Pasdois 2011 Biochem J: Difference between revisions
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|abstract=Oxidized cytochrome ''c'' is a powerful superoxide scavenger within the mitochondrial IMS (intermembrane space), but the importance of this role in situ has not been well explored. In the present study, we investigated this with particular emphasis on whether loss of cytochrome ''c'' from mitochondria during heart ischaemia may mediate the increased production of ROS (reactive oxygen species) during subsequent reperfusion that induces mPTP (mitochondrial permeability transition pore) opening. Mitochondrial cytochrome c depletion was induced ''in vitro'' with digitonin or by 30 min ischaemia of the perfused rat heart. Control and cytochrome c-deficient mitochondria were incubated with mixed respiratory substrates and an ADP-regenerating system (State 3.5) to mimic physiological conditions. This contrasts with most published studies performed with a single substrate and without significant ATP turnover. Cytochrome c-deficient mitochondria produced more H<sub>2</sub>O<sub>2</sub> than control mitochondria, and exogenous cytochrome c addition reversed this increase. In the presence of increasing [KCN] rates of H<sub>2</sub>O<sub>2</sub> production by both pre-ischaemic and end-ischaemic mitochondria correlated with the oxidized cytochrome ''c'' content, but not with rates of respiration or NAD(P)H autofluorescence. Cytochrome ''c'' loss during ischaemia was not mediated by mPTP opening (cyclosporine-A insensitive), neither was it associated with changes in mitochondrial Bax, Bad, Bak or Bid. However, bound HK2 (hexokinase 2) and Bcl-xL were decreased in end-ischaemic mitochondria. We conclude that cytochrome c loss during ischaemia, caused by outer membrane permeabilization, is a major determinant of H<sub>2</sub>O<sub>2</sub> production by mitochondria under pathophysiological conditions. We further suggest that in hypoxia, production of H<sub>2</sub>O<sub>2</sub> to activate signalling pathways may be also mediated by decreased oxidized cytochrome ''c'' and less superoxide scavenging. | |abstract=Oxidized cytochrome ''c'' is a powerful superoxide scavenger within the mitochondrial IMS (intermembrane space), but the importance of this role in situ has not been well explored. In the present study, we investigated this with particular emphasis on whether loss of cytochrome ''c'' from mitochondria during heart ischaemia may mediate the increased production of ROS (reactive oxygen species) during subsequent reperfusion that induces mPTP (mitochondrial permeability transition pore) opening. Mitochondrial cytochrome c depletion was induced ''in vitro'' with digitonin or by 30 min ischaemia of the perfused rat heart. Control and cytochrome c-deficient mitochondria were incubated with mixed respiratory substrates and an ADP-regenerating system (State 3.5) to mimic physiological conditions. This contrasts with most published studies performed with a single substrate and without significant ATP turnover. Cytochrome c-deficient mitochondria produced more H<sub>2</sub>O<sub>2</sub> than control mitochondria, and exogenous cytochrome c addition reversed this increase. In the presence of increasing [KCN] rates of H<sub>2</sub>O<sub>2</sub> production by both pre-ischaemic and end-ischaemic mitochondria correlated with the oxidized cytochrome ''c'' content, but not with rates of respiration or NAD(P)H autofluorescence. Cytochrome ''c'' loss during ischaemia was not mediated by mPTP opening (cyclosporine-A insensitive), neither was it associated with changes in mitochondrial Bax, Bad, Bak or Bid. However, bound HK2 (hexokinase 2) and Bcl-xL were decreased in end-ischaemic mitochondria. We conclude that cytochrome c loss during ischaemia, caused by outer membrane permeabilization, is a major determinant of H<sub>2</sub>O<sub>2</sub> production by mitochondria under pathophysiological conditions. We further suggest that in hypoxia, production of H<sub>2</sub>O<sub>2</sub> to activate signalling pathways may be also mediated by decreased oxidized cytochrome ''c'' and less superoxide scavenging. | ||
|keywords=Bcl-xL, cytochrome c, hexokinase (HK), mitochondrial permeability transition pore (mPTP), superoxide | |keywords=Bcl-xL, cytochrome c, hexokinase (HK), mitochondrial permeability transition pore (mPTP), superoxide | ||
|mipnetlab=UK Bristol Halestrap AP | |mipnetlab=UK Bristol Halestrap AP, FR Pessac Pasdois P | ||
}} | }} | ||
{{Labeling | {{Labeling | ||
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|preparations=Permeabilized tissue, Isolated mitochondria | |preparations=Permeabilized tissue, Isolated mitochondria | ||
|enzymes=Complex IV;cytochrome c oxidase | |enzymes=Complex IV;cytochrome c oxidase | ||
|injuries= | |injuries=Ischemia-reperfusion, Oxidative stress;RONS | ||
|topics=ATP | |topics=ATP | ||
|couplingstates=OXPHOS | |couplingstates=OXPHOS | ||
|instruments=Oxygraph-2k | |instruments=Oxygraph-2k | ||
}} | }} |
Latest revision as of 11:03, 16 December 2020
Pasdois P, Parker JE, Griffiths EJ, Halestrap AP (2011) The role of oxidized cytochrome c in regulating mitochondrial reactive oxygen species production and its perturbation in ischaemia. Biochem J 436:493-505. |
Pasdois P, Parker JE, Griffiths EJ, Halestrap AP (2011) Biochem J
Abstract: Oxidized cytochrome c is a powerful superoxide scavenger within the mitochondrial IMS (intermembrane space), but the importance of this role in situ has not been well explored. In the present study, we investigated this with particular emphasis on whether loss of cytochrome c from mitochondria during heart ischaemia may mediate the increased production of ROS (reactive oxygen species) during subsequent reperfusion that induces mPTP (mitochondrial permeability transition pore) opening. Mitochondrial cytochrome c depletion was induced in vitro with digitonin or by 30 min ischaemia of the perfused rat heart. Control and cytochrome c-deficient mitochondria were incubated with mixed respiratory substrates and an ADP-regenerating system (State 3.5) to mimic physiological conditions. This contrasts with most published studies performed with a single substrate and without significant ATP turnover. Cytochrome c-deficient mitochondria produced more H2O2 than control mitochondria, and exogenous cytochrome c addition reversed this increase. In the presence of increasing [KCN] rates of H2O2 production by both pre-ischaemic and end-ischaemic mitochondria correlated with the oxidized cytochrome c content, but not with rates of respiration or NAD(P)H autofluorescence. Cytochrome c loss during ischaemia was not mediated by mPTP opening (cyclosporine-A insensitive), neither was it associated with changes in mitochondrial Bax, Bad, Bak or Bid. However, bound HK2 (hexokinase 2) and Bcl-xL were decreased in end-ischaemic mitochondria. We conclude that cytochrome c loss during ischaemia, caused by outer membrane permeabilization, is a major determinant of H2O2 production by mitochondria under pathophysiological conditions. We further suggest that in hypoxia, production of H2O2 to activate signalling pathways may be also mediated by decreased oxidized cytochrome c and less superoxide scavenging. โข Keywords: Bcl-xL, cytochrome c, hexokinase (HK), mitochondrial permeability transition pore (mPTP), superoxide
โข O2k-Network Lab: UK Bristol Halestrap AP, FR Pessac Pasdois P
Labels:
Stress:Ischemia-reperfusion, Oxidative stress;RONS Organism: Rat Tissue;cell: Heart Preparation: Permeabilized tissue, Isolated mitochondria Enzyme: Complex IV;cytochrome c oxidase Regulation: ATP Coupling state: OXPHOS
HRR: Oxygraph-2k