Life Sciences, Vol. 48, pp. 469-498 Printed in the U.S.A.

Pergamon Press

MINIREVIEW

M O D U L A T I O N OF THE NMDA R E C E P T O R BY POLYAMINES Keith Williams1,3, Carmelo Romanol, 4, Marc A. Dichter 2, and Perry B. Molinoff 1 D e p a r t m e n t s o f 1pharmacology and 2Neurology, U n i v e r s i t y o f P e n n s y l v a n i a School o f Medicine, Philadelphia, PA 19104-6084 (Received in final form November 27, 1990)

SUMMARY Results of recent biochemical and electrophysiological studies have suggested that a recognition site for polyamines exists as part of the NMDA receptor complex. This site appears to be distinct from previously described binding sites for glutamate, glycine, Mg ++, Zn ++, and open-channel blockers such as MK-801. The endogenous polyamines spermine and spermidine increase the binding of open-channel blockers and increase NMDA-elicited currents in cultured neurons. These polyamines have been termed agonists at the polyamine recognition site. Studies of the effects of natural and synthetic polyamines on the binding of [3H]MK-801 and on NMDAelicited currents in cultured neurons have led to the identification of compounds classified as partial agonists, antagonists, and inverse agonists at the polyamine recognition site. Polyamines have also been found to affect the binding of ligands to the recognition sites for glutamate and glycine. However, these effects may be mediated at a site distinct from that at which polyamines act to modulate the binding of open-channel blockers. Endogenous polyamines may modulate excitatory synaptic transmission by acting at the polyamine recognition site of the NMDA receptor. This site could represent a novel therapeutic target for the treatment of ischemia-induced neurotoxicity, epilepsy, and neurodegenerative diseases.

INTRODUCTION The acidic amino acids glutamate and aspartate are thought to be the major fast excitatory neurotransmitters in the mammalian central nervous system (CNS). Receptors for excitatory amino acids (EAAs) are usually classified on the basis of agonists that selectively activate them. It is currently thought that there are five subtypes of receptors for EAAs. The 3Author for correspondence. 4present address: Department of Ophthalmology and Visual Sciences, Washington University Medical Center, St. Louis, MO 63110. 0024-3205/91 $3.00 +.00 Copyright (c) 1991 Pergamon Press plc

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three major subtypes of EAA receptor that have been identified are classified as NMDA (Nmethyl-D-aspartate), AMPA ((z-amino-3-hydroxy-5-methyl-4-isoxazoleproprionicacid), and kainate receptors (1-4). The AMPAreceptor is also activated by quisqualate and was formerly called the quisqualate receptor (1, 2, 4). These three subtypes of receptor are ligand-gated ion channels, probably composed of several subunits similar to the well-characterized nicotinic acetylcholine and GABAAreceptors (5-9). Another class of receptors, activated by quisqualate but not by AMPA, and coupled to the breakdown of inositol phospholipids, is called a metabotropic quisqualate receptor (3,4). An additional class of EAA receptors that is sensitive to the glutamate analogue L-2-amino-4-phosphonobutyric acid (L-APB) has been described (1-4). The L-APB-sensitive receptor may be a presynaptic autoreceptor. The NMDA receptor/ion channel complex contains an integral ion channel that gates Na +, K +, and Ca ++ ions and is blocked in a voltage-dependent manner by Mg ++ (10-13). The block of the ion channel by Mg ++ is relieved during membrane depolarization, allowing activation of the receptor complex by NMDA or glutamate. The AMPA and kainate receptors gate Na + and K+ but not Ca ++ and are not blocked by Mg ++ (2, 3, 13). NMDA receptors are involved in the generation of long-term potentiation (LTP) in the hippocampus (3, 14) and have been implicated in certain types of spatial learning (15). The voltage-dependent blockade of NMDA receptors by Mg ++ probably contributes to the associative properties of induction of LTP, which requires both activation of presynaptic elements and depolarization of the postsynaptic membrane (14, 16). The influx of Ca ++ that accompanies receptor activation is thought to be involved in many of the cellular responses mediated by NMDA receptors, including the generation of LTP (3, 14, 16). EAAs are also major neurotransmitters in the spinal cord where NMDA receptors mediate some of the responses elicited by primary afferents (17). In the developing nervous system, receptors for EAAs may be involved in defining neuronal architecture and synaptic connectivity, including experience-dependent synaptic modifications in the visual cortex (18). Stimulation of NMDA receptors has been found to promote survival, maturation, and neurite outgrowth of cultured neurons isolated from the cerebellum (19-22), hippocampus (23), or spinal cord (24). Excessive or abnormally prolonged stimulation of EAAreceptors, for example following CNS ischemia, leads to neuronal degeneration. Results of in vivo and in vitro studies have shown that stimulation of NMDA receptors and increases in the concentration of intraceUular Ca ++ are involved in this excitotoxicity, and that antagonists of NMDA receptor function can protect against the neurotoxic effects of ischemia or exposure to NMDA (25-27). NMDA receptors may also be involved in the etiology of some epilepsies. In a number of in vitro models of epilepsy, activation of NMDA receptors has been shown to be necessary for the development of susceptibility to seizures or epileptiform discharges (28-30). Other data suggest a role for NMDA receptors in the actual occurrence of epileptiform discharges (3134). The neuronal loss that sometimes occurs following status epilepticus may also be due to excitotoxicity mediated by NMDA receptors (27). Excessive activation of NMDA receptors may also be involved in brain damage following hypoglycemia and concussive head injury, and in a number of chronic neurodegenerative diseases including Huntington's disease, Alzheimer's disease, parkinsonism, and neurolathyrism (25, 26, 35). PHARMACOLOGY OF NMDA RECEPTORS The NMDA receptor complex contains a number of distinct recognition sites for endogenous and exogenous ligands (Figure 1). These include binding sites for glutamate (or NMDA), glycine (36-38), Mg ++ (10-12, 39), Zn ++ (39--42), and open-channel blockers such as PCP (phencyclidine), TCP (N- [ 1-(2-thienyl)cyclohexyl]piperidine),ketamine, and MK-801

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471

Polyamines I ANTAGONIST

ANTAGONIST

ANTAGONIST

INVERSE AGONIST

I

Figure 1. Schematic Model of the NMDA Receptor. In this model, the receptor contains distinct recogrfition sites for glutamate (GLU) and NMDA, glycine (GLY), MK-801 (and TCP), Mg ++, Zn++, and polyamines including spemaine, DET, and DA10. 7-Chlorokynurenic acid (7-CL-KYN) is a competitive antagonist at the glycine site, D-AP5 and CPP are competitive antagonists at the glutamate site, and DET is a competitive antagonist at the recognition site for polyamines. The ion channel of the receptor is blocked in a voltage-dependent manner by Mg ++. Under depolarizing conditions or in the absence of extraceUular Mg ++, glutamate and glycine cause channel opening. In biochemical assays glutamate and glycine increase the binding of [3H]MK-801. In the nominal absence of ~lutamate and glycine, Mg++ enhances the binding of [3H]MK-801. In the presence of glu~tamate and glycine, Mg++and Zn++inhibit the binding of [3H]MK-801. Spermine increases, whereas DA 10 decreases, the binding of [3H]MK-801 and the NMDA-elicited inward current in hippocampal neurons. These effects are blocked by DET. At higher concentrations, spermine can inhibit the NMDA-elicited current, an effect that is also blocked by DET. (43-46). Glycine has been shown to enhance the effects of glutamate (or NMDA) in both electrophysiological (36-38, 47, 48) and biochemical (49-51) studies. In Xenopus oocytes expressing NMDA receptors after injection of RNA from rat forebrain there is an absolute requirement for glycine in order for the channel to be opened by NMDA (48). Thus, glycine can be considered a "co-agonist" at the NMDA receptor complex. Binding of [3H]MK-801 and [3H]TCP is greatly enhanced in the presence of glutamate and glycine (45, 49, 52). Glutamate and glycine increase both the rates of association and dissociation of binding of [3H]MK-801 and [3H]TCP, with little or no effect on the apparent equilibrium dissociation constant of these ligands when studies are carried out under equilibrium conditions (53, 54). This is consistent with the hypothesis that [3H]MK-801 and [3H]TCP bind to an open-channel state of the receptor complex, probably to a site within the channel itself, and that the accessiblity of this site is increased in the presence of glutamate and glycine (Figure 1).

