMP analog also produced significant decreases in ET-1 secretion. Conversely, pretreatment with the sGC inhibitor ODQ blocked the NO-induced decrease in ET-1. Therefore, we conclude that exposure of 4-wk PAECs to exogenous NO decreases secreted ET-1 resulting from the activation of sGC and increased cGMP generation.

Topics: Amino Acid Sequence; Animals; Ascorbic Acid; Cells, Cultured; Cyclic GMP; Endothelin-1; Endothelium, Vascular; Enzyme Activation; Guanylate Cyclase; Intracellular Membranes; Membrane Potentials; Mitochondria; Molecular Sequence Data; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; Peptide Fragments; Pulmonary Artery; Sheep

Cardiac hypertrophy is a significant risk factor for the development of congestive heart failure (CHF). Mitochondrial defects are reported in CHF, but no consistent mitochondrial alterations have yet been identified in hypertrophy. In this study selective metabolic inhibitors were used to determine thresholds for respiratory inhibition and to reveal novel mitochondrial defects in hypertrophy. Cardiac hypertrophy was produced in rats by aortic banding. Mitochondria were isolated from left ventricular tissue and the effects of inhibiting respiratory complexes I and IV on mitochondrial oxygen consumption were measured. At 8 weeks post-surgery, 65+/-2% complex IV inhibition was required to inhibit respiration half maximally in control mitochondria. In contrast, only 52+/-6% complex IV inhibition was required to inhibit respiration half maximally in mitochondria from hypertrophied hearts (P=0.046). This effect persisted at 22 weeks post-surgery and was accompanied by a significant upregulation of inducible nitric oxide synthase (iNOS, 3.0+/-0.7-fold, P=0.006). We conclude that respiration is more sensitive to complex IV inhibition in hypertrophy. Nitric oxide is a well documented inhibitor of complex IV, and thus the combination of increased NO(.)from iNOS and an increased sensitivity to inhibition of one of its targets could result in a bioenergetic defect in hypertrophy that may be a harbinger of CHF.

Topics: Animals; Aorta; Ascorbic Acid; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cardiomyopathy, Hypertrophic; Constriction; Cytochromes; Dose-Response Relationship, Drug; Electron Transport Complex I; Electron Transport Complex IV; Enzyme Induction; Fatty Alcohols; Heart Failure; Male; Mitochondria, Heart; NADH, NADPH Oxidoreductases; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nitroso Compounds; Oxidative Phosphorylation; Oxygen Consumption; Rats; Rats, Sprague-Dawley; Uncoupling Agents

1. The current study explored potential redox mechanisms of nitric oxide (NO)-induced inhibition of DNA synthesis in cultured human and rat aortic smooth muscle cells. 2. Exposure to S-nitrosothiols, DETA-NONOate and NO itself inhibited ongoing DNA synthesis and S phase progression in a concentration-dependent manner, as measured by thymidine incorporation and flow cytometry. Inhibition by NO donors occurred by release of NO, as detected by chemiluminescence and judged by the effects of NO scavengers, haemoglobin and cPTIO. 3. Co-incubation with redox compounds, N-acetyl-L-cysteine, glutathione and L-ascorbic acid prevented NO inhibition of DNA synthesis. These observations suggest that redox agents may alternatively attenuate NO bioactivity extracellularly, interfere with intracellular actions of NO on the DNA synthesis machinery or restore DNA synthesis after established inhibition by NO. 4. Recovery of DNA synthesis after inhibition by NO was similar with and without redox agents suggesting that augmented restoration of DNA synthesis is an unlikely mechanism to explain redox regulation. 5. Study of extracellula interactions revealed that all redox agents potentiated S-nitrosothiol decomposition and NO release. 6. Examination of intracellular NO bioactivity showed that as opposed to attenuation of NO inhibition of DNA synthesis by redox agents, there was no inhibition (potentiation in the presence of ascorbic acid) of soluble guanylate cyclase (sGC) activation judged by cyclic GMP accumulation in rat cells. 7. These data provide evidence that NO-induced inhibition of ongoing DNA synthesis is sensitive to redox environment. Redox processes might protect the DNA synthesis machinery from inhibition by NO, in the setting of augmented liberation of biologically active NO from NO donors.

Topics: Acetylcysteine; Animals; Ascorbic Acid; Cells, Cultured; DNA; Dose-Response Relationship, Drug; Free Radical Scavengers; G1 Phase; Glutathione; Humans; Hydroxyurea; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; Oxidation-Reduction; Penicillamine; Rats; S Phase; S-Nitrosoglutathione