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 (ODxmm(2)) 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 (ODxmm(2)) 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.
Chiang
Ming-Chou
MC
Department of Pediatrics, Chang Gung University College of Medicine, Chang Gung Memorial Hospital, Taoyuan, Taiwan. cmc123@adm.cgmh.org.tw
Ashraf
Qazi M
QM
Ara
Jahan
J
Mishra
Om P
OP
Delivoria-Papadopoulos
Maria
M
D000818:Animals; D000831:Animals, Newborn; D053148:Caspase 3 / Q000378:metabolism*; D053453:Caspase 9 / Q000378:metabolism; D002540:Cerebral Cortex / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000008:administration & dosage; D000860:Hypoxia / Q000201:enzymology / Q000473:pathology*; D009842:Oligopeptides / Q000008:administration & dosage; D013552:Swine
D004791:Enzyme Inhibitors; D009842:Oligopeptides; C403754:benzyloxycarbonyl-leucyl-glutamyl-histidyl-aspartic acid fluoromethyl ketone; D053148:Caspase 3; D053453:Caspase 9