Polyamine Modulatory Site Research Articles

Confocal imaging of N-methyl- d-aspartate receptors in living cortical neurons

The fluorescence-conjugated N-methyl- d-aspartate receptor-selective antagonist, BODIPY-conantokin-G, was employed to label N-methyl- d-aspartate receptors in living neurons derived from the visual cortex of embryonic rats. The fluorescent labeling was visualized and analysed using confocal microscopy and digital imaging techniques. BODIPY-conantokin-G binding sites were homogeneously distributed across somata four days after neurons (E17–20) were placed in culture. In five-day-old cultures, BODIPY-conantokin-G binding sites became clusters of fluorescently labeled spots which were arranged irregularly on somata and proximal neurites. Distal neurites displayed fluorescent labeling after 10–15 days in culture. Displacement experiments showed that spermine and unlabeled conantokin-G compete with BODIPY-conantokin-G labeling at the N-methyl- d-aspartate receptor-associated polyamine site. The N-methyl- d-aspartate receptor antagonist 2-amino-5-phosphonovaleric acid also depressed the labeling but with a weaker effect, probably due to interactions occurring between the N-methyl- d-aspartate receptor agonist binding site and the polyamine modulatory site. The fluorescent dyes FM 1-43 and FM 4-64 were used in double-labeling studies to compare the distribution of nerve terminals with that of BODIPY-conantokin-G binding sites. BODIPY-conantokin-G binding clusters were associated with presynaptic nerve terminals while isolated BODIPY-conantokin-G binding sites were not always opposed to terminals. The aggregation of receptors to form clusters may lead to the functional formation of excitatory synapses. To investigate whether modulation of membrane potentials affected the formation of N-methyl- d-aspartate receptor clusters, cultured neurons were chronically treated for a week with either tetrodotoxin (to block membrane action potentials) or a high concentration of potassium to depolarize the membrane. While neurons in the tetrodotoxin-treated group showed a similar number of fluorescently labeled clusters compared with the control group, neurons in the high potassium group exhibited a higher number of fluorescently labeled receptor clusters. These results suggest that more active neurons may tend to form more N-methyl- d-aspartate synapses during early development.

Anticonvulsant effects of eliprodil alone or combined with the glycine B receptor antagonist L-701,324 or the competitive NMDA antagonist CGP 40116 in the amygdala kindling model in rats

The discovery that glutamate’s activity at the N-methyl- d-aspartate (NMDA) receptor is positively modulated by glycine and polyamines has led to a new pharmacological strategy that NMDA receptor-mediated events could be antagonized indirectly at the strychnine-insensitive glycine co-agonist site (glycine B receptor) and the polyamine modulatory site. Recently we demonstrated that ifenprodil and L-701,324 (7-chloro-4-hydroxy-3(3-phenoxy)phenyl-2(H)quinoline), polyamine and glycine B receptor antagonists, respectively, at subeffective doses markedly increased after-discharge threshold (ADT) when applied together in amygdala-kindled rats. Because ifenprodil and its derivative, eliprodil, exhibit different affinities for NMDA receptors composed of different subunits, our current question was whether a combination of eliprodil and the glycine B receptor antagonist, L-701,324, would produce a super-additive anticonvulsant action. In addition, we examined the combined treatment of eliprodil with a competitive NMDA receptor antagonist CGP 40116 ( d-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid) in the kindling model. Eliprodil alone (10–40 mg/kg) had no consistent ADT-increasing activity. When eliprodil was combined with an ineffective dose of L-701,324 (2.5 mg/kg), a significant rise in ADT was observed. Likewise, other measures of seizure activity such as severity and duration were modestly but significantly reduced. With respect to behavioral impairments, no signs of synergistic interaction were observed after the drug combinations. On the other hand, no anticonvulsant effects were found when CGP 40116 was administered alone at doses of 1.25–5 mg/kg or CGP 40116 1.25 mg/kg combined with eliprodil 10 mg/kg. These data suggest that combination therapy with antagonists at the polyamine and glycine sites might potentially treat therapy-resistant complex partial seizures.

