Postischemic hyperoxia reduces hippocampal pyruvate dehydrogenase activity

doi:10.1016/j.freeradbiomed.2006.01.022

Free Radical Biology and Medicine

Volume 40, Issue 11, 1 June 2006, Pages 1960-1970

https://doi.org/10.1016/j.freeradbiomed.2006.01.022 Get rights and content

Abstract

The pyruvate dehydrogenase complex (PDHC) is a mitochondrial matrix enzyme that catalyzes the oxidative decarboxylation of pyruvate and represents the sole bridge between anaerobic and aerobic cerebral energy metabolism. Previous studies demonstrating loss of PDHC enzyme activity and immunoreactivity during reperfusion after cerebral ischemia suggest that oxidative modifications are involved. This study tested the hypothesis that hyperoxic reperfusion exacerbates loss of PDHC enzyme activity, possibly due to tyrosine nitration or S-nitrosation. We used a clinically relevant canine ventricular fibrillation cardiac arrest model in which, after resuscitation and ventilation on either 100% O₂ (hyperoxic) or 21–30% O₂ (normoxic), animals were sacrificed at 2 h reperfusion and the brains removed for enzyme activity and immunoreactivity measurements. Animals resuscitated under hyperoxic conditions exhibited decreased PDHC activity and elevated 3-nitrotyrosine immunoreactivity in the hippocampus but not the cortex, compared to nonischemic controls. These measures were unchanged in normoxic animals. In vitro exposure of purified PDHC to peroxynitrite resulted in a dose-dependent loss of activity and increased nitrotyrosine immunoreactivity. These results support the hypothesis that oxidative stress contributes to loss of hippocampal PDHC activity during cerebral ischemia and reperfusion and suggest that PDHC is a target of peroxynitrite.

Section snippets

Materials

All chemicals and reagents were purchased from Sigma–Aldrich unless otherwise stated.

Canine cardiac arrest model

Animal experimentation was performed according to the guidelines of the Institutional Animal Use and Care Committee of the University of Maryland, Baltimore. The animal model used for these studies has been used extensively by us and others to study global cerebral ischemia and reperfusion [15], [16], [17], [18], [19], [20]. Adult purebred female beagles weighing 10–15 kg were anesthetized initially with an

Canine cardiac arrest and resuscitation

Whereas there were no significant differences in baseline physiologic parameters, including pH, temperature, pO₂, and pCO₂ between animal groups, the pO₂ at 30 min reperfusion for hyperoxic animals (467.6 ± 29.7 mm Hg, SE, n = 8) was significantly greater (p < 0.001) than the pO₂ of normoxic resuscitated animals (89.2 ± 4.3 mm Hg, SE, n = 8).

Postischemic hippocampal and frontal cortex PDHC enzyme activity

The values for brain tissue maximal PDHC specific activity obtained in these experiments are within the range of previously published results for canine

Discussion

The most important new observations reported in this study are that hyperoxic resuscitation after cardiac arrest reduces hippocampal PDHC enzyme activity and elevates 3-nitrotyrosine immunoreactivity compared to values obtained with sham-operated control animals or those resuscitated using normal arterial O₂ levels. The finding that hippocampal 3-nitrotyrosine immunoreactivity measured by ELISA is elevated after hyperoxic but not normoxic resuscitation is consistent with results we obtained

Acknowledgments

The authors thank Ms. Kyni Jones and Dr. Cynthia Cotta-Cumba for expert technical assistance. This work was supported by NIH NS34152, NS049425, NS050653, and AHA 0215331U and U.S. Army DAMD 17-03-1-0745.

References (78)

