In vitro effects of acetaminophen metabolites and analogs on the respiration of mouse liver mitochondria

Acetaminophen, an analgesic and antipyretic, is toxic in overdose to liver and kidney. The effects on mitochondrial respiration of acetaminophen, its less toxic analog, 3-hydroxyacetanilide, and metabolites which arise from these compounds have been investigated. The parent compounds inhibited NADH-linked respiration reversibly, whereas the metabolites inhibit all mitochondrial respiration, apparently in the Complex III region of the respiratory chain. The quinone derivatives, 4-acetamido-o-benzoquinone and 2-acetamido-p-benzoquinone, are the best inhibitors, with the onset of inhibition dependent on active respiration, suggesting interaction of these compounds with oxidized components of the electron transport chain.     

Ischemic preconditioning inhibits mitochondrial respiration,increases H2O2 release,and enhances K+ transport

Ischemic preconditioning, or the protective effect of short ischemic episodes on a longer, potentially injurious, ischemic period, is prevented by antagonists of mitochondrial ATP-sensitive K+ channels (mitoKATP) and involves changes in mitochondrial energy metabolism and reactive oxygen release after ischemia. However, the effects of ischemic preconditioning itself on mitochondria are still poorly understood. We determined the effects of ischemic preconditioning on isolated heart mitochondria and found that two brief (5 min) ischemic episodes are sufficient to induce a small but significant decrease ( approximately 25%) in mitochondrial NADH-supported respiration. Preconditioning also increased mitochondrial H2O2 release, an effect related to respiratory inhibition, because it is not observed in the presence of succinate plus rotenone and can be mimicked by chemically inhibiting complex I in the presence of NADH-linked substrates. In addition, preconditioned mitochondria presented more substantial ATP-sensitive K+ transport, indicative of higher mitoKATP activity. Thus we directly demonstrate that preconditioning leads to mitochondrial respiratory inhibition in the presence of NADH-linked substrates, increased reactive oxygen release, and activation of mitoKATP.     

Inhibition of brain mitochondrial respiration by dopamine and its metabolites: implications for Parkinson's disease and catecholamine-associated diseases

A structure-potency study examining the ability of dopamine (DA), its major metabolites and related amine and acetate congeners to inhibit NADH-linked mitochondrial O(2) consumption was carried out to elucidate mechanisms by which DA could induce mitochondrial dysfunction. In the amine studies, DA was the most potent inhibitor of respiration (IC(50) 7.0 mm) compared with 3-methoxytryramine (3-MT, IC(50) 19.6 mm), 3,4-dimethoxyphenylethylamine (IC(50) 28.6 mm), tyramine (IC(50) 40.3 mm) and phenylethylamine (IC(50) 58.7 mm). Addition of monoamine oxidase (MAO) inhibitors afforded nearly complete protection against inhibition by phenylethylamine, tyramine and 3,4-dimethoxyphenylethylamine, indicating that inhibition arose from MAO-mediated pathways. In contrast, the inhibitory effects of DA and 3-MT were only partially prevented by MAO blockade, suggesting that inhibition might also arise from two-electron catechol oxidation and quinone formation by DA and one-electron oxidation of the 4-hydroxyphenyl group of 3-MT. In the phenylacetate studies, 3,4-dihydroxyphenylacetic acid (DOPAC) was equipotent with DA in inhibiting respiration (IC(50) 7.4 mm), further implicating the catechol reaction as the cause of inhibition. All other carboxylate congeners; phenylacetic acid (IC(50) 13.0 mm), 4-hydroxyphenylacetic acid (IC(50) 12.1 mm), 4-hydroxy-3-methoxyphenylacetic acid (HVA, IC(50) 12.0 mm) and 3,4-dimethoxyphenylacetic acid (IC(50) 10.2 mm), were equipotent respiratory inhibitors and two- to fourfold more potent than their corresponding amine. These latter findings suggest that the phenylacetate ion can also contribute independently to mitochondrial inhibition. In summary, mitochondrial respiration can be inhibited by DA and its metabolites by four distinct MAO-dependent and independent mechanisms.     

