PHOSPHORYLATION OF THE ALPHA-SUBUNIT OF INITIATION-FACTOR 2 CORRELATES WITH THE INHIBITION OF TRANSLATION FOLLOWING TRANSIENT CEREBRAL-ISCHEMIA IN THE RAT

Rats were subjected to the standard four-vessel occlusion model of cerebral transient ischaemia (vertebral and carotid arteries) for 15 and 30 min. After a 30 min recirculation period, protein synthesis rate, initiation factor 2 (eIF-2) and guanine nucleotide exchange factor (GEF) activities, and the level of phosphorylation of the alpha subunit of eIF-2 (eIF-2 alpha) were determined in the neocortex region of the brain from sham-operated controls and ischaemic animals. Following reversible cerebral ischaemia, the protein synthesis rate, as measured in a cell-free system, was significantly inhibited (70%) in the ischaemic animals. eIF-2 activity, as measured by its ability to form a ternary, complex, also decrease parallel to the decrease in protein synthesis. As eIF-2 activity was assayed in the presence of Mg2+ and GTP-regenerating capacity, the decrease in ternary-complex formation indicated the possible impairment of GEF activity. Since phosphorylated eIF-2 [eIF-2(alpha P)] is a powerful inhibitor of GEF, the levels of phosphorylated eIF-2 alpha were determined, and an increase from 7% phosphorylation in sham control rats to 20% phosphorylation in 15 min and 29% phosphorylation in 30 min in ischaemic rats was observed, providing evidence for a tight correlation of phosphorylation of eIF-2 alpha and inhibition of protein synthesis. Moreover, GEF activity measured in the GDP-exchange assay was in fact inhibited in the ischaemic animals, proving that protein synthesis is impaired by the presence of eIF-2(alpha P), which blocks eIF-2 recycling.