European Journal of Pharmacology, 192 (1991) 199-200 © 1991 Elsevier Science Publishers B.V. (Biomedical Division) 0014-2999/91/$03.50 ADONIS 001429999100083C
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EJP 0285R
Rapid communication
Increased excitotoxic vulnerability of cortical cultures with reduced levels of glutathione Richard J. Bridges 1, Jae-young K o h 2, Carolyn G. Hatalski a and Carl W. C o t m a n 2 Departments of i Neurology and 2 Psychobiology, Unioersity of California, Irvine, CA 92717, U.S.A. Received 19 November 1990, accepted 20 November 1990
Considerable progress has been made in elucidating the role of the NMDA (N-methyl-D-aspartate) receptor in excitotoxic neuronal death and CNS pathology (Choi, 1988). Excessive activation of the NMDA receptor and the influx of Ca 2+ through its associated ion channel constitute the initial steps in the excitotoxic response. Much less, however, is known about the pathological processes that follow these two primary events. Abnormally high levels of Ca 2+ are postulated to initiate a number of potentially harmful biochemical schemes, including the overactivation of proteolytic enzymes, lipases and xanthine oxidase (Choi, 1988). The latter two, particularly the formation of arachidonic acid, by phospholipase, and superoxide, by xanthine oxidase, suggest that part of the neuronal injury may be attributable to oxidative damage. To explore this possibility, we examined the role of the oxidative protectant glutathione (GSH) in excitotoxic vulnerability. Glutathione (L-7-glutamyl-L-cysteinyl-glycine) participates in a number of reactions related to protection from oxidative damage (Meister and Anderson, 1983). These include the reduction of intracellular peroxides via GSH-peroxidase, non-enzymatic reduction of free radicals, and the maintenance of reduced disulfide bonds. GSH is synthesized by the consecutive action of "t-glutamyl-cysteine synthetase and GSH synthetase, the former of which can be potently inhibited with D,Lbuthionine-S,R-sulfoximine (BSO) (Meister and Anderson, 1983). We report that murine cortical cells treated with BSO exhibit both a reduced GSH content and an increased susceptibility to NMDA and L-glutamate mediated pathology. Cortical cultures (4 × 105 cells/15 mm well) were prepared from 15- to 17-day-old mouse embryos as described by Koh and Choi (1987). After 5-10 days in vitro, non-neuronal cell division was halted with 10/~M
Correspondence to: R.J. Bridges, Department of Neurology, University of California, Irvine, CA 92717, U.S.A.
cytosine arabinoside (1-3 days), and the cultures were shifted to maintenance (biweekly replacement) medium (glutamine- and bicarbonate-free MEM-Earl's salt, supplemented with 26 mM HCO3, 2 mM glutamine and 15 mM glucose). Within each experiment, sister cultures of identical ages were used to minimize variability. GSH levels were depleted by exposing the cultures to 0.5 mM BSO (kindly provided by Drs. A. Meister, O. Griffith and M. Anderson, Cornell University Medical College) 24 h prior to the excitotoxic challenge. Before the addition of excitotoxins, the cultures were rinsed to remove extracellular BSO. Total GSH (GSH + GSSG) was quantitated in cells that had been rapidly scraped into ice cold PBS and homogenized in 2 volumes of 1% sulfosalycylic acid by the GSH reductase/DTNB recycling assay as described by Griffith (1980). Cortical cultures exposed to 0.5 mM BSO for 24 h exhibited a reduction in their content of total GSH from 32.8 + 9.0 nmol/mg protein in control cultures (mean + S.D., n = 5) to 5.2 + 2.8 nmol/mg protein (mean + S.D., n = 3). When challenged with a brief (5 min) pulse of either 100 /~M L-glutamate or 30 /~M NMDA (fig. 1), those cultures that had been pretreated with BSO exhibited significantly more neuronal loss as quantitated by LDH release (Koh and Choi, 1987). Thus, when exposed to a level of L-glutamate or NMDA that produced less than 10% neural death in controls, the cultures with reduced GSH levels exhibited 28% (n = 4, P < 0.05) and 33% (n = 4, P < 0.05) cell death, respectively. Importantly cultures exposed to BSO alone did not show any evidence of injury. Thus, while low GSH levels are not inherently toxic, GSH depletion can increase the vulnerability of neurons to an excitotoxic insult. These results are consistent with both the ability of GSH to play a protective role in the latter stages of the excitotoxic process and the involvement of oxidative damage in excitotoxicity. The protective role of GSH suggested by these experiments is interesting in light of studies reporting that GSH is preferentially distributed in astrocytes (Rap et
