sodium-nitrite and 2-phenyl-4-4-5-5-tetramethylimidazoline-1-oxyl-3-oxide

Ventricular arrhythmias induced by ischemic heart disease are the main cause of sudden cardiac death. Ischemia can cause life-threatening arrhythmias by modulating connexin 43 (Cx43), a principal cardiac gap junction channel protein. The present study investigates whether nitrite can attenuate ischemia-induced ventricular arrhythmias and dephosphorylation of Cx43 in a rat model.. Rats were medicated with normal saline (control, n = 10), nitrite (0.015, 0.15, and 1.5 mg/kg, n = 9 or 10 each), and 0.15 mg/kg nitrite with either the nitric oxide scavenger 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide, sodium salt (cPTIO; n = 9) or allopurinol (xanthine oxidoreductase inhibitor, n = 9). We determined the severity of ventricular arrhythmias based on arrhythmia scores and levels of phosphorylated Cx43.. The median arrhythmia score may have been lower in the group given 0.15 mg/kg nitrite (4 [interquartile range {IQR}, 4-5]) than that in the control group (7.5 [IQR, 5.25-8]; P = 0.013). There was no difference among the control, the given 0.015 mg/kg nitrite (7 [IQR, 5-8]), and 1.5 mg/kg nitrite (7 [IQR, 5.5-7.75]; P = 0.95). The arrhythmia scores in the cPTIO (6 [IQR, 5-8]; P = 0.030) and allopurinol (7 [IQR, 5-8]; P = 0.005) groups may have been higher than that in 0.15 mg/kg nitrite group. Immunoblotting revealed that the level of phosphorylated Cx43 in the group given 0.15 mg/kg nitrite, but not in the other treated groups, was significantly higher compared with the control group (P = 0.007).. Nitrite may have attenuated acute ischemia-induced ventricular arrhythmias and Cx43 dephosphorylation in rats. Nitric oxide, which might be generated by xanthine oxidoreductase via nitrite reduction, appears to play a crucial role in this antiarrhythmic effect.

Topics: Allopurinol; Animals; Arrhythmias, Cardiac; Blood Gas Analysis; Connexin 43; Cyclic N-Oxides; Dose-Response Relationship, Drug; Enzyme Inhibitors; Free Radical Scavengers; Hemodynamics; Imidazoles; Male; Myocardial Ischemia; Nitric Oxide; Phosphorylation; Rats; Rats, Wistar; Sodium Nitrite; Ventricular Dysfunction

Recent research suggests the vivid possibility of using nitrite therapy against various pathological conditions. Moreover, chronic nitrite therapy offers protection against ischemia and augments endothelial cell proliferation through unknown mechanisms. Nitrite-mediated augmentation in the number of circulating neutrophils has also been reported; however, the exact mechanism is not known. In the present study, we have investigated the effect of nitrite (0.5-10 mM) on the proliferation of the neutrophilic cell line HL-60 and also explored the underlying mechanism. Treatment of HL-60 cells with sodium nitrite (0.5-5 mM) led to an increase in cell proliferation, which was confirmed by cell cycle analysis and 5-bromo-2-deoxyuridine and thymidine incorporation, whereas cells accumulated in the G(0)/G(1) phase after treatment with 10 mM nitrite. Experiments on the synchronized cells exhibited similar effect, which seems to be nitric oxide (NO)-dependent, because carboxyl-1H-imidazol-1-yloxy,2-(4-carboxyphenyl)-4,5-dihydro 4,4,5,5-tetramethyl-3-oxide abolished nitrite-mediated proliferative effect. Moreover, the NO donor sodium nitroprusside at micromolar concentrations also exhibited similar effects. Nitrite induced augmentation in S phase, and intracellular reactive oxygen species (ROS) generation was prevented by ROS scavenger/inhibitors. Moreover, mitochondrial blockers, rotenone and antimycin A, also reduced nitrite-mediated cell proliferation. Assessment of the cell cycle regulators cyclin-dependent kinase 2 (Cdk2), Cdk4, cyclin A, cyclin D, cyclin E, and p21 suggested augmentation in the expression and interaction of Cdk2/cyclin E and Cdk2 activity, whereas p21 was down-regulated. Indeed proliferative effect of nitrite was blocked by roscovitine, a Cdk2 inhibitor. The results obtained demonstrate that the proliferative effect of nitrite on HL-60 cells seems to be NO-mediated, redox-sensitive, and Cdk2 activation-dependent, warranting detailed studies before initiating its clinical use.

Topics: Cell Cycle; Cell Proliferation; Cell Survival; Cyclic N-Oxides; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases; Cyclins; Enzyme Activation; Free Radical Scavengers; G1 Phase; HL-60 Cells; Humans; Imidazoles; Nitric Oxide; Nitroprusside; Oxidation-Reduction; Reactive Oxygen Species; Sodium Nitrite

