Weber H, Hühns S, Lüthen F, Jonas L, Schuff-Werner P
Institute of Clinical Chemistry and Laboratory Medicine, University of Rostock, Germany.
Biochem Pharmacol. 2005 Oct 15;70(8):1241-52. doi: 10.1016/j.bcp.2005.06.028.
Oxygen radicals have been implicated as mediators in the pathogenesis of pancreatic acinar cell necrosis. However, the sequence of events between the oxidative insult and cell damage remains unclear. In the current study, we investigated whether the Ca(2+)-regulated cytosolic cysteine protease calpain is activated by oxidative stress and contributes to oxidant-induced acinar cell damage. Isolated rat pancreatic acinar cells were exposed to hydrogen peroxide (H(2)O(2))-generated oxidative stress in the presence or absence of the Ca(2+) chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM) and different calpain inhibitors including benzyloxycarbonyl-valyl-phenylalanine methyl ester. Calpain activation was studied by fluorescence spectrophotometry and immunoblotting. Cell injury was assessed by lactate dehydrogenase (LDH) release and characterization of the cellular ultrastructure including fluorescence-labeled actin filaments. Exposure of acinar cells to H(2)O(2) provoked a time- and dose-dependent increase in calpain proteolytic activity involving the ubiquitous isoforms mu- and m-calpain. The activation of calpain reflected the time course of developing cytotoxicity as demonstrated by increased LDH release. Inhibition of oxidant-induced calpain activity by BAPTA-AM and various calpain inhibitors provoked a decline in oxidant-induced cell injury. In particular, changes in the actin filament organization characterized by an increase in the basolateral actin and by a detachment of actin from the cell membrane in the region of membrane blebs were clearly reduced. In summary, our findings suggest that acinar cell damage through oxidative stress requires activation of calpain and that the actin cytoskeleton belongs to the cellular targets of the protease. The results support the hypothesis that calpain activation may play a role in the development of acute pancreatitis.
氧自由基被认为是胰腺腺泡细胞坏死发病机制中的介质。然而，氧化损伤与细胞损伤之间的事件顺序仍不清楚。在当前研究中，我们调查了钙（Ca2+）调节的胞质半胱氨酸蛋白酶钙蛋白酶是否被氧化应激激活，并导致氧化剂诱导的腺泡细胞损伤。分离的大鼠胰腺腺泡细胞在存在或不存在Ca2+螯合剂1,2-双-（邻氨基苯氧基）-乙烷-N,N,N',N'-四乙酸四乙酰甲酯（BAPTA-AM）以及不同的钙蛋白酶抑制剂（包括苄氧羰基-缬氨酰-苯丙氨酸甲酯）的情况下，暴露于过氧化氢（H2O2）产生的氧化应激中。通过荧光分光光度法和免疫印迹研究钙蛋白酶的激活。通过乳酸脱氢酶（LDH）释放以及包括荧光标记肌动蛋白丝在内的细胞超微结构特征来评估细胞损伤。腺泡细胞暴露于H2O2会引发钙蛋白酶蛋白水解活性的时间和剂量依赖性增加，涉及普遍存在的μ-和m-钙蛋白酶同工型。钙蛋白酶的激活反映了细胞毒性发展的时间进程，如LDH释放增加所示。BAPTA-AM和各种钙蛋白酶抑制剂对氧化剂诱导的钙蛋白酶活性的抑制导致氧化剂诱导的细胞损伤下降。特别是，肌动蛋白丝组织的变化明显减少，其特征是基底外侧肌动蛋白增加以及在膜泡区域肌动蛋白从细胞膜脱离。总之，我们的研究结果表明，通过氧化应激导致的腺泡细胞损伤需要钙蛋白酶的激活，并且肌动蛋白细胞骨架属于该蛋白酶的细胞靶点。这些结果支持了钙蛋白酶激活可能在急性胰腺炎发展中起作用的假说。
