ATP and cytochrome c -dependent activation of caspase-9 during hypoxia in the cerebral cortex of newborn piglets

In previous studies, we have shown that cerebral hypoxia results in increased activity of caspase-9, the initiator caspase, and caspase-3, in the cytosolic fraction of the cerebral cortex of newborn piglets. The present study examines the mechanism of caspase-9 activation during hypoxia and tests the hypothesis that the ATP and cytochrome c -dependent activation of caspase-9 increases in the cytosol of the cerebral cortex of newborn piglets. Newborn piglets were divided into normoxic (Nx, n = 4), and hypoxic (Hx, n = 4) groups. Anesthetized, ventilated animals were exposed to an FiO 2 of 0.21 (Nx) or 0.07 (Hx) for 60 min. Cerebral tissue hypoxia was documented biochemically by determining levels of ATP and phosphocreatine (PCr). Cytosolic fraction was isolated and passed through a G25-Sephadex column to remove endogenous ATP and cytochrome c. Fractions were collected and protein determined by UV spectrophotometry at 280 nm. Eluted high-molecular weight samples from normoxic and hypoxic animals were divided into four subgroups: subgroup 1 (control), incubated without added ATP and cytochrome c ; subgroup 2, incubated with added ATP; subgroup 3, incubated with added cytochrome c ; and subgroup 4, incubated with added ATP and cytochrome c. The incubation was carried out at 37 °C for 30 min. Following incubation, the protein was separated by 12% SDS–PAGE and active caspase-9 was detected using specific active caspase-9 antibody. Protein bands were detected by enhanced chemiluminescence. Protein density was determined by imaging densitometry and expressed as absorbance (OD × mm 2 ). ATP (μmol/g brain) level was 4.7 ± 0.18 in normoxic, as compared to 1.53 ± 0.16 in hypoxic ( p < 0.05 vs. Nx). PCr (μmol/g brain) level was 4.03 ± 0.11 in the normoxic and 1.1 ± 0.3 in the hypoxic brain ( p < 0.05 vs. Nx). In the normoxic preparations, active caspase-9 density increased by 9, 4 and 20% in the presence of ATP, cytochrome c and ATP + cytochrome c, respectively. In the hypoxic preparations, active caspase-9 density increased by 30, 45 and 60% in the presence of ATP, cytochrome c and ATP + cytochrome c, respectively. These results show that incubation with ATP, cytochrome c and ATP + cytochrome c result in a significantly increased activation of caspase-9 in the hypoxic group ( p < 0.05). We conclude that the ATP and cytochrome c dependent activation of caspase-9 is increased during hypoxia. We propose that the ATP and cytochrome c sites of apoptotic protease activating factor I that mediate caspase-9 activation are modified during hypoxia.