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The possible role of endogenous glutathione as an anticonvulsant in mice
Brain Research 854 Ž2000. 235–238 www.elsevier.comrlocaterbres
Short communication
The possible role of endogenous glutathione as an anticonvulsant in mice Kazuho Abe ) , Kazuko Nakanishi, Hiroshi Saito Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, The UniÕersity of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Accepted 19 October 1999
Abstract We have recently found that intracerebroventricular Ži.c.v.. administration of glutathione ŽGSH. inhibits pentylenetetrazol-induced convulsions in mice, suggesting that GSH has an anticonvulsive action. In the present study, we investigated whether endogenous GSH play a role in regulating seizure susceptibility, using L-buthionine-w S, R x-sulfoximine ŽBSO., a specific inhibitor of GSH biosynthesis. BSO treatment Ž3.2 mmol i.c.v.= 2, 48 and 24 h prior to experiments. decreased brain GSH level to 31.5% of control, and potentiated pentylenetetrazol-induced convulsions. Potentiation of convulsions by BSO treatment was recovered by supplying GSH Ž10 nmol, i.c.v... These results suggest that endogenous GSH functions as an anticonvulsant. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Glutathione; L-Buthionine-w S, R x-sulfoximine; Convulsion; Pentylenetetrazol; Mouse
The tripeptide glutathione ŽL-g-glutamyl-L-cysteinylglycine; GSH. is present in most mammalian tissues in millimolar concentrations. GSH plays an important role in the storage and interorgan transport of cysteine and in cellular defenses against free radicals, peroxides and electrophilic xenobiotics as a cofactor of glutathione peroxidase and glutathione-S-transferase. Glutathione peroxidase converts GSH into oxidized glutathione ŽGSSG. in the presence of hydrogen peroxides, and GSH is regenerated from GSSG via a process catalyzed by glutathione reductase. In the brain, GSH is localized almost exclusively in astroglial cells, though it may also exist in nerve terminals and some neurons w11,12,15x. The concentration of total GSH in the brain is approximately 2 mM, most of which is the reduced form GSH and only 1.2% or less is the oxidized form GSSG w5,13,16x. GSH is also found in the cerebrospinal fluid. The concentration of GSH in the cerebrospinal fluid was reported approximately 5 mM in rats w2x and 200 nM in humans w3x. However, its origin is not fully understood. It has been reported that GSH is released from astrocytes via a carrier-mediated mechanism w14,18x or from a neuronal compartment in response to depolarizing stimuli w19x. In addition, it is also known that GSH is transported from the plasma to the cerebrospinal fluid via epithelial cells of the
) Corresponding author. [email protected]
Fax:
q 81-3-5841-4786;
e-mail:
choroid plexus, the major sites of cerebrospinal fluid formation w2x. Anyway, the fact that GSH is released from brain cells and present at significant level in the cerebrospinal fluid implicates that GSH also functions as a neurotransmitter or neuromodulator in the brain. To explore possible role of GSH in brain neurotransmission, we have studied the effect of GSH in a number of behavioral experiments using the whole animal. Very recently, we have found that intracerebroventricular Ži.c.v.. administration of GSH inhibits pentylenetetrazol-induced convulsions in mice, suggesting that GSH has an anticonvulsive action w1x. However, it remained unknown whether endogenous GSH plays a role in regulating seizure susceptibility. Therefore, in the present study, we investigated the effect of L-buthionine-w S, R x-sulfoximine ŽBSO. on pentylenetetrazol-induced convulsions in mice. BSO is a selective inhibitor of g-glutamylcysteine synthetase, the rate limiting enzyme of GSH synthesis, and has been widely used to deplete endogenous GSH w7,8x. Male ddY mice 5 weeks old were obtained from Japan SLC, ŽShizuoka, Japan. and housed in aluminum cages for 1 week with food and water available ad libitum. All efforts were made for the care and use of animals according to the Guideline for Animal Experiment of the Faculty of Pharmaceutical Sciences, The University of Tokyo. The implantation of i.c.v. guide cannula was made as described in our previous paper w4x. In brief, mice were anesthetized with i.m. injection of ketamine and xylazine Ž41 and 3.5 mgrkg, respectively. and fixed in a stereotaxic apparatus.
