Abstract 416

Enzymes of the caspase family are involved in initiating the active phase of apoptosis in mammalian neurons. Caspase-1, also known as interleukin-1β-converting enzyme (ICE), has been shown to cleave caspase-3 into its active form, which is a key executioner of apoptosis. The present study tests the hypothesis that hypoxia results in increased activity of caspase-1 in the cytosolic fraction of the fetal guinea pig cortex. Studies were conducted in 7 pregnant guinea pigs at term, exposed for 1 hour to an FiO2 of 0.21 for the normoxic group (n = 4) or 0.07 for the hypoxic group (n = 3). 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-1 activity was determined in a 2 ml assay medium containing 50 mM Tris-HCl (pH 7.0), 1 mM dithiothreitol, 0.5 mM Na2-EDTA, 20% glycerol, 300 µg cytosolic protein and 75 µM Ac-YVAD-AMC [Acetyl-Tyrosine-Valine-Alanine-Aspartate (amino-4-methylcoumarin)], a substrate for caspase-1. The caspase-1 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. Changes in enzyme kinetics during hypoxia were evaluated by measuring caspase-1 activity in the presence of increasing concentrations of substrate (10-150 µM). 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). Caspase-1 activity increased from 3.37 ± 0.54 nmoles/mg protein/hr in the normoxic group to 4.43 ± 0.24 nmoles/mg protein/hr in the hypoxic group (p<0.05). Vmax value of the enzyme increased from 12.40 ± 0.99 nmoles/mg protein/hr in the normoxic group to 78.09 ± 9.42 nmoles/mg protein/hr in the hypoxic group (p<0.05). Similarly, the Km increased from 307.2 ± 21.7 nM in the normoxic group to 1339 ± 200 nM in the hypoxic group (p<0.05). The data demonstrate an increase in caspase-1 activity following hypoxia as well as decreased affinity of the hypoxic enzyme for its substrate. A 6.5-fold increase in maximum velocity will significantly increase the turnover number of the enzyme resulting in proteolysis of high concentrations of caspase-1 substrate. We conclude that brain tissue hypoxia modifies the active sites of caspase-1, leading to increased activity. We speculate that the increase in caspase-1 activity may in turn activate other caspases such as caspase-3 or caspase-7 resulting in the cleavage of critical cellular protein substrates such as poly(ADP-ribose) polymerase and lamins, leading to programmed cell death.

(Funded by NIH-HD-20337)