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In the presence of glutamate and glycine, Mg ++ (0.1-100 mM) inhibits the binding of [3H]MK-801 and [3H]TCP (39, 55, 56). In well-washed membranes in the absence of added amino acids, micromolar concentrations of Mg ++ enhance the binding of [3H]MK-801 and [3H]TCP, but the concentration-effect curve is biphasic and inhibition is seen at higher concentrations (0.1-100 mM) of Mg ++ (39, 55, 56). In either the absence or presence of exogenous amino acids, Zn ++inhibits the binding of [3H]MK-801 and [3H]TCP to well-washed membranes prepared from rat brain (39, 55). Both Zn++ and Mg ++ inhibit NMDA-elicited currents. Results from biochemical andelectrophysiological studies suggest that the recognition site for Zn ++ is distinct from the recognition site for Mg ++ (39-42, 55, 57). Recently, it was found that intracellular as well as extracellular Mg ++ can block the ion channel of the NMDA receptor in a voltage-dependent manner, suggesting the presence of two distinct voltagedependent binding sites for Mg ++ (58). Although the effects of Mg ++ are strongly voltagedependent (10, 11, 58), those of Zn ++ are only weakly voltage-dependent (40, 41, 59). It has been suggested that there are two binding sites for Zn ++ on the NMDA receptor complex, one within and one outside the portion of the receptor that is sensitive to membrane potential (42). POLYAMINES

The endogenous polyamines spermidine, spermine, and putrescine (Figure 2) are ubiquitously distributed in eukaryotic tissues. Putrescine (1,4-diaminobutane) is formed from ornithine by the action of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis (Figure 3). The conversions of putrescine to spermidine, and spermidine to spermine are catalyzed by aminopropyltransferases using propylamine moieties derived from decarboxylated S-adenosylmethionine (Figure 3). Polyamines have been shown to be involved in, and are thought to be required for, the normal growth and differentiation of many types of cells (60, 61). Polyamines are present in high concentrations even in the mature CNS. In the brains of various species, putrescine is present at concentrations of 4-70 nmoles/ g of tissue, and spermidine and spermine are each present at levels of 50-1400 nmoles/g of tissue (62-68). There is considerable regional variation in the concentrations of polyamines in the CNS (63, 65, 67). High-affinity uptake systems for spermine in slices of rat cerebral cortex have been reported (69), as has depolarization-induced release of tritium from slices pre-loaded with [3H]spermine (70). Electrical stimulation of the motor cortex of rhesus monkeys was found to lead to a decrease in the content of endogenous spermine and spermidine but not putrescine (67). Intracerebroventricular injection of spermine and spermidine into mice leads to a number of effects including sedation and hypothermia at low doses and to convulsions at higher doses (71). Based largely on these observations, it has been suggested that polyamines may play a role as neurotransmitters or as modulators of synaptic transmission in the CNS (70, 72), but until recently no specific neuromodulatory function of polyamines had been described. THE POLYAMINE SITE OF THE NMDA RECEPTOR The effects of polyamines on the NMDA receptor were first described by Ransom and Stec (73) who reported that spermidine and spermine increased the apparent affinity of the binding site for [3H]MK-801 in the presence or nominal absence of glutamate and glycine. Studies in our own laboratory, directed towards the identification of endogenous modulators of NMDA receptors, led to the purification of a factor from bovine brain, eventually identified as spermidine, that increased the binding of [3H]MK-801 above the level seen with maximally effective concentrations of glutamate and glycine (74). These observations indicated that polyamines are not likely to be acting at the recognition sites for glutamate or glycine (73, 74).

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Polyamine Modulation of NMDA Receptors

Putrescine H2N~NH

Spermidine

H H2N~N~NH

Spermine

2

473

2

DET

H

H2N~N~N~NH

H2N~/~N~NH2 H

2 H

DA10

Arcaine H 2 N ~ N H

2

H H N H2N~II,N ~ N ' ~ N H N

2

H

H

Argiotoxin 636

OH

•~ ~ I'-IU" v

Philanthotoxin343

.o

H Q N~bN~/~NI~/~N~N~ I~ H H H -H2N),==O

0 J

H ~II'NNH2 H NH2 N H Q

H '

"

Ifenprodil

H ~

CH3

Figure 2. Structures of Some of the Compounds Discussed in This Review. DET = diethylenetriamine, DA 10 = 1,10-diaminodecane Because polyamines are organic cations, the possibility that their effects on the binding of [3H]MK-801 are mediated at one or more of the divalent cation recognition sites on the NMDA receptor complex was considered. Evidence is now accumulating, however, from both biochemical and electrophysiological studies, which suggests that the effects of polyamines are mediated at a distinct recognition site. RADIOLIGAND BINDING STUDIES

1. Effects of Spermine and Spermidine Under non-equilibrium conditions, in the absence of added amino acids, sperrnine (Figure 4) and spermidine increase the binding of [3H]MK-801 and [3H]TCP (73, 75, 76). In the absence of added amino acids, the stimulatory effects of spermine and spermidine are greatly attenuated or are not seen when assays are carried out in the presence of the competitive glutamate antagonists D-AP5 (Figure 4), D-AP7, or CPP (73, 77) or in the presence of the competitive glycine antagonist 7-chlorokynurenic acid (77). This suggests that residual,

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Polyamlne Modulation of NMDA Receptors

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Ornithine

Putrescine

Spermidine

s~

mA

~ne_

~NIACETYLSPERMINE

Figure 3. Biosynthesis and Metabolism of Polyamines. This scheme is adapted from reference 61. The enzymes involved are ornithine decarboxylase (ODC), spermidine synthase (SPDS), spermine synthase (SPES), spermine/ spermidine-N 1-acetyltransferase (SAT), and polyamineoxidase (PAO). SAM = S-adenosylmethionine,MTA = 5'-methylthioadenosine.

endogenous glutamate and glycine remaining in the membranes are required for the stimulatory effects of the polyamines. An alternative interpretation is that spermine and spermidine act as agonists at the glutamate or glycine recognition sites with efficacies greater than those of the amino acids themselves. Several observations suggest that this is not the case. The structural requirements of compounds that are agonists at the glutamate and glycine recognition sites have been well documented and include the need for both acidic (usually carboxylic) and amino groups located at particular distances from the a-carbon (4, 78). In addition, spermine and spermidine increase the binding of [3H]glycine and have no effect or increase the binding of [3H]glutamate and [3H]CPP (79-83). If polyamines were acting at the glutamate or glycine recognition sites, they would be expected to inhibit the binding of [ 3H]glycine, [3H]glutamate, or [3H]CPP. Under equilibrium conditions, in the presence of maximally effective concentrations of glutamate and glycine, spermine and s[rermidine cause a 2- to 3-fold increase in the affinity of the binding site for [3H]MK-801 or pH]TCP (73, 74). The concentration-effect curves for spermine and spermidine are markedly biphasic in the presence of glutamate and glycine (74, 75). Maximal enhancement of the binding of [3H]MK-801 occurs at concentrations of 10-30 lxM, and the effect declines at higher concentrations of spermine or spermidine (Figure 4). Spermine has been reported to be 2- to 3-fold more potent than spermidine (73, 75, 76). The enhancement of binding of [3H]MK-801 by polyamines is due to an increase in the affinity of the binding site for [3H]MK-801, with no change in the number of binding sites (73, 74, 76). The effects of polyamines could be due to a change in the accessibility of the binding site for [3H]MK-801 or to a change in the conformation of the receptor complex such that the affinity of the binding site for [3H]MK-801 is increased, but the accessibility of the site is unchanged. Spermine (10-50 I.tM) greatly increased both the rates of association and dissociation of binding of [3H]MK-801 when these effects were measured in the presence of maximally effective concentrations of glutamate and glycine (75, 84). This suggests that spermine enhances the accessibility of the binding site for [3H]MK-801. In the absence or presence of spermine, the kinetics of association and dissociation of [ 3H]MK-801 are complex and cannot be accounted for by simple pseudo-first-order and first-order kinetics, respectively (84). It has also been reported that spermidine (100 ~tM) increased the rate of association and

Vol. 48, No. 6, 1991

Polyamine Modulation of NMDA Receptors

475

=E~ 125 ~_= ' " aD 1 g- - I ..l o u- ~ ' t ~ - a " ........ m at o ..~,_

oD~

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50

.=-'6 o~

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~

t e u ' ~ u "~-" & glycine "...............

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.

.

.

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.... -4-- Basal ....

.

-6

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[Spermine] (Log Molar) Figure 4. Effects or Spermine on the Binding of [3H]MK-S01.

The binding of [3H]MK-801 to well-washed membranes prepared from rat brain was determined in the nominal absence of glutamate and glycine (Basal, II), in the presence of 100 I.tML-glutamate and glycine (O), and in the presence of 300 [tM D-AP5 (ra). Results are expressed as a percentage of the binding of [ 3H]MK801 in the presence of L-glutamate and glycine but absence of spermine. Data are from ref. 74 and unpublished observations. decreased the rate of dissociation of [3H]TCP when experiments were carded out in the nominal absence of glutamate and glycine (76). It was concluded that spermidine altered the conformation ofthereceptor such that the affinity of the binding site for [3H]TCPwas increased but that its accessibility was unaltered (76). Thus, there may be differences in the effects of spermine compared to those of spermidine, or the effects of polyamines on the kinetics of binding of [3H]MK-801 and [3H]TCP may be dependent on the concentration of glutamate and glycine in the assay. It is conceivable that polyamines both increase the accessibility of the binding site for [3H]MK-801 and alter the conformation of the receptor such that the characteristics of the binding site are changed. It may be that in the presence of low concentrations of glutamate and glycine a conformational change that alters the characteristics of the binding site for [3H]MK-801 predominates over effects on the accessibility of the binding site. Like spermine (75, 84), Mg ++ increases both the rates of association and dissociation of binding of [3H]MK-801 when assays are carded out in the presence of glutamate and glycine (57, 84). However, the net effect of spermine is to increase and that of Mg ++ is to decrease the affinity of the binding site for [3H]MK-801. In the nominal absence of glutamate and glycine, both polyamines and Mg ++ can increase the binding of [3H]MK-801. However, spermidine produced a greater increase in the binding of [3H]MK-801 than did Mg ++, and the effects of spermidine and Mg ++ were additive (85), suggesting that Mg +÷ and spermidine do not act at the same recognition site to increase the binding of [3H]MK-801. On the other hand, a recent report has shown that the putative polyamine antagonist arcaine (see below) attenuates the stimulatory effects of both spermidine and Mg ++ in an apparently competitive manner (85a). It was suggested that in biochemical assays Mg ++ can act as a partial agonist at the polyamine recognition site (85a). Thus, in ligand binding assays, some of the effects of Mg +÷ may be mediated at the polyamine recognition site rather than at the voltage-dependent Mg ++ binding site characterized in electrophysiological studies of the NMDA receptor. Results of