4'-alkylated analogs of 1-methyl-4-phenylpyridinium ion are potent inhibitors of mitochondrial respiration

1-Methyl-4-phenylpyridinium ion, a major brain metabolite of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, is an inhibitor of Complex I of the mitochondrial respiratory chain. We have synthesized several analogs of 1-methyl-4-phenylpyridinium ion containing various alkyl groups in the 4' position of the phenyl ring and have tested them for their abilities to inhibit the oxidation of NADH-linked substrates by intact mouse liver mitochondria. These compounds are considerably more potent inhibitors than MPP+ itself, with potency increasing as the length of the alkyl chain increases. The most potent inhibitor, 1-methyl-4-(4'heptylphenyl)pyridinium ion, was about 200 times as effective as MPP+. These analogs should prove to be useful tools for studying the nature of the process whereby MPP+ and its pyridinium analogs interact with Complex I to inhibit mitochondrial respiration.     

Dopaminergic Neurotoxicity In Vivo and Inhibition of Mitochondrial Respiration In Vitro by Possible Endogenous Pyridinium-Like Substances

Elucidation of the mechanism(s) by which 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) and its active metabolite 1-methyl-4-phenylpyridinium (MPP+) cause parkinsonism in humans and other primates has prompted consideration of possible endogenous MPTP/MPP(+)-like neurotoxins in the etiology of idiopathic Parkinson's disease. Here we examined inhibition of mitochondrial respiration in vitro and neurotoxicity in rats in vivo produced by beta-carbolinium compounds that are presumed to form following Pictet-Spengler cyclization of serotonin. We also evaluated N-methylisoquinolinium, a putative endogenous neurotoxin, in the same manner. The latter compound exhibited MPP(+)-like mitochondrial respiratory inhibition, whereas the beta-carbolinium compounds, although more potent inhibitors of electron transport, exhibited weak accumulation-dependent enhancement of inhibition in intact mitochondria. It is interesting that the beta-carbolinium compounds inhibited succinate- as well as glutamate-supported respiration, and are best described as inhibitor-uncouplers. The results of partitioning experiments suggest that both the low accumulation potential and the inhibition of succinate respiration may be a consequence of the beta-carboliniums being in equilibrium with neutral "anhydro" bases. Relative to MPP+, all compounds tested had weak dopaminergic uptake activity in vitro and weak dopaminergic toxicity in vivo, consistent with other findings of relatively low neurotoxic potential for presumed endogenous pyridiniums.     

Modulation of autophagy by the novel mitochondrial complex I inhibitor Authipyrin

Autophagy ensures cellular homeostasis by the degradation of long-lived proteins, damaged organelles and pathogens. This catabolic process provides essential cellular building blocks upon nutrient deprivation. Cellular metabolism, especially mitochondrial respiration, has a significant influence on autophagic flux, and complex I function is required for maximal autophagy. In Parkinson’s disease mitochondrial function is frequently impaired and autophagic flux is altered. Thus, dysfunctional organelles and protein aggregates accumulate and cause cellular damage. In order to investigate the interdependency between mitochondrial function and autophagy, novel tool compounds are required. Herein, we report the discovery of a structurally novel autophagy inhibitor (Authipyrin) using a high content screening approach. Target identification and validation led to the discovery that Authipyrin targets mitochondrial complex I directly, leading to the potent inhibition of mitochondrial respiration as well as autophagy.     

Specific inhibition of mitochondrial protein synthesis influences the amount of complex I in mitochondria of rat liver and Neurospora crassa directly

Specific inhibition of mitochondrial protein synthesis reduces the oxidation rate of NADH-linked substrates in rat liver as well as in Neurospora crassa mitochondria. The present study shows that this is due to the fact that inhibition of mitochondrial protein synthesis leads to a decrease of the concentration of active complex I. Therefore, these results demonstrate that at least one of the genes for the subunits of complex I is localized on mitochondrial DNA.     

Inhibition of mitochondrial respiration by 1,2,3,4-tetrahydroisoquinoline-like endogenous alkaloids in mouse brain

