Effects of polyamines on the binding of [3H]MK-801 to the N-methyl-D-aspartate receptor: pharmacological evidence for the existence of a polyamine recognition site

A heat-stable factor of low molecular weight that increases the binding of [3H]MK-801 to rat brain membranes in the presence of maximally effective concentrations of L-glutamate and glycine was purified from bovine brain by reverse phase and ion-exchange high pressure liquid chromatography. The stimulatory activity was due to the presence of spermidine in the active fractions. Polyamines including spermine and spermidine are found in high concentrations in mammalian tissue. These compounds increase the affinity of N-methyl-D-aspartate (NMDA) receptors for [3H]MK-801 when assays are carried out in the presence of 100 microM L-glutamate and 100 microM glycine. At concentrations of 1 to 300 microM, a number of di- and triamines, including NH2(CH2)3NH2, NH2(CH2)3NH(CH2)2NH2, and NH2(CH2)3NH(CH2)3NH2, have partial or full agonist-like activity similar to that of spermidine. Other polyamines, including putrescine, cadaverine, NH2(CH2)2NH(CH2)2NH2, and CH3NH(CH2)3NHCH3, at concentrations of 1 to 100 microM, inhibited the binding of [3H]MK-801 in the presence of spermine, L-glutamate, and glycine but not in the presence of only L-glutamate and glycine. It is concluded that these compounds are selective antagonists of the effects of spermine at the NMDA receptor. These results suggest that there may be a polyamine recognition site on the NMDA receptor complex.     

The spider toxin, argiotoxin636, binds to a Mg2+ site on the N-methyl-D-aspartate receptor complex.

1. The mechanism of action of the arylalkylamine spider toxin, argiotoxin636, on the N-methyl-D-aspartate (NMDA) receptor was investigated by use of [3H]-dizocilpine binding to well-washed membranes obtained from rat brain. 2. Argiotoxin636 decreased [3H]-dizocilpine binding with an apparent potency of about 3 microM. The inhibition of [3H]-dizocilpine by argiotoxin636 was insensitive to the concentration of glutamate, glycine and spermidine in the assay. 3. Argiotoxin636 alone had no effect on the dissociation of [3H]-dizocilpine. However, argiotoxin636 reversed the actions of Mg2+ on the dissociation of [3H]-dizocilpine by decreasing the apparent potency of Mg2+. Argiotoxin636 also reversed the action of Ca2+ on the dissociation of [3H]-dizocilpine. 4. These results suggest that argiotoxin636 exerts a novel inhibitory effect on the NMDA receptor complex by binding to one of the Mg2+ sites located within the NMDA-operated ion channel.     

Intrathecally administered D-cycloserine produces nociceptive behavior through the activation of N-methyl-D-aspartate receptor ion-channel complex acting on the glycine recognition site

Intrathecal (i.t.) administration of D-cycloserine (100 and 300 fmol), a partial agonist of the glycine recognition site on the N-methyl-D-aspartate (NMDA) receptor ion-channel complex, produced a behavioral response mainly consisting of biting and/or licking of the hindpaw and the tail along with slight hindlimb scratching directed toward the flank in mice, which peaked at 5 - 10 min and almost disappeared at 15 min after the injection. The behavior induced by D-cycloserine (300 fmol) was dose-dependently inhibited by an intraperitoneal injection of morphine (0.5-2 mg/kg), suggesting that the behavioral response is related to nociception. The nociceptive behavior was also dose-dependently inhibited by i.t. co-administration of 7-chlorokynurenic acid (0.25-4 nmol), a competitive antagonist of the glycine recognition site on the NMDA receptor ion-channel complex; D-(-)-2-amino-5-phosphonovaleric acid (62.5-500 pmol), a competitive NMDA receptor antagonist; MK-801 (62.5-500 pmol), an NMDA ion-channel blocker; ifenprodil (0.5-8 nmol); arcaine (31-125 pmol); and agmatine (0.1-10 pmol), all being antagonists of the polyamine recognition site on the NMDA receptor ion-channel complex. However, [D-Phe7,D-His9]-substance P(6-11), a specific antagonist for substance P (NK1) receptors, and MEN-10,376, a tachykinin NK2-receptor antagonist, had no effect on D-cycloserine-induced nociceptive behavior. These results in the mouse spinal cord suggest that D-cycloserine-induced nociceptive behavior is mediated through the activation of the NMDA receptor ion-channel complex by acting on the glycine recognition site and that it does not involve the tachykinin receptor mechanism.     