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electrophysiological studies have shown that the the polyamine recognition site is distinct from the voltage-dependent Mg ++ site (see below). In biochemical assays, Zn++ decreases rather than increases both the rates of association and dissociation of [3H]MK-801 (57, 84). Thus, the effects of spermine and spermidine determined in biochemical assays are different from those of Zn ++. In the presence of glutamate and glycine, high concentrations of spermine do not enhance the binding of [3H]MK-801 to the same extent as do lower concentrations, and no effect is seen in the presence of 1 mM spermine (Figure 4). Thus, there may be an inhibitory effect of high concentrations of spermine that is superimposed on the stimulatory effects seen with lower concentrations. It is possible that the inhibitory effect of high concentrations of polyamines is due to an action at the binding sites for Mg++ or Zn ++ or even to a direct effect at the binding site for [3H]MK-801. Alternatively, the inhibitory effects of spermine may be mediated at a second polyamine recognition site associated with the NMDA receptor complex, as has been proposed based on results of electrophysiological studies (see below). Another possibility is that the inhibitory effect of high concentrations of spermine is due to a non-specific interaction with membrane phospholipids or to changes in pH. In electrophysiological studies, pH has been shown to affect the function of the NMDA receptor complex (86, 87). Spermine at concentrations up to 1 mM does not cause an appreciable change in the pH of the buffered solutions routinely used in our laboratory for binding assays with [3H]MK-801 (unpublished observations). Furthermore, inhibitory effects are not seen with a number of other polyamines that enhance the binding of [ 3H] MK-801 and are structurally similar to spermidine or spermine (e.g., Figure 5A), suggesting that the inhibitory effects of spermine are not due to non-specific membrane effects or changes in pH. However, since polyamines are highly basic, it remains possible that in weakly buffered solutions high concentrations of polyamines will cause appreciable changes in pH that could affect the properties of the NMDA receptor. This should be controlled for when carrying out studies of the effects of polyamines.

2. Antagonists at the Polyamine Recognition Site MK-801 has been shown to bind to the open (active) state of the NMDA receptor/ion channel complex. Because of their ability to enhance the binding of [3H]MK-801, spermine and spermidine were classified as agonists at the putative polyamine recognition site (74). If polyamines act at a distinct recognition site, then it is reasonable to suppose that compounds may be found that selectively and competitively antagonize the effects of spermine and spermidine at this site. To be usefully classified as antagonists at the polyamine site, such compounds should by themselves have no effect in biochemical and functional assays of NMDA receptors when polyamine agonists are not present. Thus, there is an important semantic distinction between compounds that can be classified as antagonists at the polyamine site, and compounds such as PCP and Mg ++ that are noncompetitive antagonists of the function of NMDA receptors. It is, of course, possible that antagonists at the polyamine site will have effects on the function of NMDA receptors in vivo or in intact cell systems by blocking the effects of endogenous polyamine agonists even in the absence of exogenous polyamine agonists. A number of diamines and triamines have been identified that decreased the binding of [3H]MK-801 in the presence of 10 gM spermine plus 100 gM glutamate and glycine, down to the level seen in the presence of glutamate and glycine (74). These compounds, which include the prototypical antagonist DET (Figure 2), had no effect on the equilibrium binding of [3H]MK-801 when assays were carried out in the presence of glutamate and glycine in the

Vol. 48, No. 6, 1991

Polyamine Modulation of NMDA Receptors

477

A +

ol

100

~

E _=

-7

ol

-~

-5

-4

Spermine

AGONIST

gly

I1

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+

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Spermine

•*---glu & gly

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PARTIAL AGONIST H

2

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~ H

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-4

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+

Spermine

ANTAGONIST (DET)

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100

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-6

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-4

.

[Polyamlne] (Log Molar)

Figure 5. Effects of Polyamines on the Binding of [3H]MK-801. The effects of 3,3'-iminobispropylamine (A), N-(2-aminoethyl)- 1,3-propanediamine (B), and DET (C) were determined in the presence of 100 ~tM L-glutamate and glycine ((D) and in the presence of 100 [tM L-glutamate and_glycineplus 10 ~tMspermine (e). Results are expressed as apercentage ofthe binding of [r~H]MK-801 in thepresence of L-glutamate and glycine but absence of spermine. The structures of the polyamines are shown next to each panel. Data are from ref. 74. absence of spermine (Figure 5C and ref. 74). One possible explanation for the effect of compounds like DET is that they act as competitive antagonists at the recognition site for spermine and spermidine. A second possibility is that the inhibitory effects are not due to competitive antagonism at the polyamine recognition site but are mechanistically equivalent to the inhibitory effect of high concentrations of spermine, the site and mechanism of action of which are not known. The latter hypothesis is difficult to test since the inhibitory effect of spermine cannot be studied in isolation from its stimulatory effect. A third possibility is that

478

Polyamine Modulation of NMDA Receptors

Vol. 48, No. 6, 1991

150 ' ,-"6

~

125

~i

100

¢ ~

75

.~.o

s0

DA10

N rt~

f

o

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.

. . . . . . .

.

-6

. . . . . . . .

,

-5

. . . . . . . .

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-3

Figure 6. DET Antagonizes the Effects of Spermidine and DA10 on the Binding of [3H]MK-801. Concentration-effect curves for spermidine (O,e) and DA10 (O,B) were determined in the absence (O,r3) and presence of 100 p.M DET (e) or 300 ktM DET (11). All assays contained 100 ktM L-glutamate and glycine, and data are expressed as in Figure 5. Data are from ref. 84 and unpublished.

[Spermidine or DA10] (Log Molar)

polyamine antagonists act at one or more of the divalent cation recognition sites, which may also be the site at which the inhibitory effect of high concentrations of spermine occurs. Subsequent experiments have indicated that DET does indeed act as a competitive antagonist of the effects of spermine and spermidine. In binding assays with [3H]MK-801, increasing concentrations of DET caused parallel shifts of the concentration-effect curve of spermidine when assays were carried out in the presence of 100 ~tM glutamate and glycine (Figure 6; ref. 84). These results are consistent with competitive interactions of spermine, spermidine, and DET at a recognition site that is distinct from the binding sites for glutamate, glycine, Mg ++, Zn ++, and MK-801. In the nominal absence of glutamate and glycine, or under nonequilibrium conditions, high concentrations of DET (> 100 lxM) cause a small increase in the binding of [3H]MK-801 (unpublished observations), suggesting that DET can act as a weak partial agonist at the polyamine recognition site under these conditions. The endogenous diamine, putrescine, had no effect on the binding of [3H]MK-801 or [3H]TCP in the absence of added modulators (73-76) or on the binding of [3H]MK-801 in the presence of glutamate and glycine (74, 75). Putrescine inhibited the stimulatory effects of spermine and spermidine in the absence (76) or presence (74) of maximally effective concentrations of glutamate and glycine. In the nominal absence of glutamate and glycine, the effect of putrescine on the enhancement of binding of [3H]TCP by spermidine was noncompetitive (76). It was suggested that putrescine does not act as a competitive antagonist at the proposed polyamine recognition site, and that the effects of putrescine may be mediated at the Mg ++ recognition site (76). Recently, arcaine (1,4-diguanidinobutane; Figure 2) has been reported to be an antagonist at the polyamine site (88). In the presence of maximally effective concentrations of glutamate and glycine, arcaine inhibited the binding of [3H]MK-801 to well-washed membranes prepared from rat brain with an IC50 of 1.5 ~tM. In the presence of glutamate and glycine plus 50 laM spermidine, the IC50 for arcaine was 12.9 ~tM (88). Increasing concentrations of arcaine caused a rightward shift of the spermidine concentration-effect curve (88). If arcaine is to be classified as a competitive antagonist at the polyamine site using the criteria described

