Synergistic Depletion of Astrocytic Glutathione by Glucose Deprivation and Peroxynitrite

Correlation with Mitochondrial Dysfunction and Subsequent Cell Death

Authors

  • Chung Ju,

  • Keum-Na Yoon,

  • Yu-Kyoung Oh,

  • Hyoung-Chun Kim,

  • Chan Young Shin,

  • Jae Ryun Ryu,

  • Kwang Ho Ko,

  • Won-Ki Kim


  • Lippincott Williams & Wilkins, Inc., Philadelphia

  • Abbreviations used: BSA, bovine serum albumin; L-BSO, L-buthionine-(S,R)-sulfoximine; CsA, cyclosporin A; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; GSH, reduced glutathione; GSNO, S-nitrosoglutathione; GSSG, oxidized glutathione; H2TMPyP, 5,10,15,20-tetrakis(N-methyl-4′-pyridyl)porphyrin; LDH, lactate dehydrogenase; MnTMPyP, Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin; MPT, mitochondrial permeability transition; MTP, mitochondrial transmembrane potential; NO, nitric oxide; O2[UNK]-, superoxide anion; ONOO-, peroxynitrite; PBS, phosphate-buffered saline solution; SIN-1, 3-morpholinosydnonimine; SNAP, S-nitroso-N-acetylpenicillamine; SOD, superoxide dismutase.

Address correspondence and reprint requests to Dr. W.-K. Kim at Department of Pharmacology, College of Medicine, Ewha Women's University, 911-1 Mok-6-dong, Yangchun-Ku, Seoul 158-056, Republic of Korea. E-mail: won_k@mm.ewha.ac.kr

Abstract

Abstract: Previously we reported that immunostimulated astrocytes were highly vulnerable to glucose deprivation. The augmented death was mimicked by the peroxynitrite (ONOO--producing reagent 3-morpholinosydnonimine (SIN-1). Here we show that glucose deprivation and ONOO- synergistically deplete intracellular reduced glutathione (GSH) and augment the death of astrocytes via formation of cyclosporin A-sensitive mitochondrial permeability transition (MPT) pore. Astrocytic GSH levels were only slightly decreased by glucose deprivation or SIN-1 (200 μM) alone. In contrast, a rapid and large depletion of GSH was observed in glucose-deprived/SIN-1-treated astrocytes. The depletion of GSH occurred before a significant release of lactate dehydrogenase (a marker of cell death). Superoxide dismutase and ONOO- scavengers completely blocked the augmented death, indicating that the reaction of nitric oxide with superoxide to form ONOO- was implicated. Furthermore, nitrotyrosine immunoreactivity (a marker of ONOO-) was markedly enhanced in glucose-deprived/SIN-1-treated astrocytes. Mitochondrial transmembrane potential (MTP) was synergistically decreased in glucose-deprived/SIN-1-treated astrocytes. The glutathione synthase inhibitor L-buthionine-(S,R)-sulfoximine markedly decreased the MTP and increased lactate dehydrogenase (LDH) releases in SIN-1-treated astrocytes. Cyclosporin A, an MPT pore blocker, completely prevented the MTP depolarization as well as the enhanced LDH releases in glucose-deprived/SIN-1-treated astrocytes.

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