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Specific glutathione binding sites in pig cerebral cortical synaptic membranes
Glutathione binding to synaptic membranes
Pergamon PII: S0306-4522(99)00442-X
Neuroscience Vol. 95, No. 2, pp. 617–624, 2000 617 Copyright q 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00
www.elsevier.com/locate/neuroscience
SPECIFIC GLUTATHIONE BINDING SITES IN PIG CEREBRAL CORTICAL SYNAPTIC MEMBRANES ´ KY,*† C. A. SHAW,‡ V. VARGA,*§ A. HERMANN,*§ R. DOHOVICS,*§ P. SARANSAARI* and S. S. OJA*k R. JANA *Tampere Brain Research Center, University of Tampere Medical School, Box 607, FIN-33101 Tampere, Finland ‡Department of Ophthalmology, University of British Columbia, Vancouver, British Columbia, Canada §Department of Animal Physiology, Kossuth Lajos University of Science, Debrecen, Hungary k Department of Clinical Physiology, Tampere University Hospital, Tampere, Finland
Abstract—Glutathione (g-glutamylcysteinylglycine) is a neuromodulator at glutamate receptors, but may also act as a neurotransmitter at sites of its own. The Na 1-independent binding of [ 3H]glutathione to pig cortical synaptic membranes was characterized here using glycine, cysteine analogs, dipeptides and glutathione derivatives, and ligands selective for known glutamate receptors. l-Glutamate, pyroglutamate, quinolinate, (S)-5-fluorowillardiine and 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3dione were weak inhibitors at concentrations of 0.5 or 1 mM. d-Glutamate, l- and d-aspartate, glutamine, quisqualate, kynurenate, other N-methyl-d-aspartate receptor ligands and non-N-methyl-d-aspartate receptor ligands failed to displace [ 3H]glutathione. Except for weak inhibition by d-serine (0.5 mM), glycine and other ligands of the glycine co-activatory site in the N-methyl-daspartate receptors had no displacing effect. Similarly, metabotropic glutamate group I, II and III receptor agonists and antagonists and compounds acting at the glutamate uptake sites were generally inactive. Glutathione, oxidized glutathione, S-nitrosoglutathione, g-l-glutamylcysteine, cysteinylglycine, cysteine, cysteamine and cystamine were the most potent displacers (ic50 values in the micromolar range), followed by dithiothreitol, glutathione sulfonate and the S-alkyl derivatives of glutathione (S-methyl-, -ethyl-, -propyl-, -butyl- and -pentylglutathione). l-Homocysteinate and aminomethanesulfonate exhibited a moderate efficacy. Thiokynurenate, a cysteine analog and an antagonist at the N-methyl-d-aspartate receptor glycine co-activatory site, was a potent activator of glutathione binding. At 1 mM, some dipeptides also slightly activated the binding, g-l-glutamylleucine and g-l-glutamyl-GABA being the most effective. The specific binding sites for glutathione in brain synaptic membranes are not identical to any known excitatory amino acid receptor. The cysteinyl moiety is crucial in the binding of glutathione. The oxidation or alkylation of the cysteine thiol group reduces the binding affinity. The strong activation by thiokynurenate may indicate that the glutathione receptor protein contains a modulatory site to which co-agonists may bind and allosterically activate glutathione binding. The novel population of specific binding sites of glutathione gives rise to the possibility that they may have profound effects on synaptic functions in the mammalian central nervous system. The glutathione binding sites may be an important, and for the most part unrecognized, component in signal transduction in the brain. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: glutathione binding, glutamate receptors, cerebral cortical membranes, pig.
g-Glutamylcysteinylglycine (GSH, reduced glutathione) is a phylogenetically ancient molecule. It is ubiquitous in organisms, present in virtually every cell and the most abundant peptide in the CNS. 21,34 GSH is oxidized to glutathione disulfide and thus protects cells against the harmful effects of free radicals continuously generated by metabolic processes. In addition, GSH is involved in other detoxification reactions catalysed by glutathione S-transferases. 21,34 Thus, when located intracellularly, this peptide is essential to cellular function. Each cell which contains GSH is in part protected against chemical changes and oxidizing or poisoning factors. However, if GSH is released from damaged or apoptotic cells it may serve to signal that the host has become a prey, and predators capable of perceiving this signal have an advantage in finding food. In an interesting pioneering study, Loomis 19 demonstrated, in Hydra vulgaris, apparent neurohumoral
effects of GSH which evoked a specific feeding reaction in the animal. The three constituent amino acids of the glutathione molecule are neuroactive. Two of them, glutamate and glycine, are neurotransmitters in the CNS, while cysteine acts as an excitotoxin at the N-methyl-d-aspartate (NMDA) class of ionotropic glutamate receptors. 33 The chemical structure thus tends to make GSH another neuroactive compound which may function as both neuromodulator and neurotransmitter. On the one hand, GSH modulates glutamatergic neurotransmission by interacting with ionotropic glutamate receptors and regulating the neuronal Ca 21 responses and neurotransmitter releases evoked by their selective agonists. 7–9,30,41–43 On the other, GSH has been reported to possess binding sites in the mammalian CNS. 30 In rat cerebral cortical slices, it elicits concentration-dependent excitatory field potentials which are not blocked by any antagonist of ionotropic glutamate receptors. 36,40 These findings have recently led to the hypothesis that GSH is a multifunctional molecule which, in the course of evolution, has preserved some of its signal transduction propensities. 10 The demonstration of selective Na 1-independent binding of GSH to specific receptors at synaptic plasma membranes of the CNS would be a basic proof of this hypothesis. GSH cannot be claimed to be a neurotransmitter without excluding the possible overlap
†To whom correspondence should be addressed. Tel.: 1358-3-215-6879; fax: 1358-3-215-6170. E-mail address: [email protected] (R. Jana´ky) Abbreviations: AMPA, (S)-2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate; GSH, glutathione (reduced); GSNO, S-nitrosoglutathione; gGT, g-glutamyltransferase; GYKI 53655, 1-(4-aminophenyl)-4-methyl7,8-methylenedioxy-4,5-dihydro-3-methylcarbamoyl-2,3-benzodiazepine; NMDA, N-methyl-d-aspartate. 617
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between the known ionotropic and metabotropic glutamate or inhibitory amino acid receptors and the newly discovered GSH binding sites. In order to show that GSH has receptors of its own, we studied whether or not specific ligands of glutamate receptors and glycine analogs displace [ 3H]GSH from its binding sites in pig cerebral cortical synaptic membranes. Furthermore, to shed light on the structure–function relationships, we also carried out a detailed pharmacological characterization of [ 3H]GSH binding using g-glutamyl and other dipeptides, derivatives of cysteine and analogs of GSH. EXPERIMENTAL PROCEDURES
Materials [ 3H]GSH (glycine-2-[ 3H]glutathione, 1.66 PBq/mol) was purchased from New England Nuclear (Boston, MA, U.S.A.). Some g-l-glutamyl dipeptides (g-l-glutamylcysteate, -taurine, -cholaminsulfate, -GABA, -alanine, -b-alanine, -aminoethylphosphonate, -histamine) and pyroglutamate were synthesized by the authors. The homogeneity of GSH and GSH derivatives was checked by thin-layer and ion-exchange chromatographic analyses. No measurable amounts of contaminants were detected in the preparations used and the purity of all peptides was thus greater than 99%. 1-(4-Aminophenyl)-4-methyl-7,8-methylenedioxy-4,5-dihydro-3-methylcarbamoyl-2,3-benzodiazepine (GYKI 53655) was a gift from Dr Istva´n Tarnawa (Institute for Drug Research, Budapest, Hungary) and (1)-2-aminobicyclo[3,1,0]hexane-2,6-dicarboxylate (LY 354740) was from Dr James A. Monn (Lilly Research Laboratories, Eli Lilly & Company, Indianapolis, IN, U.S.A.). All other chemicals were obtained from Sigma (St Louis, MO, U.S.A.) or Tocris Cookson (Bristol, U.K.). Pig brains obtained from a local slaughterhouse were cooled on ice. Synaptic plasma membranes were prepared from the cerebral cortex using the method of Cotman and Taylor 2 as modified by Jenei et al. 11 Binding experiments The membranes, frozen at 2208C for a few days, were thawed and washed twice with 50 mM Tris–acetate (pH 7.4). The final pellets were resuspended in the same buffer and used for [ 3H]GSH binding assays.
The membrane suspensions (300 mg protein/tube) were preincubated at 08C for 10 min in a volume of 0.5 ml with the compounds tested, then 10 nM [ 3H]GSH was added and the final incubation (60 min at 08C, if not otherwise indicated) terminated by filtration through a Whatman GF/B glass fiber filter, followed by three washes with 5 ml of ice-cold buffer using a Brandel (Gaithersburg, MD, U.S.A.) filtration system. The binding has been shown to reach an equilibrium within 60 min, independently of the incubation temperature. 30 The radioactivity bound to the membranes and trapped by the filters was measured by liquid scintillation spectrometry. Non-specific binding was determined in the presence of 1 mM GSH. [ 3H]GSH and other peptides were not broken down when incubated with synaptic membranes at 08C, except for a slight production of GSH from Snitrosoglutathione (GSNO). In the present Tris-buffered incubation medium, 500 mM GSNO yielded 6.4 mM GSH in 60 min. Even with GSNO, no measurable production of glutathione disulfide or free glutamate, glycine or cysteine was discernible. The protein concentration was measured by the method of Lowry et al. 20 The binding assays were always carried out in triplicate and the results usually expressed as mean ^ S.E.M. Calculations Statistical significance was estimated by Student’s t-test using the critical values published by Owen 35 for comparisons between several experiment means and one control. The ic50 values with their 95% confidence limits were calculated using the displacement curves fitted by the Markwardt algorithm. The same non-linear regression analysis was used in the determination of KD and Bmax in the experiments with varying concentrations of [ 3H]GSH. RESULTS
General properties of Hglutathione binding 3
Marked specific binding of [ 3H]GSH was observed in synaptic plasma membrane fractions isolated from the pig cerebral cortex (Fig. 1). The kinetic analyses within the concentration range of 1 nM to 1 mM revealed two binding components with widely differing KD and Bmax values (Table 1). The binding increased by 47.5 ^ 1.3% (mean ^ S.E.M., n 3) in the presence of 2.5 mM CaCl2 when the membranes
Fig. 1. (A) Specific binding of varying concentrations of [ 3H]GSH to pig cerebral cortical synaptic membranes. The inset shows the results in the low nanomolar concentration range. The results are mean values from three experiments. S.E.M. is shown if it exceeds the size of symbols. (B) The same data depicted in a Scatchard v/S vs v plot. The graph demonstrates that the experimental points could be fitted by two saturable binding components. The straight lines show the contribution of these high- and low-affinity bindings to the total. The inset shows the data in the low concentration range. These linear transforms are shown for illustrative purposes only, since the kinetic parameters were computed by non-linear iterative estimation.
