Dhanbhoora C M, Babson J R
Department of Pharmacology and Toxicology, College of Pharmacy, University of Rhode Island, Kingston 02881.
Arch Biochem Biophys. 1992 Feb 14;293(1):130-9. doi: 10.1016/0003-9861(92)90375-7.
Treatment of cultured neonatal cardiomyocytes with ethacrynic acid (EA) induced a rapid depletion of glutathione (GSH) that preceded a gradual elevation of cytosolic Ca2+ (monitored by phosphorylase a activation), a loss of protein thiols, and a marked inactivation of the thiol-dependent enzyme glyceraldehyde-3-phosphate dehydrogenase (G3PD). A subsequent decline of mitochondrial transmembrane potential (delta psi) and ATP occurred prior to the onset of lipid peroxidation which closely paralleled a loss of cardiomyocyte viability. The antioxidant N,N'-diphenyl-p-phenylenediamine prevented lipid peroxidation and cell death but had no effect on elevated cytosolic Ca2+, delta psi loss, GSH depletion, or G3PD inactivation. Pretreatment with the iron chelator, deferoxamine, decreased both lipid peroxidation and cell death. EA-induced lipid peroxidation and cell damage were also diminished by preincubation with acetoxymethyl esters of the Ca2+ chelators Quin-2 and ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid, even though cytosolic Ca2+ remained elevated. The extent of GSH depletion was unaltered by either chelator; however, Quin-2 did protect G3PD from inactivation by EA. An inhibitor of the mitochondrial respiratory chain, antimycin A, decreased EA-induced lipid peroxidation and cell death but had no effect on thiol depletion or elevated cytosolic Ca2+. These data suggest that cardiomyocyte thiol status may be linked to intracellular Ca2+ homeostasis and that peroxidative damage originating in the mitochondria is a major event in the onset of cell death in this cardiomyocyte model of thiol depletion.
用依他尼酸（EA）处理培养的新生心肌细胞，可导致谷胱甘肽（GSH）迅速耗竭，随后胞质Ca2+逐渐升高（通过磷酸化酶a激活监测），蛋白质硫醇丧失，以及硫醇依赖性酶甘油醛-3-磷酸脱氢酶（G3PD）显著失活。线粒体跨膜电位（δψ）和ATP随后下降，脂质过氧化开始之前，这与心肌细胞活力丧失密切相关。抗氧化剂N，N'-二苯基对苯二胺可预防脂质过氧化和细胞死亡，但对升高的胞质Ca2+、δψ丧失、GSH耗竭或G3PD失活没有影响。用铁螯合剂去铁胺预处理可降低脂质过氧化和细胞死亡。Ca2+螯合剂喹啉-2和乙二醇双（β-氨基乙基醚）N，N'-四乙酸的乙酰氧甲酯预孵育也可减少EA诱导的脂质过氧化和细胞损伤，尽管胞质Ca2+仍然升高。两种螯合剂均未改变GSH耗竭的程度；然而，喹啉-2确实保护G3PD不被EA失活。线粒体呼吸链抑制剂抗霉素A可减少EA诱导的脂质过氧化和细胞死亡，但对硫醇耗竭或升高的胞质Ca2+没有影响。这些数据表明，心肌细胞硫醇状态可能与细胞内Ca2+稳态有关，并且线粒体起源的过氧化损伤是该硫醇耗竭心肌细胞模型中细胞死亡发生的主要事件。