Concurrent activation of hippocampal glycine and polyamine sites of the N-methyl- d-aspartate receptor synergistically reverses working memory deficits in rats

Intrahippocampal administration of the non-competitive N-methyl- d-aspartate (NMDA) receptor antagonist MK-801 (0.18 μg/side) significantly increased the number of errors in the working memory task with a three-panel runway setup. The increase in working memory errors by intrahippocampal MK-801 was significantly attenuated by concurrent infusion of D-cycloserine (1.0 μg/side) or spermidine (10 μg/side), agonists of the glycine and polyamine modulatory sites on the NMDA receptor/channel complex, respectively. Combined injection of the behaviorally ineffective doses of 0.1 μg/side D-cycloserine and 0.32 μg/side spermidine synergistically reduced intrahippocampal MK-801-induced increase in working memory errors. The combination of D-cycloserine and spermidine also synergistically attenuated the increase in working memory errors resulting from intrahippocampal injection of the muscarinic acetylcholine receptor antagonist scopolamine (3.2 μg/side). These results suggest that positive modulation of the NMDA receptor/channel through activation of the glycine and polyamine sites can synergistically compensate deficiency of hippocampal NMDA and muscarinic receptor-mediated neurotransmission involved in working memory function.

Phencyclidine-like discriminative stimulus effects of polyamine modulators of N-methyl- d-aspartate receptor activity in rats

Excessive N-methyl- d-aspartate (NMDA) receptor activation has been implicated in many acute and chronic neuropathologies. NMDA antagonists might prove to be useful treatments, unfortunately, some can produce phencyclidine (PCP)-like side effects. The polyamine-site modulators, spermine (SPM) and spermidine (SPD), produce dose related biphasic modulation of NMDA channel currents while another polyamine, arcaine (ARC), produces only negative modulatory effects. The PCP-like effects of these compounds were tested in rats trained to discriminate PCP from saline in a standard two-lever drug discrimination paradigm under a fixed ratio schedule of food reinforcement. SPM, SPD and ARC occasioned little, if any, responding on the PCP-associated lever, even at response rate suppressing doses. The results provide further evidence that differences exist between the discriminative stimulus effects produced by drugs active at different sites on the NMDA receptor and suggest that the polyamine modulatory site should be a good target for development of NMDA antagonist medications with a reduced propensity for PCP-like acute behavioral effects.

Neuroprotective effect of eliprodil: attenuation of a conditioned freezing deficit induced by traumatic injury of the right parietal cortex in the rat.

We have previously demonstrated that a lateral fluid percussion-induced traumatic lesion of the right parietal cortex can lead to a deficit in a conditioned freezing response and that this deficit can be attenuated by both pre- and postlesion administration of the NMDA receptor antagonist dizocilpine. In the present study, we investigated the effects of eliprodil, a noncompetitive NMDA receptor antagonist acting at the polyamine modulatory site, which also acts as a Ca2+ channel blocker, on the trauma-induced conditioned freezing deficit. Eliprodil produced a 50% reduction in this deficit when administered as three 1 mg/kg injections i.v. at 15 min, 6 h, and 24 h following the lesion. Approximately the same degree of protection was afforded when 2 x 1.5 mg/kg were administered 6 and 24 h and equally at 12 and 24 h after surgery (56% and 59%, respectively). A single treatment (3 mg/kg) at 24 h was ineffective against the deficit. The protection afforded with treatment at 6 and 24 h after lesion was dose dependent, with a minimal active dose of 2 x 0.75 mg/kg. These data complement those previously published on the ability of eliprodil to reduce lesion volume following traumatic brain injury and show, in addition, that the neuroprotective effect has functional consequences.

