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

Abstract

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 FiO 2 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 (μmol/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 (μmol/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 (μg 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 (OD×mm 2) 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 Ca 2+-influx and fragmentation of mitochondrial DNA.