0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 2 2 6 9 - 6
236
K. Abe et al.r Brain Research 854 (2000) 235–238
A stainless steel cylindrical cannula Ž0.35 mm i.d., 5 mm in length. was implanted in each animal so that the tip of the cannula was set in the left lateral ventricle Ž1.3 mm lateral to the midline, 0.3 mm posterior to the bregma, 2.0 mm ventral to the skull surface.. The implanted cannulas were fixed with dental cement. These cannulas served as guide cannulas for i.c.v. injection of drug solution. The operated mice were allowed to recover for 1 week before experiments. To decrease endogenous GSH level in the brain, BSO was dissolved in saline and directly injected into the brain through the implanted i.c.v. guide cannula. Since it has been reported that GSH level gradually declines following administration of BSO w9x, we tested BSO treatment in four different conditions; BSO was administered at 1.6 or 3.2 mmol Ž320 or 640 mM = 5 ml. once 24 h prior to experiments or twice 48 and 24 h prior to experiments. To confirm that endogenous GSH level is successfully decreased by these procedures, total GSH ŽGSH q GSSG. level was determined by using the 5,5X-dithiobisŽ2-nitrobenzoic acid. ŽDTNB. –glutathione reductase recycling assay w6,10x. The principle of this assay is illustrated in Fig. 1A. In the presence of NADPH, glutathione reductase converts GSSG into GSH, which in turn reduces DTNB to a colored product NTB. Whole brain was quickly isolated
Fig. 1. The effect of BSO treatment on brain GSH level in mice. ŽA. Principle of a colorimetric determination of GSH concentration. In the presence of NADPH, glutathione reductase converts GSSG into GSH, which in turn reduces DTNB to 2-nitro-5-thiobenzonic acid ŽNTB.. The formation of NTB is evaluated by measuring absorbance at 412 nm. ŽB. BSO Ž1.6 or 3.2 mmol. was i.c.v. administered once 24 h prior to experiments or twice 48 and 24 h prior to experiments. For control, the vehicle Žsaline. was i.c.v. administered once or twice. Intact mice received neither i.c.v. cannula implantation nor drug administration. Total GSH ŽGSHqGSSG. was determined with the colorimetric method shown in ŽA., and divided by grams wet weight of the brain. All data are represented as the mean"S.E.M. of values obtained from five mice. UU P - 0.01 vs. intact, Duncan’s multiple range test.
Fig. 2. The effect of BSO treatment on pentylenetetrazol-induced convulsions in mice. BSO treatment was achieved as in Fig. 1B. The mice were observed for 30 min following s.c. administration of pentylenetetrazol Ž80 mgrkg., and the latency to appearance of clonic convulsion Žleft. or tonic convulsion Žright. was measured. All data are represented as the means" S.E.M. of values obtained from eight mice. U P - 0.05, UU P - 0.01 vs. intact; Duncan’s multiple range test.
and placed in ice-cold phosphate-buffered saline and homogenized with a Polytron homogenizer ŽKinematica; setting 8, 5 s = 5.. The homogenate was centrifuged at 1000 = g for 15 min, and the supernatant was added to sample buffer ŽpH 6.0. containing 20 mM DTNB, 0.1 mM NADPH and 1 mM EDTA. The reaction was started by adding glutathione reductase Ž0.5 Urml., and absorbance at 412 nm was measured over 5 min. The rate of color change was calculated, and the concentration of total GSH in samples was estimated from the standard curve constructed in each assay. As shown in Fig. 1B, total GSH level in the brain was not affected by i.c.v. administration of saline, supporting that i.c.v. cannula implantation and vehicle injection have no effect on brain GSH level. On the other hand, i.c.v. administration of BSO significantly decreased brain GSH. Of four conditions tested, the decrease in GSH level was most remarkable when 3.2 mmol BSO was administered twice 48 and 24 h prior to experiments. The BSO-treated mice showed no apparent abnormality in general behaviors. Then, pentylenetetrazol-induced convulsions were examined in BSO-treated mice. Pentylenetetrazol was dissolved in saline and subcutaneously Žs.c.. administered at a volume of 10 mlrkg. The animals were observed for 30 min following drug administration, and the following three parameters were measured to evaluate drug-induced convulsions: Ž1. latency to appearance of clonic convulsion; Ž2. latency to appearance of tonic convulsion; and Ž3. lethality. Lethality was defined as the percentage of the animals that died within 30 min after drug injection. The s.c. administration of pentylenetetrazol Ž80 mgrkg. induced both clonic and tonic convulsions. In most cases, clonic convulsions were followed by tonic convulsions. As shown in Fig. 2, the latencies to appearance of clonic and tonic convulsions were significantly smaller in BSO-treated mice than in intact or saline-treated mice, indicating that seizure susceptibility was increased by BSO treatment. Lethality was zero in all groups. Of four conditions of BSO treatment tested, the shortening of
