Camara Amadou K S, Riess Matthias L, Kevin Leo G, Novalija Enis, Stowe David F
Department of Anesthesiology, The Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Am J Physiol Heart Circ Physiol. 2004 Apr;286(4):H1289-99. doi: 10.1152/ajpheart.00811.2003. Epub 2003 Nov 26.
Hypothermic perfusion of the heart decreases oxidative phosphorylation and increases NADH. Because O(2) and substrates remain available and respiration (electron transport system, ETS) may become impaired, we examined whether reactive oxygen species (ROS) exist in excess during hypothermic perfusion. A fiberoptic probe was placed on the left ventricular free wall of isolated guinea pig hearts to record intracellular ROS, principally superoxide (O(2)(-).), and an extracellular reactive nitrogen reactant, principally peroxynitrite (ONOO(-)), a product of nitric oxide (NO.) + O(2)(-). Hearts were loaded with dihydroethidium (DHE), which is oxidized by O(2)(-). to ethidium, or were perfused with l-tyrosine, which is oxidized by ONOO(-) to dityrosine (diTyr). Shifts in fluorescence were measured online; diTyr fluorescence was also measured in the coronary effluent. To validate our methods and to examine the source and identity of ROS during cold perfusion, we examined the effects of a superoxide dismutase mimetic Mn(III) tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), the nitric oxide synthase inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME), and several agents that impair electron flux through the ETS: menadione, sodium azide (NaN(3)), and 2,3-butanedione monoxime (BDM). Drugs were given before or during cold perfusion. ROS measured by DHE was inversely proportional to the temperature between 37 degrees C and 3 degrees C. We found that perfusion at 17 degrees C increased DHE threefold versus perfusion at 37 degrees C; this was reversed by MnTBAP, but not by l-NAME or BDM, and was markedly augmented by menadione and NaN(3). Perfusion at 17 degrees C also increased myocardial and effluent diTyr (ONOO(-)) by twofold. l-NAME, MnTBAP, or BDM perfused at 37 degrees C before cooling or during 17 degrees C perfusion abrogated, whereas menadione and NaN(3) again enhanced the cold-induced increase in ROS. Our results suggest that hypothermia moderately enhances O(2)(-). generation by mitochondria, whereas O(2)(-). dismutation is markedly slowed. Also, the increase in O(2)(-). during hypothermia reacts with available NO. to produce ONOO(-), and drug-induced O(2)(-). dismutation eliminates the hypothermia-induced increase in O(2)(-).
心脏低温灌注会降低氧化磷酸化并增加NADH。由于氧气和底物仍然存在，且呼吸作用（电子传递系统，ETS）可能受损，我们研究了在低温灌注过程中活性氧（ROS）是否会过量存在。将光纤探头置于离体豚鼠心脏的左心室游离壁上，以记录细胞内ROS，主要是超氧阴离子（O₂⁻），以及细胞外活性氮反应物，主要是过氧亚硝酸盐（ONOO⁻），它是一氧化氮（NO·）与O₂⁻的产物。心脏用二氢乙锭（DHE）负载，DHE会被O₂⁻氧化为乙锭，或者用L-酪氨酸灌注，L-酪氨酸会被ONOO⁻氧化为二酪氨酸（diTyr）。在线测量荧光变化；还测量了冠状动脉流出液中的二酪氨酸荧光。为了验证我们的方法并研究冷灌注过程中ROS的来源和特性，我们研究了超氧化物歧化酶模拟物四（4-苯甲酸）锰（III）氯化卟啉（MnTBAP）、一氧化氮合酶抑制剂N⁰-硝基-L-精氨酸甲酯（L-NAME）以及几种会损害通过ETS的电子通量的试剂的作用：甲萘醌、叠氮化钠（NaN₃）和2,3-丁二酮单肟（BDM）。在冷灌注前或灌注过程中给予药物。用DHE测量的ROS在37℃至3℃之间与温度成反比。我们发现，与37℃灌注相比，17℃灌注使DHE增加了三倍；MnTBAP可使其逆转，但L-NAME或BDM则不能，而甲萘醌和NaN₃会使其显著增加。17℃灌注还使心肌和流出液中的二酪氨酸（ONOO⁻）增加了两倍。在冷却前于37℃灌注或在17℃灌注过程中灌注L-NAME、MnTBAP或BDM可消除这种影响，而甲萘醌和NaN₃再次增强了冷诱导的ROS增加。我们的结果表明，低温会适度增强线粒体产生O₂⁻的能力，而O₂⁻的歧化作用则明显减慢。此外，低温期间O₂⁻的增加会与可用的NO·反应生成ONOO⁻，药物诱导的O₂⁻歧化作用消除了低温诱导的O₂⁻增加。
