Models--Cytoprotection By Inhibition of Calpain Protease

模型——通过抑制钙蛋白酶进行细胞保护

• 批准号: 6542016
• 负责人: PAOLO B DE PETRILLO
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6542016
• 关键词: PC12 cells; alcoholism /alcohol abuse; calpain; cytoprotection; endopeptidases; enzyme inhibitors; glutamates; hippocampus; neuropharmacology; ritonavir; tissue /cell culture

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.

在啮齿动物模型中，长期给予酒精后发生的NMDA受体上调与神经细胞毒性有关，这是通过L-谷氨酸激活这些钙通道导致的钙通量增加实现的。钙蛋白酶是一类钙激活的半胱氨酸蛋白酶，已知其介导神经细胞中的钙介导的损伤。本项目的重点是：a）使用PC 12细胞模型研究L-谷氨酸介导的细胞损伤机制; B）确定钙蛋白酶活性抑制是否会导致显著的细胞保护作用; c）在L-谷氨酸细胞毒性模型中测试各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海马神经细胞损伤模型。先前在该实验室中显示，利托那韦（一种HIV蛋白酶抑制剂）也是钙蛋白酶活性的竞争性抑制剂，Ki约为10 μ M，完全在该药物用作抗逆转录病毒药物时在人体临床给药期间发现的范围内。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显示出导致钙蛋白酶活性的显着增加。钙蛋白酶激活之后是各种细胞骨架成分的降解，在该模型中，发现L-谷氨酸暴露导致肌动蛋白、tau和NF 68的显著降解，这被MK 801、钙蛋白酶抑制剂I和利托那韦阻断。在细胞毒性试验中，发现MK 801、钙蛋白酶抑制剂I和利托那韦也都抑制L-谷氨酸介导的细胞死亡。半胱天冬酶抑制剂（Z-DEVD-FMK）和DNQX（AMPA抑制剂）都没有任何保护作用，也没有阻止l-谷氨酸诱导的细胞骨架蛋白的分解。我们的结论是，l-谷氨酸介导的PC 12细胞细胞毒性是一个钙蛋白酶依赖性过程。我们还发现利托那韦对海马培养模型中氧化应激诱导的损伤具有细胞保护作用。该项目正在终止。由于这项工作，已提交两份手稿供审查，与这项工作有关的两份手稿正在编写中。