Temperature-dependent and -independent apparent binding activities of [3H]glutathione in brain synaptic membranes

Brain Research, 463 (1988) 37-46 Elsevier

37

BRE 13995

Temperature-dependent and-independent apparent binding activities of [3H]glutathione in brain synaptic membranes Kiyokazu Ogita and Yukio Yoneda Department of Pharmacology, Setsunan University, Hirakata, Osaka (Japan) (Accepted 11 May 1988)

Key words: Apparent binding of [3H]glutathione; L-Cysteine;Temperature dependency; Regional distribution; Glutamate binding site

An apparent binding activity of [3H]glutathionewas examined by using synaptic membrane preparations of the rat brain. The activity was found to be more than two times as high at 30 °C as that found at 2 °C. At 2 °C, the apparent binding sites consisted of a single component with a Kd of 0.77 ~M and a Bmaxof 5.60 pmol/mg protein. In contrast, two independent separate sites with Kds of 0.56 and 12.6 BM and BmaxSof 2.50 and 28.5 pmol/mg protein were observed at 30 °C. In vitro addition of Triton X-100 significantly inhibited the apparent binding activities detected at both temperatures, whereas pretreatment of the membranes with the detergent did not significantly affect both binding activities. Among 3 constituent amino acids of glutathione, L-cysteineinduced a selective and irreversible potentiation of the apparent activities, which occurred independently of the incubation temperature. Scatchard analysis revealed that L-cysteinedrastically increased the number of the low affinity sites without significantly altering their affinity. Apparent binding activities determined at both incubation temperatures were unevenly distributed in the central and peripheral structures. Distribution profile of the temperature-dependent activities was found to be closely related to that of the basal binding activity of [3H]L-glutamic acid, a putative central excitatory neurotransmitter. These results suggest that brain synaptic membranes may indeed contain specific binding sites of [3H]glutathione which have an interaction with the glutamate binding sites. Possible presence of two distinctly different apparent binding sites of [3H]glutathione, such as temperature-independent high affinity sites and temperature-dependent low affinity sites, is also suggested.

INTRODUCTION

brain content by ischemia 19 and a mental retardation in inherently deficient patients 21. In addition, evi-

Glutathione (y-x_-glutamyl-k-cysteinyl-glycine) is an endogenous tripeptide consisting of L-glutamic acid (Glu), L-cysteine (CySH) and glycine. Although two of these 3 constituent amino acids are believed to play neurotransmitter roles in the m a m m a l i a n central nervous system (CNS), relatively little attention has been paid to the specific functional significance of this endogenous peptide in central synaptic neurotransmission. Several lines of evidence have suggested the possible physiological importance of glutathione in the brain, other than maintenance of cellular integrity3'~7, such as an enrichment in the synaptosomal fractions 2°, involvement in convulsive seizures 2'7 and Parkinson's disease tS, a reduction of

dence that some endogenous brain peptides enriched in acidic amino acids are involved in the neurotransmission at presumed central Glu-ergic synapses is accumulating. For example, N-acetyl-L-aspartyl-L-glutamic acid ( N A A G ) has been proposed to be the neurotransmitter in the lateral olfactory tract 4. On the other hand, we have recently demonstrated that both reduced (GSH) and oxidized (GSSG) forms of glutathione potently displace Na+-dependent and -independent bindings of [3H]GIu in synaptic membrane preparations of the rat brain ~5. This displacement occurs i n d e p e n d e n t l y of the incubation temperature in a c o n c e n t r a t i o n - d e p e n d e n t m a n n e r 14. Therefore, the inhibition does not seem to be derived

Correspondence: Y. Yoneda, Department of Pharmacology, Faculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotoge-cho, Hirakata, Osaka 573-01, Japan. 0006-8993/88/$03.50© 1988 Elsevier Science Publishers B.V. (Biomedical Division)

