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Nitric oxide, cytochrome c and mitochondria
1Department of Biochemistry, University of Cambridge, U.K.
Abstract:
Nitric oxide (NO) and its derivative, peroxynitrite (ONOO-), inhibit mitochondrial respiration, and this inhibition may contribute to both the physiological and cytotoxic actions of NO. Nanomolar concentrations of NO rapidly and reversibly inhibited cytochrome oxidase in competition with oxygen, as shown with isolated cytochrome oxidase, mitochondria, brain nerve terminals and cells. Cultured astrocytes and macrophages activated (by cytokines and endotoxin) to express the inducible form of NO synthase produced up to 1 microM NO, and inhibited their own respiration and that of co-incubated cells via reversible NO inhibition of cytochrome oxidase. NO-induced inhibition of respiration in brain nerve terminals resulted in rapid glutamate release, which might contribute to the neurotoxicity of NO. NO inhibition of cytochrome oxidase is reversible; however, incubation of cells with NO donors for 4 hours resulted in an inhibition of complex I, which was reversible by light and thiol reagents and may be due to nitrosylation of thiols in complex I. NO also caused the acute inhibition of catalase, stimulation of hydrogen peroxide production by mitochondria, and reaction with hydrogen peroxide on superoxide dismutase to produce peroxynitrite. Peroxynitrite inhibited complexes I, II and V (the ATP synthase), aconitase, creatine kinase, and increases the proton leak in isolated mitochondria. Peroxynitrite also caused opening of the permeability transition pore, resulting in the release of cytochrome c, which might then trigger apoptosis. Hypoxia/ischaemia also resulted in an acute reversible inhibition of cytochrome oxidase. Heart ischaemia caused the release of cytochrome c from mitochondria into the cytosol, and at the same time caspase-3-like-protease activity was activated in the cytoplasm. Addition of cytochrome c to non-ischaemic cytosol also caused activation of this protease activity, suggesting that caspase activation and consequent apoptosis is at least partly a result of this cytochrome c release.
Insights
Nitric oxide (NO) and peroxynitrite inhibit mitochondrial respiration, impacting cellular function and potentially causing neurotoxicity and apoptosis. These molecules affect key respiratory enzymes and mitochondrial integrity.
Area of Science:
- Biochemistry
- Cell Biology
- Neuroscience
Background:
- Nitric oxide (NO) and peroxynitrite (ONOO-) are signaling molecules with significant roles in physiology and pathology.
- Mitochondrial dysfunction is implicated in various diseases, including neurodegenerative disorders.
Purpose of the Study:
- To investigate the inhibitory effects of NO and ONOO- on mitochondrial respiration.
- To elucidate the mechanisms by which NO and ONOO- induce cellular damage and apoptosis.
Main Methods:
- Experiments using isolated enzymes, mitochondria, and cell cultures (astrocytes, macrophages).
- Measurement of mitochondrial respiration, enzyme activity, and apoptosis markers.
- Utilized NO donors and hypoxia/ischemia models.
Main Results:
- NO reversibly inhibits cytochrome oxidase at nanomolar concentrations, affecting cellular respiration.
- Prolonged NO exposure leads to Complex I inhibition, potentially via thiol nitrosylation.
- ONOO- inhibits multiple mitochondrial complexes (I, II, V), aconitase, and creatine kinase, inducing mitochondrial permeability transition pore opening and cytochrome c release.
- Ischemia induces cytochrome c release and caspase activation, suggesting apoptosis.
Conclusions:
- NO and ONOO- are potent inhibitors of mitochondrial respiration, contributing to NO's physiological and cytotoxic effects.
- Mitochondrial dysfunction induced by NO and ONOO- can lead to neurotoxicity and apoptosis.
- Cytochrome c release is a key event in ischemia-induced apoptosis.