Buthionine Sulfoximine Reduces the Protective Capacity of Myocytes to Withstand Peroxide-Derived Free Radical Attack

Abstract

Mammalian heart myocytes have a limited capacity to withstand the deleterious effects of free radical generating compounds. To assess the role of the glutathione redox cycle relative to this capacity, rat heart cell cultures were subjected for 90 min to 80 μmol/1 cumene hydroperoxide (CHPO) without and with prior glutathione depletion by buthionine sulfoximine. Preincubation of cultures with 125 μmol/1 buthionine sulfoximine for 2 h and 17 h caused a reduction of glutathione by 33% and 82%, respectively, without concomitant increase of glutathione disulfide. Subsequent incubation with CHPO for 90 min caused slowing of NADPH consumption (in the first 20 min 27 pmol vs 68 pmol without pretreatment with buthionine sulfoximine for 17 h), which indicates that glutathione depletion reduced the turnover rate of the glutathione redox cycle. Pretreatment with buthionine sulfoximine for 17 h exaggerated the negative chronotropic effects of CHPO: the time elapsed to 50% of baseline contraction frequency fell from 5.7 ± 1.4 min without buthionine sulfoximine to 3.7 ± 0.4 min after pretreatment with buthionine sulfoximine (P < 0.02). The severity of CHPO-induced lipid peroxidation as assessed by malondialdehyde formation (2.23 ± 0.51 vs 0.99 ± 0.05 nmol in the first 20 min; P < 0.05) was increased by buthionine sulfoximine pretreatment, as was the extent of cell necrosis as assessed by release of α-hydroxybutyrate dehydrogenase (39.5 ± 5.1 vs 29.0 ± 12.9% in the first 45 min). A "sublethal" dose of 10 μM CHPO for 60 min caused no substantial HBDH release, no formation of malondialdehyde, and no exhaustion of cellular GSH (35 nmol/U HBDHi = 0). However, following pretreatment with buthionine sulfoximine, 10 μM CHPO for 60 min produced 12% HBDH release and extensive lipid peroxidation (1.95 nmol malondialdehyde/U HBDHi = 0). As the deleterious effects of CHPO were aggravated by glutathione depletion, we conclude that the glutathione redox cycle plays a major role in the protection of myocytes against peroxide-induced free redical attack.