Rami A, Volkmann T, Agarwal R, Schoninger S, Nürnberger F, Saido T C, Winckler J
Anatomisches Institut III, Dr. Senckenbergische Anatomie, Klinikum der JWG-Universität, Theodor-Stern-Kai 7, 60590 Frankfurt/Main, Germany.
Neurosci Res. 2003 Dec;47(4):373-82. doi: 10.1016/j.neures.2003.07.002.
In the CNS, where Ca(2+) overload has been established as a mechanism contributing to neuronal damage associated with excitotoxicity, stroke and ischemia, there is interest in understanding the role of calpain inhibition in rescuing neurons from death. In these settings, the activation of large stores of latent calpain may rapidly lead to the demise of the neuron within hours. The activity of calpain is strictly regulated by calcium concentrations and interactions with calpastatin (endogenous calpain inhibitor). The interaction between calpains and calpastatin is calcium dependent, and little is known about the regulation of the neuronal calpain-calpastatin system in vivo. It has been postulated that calpastatin can be modulated by nerve growth factors (NGFs). We have demonstrated in vitro as well as in vivo a neuroprotective effect of the beta(2)-adrenoceptor agonist clenbuterol (CLN) mediated through an increased NGF expression. In this study we attempt to find out whether CLN is capable (1) of modulating proteolysis regulated by the calpain-calpastatin system and (2) of attenuating DNA-fragmentation induced by cerebral ischemia. Rats received CLN daily for 1 week, were then subjected to ischemia and finally perfused at different times post-ischemia. The proteolytic activity of calpain was measured by the immunolocalisation of calpastatin and spectrin-breakdown products (SBP). The time course of apoptosis was assessed by terminal dUTP nick end-labeling (TUNEL)-staining. CLN reduced CA1-hippocampal cell damage by 23%, attenuated DNA-laddering and decreased proteolysis of spectrin by enhancing calpastatin activity. These results provide evidence that CLN is a potent neuroprotective substance, which through the enhancement of calpastatin synthesis attenuates the apoptotic machinery and modulates proteolysis.
在中枢神经系统中，钙（Ca2+）超载已被确认为一种导致与兴奋性毒性、中风和局部缺血相关的神经元损伤的机制，因此人们对了解钙蛋白酶抑制在挽救神经元免于死亡中的作用很感兴趣。在这些情况下，大量潜伏钙蛋白酶的激活可能会在数小时内迅速导致神经元死亡。钙蛋白酶的活性受到钙浓度以及与钙蛋白酶抑制蛋白（内源性钙蛋白酶抑制剂）相互作用的严格调节。钙蛋白酶与钙蛋白酶抑制蛋白之间的相互作用依赖于钙，而关于体内神经元钙蛋白酶 - 钙蛋白酶抑制蛋白系统的调节知之甚少。据推测，钙蛋白酶抑制蛋白可由神经生长因子（NGF）调节。我们已经在体外以及体内证明了β2 - 肾上腺素能受体激动剂克伦特罗（CLN）通过增加NGF表达介导的神经保护作用。在本研究中，我们试图弄清楚CLN是否能够（1）调节由钙蛋白酶 - 钙蛋白酶抑制蛋白系统调控的蛋白水解作用，以及（2）减轻脑缺血诱导的DNA片段化。大鼠每天接受CLN治疗1周，然后进行缺血处理，最后在缺血后的不同时间进行灌注。通过钙蛋白酶抑制蛋白和血影蛋白降解产物（SBP）的免疫定位来测量钙蛋白酶的蛋白水解活性。通过末端脱氧核苷酸转移酶介导的缺口末端标记（TUNEL）染色评估细胞凋亡的时间进程。CLN使海马CA1区细胞损伤减少了23%，通过增强钙蛋白酶抑制蛋白活性减轻了DNA梯状条带形成并减少了血影蛋白的蛋白水解。这些结果证明CLN是一种有效的神经保护物质，它通过增强钙蛋白酶抑制蛋白的合成来减弱凋亡机制并调节蛋白水解作用。
