Penna Claudia, Mancardi Daniele, Rastaldo Raffaella, Losano Gianni, Pagliaro Pasquale
Dipartimento di Scienze Cliniche e Biologiche, dell'Università di Torino, Italy.
Cardiovasc Res. 2007 Jul 1;75(1):168-77. doi: 10.1016/j.cardiores.2007.03.001. Epub 2007 Mar 12.
Postconditioning (PostC) maneuvers allow post-ischemic accumulation of autacoids, which trigger protection. We tested if PostC-triggering includes bradykinin (BK) B2 receptor activation and its downstream pathway.
Isolated rat hearts underwent 30 min ischemia and 120 min reperfusion. Infarct size was evaluated using nitro-blue tetrazolium staining. In Control hearts infarct size was 61+/-5% of risk area. PostC (5 cycles of 10 s reperfusion/ischemia) reduced infarct size to 22+/-4% (p<0.01). PostC protection was abolished by B2 BK receptor-antagonists (HOE140 or WIN64338), nitric oxide synthase-inhibitor (L-nitro-arginine-methylester), protein kinase G (PKG)-blocker (8-bromoguanosine-3',5'-cyclic-monophosphorothioate), and mitochondrial K(ATP) (mK(ATP))-blocker (5-hydroxydecanoate) each given for 3 min only. Since 3 min of BK-infusion (100 nM) did not reproduce PostC protection, protocols with Intermittent-BK infusion were used to mimic PostC: a) 5 cycles of 10 s oxygenated-no-BK/oxygenated+BK buffer; b) 5 cycles of 10 s oxygenated-no-BK/hypoxic+BK buffer. Both protocols with Intermittent-BK attenuated infarct size (36+/-5% and 38+/-4%, respectively; p<0.05 vs Control and NS vs PostC for both; NS vs each other). Intermittent-BK protection was abolished by the same antagonists used to prevent PostC protection. Intermittence of re-oxygenation only (5 cycles of 10 s oxygenated/hypoxic buffer) did not reproduce PostC. Yet, cardioprotection was triggered by intermittent mK(ATP) activation with diazoxide, but not by intermittent reactive oxygen species (ROS) generation with purine/xanthine oxidase. ROS scavengers (N-acetyl-L-cysteine or 2-mercaptopropionylglycine), given for 3 min only, abolished PostC-, Intermittent BK-and diazoxide-induced protection.
Intermittent targeting of specific cellular sites (i.e. BK B2 receptors and mK(ATP) channels) during early reperfusion triggers PostC protection via ROS signaling. Since neither intermittent oxygenation nor exogenous ROS generators can trigger protection, it is likely that intermittent autacoid accumulation and ROS compartmentalization may play a pivotal role in PostC-triggering.
后适应（PostC）操作可使缺血后内源性生物活性物质蓄积，从而触发心肌保护作用。我们测试了PostC触发机制是否包括缓激肽（BK）B2受体激活及其下游信号通路。
将离体大鼠心脏进行30分钟缺血和120分钟再灌注。采用硝基蓝四氮唑染色评估梗死面积。在对照组心脏中，梗死面积为危险区的61±5%。PostC（10秒再灌注/缺血的5个周期）使梗死面积降至22±4%（p<0.01）。B2 BK受体拮抗剂（HOE140或WIN64338）、一氧化氮合酶抑制剂（L-硝基精氨酸甲酯）、蛋白激酶G（PKG）阻滞剂（8-溴鸟苷-3',5'-环一磷酸硫代酯）和线粒体ATP敏感性钾通道（mK(ATP)）阻滞剂（5-羟基癸酸）仅给药3分钟即可消除PostC的保护作用。由于输注BK（100 nM）3分钟不能重现PostC的保护作用，因此采用间歇性BK输注方案来模拟PostC：a）10秒有氧无BK/有氧加BK缓冲液的5个周期；b）10秒有氧无BK/缺氧加BK缓冲液的5个周期。两种间歇性BK方案均减小了梗死面积（分别为36±5%和38±4%；与对照组相比p<0.05，与PostC相比均无统计学差异；两者之间无统计学差异）。间歇性BK的保护作用被用于预防PostC保护作用的相同拮抗剂所消除。仅再给氧的间歇性操作（10秒有氧/缺氧缓冲液的5个周期）不能重现PostC。然而，用二氮嗪间歇性激活mK(ATP)可触发心脏保护作用，但用嘌呤/黄嘌呤氧化酶间歇性产生活性氧（ROS）则不能。仅给药3分钟的ROS清除剂（N-乙酰-L-半胱氨酸或2-巯基丙酰甘氨酸）可消除PostC、间歇性BK和二氮嗪诱导的保护作用。
在早期再灌注期间间歇性靶向特定细胞位点（即BK B2受体和mK(ATP)通道）可通过ROS信号通路触发PostC保护作用。由于间歇性给氧和外源性ROS生成剂均不能触发保护作用，因此间歇性内源性生物活性物质蓄积和ROS区室化可能在PostC触发过程中起关键作用。