38 from the Glu released from glutathione molecule during the incubation. Consistent with these findings, we have also shown that synaptic membranes may contain specific binding sites of [3H]GSH which exhibit protein dependency, temperature sensitivity, structure selectivity, high affinity and saturability 16. This apparent binding is significantly displaced by GSH derivatives without SH-moieties such as GSSG, S-methyl-glutathione and S-hexyl-glutathione. Similarly prominent inhibition is induced by various aand y-peptides containing k-Glu, but not by those containing D-Glu 16. For instance, both a-L-Glu-L-Glu and y-L-GIu-L-Glu similarly displace the apparent binding, whereas y-L-Glu-Gly but not y-D-Glu-Gly causes a profound inhibition of [3H]GSH binding activity. These previous findings all support the proposal that the apparent binding may indeed reflect an association of the ligand with its specific binding sites, but not corresponding to either a disulfide bonding between membranous SH-residues and SHmoieties of the ligand or an association of [3H]GSH with its metabolizing enzymes. However, the possibility that the apparent binding may represent an energy- and temperature-dependent uptake process of this endogenous peptide rather than the specific binding still remains to be elucidated. In this study, we have attempted to demonstrate the apparent binding activity under the experimental conditions in which participation of the uptake system is minimized. MATERIALS AND METHODS

Materials GSH, GSSG, N A A G , L-CySH, t.-Glu, Gly and other amino acids were all purchased from Sigma Chemical (St. Louis, MO, U.S.A.). [3H]GSH (glycine-2-[3H]glutathione, 1 Ci/mmol) was obtained from New England Nuclear (Boston, MA, U.S.A.). Other chemicals used were all of the highest purity commercially available.

suspended in 0.32 M sucrose and the suspensions were frozen at -80 °C until use. Frozen suspensions were thawed at room temperature and washed twice with the buffer before each use. Apparent binding activity of [3H]GSH was stable for at least 14 days under these storage conditions. The final suspensions (approximately 350-400 #g protein) were incubated with 100 nM [3H]GSH in 500 #1 50 mM Tris-acetate buffer (pH 7.4) at 2 or 30 °C for 60 min unless indicated otherwise. Incubation was terminated by the addition of 3 ml ice-cold buffer and subsequent rapid filtration through a Whatman GF/B glass fiber filter under a constant vacuum of 15 mm Hg. For determining the binding activity in supernatant fractions, filters were soaked in 0.3% polyethyleneimine 5 h prior to use as described previously 24. The filter was rinsed with 3 ml cold buffer 4 times within 10 s, and the radioactivity trapped on the filter was measured by a liquid scintillation spectrometer (LSC 900, Aloka, Japan) using 5 ml modified Triton-toluene scintillant at a counting efficiency of 40-42% 13. More than 90% (95.7 _+ 3.7%) of the radioactivity found in the medium after incubation at 30 °C for 60 min co-migrated with the authentic GSH when analyzed by thin layer chromatography on cellulose-coated plates 1~. No significant radioactivity was detected in the spot corresponding to GSSG. For Scatchard analysis, membrane suspensions were incubated with 100 or 200 nM [3H]GSH in the presence of varying concentrations of non-radioactive GSH to cover the concentration range from 100 tot 10,000 nM. Kinetic parameters such as K d and Bma x w e r e calculated from the equation estimated by a personal computer (PC9801, NEC, Japan) with a non-linear regression analysis program. Non-specific and non-saturable binding was obtained from the radioactivity found in the presence of 1 mM GSH and subtracted from each experimental value to calculate the specific binding 16. Assays were always carried out in triplicate with a variation of less than 10%. Protein content was determined by the method of Lowry et

al. 11"

Assay of apparent [3H]GSH binding Synaptic membranes were obtained from the brains of male Wistar rats weighing 200-250 g and extensively washed with 50 mM Tris-acetate buffer (pH 7.4) by centrifuging at 50,000 g for 30 rain as described previously 12. Final membrane pellets were

Pretreatment with Triton X-100 After thawing at room temperature, membrane suspensions (approximately 8 mg protein) were preincubated with varying concentrations of Triton X100 in a total volume of 25 ml in 50 mM Tris-acetate

39 buffer at 2 °C for 10 min. This preincubation was terminated by centrifuging at 50,000 g for 30 rain. Resultant pellets were suspended in the buffer and the suspensions were again centrifuged. The final pellets were resuspended in the buffer and the suspensions were subjected to the routine binding assay.

