Nitric oxide-mediated expression of Bax protein and DNA fragmentation during hypoxia in neuronal nuclei from newborn piglets.
The present study tests the hypothesis that nitric oxide mediates the hypoxia-induced increase in expression of Bax and in DNA fragmentation in the cerebral cortex of newborn piglets, and that administration of N-nitro-L-arginine (NNLA), a nitric oxide synthase inhibitor, will prevent a change in hypoxia-induced expression of apoptotic genes and DNA damage. Piglets were assigned to normoxic, hypoxic, or NNLA-pretreated hypoxic groups. Cerebral tissue hypoxia was documented biochemically by measuring ATP and phosphocreatine (PCr) levels. Cerebral cortical neuronal nuclei were isolated and nuclear proteins were separated electrophoretically and probed with specific antibodies against Bcl-2 or Bax proteins. Neuronal nuclear DNA from normoxic, hypoxic, and NNLA-pretreated hypoxic animals was isolated, separated by electrophoresis on 1% agarose gel and stained with ethidium bromide. Cerebral hypoxia resulted in an increase in nuclear membrane Bax protein levels from 121.33+/-47.7 optical density (OD)xmm(2) in normoxic to 273.67+/-67.3 ODxmm(2) in hypoxic group (P<0.05 vs. normoxic), but levels in NNLA-pretreated hypoxic group were 155.78+/-48.3 ODxmm(2) (P<0.05 vs. hypoxic, P=NS vs. normoxic). Similarly, cerebral hypoxia resulted in the density of DNA fragments increasing from 1530.3+/-309.8 OD/mm(2) in the normoxic group to 5383.3+/-775 OD/mm(2) in the hypoxic group (P<0.05), while levels in NNLA-pretreated hypoxic group were 3574.0+/-952 OD/mm(2) (P<0.05 compared to hypoxic and normoxic groups). The data show that NNLA-pretreatment prevents the hypoxia-induced increase in Bax expression and DNA fragmentation demonstrating that the hypoxia-induced Bax gene expression and the DNA fragmentation are NO-mediated.
Zubrow
Alan B
AB
Department of Pediatrics, MCP Hahnemann University and St. Christopher's Hospital for Children, Front Street at Erie Ave., Philadelphia, PA 19134, USA. alan.zubrow@drexel.edu
Delivoria-Papadopoulos
Maria
M
Ashraf
Qazi M
QM
Ballesteros
Juan R
JR
Fritz
Karen I
KI
Mishra
Om P
OP
D000818:Animals; D000831:Animals, Newborn; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D002534:Hypoxia, Brain / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D019335:Nitroarginine / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis* / Q000187:drug effects; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis* / Q000187:drug effects; D013552:Swine; D051028:bcl-2-Associated X Protein
D004791:Enzyme Inhibitors; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D019335:Nitroarginine; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase