Kukreja R C, Kearns A A, Zweier J L, Kuppusamy P, Hess M L
Cardiology Division, Medical College of Virginia, Richmond 23298.
Circ Res. 1991 Oct;69(4):1003-14. doi: 10.1161/01.res.69.4.1003.
We investigated the role of singlet oxygen (generated from photoactivation of rose bengal) on the calcium transport and Ca(2+)-ATPase activity of cardiac sarcoplasmic reticulum (SR). Isolated cardiac SR exposed to rose bengal (10 nM) irradiated at 560 nm resulted in significant inhibition of Ca2+ uptake (from 2.27 +/- 0.05 to 0.62 +/- 0.05 mumol Ca2+/mg.min [mean +/- SEM], p less than 0.01) and Ca(2+)-ATPase activity (from 2.08 +/- 0.05 to 0.28 +/- 0.04 mumol Pi/min.mg [mean +/- SEM], p less than 0.01). The inhibition of calcium uptake and Ca(2+)-ATPase activity by rose bengal-derived activated oxygen (singlet oxygen) was dependent on the duration of exposure and intensity of light. Singlet oxygen scavengers ascorbic acid and histidine significantly protected SR Ca(2+)-ATPase against rose bengal-derived activated oxygen species, but superoxide dismutase and catalase did not attenuate the inhibition. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of SR exposed to photoactivated rose bengal for up to 14 minutes demonstrated complete loss of the Ca(2+)-ATPase monomer band, which was significantly protected by histidine. The addition of dithiothreitol (5 mM) had a slight protective effect, showing that new disulfide bond formation was not a major cause of aggregation. The results were also confirmed by high-performance liquid chromatography of the SR exposed to irradiated rose bengal. Irradiation of rose bengal also caused an 18% loss of total sulfhydryl groups of SR. On the other hand, superoxide radical (generated from xanthine oxidase action on xanthine) and hydroxyl radical (in the presence of Fe(3+)-EDTA or 0.5 mM H2O2 plus Fe(2+)-EDTA) as well as H2O2 (0.25-12 mM) were without any effect on the 97,000-d Ca(2+)-ATPase band of SR. Generation of radical species (superoxide and hydroxyl radical) from rose bengal was studied by electron paramagnetic resonance spectroscopy using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The results showed that irradiation of rose bengal formed a 1:2:2:1 quartet, characteristic of the DMPO-OH adduct, which was scavenged by ethanol but not by superoxide dismutase, catalase, or histidine. No radical species could be detected from irradiated rose bengal or irradiated DMPO under the assay conditions used. Peroxy adducts of DMPO might be produced but would be observed only at very low temperatures. Similarly, we could not detect any measurable.O2- anion from irradiation of rose bengal as indicated by either cytochrome c reduction at 550 nm or nitro blue tetrazolium reduction at 560 nm. These results show that SR is damaged most likely by singlet oxygen derived from rose bengal.(ABSTRACT TRUNCATED AT 400 WORDS)
我们研究了孟加拉玫瑰红光激活产生的单线态氧对心肌肌浆网（SR）钙转运及Ca(2+)-ATP酶活性的作用。分离出的心肌SR暴露于560nm照射的10nM孟加拉玫瑰红中，导致Ca2+摄取显著受抑（从2.27±0.05降至0.62±0.05μmol Ca2+/mg·min[平均值±标准误]，p<0.01）以及Ca(2+)-ATP酶活性受抑（从2.08±0.05降至0.28±0.04μmol Pi/min·mg[平均值±标准误]，p<0.01）。孟加拉玫瑰红衍生的活性氧（单线态氧）对钙摄取及Ca(2+)-ATP酶活性的抑制作用取决于暴露时间和光照强度。单线态氧清除剂抗坏血酸和组氨酸能显著保护SR的Ca(2+)-ATP酶免受孟加拉玫瑰红衍生的活性氧物种的影响，但超氧化物歧化酶和过氧化氢酶不能减轻这种抑制作用。对暴露于光激活的孟加拉玫瑰红长达14分钟的SR进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示，Ca(2+)-ATP酶单体条带完全消失，而组氨酸能显著保护该条带。加入二硫苏糖醇（5mM）有轻微保护作用，表明新的二硫键形成不是聚集的主要原因。暴露于照射后的孟加拉玫瑰红的SR的高效液相色谱分析也证实了这些结果。孟加拉玫瑰红的照射还导致SR总巯基基团损失18%。另一方面，黄嘌呤氧化酶作用于黄嘌呤产生的超氧自由基、（在Fe(3+)-EDTA存在下或0.5mM H2O2加Fe(2+)-EDTA时产生的）羟自由基以及H2O2（0.25 - 12mM）对SR的97,000道尔顿的Ca(2+)-ATP酶条带均无任何影响。使用自旋捕捉剂5,5 - 二甲基 - 1 - 吡咯啉 - N - 氧化物（DMPO）通过电子顺磁共振光谱研究了孟加拉玫瑰红产生的自由基物种（超氧自由基和羟自由基）。结果表明，孟加拉玫瑰红的照射形成了一个1:2:2:1的四重峰，这是DMPO - OH加合物的特征，它可被乙醇清除，但不能被超氧化物歧化酶、过氧化氢酶或组氨酸清除。在所使用的检测条件下，照射后的孟加拉玫瑰红或照射后的DMPO均未检测到任何自由基物种。DMPO的过氧加合物可能会产生，但仅在非常低的温度下才能观察到。同样，通过550nm处细胞色素c还原或560nm处硝基蓝四唑还原表明，我们未检测到孟加拉玫瑰红照射产生的任何可测量的超氧阴离子。这些结果表明，SR很可能被孟加拉玫瑰红衍生的单线态氧所损伤。（摘要截短至400字）