Differential modulation of the associated glycine recognition site by competitive N-methyl-D-aspartate receptor antagonists

The competitive N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphonopentanoate and two other five-atom linkage (C-5) omega-phosphono-alpha-amino acid analogs reduced [3H]glycine binding, in a dose-dependent manner, to a maximum of 45-55%, whereas seven-atom linkage (C-7) analogs had significantly less effect. The IC50 of the C-5 antagonists for the inhibition of [3H]glycine binding closely paralleled their potency both in displacing NMDA-selective L-[3H]glutamate binding and in negatively modulating (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne maleate ([3H]MK-801) binding. Additionally, reduction of glycine binding by the C-5 antagonists was reversed by both NMDA receptor agonists and C-7 competitive NMDA antagonists, providing evidence that the site of action of these C-5 antagonists is the NMDA recognition site, resulting in indirect modulation of the glycine site. These data imply a functional coupling between the NMDA and associated glycine recognition sites and, furthermore, suggest a differential interaction of C-5 and C-7 competitive NMDA antagonists with the NMDA receptor complex.     

The N-methyl-D-aspartate receptor channel complex and the sigma site have different target sizes

Radiation inactivation technique has been used to measure the molecular sizes of the N-methyl-D-aspartate (NMDA) receptor and the sigma recognition site. Target size analysis of [3H]MK-801 (dizocilpine maleate) binding to the NMDA receptor channel complex in rat cortical membranes has given a molecular size of 128,000 +/- 9,000 Da. This is in contrast to the target size of the sigma site in the same tissue as labelled by [3H]DTG (ditolylguanidine), giving a value of 36,000 +/- 2,900 Da. These studies have provided evidence for a clear difference in the molecular sizes of these two recognition sites.     

Spermine enhances binding to the glycine site associated with the N-methyl-D-aspartate receptor complex

Spermine enhanced strychnine-insensitive [3H]glycine binding 3-fold with an EC50 of 27 +/- 3.1 microM. Spermidine and putrescine were without effect, whereas the ethylenediamine analog of spermine had an intermediate effect. Eadie-Hofstee analysis revealed that spermine increased the affinity of glycine for its receptor without a significant change in receptor density. This effect persisted in the presence of glycine or N-methyl-D-aspartate receptor antagonists. Furthermore, spermine produced a leftward shift in the IC50 of glycine agonists in displacing [3H]glycine binding, without altering the IC50 for glycine antagonists. These data indicate that spermine interacts with the glycine receptor through a novel binding site and, further, that spermine can be used to discriminate glycine agonist and antagonist binding.     

Inhibition of cortical spreading depression by L-701,324, a novel antagonist at the glycine site of the N-methyl-D-aspartate receptor complex.