K. Abe et al.r Brain Research 854 (2000) 235–238
Fig. 3. The effect of BSO treatment was recovered by supplying exogenous GSH. BSO Ž3.2 mmol. was i.c.v. administered twice 48 and 24 h prior to experiments. Control mice received i.c.v. administration of saline 48 and 24 h prior to experiments. GSH Ž10 nmol. was i.c.v. administered 20 min before s.c. administration of pentylenetetrazol Ž80 mgrkg.. Following administration of pentylenetetrazol, the latency to appearance of clonic convulsion Žleft. or tonic convulsion Žright. was measured. All data are represented as the means"S.E.M. of eight mice. UU P - 0.01 vs. salineqsaline; aP - 0.05, aaP - 0.01 vs. BSOqsaline; Duncan’s multiple range test.
latencies to clonic and tonic convulsions was most remarkable when 3.2 mmol BSO was administered twice 48 and 24 h prior to experiments. To support that the increase of seizure susceptibility by BSO treatment is due to a decrease in GSH level, we examined if the change is recovered by exogenously supplying GSH. GSH was dissolved in saline and administered at a dose of 10 nmol Ž2 mM = 5 ml. into the brain through the implanted i.c.v. guide cannula 20 min before s.c. administration of pentylenetetrazol Ž80 mgrkg.. This dose of GSH alone had no effect on pentylenetetrazol-induced convulsions in intact mice, but significantly attenuated pentylenetetrazol-induced convulsions in BSO-treated mice ŽFig. 3.. We have found for the first time that pentylenetetrazolinduced convulsions are potentiated by treatment with the GSH biosynthesis inhibitor BSO. Comparing four different conditions of BSO treatment, the potentiation of convulsions ŽFig. 2. was parallel to the decrease in brain GSH level ŽFig. 1B.. Since it has previously been reported that BSO treatment causes a selective neuronal death w9,17x, we have also checked possible histological change by preparing paraformaldehyde-fixed, Nissl-stained brain sections. However, no apparent histological change was observed in the brain of mice employed in our present study Žunpublished data.. Furthermore, the change in seizure susceptibility produced by BSO treatment was recovered by supplying GSH. These results suggest that endogenous GSH functions as an anticonvulsant. It is generally supposed that GSH is not easily taken up by the cells. In the present study, the change produced by BSO treatment was recovered by i.c.v. administration of GSH, indicating that extracellular GSH plays a major role in the anticonvulsive action. The concentration of total GSH in the brain is reported approximately 2 mM, most of which is present in the intracellular compartment w2,3,5,13,16x. In our measurement, total GSH level in the brain was 1.49 mlrg wet weight in intact mice, and decreased to 0.47 mlrg wet
237
weight following BSO treatment ŽFig. 1B.. On the assumption that 1 g of wet weight of tissue corresponds to 1 ml of water, it is estimated that BSO treatment decreases brain GSH concentration from 1.49 to 0.47 mM. However, 10 nmol was enough for exogenous GSH to recover the functional change produced by BSO treatment. The mean wet weight of adult mouse brain used in our present study was 0.471 g. Supposing that i.c.v. administered GSH is equally distributed in the mouse brain, 10 nmol GSH is estimated to reach 21 mM. Therefore, it is likely that seizure susceptibility is regulated by micromolar concentrations of extracellular GSH, though extracellular GSH concentration may be influenced by the intracellular storage. In conclusion, we have provided evidence that endogenous GSH, especially extracellular GSH, functions as an anticonvulsant. GSH may play a role in modulating neuronal excitability in the brain. Our finding will give new insight into the possible role of GSH as a neuromodulator. Further investigations are underway in our laboratory to elucidate cellular mechanisms underlying the anticonvulsive action of GSH.
References w1x K. Abe, K. Nakanishi, H. Saito, The anticonvulsive effect of glutathione in mice, Biol. Pharm. Bull. 22 Ž1999. 1177–1179. w2x M.E. Anderson, M. Underwood, R.J. Bridges, A. Meister, Glutathione metabolism at blood–cerebrospinal fluid barrier, FASEB J. 3 Ž1989. 2527–2531. w3x F. Baronti, T.L. Davis, R.C. Boldry, M.M. Mouradian, T.N. Chase, Deprenyl effects on pharmacodynamics, mode, and free radical scavenging, Neurology 42 Ž1992. 541–544. w4x P.J. Chu, A. Shirahata, K. Samejima, H. Saito, K. Abe, N-Ž3Aminopropyl.-cyclohexylamine blocks facilitation by spermidine of N-methyl-DL-aspartate-induced seizure in mice in vivo, Eur. J. Pharmacol. 256 Ž1994. 155–160. w5x A.J.L. Cooper, W.A. Pulsineli, T.E. Duffy, Glutathione and ascorbate during ischemia and postischemic reperfusion in rat brain, J. Neurochem. 35 Ž1980. 1242–1245. w6x P. Eyer, D. Podhradsky, Evaluation of the micromethod for determination of glutathione using enzymatic cycling and Ellman’s reagent, Anal. Biochem. 153 Ž1986. 57–66. w7x O.W. Griffith, M.E. Anderson, A. Meister, Inhibition of glutathione biosynthesis by prothionine sulfoximine Ž S-n-propyl homocysteine sulfoximine., a selective inhibitor of g-glutamylcysteine synthetase, J. Biol. Chem. 254 Ž1979. 1205–1210. w8x O.W. Griffith, A. Meister, Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine Ž S-n-butyl homocysteine sulfoximine., J. Biol. Chem. 254 Ž1979. 7558–7560. w9x A. Jain, J. Martensson, E. Stole, P.A.M. Auld, A. Meister, Glutathione deficiency leads to mitochondrial damage in brain, Proc. Natl. Acad. Sci. U. S. A. 88 Ž1991. 1913–1917. w10x C.W. Owens, R.V. Belcher, A colorimetric micro-method for the determination of glutathione, Biochem. J. 94 Ž1965. 705–711. w11x M.A. Philbert, C.M. Beiswanger, D.K. Waters, K.R. Reuhl, H.E. Lowndes, Cellular and regional distribution of reduced glutathione in the nervous system of the rat: histochemical localization by mercury orange and o-phthaldialdehyde-induced histofluorescence, Toxicol. Appl. Pharmacol. 107 Ž1991. 215–227. w12x S.P. Raps, J.C.K. Lai, L. Hertz, A.J.L. Cooper, Glutathione is
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w13x
w14x w15x w16x
K. Abe et al.r Brain Research 854 (2000) 235–238 present in high concentrations in cultured astrocytes but not in cultured neurons, Brain Res. 493 Ž1989. 398–401. S. Rehncrona, J. Folbergrova, D.S. Smith, B.K. Siesjo, Influence of complete and pronounced incomplete cerebral ischemia and subsequent recirculation on cortical concentrations of oxidized and reduced glutathione in the rat, J. Neurochem. 34 Ž1980. 477–486. J. Sagara, N. Makino, S. Bannai, Glutathione efflux from cultured astrocytes, J. Neurochem. 66 Ž1996. 1876–1881. A. Slivka, C. Mytilineou, G. Cohen, Histochemical evaluation of glutathione in brain, Brain Res. 409 Ž1987. 1391–1393. A. Slivka, M.B. Spina, G. Cohen, Reduced and oxidized glutathione in human and monkey brain, Neurosci. Lett. 74 Ž1987. 112–118.
w17x U. Wullner, P.A. Loschmann, J.B. Schulz, A. Schmid, R. Dringen, F. Eblen, L. Turski, T. Klockgether, Glutathione depletion potentiates MPTP and MPPq toxicity in nigral dopaminergic neurones, NeuroReport 7 Ž1996. 921–923. w18x M. Yudkoff, D. Pleasure, L. Cregar, Z.P. Lin, I. Nissim, J. Stern, I. Nissim, Glutathione turnover in cultured astrocytes: studies with w15 Nxglutamate, J. Neurochem. 55 Ž1990. Ž1990. 137–145. w19x L. Zangerle, M. Cuenod, K.H. Winterhalter, K.Q. Do, Screening of thiol compounds: depolarization-induced release of glutathione and cysteine from rat brain slices, J. Neurochem. 59 Ž1992. 181–189.
Short communication
The possible role of endogenous glutathione as an anticonvulsant in mice Kazuho Abe ) , Kazuko Nakanishi, Hiroshi Saito Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, The UniÕersity of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Accepted 19 October 1999
Abstract We have recently found that intracerebroventricular Ži.c.v.. administration of glutathione ŽGSH. inhibits pentylenetetrazol-induced convulsions in mice, suggesting that GSH has an anticonvulsive action. In the present study, we investigated whether endogenous GSH play a role in regulating seizure susceptibility, using L-buthionine-w S, R x-sulfoximine ŽBSO., a specific inhibitor of GSH biosynthesis. BSO treatment Ž3.2 mmol i.c.v.= 2, 48 and 24 h prior to experiments. decreased brain GSH level to 31.5% of control, and potentiated pentylenetetrazol-induced convulsions. Potentiation of convulsions by BSO treatment was recovered by supplying GSH Ž10 nmol, i.c.v... These results suggest that endogenous GSH functions as an anticonvulsant. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Glutathione; L-Buthionine-w S, R x-sulfoximine; Convulsion; Pentylenetetrazol; Mouse
The tripeptide glutathione ŽL-g-glutamyl-L-cysteinylglycine; GSH. is present in most mammalian tissues in millimolar concentrations. GSH plays an important role in the storage and interorgan transport of cysteine and in cellular defenses against free radicals, peroxides and electrophilic xenobiotics as a cofactor of glutathione peroxidase and glutathione-S-transferase. Glutathione peroxidase converts GSH into oxidized glutathione ŽGSSG. in the presence of hydrogen peroxides, and GSH is regenerated from GSSG via a process catalyzed by glutathione reductase. In the brain, GSH is localized almost exclusively in astroglial cells, though it may also exist in nerve terminals and some neurons w11,12,15x. The concentration of total GSH in the brain is approximately 2 mM, most of which is the reduced form GSH and only 1.2% or less is the oxidized form GSSG w5,13,16x. GSH is also found in the cerebrospinal fluid. The concentration of GSH in the cerebrospinal fluid was reported approximately 5 mM in rats w2x and 200 nM in humans w3x. However, its origin is not fully understood. It has been reported that GSH is released from astrocytes via a carrier-mediated mechanism w14,18x or from a neuronal compartment in response to depolarizing stimuli w19x. In addition, it is also known that GSH is transported from the plasma to the cerebrospinal fluid via epithelial cells of the
) Corresponding author. [email protected]
Fax:
q 81-3-5841-4786;
e-mail:
choroid plexus, the major sites of cerebrospinal fluid formation w2x. Anyway, the fact that GSH is released from brain cells and present at significant level in the cerebrospinal fluid implicates that GSH also functions as a neurotransmitter or neuromodulator in the brain. To explore possible role of GSH in brain neurotransmission, we have studied the effect of GSH in a number of behavioral experiments using the whole animal. Very recently, we have found that intracerebroventricular Ži.c.v.. administration of GSH inhibits pentylenetetrazol-induced convulsions in mice, suggesting that GSH has an anticonvulsive action w1x. However, it remained unknown whether endogenous GSH plays a role in regulating seizure susceptibility. Therefore, in the present study, we investigated the effect of L-buthionine-w S, R x-sulfoximine ŽBSO. on pentylenetetrazol-induced convulsions in mice. BSO is a selective inhibitor of g-glutamylcysteine synthetase, the rate limiting enzyme of GSH synthesis, and has been widely used to deplete endogenous GSH w7,8x. Male ddY mice 5 weeks old were obtained from Japan SLC, ŽShizuoka, Japan. and housed in aluminum cages for 1 week with food and water available ad libitum. All efforts were made for the care and use of animals according to the Guideline for Animal Experiment of the Faculty of Pharmaceutical Sciences, The University of Tokyo. The implantation of i.c.v. guide cannula was made as described in our previous paper w4x. In brief, mice were anesthetized with i.m. injection of ketamine and xylazine Ž41 and 3.5 mgrkg, respectively. and fixed in a stereotaxic apparatus.
