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.