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Polyamine effects uponN-methyl-D -aspartate receptor functioning: differential alteration by glutamate and glycine site antagonists
Brain Research, 561 (1991) 285-291 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS ~ 9 9 3 9 1 1 7 0 5 6 J
285
BRES 17056
Polyamine effects upon Nomethyl-D-aspartate receptor functioning: differential alteration by glutamate and glycine site antagonists Ilana Zylberman Nussenzveig 1, Ratna Sircar 1'3, Ma-Li Wong 1, Mark J. Frusciante 1, Daniel C. Javitt 1'2'4 and Stephen R. Zukin 1'2'4 Departments of 1Psychiatry, 2Neuroscience and 3Neurology, Albert Einstein College of Medicine, Bronx, NY (U.S.A.) and 4Bronx Psychiatric Center, Bronx, NY 10461 (U.S.A.) (Accepted 21 May 1991) Key words: N-methyl-D-aspartate receptor ionophore complex; Glutamate; Glycine; Polyamine; Spermidine; 7-Chlorokynurenic acid; D-(-)-2-Amino-5-phosphonovaleric acid
Polyamines such as spermidine potentiate activation of the N-methyl-D-aspartate (NMDA)-type excitatory amino acid receptor. The goal of the present study was to investigate interactions between the putative polyamine binding site and previously described sites for glutamate and glycine. Binding of the high-potency PCP receptor ligand [3H]MK-801 to well-washed rat brain membranes was used as an in vitro probe of NMDA receptor activation. Spermidine concentration-response studies were performed in the absence and presence of both glutamate and glycine, with and without D-(-)-2-amino-5-phosphonovaleric acid (D(-)AP-5) or 7-chiorokynurenic acid (7C1-KYN). Incubation in the presence of spermidine alone induced a 20.4-fold increase in [3H]MK-801 binding with an ECs0 value of 13.3/~M. The mean concentration of spermidine which induced maximal stimulation of binding was 130/~M (n = 10, S.E.M. = 24.66, range = 25-250 ktM). Glutamate (10 /zM) decreased the ECs0 value for spermidine-induced stimulation of [3H]MK-801 binding to 3.4/~M. Glycine (10 ktM) did not significantly alter either maximum spermidine-induced [3H]MK-801 binding or the ECso value for spermidine-induced stimulation of [3H]MK-801 binding. Incubation in the presence of the specific glutamate antagonist D(-)AP-5 attenuated [3H]MK-801 binding in a glutamate-reversible fashion. The competitive glycine antagonist 7CI-KYN decreased maximum spermidine-induced [ 3H]MK-801 binding in a glycine-reversible fashion. In addition, 7CI-KYN increased the ECso value for spermidine-induced stimulation of [3H]MK-801 binding while D(-)AP-5 was without effect. These findings suggest that glutamate and glycine regulate the polyamine binding site differentially. PCP-like agents induce a psychotomimetic state closely resembling schizophrenia by inhibiting NMDA receptor-mediated neurotransmission. The ability of polyamines to modulate NMDA receptor functioning suggests a potential site for pharmacological intervention. INTRODUCTION N-Methyl-D-aspartate ( N M D A ) receptors play a fundamental role in the induction of long-term potentiation and frequency and amplitude-dependent gating phenomena 4. Evidence is accumulating that the brain damage associated with anoxia, stroke, hypoglycemia, epilepsy and neurodegenerative diseases (Huntington's disease) may be at least partially produced by pathological overactivation of N M D A receptors 11'24. Phencyclidine (1-(1phenylcyclohexyl)piperidine, PCP, 'angel dust'), induces prolonged psychotic reactions closely resembling schizophrenia 3. Neurobehavioral effects of PCP are mediated at a specific high-affinity binding site (PCP receptor) 26"3° located within the N M D A receptor-gated ion channel 4"9. The N M D A receptor complex is a postsynaptic ligandgated cation channel with multiple regulatory sites 22'27. The agonist site is sensitive to excitatory amino acids (Lglutamate > N M D A > ibotenate > kainate) 6"16'27. The
Hill coefficient for activation of N M D A receptors by glutamate has been shown to be significantly greater than unity suggesting that more than one molecule of agonist may be required for activation 7'8'1°'17. Glycine, acting at a strychnine-insensitive site, potentiates and may be required for N M D A receptor activation 1'12'15'23. Most recently, it has been demonstrated that polyamines stimulate N M D A receptor activation in a fashion that is additive to the effects of glutamate and giycine 19. The goal of the present study was to elucidate mechanisms involved in the interactions between polyamines and other regulators of N M D A receptor activation. L-Glutamate and D-(-)2-amino-5-phosphonovaleric acid (D(--)AP-5) were used respectively as agonist and antagonist at the excitatory amino acid recognition site 3"5'6'9'14'16'27.Glycine and 7-chlorokynurenic acid (7C1-KYN) served as putative agonist and antagonist at the glycine site 13'25. Binding of the PCP receptor ligand [3H]MK-801 was used as an in vitro probe of N M D A channel activation 14.
Correspondence: S.R. Zukin, Department of Psychiatry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, F-111, Bronx, NY 10461, U.S.A. Fax: (1) (212) 430-2488.