Pretreatment with CySH Following resuspension of the resultant pellets obtained from two washings by the centrifugation procedures, the suspensions (about 8 mg protein) were preincubated with 1 mM L-CySH at 2 or 30 °C for 60 rain in 2.5 ml buffer. The preincubation was terminated by the centrifugation and the precipitates were again washed as described above. The final pellets were resuspended in the buffer and the suspensions were incubated with 100 nM [3H]GSH at 2 or 30 °C for 60 min in the presence of varying concentrations of L-CySH. Regional distribution Each brain region was dissected on a chilled plastic plate according to the procedures described by Glowinski and Iversen 6. These central structures and peripheral tissues were individually homogenized in 50 vol. ice-cold, distilled, deionized and sterilized water with the aid of a Polytron homogenizer at setting 6 for 1 rain. Homogenates were washed 3 times with the buffer by centrifuging. Final pellets were suspended in 0.32 M sucrose and the suspensions were frozen at -80 °C for 14-16 h. Frozen suspensions were thawed and washed as above before subjecting to the routine binding assay.

vitro addition of N A A G at concentrations from 10-6 to 10-3 M (data not shown). No prominent change of the activity occurred at both incubation temperatures following the inclusion of some inorganic ions, including 20 mM NH4C1, 2.5 mM Ca(CH3COO)2, 20 mM NH4CI + 2.5 mM Ca(CH3COO)2, and 100 mM CH3COONa (data not shown). The apparent binding was saturable with increasing concentrations of [3H]GSH over the concentration range from 100 to 10,000 nM. Scatchard analysis revealed that the apparent binding consisted of a single component with a K d of 0.77 + 0.19pM and a Bma x of 5.60 _+ 1.90 pmol/ mg protein at 2 °C. At 30 °C, however, the Scatchard plots gave a hyperbola rather than a straight line, suggesting the possible presence of two independent separate elements with different affinities for the ligand, such as high affinity sites and low affinity sites (Kdl = 0.56 + 0.11/~M Bmaxl = 2.50 + 0.50 pmol/mg protein; Ka~ = 12.6 _ 3.7 pM, Bmax2 = 28.5 + 2.8 pmol/mg protein) (Fig. 1). Fig. 2 shows the effect of a non-ionic detergent, Triton X-100, on the apparent binding. In vitro addition of Triton X-100 significantly suppressed the activity at a concentration higher than 0.02%, which occurred independently of the incubation temperature for [3H]GSH binding assay (Fig. 2A). No complete abolition was induced by the detergent at the highest concentration used (0.1%). In contrast, pre-

20 o

Statistics Results were expressed as the mean + S.E.M. and the statistical significance was determined by Student's t-test. RESULTS

Biochemical characteristics A significant apparent binding activity of [3H]GSH was detected at 2 °C in the synaptic membrane preparations of the rat brain, to a lesser extent than that found at 30 °C (2 °C, 337 + 26 fmol/mg protein; 30 °C, 888 + 104 fmol/mg protein). These two apparent binding activities were not significantly affected by the in

o

15

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2

0°C

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O_o 2

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6

8

10

BoundIFree (pmolesImgproteinl.uM ) Fig. i. Scatchard plots of apparent binding of [3H]GSH. Binding assays were carried out in the presence of varying concentrations of [3H]GSH at 2 or 30 °C as described in the text. Each point indicates the mean of 8 separate experiments. 2 °C: K d = 0.77 + 0.1911M, Bmax = 5.6 4 1.9 pmol/mg protein. 30 °C: Kdl = 0.56 + 0.11, Bm~×l ~ 2.5 _+ 0.5, = 12.6 _+ 3.7, Bmax2 = 28.5 + 2.8.

Kd2

40

A

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Binding

(%

of Control } 0

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[] 10-~ • lo -3

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l

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0

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I

l

I

004

006

008

01

L-CySH

Triton X-tO0 (°~.)

B

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30"C

,___~

0

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Fig. 3. Effect of 3 constituent amino acids on apparent binding of [3H]GSH. Apparent binding activity was determined at 2 or 30 °C in the presence of varying concentrations of L-Glu, Gly or L-CySH. Each value was obtained from 6-8 separate experiments. *P < 0.05, **P < 0.01, compared with each control value. Control binding: 2 °C, 460 _+ 44; 30 °C, 1247 + 169.

t5 200

and 30 °C. A s s h o w n in Fig. 2B, this Triton-pretreat-

v "o

50 c - D - ~ .

ment did not significantly affect the apparent bind-

.c_

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A |

I

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01

Triton X - 1 0 0

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(%)

Fig. 2. Effect of Triton X- 100 on apparent binding of [3H]GSH. A: varying concentrations of Triton X-100 were directly added into the incubation mixture for the assay at 2 or 30 °C. Each value was obtained from 4 separate experiments. **P < 0.01, compared with each control value. Control binding: 2 °C, 510 __+ 60 fmol/mg protein; 30 °C, 810 _+ 80 fmol/mg protein. B: membranes were pretreated with varying concentrations of Triton X-100 at 2 °C. Following two centrifugations, binding assays were carried out at 2 or 30 °C in the absence of Triton X-100. Values were obtained from 6 independent experiments. Control binding: 2 °C, 337 _+ 26; 30 °C, 830 _+ 86.

2"C

XI~M

e "6

5

E \

0 m

i

i

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treatment with the detergent had no profound effect on the apparent binding. M e m b r a n e s were pretreated with varying concentrations of Triton X-100 at 2 °C and these Triton-pretreated m e m b r a n e s were subjected

to the determination of activities at 2

?

i%

2 3 B o u n d / F r e e (pmo~slmg protein/.uM )

*

4

100

80

c 60

Fig. 4. Scatchard analysis for CySH-induced potentiation of apparent binding of [3H]GSH. Scatchard analysis was performed at (A) 2 °C or (B) 30 °C, in the presence and absence of 1 mM L-CySH. Each point represents the mean of 6 separate experiments. (A) none: Kd = 1.15 _+ 0.32, Bin, x = 4.2 _+ 0.8. 1 mM LCySH; Kd = 5.70 + 0.84, Bin, x = 16.8 + 2.1. (B) none: Kdl = 0.76 + 0.20, Bmaxt = 4.0 _+ 0.8, g d 2 = 11.0 _+ 3.1, Bmax2 = 27.0 + 7.6.1 mM L-CySH; K d = 9.48 + 0.81, Bma x = 110 + 13.

(x

~o flD 2 4 Bound / Free

6 6 10 12 ( pmo~es / mg protein / pM )

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41 ing, with a concomitant slight reduction of the total protein content in the membranes. No significant binding activity was detected in the supernatant fractions obtained from the treatment of brain synaptic membranes with 1% Triton X-100 even after extensive dialysis against 100 vol. of the buffer containing 0.02% Triton X-100 (data not shown). These results suggest that apparent binding of [3H]GSH may indeed correspond to a real binding of the ligand to the specific binding sites, rather than reflect the uptake of [3H]GSH which is an energy-dependent, temperature-dependent and detergent-sensitive process.

Effect of L-CySH Fig. 3 shows the effect of 3 constituent amino acids on the apparent binding activities at 2 and 30 °C. Both L-Glu and Gly at the highest concentration used significantly inhibited the apparent activity at 30 °C, but not that found at 2 °C. In contrast, L-CySH potently enhanced the apparent binding at both incubation temperatures in the concentration range higher than 10.4 M. No profound change was induced by the in vitro inclusion of similar concentrations of other SH-containing compounds, such as L-cysteinesulfinic acid, L-cysteic acid, L-cystine, hypotaurine, taurine, L-cystathionine, D,L-homocysteine, L-methionine, dithiothreitol and dithioerythritol (data not shown). None of numerous other amino acids had a signifi-