1. Spreading depression (SD) is a propagating transient suppression of electrical activity, associated with cellular depolarization, which probably underlies the migraine aura and may contribute to neuronal damage in focal ischaemia. The purpose of this study was to examine whether L-701,324 (7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-2-(1H)-quinolone), a high affinity antagonist at the glycine site of the N-methyl-D-aspartate (NMDA) receptor complex, inhibits the initiation and propagation of K(+)-induced SD in the rat cerebral cortex in vivo. 2. Microdialysis probes incorporating a recording electrode were implanted in the cerebral cortex of anaesthetized rats and perfused with artificial cerebrospinal fluid (ACSF). Five episodes of repetitive SD were elicited by switching to a medium containing 130 mM K+ for 20 min, each separated by 40 min of recovery (i.e. perfusion with normal ACSF). The brief negative shifts of the extracellular direct current (d.c.) potential, characteristic of SD elicitation, were recorded with the microdialysis electrode and a reference electrode placed under the scalp. Propagation of SD was examined using glass capillary electrodes inserted about 3 mm posterior to the microdialysis electrode. L-701,324 (5 or 10 mg kg-1) or its vehicle were administered i.v. 10 min after the end of the second K(+)-stimulus. The effects of L-701,324 were compared to those of dizocilpine (MK-801; 1 mg kg-1 i.v.), a NMDA-channel blocker known to potently block SD elicitation. 3. Potassium-induced SD initiation was inhibited by 10 mg kg-1 (but not by 5 mg kg-1) of L-701,324. Thirty minutes after administration of 10 mg kg-1 L-701,324, the cumulative area of SD peaks elicited during 20 min was 15.3 +/- 2.1 mV min, versus 23.2 +/- 1.1 mV min in animals which received only the drug vehicle (P < 0.02; n = 6). The delay between application of 130 mM K+ and occurrence of the first SD was also significantly increased. It was approximately doubled in animals treated with 10 mg kg-1 of L-701,324. 4. SD propagation was more sensitive than SD elicitation to L-701,324, as both 5 and 10 mg kg-1 produced an effective inhibition. Even at the lower dose of 5 mg kg-1, L-701,324 completely blocked the propagation of SD elicited 30 min after drug administration. This differential sensitivity of SD elicitation and propagation is not specific to L-701,324 since it was previously observed with other drugs. At doses effective against SD, L-701,324 did not produce any marked alterations of the electroencephalogram. 5. L-701,324 (10 mg kg-1) and MK-801 (1 mg kg-1) had identical effects on the d.c. potential when administered during the recovery which followed the second K+ stimulus. Both drugs produced a positive shift of around 4.5 mV within 10 min of i.v. drug administration, indicating rapid drug penetration into the CNS. Paradoxically, L-701,324 (10 mg kg-1) was markedly less effective than MK-801 (1 mg kg-1) in blocking SD, since this dose of MK-801 was sufficient virtually to abolish SD initiation and completely block its propagation. The higher potency of MK-801 against SD may reflect its use-dependency, i.e. binding of MK-801 and channel blockade are enhanced when the NMDA-receptor ionophore is open. 6. Taken together, these data demonstrate that L-701,324 has an inhibitory effect on both SD initiation and propagation. This action may be beneficial in focal ischaemia, and possibly also against migraine, especially as this drug was shown to be active when administered orally.     

Biochemical and thermodynamic aspects of the binding of [3H]glycine to its strychnine-insensitive recognition site associated with the N-methyl-D-aspartate receptor complex.

The molecular mechanism of interaction between glycine and its strychnine-insensitive binding site linked to the N-methyl-D-aspartate receptor was investigated by examining on the one hand the thermodynamic properties of glycine binding, and, on the other hand, the effects of various functional group modifying agents on ligand binding. Raising the incubation temperature from 0 degrees to 37 degrees resulted in a consistent decrease of glycine binding affinity. Calculation of thermodynamic parameters from the corresponding Van't Hoff plot showed that the binding of glycine was mainly entropy-driven, the change in enthalpy contributing only little (25-30%) to the change in Gibbs free energy. Chemical modification with the sulfhydryl-directed agents p-hydroxy-mercuribenzoate and N-ethyl-maleimide showed free -SH groups to be critical for ligand binding to the receptor site. Furthermore, guanidino groups on arginyl residues, sensitive to 2,3-butanedione, were also found to participate in glycine binding. Both the -SH and the guanidino groups could be protected against their inactivation by co-incubation with glycine, indicating a direct involvement of these functional groups in the binding process. Dithiothreitol, a disulfide-reducing agent, likewise prevented [3H]glycine binding, suggesting that the glycine recognition site is stabilized by at least one disulfide bridge. It is concluded that the binding of glycine probably involves a strong ion-ion interaction between its carboxyl group and a positively charged guanidino group at the receptor site, resulting in a thermodynamically favorable increase in entropy by displacement of water molecules from the latter and a concomitant decrease in enthalpy. Furthermore, at least one free sulfhydryl group seems to participate in the binding process.     