286 MATERIALS AND METHODS
Membrane preparation Extensively washed crude synaptic membranes were prepared as described previously 7'1°. Cerebral cortex plus hippocampus from male Sprague-Dawley rats (150-200 g) were rapidly removed and homogenized in 15 vols. (v/w) of ice-cold 0.32 M sucrose, using a Teflon-glass homogenizer at 800 rpm. The homogenate was centrifuged at 1000 g for 10 min in a Sorvall RC-5B refrigerated centrifuge. The supernatant was decanted and recentrifuged at 20,000 g for 20 min. The resulting pellet was resuspended in 30 vols. (v/w) of deionized water using a Brinkman Polytron (setting 6); the resulting suspension was centrifuged at 8000 g for 20 min. The supernatant and buffy coat were decanted and centrifuged at 48,000 g for 20 min. The pellet was then resuspended in 5 mM EDTA, buffered to pH 7.4 by the addition of riffs base (approx. 15 mM), and incubated for 1 h at 37 °C. After incubation, the membrane suspension was again centrifuged at 48,000 g for 20 min. Pellets were then frozen at -4 ° for 5-46 days. On the day of the experiment, frozen pellets were thawed and suspended in 30 vols. (v/w) of deionized water for 1 h at 37 °C. The membrane suspension was recentrifuged for 20 min at 48,000 g. Pellets were then washed 3 times by resuspension in 30 vols. (v/w) of deionized water and centrifugation at 48,000 g. After the final centrifugation, pellets were suspended in 30-50 vols. (v/w) of 5 mM Tris-acetate buffer adjusted to pH 7.4.
Radioreceptor binding assay For all studies, 1-ml aliquots (138-264/~g protein) of homogenate were incubated for 90 min at 25 °C with 0.84-1 nM [3H]MK801 and increasing concentrations of spermidine. Studies were per-
1.2
formed in the absence and presence of o(-)AP-5 or 7CI-KYN, with and without L-glutamate or glycine, as indicated. All points were determined in triplicate. Non-specific binding was determined in the presence of 10 FM MK-801. [3H]MK-801 (15.0-17.8 Ci/mmol) was obtained from Dupont/NEN (Boston, MA). 7C1-KYN was purchased from Tocris (Buckhurst Hill, Essex, U.K.), and o(-)AP-5 from Cambridge Research Biochemicals (Atlantic Beach, NY). L-Glutamate, glycine and spermidine were obtained from Sigma Biochemicals (St. Louis, MO). Incubations were terminated by filtration under reduced pressure using a 24-well cell harvester (Brandel Corp., Gaithersburg, MD) and Whatman GF/B filters which had been presoaked for 30 min in 0.3% polyethyleneimine. Filter disks were placed in vials containing 4 ml Hydrofluor (National Diagnostics, Manville, NJ) and counted using an LKB 1218 Rackbeta scintillation counter at a counting efficiency of 60.11%. Dpm were determined by dividing counts per minute by calculated counting efficiency. The protein content was determined using a protein assay kit (procedure P5656, 1987) from Sigma Diagnostics (St. Louis, MO).
Data analysis The concentration of spermidine required to produce half-maximal enhancement (ECs0 value) of [3H]MK-801 binding was determined using linear regression analysis of ln-logit plots. The maximum binding of [3H]MK-801 was determined within the range of spermidine concentrations of 0 to 300-1000/~M (7CI-KYN series) or 0 to 250-300 #M (o(-)AP-5 series). Values in text represent mean -+ S.E.M. All statistical comparisons were performed using unpaired, two-tailed Student's t-tests unless otherwise indicated.
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SPERMIDINE (log [M]) Fig. 1. Concentration-response curves for stimulation of specific [3H]MK-801 binding by spermidine under conditions shown. Figure shows the pooled means from 4 separate experiments, each performed in triplicate. Error bars are the S.E.M. values.
287 RESULTS
all conditions (Figs. 1, 2). A similar biphasic effect of polyamines on [3H]MK-801 binding has been reported previously2°'28, suggesting that at high concentrations, polyamines may act at an inhibitory as well as a facilitatory binding site. In the absence of D(-)AP-5 or 7CIKYN, the mean EC50 value for stimulation by spermidine was 13.3 + 2.2/,M. Incubation in the presence of glycine did not significantly affect the spermidine ECs0 value (n = 10, t = 2.04, df -- 18, 0.1 > P > 0.05) (Figs. 5, 6). By contrast, incubation in the presence of L-glutamate significantly decreased the mean ECs0 value for stimulation of [3H]MK-801 binding by spermidine in the absence of glycine from 13.3 _+ 2.2/~M to 3.44 _+ 0.67 /*M (P < 0.001) and in the presence of glycine from 8.26 + 1.08 a M to 1.89 -+ 0.36/~M (P < 0.001) (Figs. 5, 6). In the absence of L-glutamate, with or without glycine, maximum [3H]MK-801 binding occurredat a mean spermidine concentration of 96.25 + 15.09/~M. In the presence of L-glutamate, with or without glycine, maximum [3H]MK-801 binding occurred at a mean spermidine concentration of 36.4 -+ 14.40/~M. Maximum [3H]MK-801 binding induced by spermidine was not significantly affected by the presence of L-glutamate or glycine either
Specific [3H]MK-801 binding was determined in the presence of increasing concentrations of spermidine and in the absence or presence of L-glutamate or glycine. One series of studies was performed in the presence of 7CI-KYN (10/,M), while a second series was performed in the presence of D(-)AP-5 (1/~M). No significant differences in control binding occurred between the two groups in the absence or presence of L-glutamate or glycine, but in the presence of combined L-glutamate and glycine [3H]MK-801 binding was significantly greater in the D(-)AP-5 series than in the 7CI-KYN series of experiments. Spermidine concentrations varied from 0 to 250-300/,M in the D(-)AP-5 series and 0 to 300-1000 aM in the 7CI-KYN series of experiments. In the absence of added spermidine, incubation in the presence of L-glutamate or glycine led to significant 4.69fold (P < 0.002) and 2.69-fold (P < 0.001) mean increases in specific [3H]MK-801 binding, respectively. Incubation in the presence of spermidine led to an initial concentration-dependent increase in specific [3H]MK-801 binding followed at higher doses by an inhibition, under
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Fig. 2. Concentration-response curves for stimulation of specific [3H]MK-801 binding by spermidine under conditions shown. Figure shows the pooled means from 3 separate experiments, each performed in triplicate. Error bars are the S.E.M. values.