Y.E. Bogaert et al.
Postischemic inhibition of cerebral cortex pyruvate dehydrogenase
Free Radic. Biol. Med.
(1994)
Y.E. Bogaert et al.
Neuronal subclass-selective loss of pyruvate dehydrogenase immunoreactivity following canine cardiac arrest and resuscitation
Exp. Neurol.
(2000)
N.R. Sims et al.
Mitochondrial contributions to tissue damage in stroke
Neurochem. Int.
(2002)
K.K. Haga et al.
The neuronal nitric oxide synthase inhibitor, TRIM, as a neuroprotective agent: effects in models of cerebral ischaemia using histological and magnetic resonance imaging techniques
Brain Res.
(2003)
S. Tan et al.
Hypoxia–ischemia in fetal rabbit brain increases reactive nitrogen species production: quantitative estimation of nitrotyrosine
Free Radic. Biol. Med.
(2001)
C.F. Zwemer et al.
Cardiopulmonary–cerebral resuscitation with 100% oxygen exacerbates neurological dysfunction following nine minutes of normothermic cardiac arrest in dogs
Resuscitation
(1994)
M. Krajewska et al.
Early processing of Bid and caspase-6, -8, -10, -14 in the canine brain during cardiac arrest and resuscitation
Exp. Neurol.
(2004)
L.M. Hinman et al.
An NADH-linked spectrophotometric assay for pyruvate dehydrogenase complex in crude tissue homogenates
J. Biol. Chem.
(1981)
M. Bernaudin et al.
Selective neuronal vulnerability and specific glial reactions in hippocampal and neocortical organotypic cultures submitted to ischemia
Exp. Neurol.
(1998)
M.F. Beal
Oxidatively modified proteins in aging and disease
Free Radic. Biol. Med.
(2002)

A.P. Kudin et al.

Characterization of superoxide-producing sites in isolated brain mitochondria

J. Biol. Chem.

(2004)

T.L. Vanden Hoek et al.

Reactive oxygen species released from mitochondria during brief hypoxia induce preconditioning in cardiomyocytes

J. Biol. Chem.

(1998)

J. Duranteau et al.

Intracellular signaling by reactive oxygen species during hypoxia in cardiomyocytes

J. Biol. Chem.

(1998)

H.M. Abu-Soud et al.

Nitric oxide binding to the heme of neuronal nitric-oxide synthase links its activity to changes in oxygen tension

J. Biol. Chem.

(1996)

M. Erecinska et al.

Tissue oxygen tension and brain sensitivity to hypoxia

Respir. Physiol.

(2001)

B. Lei et al.

Measurement of total nitric oxide metabolite (NO(x) (−)) levels in vivo

Brain Res. Brain Res. Protoc.

(1999)

B. Sumegi et al.

Complex I binds several mitochondrial NAD-coupled dehydrogenases

J. Biol. Chem.

(1984)

A. Denicola et al.

Peroxynitrite reaction with carbon dioxide/bicarbonate: kinetics and influence on peroxynitrite-mediated oxidations

Arch. Biochem. Biophys.

(1996)

R. Radi et al.

Peroxynitrite reactions and formation in mitochondria

Free Radic. Biol. Med.

(2002)

J. Kanski et al.

Proteomic analysis of protein nitration in aging skeletal muscle and identification of nitrotyrosine-containing sequences in vivo by nanoelectrospray ionization tandem mass spectrometry

J. Biol. Chem.

(2005)

D. Gonzalez et al.

Endogenous nitration of iron regulatory protein-1 (IRP-1) in nitric oxide-producing murine macrophages: further insight into the mechanism of nitration in vivo and its impact on IRP-1 functions

J. Biol. Chem.

(2004)

H. Ischiropoulos

Biological tyrosine nitration: a pathophysiological function of nitric oxide and reactive oxygen species

Arch. Biochem. Biophys.

(1998)

P.R. Gardner et al.

Superoxide radical and iron modulate aconitase activity in mammalian cells

J. Biol. Chem.

(1995)

J. Murray et al.

Oxidative damage to mitochondrial complex I due to peroxynitrite: identification of reactive tyrosines by mass spectrometry

J. Biol. Chem.

(2003)

D.M. Kuhn et al.

Peroxynitrite inactivates tryptophan hydroxylase via sulfhydryl oxidation: coincident nitration of enzyme tyrosyl residues has minimal impact on catalytic activity

J. Biol. Chem.

(1999)

P. Schmidt et al.

Specific nitration at tyrosine 430 revealed by high resolution mass spectrometry as basis for redox regulation of bovine prostacyclin synthase

J. Biol. Chem.

(2003)

D. Gonzalez et al.

Endogenous nitration of iron regulatory protein-1 (IRP-1) in nitric oxide-producing murine macrophages: further insight into the mechanism of nitration in vivo and its impact on IRP-1 functions

J. Biol. Chem.

(2004)

O.H. Lowry et al.

