Regional Profile of Developmental Changes in the Sensitivity of the N-Methyl-D-Aspartate Receptor to Polyamines

Abstract: The NMDA receptor exhibits increased sensitivity to stimulation during early development compared with the adult. In this study, we examined modulation of the NMDA receptor by polyamines during development to see if it correlates with differences in the functional responsiveness of the NMDA receptor. [³H]MK-801 binding was measured in discrete brain regions in the presence and absence of polyamines in 3-, 7-, 15-, 25-, and 60-day-old Sprague-Dawley rats. [³H]MK-801 binding increased between postnatal days 3 and 15, with adult levels of binding being reached between days 15 and 25. Spermidine (75 μM) caused maximal stimulation of [³H]MK-801 binding during early development, ranging from 250% in the thalamus to 450% in the caudate putamen at postnatal day 3. This effect gradually declined to levels seen in the adult by postnatal days 15–25. During all developmental stages, the stimulation seen was greater in the caudate putamen compared with the hippocampus. Diethylenetriamine (1 μM) exhibited similar developmental and regional heterogeneity in its effects on [³H]MK-801 binding, producing substantial stimulation of binding in the neonate, but not in the adult. The EC₅₀ and E_max values for the stimulatory effect of spermidine were significantly higher at day 7 compared with the adult. Unlike spermidine and diethylenetriamine, there was no regional variation in the effects of the putative “polyamine site” inverse agonist 1,10-diaminodecane at any age and only a slightly attenuated inhibition at postnatal day 3 compared with the adult. This lack of complementarity in the regional and developmental profiles of spermidine and diethylenetriamine, on the one hand, and 1,10-diaminodecane, on the other, suggests that their effects on [³H]MK-801 binding are mediated at different sites. The altered sensitivity of the NMDA receptor to polyamines during development could reflect the expression of molecular variants with different sensitivities to modulation by polyamines.     

Polyamines Modulate the Binding of [3H]MK-801 to the Solubilized N-Methyl-D-Aspartate Receptor

Spermine and spermidine enhance the binding of [³H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a, d]cyclohepten-5,10-imine ([³H]MK-801) to N-methyl-D-aspartate (NMDA) receptors in membranes prepared from rat brain. These polyamines also enhance binding of [³H]MK-801 to NMDA receptors that have been solubilized with deoxycholate. Other polyamines selectively antagonize this effect, a finding indicating that the polyamine recognition site retains pharmacological and structural specificity after solubilization. In the presence of spermidine, an increase in the affinity of the solubilized NMDA receptor for [³H]MK-801 is observed. However, the rates of both association and dissociation of [³H]MK-801 binding to solubilized NMDA receptors are accelerated when assays are carried out in the presence of spermidine. When kinetic data are transformed, pseudo-first-order association and first-order dissociation plots are nonlinear in the presence of spermidine, an observation indicating a complex binding mechanism. Effects of spermidine on solubilized NMDA receptors are similar to effects previously described in studies of membrane-bound receptors. The data indicate that polyamines interact with a specific recognition site that remains associated with other components of the NMDA receptor complex after detergent solubilization.     

Characterization of polyamines having agonist, antagonist, and inverse agonist effects at the polyamine recognition site of the NMDA receptor

The endogenous polyamines spermine and spermidine increase the binding of [3H]MK-801 to NMDA receptors. This effect is antagonized by diethylenetriamine (DET). We report here that spermine increases the rates of both association and dissociation of binding of [3H]MK-801, suggesting that it increases the accessibility of the binding site for MK-801 within the ion channel of the receptor complex. 1,10-Diaminodecane (DA10) inhibited the binding of [3H]MK-801. This effect was due to a decrease in the rate of association with no change in the rate of dissociation of [3H]MK-801. The effect of DA10 was not mediated by an action of DA10 at the binding sites for glutamate, glycine, Mg2+, or Zn2+, and was attenuated by DET. This suggests that DA10 acts at the polyamine recognition site. In hippocampal neurons the NMDA-elicited current was decreased by DA10, an effect opposite to that of spermine. The effects of spermine and DA10 were selectively blocked by DET. It is concluded that DA10 acts as a negative allosteric modulator or inverse agonist at the polyamine recognition site of the NMDA receptor.     

Characterisation of [3H]MK-801 Binding and its Cooperative Modulation by PIG Brain Membranes

Abstract

Cooperative modulation of [³H]MK-801 binding to extensively washed pig cortical brain membranes in the presence of various concentrations of L-glutamate, glycine, spermine, CPP and DCKA was evaluated in association experiments. In saturation experiments [³H]MK-801 labelled a homogeneous population of binding sites with a K_d-value of 1.26 ± 0.18 nmol 1^?1 and a B_max-value of 2130 ± 200 fmol/mg protein. The pharmacological profile of this site was further evaluated in competition experiments with known NMDA receptor channel blockers. In nonequilibrium binding experiments EC₅₀-values of reference compounds acting at the L-glutamate, at the glycine, and at the polyamine site, were determined by increasing or decreasing [³H]MK-801 binding. Ifenprodil reduced [³H]MK-801 binding in a biphasic manner. All the data obtained are in agreement with results from [³H]MK-801 binding to rodent as well as human brain membranes. This study therefore strongly suggests, that pig cortical membranes are a suitable alternative to rodent brain membranes, and an acceptable substitute for human brain membranes in [³H]MK-801 binding experiments.     

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 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 NMDA-elicited 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.     

