Dosenko V E, Nagibin V S, Tumanovskaya L V, Zagoriy V Yu, Moibenko A A, Vaage J
Department of Experimental Cardiology, Bogomoletz Institute of Physiology, Kyiv, Ukraine.
Fiziol Zh (1994). 2006;52(3):15-24.
A role of proteasomal proteolysis in the pathogenesis of ischemia-reperfusion is being actively studied. To evaluate the participation of the proteasome in postconditioning phenomenon, we used primary culture of neonatal cardiomyocytes. 30 minutes of anoxia followed by 60 minutes of reoxygenation was undergone. Postconditioning was modeled by 3 cycles of 1-minute reoxygenation followed by 1-minute anoxia, respectively. Clasto-lactacystin b-lactone, a specific proteasome inhibitor, in the dose that does not cause cell death (2.5 mM) was added to the culture medium just before the cycles of postconditioning. Percentages of living, necrotic, and apoptotic cells were determined by staining with bisBenzimide and propidium iodide. Autophagy was demonstrated by staining vacuolar structures with monodansyl cadaverine. Proteasomal activity was determined by cleavage intensity of specific fluorogenic substrates. Trypsin-like, chymotrypsin-like and peptidyl-glutamyl peptide-hydrolyzing (PGPH) activities were decreased after anoxia. Reoxygenation led to an increase in trypsin-like and chymotrypsin-like activities comparing to anoxia, but these parameters never reached the control levels. PGPH activity was restored up to the initial level. Postconditioning increased numbers of living cells and decreased that of necrotic, apoptotic and autophagic cells. Paradoxically, it was established, that proteasome inhibitors prevented the necrotic and apoptotic cell death of cardiomyocytes in anoxia-reoxygenation, but in the same concentration abolished the effects of postconditioning. The data obtained permit to suppose that proteasome inhibitors can be used for pharmacological postconditioning.
蛋白酶体蛋白水解在缺血再灌注发病机制中的作用正在被积极研究。为了评估蛋白酶体在缺血后处理现象中的参与情况，我们使用了新生大鼠心肌细胞的原代培养。经历30分钟缺氧后再进行60分钟复氧。缺血后处理通过分别进行3个循环的1分钟复氧和1分钟缺氧来模拟。在缺血后处理循环之前，将不会导致细胞死亡的剂量（2.5 mM）的蛋白酶体特异性抑制剂氯抑素b-内酯添加到培养基中。通过用双苯甲酰亚胺和碘化丙啶染色来确定活细胞、坏死细胞和凋亡细胞的百分比。通过用单丹磺酰尸胺对液泡结构进行染色来证明自噬。通过特定荧光底物的切割强度来测定蛋白酶体活性。缺氧后胰蛋白酶样、糜蛋白酶样和肽基-谷氨酰肽水解（PGPH）活性降低。与缺氧相比，复氧导致胰蛋白酶样和糜蛋白酶样活性增加，但这些参数从未达到对照水平。PGPH活性恢复到初始水平。缺血后处理增加了活细胞数量，减少了坏死、凋亡和自噬细胞数量。矛盾之处在于，已证实蛋白酶体抑制剂可预防缺氧复氧时心肌细胞的坏死和凋亡性细胞死亡，但相同浓度下会消除缺血后处理的效果。所获得的数据表明蛋白酶体抑制剂可用于药理学缺血后处理。
