Mechanism of caspase-3 activation during hypoxia in the cerebral cortex of newborn piglets

We have previously shown that the activity and the expression of caspase-9 and caspase-3 were increased during hypoxia in the cerebral cortex of newborn piglets. The present study was conducted to test the hypothesis that the hypoxia-induced activation of caspase-3 in the cerebral cortex of newborn piglets is mediated by caspase-9. Twenty-two newborn piglets were randomly assigned to four groups: normoxic (Nx), normoxic pretreated with a selective caspase-9 inhibitor, Z-Leu-Glu(OMe)-His-Asp(OMe)-Fluoromethyl ketone (Z-LEHD-FMK) (Nx + LEHD), hypoxic (Hx), and hypoxic pretreated with Z-LEHD-FMK (Hx + LEHD). Cerebral tissue hypoxia was confirmed biochemically by measuring ATP and phosphocreatine. Caspase-9 and -3 activities were determined spectrofluorometrically. The expression of caspase-9 and -3 proteins was measured by Western blot analysis using active enzyme specific antibodies. Cytosolic caspase-9 activity (nmol/mg protein/h) was 3.70 ± 0.40 in Nx, 3.56 ± 0.31 in Nx + LEHD (p = NS versus Nx), 4.99 ± 0.64 in Hx (p < 0.05 versus Nx), and 3.73 ± 0.80 in Hx + LEHD (p < 0.05 versus Hx, p = NS versus Nx). Cytosolic caspase-3 activity (nmol/mg protein/h) was 7.80 ± 1.17 in Nx, 8.15 ± 0.87 in Nx + LEHD (p = NS versus Nx), 13.07 ± 0.78 in Hx (p < 0.05 versus Nx), and 10.05 ± 2.09 in Hx + LEHD (p < 0.05 versus Hx) The density (OD × mm2) of active caspase-9 protein was 18.52 ± 1.89 in Nx, 20.53 ± 1.12 in Nx + LEHD (p = NS versus Nx), 32.36 ± 5.03 in Hx (p < 0.05 versus Nx), and 19.94 ± 3.59 in Hx + LEHD (p < 0.05 versus Hx, p = NS versus Nx). The density (OD × mm2) of active caspase-3 protein was 55.87 ± 8.73 in Nx, 55.69 ± 8.18 in Nx + LEHD (p = NS versus Nx), 94.10 ± 12.05 in Hx (p < 0.05 versus Nx), and 56.12 ± 14.56 in Hx + LEHD (p < 0.05 versus Hx, p = NS versus Nx). These data show that administration of a selective caspase-9 inhibitor, Z-LEHD-FMK, prior to hypoxia prevents the hypoxia-induced increase in caspase-3 activity and the expression of active caspase-3 protein. We conclude that the hypoxia-induced activation of caspase-3 during hypoxia in the cerebral cortex of newborn piglets is mediated by caspase-9.