Antiaggregatory and anticoagulant effects of oligoamines. 5. Alicyclic di- and triamines

Platelet Aggregation Inhibiting and Anticoagulant Effects of Oligoamines, V: Alicyclic Diamines and Triamines Seventeen oligoamines beeing derivatives of cyclohexane, adamantane, spiro[3,3]heptane and 9,10-ethanoanthracene have been synthesized. Platelet aggregation induced by collagen in vitro is inhibited by eight of them at IC₅₀ ≦ 5 μmol/L. Three compounds depress the formation of fibrin to 25 % of normal at a 50 μmol/L concentration. This was indicated by prolongation of the thromboplastin and partial thromboplastin times. The 500 MHz COSY spectrum of a spiro[3,3]heptane-2,6-dimethanamin (16) confirms that the cyclobutane rings are puckered and that the substituents are in equatorial positions.     

Barbiturate recognition site on the GABA/benzodiazepine receptor complex is distinct from the picrotoxinin/TBPS recognition site

The cage convulsant [35S]tert-butylbicyclophosphorothionate [( 35S]TBPS) labels a presumed sedative-convulsant receptor complex. The relative potencies of barbiturates in competing for [35S]TBPS binding parallels their potencies in enhancing benzodiazepine receptor binding. Barbiturates inhibit [35S]TBPS binding in a complex, mixed competitive fashion, leading to a decrease in both the apparent affinity of TBPS for its sites and the apparent number of TBPS sites. All of the barbiturates examined markedly accelerate the dissociation of [35S]TBPS from its recognition sites, while picrotoxinin does not affect the dissociation. These results suggest that the barbiturate and picrotoxinin/TBPS recognition sites are distinct but allosterically linked.     

Identification of a novel structural class of positive modulators of the N-methyl-D-aspartate receptor, with actions mediated through the glycine recognition site

This study describes a new structural class of compounds which interact at the N-methyl-D-aspartate (NMDA) receptor-associated glycine recognition site. These E-gamma-substituted vinylglycine derivatives were active in displacing [3H]glycine binding from the NMDA receptor-associated recognition site in rat forebrain synaptic plasma membranes, with Ki values in the range of 0.24-8.7 microM. Functional analyses of these compounds indicate that they positively modulate basal [3H](+)-5-methyl-10,11-dihydro-5H- [a,d]cyclohepaten-5,10-imine ([3H]MK-801) binding, consistent with their having agonist characteristics. Little stereospecificity is observed with the gamma-substituted methyl and propyl derivatives while the L-isomer of the hexyl analog is significantly more potent than the D-isomer. The D- and L-hydroxyethyl gamma-substituted vinylglycines were the most potent inhibitors of [3H]glycine binding with Ki values of 0.75 +/- 0.06 microM and 0.24 +/- 0.02 microM, respectively. The 3,4-double bond was necessary for activity in that the saturated hexyl derivative (2-aminodecanoate) was inactive. Based on the results reported herein, the hypothesis that there is a distinct size restriction for functional agonists which interact with the glycine recognition site, should be altered to include these larger extensions of vinylglycine.     