[SH]GSH Pretreatment at 2°c 2"c

O l OO500 0

500 *

Binding

cant effect on the apparent binding activities at both temperatures in the similar concentration range (10 -5 to 10-3 M): these included L-alanine, 7-aminobutyric acid, k-arginine, L-aspargine, L-aspartic acid, L-glutamine, L-histidine, L-isoleucine, k-leucine, L-lysine, L-phenylalanine, L-proline, L-threonine, L-tryptophan, k-tyrosine and L-valine (data not shown). At 2 °C, the Scatchard analysis gave a straight line in the absence of 1 mM L-CySH, whereas 1 mM L-CySH caused rather hyperbolic Scatchard plots (Fig. 4A) (none; K d = 1.15 _+ 0.32BM, Bm~,x= 4.20 + 0.80 pmol/mg protein; 1 mM CySH; K d = 5.70 + 0.84 NM, Bmax = 16.8 + 2.1 pmol/mg protein). At 30 °C, the apparent binding sites consisted of two separate components in the absence of CySH added, while CySH at 1 mM drastically increased the number of the low affinity sites with the high affinity sites being apparently eliminated (Fig. 4B) (none; Kdt = 0.76 + 0.20/~M, Bmaxl = 4.00 -- 0.80 pmol/mg protein, Kd2 = 11.0 -- 3.1 #M, Bmax2 = 27.0 + 7.6 pmol/mg protein; 1 mM CySH; K d = 9.48 + 0.81 #M, Bmax = 110 + 13 pmol/mg protein). To evaluate the reversibility of CySH-induced potentiation of [3H]GSH binding activity, synaptic membranes were pretreated with 1 mM L-CySH at 2 or 30 °C. The apparent binding was determined at 2 or 30 °C in the presence of varying concentrations of CySH from 10-5 to 10-3 M by using these CySH-pre-

( frnoles/mg protein )

Pretreatrnent at 30"C 30"C 2 "C 1000 1500 1000 500 0 0

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I Untreated

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L-CySH

L-CySH

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Treated

L-CySH (M) [--] 0

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Fig. 5. Effect of pretreatment with CySH on apparent binding of [3H]GSH. Membranes were pretreated with 1 mM L-CySHat 2 or 30 °C. The apparent binding activity was determined at 2 or 30 °C in the presence of varying concentrations of L-CySH.Values were obtained from 6 separate experiments. *P < 0.05, **P < 0.01, comparedwith each control value as indicated.

42

NONE O l O~ 3

10-3M CySH ~oo 0"C ~1

~

Fig. 6 shows the effect of an excess of non-radioactive GSH on the apparent binding. Membranes were incubated with [3H]GSH at 2 or 30 °C in the presence and absence of 1 mM CySH. Non-radioactive G S H was added 60 min after the initiation of incubation and the incubation was additionally continued for various periods. In the absence of CySH added, a relatively rapid dissociation occurred at 2 °C with the dissociation at 30 °C being considerably slow. In contrast, non-radioactive GSH failed to dissociate the apparent binding independently of incubation temperature in the presence of 1 mM CySH (Fig. 6). The data provided above suggest that L-CySH may induce a selective and irreversible potentiation of apparent binding of [3H]GSH through increasing the number of the temperature-dependent low affinity sites.

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,I

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20 I

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0

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40

60

Time (rain)

Fig. 6. Dissociation by non-radioactive GSH of apparent binding of [3H]GSH. An excess (1 mM) of non-radioactive GSH was added 60 min after the initiation of incubation at 2 or 30 °C in the presence and absence of 1 mM L-CySH. Each point represents the mean + S.E.M. obtained from 6 separate experiments. Control binding: none, 2 °C, 361 _+ 32; 30 °C, 1108 _+ 50.1 mM L-CySH, 2 °C, 530 +_47; 30 °C, 1706 + 176.

Regional variation As shown in Fig. 7, apparent binding activities of [3H]GSH were unevenly distributed in the CNS. The retina had the highest activity at 30 °C among various regions employed, with progressively lower activities in the hypothalamus, striatum, spinal cord, midbrain, medulla-pons, hippocampus, cerebellum and cerebral cortex. At 2 °C, the retina also contained the highest activity followed by the hypothalamus, hip-

treated membranes (Fig. 5). In vitro CySH significantly enhanced the apparent binding activities at both incubation temperatures in the membranes not pretreated with 1 mM CySH, but not those in the membranes pretreated with 1 mM CySH. Entirely identical results were obtained independently of the preincubation temperature (Fig. 5).

[3H]GSH Binding

( pmolcs I rng protein )

2"C 2

30"C 1

0

0

I

F Cerebral cortex -F- Striatum

2

3

--I-

Hypothalamus

E

Midbrain Hippocarnpus

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Cerebellum Medulla- Ports Spinal Cord

_+

---q. ----+ +

Retina

Fig. 7. Regional distribution of apparent binding of [3H]GSH in central structures. Assays were carried out at 2 or 30 °C. Values were obtained from 8 separate experiments.

