A Glutathione Precursor Reduces Oxidative Injury to Cultured Embryonic Cardiomyocytes

Abstract

Background: Newborn infants are highly vulnerable to oxidative stress. Following birth asphyxia, oxidative injury due to ischemia–reperfusion can result in significant brain and heart damage, leading to death or long-term disability. Study Question: The study objective was to evaluate the effectiveness of antioxidant gamma-l-glutamyl-l-cysteine (γGlu-Cys) in inhibiting oxidative injury to cultured embryonic cardiomyocytes (H9c2 cells). Study Design: Control and γGlu-Cys–treated (0.5 mM) H9c2 cells were incubated under 6-hour ischemic conditions followed by 2-hour simulated reperfusion. Measures and Outcomes: To quantify oxidative stress-induced apoptosis sustained by cardiomyocytes, lactate dehydrogenase (LDH) release and the presence of cytosolic cytochrome c were measured, as well as the number of secondary lysosomes visualized under electron microscopy. Results: Compared to controls, H9c2 cells coincubated with γGlu-Cys during ischemia–reperfusion exhibited a significant reduction in both LDH release into the incubation medium [23.88 ± 4.08 (SE) vs. 9.95 ± 1.86% of total; P = 0.02] and the number of secondary lysosomes [0.070 ± 0.009 (SD) vs. 0.043 ± 0.004 per μm2; P = 0.01]. Inhibition of LDH release with γGlu-Cys was the same (P = 0.67) as that of a caspase inhibitor. The significant increase in cytosolic cytochrome c (P = 0.01) after ischemia–reperfusion simulation further supports γGlu-Cys's role in apoptosis prevention. Conclusions: It is concluded that the glutathione precursor γGlu-Cys protects cultured embryonic cardiomyocytes from apoptosis-associated oxidative injury.

Prevention of Oxidative Injury Associated with Thrombolysis for Ischemic Stroke

“…We next provided evidence that both Na/H (NHE1) and Na/Ca (NCX 1) exchangers are present on the plasma membrane domains of these cells, and that conditions simulating ischemia and reperfusion result in elevated intracellular calcium due to activation of sodium-hydrogen ion antiport and reverse movement of sodium-calcium exchange (Figure 3). Cellular lysis following these events was significantly inhibited by adding the antioxidant γGlu-Cys [19,20,58], which is consistent with the formation of reactive oxygen species expected upon re-oxygenation in glutathione depleted cells. These findings reinforced our interpretation that a combination of elevated intracellular calcium and reactive oxygen species are involved in injury to cerebral capillary endothelial cells, under conditions of ischemia and reperfusion.…”

“…Compared to cultured cells incubated under conditions simulating ischemia (1.5 hours) and reperfusion (3 hours) in the absence of these antioxidants, 1 mM glutathione and 1 mM N-acetylcysteine inhibited mean LDH release by factors of 0.51 and 0.45, respectively. Collectively, the data indicate that injury to cultured brain capillary endothelial cells exposed to ischemia/reperfusion is significantly reduced in the presence of known antioxidants [19,20,45] including native glutathione, and its precursors N-acetylcysteine, and γ-glutamylcysteine.…”

“…These in vitro findings were consistent with our working hypothesis which predicted that elevated intracellular calcium and reactive oxygen species would activate apoptosis in cerebral capillary endothelial cells exposed to conditions of ischemia and reperfusion. Furthermore, the results suggested that: (1) inhibiting reverse movement of Na/Ca exchange with KB-R7943 [65], and (2) replenishing lost antioxidant with γGlu-Cys [19,20] would prevent reperfusion (oxidative) injury to brain capillaries. Since γGlu-Cys is a precursor of the antioxidant glutathione [59] lost during ischemia, and γGlu-Cys itself possesses antioxidant properties [19,20], it represents a reasonable antioxidant therapeutic in this setting.…”

“…Lactate dehydrogenase (LDH) release was used to determine cell lysis, and was measured colorimetrically at a wave-length of 490 nm with a commercial kit (Promega). The effectiveness of various antioxidants [19,20,45] in preventing cell death was tested by incubating the cells in the presence (1 mM) and absence of glutathione (GSH), N-acetylcysteine (NAC), and gamma-glutamylcysteine (γGlu-Cys).…”

“…In this study, we provide evidence that reperfusion injury is associated with oxidative damage to brain capillary endothelial cells in the presence of elevated calcium. Furthermore, we show that the antioxidant γ-glutamylcysteine [19,20] (a precursor of glutathione) together with an agent to prevent calcium sequestration inhibit oxidative injury to brain capillaries when co-administered immediately prior to inducing reperfusion for ischemic stroke.…”