Mechanism of Activation of Caspase-9 and Caspase-3 during Hypoxia in the Cerebral Cortex of Newborn Piglets: The Role of Nuclear Ca2+-Influx

In previous studies, we have shown that cerebral hypoxia results in increased activity of caspase-9, the initiator caspase, and caspase-3, the executioner of programmed cell death. We have also shown that cerebral hypoxia results in high affinity Ca²⁺–ATPase-dependent increase in nuclear Ca²⁺-influx in the cerebral cortex of newborn piglets. The present study tests the hypothesis that inhibiting nuclear Ca²⁺-influx by pretreatment with clonidine, an inhibitor of high affinity Ca²⁺–ATPase, will prevent the hypoxia-induced increase in caspase-9 and caspase-3 activity in the cerebral cortex of newborn piglets. Thirteen newborn piglets were divided into three groups, normoxic (Nx, n = 4), hypoxic (Hx, n = 4), and hypoxic treated with clonidine (100 mg/kg) (Hx–Cl, n = 5). Anesthetized, ventilated animals were exposed to an FiO₂ 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). Caspase-9 and -3 activity were determined spectrofluoro-metrically using specific fluorogenic synthetic substrates. ATP (μmoles/g brain) was 4.6 ± 0.3 in Nx, 1.7±0.4 in Hx (P < 0.05 vs. Nx), and 1.5 ± 0.2 in Hx–Cl (P < 0.05 vs. Nx). PCr (μmoles/g brain) was 3.6 ± 0.4 in Nx, 1.1 ± 0.3 in Hx (P < 0.05 vs. Nx), and 1.0 ± 0.2 in Hx–Cl (P < 0.05 vs. Nx). Caspase-9 activity (nmoles/mg protein/h) was 0.548 ± 0.0642 in Nx and increased to 0.808 ± 0.080 (P < 0.05 vs. Nx and Hx–Cl) in the Hx and 0.562 ± 0.050 in the Hx–Cl group (p = NS vs. Nx). Caspase-3 activity (nmoles/mg protein/h) was 22.0 ± 1.3 in Nx and 32 ± 6.3 in Hx (P < 0.05 vs. Nx) and 18.8 ± 3.2 in the Hx–Cl group (P < 0.05 vs. Hx). The data demonstrate that clonidine administration prior to hypoxia prevents the hypoxia-induced increase in the activity of caspase-9 and caspase-3. We conclude that the high afinity Ca²⁺–ATPase-dependent increased nuclear Ca²⁺ during hypoxia results in increased caspase-9 and caspase-3 activity.