288 individually or in combination (n = 10) (Figs. 3B, 4B). Incubation in the presence of a low dose of o ( - ) A P - 5 (1 /lM) alone did not significantly inhibit [3H]MK-801 binding (Fig. 3A). Even at this dose, however, D(-)AP-5 significantly decreased the maximum spermidine-induced stimulation of [3H]MK-801 binding in the absence (P < 0.02; Figs. 1A, 3B) and presence (P < 0.02; Figs. 1C, 3B) of glycine, in an L-glutamate-reversible fashion (Figs. 1B, D, 3B). Incubation in the presence of D(-)AP-5 did not significantly alter the ECs0 value for stimulation by spermidine under any condition (Figs. 1, 5). Incubation in the presence of 7CI-KYN alone decreased [3H]MK-801 binding to 23.66 - 6.10 % of control (P < 0.001) in a fashion that was reversible by glycine but not by L-glutamate (Fig. 4A). Incubation in the presence of 7C1-KYN, in the absence of added L-glutamate or glycine, also significantly decreased the maximum spermidine-stimulated [3H]MK-801 binding (P < 0.001, Figs. 2A, 4B). By contrast, no significant inhibi-
0.6
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tion of maximum binding was observed in the presence of L-glutamate, glycine or combined L-glutamate and glycine (Figs. 2, 4B). By contrast to o ( - ) A P - 5 , incubation in the presence of 7CI-KYN significantly increased the ECso value for spermidine-induced stimulation of [3H]MK-801 binding (P < 0.02, Figs. 2A, 6). This effect occurred in the presence of L-glutamate (P < 0.02, Figs. 2B, 6) but not in the presence of glycine (Fig. 2C, D). Incubation in the presence of L-glutamate significantly decreased the ECs0 value for stimulation by spermidine even in the presence of 7CI-KYN (P < 0.05, Fig. 6). DISCUSSION N M D A receptor activation is regulated by several classes of agents, including excitatory amino acids, glycine and polyamines. These agents mediate their actions at independent binding sites associated with the N M D A
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Fig. 3. A: bar chart of specific [3H]MK-801 binding in the absence of spermidine (SPD), in the presence of L-glutamate (Glu) and/or glycine (Gly) with (hatched bars) and without (open bars) 0-(-)-2amino-5-phosphonovaleric acid (o(-)AP-5). Values are means -+ S.E.M. of 4 separate experiments, each performed in triplicate. B: bar chart of maximum [3H]MK-801 binding induced by spermidine (SPD) in the presence of L-glutamate (Glu) and/or glycine (Gly) with (hatched bars) and without (open bars) o-(-)-2-amino-5phosphonovaleric acid (D(-)AP-5). Values are means -+ S.E.M. of 4 separate experiments, each performed in triplicate. *P < 0.02.
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Fig. 4. A: bar chart of specific [3H]MK-801 binding in the absence of spermidine (SPD), in the presence of u-glutamate (Glu) and/or glycine (Gly) with (hatched bars) and without (open bars) 7-chlorokynurenic acid (7CI-KYN). Values are means -+ S.E.M. of 6 separate experiments, each performed in triplicate. *P < 0.02, **P < 0.005. B: bar chart of maximum [3H]MK-801 binding induced by spermidine (SPD) in the presence of L-glutamate (Glu) and/or glycine (Gly) with (hatched bars) and without (open bars) 7-chlorokynurenic acid (7C1-KYN). Values are means -+ S.E.M. of 6 separate experiments, each performed in triplicate. ***P < 0.001.
289
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Fig. 5. Bar chart of EC5o values for stimulation by spermidine in the presence of L-glutamate (Glu) and/or glycine (Gly) with (hatched bars) and without (open bars) D-(-)-2-amino-5phosphonovaleric acid (o(-)AP-5). Values are means --- S.E.M. of 4 separate experiments, each performed in triplicate. Logarithmic values of ECso were used for statistical comparisons. *P < 0.002 vs control baseline, **P < 0.001 vs control baseline, ***P < 0.05 vs control baseline.
