Abstract
The aim of this study was to test the hypothesis that a decreased myocardial concentration of reduced glutathione (GSH) during ischemia renders the myocardium more susceptible to injury by reactive oxygen species generated during early reperfusion. To this end, rats were pretreated with L-buthionine-S,R-sulfoximine (2 mmol/kg), which depleted myocardial GSH by 55%. Isolated buffer-perfused hearts were subjected to 30 min of either hypothermic or normothermic no-flow ischemia followed by reperfusion. Prior depletion of myocardial GSH did not lead to oxidative stress during reperfusion, as myocardial concentration of glutathione disulfide (GSSG) was not increased after 5 and 30 min of reperfusion. In addition, prior depletion of GSH did not exacerbate myocardial enzyme release, nor did it impair the recoveries of tissue ATP, coronary flow rate and left ventricular developed pressure during reperfusion after either hypothermic or normothermic ischemia. Even administration of the prooxidant cumene hydroperoxide (20 μM) to postischemic GSH-depleted hearts during the first 10 min of reperfusion did not aggravate postischemic injury, although this prooxidant load induced oxidative stress, as indicated by an increased myocardial concentration of GSSG. These results do not support the hypothesis that a reduced myocardial concentration of GSH during ischemia increases the susceptibility to injury mediated by reactive oxygen species generated during reperfusion. Apparently, myocardial tissue possesses a large excess of GSH compared to the quantity of reactive oxygen species generated upon reperfusion. (Mol Cell Biochem 156: 79-85, 1996)
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References
Garlick PB, Davies MJ, Hearse DJ, Slater TF: Direct detection of free radicals in the reperfused rat heart using electron spin resonance spectroscopy. Circ Res: 61: 757–760, 1987
Kramer JH, Mišík V, Weglicki WB: Lipid peroxidation-derived free radical production and postischemic myocardial reperfusion injury. Ann N Y Acad Sci 723: 180–196, 1994
Blasig IE, Shuter S, Garlick P, Slater T: Relative time-profiles for free radical trapping, coronary flow, enzyme leakage, arrhythmias, and function during myocardial reperfusion. Free Radic Biol Med 16: 35–41, 1994
Jaeschke H, Mitchell JR: Use of isolated perfused organs in hypoxia and ischemia/reperfusion oxidant stress. Methods Enzymol 186: 752–759,1990
Meister A: Glutathione-ascorbic acid antioxidant system in animals. J Biol Chem 269: 9397–9400, 1994
Ishikawa T, Sies H: Cardiac transport of glutathione disulfide and S-conjugate: Studies with isolated perfused rat heart during hydroperoxide metabolism. J Biol Chem 259: 3838–3843, 1984
Ferrari R, Ceconi C, Curello S, Cargnoni A, Pasini E, Visioli O: The occurrence of oxidative stress during reperfusion in experimental animals and men. Cardiovasc Drugs Ther 5: 277–288, 1991
Ferrari R, Ceconi C, Curello S, Guarnieri C, Caldarera CM, Albertini A, Visioli O: Oxygen-mediated myocardial damage during ischaemia and reperfusion: Role of the cellular defences against oxygen toxicity. J Mol Cell Cardiol 17: 937–945, 1985
Arduini A, Mezzetti A, Porreca E, Lapenna D, DeJulia J, Marzio L, Polidoro G, Cuccurullo F: Effect of ischemia and reperfusion on antioxidant enzymes and mitochondrial inner membrane proteins in perfused rat heart. Biochim Biophys Acta 970: 113–121, 1988
Ceconi C, Curello S, Cargnoni A, Ferrari R, Albertini A, Visioli O: The role of glutathione status in the protection against ischaemic and reperfusion damage: Effects of N-acetyl cysteine. J Mol Cell Cardiol 20: 5–13, 1988
Singh A, Lee KJ, Lee CY, Goldfarb RD, Tsan MF: Relation between myocardial glutathione content and extent of ischemia-reperfusion injury. Circulation 80: 1795–1804, 1989
Lesnefsky EJ, Dauber IM, Horwitz LD: Myocardial sulfhydryl pool alterations occur during reperfusion after brief and prolonged myocardial ischemia in vivo. Circ Res 68: 605–613, 1991
Steare SE, Yellon DM: The potential for endogenous myocardial antioxidants to protect the myocardium against ischaemia-reperfusion injury: Refreshing the parts exogenous antioxidants cannot reach? J Mol Cell Cardiol 27: 65–74, 1995
Chatham JC, Seymour AML, Harmsen E, Radda GK: Depletion of myocardial glutathione: Its effects on heart function and metabolism during ischaemia and reperfusion. Cardiovasc Res 22: 833–839, 1988
Blaustein A, Deneke SM, Stolz RI, Baxter D, Healey N, Fanburg BL: Myocardial glutathione depletion impairs recovery after short periods of ischemia. Circulation 80: 1449–1457, 1989
Werns SW, Fantone JC, Ventura A, Lucchesi BR: Myocardial glutathione depletion impairs recovery of isolated blood-perfused hearts after global ischaemia. J Mol Cell Cardiol 24: 1215–1220, 1992
Meister A: Glutathione deficiency produced by inhibition of its synthesis, and its reversal; applications in research and therapy. Pharmacol Ther 51: 155–194, 1991
Akerboom TPM, Sies H: Assay of glutathione, glutathione disulfide, and glutathione mixed disulfides in biological samples. Methods Enzymol 77: 373–382, 1981
Tietze F: Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: Applications to mammalian blood and other tissues. Anal Biochem 27: 502–522, 1969
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265–275, 1951
Wynants J, Van Belle H: Single-run high-performance liquid chromatography of nucleotides, nucleosides, and major purine bases and its application to different tissue extracts. Anal Biochem 144: 258–266, 1985
Wettermark G, Stymne H: Substrate analyses in single cells. I. Determination of ATP. Anal Biochem 63: 293–307, 1975
Verbunt RJAM, Van Dockum WG, Bastiaanse EML, Egas JM, Van der Laarse A: Glutathione disulfide as an index of oxidative stress during postischemic reperfusion in isolated rat hearts. Mol Cell Biochem 144: 85–93, 1995
Kumar C, Okuda M, Ikai I, Chance B: Luminol enhanced chemiluminescence of the perfused rat heart during ischemia and reperfusion. FEBS Lett 272: 121–124, 1990
Henry TD, Archer SL, Nelson D, Weir EK, From AHL: Postischemic oxygen radical production varies with duration of ischemia. Am J Physiol 264: H1478-H1484, 1993
Baker JE, Felix CC, Olinger GN, Kalyanaraman B: Myocardial ischemia and reperfusion: Direct evidence for free radical generation by electron spin resonance spectroscopy. Proc Natl Acad Sci USA 85: 2786–2789, 1988
Johansson MH, Deinum J, Marklund SL, Sjöquist PO: Recombinant human extracellular superoxide dismutase reduces concentration of oxygen free radicals in the reperfused rat heart. Cardiovasc Res 24: 500–503, 1990
Powell SR, Hall D: Use of salicylate as a probe for OH formation in isolated ischemic rat hearts. Free Radic Biol Med 9: 133–141, 1990
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Verbunt, R.J.A.M., Van Dockum, W.G., Lars Bastiaanse, E.M. et al. Postischemic injury in isolated rat hearts is not aggravated by prior depletion of myocardial glutathione. Mol Cell Biochem 156, 79–85 (1996). https://doi.org/10.1007/BF00239322
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DOI: https://doi.org/10.1007/BF00239322