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above, then it is necessary to postulate that there were residual endogenous polyamines present in the membrane preparation, since arcaine inhibited the binding of [3H]MK-801 even in the absence of added spermine (88). Ifenprodil (Figure 2) and its analogue SL 82.0715 are anti-hypertensive agents that reduce or protect against the neurotoxicity that occurs after cerebral ischemia (89). These compounds are non-competitive antagonists of glutamate at the NMDA receptor complex (90), which may account for their neuroprotective effects (8 I, 90). The effects of ifenprodil on NMDA receptors are seen at concentrations of 0.1-10 lxM. At similar concentrations ifenprodil has also been reported to be an antagonist at a-adrenergic receptors and at voltagedependent Ca ++ channels in the peripheral vasculature of the rat (91-93). Nanomolar concentrations of ifenprodil and SL 82.0715 inhibited the binding of [ 3H](+)-3-ppp to g sites in membranes prepared from guinea pig or rat brain (94, 95). Results of recent studies led to the suggestion that ifenprodil and SL 82.0715 may also be antagonists at the polyamine site of the NMDA receptor (81, 96). Ifenprodil and SL 82.0715 (0.1-10 I.tM) inhibited the stimulatory effects of spermine and spermidine on the binding of [3H]TCP (81,96), although these effects did not appear to be strictly competitive. Reynolds and Miller (75) reported that ifenprodil inhibited the binding of [3H]MK-801 in the presence and in the nominal absence of glutamate and glycine. Ifenprodil caused a rightward shift of the concentration-effect curve of spermine but this effect did not ape.ear to be competitive (75). It was suggested that the effects ofifenprodil on the binding of [ H]MK-801 may be due in part to an action of ifenprodil at the recognition site for Zn ++, and in part to binding to or stabilization of an inactive form of the receptor complex (75). Studies in our own laboratory have confirmed that ifenprodil inhibits the binding of [3H]MK-801 in the absence or presence of spermine (unpublished observations). However, the potency of ifenprodil was slightly increased when assays were carried out in the presence of spermine. Ifenprodil did not appear to selectively or competitively inhibit the effects of spermine on binding of [3H]MK-801 (unpublished observations). NMDA increases the concentration of cGMP in slices prepared from rat cerebellum. Ifenprodil inhibited the NMDA-stimulated increase in cGMP levels (90). Theeffect of NMDA on cerebellar cGMP levels was potentiated by spermine (81), and the inhibitory effects of ifenprodil were markedly attenuated in the presence of high concentrations (100 I.tM-1 mM) of spermine (81). One possible explanation for these effects is that ifenprodil and spermine interact at the same site (81). An alternative interpretation is that ifenprodil is a noncompetitive antagonist that binds to a site on the NMDA receptor whose affinity for ifenprodil is reduced in the presence of spermine (75). Studies of the effects of various modulators on levels of cGMP should be interpreted with caution since the increase in cGMP caused by NMDA is several steps removed from the activation of NMDA receptors, and may involve the release or formation of nitric oxide (97, 98). Furthermore, the increase in cGMP may occur in presynaptic terminals or in glial cells rather than in the postsynaptic ceils on which the NMDA receptors are located (97, 99). The effects of ifenprodil or polyamines could occur at various points along the signalling pathways leading from receptor activation to an increase in the concentration of cGMP. Schoemaker et al. (100) have reported preliminary characterization of a high-affinity (KD = 37 nM) binding site for [3H]ifenprodil. Thus, the binding of [3H]ifenprodil can be detected at concentrations several orders of magnitude lower than those at which ifenprodil inhibits the binding of open-channel blockers to the NMDA receptor complex and at which ifenprodil inhibits the function of NMDA receptors (75, 81,96, 100). The binding of [3H]ifenprodil was inhibited by micromolar concentrations of spermine and spermidine but not by putrescine, and was partially inhibited by CPP, AP5, Mg ++, and Zn++ (100). Spermine decreased the affinity

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of the binding site for [3H]ifenprodil but had no effect on the number of sites (100). These data are consistent with a competitive interaction of ifenprodil and polyamines. However, even if ifenprodil binds to a site that is distinct from the polyamine site, the effects of modulators acting at other sites, including polyamines, could appear competitive. A similar phenomenon is seen in studies of the binding of the open-channel blockers [3H]MK-801 and [3H]TCP. In these experiments, the inhibitory effects of antagonists at the glutamate and glycine sites, of inverse agonists at the polyamine site, and of divalent cations are not due to competitive interactions at the MK-801 binding site. However, these effects appear competitive insofar as the affinity of binding of [3H]MK-801 or [3H]TCP is decreased without affecting the number of binding sites. It is possible that [3H]ifenprodil binds to the ~ site rather than the NMDA receptor complex (94, 95) The available evidence suggests that ifenprodil does not act as a purely competitive antagonist at the polyamine recognition site. However, the effects of ifenprodil at the NMDA receptor are clearly modulated by polyamines, and ifenprodil may prove to be a useful tool for studies of the mechanism of action ofpolyamines at the NMDA receptor and for characterization of other sites on the receptor complex.

3. Inverse Agonists at the Polyamine Recognition Site The diamine 1,10-diaminodecane (DA i0) inhibits the binding of [ 3 H ] ~ - 8 0 1 in the absence of polyamine agonists (Figure 6). This originally led us to conclude that DAI0 may be acting nonspecifically, possibly by a direct action at the binding sites for glutamate, glycine, MK-801 or divalent cations (74). Experiments were subsequently carried out to test these possibilities. It was found that the inhibitory effect of DA10 was not altered by a 1000-fold change in the concentrations of glutamate and glycine, indicating that the effect of DA 10 is not due to a competitive action at the binding sites for the amino acids. DA10 decreased the rate of association but had no effect on the rate of dissociation of binding of [3H]MK-801 (84). In contrast, Mg ++ increases while Zn ++ decreases the rates of both association and dissociation of binding of [3H]MK-801 (57, 84). Thus, in biochemical assays, the effect of DA10 did not appear to be mediated at the recognition sites for glutamate, glycine, Mg ++, or Zn++. Another hypothesis that may explain the effect of DA 10 is that it does act at the putative polyamine recognition site but is a negative allosteric modulator or inverse agonist that decreases rather than increases the binding of [3H]MK-801. In this case, the effects of DA10 would be formally similar to those of g-carbolines at the benzodiazepine recognition site of the GABAA receptor (101, 102). An inverse agonist is defined as a compound that acts competitively at the same site as the corresponding agonist, but produces effects opposite to those of the agonist. IfDA10 is an inverse agonist, its effect should be attenuated by a selective antagonist at the polyamine site. The effect of DA10 was attenuated by the polyamine antagonist DET (Figure 6; ref. 84) and also by a high concentration (1 mM) of spermine or N(3-aminopropyl)-1,10-diaminodecane (APDA 10), which by themselves have no net effect on the equilibrium binding of [3H]MK-801 or on the inhibition of binding of [3H]MK-801 by unlabeled MK-801 or by dextromethorphan (84, 103). Since unlabeled MK-801 and dextromethorphan act competitively at the [3H]MK-801 binding site, these data suggested that the inhibitory effects of DA 10 were not due to an action of DA10 at the MK-801 binding site. These results indicate that DA10 acts at the same polyamine recognition site as spermine, spermidine, and DET. Thus, DA10 may be a negative allosteric modulator or inverse agonist that decreases the binding of [3H]MK-801 (84). This conclusion is supported by results from electrophysiological studies in which the effect of DA10 on NMDA-induced responses was

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opposite to that of spermine (see below). These results are again consistent with the proposal that there is a distinct recognition site for polyamines associated with the NMDA receptor complex. The putative polyamine antagonist arcaine (88; and see above) inhibits the binding of [3H]MK-801 even in the absence of polyamine agonists. It is possible that arcaine interacts competitively at the polyamine recognition site but is an inverse agonist, with a mechanism of action similar to that of DA10. Studies of the effects of arcaine in the presence of DET and electrophysiological studies of the effects of arcaine may help to clarify its mechanism of action at the NMDA receptor.

4. Structure-Activity Relationships of Polyamines A well-established tenet in receptor pharmacology is that a specific recognition site has characteristics that are reflected in the structure-activity relationships of ligands that act at that site. Thus, a series of systematic changes in the structure of a compound may alter its efficacy or its potency at a given recognition site. Furthermore, the resulting compounds may have properties, for example as agonists, partial agonists, or antagonists, that are different from those of the parent molecule. In addition to the compounds described above, the effects of a number of polyamines that are structurally related to putrescine, spermidine, and spermine have been determined in binding assays with [3H]MK-801 or [3H]TCP (Table I). The agonist effects of spermine and spermidine are shared by a number of di-, tri-, and tetraamines (Table I). Of a series of linear diamines having from 3-12 methylene groups, only 1,3-diaminopropane (NH2[CH2]3NH2) enhanced the binding of [3H]MK-801 and [3H]TCP. A number of tfiamines and tetraamines containing one or more propylene-diamine moieties (NH2[CH2]3NH--) also increased the binding of [3H]MK-801 and [3H]TCP (Table I). In contrast, DET (NH2[CH2]2NH[CH2]2NH2) had no effect on the binding of [3H]MK-801 in the presence of 100 ktM glutamate and glycine, but was a competitive antagonist of the effects of spermine and spermidine (74, 84). N - ( 2 - a m i n o e t h y l ) - l , 3 - p r o p a n e d i a m i n e (NH2[CH2]3NH[CH2]2NH2) behaved as a partial agonist at the polyamine site (Figure 5B; Table I). These observations led us to suggest that the presence of two amino groups separated by three methylenes is necessary for agonist effects, and that two amino groups separated by two methylenes may be sufficient for antagonist effects of polyamines to be expressed. However, the observation that N-(2-aminoethyl)- 1,5-pentanediamine ( N H 2 [ C H 2 ] 2 N H [ C H 2 ] 5 N H 2 ) and N , N ' - b i s ( 2 - a m i n o e t h y l ) l , 3 - p r o p a n e d i a m i n e (NH2[CH2]2NH[CH2]3NH[CH2]2NH2) were agonists (Table I) suggests that this is an oversimplification. It may be that, in linear tfiamines, the overall chain length of the molecule as well as the number of atoms separating the primary and secondary amino groups is an important determinant of activity at the NMDA receptor complex. In a series of linear triamines having the structure NH2[CH2]3NH[CH2]yNH2, the activity at the polyamine site was a function of overall chain length (Table I). Compounds in which Y is 3-7 were full agonists compared to spermine when assays were carried out in the presence of glutamate and glycine, and were partial or full agonists in the absence of glutamate and glycine. When Y = 8-10, these triamines were partial agonists in the presence of glutamate and glycine, but had little or no effect on the binding of [3H]MK-801 in the absence of glutamate and glycine (Table I). One such compound, APDA 10 (Y = 10), was found to be a competitive antagonist of the effect of spermine when assays were carded out in the nominal absence of glutamate and glycine (unpublished observations). When Y = 11 or 12, the triamines inhibited the binding of [3H]MK-801 in the absence or presence of other modulators.

