Reinhardt R, Manaenko A, Guenther A, Franke H, Dickel T, Garcia de Arriba S, Muench G, Schneider D, Wagner A, Illes P
Rudolf-Boehm-Institute of Pharmacology and Toxicology, University of Leipzig, Haertelstrasse 16-18, D-04107, Leipzig, Germany.
Neurochem Int. 2003 Nov;43(6):563-71. doi: 10.1016/s0197-0186(03)00053-6.
The present study was aimed at characterizing alterations of the nucleotide content and morphological state of rat corticoencephalic cell cultures subjected to metabolic damage and treatment with modulators of mitochondrial ATP-dependent potassium channels (mitoK(ATP)). In a first series of experiments, in vitro ischemic changes of the contents of purine and pyrimidine nucleoside diphosphates and triphosphates were measured by high performance liquid chromatography (HPLC) and the corresponding histological alterations were determined by celestine blue/acid fuchsin staining. As an ischemic stimulus, incubation with a glucose-free medium saturated with argon was used. Ischemia decreased the levels of adenosine, guanine and uridine triphosphate (ATP, GTP, UTP) and increased the levels of the respective dinucleotides ADP and UDP, whereas the GDP content was not changed. Both 5-hydroxydecanoate (5-HD) and diazoxide failed to alter the contents of nucleoside diphosphates and triphosphates, when applied under normoxic conditions. 5-HD (30 microM) prevented the ischemia-induced changes of nucleotide and nucleoside levels. Diazoxide (300 microM), either alone or in combination with 5-hydroxydecanoate (30 microM) was ineffective. Pyruvate (5 mM) partially reversed the effects of ischemia or ischemia plus 2-deoxyglucose (20mM) in the incubation medium. Diazoxide (300 microM) and 5-HD (30 microM) had no effect in the presence of pyruvate (5mM) and 2-deoxyglucose (20mM). Staining the cells with celestine blue/acid fuchsin in order to classify them as intact, reversibly or profoundly injured, revealed a protective effect of 5-HD. When compared with 5-HD, diazoxide, pyruvate and 2-deoxyglucose had similar but less pronounced effects. In conclusion, these results suggest a protective role of 5-hydroxydecanoate on early corticoencephalic nucleotide and cell viability alterations during ischemia.
本研究旨在表征遭受代谢损伤并经线粒体ATP依赖性钾通道（mitoK(ATP)）调节剂处理的大鼠皮质-脑细胞培养物中核苷酸含量和形态状态的变化。在第一系列实验中，通过高效液相色谱法（HPLC）测量嘌呤和嘧啶核苷二磷酸及三磷酸含量的体外缺血变化，并通过天青石蓝/酸性品红染色确定相应的组织学改变。作为缺血刺激，使用与氩气饱和的无葡萄糖培养基孵育。缺血降低了腺苷、鸟苷和尿苷三磷酸（ATP、GTP、UTP）的水平，并增加了相应二核苷酸ADP和UDP的水平，而GDP含量未改变。在常氧条件下应用时，5-羟基癸酸酯（5-HD）和二氮嗪均未改变核苷二磷酸和三磷酸的含量。5-HD（30微摩尔）可防止缺血诱导的核苷酸和核苷水平变化。二氮嗪（300微摩尔）单独使用或与5-羟基癸酸酯（30微摩尔）联合使用均无效。丙酮酸（5毫摩尔）部分逆转了孵育培养基中缺血或缺血加2-脱氧葡萄糖（20毫摩尔）的影响。在存在丙酮酸（5毫摩尔）和2-脱氧葡萄糖（20毫摩尔）的情况下，二氮嗪（300微摩尔）和5-HD（30微摩尔）无作用。用天青石蓝/酸性品红对细胞进行染色以将其分类为完整、可逆或严重受损，结果显示5-HD具有保护作用。与5-HD相比，二氮嗪、丙酮酸和2-脱氧葡萄糖具有相似但不太明显的作用。总之，这些结果表明5-羟基癸酸酯对缺血期间早期皮质-脑核苷酸和细胞活力改变具有保护作用。
