Abstract 2062
Poster Session II, Sunday, 5/2 (poster 223)
Previous studies have shown that programmed cell death, characterized by cytoplasmic and nuclear shrinkage, chromatin condensation and DNA fragmentation in a ladder pattern, occurs following tissue hypoxia. It has also been shown that Caspase-3, a cysteine protease, is a key executioner of apoptosis and is responsible for the proteolytic cleavage of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) thereby preventing its recruitment to sites of DNA damage. The present study tests the hypothesis that caspase-3 activity increases during cerebral hypoxia. Studies were conducted on 8 pregnant guinea pigs at term, exposed for 1 hour to either an FiO2 of 0.21 for the normoxic group (n = 4) or 0.07 for the hypoxic group (n = 4). Fetal cortices were obtained, homogenized and centrifuged at 850 × g for 10 min. The supernatant was centrifuged at 100,000 × g for 1 hour and the cytosolic fraction was collected. Cortical tissue hypoxia was documented biochemically by measuring the ATP and phosphocreatine levels. The cytosolic caspase-3 activity was determined in 2 ml assay medium containing 50 mM Tris-HCl buffer (pH 7.0), 1 mM dithiothreitol, 0.5 mM Na-EDTA, 20% glycerol, 300 µg cytosolic protein and 75 µM Ac-DEVD-AMC [Acetyl-Aspartate-Glutamate-Valine-Aspartate(amino-4 -methylcoumarin)], substrate for caspase-3. The caspase-3 reaction was followed by monitoring the increase in fluorescence at 460 nm using a 380 nm excitation wavelength at 37°C for 150 seconds after the addition of the enzyme. A standard curve was prepared using solutions of AMC ranging from 0 to 50 nM. Western blot analysis was performed using a polyclonal caspase-3 (CPP32 p20) antibody at a dilution of 1:500. Protein bands were detected using enhanced chemiluminescence and analyzed by imaging densitometry. The results are expressed as absorbance (OD) × mm2. During hypoxia, ATP decreased from 4.24 ± 0.55 µmoles/g brain in the normoxic group to 1.22 ± 0.31 µmoles/g brain in the hypoxic group (p<0.001). Similarly, PCr decreased from 4.03 ± 0.67 µmoles/g brain in the normoxic group to 1.03 ± 0.34 µmoles/g brain in the hypoxic group (p<0.001). Cytosolic caspase-3 activity increased following hypoxia from 4.70 ± 0.56 to 6.96 ± 1.59 nmoles/mg protein/hr (p<0.05). Western blot analysis demonstrated a significant increase in caspase-3 protein in cytosolic fraction from hypoxic animals: 16.05 ± 1.51 vs 7.30 ± 0.12 (OD) × mm2, p<0.05. We conclude that both the activity as well as the expression of caspase-3 increase during 1 hour of hypoxia. Since activation of caspases results in the cleavage of cellular substrates, including the nuclear enzyme PARP, lamin B, pro-caspase-6 and pro-caspase-7, we speculate that in the immature brain activation of caspase-3 during hypoxia indicates an early induction of apoptotic mechanisms leading to programmed neuronal cell death.