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TABLEI Effect on Binding of

Diamines NH2(CH2)xNH2 X

MK-801 or TCP Basal or + glu, gly + glu & gly & spermine

3

1`

-

_

4, ~~

6

--

4,

7

i

m

8 O

4, 4,

4, ;

,~ 4,

4, 4,

,I,4,

4'4,

-~ 1` 1'

~ ~, -4, --

Putrescine Cadaverine

4

5

10

DA10

12

Notes

Ref.

antagonist ?

74, 76 73, 74, 75, 76 73, 74, 76

antagonist

a

76, a 76, a a

inverse agonist

74, 84, a a

Triamines NH2tCH2)xNH(CH2)yNH2 x

Y

2

2

2 3

5 2

DET

1' 1`

antagonist agonist partial agonist agonist

74, 84, a a 74, a 74, a 73, 74, 75, 76

3

3

3

4

1' 1`

--

3

5

1` 1`

--/4

3

6

1' 1`

--

3 3 3 3 3 3

7 8 9 10 11 12

I'I'

--

a

-/~

-

a

-//~ --/1' 4, 4,

,~4,

a

4, 4,

antagonist/agonist o

Spermidine

APDA10

agonist (partial) agonist

a

agonist

a

a

,~; ,~

a

Tetraamines NH2(CH2) xNH(CH2 ) Y NH(CH2)x NH2 x

Y

2

3

3

2

3 3

3 4

3 3

3

'[ 1` 1` 1`

agonist

1`1`

--__

1` 1`

--

agonist agonist

76, a 76, a 76, a 73, 74, 75, 76

6 9

'~ 1`

--

agonist

a

~ 1`

--

agonist

a

12

--/1`1`

--

Spermine

agonist

a

Table I. Activities of Polyamine Analogues Determined in Binding Assays with [3H]MK-801 or [3H]TCP. The effects of di-, tri-, and tetraamines have been determined in the nominal absence of glutamate and glycine (basal), in the presence of maximally effective concentrations of glutamate (glu) and glycine (gly), and in the presence of glutamate and glycineplus 10-50 ~tM s~rmine. T = increase, $ = decrease, and - = no change in the binding of [JH]MK-801 or [ H]TCP. References are given in the right-hand column; a, unpublished results.

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The activities of a series of linear tetraamines having the structure NH2[CH2]3NH[CH2]yNH[CH2]3NH2, where Y = 2-12, have also been determined (Table I). All of the linear tetraamines whose properties have been investigated increased the binding of [3H]MK-801 or [3H]TCP either in the presence or nominal absence of glutamate and glycine. Furthermore, at concentrations thatenhanced the bindin~ of [ 3H]MK-801 in the absence of spermine, these polyamines had no effect on the binding of [ H]MK-801 when assays were carded out in the presence of glutamate and glycine plus spermine (Table I). Thus, these tetraamines appear to be full agonists at the polyamine recognition site. Differences in the potencies of tetraamines have been reported (76) with N,N'-bis(3-aminopropyl)l,3propanediamine (NH2[CH2]3NH[CH2]3NH[CH2]3NH2) being the most potent (ECs0 = 0.8 ~tM) and N,N'-bis(2-aminoethyl) 1,3-propanediamine (NH2[CH2]2NH[CH2]3NH[CH2]2NH2) being the least potent (EC50 = 36 ~tM). This supports the conclusion that a terminal propylenediamine moiety is important for agonist activity. Linear tetraamines are potentially the most useful lead structures for the future development of potent, selective agonists at the polyamine recognition site. The addition of an aminopropyl group (NH2[CH2]3-) onto a diamine or triamine can markedly alter its activity. Thus, 1,7-diaminoheptane (NH2[CH2]TNH2) had no effect on the binding of [3H]MK-801 or [3H]TCP, while N-(3-aminopropyl)-l,7-heptanediamine (NH2[CH2]3NH[CH2]TNH2) was an agonist that increased the binding of the open channel blockers. Similarly, the addition of one aminopropyl group to a primary amine of the inverse agonist DA10 results in a compound (APDA10) having partial agonist activity in the presence of glutamate and glycine, and antagonist activity in the absence of glutamate and glycine (Table I). The importance of the presence of 2, 3, or 4 amino groups for activity at the putative polyamine recognition site is made apparent by comparison of the activities of di-, tri-, and tetraamines that have the same overall chain length. Thus, in DA10 (NH2[CH2]10NH2), N(3-aminopropyl)-l,6-hexanediamine (NH2[CH2]3NH[CH2]6NH2), and N,N'-bis(3aminopropyl)ethylenediamine(NH2[CH2]3NH[CH2]2NH[CH2]3NH2), two primary amino groups are separated by 10 atoms. DA10 is an inverse agonist that inhibits the binding of [3H]MK-801, while the tri- and tetraamine analogues are full agonists that increase the binding of [3H]MK-801 and [3H]TCP (Table I). A similar change in activity is seen when characterizing the effects of DA12 (NH2[CH2]12NH2), which inhibits the binding of [3H]MK-801, N-(3aminopropyl)-l,8-octanediamine (NH2[CH2]3NH[CH2]SNH2), which is a partial agonist, and spermine (NH2[CH2]3NH[CH2]4NH[CH2]3NH2), a full agonist (Table I). In the diamine series, only NH2[CH2]3NH2 increases the binding of [3H]MK-801 and [3H]TCP, and this diamine is a weak partial agonist. In contrast, many of the tri- and tetraamines are potent, full agonists. This suggests that the presence of three amino groups is necessary for full agonist activity at the polyamine recognition site. N-substitution at the terminal amino groups of polyamines can also alter their effects at the polyamine site of the NMDA receptor. For example, putrescine (1,4-diaminobutane) is almost inactive while arcaine (1,4-diguanidinobutane) is a potent antagonist (or possibly inverse agonis0 (88). 1,3-Diaminopropane is a weakpartial agonist, while its NdV'-dimethylated analogue is an antagonist but the N,N'-tetramethylated analogue is an agonist similar to the parent compound (74). Stimulatory activity is lost when agonist polyamines have large substitutions on the terminal amino groups. For example, N,N'-bisbenzylated derivatives of a number of linear triamines have no effect on the binding of [3H]MK-801, whereas the unsubstituted parent compounds are potent agonists (unpublished observations).

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Results of binding assays with [3H]MK-801 and [3H]TCP indicate that there is a marked structural specificity for polyamines having agonist, antagonist, partial agonist, or inverse agonist effects compared to spermine and spermidine. It is not known whether all of the effects of the polyamines listed in Table I are mediated at the same site as those of spermine, spermidine, and DET. The effects of polyamines having activities similar to those of the wellcharacterized antagonist DET and the inverse agonist DA10 should be interpreted with caution. The possibility that some polyamines may interact with multiple sites on the NMDA receptor complex or may have nonspecific effects at the plasma membrane has not been rigorously investigated. The activities of only a few polyamines have been determined in electrophysiological studies of NMDA receptors (see below). Nonetheless, the spectrum of activities described in Table I and the systematic changes in activity that are seen with changes in the structures of polyamines are consistent with effects of polyamines at a specific and distinct recognition site associated with the NMDA receptor complex.

5. Spider and Wasp Toxins Containing Polyamine Moieties A number of spider toxins have been found to be antagonists of excitatory amino acid receptors. These include the Joro spider toxins (JSTX) isolated from Nephila clavata and the argiotoxins isolated from Argiope trifasciata andArgiope lobata (Figure 2; refs. 104, 105). The argiotoxins and JSTX contain polyamine moieties similar to spermine and spermidine (104, 106, 107). These toxins inhibit glutamatergic transmission at synapses in a number of invertebrate preparations (108-112) and in the mammalian hippocampus (113-116). Results obtained in a number of studies suggested that in the mammalian CNS the toxins may function as open-channel blockers of quisqualate (AMPA) and kainate receptors (114, 116, 117). A similar mechanism has been proposed to explain the effects of spider toxins at glutamate receptors of the neuromuscular junction in insects (104, 105). Kemp et al. (118, 119) reported that argiotoxins can also block NMDA receptors in rat cortical neurons in a use- and voltagedependent manner. One toxin, argiotoxin636, showed a 30-fold selectivity for NMDA receptors over quisqualate and kainate receptors (119). We have found that argiotoxins inhibited the binding of [3H] MK-801 with similar potencies in the absence or presence of spermine, and that the inhibitory effects were not attenuated by the polyamine antagonist DET (unpublished observations). Thus, the effects of argiotoxins are different from those of polyamines that have been classified as antagonists and inverse agonists at the polyamine site. Taken together, these data suggest that spider toxins containing polyamine moieties can inhibit the function of NMDA receptors but that this is probably due to a direct, activity-dependent block of the ion channel rather than to an action at the polyamine recognition site. It remains possible that the toxins also interact at the polyamine recognition site, but that this effect cannot be measured because the open-channel blocking effect of the toxins predominates over their effects at the polyamine recognition site. Venom from the wasp Philanthus triangulum also contains a polyamine-derived toxin, philanthotoxin-433 (PhTX-433), which interacts with EAA receptors (120, 121). At concentrations of 30-100 ~tM the toxin is a noncompetitive inhibitor of the effects of NMDA and kainate on EAA receptors expressed in Xenopus oocytes following injection of rat brain RNA (121). Low concentrations (1-10 ~M) of PhTX-433 enhanced while higher concentrations (20-1000 ~tM) inhibited the binding of [3H]MK-801 to NMDA receptors (121). The inhibition may be due to a direct channel-blocking effect of the toxin (121). PhTX-433 (1-20 ~tM) is also an antagonist of the nicotinic acetylcholine receptor, probably acting as an open-channel blocker (122). PhTX-343 (Figure 2), a synthetic analogue of PhTX-433, has been found to

