Weinberg J M, Venkatachalam M A, Roeser N F, Davis J A, Varani J, Johnson K J
Department of Internal Medicine, University of Michigan, Ann Arbor.
Lab Invest. 1991 Dec;65(6):671-8.
Treatment of two cultured renal tubule epithelial cell lines, MDCK and LLC-PK1, with ionomycin produced rapidly evolving models of lethal cell injury characterized by increases of cytosolic free calcium to the microM level within 15 minutes followed by lactate dehydrogenase release and failure to exclude vital dyes that began between 30 and 60 minutes and became extensive after 60 minutes. The pattern of injury was similar when the mitochondrial uncoupler, carbonyl cyanide-m-chlorophenylhydrazone, was added to ionomycin. Carbonyl cyanide-m-chlorophenylhydrazone alone produced severe ATP depletion but not lactate dehydrogenase release. Inclusion of glycine in the experimental medium at concentrations ranging from 0.25 mM to 5 mM did not affect the increases of cytosolic free calcium or ATP depletion but was protective against enzyme release and failure to exclude vital dyes for 180 minutes. Maximal protection was achieved at glycine concentrations between 1 and 5 mM. Several other small neutral amino acids including alanine, beta-alanine, L-serine, 1-aminocyclopropane-1-carboxylic acid, and alpha-aminoisobutyric acid also had protective effects but, glucose, pyruvate, glutamate, glutamine, leucine, valine, and taurine did not. These data indicate that potent protective effects of glycine and other small neutral amino acids previously shown in fresh tubule preparations are fully expressed in cultured tubule cells of diverse origin when appropriate acute injury models are used and the protective effects are sustained for long durations. The suitability of cultured cell lines for prolonged exposure studies will provide a powerful way of further exploring mechanisms of these effects.
用离子霉素处理两种培养的肾小管上皮细胞系MDCK和LLC-PK1，可产生快速演变的致死性细胞损伤模型，其特征为15分钟内胞质游离钙增加至微摩尔水平，随后乳酸脱氢酶释放，30至60分钟开始出现不能排除活体染料的情况，60分钟后变得广泛。当线粒体解偶联剂羰基氰化物-间氯苯腙加入离子霉素时，损伤模式相似。单独使用羰基氰化物-间氯苯腙会导致严重的ATP耗竭，但不会导致乳酸脱氢酶释放。在实验培养基中加入浓度范围为0.25 mM至5 mM的甘氨酸，不会影响胞质游离钙的增加或ATP耗竭，但可保护细胞180分钟不发生酶释放和不能排除活体染料的情况。在甘氨酸浓度为1至5 mM时可实现最大保护。其他几种小的中性氨基酸，包括丙氨酸、β-丙氨酸、L-丝氨酸、1-氨基环丙烷-1-羧酸和α-氨基异丁酸也有保护作用，但葡萄糖、丙酮酸、谷氨酸、谷氨酰胺、亮氨酸、缬氨酸和牛磺酸则没有。这些数据表明，甘氨酸和其他小的中性氨基酸先前在新鲜肾小管制剂中显示出的强大保护作用，在使用适当的急性损伤模型时，在不同来源的培养肾小管细胞中得到充分表达，并且保护作用可持续很长时间。培养细胞系适用于长时间暴露研究，这将为进一步探索这些作用的机制提供有力方法。
