ABSTRACT
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.
Subject(s)
Chemical Warfare Agents/toxicity , Dopamine/physiology , Electron Transport Complex IV/antagonists & inhibitors , Hydrogen Cyanide/toxicity , Neurons/drug effects , Nitric Oxide Donors/pharmacology , Sodium Nitrite/pharmacology , Animals , Cell Line, Transformed , Cyclic N-Oxides/pharmacology , Drug Antagonism , Electron Transport Complex IV/metabolism , Free Radical Scavengers/pharmacology , Hydrogen Cyanide/metabolism , Imidazoles/pharmacology , Neurons/metabolism , Nitric Oxide/metabolism , RatsABSTRACT
Acute cyanide toxicity is attributed to inhibition of cytochrome c oxidase (CcOX), the oxygen-reducing component of mitochondrial electron transport; however, the mitochondrial action of cyanide is complex and not completely understood. State-3 oxygen consumption and CcOX activity were studied in rat N27 mesencephalic cells to examine the functional interaction of cyanide and nitric oxide (NO). KCN produced a concentration-dependent inhibition of cellular respiration. Cyanide's median inhibitory concentration (IC50) of oxygen consumption (13.2 +/- 1.8microM) was higher than the CcOX IC50 (7.2 +/- 0.1microM). Based on respiratory threshold analysis, 60% inhibition of CcOX was necessary before oxygen consumption was decreased. Addition of high levels of exogenous NO (100microM S-nitroso-N-acetyl-DL-penicillamine) attenuated cyanide inhibition of both respiration and CcOX. On the other hand, when endogenous NO generation was blocked by an NOS inhibitor (N(omega)-monomethyl-L-arginine ester), the cyanide IC50 for both respiration and CcOX increased to 59.6 +/- 0.9microM and 102 +/- 10microM, respectively, thus showing constitutive, low-level NO production enhanced cyanide inhibition. Laser scanning cytometry showed that cyanide elevated mitochondrial NO, which then was available to interact with CcOX to enhance the inhibition. It is concluded that the rapid, potent action of cyanide is due in part to mitochondrial generation of NO, which enhances inhibition of CcOX. At low mitochondrial oxygen tensions, the cyanide-NO interaction would be increased. Also, the antidotal action of sodium nitrite is partly explained by generation of high mitochondrial levels of NO, which antagonizes the CcOX inhibition.