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Glutathione binding to synaptic membranes Table 1. Kinetic parameters of [ 3H]glutathione binding to pig cerebral cortical synaptic membranes
Table 3. Effects of N-methyl-d-aspartate and glycine site ligands on the binding of [ 3H]glutathione to pig cerebral cortical synaptic membranes
Component
KD (nM)
Bmax (mmol/kg protein)
Effectors
High affinity Low affinity
9.60 ^ 2.26 1880 ^ 330
0.208 ^ 0.070 13.8 ^ 1.3
The parameters with their 95% confidence limits were estimated from the results shown in Fig. 1A by non-linear optimization using the Markwardt algorithm.
were incubated at 08C. When the incubation temperature was elevated to 378C, the specific binding of 10 nM [ 3H]GSH increased by 120.0 ^ 4.5% (mean ^ S.E.M., n 3) in the absence of CaCl2 and by 186.1 ^ 10.2% (mean ^ S.E.M., n 3) in the presence of 2.5 mM CaCl2. The ic50 values with their 95% confidence limits for non-radioactive GSH were 2.5 (1.7–3.8), 3.9 (2.9–5.2) and 7.4 (5.4–10.2) at 0 and 378C without CaCl2 and at 378C with 2.5 mM CaCl2, respectively. Acivicin, an inhibitor of g-glutamyltransferase (g-GT; EC 2.3.2.2, 0.1 and 1 mM) failed to affect [ 3H]GSH binding (data not shown). Effects of glutamate derivatives and glutamate receptor ligands Of the glutamate derivatives and mixed glutamate receptor ligands (all 1 mM), l- and d-aspartate, d-glutamate, l-glutamine and, quisqualate had no effect on [ 3H]GSH binding to pig cerebral cortical synaptic membranes. With l-glutamate, kynurenate and pyroglutamate, there was slight but significant inhibition (Table 2). Of the NMDA ligands acting at the glutamate agonist or competitive antagonist sites, quinolinate (1 mM) had a significant displacing effect (Table 3). NMDA (1 mM), 1-aminocyclobutane-cis-1,3-dicarboxylate (0.5 mM), 3-[(R)-2-carboxypiperazin-4-yl]propyl-1phosphonate (0.5 mM) and l(1)-2-amino-5-phosphonopentanoate (1 mM) failed to affect [ 3H]GSH binding. Glycine (0.5 and 1 mM) and ligands of the glycine co-activatory site in the NMDA receptors [(1)-1-hydroxy-3-amino-2-pyrrolidone, 1-aminocyclobutanecarboxylate, 7-chlorokynurenate and (RS)-(tetrazol-5-yl)glycine; all 0.5 mM] likewise had no displacing effects (Table 3). In contrast, d-serine (0.5 mM) slightly but significantly diminished the binding. Of the non-NMDA receptor ligands, kainate (0.5 mM), (S)2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA;
NMDA site ligands NMDA cis-ACBD Quinolinate l-AP5 R-CPP Glycine site ligands Glycine d-Serine HA-966 ACBC (RS)-(Tetrazol-5-yl)glycine 7-Chlorokynurenate
Effectors, 1 mM l-Glutamate d-Glutamate l-Aspartate d-Aspartate l-Glutamine Quisqualate Kynurenate Pyroglutamate
Percentage specific binding ^ S.E.M. 90.8 ^ 0.4* 104.6 ^ 4.1 92.8 ^ 4.7 89.8 ^ 3.0 95.9 ^ 0.9 92.9 ^ 0.9 72.3 ^ 10.7** 69.9 ^ 7.8**
The control specific binding in the absence of any displacer was 31.3 ^ 1.1 nmol/kg protein (mean ^ S.E.M., n 30). Mean values^ S.E.M. of three experiments. Significantly different from control: *P , 0.05; **P , 0.01.
103.2 ^ 2.0 99.1 ^ 3.8 59.4 ^ 3.6** 93.0 ^ 9.7 104.0 ^ 3.1 96.6 ^ 2.6 90.7 ^ 1.0* 95.9 ^ 1.4 92.7 ^ 1.6 96.4 ^ 2.0 99.7 ^ 1.5
Abbreviations and concentrations: 1 mM NMDA; cis-ACBD, 0.5 mM 1aminocyclobutane-cis-1,3-dicarboxylate; 1 mM quinolinate; l-AP5, 1 mM l(1)-2-amino-5-phosphonopentanoate; R-CPP, 0.5 mM 3-[(R)2-carboxypiperazin-4-yl]propyl-1-phosphonate; 0.5 mM glycine; 0.5 mM d-serine; HA-966, 0.5 mM (1)-1-hydroxy-3-amino-2-pyrrolidone; ACBC, 0.5 mM 1-aminocyclobutanecarboxylate; 0.5 mM (RS)-(tetrazol-5-yl)glycine; 0.5 mM 7-chlorokynurenate. Mean values ^ S.E.M. of three experiments. Significantly different from the corresponding control: *P , 0.05; **P , 0.01.
0.5 mM), 6-cyano-7-nitroquinoxaline-2,3-dione (0.1 mM), 6,7-dinitroquinoxaline-2,3-dione (0.1 mM), 6,7-dichloroquinoxaline-2,3-dione (0.1 mM) and 5,7-dinitroquinoxaline-2,3dione (0.1 mM) had no effects (Table 4). (S)-5-Fluorowillardiine (0.5 mM) and 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione (0.5 mM dissolved in 4.8% dimethylsulfoximine) slightly but significantly displaced the binding. The binding was seemingly increased at the 0.5 mM concentration of quinoxalines (6-cyano-7-nitroquinoxaline2,3-dione, 6,7-dinitroquinoxaline-2,3-dione and 6,7-dichloroquinoxaline-2,3-dione) and GYKI 53655 (data not shown). However, this was attributable to the 20% dimethylsulfoximine used as solvent in these experiments, because it alone increased the binding equally by 43.3 ^ 13.4% (mean^ S.E.M., n 3). Many agonists or antagonists of the group I, II and III Table 4. Effects of non-N-methyl-d-aspartate receptor ligands on the binding of [ 3H]glutathione to pig cerebral cortical synaptic membranes Effectors
Table 2. Effects of excitatory amino acids, their derivatives and broadspectrum glutamate receptor agonists and antagonists on the binding of [ 3H]glutathione to pig cerebral cortical synaptic membranes
Percentage specific binding ^ S.E.M.
Kainate AMPA (S)-5-Fluorowillardiine CNQX DNQX NBQX DCQX MNQX
Percentage specific binding ^ S.E.M. 95.8 ^ 0.7 103.5 ^ 3.8 89.5 ^ 0.7* 107.3 ^ 5.3 106.7 ^ 4.5 84.4 ^ 1.3** 101.7 ^ 1.8 99.2 ^ 2.7
Abbreviations and concentrations: 0.5 mM kainate; 0.5 mM AMPA; 0.5 mM (S)-5-fluorowillardiine; CNQX, 0.1 mM 6-cyano-7-nitroquinoxaline-2,3-dione; DNQX, 0.1 mM 6,7-dinitroquinoxaline-2,3-dione; NBQX, 0.5 mM 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione; DCQX, 0.1 mM 6,7-dichloroquinoxaline-2,3-dione; MNQX, 0.1 mM 5,6-dinitroquinoxaline-2,3-dione. DCQX and MNQX are also active at the glycine co-activatory sites in the NMDA receptors. Mean values^ S.E.M. of three experiments. Significantly different from control: *P , 0.05; **P , 0.01.
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Table 5. Effects of metabotropic glutamate receptor ligands on the binding of [ 3H]glutathione to pig cerebral cortical synaptic membranes Effectors, 0.5 mM Group I receptor ligands 1S,3R-ACPD S-DHPG l-AP3 S-4CPG AIDA
Percentage specific binding ^ S.E.M.
99.5 ^ 2.0 94.0 ^ 1.9 75.7 ^ 1.4* 94.5 ^ 1.2 93.1 ^ 1.2
Group II receptor ligands LY 354740 DCG IV EGLU MTPG CPPG
94.8 ^ 0.9 91.3 ^ 5.3 108.1 ^ 4.5 106.4 ^ 4.4 93.8 ^ 1.8
Group III receptor ligands l-AP4 l-SOP MSOP MPPG
106.1 ^ 4.7 100.0 ^ 1.7 100.4 ^ 1.9 100.8 ^ 3.5
Glutamate uptake ligands l-trans-2,4-PDC trans-ACBD
136.3 ^ 7.5* 92.6 ^ 4.7
Abbreviations: 1S,3R-ACPD, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate; S-DHPG, (S)-3,5-dihydroxyphenylglycine; l-AP3, l(1)-2-amino3-phosphonopropionate; S-4CPG, (S)-4-carboxyphenylglycine; AIDA, (RS)-1-aminoindan-1,5-dicarboxylate; LY 354740, (1)-2-aminobicyclo[3,1,0]hexane-2,6-dicarboxylate; DCG IV, (2S,2 0 R,3 0 R)-2-(2 0 ,3 0 dicarboxycyclopropyl)glycine; EGLU, (2S)-2-ethylglutamate; MTPG, (RS)-2-methyl-4-tetrazolylphenylglycine; CPPG, (RS)-2-cyclopropyl-4phosphonophenylglycine; l-AP4, l(1)-2-amino-4-phosphonobutyrate; l-SOP, l-serine-O-phosphate; MSOP, (RS)-2-methylserine-O-phosphate; MPPG, (RS)-2-methyl-4-phosphonophenylglycine; l-trans-2,4PDC, l-trans-pyrrolidine-2,4-dicarboxylate; trans-ACBD, 1-aminocyclobutane-trans-1,3-dicarboxylate. 1S,3R-ACPD also activates group II receptors. DCG IV is also an agonist at NMDA receptors at this concentration. MTPG and CPPG are also ligands of group III receptors. Mean values ^ S.E.M. of three experiments. Significantly different from control: *P , 0.01.
metabotropic glutamate receptors were also tested (Table 5). Of these, l(1)-2-amino-3-phosphonopropionate (0.5 mM) was an effective but weak displacer. All other metabotropic glutamate receptor ligands proved to be inactive. Two compounds affecting the glutamate uptake sites, 1aminocyclobutane-trans-1,3-dicarboxylate (0.5 mM) and ltrans-pyrrolidine-2,4-dicarboxylate (0.5 mM), were also tested. The former was inactive, while the latter enhanced the binding by 36.3 ^ 7.5% (mean ^ S.E.M., n 3; Table 5). Effects of cysteine derivatives and sulfhydryl compounds Thiokynurenate had a striking activatory effect on [ 3H]GSH binding which was clearly concentration dependent (Fig. 2A). The effects of the other cysteine derivatives tested (1 mM) are shown in Table 6. l-Cysteine, l-cysteamine, l-cystamine and dithiothreitol were the most potent displacers of [ 3H]GSH binding. Their effects were concentration dependent, as shown in Fig. 2B with cysteine and cysteamine. The displacement curve for cystamine was very similar (not shown). The ic50 values of these most active compounds were in the high micromolar range. l-Homocysteinate and aminomethanesulfonate (1 mM) exhibited only moderate efficacy (35–38%) as displacers (Table 6). l-Cysteate, taurine, homotaurine, cystathionine (all 1 mM) and l-cystine (0.2 mM) were inactive. Hypotaurine slightly but significantly enhanced the binding. Effects of dipeptides and glutathione derivatives Most of the g-glutamyl dipeptides tested were without significant effect (Table 7). g-d-Glutamylglycine and g-lglutamylphenylalanine were weak and g-l-glutamylcysteine and l-cysteinylglycine fairly strong displacers. Glycylglycine, g-l-glutamylcysteate, g-l-glutamyl-GABA and g-l-glutamylleucine enhanced the binding in this order of increasing potency (Table 7). The enhancements by g-l-glutamyl-GABA and gl-glutamylleucine were concentration dependent (Fig. 2A). All GSH derivatives tested effectively displaced [ 3H]GSH (Table 8). GSH and GSNO were the most potent (ic50 values
Fig. 2. (A) Concentration dependence of the activation of [ 3H]GSH binding to pig cerebral cortical membranes by thiokynurenate (W), g-l-glutamyl-l-leucine (X) and g-l-glutamyl-GABA (K). (B) Displacement of [ 3H]GSH binding by GSNO (W), l-cysteine (X) and l-cysteamine (K). The results are mean values of three experiments with S.E.M. less than 5% of the mean for each experimental point.
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Glutathione binding to synaptic membranes Table 6. Effects of cysteine derivatives and sulfhydryl compounds on the binding of [ 3H]glutathione to pig cerebral cortical synaptic membranes Effectors, 1 mM
Percentage specific binding ^ S.E.M.
l-Cysteine l-Cystine† l-Cysteate l-Homocysteate Aminomethanesulfonate Hypotaurine Taurine Homotaurine l-Cysteamine l-Cystamine l-Cystathione Dithiothreitol
31.3 ^ 0.6* 91.6 ^ 3.6 96.3 ^ 3.6 61.8 ^ 1.1* 65.4 ^ 2.6* 123.0 ^ 4.3* 96.0 ^ 3.6 96.4 ^ 2.4 22.0 ^ 0.5* 12.9 ^ 1.6* 95.8 ^ 14.9 35.1 ^ 4.6*
Mean values ^ S.E.M. of three experiments. Significantly different from control: *P , 0.01. †The concentration of cystine was 0.2 mM.
in the low micromolar range), followed by g-l-glutamylcysteine and l-cysteine (ic50 values in the high micromolar range) (Table 9). Glutathione sulfonate and the S-alkyl derivatives of glutathione exhibited moderate efficacy (Table 8). The displacing effects were concentration dependent, as shown in Fig. 2B with GSNO. Effects of inhibitory glycine and GABA receptor ligands Some constituent amino acids of the g-glutamyl dipeptides tested, known to be active at other neurotransmitter receptors, were also screened for their displacing efficacy, but none of them was found to be active. For instance, GABA, 5-aminopentanoate, b-alanine, l-alanine, l-serine and proline (all 1 mM) exhibited no effect (data not shown). DISCUSSION
In the present article, specific binding sites for GSH in the Table 7. Effects of dipeptides on the binding of [ 3H]glutathione to pig cerebral cortical synaptic membranes Effectors, 1 mM g-l-Glutamylglycine g-d-Glutamylglycine g-l-Glutamylalanine g-l-Glutamyl-b-alanine g-l-Glutamyl-GABA g-l-Glutamylleucine a-l-Glutamylglutamate g-l-Glutamylglutamate g-l-Glutamylcysteine g-l-Glutamylcysteate g-l-Glutamyltaurine g-d-Glutamyltaurine g-l-Glutamylcholaminsulfate g-l-Glutamyl-AEP g-l-Glutamyltyrosine g-l-Glutamylhistidine g-l-Glutamylhistamine g-l-Glutamylphenylalanine Glycylglycine l-Cysteinylglycine
Percentage specific binding ^ S.E.M. 102.3 ^ 1.8 85.7 ^ 3.7* 87.3 ^ 4.4 93.2 ^ 3.4 170.1 ^ 4.0** 208.9 ^ 1.1** 102.3 ^ 5.0 105.4 ^ 3.5 22.2 ^ 0.3** 118.9 ^ 3.0** 97.6 ^ 6.3 101.1 ^ 1.4 91.7 ^ 3.4 99.5 ^ 2.7 110.3 ^ 3.1 99.7 ^ 3.1 86.9 ^ 4.4 83.6 ^ 1.9** 116.9 ^ 2.9* 50.3 ^ 0.8**
g-l-Glutamyl-AEP, g-l-glutamylaminoethylphosphate. Mean values ^ S.E.M. of three experiments. Significantly different from control: *P , 0.05; **P , 0.01.
Table 8. Effects of glutathione derivatives on the binding of [ 3H]glutathione to pig cerebral cortical synaptic membranes Effectors, 1 mM Oxidized glutathione S-Nitrosoglutathione Glutathione sulfonate S-Methylglutathione S-Ethylglutathione S-Propylglutathione S-Butylglutathione S-Pentylglutathione
Percentage specific binding ^ S.E.M. 34.3 ^ 1.4 0.0 ^ 0.9 74.2 ^ 4.1 56.6 ^ 2.6 68.4 ^ 1.4 44.4 ^ 1.5 70.2 ^ 4.5 49.1 ^ 1.6
Mean values ^ S.E.M. of four to eight experiments. All derivatives significantly (P , 0.01) diminished the binding.
porcine cortex were demonstrated which are qualitatively similar to those described by other researchers in rodent brains. 4,5,14,15,25–28 In our preparation, GSH bound at 08C to two populations of binding sites. This finding is similar to that of Ogita and Yoneda, 26 who reported that [ 3H]GSH binding consists of two components, one temperature-independent high affinity and the other temperature-dependent low affinity. In the present study, the very-low-affinity, high-capacity binding at 378C, enhanced in the presence of Ca 21 and not saturated at concentrations below 1 mM, may represent binding of GSH to sites other than plasma membrane receptors. These sites may be GSH transporters in plasma membranes or, although our preparation was highly purified, even transport sites in mitochondrial membranes. To exclude the binding of [ 3H]GSH to transporters, we carried out all subsequent binding assays at 08C in Tris–acetate buffer in the absence of Na 1 and Ca 21 ions. Immunocytochemically, GSH has been located in the CNS in both neurons and glia, although in none of the brain regions studied did the glial location predominate. 6 Within the brain, marked regional heterogeneity with respect to autoradiographically visualized [ 3H]GSH binding sites exists. 39 GSH binding sites have also been found in primary cultures of rat cortical astrocytes, 4 as well as in certain peripheral tissues, 30 but these elements did not constitute any major source of GSH binding sites in our synaptic membrane preparations. In order to ensure that the detected binding sites represent neurotransmitter receptors, it is first necessary to establish whether GSH could be bound to other proteins in the CNS and to take steps to exclude this possibility. In the mammalian CNS, radiolabeled GSH could potentially bind Table 9. Displacement of [ 3H]glutathione by glutathione and cysteine derivatives in pig cerebral cortical synaptic membranes Displacers Reduced glutathione Oxidized glutathione S-Nitrosoglutathione g-l-Glutamylcysteine l-Cysteinylglycine l-Cysteine l-Cysteamine l-Cystamine
ic50, mM (95% confidence limit) 2.49 (2.10–2.94) 380 (331–431) 12.5 (10.9–14.3) 81.8 (49.7–135.7) 190 (151–240) 96.6 (69.7–133.8) 139 (113–171) 331 (297–368)
The membranes (0.3 mg protein/tube) were preincubated with different (100 nM to 1 mM) concentrations of the displacers at 08C for 10 min, whereafter 10 nM [ 3H]GSH was added and the incubation continued for a further 60 min.
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to non-receptor binding sites in the membranes, for example Na 1-dependent and -independent GSH transport proteins 12 and membrane-bound cell surface enzymes such as glutathione transferases (EC 2.5.1.18) and g-GT. Since the binding assays were performed in Tris–acetate buffer, GSH binding does not require the Na 1 and Cl 2 ions which provide the driving force for transmembrane transport of many neurotransmitters and amino acids. Moreover, of the two ligands of the Na 1-dependent high-affinity glutamate uptake sites tested, 1-aminocyclobutane-trans-1,3-dicarboxylate 3 was totally inactive and l-trans-pyrrolidine-2,4-dicarboxylate 23 slightly enhancing. Radiolabeled GSH thus labels sites different from glutamate transporter proteins. Pasqualotto et al. 36 have shown recently that [ 3H]GSH also does not label glutathione S-transferases in the rodent brain. In the present set of experiments acivicin, an inhibitor of g-GT, likewise failed to displace [ 3H]GSH. Similarly, many g-glutamyl dipeptides, known to be substrates of this enzyme, also failed to affect GSH binding, demonstrating that [ 3H]GSH is also not bound to the active site of g-GT. Since [ 3H]GSH was not broken down upon incubations, the bound radioactivity represents GSH binding and not that of a labeled glycine moiety liberated from the GSH molecule. The insensitivity of binding to glycine and glycinergic compounds also strongly corroborates this inference. Of all peptides tested, only the effect of GSNO could be partly attributable to the breakdown product of the parent compound. Since GSH at micromolar concentrations displaces ligands of all ionotropic glutamate receptors, and at millimolar concentrations also glycine, from the co-agonist site in the NMDA receptors, 7,9,11,29–32,41,43 it is obvious that [ 3H]GSH could label glutamate receptors. Such binding would be consistent with the neuromodulatory but not the neurotransmitter role of GSH. To exclude the possible overlap between the known glutamate receptors and the novel GSH binding sites, we tested glutamate analogs, mixed glutamate receptor ligands, NMDA receptor ligands, glycine, the ligands of the glycine co-activatory site in NMDA receptors, non-NMDA receptor ligands, agonists or antagonists at group I, II and III metabotropic glutamate receptors, and inhibitory amino acids. All were either totally inactive or exhibited only minor effects. Only the NMDA receptor subtype-specific agonist quinolinate 24,37 was a moderate displacer. GSH thus displaces glutamate, but glutamate not GSH, from specific binding sites. Thiokynurenate, which can be designated a cysteine derivative, had a strikingly strong concentration-dependent activatory effect, while kynurenate was inactive. These results from our displacement experiments show that GSH possesses specific binding sites in brain synaptic membranes which are not identical to any known glutamate receptor. Evidence for an independent GSH receptor population(s) has also been provided in a series of electrophysiological studies. Using field potential and single-unit recording methods, Shaw and colleagues examined the responses to bath-applied GSH on rat cortical slices. 1,10,36,40 In brief, it emerged that GSH is able to elicit a dose-dependent depolarization which appears to be Na 1 dependent. This response, unlike that for NMDA or cysteine, cannot be blocked by co-application of NMDA antagonists or by the removal of external Ca 21. Nor is it affected by the AMPA receptor antagonists 6,7-dinitroquinoxaline-2,3-dione or 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione. In keeping with the present strong activation
of [ 3H]GSH binding, thiokynurenate also greatly potentiates GSH-evoked depolarization in the electrophysiological studies described above. 10 When the pharmacological profiles of GSH binding in different synaptic plasma membrane preparations in different species and brain regions are compared, 10 they seem to exhibit a common feature, namely insensitivity to glutamate analogs and glutamate receptor ligands. This again corroborates the view that no overlap exists between the known glutamate receptors and the novel GSH sites. The slight pharmacological differences in different species and brain regions 10 may be indicative of the existence of more than one subtype of GSH binding sites. One striking difference is the relative sensitivity of GSH binding to metabotropic glutamate receptor ligands [e.g., quisqualate and l-(1)-2-amino-4butyrate] when tested in synaptic membranes isolated from the whole brain. The GSH receptors in subcortical regions may thus have a pharmacological profile distinct from that of cortical receptors. The displacement of the binding by l-glutamate itself in all preparations must draw our attention to the possibility that the GSH receptor protein exhibits structural similarities to glutamate receptors, in spite of it differing from any known glutamate receptor. In this respect it may be of significance that two glutamate receptor channel subunits (d1 and d2, the “orphan” subunits) so far remain unassigned. 18,36,38,44 The present binding data and those on the rat cortex 36 are strikingly consistent. In addition, the basic pharmacology of the GSH binding sites described in these two preparations is mirrored by the results of field potential recordings from rat cortical slices cited above. Together, these data appear to support the hypothesis that GSH acts as a neurotransmitter at its own population(s) of receptors. In this, GSH appears to have preserved its ancient role as a signal transduction molecule throughout phylogeny, 16 in addition to its roles as an antioxidant and detoxifier. If this is the case, it functions as a neurotransmitter and GSH receptors participate in information transfer in the mammalian CNS. The next question is whether or not different thiol-containing compounds, cysteine analogs, dipeptides and tripeptide derivatives of glutathione displace [ 3H]GSH from its specific binding sites. Testing their effects on the binding revealed a number of important structure–function relationships. Of the cysteine derivatives, cysteine, cysteamine and cystamine were the most potent displacers, with ic50 values in the micromolar range, and only those dipeptides which contain a cysteinyl moiety displace GSH. Likewise, only dipeptides with a similar composition have produced GSH-like excitatory field potentials in cortical-wedge recordings. 36 It may thus be inferred that the cysteinyl moiety in the molecule is crucial in GSH binding. Dithiothreitol, a thiol-reducing agent, was a relatively strong displacer, which would also point to the importance of the free thiol group in GSH binding. The oxidation or alkylation of the free thiol group in the GSH molecule diminishes the binding affinity. This in turn indicates that the neuromodulatory and neurotransmitter functions of the GSH molecule stem from different constituents. There is overwhelming evidence on redox modulation of glutamate receptors by the sulfhydryl group in GSH. 9,17,30 The g-glutamyl moiety is crucial in GSH binding to glutamate receptors, 11,41,43 while the cysteinyl residue forms the basis of binding to specific GSH sites. The strong activation by thiokynurenate but not by kynurenate may indicate that the GSH
Glutathione binding to synaptic membranes
receptor protein contains a strictly selective modulatory site to which co-agonists may bind and allosterically activate GSH binding. However, the nature of this activation calls for further elucidation. The possible existence of endogenous co-agonist(s), structurally related to synthetic thiokynurenate, which could allosterically activate GSH binding, is another open question warranting investigation. GSH has evinced a number of roles in biological systems. 13 Crucial amongst these are actions as an antioxidant and free radical scavenger, 22 and in the modulation of ionotropic glutamate receptor functions. 9,42,43 The present results add another role, that of a neurotransmitter, a role which alone or in concert with those already demonstrated may alter our current conceptions of its synaptic functions in the CNS. CONCLUSIONS
The specific binding sites for GSH differ from any known
623
excitatory or inhibitory amino acid receptor. The cysteinyl moiety is crucial in the binding process, since oxidation or alkylation of the cysteine thiol group diminishes the binding affinity. The receptor protein may contain a site to which coagonists bind and allosterically activate the binding. The present data suggest that GSH binding sites are distinct receptors in some regions of the mammalian CNS. Such a conclusion leads to a further one, namely that GSH can act as a neurotransmitter on cells possessing such receptors. GSH binding sites may thus be an important, largely unrecognized, component in signal transduction in some neural circuits.
Acknowledgements—The skilful technical assistance of Ms Oili Pa¨a¨kko¨nen and Ms Sari Luokkala, and the financial support of the Medical Research Fund of Tampere University Hospital, the Academy of Finland and OTKA-23888, Hungary, are gratefully acknowledged.
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P., Rinvik E., Huster D., Reichelt W. and Ottersen O. P. (1998) Antibodies to glutathione: production, characterization, and immunocytochemical application to the central nervous system. In Glutathione in the Nervous System (ed. Shaw C. A.), pp. 63–88. Taylor & Francis, Washington, DC. Jana´ky R., Varga V., Saransaari P. and Oja S. S. (1993) Glutathione modulates the N-methyl-d-aspartate receptor-activated calcium influx into cultured rat cerebellar granule cells. Neurosci. Lett. 156, 153–157. Jana´ky R., Varga V., Saransaari P. and Oja S. S. (1994) Release of [ 3H]GABA evoked by glutamate agonists from hippocampal slices: effects of dithiothreitol and glutathione. Neurochem. Int. 24, 575–582. Jana´ky R., Varga V., Jenei Zs., Saransaari P. and Oja S. S. (1998) Glutathione and glutathione derivatives: possible modulators of ionotropic glutamate receptors. In Glutathione in the Nervous System (ed. Shaw C. A.), pp. 163–196. Taylor & Francis, Washington, DC. 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A., Wagey R. and Krieger C. (1994) Characterization, distribution, and protein kinase C-mediated regulation of [ 35S]glutathione binding sites in mouse and human spinal cord. J. Neurochem. 63, 155–160. Lenhoff H. M. (1998) The discovery of the GSH receptor in hydra and its evolutionary significance. In Glutathione in the Nervous System (ed. Shaw C. A.), pp. 25–43. Taylor & Francis, Washington, DC. Lipton S. A. and Stamler J. S. (1994) Actions of redox-related congeners of nitric oxide at the NMDA receptor. Neuropharmacology 33, 1229–1233. Lomeli H., Sprengel R., Laurie D. J., Ko¨hr G., Herb A., Seeburg P. H. and Wisden W. (1993) The rat delta-1 and delta-2 subunits extend the excitatory amino acid receptor family. Fedn Eur. biochem. Socs Lett. 315, 318–322. Loomis W. F. (1955) Glutathione control of the specific feeding reactions of hydra. Ann. N. Y. Acad. Sci. 62, 209–228. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265–275. Martin H. and MacIlwain H. (1959) Glutathione, oxidized and reduced, in the brain and isolated cerebral tissue. Biochem. J. 71, 275–280. Meister A. and Anderson M. E. (1983) Glutathione. A. Rev. Biochem. 52, 711–760. Mitrovic A. D. and Johnston G. A. (1994) Regional differences in the inhibition of l-glutamate and l-aspartate sodium-dependent high affinity uptake systems in rat CNS synaptosomes by l-trans-pyrrolidine-2,4-dicarboxylic, threo-3-hydroxy-d-aspartate and d-aspartate. Neurochem. Int. 24, 583–588. Monaghan D. T. and Beaton J. A. (1991) Quinolinate differentiates between forebrain and cerebellar NMDA receptors. Eur. J. Pharmac. 194, 123–125. Ogita K. and Yoneda Y. (1987) Possible presence of [ 3H]glutathione (GSH) binding sites in synaptic membranes from rat brain. Neurosci. Res. 4, 486–496. Ogita K. and Yoneda Y. (1988) Temperature-dependent and -independent apparent binding activities of [ 3H]glutathione in brain synaptic membranes. Brain Res. 463, 37–46. Ogita K. and Yoneda Y. (1989) Selective potentiation by l-cysteine of apparent binding activity of [ 3H]glutathione in synaptic membranes of rat brain. Biochem. Pharmac. 38, 1499–1505. Ogita K., Ogawa Y. and Yoneda Y. (1988) Apparent binding activities of [ 3H]glutathione in rat central and peripheral tissues. Neurochem. Int. 13, 493–497. Ogita K., Enomoto R., Nakahara F., Ishitsubo N. and Yoneda Y. (1995) A possible role of glutathione as an endogenous agonist at the N-methyl-daspartate recognition domain in rat brain. J. Neurochem. 64, 1088–1096. Ogita K., Shuto M., Maeda H., Minami T. and Yoneda Y. (1998) Possible modulation by glutathione of glutamatergic neurotransmission. In Glutathione in the Nervous System (ed. Shaw C. A.), pp. 137–161. Taylor & Francis, Washington, DC.
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Pergamon PII: S0306-4522(99)00442-X
Neuroscience Vol. 95, No. 2, pp. 617–624, 2000 617 Copyright q 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00
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SPECIFIC GLUTATHIONE BINDING SITES IN PIG CEREBRAL CORTICAL SYNAPTIC MEMBRANES ´ KY,*† C. A. SHAW,‡ V. VARGA,*§ A. HERMANN,*§ R. DOHOVICS,*§ P. SARANSAARI* and S. S. OJA*k R. JANA *Tampere Brain Research Center, University of Tampere Medical School, Box 607, FIN-33101 Tampere, Finland ‡Department of Ophthalmology, University of British Columbia, Vancouver, British Columbia, Canada §Department of Animal Physiology, Kossuth Lajos University of Science, Debrecen, Hungary k Department of Clinical Physiology, Tampere University Hospital, Tampere, Finland
Abstract—Glutathione (g-glutamylcysteinylglycine) is a neuromodulator at glutamate receptors, but may also act as a neurotransmitter at sites of its own. The Na 1-independent binding of [ 3H]glutathione to pig cortical synaptic membranes was characterized here using glycine, cysteine analogs, dipeptides and glutathione derivatives, and ligands selective for known glutamate receptors. l-Glutamate, pyroglutamate, quinolinate, (S)-5-fluorowillardiine and 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3dione were weak inhibitors at concentrations of 0.5 or 1 mM. d-Glutamate, l- and d-aspartate, glutamine, quisqualate, kynurenate, other N-methyl-d-aspartate receptor ligands and non-N-methyl-d-aspartate receptor ligands failed to displace [ 3H]glutathione. Except for weak inhibition by d-serine (0.5 mM), glycine and other ligands of the glycine co-activatory site in the N-methyl-daspartate receptors had no displacing effect. Similarly, metabotropic glutamate group I, II and III receptor agonists and antagonists and compounds acting at the glutamate uptake sites were generally inactive. Glutathione, oxidized glutathione, S-nitrosoglutathione, g-l-glutamylcysteine, cysteinylglycine, cysteine, cysteamine and cystamine were the most potent displacers (ic50 values in the micromolar range), followed by dithiothreitol, glutathione sulfonate and the S-alkyl derivatives of glutathione (S-methyl-, -ethyl-, -propyl-, -butyl- and -pentylglutathione). l-Homocysteinate and aminomethanesulfonate exhibited a moderate efficacy. Thiokynurenate, a cysteine analog and an antagonist at the N-methyl-d-aspartate receptor glycine co-activatory site, was a potent activator of glutathione binding. At 1 mM, some dipeptides also slightly activated the binding, g-l-glutamylleucine and g-l-glutamyl-GABA being the most effective. The specific binding sites for glutathione in brain synaptic membranes are not identical to any known excitatory amino acid receptor. The cysteinyl moiety is crucial in the binding of glutathione. The oxidation or alkylation of the cysteine thiol group reduces the binding affinity. The strong activation by thiokynurenate may indicate that the glutathione receptor protein contains a modulatory site to which co-agonists may bind and allosterically activate glutathione binding. The novel population of specific binding sites of glutathione gives rise to the possibility that they may have profound effects on synaptic functions in the mammalian central nervous system. The glutathione binding sites may be an important, and for the most part unrecognized, component in signal transduction in the brain. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: glutathione binding, glutamate receptors, cerebral cortical membranes, pig.
g-Glutamylcysteinylglycine (GSH, reduced glutathione) is a phylogenetically ancient molecule. It is ubiquitous in organisms, present in virtually every cell and the most abundant peptide in the CNS. 21,34 GSH is oxidized to glutathione disulfide and thus protects cells against the harmful effects of free radicals continuously generated by metabolic processes. In addition, GSH is involved in other detoxification reactions catalysed by glutathione S-transferases. 21,34 Thus, when located intracellularly, this peptide is essential to cellular function. Each cell which contains GSH is in part protected against chemical changes and oxidizing or poisoning factors. However, if GSH is released from damaged or apoptotic cells it may serve to signal that the host has become a prey, and predators capable of perceiving this signal have an advantage in finding food. In an interesting pioneering study, Loomis 19 demonstrated, in Hydra vulgaris, apparent neurohumoral
effects of GSH which evoked a specific feeding reaction in the animal. The three constituent amino acids of the glutathione molecule are neuroactive. Two of them, glutamate and glycine, are neurotransmitters in the CNS, while cysteine acts as an excitotoxin at the N-methyl-d-aspartate (NMDA) class of ionotropic glutamate receptors. 33 The chemical structure thus tends to make GSH another neuroactive compound which may function as both neuromodulator and neurotransmitter. On the one hand, GSH modulates glutamatergic neurotransmission by interacting with ionotropic glutamate receptors and regulating the neuronal Ca 21 responses and neurotransmitter releases evoked by their selective agonists. 7–9,30,41–43 On the other, GSH has been reported to possess binding sites in the mammalian CNS. 30 In rat cerebral cortical slices, it elicits concentration-dependent excitatory field potentials which are not blocked by any antagonist of ionotropic glutamate receptors. 36,40 These findings have recently led to the hypothesis that GSH is a multifunctional molecule which, in the course of evolution, has preserved some of its signal transduction propensities. 10 The demonstration of selective Na 1-independent binding of GSH to specific receptors at synaptic plasma membranes of the CNS would be a basic proof of this hypothesis. GSH cannot be claimed to be a neurotransmitter without excluding the possible overlap
†To whom correspondence should be addressed. Tel.: 1358-3-215-6879; fax: 1358-3-215-6170. E-mail address: [email protected] (R. Jana´ky) Abbreviations: AMPA, (S)-2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate; GSH, glutathione (reduced); GSNO, S-nitrosoglutathione; gGT, g-glutamyltransferase; GYKI 53655, 1-(4-aminophenyl)-4-methyl7,8-methylenedioxy-4,5-dihydro-3-methylcarbamoyl-2,3-benzodiazepine; NMDA, N-methyl-d-aspartate. 617
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between the known ionotropic and metabotropic glutamate or inhibitory amino acid receptors and the newly discovered GSH binding sites. In order to show that GSH has receptors of its own, we studied whether or not specific ligands of glutamate receptors and glycine analogs displace [ 3H]GSH from its binding sites in pig cerebral cortical synaptic membranes. Furthermore, to shed light on the structure–function relationships, we also carried out a detailed pharmacological characterization of [ 3H]GSH binding using g-glutamyl and other dipeptides, derivatives of cysteine and analogs of GSH. EXPERIMENTAL PROCEDURES
Materials [ 3H]GSH (glycine-2-[ 3H]glutathione, 1.66 PBq/mol) was purchased from New England Nuclear (Boston, MA, U.S.A.). Some g-l-glutamyl dipeptides (g-l-glutamylcysteate, -taurine, -cholaminsulfate, -GABA, -alanine, -b-alanine, -aminoethylphosphonate, -histamine) and pyroglutamate were synthesized by the authors. The homogeneity of GSH and GSH derivatives was checked by thin-layer and ion-exchange chromatographic analyses. No measurable amounts of contaminants were detected in the preparations used and the purity of all peptides was thus greater than 99%. 1-(4-Aminophenyl)-4-methyl-7,8-methylenedioxy-4,5-dihydro-3-methylcarbamoyl-2,3-benzodiazepine (GYKI 53655) was a gift from Dr Istva´n Tarnawa (Institute for Drug Research, Budapest, Hungary) and (1)-2-aminobicyclo[3,1,0]hexane-2,6-dicarboxylate (LY 354740) was from Dr James A. Monn (Lilly Research Laboratories, Eli Lilly & Company, Indianapolis, IN, U.S.A.). All other chemicals were obtained from Sigma (St Louis, MO, U.S.A.) or Tocris Cookson (Bristol, U.K.). Pig brains obtained from a local slaughterhouse were cooled on ice. Synaptic plasma membranes were prepared from the cerebral cortex using the method of Cotman and Taylor 2 as modified by Jenei et al. 11 Binding experiments The membranes, frozen at 2208C for a few days, were thawed and washed twice with 50 mM Tris–acetate (pH 7.4). The final pellets were resuspended in the same buffer and used for [ 3H]GSH binding assays.