receptor complex. It is now well established that glutamate and glycine act in concert with polyamines to regulate N M D A channel function. The present data demonstrate that occupation of the glutamate and glycine sites by agonists or antagonists differentially alters the effects of interaction of polyamines with their unique binding site. Moreover, glutamate- and glycine-site antagonists differentially affect polyamine-stimulated [3H]MK-801 binding, suggesting that the glutamate and glycine sites are linked differentially with the polyamine site. The major finding of the present study is that glutamate- and glycine-site antagonists differentially affect polyamine-induced stimulation of N M D A receptor activation. D(-)AP-5, a glutamate site antagonist, inhibited maximum polyamine-induced [3H]MK-801 binding without affecting the EC50 value for spermidine-induced stimulation of [3H]MK-801 binding. This finding agrees with an earlier report that CPP prevents polyamine-induced enhancement of PCP receptor binding 2~ and suggests that glutamate antagonists exert non-competitive inhibition with respect to the polyamine site. By contrast, 7C1-KYN, a glycine site antagonist, both decreased the maximum binding and increased the ECso value for stimulation of [3H]MK-801 binding by spermidine, .suggesting that it exerts uncompetitive antagonism of polyamine-stimulated [3H]MK-801 binding. These findings are consistent with a model in which the polyamine recognition site is mechanistically distinct from the glutamate site but may partially overlap the recognition site
-6.0
//// Baseline
10//.M ,.Gltllan-,ate
10,u.M Glycine
10/J.M+Glycine 10/zM L.Glutamate
Fig. 6. Bar chart of EC5o values for stimulation by spermidine in the presence of L-glutamate(Glu) and/or glycine(Gly)with (hatched bars) and without (open bars) 7-chlorokynurenic acid (7CI-KYN). Values are means +-- S.E.M. of 6 separate experiments, each performed in triplicate. Logarithmic values of ECs0 were used for statistical calculations. *P < 0.02, # P < 0.01 vs control baseline, # # P < 0.001 vs control baseline, # # # P < 0.02 vs control baseline. Glycine significantly reduced the ECso value compared to the baseline value, both obtained in the presence of 7-chlorokynurenic acid (7CI-KYN), P < 0.02.
for glycine. It has been shown that spermine decreases the K d for binding of [3H]glycine to its recognition site and decreased the K i for inhibition of [3H]glycine binding by glycine agonists but not by antagonists TM. Those data further support a model in which polyamine and glycine sites are closely linked. Differential effects were also observed in the manner in which glutamate and glycine potentiated polyamineinduced N M D A receptor activation. Neither agent stimulated [3H]MK-801 binding over the maximal level induced by spermidine alone. However, glutamate but not glycine decreased the ECs0 value for stimulation of [3H]MK-801 binding by spermidine. These data differ from those of Ransom and Stec 19 who reported effects of glutamate and glycine that were additive to those of spermidine. However, these data are consistent with a later report 2~ showing a similar level of polyamine-stimulated binding in the presence and absence of glutamate and glycine. A consequence of the differential effects of glutamate and glycine on the polyamine dose-response curve is that glutamate significantly increases [3H]MK801 binding in the presence of non-saturating spermidine concentrations whereas glycine does not. Total brain spermidine concentrations are reported to be in the range of 20-100 #g/g wet tissue 29. However, the effective extracellular concentration is unknown and may be several orders of magnitude lower. The mutually facilitatory effect of L-glutamate and subsaturating concentrations of
290
spermidine may therefore be of physiological relevance. The similar levels of maximum polyamine-stimulated [3H]MK-801 binding in the presence or absence of L-glutamate or glycine found in the present study need not imply similar levels of channel activation. We have previously suggested that PCP receptor ligands may gain access to their receptor via two paths: a fast, hydrophilic path associated with the open NMDA channel and a slow, hydrophobic path representing diffusion through the lipid bilayer or hydrophobic domains of the closed NMDA receptor complex 7"8"t°. It is therefore possible that polyamines increase binding primarily by facilitating entry of [3H]MK-801 via the slow diffusion path so that data obtained at a single time point would be unable to distinguish binding to open as compared to closed channels. A kinetic approach in which the slow and fast components of binding can be resolved independently would
be required to compare the influences of L-glutamate and glycine upon the degree of channel activation in the presence of saturing concentrations of polyamines. It has been proposed that NMDA antagonists might be useful in the treatment of excitotoxic brain disorders while positive allosteric modulators might be effective in the treatment of dementing illnesses such as Alzheimer's disease or schizophrenia. Further analysis of the interactions between recognition sites of the NMDA receptor complex will be crucial to the development of specific neurochemical manipulations of NMDA receptor functioning in such clinical situations.
Acknowledgements.This study was supported by Grants USPHS DA-03383, Riner Foundation (S.R.Z.); USPHS MH-00631 (D.C.J.), NARSAD (D.C.J., R.S.); and the Department of Psychiatry, AECOM (Dr.H.M. van Praag, Chairman).