Spermidine, a polyamine site agonist, attenuates working memory deficits caused by blockade of hippocampal muscarinic receptors and mGluRs in rats

Spermidine, an agonist of the polyamine site on the NMDA receptor/channel complex, did not affect the number of errors (attempts to pass through two incorrect panels of the three panel-gates at four choice points) in the working memory task with a three-panel runway setup, when injected bilaterally at 1 or 10 μg/side into the dorsal hippocampus. Intrahippocampal administration of the muscarinic receptor antagonist scopolamine (3.2 μg/side) significantly increased the number of working memory errors. The increase in working memory errors by intrahippocampal 3.2 μg/side scopolamine was significantly reduced by concurrent infusion of 1 and 10 μg/side spermidine. Likewise, spermidine (10 μg/side) was effective in attenuating the increase in working memory errors induced by intrahippocampal administration of AIDA (3.2 μg/side), a potent and selective antagonist of the class I metabotropic glutamate receptor (mGluR). These results suggest that enhanced NMDA function via polyamine modulatory site on the NMDA receptor/channel can compensate dysfunction of hippocampal cholinergic and mGluR-mediated neurotransmission involved in working memory function.

Protective effect of ifenprodil against glucose deprivation-induced damage in cultured rat hippocampal neurons.

When hippocampal cultures were deprived of glucose, massive release of lactate dehydrogenase (LDH), an indicator of neuronal death, occurred 24 hr following the onset of hypoglycemic insult via N-methyl-D-aspartate (NMDA)-type glutamate receptor activation. Ifenprodil (0.1 and 1 M) significantly inhibited LDH release, which was antagonized by polyamines. These results suggest that ifenprodil protects neurons from glucose deprivation by antagonizing the effects of glutamate via selective interaction with polyamine modulatory sites on the NMDA receptor complex. The observed phenomenon further indicate that ifenprodil might be used prophylactically against neuronal death induced by excitotoxic disorders.

Cognitive enhancement. New strategies for stimulating cholinergic, glutamatergic, and nitric oxide systems.

The development of treatments for AD is being pursued along many diverse lines. While the ACh hypothesis has generated abundant development efforts, little clinical progress has been achieved to date. Recent efforts aimed at developing more potent, more specific, and safer ChE inhibitors appear to offer greater potential for therapeutic success than achieved to date. Treatments aimed at the NMDA Glu system lag much further behind in their development. Progress in this area must be tempered by the potential for glutamate excitotoxicity mediated through this neurotransmitter system. Development of indirect agonists operating at the glycine and polyamine modulatory sites on the NMDA receptor might offer the safest alternative to applying more direct agonists. While a great degree of interest had been generated by the reports of NO involvement in signal transduction through the NMDA system, this area of research has been complicated by conflicting reports regarding NO involvement in learning and LTP. Moreover, the interaction of drugs acting on NOS with the vascular effects mediated by eNOS has also complicated development of drugs that act specifically on the neural actions of NO. This area will continue to receive extensive research attention; but similar to the development of Glu agonists, attention must be given to the potential neurotoxic effects of overstimulating this system. Perhaps targeting other presynaptic mechanisms that effect glutamate release might be a safer strategy to pursue. Considerable progress has been made over the last two decades in identifying the genetic and neural mechanisms involved in AD. Progress in developing treatments will remain highly correlated with this effort, and with basic research geared to comprehending how memories are formed and why neurons degenerate and regenerate.

Synthesis of N,N'-substituted piperazine and homopiperazine derivatives with polyamine-like actions at N-methyl-D-aspartate receptors.

A series of N,N'-substituted piperazine and homopiperazine derivatives have been synthesized with the objective of producing compounds that interact with polyamine modulatory sites on N-methyl-D-aspartate (NMDA) receptors. These novel compounds exhibited polyamine-like actions, enhancing [3H]MK-801 binding to NMDA receptors in rat forebrain membranes. The potencies of N,N'-bis(2-aminoacetyl)homopiperazine (15), N,N'-bis(N-methyl-4-aminobutyl)-piperazine (7), and N,N'-bis(3-aminopropyl)homopiperazine (11) (EC50 18.0, 21.3, and 24.4 microM, respectively) to enhance [3H]MK-801 binding were comparable to that of spermine (EC50 5.2 microM). However, the efficacies of 15, 7, and 11 in this measure were lower (by approximately 40%, 32%, and 24%, respectively) than spermine, which may be indicative of partial agonist actions. Like spermine, the ability of these piperazine and homopiperazine derivatives to enhance [3H]MK-801 binding could be inhibited by both a competitive polyamine antagonist (arcaine) and a specific, noncompetitive polyamine antagonist (conantokin-G). However, unlike endogenous polyamines, high concentrations (up to 1 mM) of these novel polyamine-like compounds did not inhibit [3H]MK-801 binding. N,N'-Aminoalkylated and aminoacylated piperazine and homopiperazine derivatives may prove useful for studying polyamine recognition sites associated with NMDA receptors.

