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 degrees 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 x mm(2)).
ATP (mumol/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 (mumol/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.