Since the discovery of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism, it has been postulated that (a) MPTP-like toxin(s) such as 1,2,3,4-tetrahydroisoquinoline (TIQ) may induce Parkinson's disease. As the neuronal degeneration in MPTP-induced parkinsonism is thought to be caused by the inhibition of the mitochondrial respiration by 1-methyl-4-phenylpyridinium ion (MPP⁺), we studied the effects of TIQ-like alkaloids including dopaminederived ones on the mitochondrial respiration using mouse brains. TIQ, tetrahydropapaveroline (THP), and tetrahydropapaverine (THPV) produced significant inhibition of the state 3 and 4 respiration and respiratory control ratio supported by glutamate + malate, the activity of Complex 1 and the ATP synthesis. Among those compounds, THPV was most potent. Toxic properties of these compounds on mitochondria were quite similar to that of MPP⁺. Our results support the hypothesis that (a) MPTP- or MPP⁺-like substance(s) may be responsible for the nigral degeneration in Parkinson's disease.Abbreviations used MPTP 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine - MPP⁺ 1-methyl-4-phenylpyridinium ion - ATP adenosine triphosphate - ADP Adenosine diphosphate - TCL tricarboxylic acid - TIQ cycle: 1,2,3,4-Tetrahydroisoquinoline - THPV Tetrahydropapaverine - THP Tetrahydropaveroline     

Mitochondrial defects and heterogeneous cytochrome c release after cardiac cold ischemia and reperfusion

Mitochondria play a critical role in myocardial cold ischemia-reperfusion (CIR) and induction of apoptosis. The nature and extent of mitochondrial defects and cytochrome c (Cyt c) release were determined by high-resolution respirometry in permeabilized myocardial fibers. CIR in a rat heart transplant model resulted in variable contractile performance, correlating with the decline of ADP-stimulated respiration. Respiration with succinate or N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (substrates for complexes II and IV) was partially restored by added Cyt c, indicating Cyt c release. In contrast, NADH-linked respiration (glutamate+malate) was not stimulated by Cyt c, owing to a specific defect of complex I. CIR but not cold ischemia alone resulted in the loss of NADH-linked respiratory capacity, uncoupling of oxidative phosphorylation and Cyt c release. Mitochondria depleted of Cyt c by controlled hypoosmotic shock provided a kinetic model of homogeneous Cyt c depletion. Comparison to Cyt c control of respiration in CIR-injured myocardial fibers indicated heterogeneity of Cyt c release. The complex I defect and uncoupling correlated with heterogeneous Cyt c release, the extent of which increased with loss of cardiac performance. These results demonstrate a complex pattern of multiple mitochondrial damage as determinants of CIR injury of the heart.     

Dopamine Oxidation Alters Mitochondrial Respiration and Induces Permeability Transition in Brain Mitochondria

Both reactive dopamine metabolites and mitochondrial dysfunction have been implicated in the neurodegeneration of Parkinson's disease. Dopamine metabolites, dopamine quinone and reactive oxygen species, can directly alter protein function by oxidative modifications, and several mitochondrial proteins may be targets of this oxidative damage. In this study, we examined, using isolated brain mitochondria, whether dopamine oxidation products alter mitochondrial function. We found that exposure to dopamine quinone caused a large increase in mitochondrial resting state 4 respiration. This effect was prevented by GSH but not superoxide dismutase and catalase. In contrast, exposure to dopamine and monoamine oxidase-generated hydrogen peroxide resulted in a decrease in active state 3 respiration. This inhibition was prevented by both pargyline and catalase. We also examined the effects of dopamine oxidation products on the opening of the mitochondrial permeability transition pore, which has been implicated in neuronal cell death. Dopamine oxidation to dopamine quinone caused a significant increase in swelling of brain and liver mitochondria. This was inhibited by both the pore inhibitor cyclosporin A and GSH, suggesting that swelling was due to pore opening and related to dopamine quinone formation. In contrast, dopamine and endogenous monoamine oxidase had no effect on mitochondrial swelling. These findings suggest that mitochondrial dysfunction induced by products of dopamine oxidation may be involved in neurodegenerative conditions such as Parkinson's disease and methamphetamine-induced neurotoxicity.     

Inhibition of mitochondrial succinate oxidation--similarities and differences between N-methylated beta-carbolines and MPP+.

