title: Mechanisms of expression of apoptotic protease activating factor-1 (Apaf-1) in nuclear, mitochondrial and cytosolic fractions of the cerebral cortex of newborn piglets.
abstract: Apoptotic protease activating factor-1 (Apaf-1) is a critical regulator of apoptosis and a crucial part of the apoptosome that is assembled in response to several cellular stresses like hypoxia. We have previously shown that hypoxia results in increased influx of nuclear Ca(2+) and increased expression of nuclear apoptotic proteins. The present study investigates that Apaf-1 is expressed during hypoxia in the cerebral cortex of newborn piglets and that administration of clonidine prevents the hypoxia induced increase expression of Apaf-1. Studies were conducted in 19 newborn piglets, 6 normoxic (Nx), 7 hypoxic (Hx FiO(2) of 0.05-0.07 for 1h) and 6 clonidine-treated hypoxic (Hx-Clo) piglets. Tissue hypoxia was confirmed biochemically by determining the levels of high energy phosphates ATP and phosphocreatine (PCr). Neuronal nuclei, mitochondrial membranes and cytosolic fractions were isolated and separated by 12% SDS-PAGE and probed with specific antibodies to Apaf-1. The expression of Apaf-1 in neuronal nuclei was 48.86+/-5.27 in Nx, 108.43+/-6.37 in Hx and 78.53+/-7.00 in Hx-Clo. The Apaf-1 expression of in mitochondrial fraction was 72.73+/-11.76 in Nx, 132.27+/-16.15 in Hx and 85.17+/-5.64 in Hx-Clo. Similarly, the expression of Apaf-1 in cytosolic fraction was 86.79+/-6.97 in Nx, 193.95+/-15.41 in Hx and 111.07+/-7.91 in Hx-Clo. In summary, the results show that hypoxia results in increased expression of Apaf-1 proteins in neuronal nuclear, mitochondrial and cytosolic fractions. Administration of a high affinity Ca(2+)-ATPase, prevented the hypoxia induced increased expression of Apaf-1 protein, suggesting that the hypoxia-induced increased expression of Apaf-1 proteins is nuclear Ca(2+)-influx mediated. We conclude that cerebral hypoxia-induced increase in Apaf-1 protein will lead to increased activation of procaspase-9 to caspase-9 in the cytosolic compartment leading to a cascade of hypoxic neuronal death.
authors.affiliation: Department of Pediatrics, Division of Neonatology, Drexel University College of Medicine and St. Christopher's Hospital for Children, 245 N. 15th Street, Philadelphia, PA 19102, USA. qazi@drexel.edu
authors.forename: Qazi M
authors.initials: QM
authors.lastname: Ashraf
authors.forename: Om P
authors.initials: OP
authors.lastname: Mishra
authors.forename: Maria
authors.initials: M
authors.lastname: Delivoria-Papadopoulos
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000502:physiology*; D053477:Apoptotic Protease-Activating Factor 1 / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002540:Cerebral Cortex / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D002534:Hypoxia, Brain / Q000378:metabolism* / Q000503:physiopathology; D008928:Mitochondria / Q000378:metabolism; D009410:Nerve Degeneration / Q000378:metabolism* / Q000503:physiopathology; D009474:Neurons / Q000378:metabolism* / Q000648:ultrastructure; D013347:Subcellular Fractions / Q000378:metabolism*; D034421:Sus scrofa; D015854:Up-Regulation / Q000502:physiology
chemical_list: D053477:Apoptotic Protease-Activating Factor 1; D000252:Calcium-Transporting ATPases