In contrast to other metal-dithiocarbamate [DEDTC] complexes, the copper-DEDTC complex is highly cytotoxic, inducing oxidative stress, preferentially in tumor cells. Because nitric oxide (NO) forms adducts with Cu[DEDTC](2), we investigated whether NO donors like S-nitroso-N-acetyl penicillamine (SNAP) or sodium nitroprusside (SNP), and nitrite, a NO decomposition product, modulate Cu[DEDTC](2) cytotoxicity against human tumor cells. We show that apoptosis-associated PARP cleavage and inducible nitric oxide synthase (iNOS) down-regulation induced by nanomolar Cu[DEDTC](2), are counteracted by 50 muM SNAP, SNP, or CoCl(2), an inducer of hypoxia and NO signaling. Nitrite was stochiometrically effective in antagonizing Cu[DEDTC](2) cytotoxicity and inducing shifts in the absorption spectrum of the binary complex in the 280 and 450 nm regions. Subtoxic concentrations of Cu[DEDTC](2) became lethal when tumor cells were pretreated with c-PTIO, a membrane-impermeable scavenger for extracellular NO. Our results suggest that: (a) reactive oxygen species induced by Cu[DEDTC](2) are scavenged by nitrite released from NO, (b) the extent of lethality of Cu[DEDTC](2) is dependent on the reciprocal formation of an inactive ternary Cu[DEDTC](2)NO copper-nitrosyl complex.

Topics: Cell Death; Cell Line, Tumor; Cell Survival; Cobalt; Cyclic N-Oxides; Down-Regulation; Free Radical Scavengers; Humans; Imidazoles; Melanoma; Mitochondria; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase Type II; Nitrites; Nitroprusside; Nitroso Compounds; Organometallic Compounds; Poly(ADP-ribose) Polymerases; S-Nitroso-N-Acetylpenicillamine; Sodium Nitrite

Cyanide inhibits aerobic metabolism by binding to the binuclear heme center of cytochrome c oxidase (CcOX). Amyl nitrite and sodium nitrite (NaNO(2)) antagonize cyanide toxicity in part by oxidizing hemoglobin to methemoglobin (mHb), which then scavenges cyanide. mHb generation is thought to be a primary mechanism by which the NO(2)(-) ion antagonizes cyanide. On the other hand, NO(2)(-) can undergo biotransformation to generate nitric oxide (NO), which may then directly antagonize cyanide inhibition of CcOX. In this study, nitrite-mediated antagonism of cyanide inhibition of oxidative phosphorylation was examined in rat dopaminergic N27 cells. NaNO(2) produced a time- and concentration-dependent increase in whole-cell and mitochondrial levels of NO. The NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxy 3-oxide (PTIO) reversed this increase in cellular and mitochondrial NO. NO generated from NaNO(2) decreased cellular oxygen consumption and inhibited CcOX activity. PTIO reversed the NO-mediated inhibition, thus providing strong evidence that NO mediates the action of NaNO(2). Under similar conditions, KCN (20muM) inhibited cellular state-3 oxygen consumption and CcOX activity. Pretreatment with NaNO(2) reversed KCN-mediated inhibition of both oxygen consumption and CcOX activity. The NaNO(2) antagonism of cyanide was blocked by pretreatment with the NO scavenger PTIO. It was concluded that NaNO(2) antagonizes cyanide inhibition of CcOX by generating of NO, which then interacts directly with the binding of KCN x CcOX to reverse the toxicity. In vivo antagonism of cyanide by NO(2)(-) appears to be due to both generation of mHb and direct displacement of cyanide from CcOX by NO.

Topics: Animals; Cell Line, Transformed; Chemical Warfare Agents; Cyclic N-Oxides; Dopamine; Drug Antagonism; Electron Transport Complex IV; Free Radical Scavengers; Hydrogen Cyanide; Imidazoles; Neurons; Nitric Oxide; Nitric Oxide Donors; Rats; Sodium Nitrite

Chronic tissue ischemia due to defective vascular perfusion is a hallmark feature of peripheral artery disease for which minimal therapeutic options exist. We have reported that sodium nitrite therapy exerts cytoprotective effects against acute ischemia/reperfusion injury in both heart and liver, consistent with the model of bioactive NO formation from nitrite during ischemic stress. Here, we test the hypothesis that chronic sodium nitrite therapy can selectively augment angiogenic activity and tissue perfusion in the murine hind-limb ischemia model. Various therapeutic doses (8.25-3,300 mug/kg) of sodium nitrite or PBS were administered. Sodium nitrite significantly restored ischemic hind-limb blood flow in a time-dependent manner, with low-dose sodium nitrite being most effective. Nitrite therapy significantly increased ischemic limb vascular density and stimulated endothelial cell proliferation. Remarkably, the effects of sodium nitrite therapy were evident within 3 days of the ischemic insult demonstrating the potency and efficacy of chronic sodium nitrite therapy. Sodium nitrite therapy also increased ischemic tissue nitrite and NO metabolites compared to nonischemic limbs. Use of the NO scavenger carboxy PTIO completely abolished sodium nitrite-dependent ischemic tissue blood flow and angiogenic activity consistent with nitrite reduction to NO being the proangiogenic mechanism. These data demonstrate that chronic sodium nitrite therapy is a recently discovered therapeutic treatment for peripheral artery disease and critical limb ischemia.

Topics: Animals; Arteries; Cyclic N-Oxides; Cytoprotection; Disease Models, Animal; Endothelium, Vascular; Free Radical Scavengers; Hindlimb; Imidazoles; Ischemia; Male; Mice; Mice, Inbred C57BL; Neovascularization, Physiologic; Nitric Oxide; Peripheral Vascular Diseases; Sodium Nitrite