Protein measurement with the Folin phenol reagent

J. Biol. Chem.

(1951)

Y. Takeda et al.

Mitochondria consume energy and compromise cellular membrane potential by reversing ATP synthetase activity during focal ischemia in rats

J. Cereb. Blood Flow Metab.

(2004)

O. Hurtado et al.

Inhibition of glutamate release by delaying ATP fall accounts for neuroprotective effects of antioxidants in experimental stroke

FASEB J.

(2003)

D. Liu et al.

Activation of mitochondrial ATP-dependent potassium channels protects neurons against ischemia-induced death by a mechanism involving suppression of Bax translocation and cytochrome c release

J. Cereb. Blood Flow Metab.

(2002)

B.M. Polster et al.

Mitochondrial mechanisms of neural cell apoptosis

J. Neurochem.

(2004)

E. Zaidan et al.

The pyruvate dehydrogenase complex is partially inactivated during early recirculation following short-term forebrain ischemia in rats

J. Neurochem.

(1998)

T. Sasaki et al.

Inhibition of nitric oxide production during global ischemia ameliorates ischemic damage of pyramidal neurons in the hippocampus

Keio J. Med.

(2001)

S.S. Sharma et al.

Neuroprotective effect of peroxynitrite decomposition catalyst and poly(adenosine diphosphate-ribose) polymerase inhibitor alone and in combination in rats with focal cerebral ischemia

J. Neurosurg.

(2004)

D.S. Warner et al.

Oxidants, antioxidants and the ischemic brain

J. Exp. Biol.

(2004)

Vereczki, V.; Martin, E.; Rosenthal, R. E.; Hof, P. R.; Hoffman, G. E.; Fiskum, G. Normoxic resuscitation after cardiac...

Y. Liu et al.

Normoxic ventilation after cardiac arrest reduces oxidation of brain lipids and improves neurological outcome

Stroke

(1998)

Y. Leonov et al.

Mild cerebral hypothermia during and after cardiac arrest improves neurologic outcome in dogs

J. Cereb. Blood Flow Metab.

(1990)

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Original ContributionPostischemic hyperoxia reduces hippocampal pyruvate dehydrogenase activity

Abstract

Section snippets

Materials

Canine cardiac arrest model

Canine cardiac arrest and resuscitation

Postischemic hippocampal and frontal cortex PDHC enzyme activity

Discussion

Acknowledgments

Free Radic. Biol. Med.

Exp. Neurol.

Neurochem. Int.

Brain Res.

Free Radic. Biol. Med.

Resuscitation

Exp. Neurol.

J. Biol. Chem.

Exp. Neurol.

Free Radic. Biol. Med.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Respir. Physiol.

Brain Res. Brain Res. Protoc.

J. Biol. Chem.

Arch. Biochem. Biophys.

Free Radic. Biol. Med.

J. Biol. Chem.

J. Biol. Chem.

Arch. Biochem. Biophys.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Mitochondria consume energy and compromise cellular membrane potential by reversing ATP synthetase activity during focal ischemia in rats

J. Cereb. Blood Flow Metab.

Inhibition of glutamate release by delaying ATP fall accounts for neuroprotective effects of antioxidants in experimental stroke

FASEB J.

Activation of mitochondrial ATP-dependent potassium channels protects neurons against ischemia-induced death by a mechanism involving suppression of Bax translocation and cytochrome c release

J. Cereb. Blood Flow Metab.

Mitochondrial mechanisms of neural cell apoptosis

J. Neurochem.

The pyruvate dehydrogenase complex is partially inactivated during early recirculation following short-term forebrain ischemia in rats

J. Neurochem.

Inhibition of nitric oxide production during global ischemia ameliorates ischemic damage of pyramidal neurons in the hippocampus

Keio J. Med.

Neuroprotective effect of peroxynitrite decomposition catalyst and poly(adenosine diphosphate-ribose) polymerase inhibitor alone and in combination in rats with focal cerebral ischemia

J. Neurosurg.

Oxidants, antioxidants and the ischemic brain

J. Exp. Biol.

Normoxic ventilation after cardiac arrest reduces oxidation of brain lipids and improves neurological outcome

Stroke

Mild cerebral hypothermia during and after cardiac arrest improves neurologic outcome in dogs

J. Cereb. Blood Flow Metab.

Original Contribution
Postischemic hyperoxia reduces hippocampal pyruvate dehydrogenase activity