43

[3HIGSH Binding

( pmoles I m g protein )

2"(2 4

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1

0

0

1

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Heart Lung Pancreas

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Fig. 8. Regional distribution of apparent binding of [3H]GSH in peripheral tissues. The activity was determined at 2 or 30 °C. Each value was obtained from 8 separate experiments, n.d., not detectable.

pocampus, striatum, cerebral cortex, cerebellum, medulla-pons, midbrain and spinal cord. In addition to these central structures, an apparent binding activity of [3H]GSH was detected at both incubation tern-

peratures in some peripheral tissues (Fig. 8). The pituitary possessed much higher activity than that found in the retina, accompanied by the adrenal, liver, spleen, skeletal muscle and heart. Relatively

[3H]GSH Binding ( pmoLes/rngprotein 0 Cerebral cortex

3

Striatum Hypothalamus Midbrain Hippocarnpus CerebeUum Mmdutta- Ports Spinal cord R~tina

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I

Pituitary

+

Heart

]-

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! f---~

Lung

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Pancreas Spleen

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----4_

Kidney

n.d

Intestinal mucosa

I I

Testis Skeletal muscle

--jr

Fig. 9. Regional distribution of temperature-dependent activity of apparent binding of [3H]GSH in central and peripheral structures. Values were obtained from 8 independent experiments, n.d., not detectable.

44 low activities were found in the lung, intestinal mucosa, testis and pancreas, but the kidney did not have any detectable activity. A similar distribution profile was observed independently of the incubation temperature. Fig. 9 shows the regional distribution of the temperature-dependent activity of the apparent binding in the central and peripheral structures. In the CNS, the temperature-dependent component exhibited a distribution profile identical to that of the apparent binding determined at 30 °C. However, peripheral temperature-dependent activity was distributed somewhat differently from the apparent activity detected at 30 °C. In particular, the liver had a very low temperature-dependent portion of the apparent binding, and the testicular and pulmonary activities were inversely dependent on the incubation temperature (Fig. 9). These results suggest that temperature-dependent and -independent apparent binding activities of [3H]GSH may be similarly distributed in the central structures, but not in the peripheral tissues. DISCUSSION Although there is no direct evidence for glutathione as a neuroactive endogenous peptide, the data provided in this study support the proposal that glutathione may play some specific and positive functional role other than the maintenance of cellular integrity in the central synaptic neurotransmission through associating with its apparent binding sites. One possible interpretation is that the apparent binding may reflect the transport of [3H]GSH into resealed vesicular components in the synaptic membranous preparations. However, the apparent binding indeed occurred at a low incubation temperature in the membranes previously treated with Triton X-100. Therefore, the inhibition by in vitro Triton seems to be due to the GSH and/or GSSG released from the membrane preparations during the incubation in the presence of the detergent. Another possibility that the apparent binding may represent an association of [3H]GSH with its metabolizing enzymes having a considerably high affinity for glutathione in a membrane-bound form, including GSH peroxidase, GSH-S-transferase and 7-glutamyltransferase, is excluded by taking into consider-

ation the findings, such as no significant formation of [3H]GSSG during the incubation, no necessity of 7-peptidic structure 16 and complete abolition by both GSH and GSSG 14. The absence of an apparent binding activity from the kidney is also against these two aforementioned possibilities. Since a complete abolition of the apparent binding is induced by some GSHderivatives without SH-moieties 16, it is evident that the apparent binding is not derived from the disulfide binding between membranous SH-residues and SHmoieties of the radioactive ligand. The apparent binding seems to consist of two independent separate constituents such as high affinity sites and low affinity sites. The high affinity sites exhibited a temperature-independency, while the low affinity sites wore entirely dependent on the incubation temperature. In addition, L-CySH caused a selective and irreversible increment of the number of ihese low affinity and temperature-dependent sites, without affecting the high affinity and temperatureindependent sites. In addition, L-CySH disclosed the low affinity sites even at a low incubation temperature. Considering these results, along with the fact that S-methyl-glutathione selectively eliminates the high affinity sites 16, it seems possible to speculate that the low affinity sites may play a physiological role distinctly different from that of the high affinity sites in the brain. It is still conceivable that these low affinity and temperature-dependent sites may be intimately related to an energy- and temperature-dependent low affinity uptake process of GSH 8. If this is the case, dissociation of the apparent binding may be inevitably slower at 30 °C than at 2 °C. In addition, no rapid dissociation occurred following the addition of non-radioactive GSH in the presence of 1 mM CySH independently of the incubation temperature. Therefore, addition of L-CySH might elicit a significant effect similar to elevating the incubation temperature in terms of a disclosure of the temperature-dependent low affinity sites. At any rate, it is peculiar that L-CySH not only increases the number of temperature-dependent low affinity sites, but also reduces the dissociation rate of the apparent binding at both incubation temperatures. The exact molecular mechanism underlying the CySH-induced potentiation as well as its functional significance remains to be elucidated. A prominent enhancement of the apparent binding

