Le C T, Hollaar L, van der Valk E J, van der Laarse A
Department of Cardiology, University Hospital, Leiden, The Netherlands.
J Mol Cell Cardiol. 1993 May;25(5):519-28. doi: 10.1006/jmcc.1993.1062.
Mammalian heart myocytes have a limited capacity to withstand the deleterious effects of free radical generating compounds. To assess the role of the glutathione redox cycle relative to this capacity, rat heart cell cultures were subjected for 90 min to 80 mumol/l cumene hydroperoxide (CHPO) without and with prior glutathione depletion by buthionine sulfoximine. Preincubation of cultures with 125 mumol/l buthionine sulfoximine for 2 h and 17 h caused a reduction of glutathione by 33% and 82%, respectively, without concomitant increase of glutathione disulfide. Subsequent incubation with CHPO for 90 min caused slowing of NADPH consumption (in the first 20 min 27 pmol vs 68 pmol without pretreatment with buthionine sulfoximine for 17 h), which indicates that glutathione depletion reduced the turnover rate of the glutathione redox cycle. Pretreatment with buthionine sulfoximine for 17 h exaggerated the negative chronotropic effects of CHPO: the time elapsed to 50% of baseline contraction frequency fell from 5.7 +/- 1.4 min without buthionine sulfoximine to 3.7 +/- 0.4 min after pretreatment with buthionine sulfoximine (P < 0.02). The severity of CHPO-induced lipid peroxidation as assessed by malondialdehyde formation (2.23 +/- 0.51 vs 0.99 +/- 0.05 nmol in the first 20 min; P < 0.05) was increased by buthionine sulfoximine pretreatment, as was the extent of cell necrosis as assessed by release of alpha-hydroxybutyrate dehydrogenase (39.5 +/- 5.1 vs 29.0 +/- 12.9% in the first 45 min). A "sublethal" dose of 10 microM CHPO for 60 min caused no substantial HBDH release, no formation of malondialdehyde, and no exhaustion of cellular GSH (35 nmol/U HBDHt = 0). However, following pretreatment with buthionine sulfoximine, 10 microM CHPO for 60 min produced 12% HBDH release and extensive lipid peroxidation (1.95 nmol malondialdehyde/U HBDHt = 0). As the deleterious effects of CHPO were aggravated by glutathione depletion, we conclude that the glutathione redox cycle plays a major role in the protection of myocytes against peroxide-induced free radical attack.
哺乳动物心脏心肌细胞抵抗自由基生成化合物有害作用的能力有限。为了评估谷胱甘肽氧化还原循环相对于该能力的作用，将大鼠心脏细胞培养物在无谷胱甘肽消耗和经丁硫氨酸亚砜亚胺使谷胱甘肽预先耗竭的情况下，用80μmol/L的氢过氧化异丙苯（CHPO）处理90分钟。用125μmol/L丁硫氨酸亚砜亚胺对培养物进行2小时和17小时的预孵育，分别使谷胱甘肽减少33%和82%，而谷胱甘肽二硫化物没有相应增加。随后用CHPO孵育90分钟导致NADPH消耗减慢（在最初20分钟内为27pmol，而未经丁硫氨酸亚砜亚胺17小时预处理时为68pmol），这表明谷胱甘肽耗竭降低了谷胱甘肽氧化还原循环的周转率。用丁硫氨酸亚砜亚胺预处理17小时会夸大CHPO的负性变时作用：达到基线收缩频率50%所需的时间从无丁硫氨酸亚砜亚胺时的5.7±1.4分钟降至丁硫氨酸亚砜亚胺预处理后的3.7±0.4分钟（P<0.02）。通过丙二醛形成评估的CHPO诱导的脂质过氧化严重程度（最初20分钟内为2.23±0.51对0.99±0.05nmol；P<0.05）因丁硫氨酸亚砜亚胺预处理而增加，通过α-羟丁酸脱氢酶释放评估的细胞坏死程度（最初45分钟内为39.5±5.1对29.0±12.9%）也增加。10μM CHPO的“亚致死”剂量处理60分钟未导致大量HBDH释放、未形成丙二醛且细胞内谷胱甘肽未耗尽（35nmol/U HBDHt = 0）。然而，在用丁硫氨酸亚砜亚胺预处理后，10μM CHPO处理60分钟导致12%的HBDH释放和广泛的脂质过氧化（1.95nmol丙二醛/U HBDHt = 0）。由于CHPO的有害作用因谷胱甘肽耗竭而加剧，我们得出结论，谷胱甘肽氧化还原循环在保护心肌细胞免受过氧化物诱导的自由基攻击中起主要作用。
