Mehendale H M, Svensson S A, Baldi C, Orrenius S
Eur J Biochem. 1985 May 15;149(1):201-6. doi: 10.1111/j.1432-1033.1985.tb08912.x.
Previous studies have indicated that the presence of cytotoxic levels of menadione (2-methyl-1,4-naphthoquinone) causes rapid changes in intracellular thiol and Ca2+ homeostasis in isolated rat hepatocytes. The present investigation was undertaken to examine these effects in the intact liver. Rat livers were therefore perfused with Krebs-Henseleit buffer containing 1.3 mM Ca2+ using a single-pass mode, and the perfusate Ca2+ level was monitored with an on-line Ca2+-selective electrode. Infusion of menadione elicited an increased O2 uptake by the liver, followed by a dose-dependent decrease in the perfusate level of Ca2+. Hepatic accumulation of Ca2+ was accompanied by stimulation of cytosolic phosphorylase a activity. Cessation of menadione infusion resulted in gradual recovery of perfusate Ca2+ to base levels. Ca2+ uptake was not accompanied by decreases in reduced pyridine nucleotide or ATP levels in the liver as evidenced by measurements either during maximal Ca2+ uptake or after recovery. However, Ca2+ uptake was correlated with decreased glutathione and increased glutathione disulfide levels in the liver, both of which reversed during recovery from Ca2+ uptake. Moreover, depletion of hepatic glutathione by pretreatment with diethylmaleate resulted in increased Ca2+ uptake during menadione infusion. The amount of protein-bound mixed disulfides showed a particularly striking relationship to Ca2+ uptake, reaching a maximal level during Ca2+ uptake and reversing toward normal value during recovery from Ca2+ accumulation. The present findings suggest that menadione-induced Ca2+ uptake is due to plasma membrane dysfunction as a result of loss of protein thiol groups critical for maintaining the plasma membrane Ca2+ extrusion mechanism. Our model offers a particularly useful opportunity to study mechanisms underlying toxic disturbances in Ca2+ homeostasis in the intact liver, since Ca2+ fluxes can be monitored under conditions in which cellular control mechanisms are not obliterated by excessive toxicity.
先前的研究表明，细胞毒性水平的甲萘醌（2-甲基-1,4-萘醌）的存在会导致分离的大鼠肝细胞内的硫醇和Ca2+稳态迅速变化。本研究旨在检查完整肝脏中的这些影响。因此，使用单通道模式用含有1.3 mM Ca2+的Krebs-Henseleit缓冲液灌注大鼠肝脏，并用在线Ca2+选择性电极监测灌注液中的Ca2+水平。注入甲萘醌会引起肝脏对O2的摄取增加，随后灌注液中Ca2+水平呈剂量依赖性下降。Ca2+在肝脏中的积累伴随着细胞溶质磷酸化酶a活性的刺激。停止注入甲萘醌会导致灌注液中的Ca2+逐渐恢复到基线水平。无论是在最大Ca2+摄取期间还是在恢复后进行测量，都表明Ca2+摄取并未伴随着肝脏中还原型吡啶核苷酸或ATP水平的降低。然而，Ca2+摄取与肝脏中谷胱甘肽水平降低和谷胱甘肽二硫化物水平升高相关，这两者在从Ca2+摄取中恢复期间都会逆转。此外，用马来酸二乙酯预处理使肝脏谷胱甘肽耗竭会导致注入甲萘醌期间Ca2+摄取增加。蛋白质结合的混合二硫化物的量与Ca2+摄取呈现出特别显著的关系，在Ca2+摄取期间达到最高水平，并在从Ca2+积累中恢复期间恢复到正常值。目前的研究结果表明，甲萘醌诱导的Ca2+摄取是由于质膜功能障碍，这是由于对维持质膜Ca2+外排机制至关重要的蛋白质硫醇基团丧失所致。我们的模型为研究完整肝脏中Ca2+稳态中毒性干扰的潜在机制提供了一个特别有用的机会，因为可以在细胞控制机制不会因过度毒性而被消除的条件下监测Ca2+通量。
