Hypoxia-induced Bax and Bcl-2 protein expression, caspase-9 activation, DNA fragmentation, and lipid peroxidation in mitochondria of the cerebral cortex of newborn piglets: the role of nitric oxide.
The present study tests the hypothesis that cerebral hypoxia results in increased ratio of Bax/Bcl-2, activation of caspase-9, lipid peroxidation, and DNA fragmentation in mitochondria of the cerebral cortex of newborn piglets and that the inhibition of nitric oxide synthase by N-nitro-L-arginine during hypoxia will prevent the events leading to mitochondrial DNA fragmentation. To test this hypothesis, six piglets, 3-5 days old, were divided into three groups: normoxic (n=5), hypoxic (n=5), and hypoxic-nitric oxide synthase (n=4). Hypoxic animals were exposed to a FiO2 of 0.6 for 60 min. Nitric oxide synthase (40 mg/kg) was infused over 60 min prior to hypoxia. Tissue hypoxia was confirmed by measuring levels of ATP and phosphocreatine. Cerebral cortical tissue mitochondria were isolated and purified using a discontinuous ficoll gradient. Mitochondrial Bax and Bcl-2 proteins were determined by Western blot. Caspase-9 activity in mitochondria was determined spectro-fluorometrically using fluorogenic substrate for caspase-9. Fluorescent compounds, an index of mitochondrial membrane lipid peroxidation, were determined spectrofluorometrically. Mitochondrial DNA was isolated and separated by electrophoresis on 1% agarose gel and stained with ethidium bromide. ATP levels (micromol/g brain) were 4.52+/-0.34 in normoxic, 1.18+/-0.29 in hypoxic (P<0.05) and 1.00+/-0.26 in hypoxic-nitric oxide synthase animals (P<0.05 vs. normoxic). Phosphocreatine levels (micromol/g brain) were 3.61+/-0.33 in normoxic, 0.70+/-0.20 in hypoxic (P<0.05 vs. normoxic) and 0.57+/-0.14 in hypoxic-nitric oxide synthase animals (P<0.05 vs. normoxic, P=NS vs. hypoxic). Bax density in mitochondrial membranes was 160+/-28 in normoxic and 324+/-65 in hypoxic (P<0.001 vs. normoxic). Bcl-2 density mitochondria was 96+/-18 in normoxic and 98+/-20 in hypoxic (P=NS vs. normoxic). Mitochondrial caspase-9 activity (nmol/mg protein/h) was 1.32+/-0.23 in normoxic and 2.25+/-0.24 in hypoxic (P<0.01 vs. normoxic). Levels of fluorescent compounds (microg of quinine sulfate/g protein) were 12.48+/-4.13 in normoxic and 37.92+/-7.62 in hypoxic (P=0.003 vs. normoxic). Densities (ODxmm2) of low molecular weight DNA fragments were 143+/-38 in normoxic, 365+/-152 in hypoxic, (P<0.05 vs. normoxic) and 163+/-25 in hypoxic-nitric oxide synthase animals (P<0.05 vs. hypoxic, P=NS vs. normoxic). The data demonstrate that hypoxia results in increased mitochondrial proapoptotic protein Bax, increased mitochondrial caspase-9 activity, increased mitochondrial lipid peroxidation, and increased fragmentation of DNA in mitochondria of the cerebral cortex of newborn piglets. The administration of a nitric oxide synthase inhibitor, nitric oxide synthase, prior to hypoxia prevented fragmentation of mitochondrial DNA, indicating that the hypoxia-induced mitochondrial DNA fragmentation is NO-mediated. We propose that NO free radicals generated during hypoxia lead to NO-mediated altered expression of Bax leading to increased ratio of pro-apoptotic/anti-apoptotic protein resulting in modification of mitochondrial membrane, and subsequently Ca2+-influx and fragmentation of mitochondrial DNA.
Mishra
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Department of Pediatrics, Room 701, 7th Floor Heritage Building, Drexel University College of Medicine, Philadelphia, PA 19129, USA. Mishra@drexel.edu
Randis
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Ashraf
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Delivoria-Papadopoulos
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western / Q000379:methods; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D002540:Cerebral Cortex / Q000473:pathology*; D053938:DNA Fragmentation / Q000502:physiology*; D004789:Enzyme Activation / Q000502:physiology; D015870:Gene Expression / Q000502:physiology*; D000860:Hypoxia / Q000473:pathology*; D015227:Lipid Peroxidation / Q000502:physiology; D008928:Mitochondria / Q000378:metabolism*; D009569:Nitric Oxide / Q000502:physiology*; D010725:Phosphocreatine / Q000031:analogs & derivatives / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D013552:Swine; D051028:bcl-2-Associated X Protein / Q000378:metabolism*
D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D010725:Phosphocreatine; D009569:Nitric Oxide; C025403:phosphocreatinine; D000255:Adenosine Triphosphate; D053453:Caspase 9; D020169:Caspases