N-Methylated beta-carbolinium compounds (N-Me-BCs), including 2-N-methyl and 2,9-N,N-dimethyl analogs, structural analogs of 1-methyl-4-phenylpyridinium (MPP+), may be endogenously bioactivated, MPP(+)-like toxins, capable of inducing parkinsonism. Both MPP+ and selected N-Me-BCs inhibit NADH-linked mitochondrial respiration (Complex I). We now show that both also inhibit succinate-supported (Complex II) respiration, the greatest inhibition (80%) being seen for 2,9-dimethylharmanium. Complex I inhibition occurs at MPP+ concentrations (IC50 = 0.17 mM) about one order of magnitude lower than Complex II inhibition (greater than 1.2 mM). In contrast, Complex I and Complex II inhibition by the N-Me-BCs tested occurred at similar concentrations (I, 0.1 mM; II, 0.25 mM) and concentrations similar to Complex I inhibition by MPP+. 2,9-N,N-Dimethyl-BCs, which are the permanently charged BC analogs of MPP+, show inhibitory characteristics similar to MPP+: slow onset of inhibition, potentiation by TPB, and reversal by DNP. The fact that succinate oxidation cannot bypass the Complex II inhibition by N-Me-BCs could enhance any chronic neurotoxicity of N-Me-BCs.     

Anatomic and Disease Specificity of NADH CoQ1 Reductase (Complex I) Deficiency in Parkinson''s Disease

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is thought to produce parkinsonism in humans and other primates through its inhibition of complex I. The recent discovery of mitochondrial complex I deficiency in the substantia nigra of patients with Parkinson's disease has provided a remarkable link between the idiopathic disease and the action of the neurotoxin MPTP. This article shows that complex I deficiency in Parkinson's disease is anatomically specific for the substantia nigra, and is not present in another neurodegenerative disorder involving the substantia nigra. Evidence is also provided to show that there is no correlation between L-3,4-dihydroxyphenylalanine therapy and complex I deficiency. These results suggest that complex I deficiency may be the underlying cause of dopaminergic cell death in Parkinson's disease.     

Acetaminophen toxicity results in site-specific mitochondrial damage in isolated mouse hepatocytes

Exposure of isolated mouse hepatocytes to a toxic concentration of acetaminophen (5 mM) resulted in damage to the mitochondrial respiratory apparatus. The nature of this damage was investigated by measuring respiration stimulated by site-specific substrates in digitonin-permeabilized hepatocytes after acetaminophen exposure. Respiration stimulated by succinate at energy-coupling site 2 was most sensitive to inhibition and was decreased by 47% after 1 h. Respiration supported by NADH-linked substrates (site 1) was also decreased but to a lesser extent, while there was no decrease in the rate of ascorbate + N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD)-supported respiration (site 3). The loss of mitochondrial respiratory function was accompanied by a decrease in ATP levels and ATP/ADP ratios in the cytosolic compartment and was preceded by a loss of reduced glutathione in both the cytosol and mitochondria. All these effects occurred well before the loss of cell membrane integrity. The putative toxic metabolite of acetaminophen, N-acetyl-p-benzoquinonimine (NAPQI), produced a similar pattern of respiratory dysfunction in isolated hepatic mitochondria. Respiration stimulated by succinate- and NADH-linked substrates was very sensitive to 50 microM NAPQI, while ascorbate + TMPD-supported respiration was unaffected. The interaction between NAPQI and the respiratory chain was further investigated using submitochondrial particles. Succinate dehydrogenase (associated with respiratory complex II) was found to be very sensitive to NAPQI, while NADH dehydrogenase (respiratory complex I) was inhibited to a lesser extent. Our results indicate that a loss of the ability to utilize succinate- and NADH-linked substrates due to attack of the respiratory chain by NAPQI causes a disruption of energy homeostasis in acetaminophen hepatotoxicity.     

Cubebin and derivatives as inhibitors of mitochondrial complex I. Proposed interaction with subunit B8

The effects on mitochondrial respiration and complex I NADH oxidase activity of cubebin and derivatives were evaluated. The compounds inhibited the state 3 glutamate/malate-supported respiration of hamster liver mitochondria with IC(50) values ranging from 12.16 to 83.96 microM. NADH oxidase reaction was evaluated in submitochondrial particles. The compounds also inhibited this activity, showing the same order of potency observed for effects on state 3 respiration, as well as a tendency towards a non-competitive type of inhibition (K(I) values ranging from 0.62 to 16.1 microM). A potential binding mode of these compounds with complex I subunit B8, assessed by docking calculations, is proposed.     

