Heinen André, Camara Amadou K S, Aldakkak Mohammed, Rhodes Samhita S, Riess Matthias L, Stowe David F
Anesthesiology Research Laboratories, Department of Anesthesiology, Medical College of Wisconsin, M4280, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
Am J Physiol Cell Physiol. 2007 Jan;292(1):C148-56. doi: 10.1152/ajpcell.00215.2006. Epub 2006 Jul 26.
We recently demonstrated a role for altered mitochondrial bioenergetics and reactive oxygen species (ROS) production in mitochondrial Ca(2+)-sensitive K(+) (mtK(Ca)) channel opening-induced preconditioning in isolated hearts. However, the underlying mitochondrial mechanism by which mtK(Ca) channel opening causes ROS production to trigger preconditioning is unknown. We hypothesized that submaximal mitochondrial K(+) influx causes ROS production as a result of enhanced electron flow at a fully charged membrane potential (DeltaPsi(m)). To test this hypothesis, we measured effects of NS-1619, a putative mtK(Ca) channel opener, and valinomycin, a K(+) ionophore, on mitochondrial respiration, DeltaPsi(m), and ROS generation in guinea pig heart mitochondria. NS-1619 (30 microM) increased state 2 and 4 respiration by 5.2 +/- 0.9 and 7.3 +/- 0.9 nmol O(2).min(-1).mg protein(-1), respectively, with the NADH-linked substrate pyruvate and by 7.5 +/- 1.4 and 11.6 +/- 2.9 nmol O(2).min(-1).mg protein(-1), respectively, with the FADH(2)-linked substrate succinate (+ rotenone); these effects were abolished by the mtK(Ca) channel blocker paxilline. DeltaPsi(m) was not decreased by 10-30 microM NS-1619 with either substrate, but H(2)O(2) release was increased by 44.8% (65.9 +/- 2.7% by 30 muM NS-1619 vs. 21.1 +/- 3.8% for time controls) with succinate + rotenone. In contrast, NS-1619 did not increase H(2)O(2) release with pyruvate. Similar results were found for lower concentrations of valinomycin. The increase in ROS production in succinate + rotenone-supported mitochondria resulted from a fully maintained DeltaPsi(m), despite increased respiration, a condition that is capable of allowing increased electron leak. We propose that mild matrix K(+) influx during states 2 and 4 increases mitochondrial respiration while maintaining DeltaPsi(m); this allows singlet electron uptake by O(2) and ROS generation.
我们最近证明了线粒体生物能量学改变和活性氧（ROS）生成在离体心脏中线粒体钙敏感钾（mtK(Ca)）通道开放诱导的预处理中的作用。然而，mtK(Ca)通道开放导致ROS生成从而触发预处理的潜在线粒体机制尚不清楚。我们假设，在膜电位完全充电（ΔΨm）时，亚最大线粒体钾内流由于电子流增强而导致ROS生成。为了验证这一假设，我们测量了假定的mtK(Ca)通道开放剂NS-1619和钾离子载体缬氨霉素对豚鼠心脏线粒体呼吸、ΔΨm和ROS生成的影响。NS-1619（30 μM）分别使以NADH连接底物丙酮酸时的状态2和状态4呼吸增加5.2±0.9和7.3±0.9 nmol O₂·min⁻¹·mg蛋白⁻¹，以FADH₂连接底物琥珀酸（+鱼藤酮）时分别增加7.5±1.4和11.6±2.9 nmol O₂·min⁻¹·mg蛋白⁻¹；这些作用被mtK(Ca)通道阻滞剂放线菌酮消除。10 - 30 μM NS-1619在两种底物存在时均未降低ΔΨm，但在琥珀酸+鱼藤酮存在时，H₂O₂释放增加了44.8%（30 μM NS-1619时为65.9±2.7%，而时间对照为21.1±3.8%）。相比之下，NS-1619在丙酮酸存在时未增加H₂O₂释放。较低浓度的缬氨霉素也得到了类似结果。在琥珀酸+鱼藤酮支持的线粒体中ROS生成增加是由于尽管呼吸增加但ΔΨm完全维持，这种情况能够允许增加的电子泄漏。我们提出，在状态2和状态4期间轻度的基质钾内流增加线粒体呼吸同时维持ΔΨm；这允许氧气摄取单电子并生成ROS。
