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.