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27
N-methyl-o-aspartate receptor complex, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 6547-6550. Kemp, J.A., Foster, A.C. and Wong, E.H., Non-competitive antagonists of excitatory amino acid receptors, Trends Neurosci., 10 (1987) 294-298. Kleckner, N.W. and Dingledine, R., Requirement for glycine in activation of NMDA-receptors expressed in Xenopus oocytes, Science, 241 (1988) 835-837. Olverman, H.J., Jones, A.W. and Watkins, J.C., L-Glutamate has higher affinity than other amino acids for [3H]-D-AP5 binding sites in rat brain membranes, Nature, 307 (1984) 460-462. Patneau, D.K and Mayer, M.L., Structure-activity relationships for amino acid transmitter candidates acting at N-methylD-aspartate and quisqualate receptors, J. Neurosci., 10 (1990) 2385-2399. Ransom, R.W. and Deschenes, N.L., Polyamines regulate glycine interaction with the N-methyl-D-aspartate receptor, Synapse, 5 (1990) 294-298. Ransom, R.W. and Stec, N.L., Cooperative modulation of [3H]MK-801 binding to the N-methyl-D-aspartate receptor-ion channel complex by L-glutamate, glycine, and polyamines, J. Neurochem., 51 (1988) 830-836. Reynolds, l.J., Arcaine uncovers dual interactions of polyamines with the N-methyl-D-aspartate receptor, J. Pharmacol. Exp. Ther., 255 (1990) 1001-1007. Reynolds, l.J. and Miller, R.J., lfenprodil is a novel type of N-methyl-D-aspartate receptor antagonist: interaction with polyamines, Mol. Pharmacol., 36 (1989) 758-765. Reynolds, I.J. and Miller, R.J., Multiple sites for the regulation of the N-methyl-D-aspartate receptor, Mol. Pharmacol., 33 (1988) 581-584. Reynolds, l.J., Murphy, S.N. and Miller, R.J., 3H-labeled MK801 binding to the excitatory amino acid receptor complex from rat brain is enhanced by glyeine, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 7744-7748. Rotbman, S.M. and Olney, W., Excitotoxicity and the NMDA receptor, Trends Neurosci., 10 (1987) 299-302. Sircar, R., Frusciante, M.J., Javitt, D.C. and Zukin, S.R., Glycine reverses 7-chlorokynurenic acid-indueed inhibition of [3H]MK-801 binding, Brain Research, 504 (1989) 325-327. Vincent, J.P., Kartalovski, B., Geneste, P., Kamenka, J.M. and Lazdunski, M., Interaction of pheneyelidine ('angel dust') with a specific receptor in rat brain membranes, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 4678-4682. Watkins, J.C. and Olverman, H.J., Agonists and antagonists for
291 excitatory amino acid receptors, Trends Neurosci., 10 (1987) 265-272. 28 Williams, K., Romano, C., Dichter, M.A. and Molinoff, P.B., Modulation of the NMDA receptor by polyamines, Life Sci., 48 (1991) 469-498. 29 Williams, K., Romano, C. and Molinoff, P.B., Effects of polyamines on the binding of [3H]MK-801 to the N-methyl-D-aspar-
tate receptor: pharmacological evidence for the existence of a polyamine recognition site, Mol. Pharmacol., 36 (1989) 575581. 30 Zukin, S.R. and Zukin, R.S., Specific [3H]phencyclidine binding in rat central nervous system, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 5372-5376.
285
BRES 17056
Polyamine effects upon Nomethyl-D-aspartate receptor functioning: differential alteration by glutamate and glycine site antagonists Ilana Zylberman Nussenzveig 1, Ratna Sircar 1'3, Ma-Li Wong 1, Mark J. Frusciante 1, Daniel C. Javitt 1'2'4 and Stephen R. Zukin 1'2'4 Departments of 1Psychiatry, 2Neuroscience and 3Neurology, Albert Einstein College of Medicine, Bronx, NY (U.S.A.) and 4Bronx Psychiatric Center, Bronx, NY 10461 (U.S.A.) (Accepted 21 May 1991) Key words: N-methyl-D-aspartate receptor ionophore complex; Glutamate; Glycine; Polyamine; Spermidine; 7-Chlorokynurenic acid; D-(-)-2-Amino-5-phosphonovaleric acid
Polyamines such as spermidine potentiate activation of the N-methyl-D-aspartate (NMDA)-type excitatory amino acid receptor. The goal of the present study was to investigate interactions between the putative polyamine binding site and previously described sites for glutamate and glycine. Binding of the high-potency PCP receptor ligand [3H]MK-801 to well-washed rat brain membranes was used as an in vitro probe of NMDA receptor activation. Spermidine concentration-response studies were performed in the absence and presence of both glutamate and glycine, with and without D-(-)-2-amino-5-phosphonovaleric acid (D(-)AP-5) or 7-chiorokynurenic acid (7C1-KYN). Incubation in the presence of spermidine alone induced a 20.4-fold increase in [3H]MK-801 binding with an ECs0 value of 13.3/~M. The mean concentration of spermidine which induced maximal stimulation of binding was 130/~M (n = 10, S.E.M. = 24.66, range = 25-250 ktM). Glutamate (10 /zM) decreased the ECs0 value for spermidine-induced stimulation of [3H]MK-801 binding to 3.4/~M. Glycine (10 ktM) did not significantly alter either maximum spermidine-induced [3H]MK-801 binding or the ECso value for spermidine-induced stimulation of [3H]MK-801 binding. Incubation in the presence of the specific glutamate antagonist D(-)AP-5 attenuated [3H]MK-801 binding in a glutamate-reversible fashion. The competitive glycine antagonist 7CI-KYN decreased maximum spermidine-induced [ 3H]MK-801 binding in a glycine-reversible fashion. In addition, 7CI-KYN increased the ECso value for spermidine-induced stimulation of [3H]MK-801 binding while D(-)AP-5 was without effect. These findings suggest that glutamate and glycine regulate the polyamine binding site differentially. PCP-like agents induce a psychotomimetic state closely resembling schizophrenia by inhibiting NMDA receptor-mediated neurotransmission. The ability of polyamines to modulate NMDA receptor functioning suggests a potential site for pharmacological intervention. INTRODUCTION N-Methyl-D-aspartate ( N M D A ) receptors play a fundamental role in the induction of long-term potentiation and frequency and amplitude-dependent gating phenomena 4. Evidence is accumulating that the brain damage associated with anoxia, stroke, hypoglycemia, epilepsy and neurodegenerative diseases (Huntington's disease) may be at least partially produced by pathological overactivation of N M D A receptors 11'24. Phencyclidine (1-(1phenylcyclohexyl)piperidine, PCP, 'angel dust'), induces prolonged psychotic reactions closely resembling schizophrenia 3. Neurobehavioral effects of PCP are mediated at a specific high-affinity binding site (PCP receptor) 26"3° located within the N M D A receptor-gated ion channel 4"9. The N M D A receptor complex is a postsynaptic ligandgated cation channel with multiple regulatory sites 22'27. The agonist site is sensitive to excitatory amino acids (Lglutamate > N M D A > ibotenate > kainate) 6"16'27. The
Hill coefficient for activation of N M D A receptors by glutamate has been shown to be significantly greater than unity suggesting that more than one molecule of agonist may be required for activation 7'8'1°'17. Glycine, acting at a strychnine-insensitive site, potentiates and may be required for N M D A receptor activation 1'12'15'23. Most recently, it has been demonstrated that polyamines stimulate N M D A receptor activation in a fashion that is additive to the effects of glutamate and giycine 19. The goal of the present study was to elucidate mechanisms involved in the interactions between polyamines and other regulators of N M D A receptor activation. L-Glutamate and D-(-)2-amino-5-phosphonovaleric acid (D(--)AP-5) were used respectively as agonist and antagonist at the excitatory amino acid recognition site 3"5'6'9'14'16'27.Glycine and 7-chlorokynurenic acid (7C1-KYN) served as putative agonist and antagonist at the glycine site 13'25. Binding of the PCP receptor ligand [3H]MK-801 was used as an in vitro probe of N M D A channel activation 14.
