Zucchi R, Yu G, Galbani P, Mariani M, Ronca G, Ronca-Testoni S
Scuola Superiore St Anna, Dipartimento di Scienze dell'Uomo e dell'Ambiente, University of Pisa, Italy.
Circ Res. 1998 Nov 2;83(9):908-15. doi: 10.1161/01.res.83.9.908.
We investigated the effect of sulfhydryl and disulfide reagents on ischemic preconditioning and on sarcoplasmic reticulum Ca2+ release. Isolated working rat hearts were subjected to ischemic preconditioning (three 3-minute periods of global ischemia) or to control aerobic perfusion, which was followed by 30 minutes of global ischemia and 120 minutes of retrograde reperfusion. Necrosis was evaluated on the basis of lactate dehydrogenase release and triphenyltetrazolium chloride staining. In parallel experiments, sarcoplasmic reticulum Ca2+ release and [3H]-ryanodine binding were determined before the sustained ischemia. Ischemic preconditioning was associated with protection versus ischemic injury, decreased Ca2+ release and reduced [3H]-ryanodine binding. The disulfide reducing agent dithiothreitol (1 mmol/L) removed the protection provided by ischemic preconditioning, if added to the perfusion buffer either before or after the preconditioning procedure. In preconditioned hearts, dithiothreitol increased sarcoplasmic reticulum Ca2+ release and ryanodine binding, whereas in control hearts it had no effect on either tissue injury or sarcoplasmic reticulum function. Perfusion of control hearts with the sulfhydryl blocking agents 4,4'-dithiodipyridine (25 micromol/L) and N-ethylmaleimide (16 micromol/L) increased the resistance to ischemia and reduced sarcoplasmic reticulum Ca2+ release and [3H]-ryanodine binding. These effects were not additive with those induced by preconditioning. Sulfhydryl and disulfide reagents produced similar effects on Ca2+ release and [3H]-ryanodine binding if added in vitro to preparations obtained from control and preconditioned hearts. We conclude that ischemic preconditioning is associated with the oxidation of sulfhydryl groups involved in the modulation of sarcoplasmic reticulum Ca2+ release.
我们研究了巯基和二硫键试剂对缺血预处理及肌浆网Ca2+释放的影响。将离体工作大鼠心脏进行缺血预处理（3次3分钟的全心缺血）或对照有氧灌注，随后进行30分钟全心缺血和120分钟逆行再灌注。根据乳酸脱氢酶释放和氯化三苯基四氮唑染色评估坏死情况。在平行实验中，在持续缺血前测定肌浆网Ca2+释放及[3H]-ryanodine结合。缺血预处理与对缺血损伤的保护作用、Ca2+释放减少及[3H]-ryanodine结合降低相关。二硫键还原剂二硫苏糖醇（1 mmol/L），若在预处理程序之前或之后添加到灌注缓冲液中，会消除缺血预处理提供的保护作用。在预处理的心脏中，二硫苏糖醇增加了肌浆网Ca2+释放和ryanodine结合，而在对照心脏中，它对组织损伤或肌浆网功能均无影响。用巯基阻断剂4,4'-二硫代二吡啶（25 μmol/L）和N-乙基马来酰亚胺（16 μmol/L）灌注对照心脏，增加了对缺血的耐受性，并减少了肌浆网Ca2+释放和[3H]-ryanodine结合。这些作用与预处理诱导的作用并非相加性的。如果在体外添加到从对照和预处理心脏获得的制剂中，巯基和二硫键试剂对Ca2+释放和[3H]-ryanodine结合产生相似的作用。我们得出结论，缺血预处理与参与调节肌浆网Ca2+释放的巯基氧化有关。