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both potentiate and inhibit responses to NMDA in Xenopus oocytes expressing EAA receptors (123), suggesting that this substance may actually interact at the polyamine site in addition to having properties as an open-channel blocker (see below). It is of interest that the argiotoxins and philanthotoxins, in addition to containing polyamine moieties, also contain a hydroxyphenolic group separated from a nitrogen atom by two carbons. A similar group is found in the non-competitive NMDA receptor antagonist ifenprodil (Figure 2). Interactions between ifenprodil and polyamines at the NMDA receptor complex have been reported (75, 81, 96, 100; and see above). It is possible that the hydroxyphenol portions of ifenprodil and of the polyamine-derived toxins bind to the same site, and that this site is close to the site at which the polyamine portions of the toxins interact. If the binding site for the hydroxyphenol moieties is involved in the voltage- and activitydependent block of the ion channel by argiotoxins (119), this site may be within the outer mouth of the channel or deeper within the channel pore. The adjacent polyamine site, which is not voltage-dependent (see below), would presumably be located close to but outside the ion channel pore.

6. Effects of Polyamines on Solubilized NMDA Receptors After solubilization with sodium deoxycholate or sodium cholate, the NMDA receptor complex retains its sensitivity to the modulatory effects of glutamate, glycine, Mg ++, and Zn ++ measured in binding assays with [3HI MK-801 or [3H]TCP (124,125). The effects ofpolyamines have been investigated after solubilization of NMDA receptors with sodium deoxycholate (126, 126a). Spermine, spermidine, and 3,3'-iminobispropylamine increased the binding of [3H]MK-801 with potencies similar to those seen in experiments with membrane-bound receptors. Under equilibrium conditions, a maximally effective concentration of spermidine (100 gM) caused a two-fold increase in the affinity of the binding site for [3H]MK-801, identical to the effect of spermine on membrane-bound receptors (74, 126, 126a). The effects of polyamine agonists on solubilized receptors could be selectively inhibited by APDA10. However, the potency of DET as an antagonist of the effect of spermine was greatly reduced after solubilization. Spermidine increased both the rates of association and dissociation of binding of [3H]MK-801 to solubilized receptors (126), similar to the effects of spermine seen in studies of membrane-bound receptors (84). These results suggest that the polyamine recognition site is tightly associated with the other components of the NMDA receptor complex. The inhibitory phase of the polyamine concentration-effect curves was attenuated after solubilization of receptors with deoxycholate (126). This suggests that the inhibitory effects of high concentrations of spermine may be due to interactions with the native membrane structure, perhaps with membrane phospholipids, rather than with a specific site on the NMDA receptor complex.

7. Effects of Polyamines on the Binding of [3H]Glycine Effects of polyamines on the binding of [3H]glycine to the glycine recognition site of the NMDA receptor have been reported. Spermine was found to enhance the binding of [3H]glycine with an EC50 of 14-27 gM (79, 80). Spermidine has also been reported to enhance the binding of [3H]glycine but less potently and to a lesser extent than did spermine (80). However, spermine (8 I, 82) and spermidine (79) have also been reported to have no effect on the binding of [3H]glycine. There is evidence for reciprocal allosteric interactions between the binding sites for glutamate and glycine (73, 80, 127-130), and glutamate or glycine and polyamines (73, 79, 80). Thus, it is possible that the reported differences in the effects of polyamines on the binding of [3H]glycine are due to differences in the concentration of glutamate remaining in well-washed membranes as prepared in different laboratories. When

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effects of spermine on the binding of [3H]glycine were seen, they were due to an increase in affinity with no change in the number of binding sites (79, 80). Furthermore, spermine increased the affinity of the glycine recognition site for agonists including glycine, D-alanine, and D-serine but not for the antagonists 7-chlorokynurenic acid and HA-966 (79, 80). Spermine decreased both the rates of association and dissociation of binding of [ 3H]glycine, with the effect on dissociation predominating over that on association (80). Maximal effects of spermine were observed at concentrations of 1 mM (79, 80) and were additive with the effects of 50 ~tM glutamate, which also increased the affinity for binding of [3H]glycine (80). Effects of spermine on the binding of [3H]~lycineoccurred at concentrations 30- to 100-fold higher than those thatincreased the binding of [ H]MK-801, but at concentrations similar to those that inhibited binding of [3H]MK-801. DET and putrescine inhibited the effect ofspermine on the binding of [3H]MK-801 (74, 76), but not on the binding of [ 3H]glycine (131; and unpublished observations). This suggests that the effects of spermine on the binding of [3H]glycine are mediated at a site different from that responsible for the effects of polyamines on the binding of [3H]MK-801. The effects ofpolyamines on the binding of [3H]glycine may be due to an action at one of the divalent cation recognition sites since Mg ++ has also been shown to enhance the binding of [3H]glycine to the NMDA receptor complex (132, 133). 8. Effects of Polyamines on the Glutamate Recognition Site Spermine and spermidine have been reported to increase binding of the glutamate site antagonist [3H]CPP (81,83). The effects of polyamines on the binding of [3H]CPP were seen at concentrations 30- to 100-fold higher than those which increased the binding of [ 3H]MK-801 and [3H]TCP. Several observations suggest that the effect of polyamines on the binding of [3H]CPP may be mediated at a site different from that which mediates effects on the binding of [3H]MK-801 and [3H]TCP. The polyamine antagonist DET did not block the effects of spermine and spermidine on the binding of [3H]CPP (L.M. Pullan, personal communication). The triamine APDA10 had partial agonist effects on the binding of [3H]MK-801 when assays were carried out in the presence of glutamate and glycine, and was an antagonist at the polyamine recognition site in the nominal absence of glutamate and glycine (unpublished observations). However, APDA 10 increased the binding of [3H]CPP, and was more potent and produced a greater increase in the binding of [ 3H]CPP than did spermine (L.M. PuUan, personal communication). It has been reported that antagonists at the glycine site such as HA-966 increase the binding of [3H]CPP to the glutamate recognition site and increase the potency of CPP for inhibiting the binding of [3H]MK-801 (128, 134). There are differences in the regional distributions of the binding sites for [3H]CPP and [3H]glutamate in the CNS and in their sensitivities to the modulatory effects of glycine (127). It has been suggested that the glutamate recognition site of the NMDA receptor complex may exist in agonist- and antagonistpreferring conformations, and that compounds acting at the glycine site can promote interconversion of the agonist- and antagonist-preferring states (127, 128, 134). Thus, the effects of polyamines on the characteristics of the glutamate site could be mediated by an action at the glycine site. We have found that APDAI0 greatly increased the potencies of CPP and D-AP5 for inhibiting the binding of [3H]MK-801, consistent with the observed effects of APDA10 on the binding of [3H]CPP. However, the effect of APDA10 on the affinity of antagonists at the glutamate site was not altered when assays were carded out in the presence of varying concentrations of glycine (unpublished observations), suggesting that polyamines were not acting at the glycine recognition site. This conclusion is supported by the observation that APDA10 had no effect on the binding of [3H]glycine to the NMDA receptor complex (unpublished observations). High concentrations (0.1-1 mM) of some polyamines, including

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spermine and APDA 10, increase the binding of [3H]glutamate to NMDA receptors, an effect that is not blocked by DET (unpublished observations). The effects of polyamines on the binding of [3H]CPP and [3H]glutamate could be mediated at the same site that is responsible for their effects on the binding of [3H]glycine. This may be one of the recognition sites for divalent cations (see above). In summary, the effects of polyamines on the binding of [3H]glutamate, [3H]CPP, and [3H]glycine occur at concentrations higher than those needed to increase the binding of [3H]MK-801 and these effects are not blocked by antagonists like DET. Furthermore, pronounced effects ofpolyamines on the binding of [ 3H]MK-801 were seen even in the presence of maximally effective concentrations of glutamate and glycine. Thus, it is unlikely that an effect of spermine on the affinity of binding of agonists at the glutamate or glycine recognition sites contributes markedly to its modulation of [3H]MK-801 binding. FUNCTIONAL STUDIES

