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.
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.
Mishra
Om Prakash
OP
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
Zubrow
Alan B
AB
Ashraf
Qazi M
QM
Delivoria-Papadopoulos
Maria
M
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*
D007191:Indazoles; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D019001:Nitric Oxide Synthase; D010100:Oxygen; C080122:7-nitroindazole