Recent studies suggest that nuclear factor
NF-kappaB may be involved in
excitotoxin-induced cell apopotosis. To analyze the variation of
NF-kappaB, levels of
NF-kappaB were measured after the rats were subjected to 30 min of four-vessel occlusion and sacrificed in selected reperfusion time points. Induction of
NF-kappaB consisting mainly of p65 and p50 subunits was detected by Western blot with anti p65, p50
antibodies, respectively.
DNA binding activity of
NF-kappaB was performed by electrophoretic mobility-shift analysis. Our studies indicate that
ischemia-induced
NF-kappaB nuclear translocation is time-dependent. Inductions or binding activity of
NF-kappaB in nucleus increased about 10-fold from 6 to 12 h as compared with that of the control group, then gradually declined in the following 24, 72 h. To further analyze the regulation by
ionotropic glutamate receptor and L-type voltage-gated Ca(2+) channel (L-VGCC) in vivo,
N-methyl-D-aspartate (
NMDA) receptor antagonist
ketamine, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/
kainate receptor antagonist 6,7-dinitroquinoxaline-2,3 (1H,4H)-dione and L-VGCC antagonist
nifedipine were given 20 min prior to 30 min of
ischemia. The
NF-kappaB nuclear translocation was completely blocked by these three antagonists in a dose-dependent manner after
ischemia/reperfusion 6 h. Increased phosphorylation of the
NF-kappaB regulatory unit
IkappaB-alpha was detected by Western blot. Decrement of
IkappaB-alpha was found after 3 h reperfusion in the cytoplasm following global
ischemia, which was also blocked by such three antagonists. These results illustrate that
glutamate-gated ionotropic
NMDA or non-
NMDA receptors and voltage-gated Ca(2+) channels are important routes to mediate
NF-kappaB activation during brain ischemic injury. Active
NF-kappaB may attend the
excitotoxin-induced cell death in turn. Our studies also suggest that
IkappaB-alpha is an important regulatory unit that controls the activation of
NF-kappaB after its phosphorylation and degradation and resynthesis in rat hippocampus following global
ischemia.