Lund L G, Paraidathathu T, Kehrer J P
Division of Pharmacology and Toxicology, College of Pharmacy, University of Texas at Austin 78712-1074.
Toxicology. 1994 Nov 11;93(2-3):249-62. doi: 10.1016/0300-483x(94)90082-5.
A tissue's response to an oxidative stress is related to its capacity to supply reducing equivalents and may be affected by energy levels. The ability of intact rat heart tissue to supply NADPH and reduce glutathione disulfide (GSSG) produced by diamide was determined under normoxic or hypoxic conditions with and without glycolytic energy production. Cardiac ATP and phosphocreatine (PCr) levels remained relatively constant (approximately 20 nmol/mg dry weight) during a 60 min perfusion with oxygenated Krebs-Henseleit buffer containing glucose. Levels of ATP and PCr were depleted 85-92% following 60 min of hypoxia. A 5 min infusion of 800 microM diamide, after 60 min of normoxia or hypoxia, oxidized 70-80% of cardiac glutathione (GSH), but had no effect on total glutathione. After a subsequent 25 min diamidefree perfusion, 75-85% of the GSSG formed was reduced in both normoxic and hypoxic hearts. The removal of glucose, or the inhibition of glycolysis with 2-deoxy-D-glucose, did not affect GSSG reduction. Cardiac NADH levels were increased from 0.05 to 0.5 nmol/mg dry weight after 60 min hypoxia in hearts perfused with or without glucose. A 5 min infusion of diamide in hypoxic hearts slightly decreased NADH levels, but there was no further change after a subsequent 25 min diamide-free period. Inhibition of glutathione reductase with 1,3-bis(2-chloroethyl)-1-nitrosourea prevented GSSG reduction, showing NADPH was required. However, NADPH levels were not affected by hypoxia or diamide infusion and remained constant at 0.2 nmol/mg dry weight in hearts perfused with or without glucose. Inhibition of glycolysis with 2-deoxy-D-glucose also did not affect NADPH levels. These results demonstrate that hypoxia did not affect the ability of oxidatively stressed, intact heart tissue to supply NADPH for the reduction of GSSG. In addition, GSSG reduction was independent of energy levels and appeared to be unaffected by glucose availability. NADH may be involved in maintaining NADPH levels through interconversion pathways.
组织对氧化应激的反应与其提供还原当量的能力有关，并且可能受到能量水平的影响。在有氧或缺氧条件下，在有或没有糖酵解能量产生的情况下，测定完整大鼠心脏组织提供NADPH和还原由二酰胺产生的谷胱甘肽二硫化物（GSSG）的能力。在用含葡萄糖的充氧Krebs-Henseleit缓冲液灌注60分钟期间，心脏ATP和磷酸肌酸（PCr）水平保持相对恒定（约20 nmol/mg干重）。缺氧60分钟后，ATP和PCr水平降低了85-92%。在常氧或缺氧60分钟后，输注5分钟800 microM二酰胺，氧化了70-80%的心脏谷胱甘肽（GSH），但对总谷胱甘肽没有影响。在随后25分钟无二酰胺灌注后，常氧和缺氧心脏中形成的GSSG的75-85%被还原。去除葡萄糖或用2-脱氧-D-葡萄糖抑制糖酵解，并不影响GSSG的还原。在有或没有葡萄糖灌注的心脏中，缺氧60分钟后，心脏NADH水平从0.05增加到0.5 nmol/mg干重。在缺氧心脏中输注5分钟二酰胺会使NADH水平略有下降，但在随后25分钟无二酰胺期间没有进一步变化。用1,3-双（2-氯乙基）-1-亚硝基脲抑制谷胱甘肽还原酶可阻止GSSG还原，表明需要NADPH。然而，NADPH水平不受缺氧或二酰胺输注的影响，在有或没有葡萄糖灌注的心脏中保持恒定，为0.2 nmol/mg干重。用2-脱氧-D-葡萄糖抑制糖酵解也不影响NADPH水平。这些结果表明，缺氧并不影响氧化应激的完整心脏组织提供NADPH以还原GSSG的能力。此外，GSSG的还原与能量水平无关，似乎不受葡萄糖可用性的影响。NADH可能通过相互转化途径参与维持NADPH水平。