0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 2 2 6 9 - 6
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K. Abe et al.r Brain Research 854 (2000) 235–238
A stainless steel cylindrical cannula Ž0.35 mm i.d., 5 mm in length. was implanted in each animal so that the tip of the cannula was set in the left lateral ventricle Ž1.3 mm lateral to the midline, 0.3 mm posterior to the bregma, 2.0 mm ventral to the skull surface.. The implanted cannulas were fixed with dental cement. These cannulas served as guide cannulas for i.c.v. injection of drug solution. The operated mice were allowed to recover for 1 week before experiments. To decrease endogenous GSH level in the brain, BSO was dissolved in saline and directly injected into the brain through the implanted i.c.v. guide cannula. Since it has been reported that GSH level gradually declines following administration of BSO w9x, we tested BSO treatment in four different conditions; BSO was administered at 1.6 or 3.2 mmol Ž320 or 640 mM = 5 ml. once 24 h prior to experiments or twice 48 and 24 h prior to experiments. To confirm that endogenous GSH level is successfully decreased by these procedures, total GSH ŽGSH q GSSG. level was determined by using the 5,5X-dithiobisŽ2-nitrobenzoic acid. ŽDTNB. –glutathione reductase recycling assay w6,10x. The principle of this assay is illustrated in Fig. 1A. In the presence of NADPH, glutathione reductase converts GSSG into GSH, which in turn reduces DTNB to a colored product NTB. Whole brain was quickly isolated
Fig. 1. The effect of BSO treatment on brain GSH level in mice. ŽA. Principle of a colorimetric determination of GSH concentration. In the presence of NADPH, glutathione reductase converts GSSG into GSH, which in turn reduces DTNB to 2-nitro-5-thiobenzonic acid ŽNTB.. The formation of NTB is evaluated by measuring absorbance at 412 nm. ŽB. BSO Ž1.6 or 3.2 mmol. was i.c.v. administered once 24 h prior to experiments or twice 48 and 24 h prior to experiments. For control, the vehicle Žsaline. was i.c.v. administered once or twice. Intact mice received neither i.c.v. cannula implantation nor drug administration. Total GSH ŽGSHqGSSG. was determined with the colorimetric method shown in ŽA., and divided by grams wet weight of the brain. All data are represented as the mean"S.E.M. of values obtained from five mice. UU P - 0.01 vs. intact, Duncan’s multiple range test.
Fig. 2. The effect of BSO treatment on pentylenetetrazol-induced convulsions in mice. BSO treatment was achieved as in Fig. 1B. The mice were observed for 30 min following s.c. administration of pentylenetetrazol Ž80 mgrkg., and the latency to appearance of clonic convulsion Žleft. or tonic convulsion Žright. was measured. All data are represented as the means" S.E.M. of values obtained from eight mice. U P - 0.05, UU P - 0.01 vs. intact; Duncan’s multiple range test.
and placed in ice-cold phosphate-buffered saline and homogenized with a Polytron homogenizer ŽKinematica; setting 8, 5 s = 5.. The homogenate was centrifuged at 1000 = g for 15 min, and the supernatant was added to sample buffer ŽpH 6.0. containing 20 mM DTNB, 0.1 mM NADPH and 1 mM EDTA. The reaction was started by adding glutathione reductase Ž0.5 Urml., and absorbance at 412 nm was measured over 5 min. The rate of color change was calculated, and the concentration of total GSH in samples was estimated from the standard curve constructed in each assay. As shown in Fig. 1B, total GSH level in the brain was not affected by i.c.v. administration of saline, supporting that i.c.v. cannula implantation and vehicle injection have no effect on brain GSH level. On the other hand, i.c.v. administration of BSO significantly decreased brain GSH. Of four conditions tested, the decrease in GSH level was most remarkable when 3.2 mmol BSO was administered twice 48 and 24 h prior to experiments. The BSO-treated mice showed no apparent abnormality in general behaviors. Then, pentylenetetrazol-induced convulsions were examined in BSO-treated mice. Pentylenetetrazol was dissolved in saline and subcutaneously Žs.c.. administered at a volume of 10 mlrkg. The animals were observed for 30 min following drug administration, and the following three parameters were measured to evaluate drug-induced convulsions: Ž1. latency to appearance of clonic convulsion; Ž2. latency to appearance of tonic convulsion; and Ž3. lethality. Lethality was defined as the percentage of the animals that died within 30 min after drug injection. The s.c. administration of pentylenetetrazol Ž80 mgrkg. induced both clonic and tonic convulsions. In most cases, clonic convulsions were followed by tonic convulsions. As shown in Fig. 2, the latencies to appearance of clonic and tonic convulsions were significantly smaller in BSO-treated mice than in intact or saline-treated mice, indicating that seizure susceptibility was increased by BSO treatment. Lethality was zero in all groups. Of four conditions of BSO treatment tested, the shortening of