Pregnenolone sulfate potentiation of NMDA-mediated increases in intracellular calcium in cultured chick cortical neurons

Pregnenolone sulfate (PS) has been reported to selectively augment glutamate-induced depolarizations mediated by the NMDA subtype of the glutamate receptor. The present study examines the ability of this neuroactive steroid to potentiate NMDA-mediated increases in intracellular calcium in cultured chick cortical neurons using the fluorescent dye Fura2. PS, in the absence of NMDA and glycine, significantly elevated intracellular calcium at 250 and 500 μM. This increase in free calcium was significantly attenuated at 250 μM PS by the prior addition of 50 μM CNQX, 10 μM dizocilpine or 1 μM nimodipine. NMDA and glycine, when added to the cells in saturating concentrations of 500 and 50 μM, respectively, consistently increased intracellular free calcium over baseline levels. In the presence of NMDA and glycine, both 50 and 100 μM PS produced a further significant rise in intracellular free calcium. The prior addition of CNQX, dizocilpine or both compounds together significantly inhibited this elevation in free calcium. The application of the endogenous polyamine spermine (250 μM) significantly potentiated the response of chick cortical neuronal cells to NMDA and glycine. PS, in the presence of NMDA, glycine and spermine, produced a further increase in intracellular free calcium at concentrations of 50 and 100 μM. The prior application of CNQX, dizocilpine or both compounds together significantly attenuated this rise in free calcium. These data confirm that PS is a positive allosteric modulator of the NMDA receptor and provide evidence that this neurosteroid does not interact with the polyamine modulatory site.

Effect of eliprodil, an NMDA receptor antagonist acting at the polyamine modulatory site, on local cerebral glucose use in the rat brain

The present investigation examined the effect of eliprodil, an atypical NMDA receptor antagonist that acts at the polyamine modulatory site, on local cerebral glucose utilization using the quantitative autoradiographic 2-[ 14C]deoxyglucose method in the conscious rat. Eliprodil, at doses of 3, 10 and 30 mg/kg i.p., did not increase cerebral glucose use in any of the 82 different brain regions studied. However, in some of the regions examined, local cerebral glucose utilization was slightly reduced, the most pronounced decreases being measured in some extrapyramidal, sensorimotor and limbic areas (dentate gyrus, septum, lateral habenula, amygdala). This decrease in glucose utilization was dose-dependent: no significant change was noted after 3 mg/kg i.p. of eliprodil, while 18 (at 10 mg/kg, i.p.) and 29 (at 30 mg/kg, i.p.) regions displayed a moderate (20–25%) though significant decrease in glucose use. These data demonstrate that the pattern of alterations in glucose use produced by eliprodil is different from that induced by NMDA channel blockers or competitive NMDA receptor antagonists. The fact that blockade of the polyamine modulatory site is not associated with an activation of specific limbic circuits may explain why, at neuroprotective doses, eliprodil is devoid of those unwanted side effects (including intrinsic neurotoxicity on cortical neurons) associated with NMDA channel blockers.

The NMDA receptor complex.

