Burda J, Martín M E, Gottlieb M, Chavko M, Marsala J, Alcázar A, Pavón M, Fando J L, Salinas M
Department of Neurochemistry, Institute of Neurobiology, Slovak Academy of Sciences, Kosice, Slovak Republic.
J Cereb Blood Flow Metab. 1998 Jan;18(1):59-66. doi: 10.1097/00004647-199801000-00006.
Rats were subjected to the standard four-vessel occlusion model of transient cerebral ischemia (vertebral and carotid arteries). The effects of normothermic ischemia (37 degrees C) followed or not by 30-minute reperfusion, as well as 30-minute postdecapitative ischemia, on translational rates were examined. Protein synthesis rate, as measured in a cell-free system, was significantly inhibited in ischemic rats, and the extent of inhibition strongly depended on duration and temperature, and less on the model of ischemia used. The ability of reinitiation in vitro (by using aurintricarboxylic acid) decreased after ischemia, suggesting a failure in the synthetic machinery at the initiation level. Eukaryotic initiation factor 2 (eIF-2) presented almost basal activity and levels after 30-minute normothermic ischemia, and the amount of phosphorylated eIF-2 alpha in these samples, as well as in sham-control samples, was undetectable. The decrease in the levels of phosphorylated initiation factor 4E (eIF-4E) after 30-minute ischemia (from 32% to 16%) could explain, at least partially, the impairment of initiation during transient cerebral ischemia. After reperfusion, eIF-4E phosphorylation was almost completely restored to basal levels (29%), whereas the level of phosphorylated eIF-2 alpha was higher (13%) than in controls and ischemic samples (both less than 2%). eIF-2 alpha kinase activity in vitro as measured by phosphorylation of endogenous eIF-2 in the presence of ATP/Mg2+, was higher in ischemic samples (8%) than in controls (4%). It seems probable that the failure of the kinase in phosphorylating eIF-2 in vivo during ischemia is due to the depletion of ATP stores. The levels of the double-stranded activated eIF-2 alpha kinase were slightly higher in ischemic animals than in controls. Our results suggest that the modulation of eIF-4E phosphorylation could be implicated in the regulation of translation during ischemia. On the contrary, phosphorylation of eIF-2 alpha, by an eIF-2 alpha kinase already activated during ischemia, represents a plausible mechanism for explaining the inhibition of translation during reperfusion.
将大鼠置于短暂性脑缺血（椎动脉和颈动脉）的标准四血管闭塞模型中。研究了常温缺血（37℃）后再灌注30分钟以及断头后缺血30分钟对翻译速率的影响。在无细胞系统中测量的蛋白质合成速率在缺血大鼠中显著受到抑制，抑制程度强烈依赖于持续时间和温度，而较少依赖于所使用的缺血模型。缺血后体外重新起始的能力（通过使用金精三羧酸）下降，这表明在起始水平上合成机制存在缺陷。在30分钟常温缺血后，真核起始因子2（eIF-2）呈现出几乎基础的活性和水平，并且在这些样品以及假手术对照样品中，磷酸化的eIF-2α的量无法检测到。缺血30分钟后，磷酸化起始因子4E（eIF-4E）水平的降低（从32%降至16%）至少可以部分解释短暂性脑缺血期间起始的受损情况。再灌注后，eIF-4E磷酸化几乎完全恢复到基础水平（29%），而磷酸化的eIF-2α水平比对照组和缺血样品更高（13%）（两者均小于2%）。在ATP/Mg2+存在的情况下，通过内源性eIF-2的磷酸化测量的体外eIF-2α激酶活性在缺血样品中（8%）高于对照组（4%）。缺血期间激酶在体内磷酸化eIF-2失败似乎可能是由于ATP储备的耗尽。双链活化的eIF-2α激酶水平在缺血动物中比对照组略高。我们的结果表明，eIF-4E磷酸化的调节可能与缺血期间的翻译调节有关。相反，在缺血期间已经活化的eIF-2α激酶对eIF-2α的磷酸化是解释再灌注期间翻译抑制的一种合理机制。
