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: Om Prakash Mishra, Alan B Zubrow, Qazi M Ashraf, Maria Delivoria-Papadopoulos
• Journal: Brain research (Brain Res) Vol. 1101 Issue 1 Pg. 20-8 (Jul 26 2006) ISSN: 0006-8993 [Print] Netherlands
• PMID: 16781684 (Publication Type: Comparative Study, Journal Article, Research Support, N.I.H., Extramural)

Chemical References

• Indazoles
• Proto-Oncogene Proteins c-bcl-2
• bcl-2-Associated X Protein
• Nitric Oxide Synthase
• Oxygen
• 7-nitroindazole

Topics

• Analysis of Variance
• Animals
• Animals, Newborn
• Blotting, Southern (methods)
• Blotting, Western (methods)
• Cell Nucleus (drug effects, metabolism)
• DNA Fragmentation (drug effects, physiology)
• Gene Expression Regulation, Developmental (drug effects, physiology)
• Hypoxia, Brain (metabolism, physiopathology)
• Indazoles (pharmacology)
• Neurons (cytology)
• Nitric Oxide Synthase (antagonists & inhibitors)
• Oxygen (pharmacology)
• Proto-Oncogene Proteins c-bcl-2 (metabolism)
• Swine
• bcl-2-Associated X Protein (metabolism)