1. Electrophysiological Studies a. Effects of Polvamines on EAA-Induced Currents The effects of polyamines on NMDA-elicited currents have been determined in hippocampal neurons maintained in primary culture (84, 135). Low concentrations (0.2-10 ~tM) of spermine enhanced the currents elicited by NMDA in medium containing 1 ~tM glycine without added Mg ++, while higher concentrations of spermine (> 35 ~tM) inhibited the currents (Figure 7). Spermine (1-100 lxM) by itself did not elicit any detectable current nor did it alter the holding current. Polyamines did not affect currents elicited by kainate, quisqualate, or GABA in hippocampal neurons, indicating that the effects of polyamines are selective for NMDA receptors (84, 135). Neither the stimulatory nor the inhibitory effects of spermine on NMDA-elicited responses were voltage-dependent, and both effects were blocked by DET (Figure 7). The polyamine antagonist DET by itself did not alter the NMDA-evoked current and had no effect on the holding current (84, 135). The increase in NMDA-elicited currents in the presence of low concentrations of spermine is consistent with the suggestion that polyamines increase the accessibility of the ion channel for [3H]MK-801, possibly reflecting an increase in the mean channel open time or the frequency of channel opening (84, 135). Spermidine (1-100 ~tM) has also been reported to potentiate NMDA-induced currents in cultured striatal neurons (136) and in hippocampal neurons (135). In striatal neurons the effect of spermidine was additive with that of glycine. Responses to NMDA were markedly inhibited in the presence of the glycine antagonist 7-chlorokynurenic acid. Glycine, but not spermidine, could overcome the inhibitory effects of 7-chlorokynurenic acid, suggesting that the effects of spermidine were not mediated at the glycine site (136). DA10, which was characterized as an inverse agonist in radioligand binding studies (see above), inhibited the NMDA-induced current in hippocampal neurons. This inhibition was not voltage-dependent and was blocked by DET (Figure 7; and ref. 84). In contrast, the inhibitory effects of Mg ++ (10, 11) and of MK-801 (46) at the NMDA receptor complex are voltage-dependent. DA10 did not affect responses to kainate or quisqualate in hippocampal neurons (84). These results support the conclusion that DA10 acts as an inverse agonist at the polyamine recognition site.

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NMOA

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NMDA + Spermine (1 ~M) . DET (100 t=M)

201~c

B

NMDA

NMDA + Spermine (1.00 ~M)

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NMDA + Spernline (100 pM) + DET (150 pM)

10~,ec

C

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NMDA + DA10 (100 pM)

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NMDA + DAIO (100 p.M) + DET (150 FM)

[

~OpA

lOst~

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P'~ Control [ ] + 50 p.M D E T / [ ] + 150 p.M DETJ

I m

,ili]i 1// NMDA

NMDA + I tiM Spermine

NMDA + 100 tiM Spermine

NMDA + 100~tM DA10

Figure 7. Effects of Polyamines on NMDA-Induced Currents in Hippocampal Neurons. A-C: Pen recorder traces of inward currents elicited by 1O0 ~M NMDA in the presence of 1 I.tMspermine (A), 100 ~tMspermine (B), and 100 txMDA 10 (C), in the absence and presence of DET. D: Effects of DET, spermine, and DA10 on currents elicited by 100 IsM NMDA in 6-16 cells. Results are expressed as a percentage of the control response measured in the absence of polyamines. Note that DET by itself has no effect on NMDA-induced currents but blocks the stimulatory and inhibitory effects of spermine and DA10. Data are taken in part from refs. 84 and 135. Since the effects of polyamines are not voltage-dependent, they are unlikely to be mediated at the binding sites for Mg ++ or Zn ++. The blockade of the NMDA receptor by Mg ++ is strongly voltage-dependent and the blockade by Zn ++ is weakly voltage-dependent (10, 11, 40, 59). This may be a consequence of the presence of two recognition sites for Zn++ on the receptor complex, one of which is voltage-dependent and the other is voltage-independent (42). The possibility that polyamines act at the voltage-independentZn ++ recognition site cannot be excluded. However, in binding assays with [3H]MK-801, the effects of spermine and of

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DA10 are mechanistically different from those of Zn ++. The available evidence suggests that spermine, spermidine, DET, and DA 10 act competitively at a common recognition site. Taken together, these results suggest that the polyamine recognition site is distinct from all three of the proposed divalent cation recognition sites, and that the effects of polyamines on NMDA receptors on hippocampal neurons are mediated at the polyamine recognition site characterized in studies of the binding of [3H]MK-801 and [3H]TCP. The pharmacology of the effects of spermine, spermidine, and DET was similar in electrophysiological and biochemical studies. However, in electrophysiological experiments the stimulatory and inhibitory effects of spermine were seen at concentrations 10-30 fold lower than those required for the corresponding effects in biochemical assays. DET blocked both the stimulatory and the inhibitory effects of spermine on NMDA-elicited currents in cultured hippocampal neurons. In biochemical assays, concentrations of DET up to 300 ~tM attenuated the stimulatory but not the inhibitory effects of spermine on binding of [3H]MK-801. The failure to observe an effect of DET on the inhibitory portion of the spermine curve in binding assays with [3H]MK-801 may be due to the high concentrations of spermine that are required. A more rigorous comparison of the inhibitory effects of spermine in binding assays and in electrophysiological assays will require the development and use of more potent antagonists at the polyamine recognition site. Since one polyamine (spermine) can both enhance and inhibit NMDA-evoked currents in hippocampal neurons (135), it is possible that polyamines act at at two or more recognition sites associated with NMDA receptors. If there are two discrete polyamine recognition sites, both of which are blocked by DET, then it is possible that the "agonist" effects of spermine are mediated at one site and the "inverse agonist" effects of spermine and of DA10 are mediated at a second site. Another possibility is that a single polyamine recognition site has two functional domains at which spermine interacts to modulate the NMDA receptor complex. The effects of polyamines have also been investigated in Xenopus oocytes expressing NMDA receptors after injection of RNA from rat brain. The oocyte expression system has proven to be useful for studies of ion channels and receptors from the CNS (137). NMDA receptors expressed inXenopus oocytes have characteristics that are similar to their counterparts in situ with respect to the voltage-dependent blockade by Mg ++, blockade by Zn ++, sensitivity to agonists and antagonists at the glutamate and glycine recognition sites, and sensitivity to open-channel blockers such as MK-801 (48, 138-142). Two groups have reported that spermine potentiated NMDA-induced currents, measured by the whole-cell voltage-clamp technique, in Xenopus oocytes expressing NMDA receptors after injection of RNA from rat or chick brain (123,143). Spermine did not affect responses to kainate or to quisqualate acting at ion-channel-coupled (AMPA) receptors or receptors linked to inositol phospholipid hydrolysis (143). By itself, spermine did not induce currents or alter membrane resistance (143). A maximal effect of spermine was seen at 250 lxM, and the effect decreased at higher concentrations (143). Spermine did not alter the apparent affinity or the Hill slope for NMDA, but increased the maximum response to NMDA (123, 143). Spermine increased the apparent affinity for glycine at the NMDA receptor by about 3-fold (143). It was suggested that the effect of spermine may be due in part to an increase in the affinity of the binding site for glycine (143) as has been observed in binding assays with [3H]glycine (79, 80; and see above). However, the observation that spermine can increase NMDA-induced currents and can increase the binding of [3H]MK-801 even in the presence of supra-saturating concentrations of glycine (73-75, 135, 143) indicates that changes in the affinity of the glycine binding site cannot account for all of the stimulatory effects of

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polyamines. It remains to be determined whether polyamines have other effects on the interaction of glycine with the NMDA receptor complex, for example by altering the effects of glycine on desensitization of NMDA-mediated responses. In another report, micromolar concentrations of spermine and spermidine decreased rather than increased NMDA-elicited currents in voltage-clamped Xenopus oocytes injected with RNA from whole rat brain (144). Polyamines had no effect on currents elicited by kainate (144). The inhibitory effects of spermine and spermidine were rapidly reversible, were not voltage-dependent, and were attenuated by DET (1 44). DET by itself had no effect on NMDAinduced currents or on the holding current. The magnitude oftheinhibitory effects of spermine was independent of the concentration of NMDA (0.1 lxM-1 mM) and of glycine (0.1-10 I.tM), suggesting that this inhibition was not due to a competitive action at the NMDA (glutamate) or glycine recognition sites, and was indeed mediated at a distinct polyamine recognition site. The effects of polyamines determined in electrophysiological studies of hippocampal neurons may be mediated at two recognition sites associated with the NMDA receptor complex (see above). Both of these proposed sites are blocked by DET. One site may mediate the stimulatory effects and the other site the inhibitory effects of polyamines. It is possible that NMDA receptors expressed in oocytes as studied in different laboratories contain only one of the two polyamine recognition sites or that these two sites are expressed in varying proportions in heterogeneous populations of receptors under different experimental conditions. This could be due to alterations in the biochemical processing of the receptor proteins or to the expression of receptors composed of different combinations of subunits. It is likely that the NMDA receptor is a multi-subunit complex analogous to the nicotinic acetylcholine receptor, the GABAA receptor, and the inhibitory glycine receptor (6, 7, 9, 145). It is also possible that multiple isoforms of the subunits of NMDA receptors exist, as has been reported for acetylcholine and for GABAA receptors (146, 147). This could lead to the expression of different forms of the NMDA receptor complex in neurons and in Xenopus oocytes after different methods of preparation of RNA or through variability between oocytes. b. Effects of Polvamines on Excitatory Svnamic Transmission Several preliminary studies of the effects of polyamines on the function of NMDA receptors in vivo have been carded out. Spermidine enhanced the effects of NMDA determined in single-unit recordings of neurons in the red nucleus of the rat (82). The effects of spermidine were seen even in the presence of maximally effective concentrations of D-serine, an agonist at the glycine recognition site of the NMDA receptor (82). Spermine and spermidine have also been reported to increase the spontaneous activity of some neurons and decrease the activity of others in the brainstem of the rat and cat (148), but it is not known whether this is due to effects on NMDA receptors. High concentrations of spermine (100 BM-1 mM) have been shown to increase spontaneous epileptiform discharges in the rat cortical wedge preparation (149). This spontaneous activity is known to be mediated in part by activation of NMDA receptors. However, in the same preparation, spermine also directly depolarized the tissue, an effect that appeared to be unrelated to an interaction with NMDA receptors (149). To more directly assess the effects of polyamines on excitatory synaptic transmission, spontaneous excitatory postsynaptic currents (EPSCs) were recorded from hippocampal neurons maintained in dissociated cell culture, and the effects of various polyamines were determined (150). In Mg++-free buffer containing 1 lxM glycine, spermine at a concentration of 1 I.tMincreased the amplitude of EPSCs, and this potentiation was blocked by DET (50 I.tM). At a concentration of 100 l.tM, spermine decreased the amplitude of EPSCs, an effect that was also blocked by DET (150 l.tM).