Correspondence: S.R. Zukin, Department of Psychiatry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, F-111, Bronx, NY 10461, U.S.A. Fax: (1) (212) 430-2488.
286 MATERIALS AND METHODS
Membrane preparation Extensively washed crude synaptic membranes were prepared as described previously 7'1°. Cerebral cortex plus hippocampus from male Sprague-Dawley rats (150-200 g) were rapidly removed and homogenized in 15 vols. (v/w) of ice-cold 0.32 M sucrose, using a Teflon-glass homogenizer at 800 rpm. The homogenate was centrifuged at 1000 g for 10 min in a Sorvall RC-5B refrigerated centrifuge. The supernatant was decanted and recentrifuged at 20,000 g for 20 min. The resulting pellet was resuspended in 30 vols. (v/w) of deionized water using a Brinkman Polytron (setting 6); the resulting suspension was centrifuged at 8000 g for 20 min. The supernatant and buffy coat were decanted and centrifuged at 48,000 g for 20 min. The pellet was then resuspended in 5 mM EDTA, buffered to pH 7.4 by the addition of riffs base (approx. 15 mM), and incubated for 1 h at 37 °C. After incubation, the membrane suspension was again centrifuged at 48,000 g for 20 min. Pellets were then frozen at -4 ° for 5-46 days. On the day of the experiment, frozen pellets were thawed and suspended in 30 vols. (v/w) of deionized water for 1 h at 37 °C. The membrane suspension was recentrifuged for 20 min at 48,000 g. Pellets were then washed 3 times by resuspension in 30 vols. (v/w) of deionized water and centrifugation at 48,000 g. After the final centrifugation, pellets were suspended in 30-50 vols. (v/w) of 5 mM Tris-acetate buffer adjusted to pH 7.4.
Radioreceptor binding assay For all studies, 1-ml aliquots (138-264/~g protein) of homogenate were incubated for 90 min at 25 °C with 0.84-1 nM [3H]MK801 and increasing concentrations of spermidine. Studies were per-
1.2
formed in the absence and presence of o(-)AP-5 or 7CI-KYN, with and without L-glutamate or glycine, as indicated. All points were determined in triplicate. Non-specific binding was determined in the presence of 10 FM MK-801. [3H]MK-801 (15.0-17.8 Ci/mmol) was obtained from Dupont/NEN (Boston, MA). 7C1-KYN was purchased from Tocris (Buckhurst Hill, Essex, U.K.), and o(-)AP-5 from Cambridge Research Biochemicals (Atlantic Beach, NY). L-Glutamate, glycine and spermidine were obtained from Sigma Biochemicals (St. Louis, MO). Incubations were terminated by filtration under reduced pressure using a 24-well cell harvester (Brandel Corp., Gaithersburg, MD) and Whatman GF/B filters which had been presoaked for 30 min in 0.3% polyethyleneimine. Filter disks were placed in vials containing 4 ml Hydrofluor (National Diagnostics, Manville, NJ) and counted using an LKB 1218 Rackbeta scintillation counter at a counting efficiency of 60.11%. Dpm were determined by dividing counts per minute by calculated counting efficiency. The protein content was determined using a protein assay kit (procedure P5656, 1987) from Sigma Diagnostics (St. Louis, MO).
Data analysis The concentration of spermidine required to produce half-maximal enhancement (ECs0 value) of [3H]MK-801 binding was determined using linear regression analysis of ln-logit plots. The maximum binding of [3H]MK-801 was determined within the range of spermidine concentrations of 0 to 300-1000/~M (7CI-KYN series) or 0 to 250-300 #M (o(-)AP-5 series). Values in text represent mean -+ S.E.M. All statistical comparisons were performed using unpaired, two-tailed Student's t-tests unless otherwise indicated.
