Models--Cytoprotection By Inhibition of Calpain Protease

在啮齿类动物模型中，长期饮酒后发生的NMDA受体上调与神经细胞毒性有关，通过l-谷氨酸激活这些钙通道导致钙通量增加。钙蛋白酶是一类钙活化的半胱氨酸蛋白酶，已知可介导神经细胞钙介导的损伤。本项目的重点是：a)利用PC12细胞模型研究l-谷氨酸介导的细胞损伤机制；B)确定抑制calpain活性是否会产生显著的细胞保护作用；C)在l-谷氨酸细胞毒性模型中测试多种钙蛋白酶抑制剂作为细胞保护剂；D)将这些观察结果扩展到神经细胞损伤的海马模型。该实验室先前的研究表明，HIV蛋白酶抑制剂利托那韦（ritonavir）也是一种竞争性钙蛋白酶活性抑制剂，其Ki值约为10微米，完全在人类临床给药时用作抗逆转录病毒药物的范围内。使用fura-2，发现l-谷氨酸显著增加细胞内游离钙浓度。l-谷氨酸诱导的细胞内游离钙的增加也导致钙蛋白酶活性的显著增加。Calpain激活之后是多种细胞骨架成分的降解，在该模型中发现，l-谷氨酸暴露导致actin、tau和NF68的显著降解，而MK801、Calpain抑制剂I和利托那韦会阻断这一降解。在细胞毒性试验中发现，MK801、calpain抑制剂I和利托那韦也均抑制l-谷氨酸介导的细胞死亡。caspase抑制剂（Z-DEVD-FMK）和DNQX （AMPA抑制剂）都没有任何保护作用，也不能阻止l-谷氨酸诱导的细胞骨架蛋白分解。我们得出结论，l-谷氨酸介导的PC12细胞毒性是一个calpain依赖的过程。我们还发现利托那韦在海马培养模型中对氧化应激诱导的损伤具有细胞保护作用。这个项目即将终止。由于这项工作，已经提交了两份手稿供审查，两份与工作有关的手稿正在准备中。

NMDA receptor upregulation occurring after prolonged alcohol administration in rodent models has been implicated in neural cytotoxicity, via an increase in calcium-flux resulting from l-glutamate activation of these calcium channels. Calpains are a class of calcium-activated cysteine proteases that are known to mediate calcium-mediated injury in neural cells. The focus of this project is to a) examine the mechanism of l-glutamate mediated cell injury using a PC12 cell model; b) determine whether inhibition of calpain activity would result in significant cytoprotection; c) test a variety of calpain inhibitors as cytoprotectants in an l-glutamate model of cytotoxicity; d) extend these observations to a hippocmpal model of neural cell injury. It was previously shown in this laboratory that ritonavir, an HIV protease inhibitor, is also a competitive inhibitor of calpain activity, with a Ki of about 10 microM, well within range found during clinical dosing of the drug in humans when used as an anti-retroviral. Using fura-2, it was found that l-glutamate singificantly increased intracellular concentrations of free calcium. The increase in intracellular free calcium induced by l-glutamate was also shown to result in a signficant increase in calpain protease activity. Calpain activation is followed by degradation of a variety of cytoskeletal components, and in this model it was found that l-glutamate exposure resulted in significant degradation of actin, tau, and NF68, which was blocked by MK801, calpain inhibitor I and ritonavir. In cytotoxicity tests, it was found that MK801, calpain inhibitor I, and ritonavir also all inhibited l-glutamate mediated cell death. Neither a caspase inhibitor (Z-DEVD-FMK) nor DNQX (AMPA inhibitor) had any protectant effects, nor did they prevent l-glutamate induced breakdown of cytoskeletal proteins. We conclude that l-glutamate mediated cytotoxicity in PC12 cells is a calpain-dependent process. We also found that ritonavir was cytoprotective against oxidative stress-induced injury in a hippocampal culture model. The project is being terminated. As a result of this work, two manuscripts have been submitted for review, and two manuscripts related to the work are in preparation.