The membrane suspensions (300 mg protein/tube) were preincubated at 08C for 10 min in a volume of 0.5 ml with the compounds tested, then 10 nM [ 3H]GSH was added and the final incubation (60 min at 08C, if not otherwise indicated) terminated by filtration through a Whatman GF/B glass fiber filter, followed by three washes with 5 ml of ice-cold buffer using a Brandel (Gaithersburg, MD, U.S.A.) filtration system. The binding has been shown to reach an equilibrium within 60 min, independently of the incubation temperature. 30 The radioactivity bound to the membranes and trapped by the filters was measured by liquid scintillation spectrometry. Non-specific binding was determined in the presence of 1 mM GSH. [ 3H]GSH and other peptides were not broken down when incubated with synaptic membranes at 08C, except for a slight production of GSH from Snitrosoglutathione (GSNO). In the present Tris-buffered incubation medium, 500 mM GSNO yielded 6.4 mM GSH in 60 min. Even with GSNO, no measurable production of glutathione disulfide or free glutamate, glycine or cysteine was discernible. The protein concentration was measured by the method of Lowry et al. 20 The binding assays were always carried out in triplicate and the results usually expressed as mean ^ S.E.M. Calculations Statistical significance was estimated by Student’s t-test using the critical values published by Owen 35 for comparisons between several experiment means and one control. The ic50 values with their 95% confidence limits were calculated using the displacement curves fitted by the Markwardt algorithm. The same non-linear regression analysis was used in the determination of KD and Bmax in the experiments with varying concentrations of [ 3H]GSH. RESULTS
General properties of Hglutathione binding 3
Marked specific binding of [ 3H]GSH was observed in synaptic plasma membrane fractions isolated from the pig cerebral cortex (Fig. 1). The kinetic analyses within the concentration range of 1 nM to 1 mM revealed two binding components with widely differing KD and Bmax values (Table 1). The binding increased by 47.5 ^ 1.3% (mean ^ S.E.M., n 3) in the presence of 2.5 mM CaCl2 when the membranes
Fig. 1. (A) Specific binding of varying concentrations of [ 3H]GSH to pig cerebral cortical synaptic membranes. The inset shows the results in the low nanomolar concentration range. The results are mean values from three experiments. S.E.M. is shown if it exceeds the size of symbols. (B) The same data depicted in a Scatchard v/S vs v plot. The graph demonstrates that the experimental points could be fitted by two saturable binding components. The straight lines show the contribution of these high- and low-affinity bindings to the total. The inset shows the data in the low concentration range. These linear transforms are shown for illustrative purposes only, since the kinetic parameters were computed by non-linear iterative estimation.
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Glutathione binding to synaptic membranes Table 1. Kinetic parameters of [ 3H]glutathione binding to pig cerebral cortical synaptic membranes
Table 3. Effects of N-methyl-d-aspartate and glycine site ligands on the binding of [ 3H]glutathione to pig cerebral cortical synaptic membranes
Component
KD (nM)
Bmax (mmol/kg protein)
Effectors
High affinity Low affinity
9.60 ^ 2.26 1880 ^ 330
0.208 ^ 0.070 13.8 ^ 1.3
The parameters with their 95% confidence limits were estimated from the results shown in Fig. 1A by non-linear optimization using the Markwardt algorithm.
were incubated at 08C. When the incubation temperature was elevated to 378C, the specific binding of 10 nM [ 3H]GSH increased by 120.0 ^ 4.5% (mean ^ S.E.M., n 3) in the absence of CaCl2 and by 186.1 ^ 10.2% (mean ^ S.E.M., n 3) in the presence of 2.5 mM CaCl2. The ic50 values with their 95% confidence limits for non-radioactive GSH were 2.5 (1.7–3.8), 3.9 (2.9–5.2) and 7.4 (5.4–10.2) at 0 and 378C without CaCl2 and at 378C with 2.5 mM CaCl2, respectively. Acivicin, an inhibitor of g-glutamyltransferase (g-GT; EC 2.3.2.2, 0.1 and 1 mM) failed to affect [ 3H]GSH binding (data not shown). Effects of glutamate derivatives and glutamate receptor ligands Of the glutamate derivatives and mixed glutamate receptor ligands (all 1 mM), l- and d-aspartate, d-glutamate, l-glutamine and, quisqualate had no effect on [ 3H]GSH binding to pig cerebral cortical synaptic membranes. With l-glutamate, kynurenate and pyroglutamate, there was slight but significant inhibition (Table 2). Of the NMDA ligands acting at the glutamate agonist or competitive antagonist sites, quinolinate (1 mM) had a significant displacing effect (Table 3). NMDA (1 mM), 1-aminocyclobutane-cis-1,3-dicarboxylate (0.5 mM), 3-[(R)-2-carboxypiperazin-4-yl]propyl-1phosphonate (0.5 mM) and l(1)-2-amino-5-phosphonopentanoate (1 mM) failed to affect [ 3H]GSH binding. Glycine (0.5 and 1 mM) and ligands of the glycine co-activatory site in the NMDA receptors [(1)-1-hydroxy-3-amino-2-pyrrolidone, 1-aminocyclobutanecarboxylate, 7-chlorokynurenate and (RS)-(tetrazol-5-yl)glycine; all 0.5 mM] likewise had no displacing effects (Table 3). In contrast, d-serine (0.5 mM) slightly but significantly diminished the binding. Of the non-NMDA receptor ligands, kainate (0.5 mM), (S)2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA;
NMDA site ligands NMDA cis-ACBD Quinolinate l-AP5 R-CPP Glycine site ligands Glycine d-Serine HA-966 ACBC (RS)-(Tetrazol-5-yl)glycine 7-Chlorokynurenate
Effectors, 1 mM l-Glutamate d-Glutamate l-Aspartate d-Aspartate l-Glutamine Quisqualate Kynurenate Pyroglutamate
Percentage specific binding ^ S.E.M. 90.8 ^ 0.4* 104.6 ^ 4.1 92.8 ^ 4.7 89.8 ^ 3.0 95.9 ^ 0.9 92.9 ^ 0.9 72.3 ^ 10.7** 69.9 ^ 7.8**
The control specific binding in the absence of any displacer was 31.3 ^ 1.1 nmol/kg protein (mean ^ S.E.M., n 30). Mean values^ S.E.M. of three experiments. Significantly different from control: *P , 0.05; **P , 0.01.
103.2 ^ 2.0 99.1 ^ 3.8 59.4 ^ 3.6** 93.0 ^ 9.7 104.0 ^ 3.1 96.6 ^ 2.6 90.7 ^ 1.0* 95.9 ^ 1.4 92.7 ^ 1.6 96.4 ^ 2.0 99.7 ^ 1.5
Abbreviations and concentrations: 1 mM NMDA; cis-ACBD, 0.5 mM 1aminocyclobutane-cis-1,3-dicarboxylate; 1 mM quinolinate; l-AP5, 1 mM l(1)-2-amino-5-phosphonopentanoate; R-CPP, 0.5 mM 3-[(R)2-carboxypiperazin-4-yl]propyl-1-phosphonate; 0.5 mM glycine; 0.5 mM d-serine; HA-966, 0.5 mM (1)-1-hydroxy-3-amino-2-pyrrolidone; ACBC, 0.5 mM 1-aminocyclobutanecarboxylate; 0.5 mM (RS)-(tetrazol-5-yl)glycine; 0.5 mM 7-chlorokynurenate. Mean values ^ S.E.M. of three experiments. Significantly different from the corresponding control: *P , 0.05; **P , 0.01.
0.5 mM), 6-cyano-7-nitroquinoxaline-2,3-dione (0.1 mM), 6,7-dinitroquinoxaline-2,3-dione (0.1 mM), 6,7-dichloroquinoxaline-2,3-dione (0.1 mM) and 5,7-dinitroquinoxaline-2,3dione (0.1 mM) had no effects (Table 4). (S)-5-Fluorowillardiine (0.5 mM) and 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione (0.5 mM dissolved in 4.8% dimethylsulfoximine) slightly but significantly displaced the binding. The binding was seemingly increased at the 0.5 mM concentration of quinoxalines (6-cyano-7-nitroquinoxaline2,3-dione, 6,7-dinitroquinoxaline-2,3-dione and 6,7-dichloroquinoxaline-2,3-dione) and GYKI 53655 (data not shown). However, this was attributable to the 20% dimethylsulfoximine used as solvent in these experiments, because it alone increased the binding equally by 43.3 ^ 13.4% (mean^ S.E.M., n 3). Many agonists or antagonists of the group I, II and III Table 4. Effects of non-N-methyl-d-aspartate receptor ligands on the binding of [ 3H]glutathione to pig cerebral cortical synaptic membranes Effectors
Table 2. Effects of excitatory amino acids, their derivatives and broadspectrum glutamate receptor agonists and antagonists on the binding of [ 3H]glutathione to pig cerebral cortical synaptic membranes
Percentage specific binding ^ S.E.M.
Kainate AMPA (S)-5-Fluorowillardiine CNQX DNQX NBQX DCQX MNQX
Percentage specific binding ^ S.E.M. 95.8 ^ 0.7 103.5 ^ 3.8 89.5 ^ 0.7* 107.3 ^ 5.3 106.7 ^ 4.5 84.4 ^ 1.3** 101.7 ^ 1.8 99.2 ^ 2.7
Abbreviations and concentrations: 0.5 mM kainate; 0.5 mM AMPA; 0.5 mM (S)-5-fluorowillardiine; CNQX, 0.1 mM 6-cyano-7-nitroquinoxaline-2,3-dione; DNQX, 0.1 mM 6,7-dinitroquinoxaline-2,3-dione; NBQX, 0.5 mM 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione; DCQX, 0.1 mM 6,7-dichloroquinoxaline-2,3-dione; MNQX, 0.1 mM 5,6-dinitroquinoxaline-2,3-dione. DCQX and MNQX are also active at the glycine co-activatory sites in the NMDA receptors. Mean values^ S.E.M. of three experiments. Significantly different from control: *P , 0.05; **P , 0.01.
