CELLULAR REDUCING EQUIVALENTS AND OXIDATIVE STRESS

细胞和组织抵御氧化应激的能力是 由还原当量的供应决定。我们的数据表明 细胞的能量水平并不直接关系到 心脏组织提供还原当量，以响应氧化 应力然而，这种供应所涉及的途径仍然不清楚， 特别是如果完成还原当量的反应，例如 ATP合成活跃。心脏研究之所以有趣，是因为 这个器官持续的高呼吸，以及氧化的可能性， 梗死或手术后再灌注期间的损伤。谷胱甘肽 (GSH)是细胞抗氧化系统的主要组成部分。gsh是 通过谷胱甘肽还原酶保持还原态。虽然这 酶是特异性的NADPH，完整细胞的能力，分离 线粒体（线粒体是自由基的主要来源， 抗氧化系统独立于细胞的其余部分），和整个 组织提供还原当量和维持正常水平的GSH 似乎涉及到NADH。待检验的具体假设是： 用于还原外源性氧化剂的还原当量可以 由NADH连接的底物提供，2）还原当量的供应 防御氧化应激的能力在状态4下增强， 在状态3呼吸下减少，3）完整的器官可以供应 更有效地减少对氧化应激反应的当量 而不是孤立的细胞。这些假设的有效性将通过以下方式进行检验： 使用完整的心脏组织，分离的心脏线粒体， 心肌细胞，提供有以下能量相关底物： 葡萄糖、乙酸盐、乳酸盐或丙酮酸盐（完整心脏和心肌细胞）; 苹果酸盐/谷氨酸盐、琥珀酸盐或辛酸盐（线粒体）。在这些 ATP、磷酸肌酸、NAD、NADH、NADP和NADPH的含量 将被确定，并与GSH，GSSH，蛋白质 硫醇和蛋白质GSH混合二硫化物。氧化的影响 用二酰胺（直接硫醇氧化剂）或叔丁基 氢过氧化物将被测定。减少的途径 提供的等同物将通过提供 不同的能量连接的底物在抑制剂的存在下， 呼吸（鱼藤酮，抗霉素A或FCCP [羰基氰对- 三氟甲氧基苯腙]）。线粒体底物和抑制剂 研究将在状态4（无ADP）和状态3（有ADP）下进行。 ADP）呼吸。这些数据将提供重要的新信息， 组织提供还原当量的途径，并将揭示 是否存在差异，这似乎存在于一个完整的器官和 分离的细胞是真实的。这些结果可能具有重要的临床意义。 在制定战略以加强 组织保护自身免受氧化应激诱导的能力 通过再灌注或外源性物质。

The ability of cells and tissues to defend against oxidative stress is determined by the supply of reducing equivalents. Our data demonstrate that cellular energy levels are not directly related to the ability of heart tissue to supply reducing equivalents in response to an oxidative stress. However, the pathways involved in this supply remain unclear, particularly if reactions which complete for reducing equivalents, such as ATP synthesis, are active. Studies in heart are of interest because of the sustained high respiration of this organ, and the potential for oxidative injury during reperfusion subsequent to infarction or surgery. Glutathione (GSH) is a major component of cellular antioxidant systems. GSH is maintained in the reduced form by glutathione reductase. Although this enzyme is specific for NADPH, the ability of intact cells, isolated mitochondria (which are a major source of free radicals and contain antioxidant systems independent of the rest of the cell), and whole tissues to supply reducing equivalents and maintain normal levels of GSH appears to involve NADH. The specific hypotheses to be tested are that: l) reducing equivalents used to reduce exogenous oxidizing agents can be supplied by NADH-linked substrates, 2) the supply of reducing equivalents to defend against oxidative stress is augmented under state 4 and diminished under state 3 respiration, and 3) intact organs can supply reducing equivalents in response to an oxidative stress more effectively than isolated cells. The validity of these hypotheses will be tested by using intact heart tissue, isolated cardiac mitochondria and cardiomyocytes provided with the following energy-linked substrates: glucose, acetate, lactate or pyruvate (intact heart and cardiomyocytes); malate/glutamate, succinate or octanoate (mitochondria). In each of these systems, the contents of ATP, phosphocreatine, NAD, NADH, NADP, and NADPH will be determined and correlated with levels of GSH, GSSH, protein thiols, and protein GSH-mixed disulfides. The effect of oxidatively stressing these systems with diamide (a direct thiol oxidant) or t-butyl hydroperoxide will be determined. The pathways by which reducing equivalents are supplied will be further assessed by providing the different energy-linked substrates in the presence of inhibitors of respiration (rotenone, antimycin A or FCCP [carbonyl cyanide p- trifluoromethoxyphenylhydrazone]). Mitochondrial substrate and inhibitor studies will be performed in both state 4 (without ADP) and state 3 (with ADP) respiration. These data will provide important new information on the pathways used by tissues to supply reducing equivalents and will reveal whether differences, which appear to exist between an intact organ and isolated cells, are real. The results may have important clinical relevance in the context of designing strategies for augmenting the ability of tissues to defend themselves against oxidative stress induced by reperfusion or xenobiotics.