Hypoxia-induced caspase-3 activation and DNA fragmentation in cortical neurons of newborn piglets: role of nitric oxide.
Hypoxia results in generation of nitric oxide (NO) free radicals, activation of caspase-3, and genomic DNA fragmentation. The present study tests the hypothesis that hypoxia-induced caspase-3 activation and DNA fragmentation are nitric oxide mediated. Studies were conducted in newborn piglets, divided into normoxic (n = 5), hypoxic (n = 5), and hypoxic-7-NINA (n = 6). Hypoxic-7-NINA group received the neuronal nitric oxide synthase inhibitor, 7-Nitroindazole (7-NINA). Caspase-3 activity was determined spectrofluorometrically using enzyme-specific substrates. Sections from the neocortex were stained with an antiserum recognizing active caspase-3. Purified DNA was separated by gel electrophoresis. Administration of 7-NINA resulted in decreased immunoreactivity of caspase-3 (mean LI: 20.2%) as compared to the untreated hypoxia group (mean LI: 57.5%) (P < 0.05). 7-NINA attenuated caspase-3 enzymatic activity as well in comparison to the untreated hypoxia group (P < 0.05). Furthermore, multiple low molecular weight bands corresponding to DNA fragments were present in the hypoxic but not in the normoxic or hypoxic-7-NINA groups. Inhibition of nNOS abates the hypoxia-induced increase in active caspase-3 immunoreactivity, as well as enzymatic activity in cortical neurons, and DNA fragmentation in brain homogenates. We conclude that the coordinate increase of capase-3 activity and fragmentation of nuclear DNA in the hypoxic newborn piglet brain are NO mediated.
Parikh
N A
NA
Department of Pediatrics, Jefferson Medical College, Thomas Jefferson University Hospital, Philadelphia, Pennsylvania, USA. Nehal.A.Parikh@uth.tmc.edu
Katsetos
C D
CD
Ashraf
Q M
QM
Haider
S H
SH
Legido
A
A
Delivoria-Papadopoulos
M
M
Mishra
O P
OP
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002540:Cerebral Cortex / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D053938:DNA Fragmentation; D004587:Electrophoresis, Agar Gel; D004789:Enzyme Activation; D000860:Hypoxia / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D007150:Immunohistochemistry; D009474:Neurons / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D009569:Nitric Oxide / Q000378:metabolism*; D010725:Phosphocreatine / Q000378:metabolism; D013552:Swine
D010725:Phosphocreatine; D009569:Nitric Oxide; D000255:Adenosine Triphosphate; D053148:Caspase 3; D020169:Caspases