R. Jana´ky et al.
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Table 5. Effects of metabotropic glutamate receptor ligands on the binding of [ 3H]glutathione to pig cerebral cortical synaptic membranes Effectors, 0.5 mM Group I receptor ligands 1S,3R-ACPD S-DHPG l-AP3 S-4CPG AIDA
Percentage specific binding ^ S.E.M.
99.5 ^ 2.0 94.0 ^ 1.9 75.7 ^ 1.4* 94.5 ^ 1.2 93.1 ^ 1.2
Group II receptor ligands LY 354740 DCG IV EGLU MTPG CPPG
94.8 ^ 0.9 91.3 ^ 5.3 108.1 ^ 4.5 106.4 ^ 4.4 93.8 ^ 1.8
Group III receptor ligands l-AP4 l-SOP MSOP MPPG
106.1 ^ 4.7 100.0 ^ 1.7 100.4 ^ 1.9 100.8 ^ 3.5
Glutamate uptake ligands l-trans-2,4-PDC trans-ACBD
136.3 ^ 7.5* 92.6 ^ 4.7
Abbreviations: 1S,3R-ACPD, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate; S-DHPG, (S)-3,5-dihydroxyphenylglycine; l-AP3, l(1)-2-amino3-phosphonopropionate; S-4CPG, (S)-4-carboxyphenylglycine; AIDA, (RS)-1-aminoindan-1,5-dicarboxylate; LY 354740, (1)-2-aminobicyclo[3,1,0]hexane-2,6-dicarboxylate; DCG IV, (2S,2 0 R,3 0 R)-2-(2 0 ,3 0 dicarboxycyclopropyl)glycine; EGLU, (2S)-2-ethylglutamate; MTPG, (RS)-2-methyl-4-tetrazolylphenylglycine; CPPG, (RS)-2-cyclopropyl-4phosphonophenylglycine; l-AP4, l(1)-2-amino-4-phosphonobutyrate; l-SOP, l-serine-O-phosphate; MSOP, (RS)-2-methylserine-O-phosphate; MPPG, (RS)-2-methyl-4-phosphonophenylglycine; l-trans-2,4PDC, l-trans-pyrrolidine-2,4-dicarboxylate; trans-ACBD, 1-aminocyclobutane-trans-1,3-dicarboxylate. 1S,3R-ACPD also activates group II receptors. DCG IV is also an agonist at NMDA receptors at this concentration. MTPG and CPPG are also ligands of group III receptors. Mean values ^ S.E.M. of three experiments. Significantly different from control: *P , 0.01.
metabotropic glutamate receptors were also tested (Table 5). Of these, l(1)-2-amino-3-phosphonopropionate (0.5 mM) was an effective but weak displacer. All other metabotropic glutamate receptor ligands proved to be inactive. Two compounds affecting the glutamate uptake sites, 1aminocyclobutane-trans-1,3-dicarboxylate (0.5 mM) and ltrans-pyrrolidine-2,4-dicarboxylate (0.5 mM), were also tested. The former was inactive, while the latter enhanced the binding by 36.3 ^ 7.5% (mean ^ S.E.M., n 3; Table 5). Effects of cysteine derivatives and sulfhydryl compounds Thiokynurenate had a striking activatory effect on [ 3H]GSH binding which was clearly concentration dependent (Fig. 2A). The effects of the other cysteine derivatives tested (1 mM) are shown in Table 6. l-Cysteine, l-cysteamine, l-cystamine and dithiothreitol were the most potent displacers of [ 3H]GSH binding. Their effects were concentration dependent, as shown in Fig. 2B with cysteine and cysteamine. The displacement curve for cystamine was very similar (not shown). The ic50 values of these most active compounds were in the high micromolar range. l-Homocysteinate and aminomethanesulfonate (1 mM) exhibited only moderate efficacy (35–38%) as displacers (Table 6). l-Cysteate, taurine, homotaurine, cystathionine (all 1 mM) and l-cystine (0.2 mM) were inactive. Hypotaurine slightly but significantly enhanced the binding. Effects of dipeptides and glutathione derivatives Most of the g-glutamyl dipeptides tested were without significant effect (Table 7). g-d-Glutamylglycine and g-lglutamylphenylalanine were weak and g-l-glutamylcysteine and l-cysteinylglycine fairly strong displacers. Glycylglycine, g-l-glutamylcysteate, g-l-glutamyl-GABA and g-l-glutamylleucine enhanced the binding in this order of increasing potency (Table 7). The enhancements by g-l-glutamyl-GABA and gl-glutamylleucine were concentration dependent (Fig. 2A). All GSH derivatives tested effectively displaced [ 3H]GSH (Table 8). GSH and GSNO were the most potent (ic50 values
Fig. 2. (A) Concentration dependence of the activation of [ 3H]GSH binding to pig cerebral cortical membranes by thiokynurenate (W), g-l-glutamyl-l-leucine (X) and g-l-glutamyl-GABA (K). (B) Displacement of [ 3H]GSH binding by GSNO (W), l-cysteine (X) and l-cysteamine (K). The results are mean values of three experiments with S.E.M. less than 5% of the mean for each experimental point.
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Glutathione binding to synaptic membranes Table 6. Effects of cysteine derivatives and sulfhydryl compounds on the binding of [ 3H]glutathione to pig cerebral cortical synaptic membranes Effectors, 1 mM
Percentage specific binding ^ S.E.M.
l-Cysteine l-Cystine† l-Cysteate l-Homocysteate Aminomethanesulfonate Hypotaurine Taurine Homotaurine l-Cysteamine l-Cystamine l-Cystathione Dithiothreitol
31.3 ^ 0.6* 91.6 ^ 3.6 96.3 ^ 3.6 61.8 ^ 1.1* 65.4 ^ 2.6* 123.0 ^ 4.3* 96.0 ^ 3.6 96.4 ^ 2.4 22.0 ^ 0.5* 12.9 ^ 1.6* 95.8 ^ 14.9 35.1 ^ 4.6*
Mean values ^ S.E.M. of three experiments. Significantly different from control: *P , 0.01. †The concentration of cystine was 0.2 mM.
in the low micromolar range), followed by g-l-glutamylcysteine and l-cysteine (ic50 values in the high micromolar range) (Table 9). Glutathione sulfonate and the S-alkyl derivatives of glutathione exhibited moderate efficacy (Table 8). The displacing effects were concentration dependent, as shown in Fig. 2B with GSNO. Effects of inhibitory glycine and GABA receptor ligands Some constituent amino acids of the g-glutamyl dipeptides tested, known to be active at other neurotransmitter receptors, were also screened for their displacing efficacy, but none of them was found to be active. For instance, GABA, 5-aminopentanoate, b-alanine, l-alanine, l-serine and proline (all 1 mM) exhibited no effect (data not shown). DISCUSSION
In the present article, specific binding sites for GSH in the Table 7. Effects of dipeptides on the binding of [ 3H]glutathione to pig cerebral cortical synaptic membranes Effectors, 1 mM g-l-Glutamylglycine g-d-Glutamylglycine g-l-Glutamylalanine g-l-Glutamyl-b-alanine g-l-Glutamyl-GABA g-l-Glutamylleucine a-l-Glutamylglutamate g-l-Glutamylglutamate g-l-Glutamylcysteine g-l-Glutamylcysteate g-l-Glutamyltaurine g-d-Glutamyltaurine g-l-Glutamylcholaminsulfate g-l-Glutamyl-AEP g-l-Glutamyltyrosine g-l-Glutamylhistidine g-l-Glutamylhistamine g-l-Glutamylphenylalanine Glycylglycine l-Cysteinylglycine
Percentage specific binding ^ S.E.M. 102.3 ^ 1.8 85.7 ^ 3.7* 87.3 ^ 4.4 93.2 ^ 3.4 170.1 ^ 4.0** 208.9 ^ 1.1** 102.3 ^ 5.0 105.4 ^ 3.5 22.2 ^ 0.3** 118.9 ^ 3.0** 97.6 ^ 6.3 101.1 ^ 1.4 91.7 ^ 3.4 99.5 ^ 2.7 110.3 ^ 3.1 99.7 ^ 3.1 86.9 ^ 4.4 83.6 ^ 1.9** 116.9 ^ 2.9* 50.3 ^ 0.8**
g-l-Glutamyl-AEP, g-l-glutamylaminoethylphosphate. Mean values ^ S.E.M. of three experiments. Significantly different from control: *P , 0.05; **P , 0.01.
