Stoyanovsky D A, Salama G, Kagan V E
Department of Environmental and Occupational Health, University of Pittsburgh, Pennsylvania 15238.
Arch Biochem Biophys. 1994 Jan;308(1):214-21. doi: 10.1006/abbi.1994.1030.
Ca(2+)-release channel or ryanodine receptor is known to be involved in physiologic Ca(2+)-release from sarcoplasmic reticulum in skeletal and cardiac muscle. A variety of chemical oxidants and in particular SH-oxidizing reagents have been shown to activate Ca2+ release. However, the role of the oxidative modification of the channel in the physiologic mechanism(s) of Ca2+ release and in pathologic states of the muscle remains to be elucidated. Ascorbate/iron redox couple is known to be an efficient generator of oxygen radicals and semidehydroascorbyl radicals. Ascorbate/iron was shown to be released from cardiomyocytes during ischemia-reperfusion and was suggested to be involved in the ischemia-reperfusion injury and cardiomyocyte death. To understand the potential contribution of ascorbate/iron to Ca2+ release mechanism(s), calcium release channels from skeletal sarcoplasmic reticulum (SR) were reconstituted in artificial planar bilayers to examine the effects of this redox couple on the channel activity. Ascorbate elicited a transient (about 2 min) but dramatic increase of open-time probability of the channel. At pCa = 7.0, the presence of EGTA blocked ascorbate induced activation of release channels. However, when exogenous iron was added, ascorbate activated Ca2+ release channels, even in the presence of EGTA. ESR measurements demonstrated that semidehydroascorbyl radicals were generated from ascorbate in the absence of EGTA. The semidehydroascorbyl radical ESR signal was quenched by EGTA in the absence (but not in the presence) of exogenous iron. Thus, the production of ascorbyl radicals was associated with increased channel activity. In the presence of heparin, ascorbate plus iron elicited a long-lasting activation of the channel which had conductance gCa2+ = 100 pS characteristic for the ryanodine receptor and which could be blocked by the ryanodine channel inhibitor, ruthenium red. In conclusion the physiologically relevant redox couple--ascorbate/iron--at physiologic concentrations can activate Ca2+ channels in sarcoplasmic reticulum vesicles.
已知钙离子释放通道或雷诺丁受体参与骨骼肌和心肌肌浆网的生理性钙离子释放。多种化学氧化剂，尤其是巯基氧化试剂，已被证明可激活钙离子释放。然而，通道的氧化修饰在钙离子释放的生理机制以及肌肉病理状态中的作用仍有待阐明。抗坏血酸/铁氧化还原对是已知的氧自由基和半脱氢抗坏血酸自由基的有效生成剂。抗坏血酸/铁在缺血再灌注期间从心肌细胞中释放出来，并被认为与缺血再灌注损伤和心肌细胞死亡有关。为了了解抗坏血酸/铁对钙离子释放机制的潜在贡献，将骨骼肌肌浆网（SR）的钙释放通道重建于人工平面双层膜中，以研究该氧化还原对通道活性的影响。抗坏血酸引起通道开放时间概率短暂（约2分钟）但显著增加。在pCa = 7.0时，EGTA的存在可阻断抗坏血酸诱导的释放通道激活。然而，当加入外源性铁时，即使存在EGTA，抗坏血酸仍可激活钙离子释放通道。电子自旋共振（ESR）测量表明，在不存在EGTA的情况下，抗坏血酸会生成半脱氢抗坏血酸自由基。在不存在（但存在）外源性铁的情况下，EGTA可淬灭半脱氢抗坏血酸自由基的ESR信号。因此，抗坏血酸自由基的产生与通道活性增加相关。在肝素存在的情况下，抗坏血酸加铁可引起通道的持久激活，该通道具有雷诺丁受体特征性的钙离子电导gCa2+ = 100 pS，且可被雷诺丁通道抑制剂钌红阻断。总之，生理浓度下具有生理相关性的氧化还原对——抗坏血酸/铁——可激活肌浆网小泡中的钙离子通道。
