Nuclear factor κB activation is mediated by NMDA and non-NMDA receptor and L-type voltage-gated Ca2+ channel following severe global ischemia in rat hippocampus

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 Ca2+ 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 Ca2+ channels are important routes to mediate NF-kappaB activation during brain isehemic 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. (C) 2002 Elsevier Science B.V. All rights reserved.