Morris T E, Sulakhe P V
Department of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, Canada.
Free Radic Biol Med. 1997;22(1-2):37-47. doi: 10.1016/s0891-5849(96)00238-9.
The effects of hydroxyl radical exposure of intact cardiomyocytes on sarcoplasmic reticulum (SR) function were investigated. For this purpose, isolated rat heart myocytes were exposed briefly (1 min) to the hydroxyl radical generating system (H2O2/FeCl2 or FeSO4) or 5-5'-dithiobis-nitrobenzoic acid (DTNB), a sulfhydryl oxidizing reagent, and following this a SR-enriched fraction was isolated. Marked decreases in the SR calcium uptake activities were seen in the myocytes exposed to either the hydroxyl radical-generating system or DTNB. The exposure of myocytes to the hydroxyl radical, but not DTNB, markedly increased the amount of malonyldialdehyde (MDA) in the subsequently isolated SR. Total sulfhydryl group content in SR was decreased by exposure of myocytes to DTNB. Further, there was a significant decrease in [3H]-NEM binding to SR isolated from the hydoxyl radical-treated myocytes indicating that sulfhydryl groups are affected (oxidized). Both mannitol and catalase were found to offer complete protection against the inhibitory effect of peroxide +/- iron on calcium uptake. Also the above-mentioned alterations in both MDA and sulfhydryl group content were prevented by mannitol and catalase. Exogenously added cyclic AMP-dependent protein kinase (A-PK) or calmodulin (CAM) increased SR calcium uptake activity. In the SR isolated from the treated myocytes, the stimulatory effects of A-PK and CAM were also seen, although under all assay conditions calcium uptakes were of lower magnitude. The findings are consistent with the view that the damaging effect of the hydroxyl radical and DTNB on the functioning of SR occurs rapidly in the intact cardiomyocytes. The hydroxyl radical-provoked damage involves both protein sulfhydryl and lipid oxidation.
研究了完整心肌细胞暴露于羟基自由基对肌浆网（SR）功能的影响。为此，将分离的大鼠心脏肌细胞短暂（1分钟）暴露于羟基自由基生成系统（H2O2/FeCl2或FeSO4）或5-5'-二硫代双硝基苯甲酸（DTNB，一种巯基氧化试剂），然后分离富含SR的部分。暴露于羟基自由基生成系统或DTNB的肌细胞中，SR钙摄取活性显著降低。肌细胞暴露于羟基自由基而非DTNB后，在随后分离的SR中丙二醛（MDA）含量显著增加。肌细胞暴露于DTNB会降低SR中的总巯基含量。此外，从羟基自由基处理的肌细胞中分离的SR与[3H]-NEM的结合显著减少，表明巯基受到影响（氧化）。发现甘露醇和过氧化氢酶都能完全保护细胞免受过氧化氢+/-铁对钙摄取的抑制作用。甘露醇和过氧化氢酶还能防止上述MDA和巯基含量的变化。外源添加的环磷酸腺苷依赖性蛋白激酶（A-PK）或钙调蛋白（CAM）可增加SR钙摄取活性。在从处理过的肌细胞中分离的SR中，也观察到了A-PK和CAM的刺激作用，尽管在所有测定条件下钙摄取量都较低。这些发现与以下观点一致：羟基自由基和DTNB对SR功能的破坏作用在完整心肌细胞中迅速发生。羟基自由基引发的损伤涉及蛋白质巯基和脂质氧化。
