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Camara AK, Riess ML, Kevin LG, Novalija E, Stowe DF: Hypothermia augments reactive oxygen species detected in the guinea pig isolated perfused heart. Am J Physiol Heart Circ Physiol. 2004 Apr;286(4):H1289-99. 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)(-). |
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