Cell death, calcium mobilization, and immunostaining for phosphorylated eukaryotic initiation factor 2-alpha (eIF2alpha) in neuronally differentiated NB-104 cells: arachidonate and radical-mediated injury mechanisms.
These experiments examine the effects of arachidonate with respect to cell death, radical-mediated injury, Ca2+ mobilization, and formation of ser-51-phosphorylated eukaryotic initiation factor 2alpha [eIF2alpha(P)]. It is known that during brain ischemia the concentration of free arachidonate can reach 180 microM, and during reperfusion oxidative metabolism of arachidonate leads to generation of superoxide that can reduce stored ferric iron and promote lipid peroxidation. During early brain reperfusion, we have shown an approximately 20-fold increase in eIF2alpha(P) which maps to vulnerable neurons that display inhibition of protein synthesis. Here in neuronally differentiated NB-104 cells, equivalent cell death (assessed by LDH release) was induced by 40 microM arachidonate and 20 microM cumene hydroperoxide (CumOOH, a known alkoxyl radical generator). In these injury models (1) radical inhibitors (BHA, BHT, and the lipophilic iron chelator EMHP) block CumOOH-induced cell death but do not block arachidonate-induced death; (2) 40 microM arachidonate (but not up to 40 microM CumOOH) rapidly induces Ca2+ release from intracellular stores; (3) both 40 microM arachidonate and 20 microM CumOOH induce intense immunostaining for eIF2alpha(P); and (4) the elF2alpha(P) immunostaining induced by CumOOH but not that induced by arachidonate is completely blocked by anti-radical intervention with EMHP. Arachidonate-induced formation of eIF2alpha(P) and cell death do not require iron-mediated radical mechanisms and are associated with Ca2+ release from intracellular stores; however, radical-mediated injury also induces both eIF2alpha(P) and cell death without release of intracellular Ca2+. Our data link eIF2alpha(P) formation during brain reperfusion to two established injury mechanisms that may operate concurrently.
O'Neil
B J
BJ
Department of Emergency Medicine, Wayne State University School of Medicine, MI 48201, USA. boneil@med.wayne.edu
McKeown
T R
TR
DeGracia
D J
DJ
Alousi
S S
SS
Rafols
J A
JA
White
B C
BC
D000818:Animals; D016718:Arachidonic Acid / Q000494:pharmacology*; D001555:Benzene Derivatives / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D016923:Cell Death; D002478:Cells, Cultured; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D005609:Free Radicals; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D016877:Oxidants / Q000494:pharmacology; D051381:Rats
D001555:Benzene Derivatives; D015852:Eukaryotic Initiation Factor-2; D005609:Free Radicals; D016877:Oxidants; D016718:Arachidonic Acid; C007164:cumene hydroperoxide; D002118:Calcium