Division of Cardiothoracic Surgery, The Ohio State University Medical Center, Columbus, Ohio 43210, USA.
J Surg Res. 2011 Jan;165(1):5-14. doi: 10.1016/j.jss.2010.09.018. Epub 2010 Oct 13.
Proton leak (H(+) leak) dissipates mitochondrial membrane potential (mΔΨ) through the re-entry of protons into the mitochondrial matrix independent of ATP synthase. Changes in H(+) leak may affect reactive oxygen species (ROS) production. We measured H(+) leak and ROS production during ischemia-reperfusion and ischemic preconditioning (IPC) and examined how changing mitochondrial respiration affected mΔΨ and ROS production.
Isolated rat hearts (n = 6/group) were subjected to either control-IR or IPC. Rate pressure product (RPP) was measured. Mitochondria were isolated at end reperfusion. Respiration was measured by polarography and titrated with increasing concentrations of malonate (0.5-2 mM). mΔΨ was measured using a tetraphenylphosphonium electrode. H(+) leak is the respiratory rate required to maintain membrane potential at -150 mV in the presence of oligomycin-A. Mitochondrial complex III ROS production was measured by fluorometry using Amplex-red.
IPC improved recovery of RPP at end reperfusion (63% ± 4% versus 21% ± 2% in control-IR, P < 0.05). Ischemia-reperfusion caused increased H(+) leak (94 ± 12 versus 31 ± 1 nmol O/mg protein/min in non-ischemic control, P < 0.05). IPC attenuates these increases (55 ± 9 nmol O/mg protein/min, P < 0.05 versus control-IR). IPC reduced mitochondrial ROS production compared with control-IR (31 ± 2 versus 40 ± 3 nmol/mg protein/min, P < 0.05). As mitochondrial respiration decreased, mΔΨ and mitochondrial ROS production also decreased. ROS production remained lower in IPC than in control-IR for all mΔΨ and respiration rates.
Increasing H(+) leak is not associated with decreased ROS production. IPC decreases both the magnitude of H(+) leak and ROS production after ischemia-reperfusion.
质子泄漏（H+泄漏）通过质子独立于 ATP 合酶重新进入线粒体基质来耗散线粒体膜电位（mΔΨ）。H+泄漏的变化可能会影响活性氧（ROS）的产生。我们在缺血再灌注和缺血预处理（IPC）期间测量了 H+泄漏和 ROS 的产生，并研究了改变线粒体呼吸如何影响 mΔΨ和 ROS 的产生。
分离的大鼠心脏（每组 n=6）分别进行对照-IR 或 IPC。测量心率压力产物（RPP）。在再灌注末期分离线粒体。通过极谱法测量呼吸，并通过增加丙二酸浓度（0.5-2 mM）滴定。使用四苯基膦电极测量 mΔΨ。在寡霉素 A 存在下，维持膜电位为-150 mV 所需的呼吸速率为 H+泄漏。通过使用 Amplex-red 的荧光法测量线粒体复合物 III ROS 的产生。
IPC 改善了再灌注末期 RPP 的恢复（对照-IR 为 63%±4%，而非缺血对照为 21%±2%，P<0.05）。缺血再灌注导致 H+泄漏增加（与非缺血对照相比为 94±12 与 31±1 nmol O/mg 蛋白/min，P<0.05）。IPC 减轻了这些增加（55±9 nmol O/mg 蛋白/min，P<0.05 与对照-IR 相比）。与对照-IR 相比，IPC 减少了线粒体 ROS 的产生（31±2 与 40±3 nmol/mg 蛋白/min，P<0.05）。随着线粒体呼吸的降低，mΔΨ 和线粒体 ROS 的产生也降低。在所有 mΔΨ 和呼吸速率下，IPC 中的 ROS 产生仍然低于对照-IR。
增加 H+泄漏与 ROS 产生减少无关。IPC 可降低缺血再灌注后 H+泄漏和 ROS 产生的幅度。