The synaptic responses elicited by glutamate and aspartate in the CNS are mediated by distinct groups of receptors which include the ionotropic NMDA receptor. The NMDA receptor is activated by high-strength synaptic input and produces relatively sustained depolarization which can lead to repetitive burst firing. These characteristics allow it to be involved in the maintainance of rhythmic neuronal activity and in the modulation of synaptic efficacy and plasticity. Overstimulation of the NMDA receptor appears to play a pivotal role in the physiopathology of ischemic brain injury. The NMDA receptor contains an integral cationic channel which is highly permeable to Ca2+ as well as to Na+ and K+. This receptor has several domains in addition to the NMDA recognition site: i) a divalent cation binding site within the channel pore, at which Mg2+ ions bind, ii) a binding site recognized by dissociative anesthetics and MK-801 within the channel; and iii) modulatory sites sensitive to glycine, Zn2+ and polyamines. The NMDA receptor is strictly controlled by Mg2+ ions in a voltage-dependent manner. Moreover, it is modulated by protons, by changes in the redox state and by endogenous physiological substances, eg NO and arachidonic acid. Selective antagonists now exist for the NMDA recognition site and glycine and polyamine modulatory sites. Molecular cloning of the NMDA receptor has identified a subunit termed NMDA-R1 and four additional subunits (NMDA-R2A through NMDA-R2D). Functionally distinct NMDA receptor subtypes are formed by heteromeric assembly of NMDA-R1 with NMDA-R2 subunits. NMDA receptor subunits contain consensus phosphorylation sites for protein kinases at the cytoplasmic domain. The high Ca2+ permeability and sensitivity to channel block by Mg2+ are imparted by asparagine residues in a putative channel-forming segment of the protein transmembrane 2. The knowledge of the molecular structure of the NMDA receptor will help to understand the molecular mechanisms responsible for its regulatory features and the molecular bases of neurotoxicity.

Substitution by NMDA antagonists and other drugs in rats trained to discriminate ethanol

It has been reported that, in animals trained to discriminate ethanol, stimulus control generalized to the non-competitive NMDA antagonists phencyclidine, ketamine and dizocilpine. In the present study, rats were trained to discriminate a dose of ethanol (1g/kg, i.p.) and substitution tests were carried out with phencyclidine, dizocilpine, CGS 19755, eliprodil, triazolam, chlordiazepoxide, abecarnil, alpidem and d-amphetamine. Phencyclidine and dizocilpine produce dose-related substitution for ethanol as did the competitive NMDA antagonist, CGS 19755, and the benzodiazepines, triazolam and chlordiazepoxide. Eliprodil, an NMDA antagonist acting through the polyamine modulatory site, neither substituted for ethanol nor modified the ethanol dose-response curve. d-Amphetamine, and the non-benzodiazepine anxiolytics, alpidem and abecarnil, did not substitute for ethanol. The results show that both NMDA antagonists and compounds acting through (GABA receptors (benzodiazepines) can substitute for ethanol, emphasizing that the ethanol cue may involve several mechanisms. As all the drugs substituting for ethanol, like ethanol itself, are known to produce ataxia and muscle relaxation, it is proposed that this property may be an important aspect of the ethanol cue.

Prevention by eliprodil (SL 82.0715) of traumatic brain damage in the rat. Existence of a large (18h) therapeutic window

The neuroprotective potential of eliprodil (SL 82.0715), an N-methyl- d-aspartate (NMDA) receptor antagonist acting at the polyamine modulatory site, in brain trauma was examined in a rat model of lateral fluid-percussion brain injury. The volume of the lesion was assessed histologically by measuring, at 7-days post-injury, the area of brain damage at 10 coronal planes. Eliprodil (10 mg/kg i.p.) when given 15 min, 6 h and 24 h after fluid percussion (1.6 atm) and then b.i.d. for the following 6 days, reduced by 60% the volume of cortical damage. A similar neuroprotection was obtained when the first administration of eliprodil was delayed by up to 12 h after the brain insult. Moreover, when the treatment with this compound was started at 18 h post-injury, cortical damage was still significantly reduced by 33%. Autoradiographic studies showed that eliprodil treatment (10 mg/kg, i.p.), initiated 15 min after the trauma, also caused a marked reduction of the loss of the neuronal markerω 1−2 (central benzodiazepine) binding sites of the increase in the glial/macrophage marker peripheral type benzodiazepine binding sites in the cerebral cortex. In contrast, dizocilpine (a blocker of the cationic channel coupled to the NMDA receptor) when administered 6 h and 24 h after fluid percussion and then b.i.d. for the following 6 days induced a non significant reduction of the volume of the lesion at the highest tolerated dose (0.6 mg/kg i.p.). These results demonstrate the neuroprotective activity of eliprodil in experimental brain trauma using neuropathology as an endpoint and indicate that three is a very large time window for therapeutic intervention, consistent with the delayed nature of the neuronal loss, in this condition.