An in vitro model of
ischemia was obtained by subjecting PC12 cells differentiated with
nerve growth factor to a combination of
glucose deprivation plus
anoxia. Immediately after the ischemic period, the
protein synthesis rate was significantly inhibited (80%) and western blots of
cell extracts revealed a significant accumulation of phosphorylated
eukaryotic initiation factor 2, alpha subunit,
eIF2(alphaP) (42%). Upon recovery,
eIF2(alphaP) levels returned to control values after 30 min, whereas
protein synthesis was still partially inhibited (33%) and reached almost control values within 2 h. The activities of the mammalian eIF2alpha
kinases,
double-stranded RNA-activated
protein kinase, mammalian GCN2 homologue, and endoplasmic reticulum-resident
kinase, were determined. None of the eIF2alpha
kinases studied showed increased activity in ischemic cells as compared with controls. Exposure of cells to cell-permeable inhibitors of
protein phosphatases 1 and 2A,
calyculin A or
tautomycin, induced dose- and time-dependent accumulation of
eIF2(alphaP), mimicking an ischemic effect.
Protein phosphatase activity, as measured with [(32)P]
phosphorylase a as a substrate, diminished during
ischemia and returned to control levels upon 30-min recovery. In addition, the rate of
eIF2(alphaP) dephosphorylation was significantly lower in ischemic cells, paralleling both the greatest translational inhibition and the highest
eIF2(alphaP) levels. The endogenous
phosphatase activity from control and ischemic extracts showed different sensitivity to inhibitor 2 and
fostriecin in in vitro assays, inhibitor-2 effect in ischemic cells being lower than in control cells. Together these results indicate that an eIF2alpha
phosphatase, probably
protein phosphatase 1, is implicated in the
ischemia-induced
eIF2(alphaP) accumulation in PC12 cells.