Table 8. Effects of glutathione derivatives on the binding of [ 3H]glutathione to pig cerebral cortical synaptic membranes Effectors, 1 mM Oxidized glutathione S-Nitrosoglutathione Glutathione sulfonate S-Methylglutathione S-Ethylglutathione S-Propylglutathione S-Butylglutathione S-Pentylglutathione
Percentage specific binding ^ S.E.M. 34.3 ^ 1.4 0.0 ^ 0.9 74.2 ^ 4.1 56.6 ^ 2.6 68.4 ^ 1.4 44.4 ^ 1.5 70.2 ^ 4.5 49.1 ^ 1.6
Mean values ^ S.E.M. of four to eight experiments. All derivatives significantly (P , 0.01) diminished the binding.
porcine cortex were demonstrated which are qualitatively similar to those described by other researchers in rodent brains. 4,5,14,15,25–28 In our preparation, GSH bound at 08C to two populations of binding sites. This finding is similar to that of Ogita and Yoneda, 26 who reported that [ 3H]GSH binding consists of two components, one temperature-independent high affinity and the other temperature-dependent low affinity. In the present study, the very-low-affinity, high-capacity binding at 378C, enhanced in the presence of Ca 21 and not saturated at concentrations below 1 mM, may represent binding of GSH to sites other than plasma membrane receptors. These sites may be GSH transporters in plasma membranes or, although our preparation was highly purified, even transport sites in mitochondrial membranes. To exclude the binding of [ 3H]GSH to transporters, we carried out all subsequent binding assays at 08C in Tris–acetate buffer in the absence of Na 1 and Ca 21 ions. Immunocytochemically, GSH has been located in the CNS in both neurons and glia, although in none of the brain regions studied did the glial location predominate. 6 Within the brain, marked regional heterogeneity with respect to autoradiographically visualized [ 3H]GSH binding sites exists. 39 GSH binding sites have also been found in primary cultures of rat cortical astrocytes, 4 as well as in certain peripheral tissues, 30 but these elements did not constitute any major source of GSH binding sites in our synaptic membrane preparations. In order to ensure that the detected binding sites represent neurotransmitter receptors, it is first necessary to establish whether GSH could be bound to other proteins in the CNS and to take steps to exclude this possibility. In the mammalian CNS, radiolabeled GSH could potentially bind Table 9. Displacement of [ 3H]glutathione by glutathione and cysteine derivatives in pig cerebral cortical synaptic membranes Displacers Reduced glutathione Oxidized glutathione S-Nitrosoglutathione g-l-Glutamylcysteine l-Cysteinylglycine l-Cysteine l-Cysteamine l-Cystamine
ic50, mM (95% confidence limit) 2.49 (2.10–2.94) 380 (331–431) 12.5 (10.9–14.3) 81.8 (49.7–135.7) 190 (151–240) 96.6 (69.7–133.8) 139 (113–171) 331 (297–368)
The membranes (0.3 mg protein/tube) were preincubated with different (100 nM to 1 mM) concentrations of the displacers at 08C for 10 min, whereafter 10 nM [ 3H]GSH was added and the incubation continued for a further 60 min.
622
R. Jana´ky et al.
to non-receptor binding sites in the membranes, for example Na 1-dependent and -independent GSH transport proteins 12 and membrane-bound cell surface enzymes such as glutathione transferases (EC 2.5.1.18) and g-GT. Since the binding assays were performed in Tris–acetate buffer, GSH binding does not require the Na 1 and Cl 2 ions which provide the driving force for transmembrane transport of many neurotransmitters and amino acids. Moreover, of the two ligands of the Na 1-dependent high-affinity glutamate uptake sites tested, 1-aminocyclobutane-trans-1,3-dicarboxylate 3 was totally inactive and l-trans-pyrrolidine-2,4-dicarboxylate 23 slightly enhancing. Radiolabeled GSH thus labels sites different from glutamate transporter proteins. Pasqualotto et al. 36 have shown recently that [ 3H]GSH also does not label glutathione S-transferases in the rodent brain. In the present set of experiments acivicin, an inhibitor of g-GT, likewise failed to displace [ 3H]GSH. Similarly, many g-glutamyl dipeptides, known to be substrates of this enzyme, also failed to affect GSH binding, demonstrating that [ 3H]GSH is also not bound to the active site of g-GT. Since [ 3H]GSH was not broken down upon incubations, the bound radioactivity represents GSH binding and not that of a labeled glycine moiety liberated from the GSH molecule. The insensitivity of binding to glycine and glycinergic compounds also strongly corroborates this inference. Of all peptides tested, only the effect of GSNO could be partly attributable to the breakdown product of the parent compound. Since GSH at micromolar concentrations displaces ligands of all ionotropic glutamate receptors, and at millimolar concentrations also glycine, from the co-agonist site in the NMDA receptors, 7,9,11,29–32,41,43 it is obvious that [ 3H]GSH could label glutamate receptors. Such binding would be consistent with the neuromodulatory but not the neurotransmitter role of GSH. To exclude the possible overlap between the known glutamate receptors and the novel GSH binding sites, we tested glutamate analogs, mixed glutamate receptor ligands, NMDA receptor ligands, glycine, the ligands of the glycine co-activatory site in NMDA receptors, non-NMDA receptor ligands, agonists or antagonists at group I, II and III metabotropic glutamate receptors, and inhibitory amino acids. All were either totally inactive or exhibited only minor effects. Only the NMDA receptor subtype-specific agonist quinolinate 24,37 was a moderate displacer. GSH thus displaces glutamate, but glutamate not GSH, from specific binding sites. Thiokynurenate, which can be designated a cysteine derivative, had a strikingly strong concentration-dependent activatory effect, while kynurenate was inactive. These results from our displacement experiments show that GSH possesses specific binding sites in brain synaptic membranes which are not identical to any known glutamate receptor. Evidence for an independent GSH receptor population(s) has also been provided in a series of electrophysiological studies. Using field potential and single-unit recording methods, Shaw and colleagues examined the responses to bath-applied GSH on rat cortical slices. 1,10,36,40 In brief, it emerged that GSH is able to elicit a dose-dependent depolarization which appears to be Na 1 dependent. This response, unlike that for NMDA or cysteine, cannot be blocked by co-application of NMDA antagonists or by the removal of external Ca 21. Nor is it affected by the AMPA receptor antagonists 6,7-dinitroquinoxaline-2,3-dione or 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione. In keeping with the present strong activation
of [ 3H]GSH binding, thiokynurenate also greatly potentiates GSH-evoked depolarization in the electrophysiological studies described above. 10 When the pharmacological profiles of GSH binding in different synaptic plasma membrane preparations in different species and brain regions are compared, 10 they seem to exhibit a common feature, namely insensitivity to glutamate analogs and glutamate receptor ligands. This again corroborates the view that no overlap exists between the known glutamate receptors and the novel GSH sites. The slight pharmacological differences in different species and brain regions 10 may be indicative of the existence of more than one subtype of GSH binding sites. One striking difference is the relative sensitivity of GSH binding to metabotropic glutamate receptor ligands [e.g., quisqualate and l-(1)-2-amino-4butyrate] when tested in synaptic membranes isolated from the whole brain. The GSH receptors in subcortical regions may thus have a pharmacological profile distinct from that of cortical receptors. The displacement of the binding by l-glutamate itself in all preparations must draw our attention to the possibility that the GSH receptor protein exhibits structural similarities to glutamate receptors, in spite of it differing from any known glutamate receptor. In this respect it may be of significance that two glutamate receptor channel subunits (d1 and d2, the “orphan” subunits) so far remain unassigned. 18,36,38,44 The present binding data and those on the rat cortex 36 are strikingly consistent. In addition, the basic pharmacology of the GSH binding sites described in these two preparations is mirrored by the results of field potential recordings from rat cortical slices cited above. Together, these data appear to support the hypothesis that GSH acts as a neurotransmitter at its own population(s) of receptors. In this, GSH appears to have preserved its ancient role as a signal transduction molecule throughout phylogeny, 16 in addition to its roles as an antioxidant and detoxifier. If this is the case, it functions as a neurotransmitter and GSH receptors participate in information transfer in the mammalian CNS. The next question is whether or not different thiol-containing compounds, cysteine analogs, dipeptides and tripeptide derivatives of glutathione displace [ 3H]GSH from its specific binding sites. Testing their effects on the binding revealed a number of important structure–function relationships. Of the cysteine derivatives, cysteine, cysteamine and cystamine were the most potent displacers, with ic50 values in the micromolar range, and only those dipeptides which contain a cysteinyl moiety displace GSH. Likewise, only dipeptides with a similar composition have produced GSH-like excitatory field potentials in cortical-wedge recordings. 36 It may thus be inferred that the cysteinyl moiety in the molecule is crucial in GSH binding. Dithiothreitol, a thiol-reducing agent, was a relatively strong displacer, which would also point to the importance of the free thiol group in GSH binding. The oxidation or alkylation of the free thiol group in the GSH molecule diminishes the binding affinity. This in turn indicates that the neuromodulatory and neurotransmitter functions of the GSH molecule stem from different constituents. There is overwhelming evidence on redox modulation of glutamate receptors by the sulfhydryl group in GSH. 9,17,30 The g-glutamyl moiety is crucial in GSH binding to glutamate receptors, 11,41,43 while the cysteinyl residue forms the basis of binding to specific GSH sites. The strong activation by thiokynurenate but not by kynurenate may indicate that the GSH
Glutathione binding to synaptic membranes
receptor protein contains a strictly selective modulatory site to which co-agonists may bind and allosterically activate GSH binding. However, the nature of this activation calls for further elucidation. The possible existence of endogenous co-agonist(s), structurally related to synthetic thiokynurenate, which could allosterically activate GSH binding, is another open question warranting investigation. GSH has evinced a number of roles in biological systems. 13 Crucial amongst these are actions as an antioxidant and free radical scavenger, 22 and in the modulation of ionotropic glutamate receptor functions. 9,42,43 The present results add another role, that of a neurotransmitter, a role which alone or in concert with those already demonstrated may alter our current conceptions of its synaptic functions in the CNS. CONCLUSIONS
The specific binding sites for GSH differ from any known
623
excitatory or inhibitory amino acid receptor. The cysteinyl moiety is crucial in the binding process, since oxidation or alkylation of the cysteine thiol group diminishes the binding affinity. The receptor protein may contain a site to which coagonists bind and allosterically activate the binding. The present data suggest that GSH binding sites are distinct receptors in some regions of the mammalian CNS. Such a conclusion leads to a further one, namely that GSH can act as a neurotransmitter on cells possessing such receptors. GSH binding sites may thus be an important, largely unrecognized, component in signal transduction in some neural circuits.
Acknowledgements—The skilful technical assistance of Ms Oili Pa¨a¨kko¨nen and Ms Sari Luokkala, and the financial support of the Medical Research Fund of Tampere University Hospital, the Academy of Finland and OTKA-23888, Hungary, are gratefully acknowledged.
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