Ramirez-Ortega Margarita, Zarco Gabriela, Maldonado Vilma, Carrillo Jose F, Ramos Pilar, Ceballos Guillermo, Melendez-Zajgla Jorge, Garcia Noemí, Zazueta Cecilia, Chanona Jose, Suarez Jorge, Pastelin Gustavo
Departamento de Farmacologia, Instituto Nacional de Cardiologia Ignacio Chavez, Juan Badiano 1, Col. Seccion XVI, 14080 Mexico, D. F., Mexico.
Eur J Pharmacol. 2007 Jul 2;566(1-3):34-42. doi: 10.1016/j.ejphar.2007.03.033. Epub 2007 Mar 30.
Our aim in performing this study was to analyze in vivo the cell death mechanism induced by toxic doses of digitalis compounds on guinea-pig cardiomyocytes. We analyzed three study groups of five male guinea pigs each. Guinea pigs were intoxicated under anesthesia with ouabain or digoxin (at a 50-60% lethal dose); the control group did not receive digitalis. A 5-hours period elapsed before guinea pig hearts were extracted to obtain left ventricle tissue. We carried out isolation of mitochondria and cytosol, cytochrome c and caspase-3 and -9 determination, and electrophoretic analysis of nuclear DNA. TdT-mediated DUTP-X nick end labeling (TUNEL) reaction was performed in histologic preparations to identify in situ apoptotic cell death. Ultrastructural analysis was performed by electron microscopy. Electrophoretic analysis of DNA showed degradation into fragments of 200-400 base pairs in digitalis-treated groups. TUNEL reaction demonstrated the following: in the control group, <10 positive nuclei per field; in the digoxin-treated group, 2-14 positive nuclei per field, while in the ouabain-treated group counts ranged from 9-30 positive nuclei per field. Extracts from ouabain-treated hearts had an elevation of cytochrome c in cytosol and a corresponding decrease in mitochondria; this release of cytochrome c provoked activation of caspase-9 and -3. Electron microscopy revealed presence of autophagic vesicles in cytoplasm of treated hearts. Toxic dosages of digitalis at 50-60% of the lethal dose are capable of inducing cytochrome c release from mitochondria, processing of procaspase-9 and -3, and DNA fragmentation; these observations are mainly indicative of apoptosis, although a mixed mechanism of cell death cannot be ruled out.
我们开展这项研究的目的是在体内分析洋地黄化合物的毒性剂量对豚鼠心肌细胞诱导的细胞死亡机制。我们分析了三个研究组，每组有五只雄性豚鼠。豚鼠在麻醉状态下用哇巴因或地高辛（致死剂量的50 - 60%）进行中毒处理；对照组未接受洋地黄治疗。在提取豚鼠心脏以获取左心室组织之前经过了5小时。我们进行了线粒体和胞质溶胶的分离、细胞色素c以及半胱天冬酶-3和-9的测定，以及核DNA的电泳分析。在组织学制剂中进行TdT介导的dUTP-X缺口末端标记（TUNEL）反应以鉴定原位凋亡细胞死亡。通过电子显微镜进行超微结构分析。DNA的电泳分析显示在洋地黄处理组中DNA降解为200 - 400个碱基对的片段。TUNEL反应显示如下：在对照组中，每视野<10个阳性细胞核；在地高辛处理组中，每视野有2 - 14个阳性细胞核，而在哇巴因处理组中，每视野阳性细胞核数范围为9 - 30个。哇巴因处理的心脏提取物中胞质溶胶中的细胞色素c升高，而线粒体中相应降低；这种细胞色素c的释放引发了半胱天冬酶-9和-3的激活。电子显微镜显示处理过的心脏细胞质中存在自噬泡。致死剂量50 - 60%的洋地黄毒性剂量能够诱导细胞色素c从线粒体释放、前半胱天冬酶-9和-3的加工以及DNA片段化；这些观察结果主要表明存在凋亡，尽管不能排除细胞死亡的混合机制。
