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In Vitro And In Vivo Models Of Cytoprotection By Inhibit

抑制细胞保护的体外和体内模型

基本信息

批准号：
6677071
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
项目类别：
财政年份：
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/6677071
关键词：
PC12 cells alcoholism /alcohol abuse calpain cytoprotection endopeptidases enzyme inhibitors glutamates hippocampus neuropharmacology ritonavir tissue /cell culture

项目摘要

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.

在啮齿动物模型中，长期饮酒后发生的 NMDA 受体上调与神经细胞毒性有关，这是由于这些钙通道的 L-谷氨酸激活导致钙通量增加。钙蛋白酶是一类钙激活的半胱氨酸蛋白酶，已知可介导钙介导的神经细胞损伤。该项目的重点是 a) 使用 PC12 细胞模型检查 L-谷氨酸介导的细胞损伤机制； b) 确定抑制钙蛋白酶活性是否会导致显着的细胞保护作用； 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。该实验室此前已证明，利托那韦（一种 HIV 蛋白酶抑制剂）也是一种钙蛋白酶活性的竞争性抑制剂，Ki 约为 10 microM，完全在该药物用作抗逆转录病毒药物时在人体临床给药时发现的范围内。使用fura-2，发现L-谷氨酸显着增加细胞内游离钙的浓度。 L-谷氨酸诱导的细胞内游离钙的增加也被证明导致钙蛋白酶活性显着增加。钙蛋白酶激活后，多种细胞骨架成分被降解，在该模型中发现，L-谷氨酸暴露导致肌动蛋白、tau 和 NF68 显着降解，而这种降解被 MK801、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MK801、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。 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 细胞细胞毒性是钙蛋白酶依赖性过程。我们还发现，在海马培养模型中，利托那韦对氧化应激诱导的损伤具有细胞保护作用。