Ubiquinone (coenzyme q10) and mitochondria in oxidative stress of parkinson's disease

Parkinson's disease is the second most common neurodegenerative disorder after Alzheimer's disease affecting approximately1% of the population older than 50 years. There is a worldwide increase in disease prevalence due to the increasing age of human populations. A definitive neuropathological diagnosis of Parkinson's disease requires loss of dopaminergic neurons in the substantia nigra and related brain stem nuclei, and the presence of Lewy bodies in remaining nerve cells. The contribution of genetic factors to the pathogenesis of Parkinson's disease is increasingly being recognized. A point mutation which is sufficient to cause a rare autosomal dominant form of the disorder has been recently identified in the alpha-synuclein gene on chromosome 4 in the much more common sporadic, or 'idiopathic' form of Parkinson's disease, and a defect of complex I of the mitochondrial respiratory chain was confirmed at the biochemical level. Disease specificity of this defect has been demonstrated for the parkinsonian substantia nigra. These findings and the observation that the neurotoxin 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP), which causes a Parkinson-like syndrome in humans, acts via inhibition of complex I have triggered research interest in the mitochondrial genetics of Parkinson's disease. Oxidative phosphorylation consists of five protein-lipid enzyme complexes located in the mitochondrial inner membrane that contain flavins (FMN, FAD), quinoid compounds (coenzyme Q10, CoQ10) and transition metal compounds (iron-sulfur clusters, hemes, protein-bound copper). These enzymes are designated complex I (NADH:ubiquinone oxidoreductase, EC 1.6. 5.3), complex II (succinate:ubiquinone oxidoreductase, EC 1.3.5.1), complex III (ubiquinol:ferrocytochrome c oxidoreductase, EC 1.10.2.2), complex IV (ferrocytochrome c:oxygen oxidoreductase or cytochrome c oxidase, EC 1.9.3.1), and complex V (ATP synthase, EC 3.6.1.34). A defect in mitochondrial oxidative phosphorylation, in terms of a reduction in the activity of NADH CoQ reductase (complex I) has been reported in the striatum of patients with Parkinson's disease. The reduction in the activity of complex I is found in the substantia nigra, but not in other areas of the brain, such as globus pallidus or cerebral cortex. Therefore, the specificity of mitochondrial impairment may play a role in the degeneration of nigrostriatal dopaminergic neurons. This view is supported by the fact that MPTP generating 1-methyl-4-phenylpyridine (MPP(+)) destroys dopaminergic neurons in the substantia nigra. Although the serum levels of CoQ10 is normal in patients with Parkinson's disease, CoQ10 is able to attenuate the MPTP-induced loss of striatal dopaminergic neurons.     

Mechanism of toxicity of pesticides acting at complex I: relevance to environmental etiologies of Parkinson's disease

Parkinson's disease (PD) has been linked to mitochondrial dysfunction and pesticide exposure. The pesticide rotenone (ROT) inhibits complex I and reproduces features of PD in animal models, suggesting that environmental agents that inhibit complex I may contribute to PD. We have previously demonstrated that ROT toxicity is dependent upon complex I inhibition and that oxidative stress is the primary mechanism of toxicity. In this study, we examined the in vitro toxicity and mechanism of action of several putative complex I inhibitors that are commonly used as pesticides. The rank order of toxicity of pesticides to neuroblastoma cells was pyridaben > rotenone > fenpyroximate > fenazaquin > tebunfenpyrad. A similar order of potency was observed for reduction of ATP levels and competition for (3)H-dihydrorotenone (DHR) binding to complex I, with the exception of pyridaben (PYR). Neuroblastoma cells stably expressing the ROT-insensitive NADH dehydrogenase of Saccharomyces cerevisiae (NDI1) were resistant to these pesticides, demonstrating the requirement of complex I inhibition for toxicity. We further found that PYR was a more potent inhibitor of mitochondrial respiration and caused more oxidative damage than ROT. The oxidative damage could be attenuated by NDI1 or by the antioxidants alpha-tocopherol and coenzyme Q(10). PYR was also highly toxic to midbrain organotypic slices. These data demonstrate that, in addition to ROT, several commercially used pesticides directly inhibit complex I, cause oxidative damage, and suggest that further study is warranted into environmental agents that inhibit complex I for their potential role in PD.     