K. Abe et al.r Brain Research 854 (2000) 235–238
Fig. 3. The effect of BSO treatment was recovered by supplying exogenous GSH. BSO Ž3.2 mmol. was i.c.v. administered twice 48 and 24 h prior to experiments. Control mice received i.c.v. administration of saline 48 and 24 h prior to experiments. GSH Ž10 nmol. was i.c.v. administered 20 min before s.c. administration of pentylenetetrazol Ž80 mgrkg.. Following administration of pentylenetetrazol, the latency to appearance of clonic convulsion Žleft. or tonic convulsion Žright. was measured. All data are represented as the means"S.E.M. of eight mice. UU P - 0.01 vs. salineqsaline; aP - 0.05, aaP - 0.01 vs. BSOqsaline; Duncan’s multiple range test.
latencies to clonic and tonic convulsions was most remarkable when 3.2 mmol BSO was administered twice 48 and 24 h prior to experiments. To support that the increase of seizure susceptibility by BSO treatment is due to a decrease in GSH level, we examined if the change is recovered by exogenously supplying GSH. GSH was dissolved in saline and administered at a dose of 10 nmol Ž2 mM = 5 ml. into the brain through the implanted i.c.v. guide cannula 20 min before s.c. administration of pentylenetetrazol Ž80 mgrkg.. This dose of GSH alone had no effect on pentylenetetrazol-induced convulsions in intact mice, but significantly attenuated pentylenetetrazol-induced convulsions in BSO-treated mice ŽFig. 3.. We have found for the first time that pentylenetetrazolinduced convulsions are potentiated by treatment with the GSH biosynthesis inhibitor BSO. Comparing four different conditions of BSO treatment, the potentiation of convulsions ŽFig. 2. was parallel to the decrease in brain GSH level ŽFig. 1B.. Since it has previously been reported that BSO treatment causes a selective neuronal death w9,17x, we have also checked possible histological change by preparing paraformaldehyde-fixed, Nissl-stained brain sections. However, no apparent histological change was observed in the brain of mice employed in our present study Žunpublished data.. Furthermore, the change in seizure susceptibility produced by BSO treatment was recovered by supplying GSH. These results suggest that endogenous GSH functions as an anticonvulsant. It is generally supposed that GSH is not easily taken up by the cells. In the present study, the change produced by BSO treatment was recovered by i.c.v. administration of GSH, indicating that extracellular GSH plays a major role in the anticonvulsive action. The concentration of total GSH in the brain is reported approximately 2 mM, most of which is present in the intracellular compartment w2,3,5,13,16x. In our measurement, total GSH level in the brain was 1.49 mlrg wet weight in intact mice, and decreased to 0.47 mlrg wet
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weight following BSO treatment ŽFig. 1B.. On the assumption that 1 g of wet weight of tissue corresponds to 1 ml of water, it is estimated that BSO treatment decreases brain GSH concentration from 1.49 to 0.47 mM. However, 10 nmol was enough for exogenous GSH to recover the functional change produced by BSO treatment. The mean wet weight of adult mouse brain used in our present study was 0.471 g. Supposing that i.c.v. administered GSH is equally distributed in the mouse brain, 10 nmol GSH is estimated to reach 21 mM. Therefore, it is likely that seizure susceptibility is regulated by micromolar concentrations of extracellular GSH, though extracellular GSH concentration may be influenced by the intracellular storage. In conclusion, we have provided evidence that endogenous GSH, especially extracellular GSH, functions as an anticonvulsant. GSH may play a role in modulating neuronal excitability in the brain. Our finding will give new insight into the possible role of GSH as a neuromodulator. Further investigations are underway in our laboratory to elucidate cellular mechanisms underlying the anticonvulsive action of GSH.
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