Cell death, calcium mobilization, and immunostaining for phosphorylated eukaryotic initiation factor 2-alpha (eIF2 alpha) 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 2 alpha [eIF2 alpha(P)]. It is known that during brain ischemia the concentration of free arachidonate can reach 180 mu M, and during reperfusion oxidative metabolism of arachidonat e leads to generation of superoxide that can reduce stored ferric iron and promote lipid peroxidation. During early brain reperfusion, we have shown a n similar to 20-fold increase in eIF2 alpha(P) which maps to vulnerable neu rons that display inhibition of protein synthesis. Here in neuronally diffe rentiated NB-104 cells, equivalent cell death (assessed by LDH release) was induced by 40 mu M arachidonate and 20 mu M cumene hydroperoxide (CumOOH, a known alkoxyl radical generator). In these injury models (1) radical inhi bitors (BHA, BHT, and the lipophilic iron chelator EMHP) block CumOOH-induc ed cell death but do not block arachidonate-induced death; (2) 40 mu M arac hidonate (but not up to 40 mu M CumOOH) rapidly induces Ca2+ release from i ntracellular stores; (3) both 40 mu M arachidonate and 20 mu M CumOOH induc e intense immunostaining for eIF2 alpha(P); and (4) the eIF2 alpha(P) immun ostaining induced by CumOOH but not that induced by arachidonate is complet ely blocked by anti-radical intervention with EMHP. Arachidonate-induced fo rmation of eIF2 alpha(P) and cell death do mot require iron-mediated radica l mechanisms and are associated with Ca2+ release from intracellular stores ; however, radical-mediated injury also induces both eIF2 alpha(P) and cell death without release of intracellular Ca2+. Our data link eIF2 alpha(P) f ormation during brain reperfusion to two established injury mechanisms that may operate concurrently. (C) 1999 Elsevier Science Ireland Ltd. All right s reserved.