Effects of L-701,324, a high-affinity antagonist at the N-methyl-D-aspartate (NMDA) receptor glycine site, on the rat electroencephalogram

L-701,324 (7-chloro-4-hydroxy-3-(3-phenoxy) phenyl-2-(1H)-quinolone) is a novel, orally active antagonist at the N-methyl-D-aspartate (NMDA) receptor glycine site. As NMDA receptor antagonism is generally associated with anaesthetic effects, we have examined the electroencephalographic alterations produced by doses of L-701,324 that effectively reduce NMDA-evoked responses in vivo. Microdialysis probes incorporating an electrode were implanted in the striatum of rats and perfused with artificial cerebrospinal fluid (ACSF). Under light halothane anaesthesia, 12 consecutive depolarizations were elicited by switching to ACSF containing 200 μM NMDA for 2 or 3 min, every 20 min. NMDA-evoked depolarizations and EEG were recorded with the microdialysis electrode. L-701,324 (5 or 10 mg kg^-1 i.v.) or vehicle were administered 5 min after the 3rd NMDA stimulus. L-701,324 dose-dependently inhibited NMDA-evoked depolarizations, with 10 mg kg^-1 reducing these responses by 50 % for at least 3 h. The average amplitude of the EEG in the window 0.25-6 Hz (low frequencies) and 6-21 Hz (high frequencies) did not change in the control group. At the higher dose of 10 mg kg^-1 L-701,324 transiently increased the amplitude of low frequencies by around 20 %. In contrast, both 5 and 10 mg kg^-1 significantly reduced the high frequencies to around 70 % of control, and this action was sustained with the higher dose. Analysis of the relative EEG power spectra confirmed a small, but persistent shift from high to low EEG frequencies. Our results suggest that L-701,324 slightly strengthened halothane anaesthesia at doses inhibiting effectively NMDA receptor function. Accordingly, the resulting anticonvulsant and neuroprotective actions of L-701,324 may not be associated with marked anaesthesia-like side-effects. Received: 27 December 1996 / Accepted: 14 February 1997     

1,5-(Diethylamino)piperidine, a novel spermidine analogue that more specifically activates the N-methyl-D-aspartate receptor-associated polyamine site.

We have investigated the action of 1,5-(diethylamino)piperidine (DEAP), a novel spermidine analogue that activates the polyamine site associated with the N-methyl-D-aspartate receptor. DEAP increased [3H]dizocilpine ([3H]MK801) binding to rat brain membranes with a potency similar to that of spermine and spermidine, but with a somewhat greater efficacy. Unlike other polyamines, however, DEAP did not exhibit low affinity inhibition of [3H] dizocilpine binding, suggesting that it binds more selectively to the polyamine site. DEAP increased the equilibrium affinity of [3H]dizocilpine. The increase in affinity was due to slowed dissociation, as well as a small increase in the association rate of [3H]dizocilpine. All of these effects of DEAP could be reversed by arcaine. These data illustrate the utility of DEAP as a novel polyamine agonist at the N-methyl-D-aspartate receptor complex. However, these data fail to support the hypothesis that polyamines activate the N-methyl-D-aspartate receptor by a mechanism similar to that of glutamate and glycine.     

The anticonvulsant and behavioural profile of L-687,414, a partial agonist acting at the glycine modulatory site on the N-methyl-D-aspartate (NMDA) receptor complex.

