Changes in the phosphorylation of initiation factor eIF-2 alpha, elongation factor eEF-2 and p70 S6 kinase after transient focal cerebral ischaemia in mice

Mice were subjected to 60 min occlusion of the left middle cerebral artery (MCA) followed by 1-6 h of reperfusion. Tissue samples were taken from the MCA territory of both hemispheres to analyse ischaemia-induced changes in t he phosphorylation of the initiation factor eIF-2 alpha, the elongation fac tor eEF-2 and p70 S6 kinase by western blot analysis. Tissue sections from additional animals were taken to evaluate ischaemia-induced changes in glob al protein synthesis by autoradiography and changes in eIF-2 alpha phosphor ylation by immunohistochemistry. Transient MCA occlusion induced a persiste nt suppression of protein synthesis. Phosphorylation of eIF-2 alpha was sli ghtly increased during ischaemia, it was markedly up-regulated after 1 h of reperfusion and it normalized after 6 h of recirculation despite ongoing s uppression of protein synthesis. Similar changes in eIF-2 alpha phosphoryla tion were induced in primary neuronal cell cultures by blocking of endoplas mic reticulum (ER) calcium pump, suggesting that disturbances of ER calcium homeostasis may play a role in ischaemia-induced changes in eIF-2 alpha ph osphorylation. Dephosphorylation of eIF-2 alpha was not paralleled by a ris e in levels of p67, a glycoprotein that protects eIF-2 alpha from phosphory lation, even in the presence of active eIF-2 alpha kinase. Phosporylation o f eEF-2 rose moderately during ischaemia, but returned to control levels af ter 1 h of reperfusion and declined markedly below control levels after 3 a nd 6 h of recirculation. In contrast to the only shortlasting phosphorylati on of eIF-2 alpha and eEF-2, transient focal ischaemia induced a long-lasti ng dephosphorylation of p70 S6 kinase. The results suggest that blocking of elongation does not play a major role in suppression of protein synthesis induced by transient focal cerebral ischaemia. Investigating the factors in volved in ischaemia-induced suppression of the initiation step of protein s ynthesis and identifying the underlying mechanisms may help to further eluc idate those disturbances directly related to the pathological process trigg ered by transient cerebral ischaemia and leading to neuronal cell injury.