45 was induced by the in vitro addition of L-CySH, but not by that of structurally related amino acids, sulfurcontaining compounds or numerous other amino acids. These results clearly indicate that the SHmoiety is not involved in the potentiation of [3H]GSH binding activity by L-CySH. L-CySH has been proposed to be transported into various cells via the ASC transport system 5'1°. Since none of other substrates for this ASC transport system, including L-alanine, I_serine and L-glutamine, affected the apparent binding, it seems unlikely that the ASC amino acid transport system may be responsible for the selective and irreversible augmentation by L-CySH. Dimeric form of L-CySH, L-cystine, is exchanged with L-Glu across plasma membranes in the human fibroblasts 1. L-Cystine is also shown to potently inhibit the Cl--dependent binding of [3H]Glu, which may reflect the amino acid transport system (x c )9. These reports, together with our previous findings that various displacers of [3H]Glu binding invariably diminish the apparent binding of [3H]GSH 16, suggest the possible interaction of [3H]Glu binding activity with some endogenous sulfur amino acids such as L-CySH, L-cystine and glutathione. One of the interesting findings obtained in the present study is that the apparent binding activities were unevenly distributed in the peripheral tissues in addition to the CNS. Similar central distribution profile is observed with [3H]Glu binding 22,23. The retina has the highest activities of [3H]GIu binding as well as apparent [3H]GSH binding amongst various central structures examined, with progressively lower activities in both the hypothalamus and striatum. Relatively high activity of apparent binding of [3H]GSH was also detected in some peripheral excitable tissues having a considerably high activity of [3H]Glu bind-

REFERENCES 1 Bannai, S., Exchange of cystine and glutamate across plasma membrane of human fibroblasts, J. Biol. Chem., 261 (1986) 2256-2263. 2 Berl, S., Purpura, D.P., Girado, M. and Waelsch, H., Amino acid metabolism in epileptogenic and non-epileptogenic lesions of the neocortex (cat), J. Neurochem., 4 (1959) 311-317. 3 Christopherson, B.O., The inhibitory effect of reduced glutathione on the lipid peroxidation of the microsomal fraction and mitochondria, Biochem. J., 106 (1968) 515-522. 4 Ffrench-Mullen, J.M.H., Koller, K., Zaczek, R., Coyle,

ing, such as the pituitary and adrenal 23. These data again give strong support for the possible interaction of apparent binding sites of [3H]GSH with [3H]GIu binding sites. In fact, [3H]GSH binding activity is significantly inhibited by the addition of a relatively high concentration of an agonist (quisqualic acid) and an antagonist (D,L-2-amino-4-phosphonobutyric acid) of the central Glu receptors in brain synaptic membranes, but not by that of the other agonists such as N-methyl-D-aspartic acid and kainic acid 16. However, the peripheral distribution profile of the apparent binding is somewhat different from that of [3H]Glu binding. No significant binding of [3H]GIu is detected in the liver, pancreas and kidney 23. Considering these previous findings together with the present results, it is conceivable that [3H]GSH may associate with its own binding sites in addition to interacting with [3H]Glu binding sites. The possibility that peripheral apparent binding sites may be distinctly different from the central sites cannot be excluded at present. It thus appears that synaptic membranes may contain two independent separate constituents of [3H]GSH binding, such as temperature-independent high affinity sites and temperature-dependent low affinity sites. Functional significance of these apparent binding sites as well as some endogenous sulfur amino acids including L-CySH and L-cystine in the brain, may need to be re-evaluated.

ACKNOWLEDGEMENTS This work was supported in part by a Grant-in-Aid for Scientific Research 62771981 to K.O. from the Ministry of Education, Science and Culture, Japan.

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