Acetylsalicylic acid and acetaminophen protect against MPP+-induced mitochondrial damage and superoxide anion generation

The effects of 1-methyl-4-phenylpyridinium (MPP+) has been extensively researched due to its selective toxicity to dopaminergic neurons. Mitochondrial dysfunction which is common in the etiology of Parkinson's disease (PD), has been widely implicated in MPP+-induced toxicity. MPP+-induced mitochondrial dysfunction is believed to result in the generation of free radicals. This study was therefore performed to assess the effect of MPP+ on mitochondrial function and the ability of MPP+ to generate superoxide free radicals. Furthermore, we assessed the ability of the non-narcotic analgesics, acetaminophen and acetylsalicylic acid to prevent any diliterious effects of the potent neurotoxin, MPP+, on mitochondrial function and superoxide anion generation, in vivo. Acetylsalicylic acid and acetaminophen prevented the MPP+-induced inhibition of the electron transport chain and complex I activity. In addition, acetylsalicylic acid and acetaminophen significantly attenuated the MPP+-induced superoxide anion generation. Furthermore the results provide novel data explaining the ability of these agents to prevent MPP+-induced mitochondrial dysfunction and subsequent reactive oxygen species generation. While these findings suggest the usefulness of non-narcotic analgesics in neuroprotective therapy in neurodegenerative diseases, acetylsalicylic acid appears to be a potential candidate in prophylactic as well as in adjuvant therapy in Parkinson's disease.     

Rotenone induces reductive stress and triacylglycerol deposition in C2C12 cells

Environmental rotenone is associated with Parkinson's disease due to its inhibitory property to the complex I of mitochondrial respiration chain. Although environmental pollution has been postulated as a causal factor for the increasing prevalence of obesity, the role of rotenone in the pathogenesis of obesity has not been studied. We employed muscle-derived cell C2C12 as a model and shotgun lipidomics as a tool for lipid analysis and found that treatment with rotenone led to the profound deposition of intracellular triacylglycerol (TAG) in a time- and dose-dependent fashion. The TAG deposition resulted from complex I inhibition. Further studies revealed that rotenone induced mitochondrial stress as shown by decreased mitochondrial oxygen consumption rate, increased NADH/NAD+ ratio (i.e., reductive stress) and mitochondrial metabolites. We demonstrated that rotenone activated fatty acid de novo synthesis and TAG synthesis and ultimately resulted in intracellular TAG deposition. These studies suggested that increased mitochondrial stresses might be an underlying mechanism responsible for TAG accumulation manifest in obesity.     

Cubebin and derivatives as inhibitors of mitochondrial complex I. Proposed interaction with subunit B8

The effects on mitochondrial respiration and complex I NADH oxidase activity of cubebin and derivatives were evaluated. The compounds inhibited the state 3 glutamate/malate-supported respiration of hamster liver mitochondria with IC₅₀ values ranging from 12.16 to 83.96 μM. NADH oxidase reaction was evaluated in submitochondrial particles. The compounds also inhibited this activity, showing the same order of potency observed for effects on state 3 respiration, as well as a tendency towards a non-competitive type of inhibition (K_I values ranging from 0.62 to 16.1 μM). A potential binding mode of these compounds with complex I subunit B8, assessed by docking calculations, is proposed.     

Reversal of nitric oxide-, peroxynitrite- and S-nitrosothiol-induced inhibition of mitochondrial respiration or complex I activity by light and thiols

Nitric oxide (NO) and its derivatives peroxynitrite and S-nitrosothiols inhibit mitochondrial respiration by various means, but the mechanisms and/or the reversibility of such inhibitions are not clear. We find that the NO-induced inhibition of respiration in isolated mitochondria due to inhibition of cytochrome oxidase is acutely reversible by light. Light also acutely reversed the inhibition of respiration within iNOS-expressing macrophages, and this reversal was partly due to light-induced breakdown of NO, and partly due to reversal of the NO-induced inhibition of cytochrome oxidase. NO did not cause inhibition of complex I activity within isolated mitochondria, but 0.34 mM peroxynitrite, 1 mM S-nitroso-N-acetylpenicillamine or 1 mM S-nitrosoglutathione did cause substantial inhibition of complex I activity. Inhibition by these reagents was reversed by light, dithiothreitol or glutathione-ethyl ester, either partially or completely, depending on the reagent used. The rapid inhibition of complex I activity by S-nitroso-N-acetylpenicillamine also occurred in conditions where there was little or no release of free NO, suggesting that the inhibition was due to transnitrosylation of the complex. These findings have implications for the physiological and pathological regulation of respiration by NO and its derivatives.