200 BSO POTENTIATES EXCITOTOXICITY
40'
199
EJP 0285R
Rapid communication
Increased excitotoxic vulnerability of cortical cultures with reduced levels of glutathione Richard J. Bridges 1, Jae-young K o h 2, Carolyn G. Hatalski a and Carl W. C o t m a n 2 Departments of i Neurology and 2 Psychobiology, Unioersity of California, Irvine, CA 92717, U.S.A. Received 19 November 1990, accepted 20 November 1990
Considerable progress has been made in elucidating the role of the NMDA (N-methyl-D-aspartate) receptor in excitotoxic neuronal death and CNS pathology (Choi, 1988). Excessive activation of the NMDA receptor and the influx of Ca 2+ through its associated ion channel constitute the initial steps in the excitotoxic response. Much less, however, is known about the pathological processes that follow these two primary events. Abnormally high levels of Ca 2+ are postulated to initiate a number of potentially harmful biochemical schemes, including the overactivation of proteolytic enzymes, lipases and xanthine oxidase (Choi, 1988). The latter two, particularly the formation of arachidonic acid, by phospholipase, and superoxide, by xanthine oxidase, suggest that part of the neuronal injury may be attributable to oxidative damage. To explore this possibility, we examined the role of the oxidative protectant glutathione (GSH) in excitotoxic vulnerability. Glutathione (L-7-glutamyl-L-cysteinyl-glycine) participates in a number of reactions related to protection from oxidative damage (Meister and Anderson, 1983). These include the reduction of intracellular peroxides via GSH-peroxidase, non-enzymatic reduction of free radicals, and the maintenance of reduced disulfide bonds. GSH is synthesized by the consecutive action of "t-glutamyl-cysteine synthetase and GSH synthetase, the former of which can be potently inhibited with D,Lbuthionine-S,R-sulfoximine (BSO) (Meister and Anderson, 1983). We report that murine cortical cells treated with BSO exhibit both a reduced GSH content and an increased susceptibility to NMDA and L-glutamate mediated pathology. Cortical cultures (4 × 105 cells/15 mm well) were prepared from 15- to 17-day-old mouse embryos as described by Koh and Choi (1987). After 5-10 days in vitro, non-neuronal cell division was halted with 10/~M
Correspondence to: R.J. Bridges, Department of Neurology, University of California, Irvine, CA 92717, U.S.A.
cytosine arabinoside (1-3 days), and the cultures were shifted to maintenance (biweekly replacement) medium (glutamine- and bicarbonate-free MEM-Earl's salt, supplemented with 26 mM HCO3, 2 mM glutamine and 15 mM glucose). Within each experiment, sister cultures of identical ages were used to minimize variability. GSH levels were depleted by exposing the cultures to 0.5 mM BSO (kindly provided by Drs. A. Meister, O. Griffith and M. Anderson, Cornell University Medical College) 24 h prior to the excitotoxic challenge. Before the addition of excitotoxins, the cultures were rinsed to remove extracellular BSO. Total GSH (GSH + GSSG) was quantitated in cells that had been rapidly scraped into ice cold PBS and homogenized in 2 volumes of 1% sulfosalycylic acid by the GSH reductase/DTNB recycling assay as described by Griffith (1980). Cortical cultures exposed to 0.5 mM BSO for 24 h exhibited a reduction in their content of total GSH from 32.8 + 9.0 nmol/mg protein in control cultures (mean + S.D., n = 5) to 5.2 + 2.8 nmol/mg protein (mean + S.D., n = 3). When challenged with a brief (5 min) pulse of either 100 /~M L-glutamate or 30 /~M NMDA (fig. 1), those cultures that had been pretreated with BSO exhibited significantly more neuronal loss as quantitated by LDH release (Koh and Choi, 1987). Thus, when exposed to a level of L-glutamate or NMDA that produced less than 10% neural death in controls, the cultures with reduced GSH levels exhibited 28% (n = 4, P < 0.05) and 33% (n = 4, P < 0.05) cell death, respectively. Importantly cultures exposed to BSO alone did not show any evidence of injury. Thus, while low GSH levels are not inherently toxic, GSH depletion can increase the vulnerability of neurons to an excitotoxic insult. These results are consistent with both the ability of GSH to play a protective role in the latter stages of the excitotoxic process and the involvement of oxidative damage in excitotoxicity. The protective role of GSH suggested by these experiments is interesting in light of studies reporting that GSH is preferentially distributed in astrocytes (Rap et
200 BSO POTENTIATES EXCITOTOXICITY
40'