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287 RESULTS
all conditions (Figs. 1, 2). A similar biphasic effect of polyamines on [3H]MK-801 binding has been reported previously2°'28, suggesting that at high concentrations, polyamines may act at an inhibitory as well as a facilitatory binding site. In the absence of D(-)AP-5 or 7CIKYN, the mean EC50 value for stimulation by spermidine was 13.3 + 2.2/,M. Incubation in the presence of glycine did not significantly affect the spermidine ECs0 value (n = 10, t = 2.04, df -- 18, 0.1 > P > 0.05) (Figs. 5, 6). By contrast, incubation in the presence of L-glutamate significantly decreased the mean ECs0 value for stimulation of [3H]MK-801 binding by spermidine in the absence of glycine from 13.3 _+ 2.2/~M to 3.44 _+ 0.67 /*M (P < 0.001) and in the presence of glycine from 8.26 + 1.08 a M to 1.89 -+ 0.36/~M (P < 0.001) (Figs. 5, 6). In the absence of L-glutamate, with or without glycine, maximum [3H]MK-801 binding occurredat a mean spermidine concentration of 96.25 + 15.09/~M. In the presence of L-glutamate, with or without glycine, maximum [3H]MK-801 binding occurred at a mean spermidine concentration of 36.4 -+ 14.40/~M. Maximum [3H]MK-801 binding induced by spermidine was not significantly affected by the presence of L-glutamate or glycine either
Specific [3H]MK-801 binding was determined in the presence of increasing concentrations of spermidine and in the absence or presence of L-glutamate or glycine. One series of studies was performed in the presence of 7CI-KYN (10/,M), while a second series was performed in the presence of D(-)AP-5 (1/~M). No significant differences in control binding occurred between the two groups in the absence or presence of L-glutamate or glycine, but in the presence of combined L-glutamate and glycine [3H]MK-801 binding was significantly greater in the D(-)AP-5 series than in the 7CI-KYN series of experiments. Spermidine concentrations varied from 0 to 250-300/,M in the D(-)AP-5 series and 0 to 300-1000 aM in the 7CI-KYN series of experiments. In the absence of added spermidine, incubation in the presence of L-glutamate or glycine led to significant 4.69fold (P < 0.002) and 2.69-fold (P < 0.001) mean increases in specific [3H]MK-801 binding, respectively. Incubation in the presence of spermidine led to an initial concentration-dependent increase in specific [3H]MK-801 binding followed at higher doses by an inhibition, under
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Fig. 2. Concentration-response curves for stimulation of specific [3H]MK-801 binding by spermidine under conditions shown. Figure shows the pooled means from 3 separate experiments, each performed in triplicate. Error bars are the S.E.M. values.
288 individually or in combination (n = 10) (Figs. 3B, 4B). Incubation in the presence of a low dose of o ( - ) A P - 5 (1 /lM) alone did not significantly inhibit [3H]MK-801 binding (Fig. 3A). Even at this dose, however, D(-)AP-5 significantly decreased the maximum spermidine-induced stimulation of [3H]MK-801 binding in the absence (P < 0.02; Figs. 1A, 3B) and presence (P < 0.02; Figs. 1C, 3B) of glycine, in an L-glutamate-reversible fashion (Figs. 1B, D, 3B). Incubation in the presence of D(-)AP-5 did not significantly alter the ECs0 value for stimulation by spermidine under any condition (Figs. 1, 5). Incubation in the presence of 7CI-KYN alone decreased [3H]MK-801 binding to 23.66 - 6.10 % of control (P < 0.001) in a fashion that was reversible by glycine but not by L-glutamate (Fig. 4A). Incubation in the presence of 7C1-KYN, in the absence of added L-glutamate or glycine, also significantly decreased the maximum spermidine-stimulated [3H]MK-801 binding (P < 0.001, Figs. 2A, 4B). By contrast, no significant inhibi-
0.6
A [~
tion of maximum binding was observed in the presence of L-glutamate, glycine or combined L-glutamate and glycine (Figs. 2, 4B). By contrast to o ( - ) A P - 5 , incubation in the presence of 7CI-KYN significantly increased the ECso value for spermidine-induced stimulation of [3H]MK-801 binding (P < 0.02, Figs. 2A, 6). This effect occurred in the presence of L-glutamate (P < 0.02, Figs. 2B, 6) but not in the presence of glycine (Fig. 2C, D). Incubation in the presence of L-glutamate significantly decreased the ECs0 value for stimulation by spermidine even in the presence of 7CI-KYN (P < 0.05, Fig. 6). DISCUSSION N M D A receptor activation is regulated by several classes of agents, including excitatory amino acids, glycine and polyamines. These agents mediate their actions at independent binding sites associated with the N M D A
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Fig. 3. A: bar chart of specific [3H]MK-801 binding in the absence of spermidine (SPD), in the presence of L-glutamate (Glu) and/or glycine (Gly) with (hatched bars) and without (open bars) 0-(-)-2amino-5-phosphonovaleric acid (o(-)AP-5). Values are means -+ S.E.M. of 4 separate experiments, each performed in triplicate. B: bar chart of maximum [3H]MK-801 binding induced by spermidine (SPD) in the presence of L-glutamate (Glu) and/or glycine (Gly) with (hatched bars) and without (open bars) o-(-)-2-amino-5phosphonovaleric acid (D(-)AP-5). Values are means -+ S.E.M. of 4 separate experiments, each performed in triplicate. *P < 0.02.
