The role of nitric oxide in the cyanide-mediated inhibition of cytochrome c oxidase

Heather B Leavesley, Purdue University

Abstract

Cyanide causes rapid-onset neurotoxicity by inhibiting cellular respiration at cytochrome c oxidase (CcOX). Cyanide primarily targets the brain, where it promotes dopaminergic cell death. Acute cyanide toxicity has been associated with a late-onset Parkinson-like neuropathy. Nitric oxide (NO), an endogenous CcOX inhibitor, has been implicated in the cyanide-induced loss of dopaminergic neurons. We recently found that the cyanide-mediated increase in mitochondrial NO (NOmt) was associated with CcOX inhibition. Treatment with a nitric oxide synthase (NOS) inhibitor significantly attenuated the CcOX inhibition. Mitochondrial NOS (mtNOS) activity may play a key role in the cyanide inhibition of CcOX. On the other hand, high concentrations of NO antagonize the cyanide inhibition of CcOX. The sodium nitrite antidote, an NO donor, may attenuate cyanide toxicity through NO generation. In the current study, we examined the role of endogenous and exogenous NO on the cyanide (1-60 μM) inhibition of CcOX. First, we examined the mechanism by which NOmt promotes cyanide inhibition of CcOX. Cyanide is known to increase intracellular Ca2+, which increases mtNOS activity. Therefore, we hypothesized that cyanide increases NOmt via a Ca2+-induced increase in mtNOS activity. Ca 2+-mediated changes in mtNOS activity were examined using fluorometric analyses of Ca2+ and NO in N27 dopaminergic cells. 10-s and 15-min cyanide treatments were used to examine acute and latent effects on mtNOS activity, respectively. Within seconds, 8 μM KCN produced a significant increase in Ca2+ mt, NOmt, and mtNOS. These effects were blocked by the the Ca2+ uniport inhibitor ruthenium red (RuRed, 5 μM). The increase in mtNOS activity was associated with a decrease in Δψ m and CcOX activity (IC50 = 7.8 μM), which were both reversed by RuRed. After 15 min, however, the increase in NOmt by cyanide was associated with an increase in cytosolic Ca2+. The findings suggest that the cyanide-mediated increase in NOmt and CcOX inhibition are facilitated by a rapid rise in mtNOS activity. Next, we examined the role of sodium nitrite (NaNO2) in the reversal of cyanide inhibition of CcOX. In N27 cells, 100 μM NaNO2 inhibited cellular respiration and CcOX function via an increase in NOmt, as determined by fluorometric and enzymatic analyses. KCN (20 μM) also inhibited cellular respiration. However, in the presence of nitrite (100 μM), the cyanide inhibition of CcOX and cellular respiration was reversed. Treatment with the NO scavenger PTIO (200 μM) prevented the nitrite antagonism of cyanide. In addition, cobinamide (250 μM), which scavenges NO and cyanide, stimulated cellular respiration above control (untreated cells). The data suggest that sodium nitrite antagonizes cyanide inhibition of CcOX in part via exogenous NO. The current studies indicate that cyanide increases mtNOS activity to facilitate rapid-onset CcOX inhibition, which can be reversed by exogenous NO. Therefore, Ca2+mt uniport inhibitors and NO donors are potential therapeutic candidates for treating cyanide-induced parkinsonism.

Degree

Ph.D.

Advisors

Isom, Purdue University.

Subject Area

Neurosciences|Toxicology|Surgery|Pharmacology

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