title: Effect of nitric oxide synthase inhibition during post-hypoxic reoxygenation on Bax and Bcl-2 protein expression and DNA fragmentation in neuronal nuclei of newborn piglets.
abstract: Previous studies have shown that cerebral tissue hypoxia results in increased generation of oxygen-free radicals including nitric oxide (NO), expression of the proapoptotic protein Bax and fragmentation of nuclear DNA. The present study tests the hypothesis that post-hypoxic reoxygenation for 6 h following hypoxia (FiO2=0.06 for 1 h) results in continued hypoxia-induced, NO-mediated expression of the Bax protein and nuclear DNA fragmentation in the cerebral cortex of newborn piglets. Piglets were divided into normoxic (Nx), hypoxic (Hx, FiO2=0.06 for 1 h), hypoxic with 6 h reoxygenation (Hx+reox) and hypoxic with 6 h reoxygenation injected with 7-nitroindazole sodium salt (7-NINA), a selective nNOS inhibitor, immediately after hypoxia (Hx+7-NINA). Cerebral tissue hypoxia was documented by levels of ATP and phosphocreatine (PCr). Bax and Bcl-2 were analyzed by Western blot and DNA fragmentation was determined by agarose gel electrophoresis. ATP and PCr values in Hx, Hx+reox and Hx+7-NINA were significantly different from Nx (P<0.05 vs. Nx). Bax protein (ODxmm2) was 128.9+/-38.7 in Nx; 223.6+/-45.8 in Hx (P<0.05 vs. Nx); 340.5+/-73.2 in Hx+reox (P<0.05 vs. Nx, Hx and Hx+7-NINA); and 202.2+/-34.8 in Hx+7-NINA (P=NS vs. Hx). Bcl-2 protein (ODxmm2) was 14.9+/-2.7 in Nx, 12.4+/-2.1 in Hx, (P<0.05 vs. Nx), 15.7+/-3.8 in Hx+reox, (P<0.05 vs. Hx) and 13.1+/-2.2 in Hx+7-NINA (P=NS among groups). Nuclear DNA fragmentation (ODxmm2) was 147+/-15 in Nx; 797+/-84 in Hx (P<0.05 vs. Nx); 1134+/-127 in Hx+reox (P<0.05 vs. Nx, Hx and Hx+7-NINA); and 778+/-146 in Hx+7-NINA (P=NS vs. Hx, P<0.05 vs. Hx+reox). The results show that post-hypoxic reoxygenation results in increased expression of Bax protein without affecting Bcl-2 protein and increased fragmentation of nuclear DNA, which are prevented by 7-NINA. We conclude that during post-hypoxic reoxygenation the increase in Bax protein expression and fragmentation of nuclear DNA are mediated by NO derived from nNOS. We propose that in addition to NO-mediated nuclear DNA damage, the hypoxia-induced increased ratio of Bax/Bcl-2 protein will lead to caspase-activated cascade of hypoxic neuronal death during post-hypoxic reoxygenation.
authors.affiliation: Department of Pediatrics, Drexel University College of Medicine and St. Christopher's Hospital for Children, Room 701, 7th Floor Heritage Building, 3300 Henry Avenue, Philadelphia, PA 19129, USA. Mishra@drexel.edu
authors.forename: Om Prakash
authors.initials: OP
authors.lastname: Mishra
authors.forename: Alan B
authors.initials: AB
authors.lastname: Zubrow
authors.forename: Qazi M
authors.initials: QM
authors.lastname: Ashraf
authors.forename: Maria
authors.initials: M
authors.lastname: Delivoria-Papadopoulos
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D015139:Blotting, Southern / Q000379:methods; D015153:Blotting, Western / Q000379:methods; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D053938:DNA Fragmentation / Q000187:drug effects / Q000502:physiology*; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects / Q000502:physiology; D002534:Hypoxia, Brain / Q000378:metabolism* / Q000503:physiopathology; D007191:Indazoles / Q000494:pharmacology; D009474:Neurons / Q000166:cytology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors*; D010100:Oxygen / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D013552:Swine; D051028:bcl-2-Associated X Protein / Q000378:metabolism*
chemical_list: D007191:Indazoles; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D019001:Nitric Oxide Synthase; D010100:Oxygen; C080122:7-nitroindazole