1. The anticonvulsant and behavioural effects of the glycine/NMDA receptor partial agonist, L-687,414 (R(+)-cis-beta-methyl-3-amino-1-hydroxypyrrolid-2-one) have been investigated in rodents. 2. L-687,414 dose-dependently antagonized seizures induced by N-methyl-D,L- aspartic acid (NMDLA, ED50 = 19.7 mg kg-1), pentylenetetrazol (PTZ, ED50 = 13.0 mg kg-1) and electroshock (ED50 = 26.1 mg kg-1) when given intravenously 15 min before test, in male Swiss Webster mice but was most potent against audiogenic seizures induced by a 120 dB bell in DBA/2 mice (ED50 = 5.1 mg kg-1, i.p., 30 min before test). 3. L-687,414 also induced impairments of performance in a rotarod test in both Swiss Webster and DBA/2 mice and the ratio [rotarod MED:anticonvulsant ED50] varied between 0.9 and 5, depending on the convulsant used. 4. Similar behaviours to those seen after administration of the non-competitive NMDA receptor antagonist, MK-801 (head weaving, body rolling, hyperlocomotion) were seen in the mouse after giving L-687,414, although the peak effect occurred at a dose (100 mg kg-1) which was 5-20 times the anticonvulsant ED50S, depending on the convulsant used. Unlike MK-801, however, doses of L-687,414 that were behaviourally stimulant did not increase dopamine turnover in the nucleus accumbens. 5. Consistent with the interaction of L-687,414 with the glycine/NMDA receptor, the anticonvulsant, ataxic and motor stimulant effects of the compound were significantly attenuated by the glycine/NMDA receptor agonist, D-serine (10-100 micrograms per mouse, i.c.v.).(ABSTRACT TRUNCATED AT 250 WORDS)     

Anticonvulsant effects of antagonists of the N-methyl-D-aspartate receptor complex in a genetic model of epilepsy: the quaking mouse

Tonic-clonic convulsions of mutant quaking mice were antagonized by the intracerebroventricular injection of N-methyl-D-aspartate receptor antagonists. The competitive antagonists, CPP (3-((+/-)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid) and CGS 19755 (cis-4-(phosphonomethyl)-2-piperidine carboxylic acid), exerted a partial anticonvulsant action, with ED50S of 0.115 and 0.076 nmol, respectively. The non-competitive antagonists, TCP (1-(1-(2-thienyl)cyclohexyl)piperidine) and MK-801 [+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine), provided full protection, with ED50s of 4.49 and 2.67 nmol, respectively. The competitive antagonists elicited a marked ataxia whereas the non-competitive antagonists did not have side-effects. These results might reflect the involvement of glutamatergic neurotransmission in the convulsions of the quaking mutants.     

Effects of memantine, a non-competitive N-methyl-D-aspartate receptor antagonist, on genomic stability

Memantine is an aminoadamantane drug useful in neurodegenerative diseases, with beneficial effects on cognitive functions. Some studies have shown that memantine protects brain cells, thereby decreasing glutamate excitotoxicity. This study evaluated the genotoxic/antigenotoxic and mutagenic effects of memantine in CF-1 mice, following standardized protocols. Memantine was administered i.p. at 7.5, 15 or 30 mg/kg for three consecutive days. Blood and brain samples were collected to assess DNA damage using the alkaline comet assay. The mutagenic effect was assessed using the bone marrow micronucleus test. In addition, possible antioxidant effects were evaluated measuring the survival of Saccharomyces cerevisiae yeast strains [wild-type (WT) and isogenic mutants lacking superoxide dismutase] to cotreatment of memantine plus hydrogen peroxide. Memantine decreased DNA oxidative damage mainly in brain tissue. This antigenotoxic effect corroborated an increase observed in the survival of S. cerevisiae WT strain against hydrogen peroxide-induced damage. Furthermore, memantine did not increase the micronucleus frequency. The overall results indicate that memantine showed no mutagenic activity, did not cause DNA damage in the blood and brain tissues and showed antigenotoxic effects in brain tissue.     

The polyamine spermine has multiple actions on N-methyl-D-aspartate receptor single-channel currents in cultured cortical neurons.

Spermine potentiates the action of N-methyl-D-aspartate (NMDA) at micromolar concentrations but is less effective at millimolar concentrations. In cultured cortical neurons we demonstrate that spermine enhances NMDA receptor currents in a unique manner. At low concentrations (1-10 microM) spermine enhances NMDA receptor current by increasing channel opening frequency, and at higher concentrations (greater than 10 microM) it produces, in addition, a voltage-dependent decrease in channel amplitude and average open time that limits its enhancing action. It is likely that these two actions of spermine, due to differences in concentration and voltage dependence, are mediated by independent sites on the NMDA receptor complex.