M Gómez-Calcerrada | PubMed Health

PubMed2006

Calpain-induced proteolysis after transient global cerebral ischemia and ischemic tolerance in a rat model.

The activation of the [Ca(2+)]-dependent cysteine protease calpain plays an important role in ischemic injury. Here, the levels of two calpain-specific substrates, p35 protein and eukaryotic initiation factor 4G (eIF4G), as well as its physiological regulator calpastatin, were investigated in a rat model of transient global cerebral ischemia with or without ischemic tolerance (IT). Extracts of the cerebral cortex, whole hippocampus and hippocampal subregions after 30 min of ischemia and different reperfusion times (30 min and 4 h) were used. In rats without IT, the p35 levels slightly decreased after ischemia or reperfusion, whereas the levels of p25 (the truncated form of p35) were much higher than those in sham control rats after ischemia and remained elevated during reperfusion. The eIF4G levels deeply diminished after reperfusion and the decrease was significantly greater in CA1 and the rest of the hippocampus than in the cortex. By contrast, the calpastatin levels did not significantly decrease during ischemia or early reperfusion, but were upregulated after 4 h of reperfusion in the cortex. Although IT did not promote significant changes in p35 and p25 levels, it induced a slight increase in calpastatin and eIF4G levels in the hippocampal subregions after 4 h of reperfusion.

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PubMed1996

Changes in the phosphorylation of eukaryotic initiation factor 2 alpha, initiation factor 2B activity and translational rates in primary neuronal cultures under different physiological growing conditions.

Phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF-2) is one of the best known mechanisms regulating protein synthesis in a wide range of eukaryotic cells, from yeast to human. To determine whether this mechanism operates in primary neuronal cells, we have cultured primary neuronal cells for 7 days under two optimal growing conditions, complete medium (containing 15% serum) and serum-free medium, and determined the protein synthesis rate, eukaryotic initiation 2 and 2B (eIF-2B) activities, as well as the level of phosphorylation of eIF-2. Cells cultured in serum-free medium exhibited a lower rate of protein synthesis (75%), concomitant to a decreased eIF-2 activity (71%), and slightly higher eIF-2(alpha P) levels (from 10 to 16% of total eIF-2) with respect to cells cultured in complete media. eIF-2B activity, as measured at saturating eIF-2. GDP concentrations (assay independent on the presence of eIF-2(alpha P)) was similar under the two culture conditions. When neurons cultured in serum-free medium are exposed to complete medium for only 24 h, there is a clear decrease in the phosphorylation of eIF-2 alpha (16-3%). This decrease correlates in time with an increase in the protein synthesis rate (154%), as well as eIF-2 activity (236%). The increased levels of eIF-2(alpha P), a competitive inhibitor of eIF-2B in the guanine-exchange reaction, are responsible for the decreased eIF-2B activity found in the neurons cultured in serum-free medium. Additionally, eIF-2(alpha P) is accountable for the lower effect of exogenous eIF-2B in ternary complex formation from preformed eIF-2. GDP in the serum-free media. These changes in phosphorylation of eIF-2 alpha in normal mammalian cells in response to changes in the extracellular medium are reported here for the first time.

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