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Fig. 4. A: bar chart of specific [3H]MK-801 binding in the absence of spermidine (SPD), in the presence of u-glutamate (Glu) and/or glycine (Gly) with (hatched bars) and without (open bars) 7-chlorokynurenic acid (7CI-KYN). Values are means -+ S.E.M. of 6 separate experiments, each performed in triplicate. *P < 0.02, **P < 0.005. B: bar chart of maximum [3H]MK-801 binding induced by spermidine (SPD) in the presence of L-glutamate (Glu) and/or glycine (Gly) with (hatched bars) and without (open bars) 7-chlorokynurenic acid (7C1-KYN). Values are means -+ S.E.M. of 6 separate experiments, each performed in triplicate. ***P < 0.001.
289
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receptor complex. It is now well established that glutamate and glycine act in concert with polyamines to regulate N M D A channel function. The present data demonstrate that occupation of the glutamate and glycine sites by agonists or antagonists differentially alters the effects of interaction of polyamines with their unique binding site. Moreover, glutamate- and glycine-site antagonists differentially affect polyamine-stimulated [3H]MK-801 binding, suggesting that the glutamate and glycine sites are linked differentially with the polyamine site. The major finding of the present study is that glutamate- and glycine-site antagonists differentially affect polyamine-induced stimulation of N M D A receptor activation. D(-)AP-5, a glutamate site antagonist, inhibited maximum polyamine-induced [3H]MK-801 binding without affecting the EC50 value for spermidine-induced stimulation of [3H]MK-801 binding. This finding agrees with an earlier report that CPP prevents polyamine-induced enhancement of PCP receptor binding 2~ and suggests that glutamate antagonists exert non-competitive inhibition with respect to the polyamine site. By contrast, 7C1-KYN, a glycine site antagonist, both decreased the maximum binding and increased the ECso value for stimulation of [3H]MK-801 binding by spermidine, .suggesting that it exerts uncompetitive antagonism of polyamine-stimulated [3H]MK-801 binding. These findings are consistent with a model in which the polyamine recognition site is mechanistically distinct from the glutamate site but may partially overlap the recognition site
-6.0
//// Baseline
10//.M ,.Gltllan-,ate
10,u.M Glycine
10/J.M+Glycine 10/zM L.Glutamate
Fig. 6. Bar chart of EC5o values for stimulation by spermidine in the presence of L-glutamate(Glu) and/or glycine(Gly)with (hatched bars) and without (open bars) 7-chlorokynurenic acid (7CI-KYN). Values are means +-- S.E.M. of 6 separate experiments, each performed in triplicate. Logarithmic values of ECs0 were used for statistical calculations. *P < 0.02, # P < 0.01 vs control baseline, # # P < 0.001 vs control baseline, # # # P < 0.02 vs control baseline. Glycine significantly reduced the ECso value compared to the baseline value, both obtained in the presence of 7-chlorokynurenic acid (7CI-KYN), P < 0.02.
for glycine. It has been shown that spermine decreases the K d for binding of [3H]glycine to its recognition site and decreased the K i for inhibition of [3H]glycine binding by glycine agonists but not by antagonists TM. Those data further support a model in which polyamine and glycine sites are closely linked. Differential effects were also observed in the manner in which glutamate and glycine potentiated polyamineinduced N M D A receptor activation. Neither agent stimulated [3H]MK-801 binding over the maximal level induced by spermidine alone. However, glutamate but not glycine decreased the ECs0 value for stimulation of [3H]MK-801 binding by spermidine. These data differ from those of Ransom and Stec 19 who reported effects of glutamate and glycine that were additive to those of spermidine. However, these data are consistent with a later report 2~ showing a similar level of polyamine-stimulated binding in the presence and absence of glutamate and glycine. A consequence of the differential effects of glutamate and glycine on the polyamine dose-response curve is that glutamate significantly increases [3H]MK801 binding in the presence of non-saturating spermidine concentrations whereas glycine does not. Total brain spermidine concentrations are reported to be in the range of 20-100 #g/g wet tissue 29. However, the effective extracellular concentration is unknown and may be several orders of magnitude lower. The mutually facilitatory effect of L-glutamate and subsaturating concentrations of
290
spermidine may therefore be of physiological relevance. The similar levels of maximum polyamine-stimulated [3H]MK-801 binding in the presence or absence of L-glutamate or glycine found in the present study need not imply similar levels of channel activation. We have previously suggested that PCP receptor ligands may gain access to their receptor via two paths: a fast, hydrophilic path associated with the open NMDA channel and a slow, hydrophobic path representing diffusion through the lipid bilayer or hydrophobic domains of the closed NMDA receptor complex 7"8"t°. It is therefore possible that polyamines increase binding primarily by facilitating entry of [3H]MK-801 via the slow diffusion path so that data obtained at a single time point would be unable to distinguish binding to open as compared to closed channels. A kinetic approach in which the slow and fast components of binding can be resolved independently would
be required to compare the influences of L-glutamate and glycine upon the degree of channel activation in the presence of saturing concentrations of polyamines. It has been proposed that NMDA antagonists might be useful in the treatment of excitotoxic brain disorders while positive allosteric modulators might be effective in the treatment of dementing illnesses such as Alzheimer's disease or schizophrenia. Further analysis of the interactions between recognition sites of the NMDA receptor complex will be crucial to the development of specific neurochemical manipulations of NMDA receptor functioning in such clinical situations.
Acknowledgements.This study was supported by Grants USPHS DA-03383, Riner Foundation (S.R.Z.); USPHS MH-00631 (D.C.J.), NARSAD (D.C.J., R.S.); and the Department of Psychiatry, AECOM (Dr.H.M. van Praag, Chairman).
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