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Spontaneous EPSCs recorded in medium nominally free of Mg++ but containing 1 ~tM glycine have components that are mediated by both non-NMDA (AMPA and kainate) and NMDA receptors. Non-NMDA receptor-mediated EPSCs can be studied in isolation by recording in extracellular solutions containing 1 mM Mg ++ without added glycine. Under these conditions, spermine (1-100 ktM) had no effect on the amplitude of EPSCs. NMDA receptor-mediated EPSCs can be studied in isolation by recording in Mg++-free medium containing glycine and the AMPA/kainate receptor antagonist CNQX (6-cyano-7nitroquinoxaline-2,3-dione). Under these conditions, spermine (1 ~tM) markedly enhanced the size of spontaneous EPSCs (unpublished observations). Thus, the effect of spermine on EPSCs was similar to its effect on NMDA-induced inward currents and was specific for EPSCs mediated by NMDA receptors. To determine whether endogenous polyamines could tonically activate the polyamine recognition site during synaptic transmission in culture, the polyamine antagonist DET was applied alone to neurons. The effects of DET on the size, duration, and frequency of EPSCs were assessed. DET does not have direct effects on the membrane holding current, or on NMDA-induced responses measured in the absence of a polyamine agonist or inverse agonist (84, 135; and see above). DET did, however, cause a reversible decrease in the size of spontaneously occurring EPSCs mediated by NMDA receptors (unpublished observations). This suggests that endogenously released polyamines may be involved in the generation of spontaneous postsynaptic events in these neurons. These results also suggest that under basal physiological conditions, excitatory synaptic transmission could be either up- or downregulated by changes in the levels of endogenous polyamines. Furthermore, in light of such tonic activity, administration of drugs that act as agonists, inverse agonists, or antagonists at the polyamine recognition site could have significant effects on a variety of normal and pathophysiological states.

2. Biochemical and Behavioral Studies

There have been several reports of experiments designed to determine the effects of polyamines on the function of NMDA receptors using biochemical assays. Ransom and Stec (73) did not observe effects ofspermidine on 22Na+ effhx stimulated by NMDAin hippocampal slices prepared from rat brain. However, glycine had no effect on NMDA responses in this system, suggesting that endogenous levels of the modulators may already be having maximal effects at their respective recognition sites (73). Spermine and spermidine had no effect by themselves on the level of cGMP in the cerebellum when administered in vivo (151) or applied to cerebellar slices in vitro (81). Polyamines have been reported, however, to potentiate the stimulatory effects of NMDA on cGMP levels in cerebellar slices (81), although these effects occur at high (100 ~tM-1 mM) concentrations of polyamine. On the other hand, spermine and spermidine attenuated the increase in the concentration of cGMP caused by D-serine or quisqualate after intracerebellar injection in vivo (151). It is not known if these effects are related to an action of polyamines at the NMDA receptor. The difficulties in interpreting studies of the effects ofpolyamines on levels ofcGMP have been discussed above. Spermidine had no effect on the NMDA-evoked release of [3H] norepinephrine in rat hippocampal minces pre-loaded with [3H]norepinephrine (152). In the same preparation, arcaine was a noncompetitive antagonist of the NMDA-evoked release of [3H]norepinephrine. The effect of arcaine was reversed by high (1-3 mM) concentrations of spermidine in the presence of 5% dimethylsulfoxide (152).

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It has recently been reported that spermidine (1-2 gmol) administered intracerebroventricularly decreased the latency for induction of tonic seizures following subcutaneous injection of N-methyl-DL-aspartate (NMDLA) (153). Spermidine (2 g mol) decreased the ED50 for NMDLA by about 3 fold but had no effect on the latency of seizures induced by pentylenetetrazole. At this dose, spermidine did not induce seizure activity in the absence of NMDLA (153). LOCATION OF THE POLYAMINE RECOGNITION SITE The polyamine recognition site of the NMDA receptor could be located on an intracellular or extracellular domain of the receptor complex, in the membrane-spanning regions, or on phospholipids associated with the receptor. Results of studies of solubilized NMDA receptors suggest that the polyamine recognition site is tightly associated with the other components of the receptor complex, and is located on the receptor protein rather than on membrane phospholipids. In electrophysiological studies, effects of polyamines were seen immediately after co-application of polyamines and NMDA to the extracellular surface of neurons or oocytes, suggesting an extracellular site of action of polyamines. However, it is possible that exogenously applied polyamines are rapidly taken up by the cells and act at a polyamine recognition site located on an intracellular domain of the receptor. A more definitive localization of the polyamine site will require detailed biochemical and molecular biological studies of the proteins that make up the receptor complex, and single channel recordings using a variety of patch configurations. CONCLUSIONS Results of biochemical and electrophysiological studies indicate the existence of a specific recognition site for polyamines on the NMDA receptor complex. Compounds that have been classified as agonists, partial agonists, antagonists, and inverse agonists at this recognition site have been identified. Polyamines acting at this site modulate the binding of open-channel blockers to the receptor complex and modulate NMDA-induced currents. Polyamines may prove to be useful tools for studies of the function and regulation of NMDA receptors and of excitatory synaptic transmission in vivo and in vitro. Polyamines can also alter the binding of ligands to the recognition sites for glutamate and glycine but these effects may be mediated by an action of polyamines at a different site. The identification of a polyamine recognition site on the NMDA receptor complex suggests that endogenous polyamines may be involved in the regulation of excitatory synaptic transmission by acting at this site. Many of the well-documented effects of polyamines on cellular growth and differentiation appear to be mediated at intracellular sites (60, 61). Some of these effects involve the rapid induction of ODC activity and subsequent synthesis of polyamines (60, 61, 154). However, in the CNS, polyamines are also present in high concentrations in pools that show marked regional variation. It is possible that polyamines are selectively released from endogenous stores into the extracellular space or synaptic cleft where they act on the NMDA receptor complex. It is also possible that spermine and spermidine are not the endogenous ligands that interact with the NMDA receptor, but that other, as yet unidentified, polyamines exist in brain that interact with the NMDA receptor complex in vivo. Studies directed towards the identification and characterization of a possible role ofpolyamines in synaptic transmission will provide valuable information in this respect.

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The levels of endogenous polyamines and of ODC activity in the brain have been reported to be increased following transient cerebral ischemia (155, 156). It is possible that excessive release of endogenous polyamines following ischemia contributes to neurotoxicity mediated by stimulation of NMDA receptors. Conversely, high extracellular concentrations of polyamines could attenuate the neurotoxic effects of excitatory amino acids by reducing activation of NMDA receptors. Finally, the polyamine recognition site of the NMDA receptor may represent a novel target for the treatment or prevention of neurotoxicity, epilepsy, and neurodegenerative diseases. The spectrum of pharmacological activities that has already been described for polyamines at the NMDA receptor suggests that it may be possible to develop compounds that act at the polyamine recognition site and are highly selective and potent modulators of the function of NMDA receptors. Given both the positive and negative modulatory effects of polyamines that have been described, it is possible that compounds having mixed agonist, antagonist, or inverse agonist effects could be devised. Such compounds may provide a means of pharmacologically altering the activity of NMDA receptors without some of the side effects associated with compounds acting at the other binding sites, which generally have an all-ornone effect on receptor function. ACKNOWLEDGEMENTS We are grateful to colleagues for providing us with preprints of papers and abstracts. We thank Lotte Gottschlich and young Catherine Buettner for secretarial assistance. Work in the authors' laboratories was supported by grants from the Epilepsy Foundation of America, ICI Pharmaceuticals Group of ICI Americas, the USPHS (GM 34781 and NS 24927), and the Pew Charitable Trusts. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

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Modulation of the NMDA receptor by polyamines.

Results of recent biochemical and electrophysiological studies have suggested that a recognition site for polyamines exists as part of the NMDA recept...
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