[3H]MK-801 Binding to N-Methyl-d-Aspartate Receptors Solubilized from Rat Brain: Effects of Glycine Site Ligands, Polyamines, Ifenprodil, and Desipramine

The N-methyl-D-aspartate (NMDA) receptor is thought to contain several distinct binding sites that can regulate channel opening. In the present experiments, the effects of ligands for these sites have been examined on [3H]MK-801 binding to a soluble receptor preparation, which had been passed down a gel filtration column to reduce the levels of endogenous small-molecular-weight substances. Glycine site agonists, partial agonists, and antagonists gave effects similar to those observed in membranes [EC50 values (in microM): glycine, 0.31; D-serine, 0.20; D-cycloserine, 1.46; (+)-HA-966, 4.06; and 7-chlorokynurenic acid, 1.81]. Spermine and spermidine enhanced [3H]MK-801 binding to the soluble receptor preparation (EC50, 4.3 and 20.1 microM, respectively), whereas putrescine and cadaverine gave small degrees of inhibitions. When spermine and spermidine were tested under conditions where [3H]MK-801 binding approached equilibrium, their ability to enhance [3H]MK-801 binding was much reduced, a result suggesting that the polyamines increase the rate to equilibrium. Putrescine antagonised the effects of spermine. Ifenprodil reduced [3H]MK-801 binding under both equilibrium and nonequilibrium conditions, although the high-affinity component of inhibition described in membranes was not observed. Ifenprodil antagonised spermine effects in an apparently noncompetitive manner. Desipramine was able to give total inhibition of specific [3H]MK-801 binding under nonequilibrium conditions with an IC50 of 4 microM, and this value was unaltered when [3H]MK-801 binding was allowed to reach equilibrium. These results suggest that the sites mediating the effects of glycine and its analogues, polyamines and desipramine are integral components of the NMDA receptor protein.     

Preservation of Redox, Polyamine, and Glycine Domains of the N-Methyl-D-Aspartate in Alzheimer's Disease

Abstract: This study used [³H] dizocilpine ([³H] MK-801) binding to the N-methyl-D-aspartate (NMDA) receptor to examine redox, polyamine, and glycine modulatory sites in membranes derived from the superior frontal and the superior temporal cortex of patients with Alzheimer's disease. In control subjects the competitive polyamine site antagonist arcaine inhibited [³H] dizocilpine binding in a dose-dependent fashion and this curve was shifted to the right by the addition of 50 μM spermidine. Arcaine inhibition of binding was more potent in the temporal cortex than in the frontal cortex, in both the absence and presence of 50 μMspermidine. In Alzheimer's disease, arcaine inhibition of [³H] dizocilpine binding (in both the absence and the presence of spermidine) was not different from control in either of the two brain areas examined. The sulfhydryl redox site of the NMDA receptor was assessed using the oxidizing agent 5, 5′-dithio-bis(2-nitrobenzoic acid), which inhibited binding in a dose-dependent fashion. This inhibition was similar in patients with Alzheimer's disease and control subjects. Glycine-stimulated [³H] dizocilpine binding was also unaffected in patients with Alzheimer's disease. However, in the temporal cortex there was a significant age-associated decline in [³H] dizocilpine binding in the presence of 100 μM glutamate (R₈=-0.71) and 100 μM glutamate plus 30 μM glycine (R₈=?0.90). There was also an age-related increase in arcaine IC₅₀ (which reflects an age-related decrease in arcaine affinity) in the frontal cortex, determined both in the absence (R₈= 0.83) and the presence (R₈= 0.79) of spermidine. These data indicate that the NMDA receptor and its modulatory redox, polyamine, and glycine subsites are intact in patients with Alzheimer's disease and that the modulatory activity of polyamine and glycine sites decline with aging.     

β-Amyloid (25-35) or Substance P Stimulates [3H]MK-801. Binding to Rat Cortical Membranes in the Presence of Glutamate and Glycine

Abstract: Micromolar concentrations of β-amyloid (25–35) or substance P stimulated [³H] MK-801 binding in the presence of low concentrations of glutamate (1 γM) and glycine (0.02 γM). Unlike polyamines spermine and spermidine, neither β-amyloid (25–35) nor substance P increased [³H] MK-801 binding in the presence of maximally stimulating concentrations of glutamate and glycine. 5,7-Dichloro-kynurenic acid, CGS-19755, and arcaine completely inhibited the stimulated [³H] MK-801 binding. There was an apparent decreased potency of the [³H] MK-801 binding inhibition curve for 5,7-dichlorokynurenic acid, but not CGS-19755 or arcaine, in the presence of either β-amyloid (25–35) or substance P. The compounds do not appear to act through the strychnine-insensitive glycine binding site because neither β-amyloid (25–35) nor substance P displaced [³H] glycine binding. Full-length β-amyloid (1-40), up to 10 γM, did not stimulate [³H] MK-801 binding. Concentrations >10 γM could not be tested because they formed large aggregate precipitates in the assay. The data indicate that β-amyloid (25–35) or substance P does not stimulate [³H] MK-801 binding at either the N-methyl-D-aspartate, glycine, or polyamine binding sites. Furthermore, the nonpeptide substance P receptor (NK,) antagonist, CP-96,345, did not block β-amyloid (25–35)- or substance P-stimulated [³H] MK-801 binding. Therefore, the effect is not due to an interaction between the substance P receptors and the N-methyl-D-aspartate receptor-operated ionophore. Finally, if these observations can be verified using single-channel recording techniques, they may have implications in the pattern of selective neuronal loss observed in patients with neurodegenerative processes such as Alzheimer's, Parkinson's, and Huntington's diseases.     

A Possible Role of Glutathione as an Endogenous Agonist at the N-Methyl-d-Aspartate Recognition Domain in Rat Brain

Abstract: Glutathione, both reduced (GSH) and oxidized (GSSG), was effective in displacing binding of l -[³H]-glutamic acid (l -[³H]Glu) and dl -(E)-2-[³H]amino-4-propyl-5-phosphono-3-pentenoic acid ([³H]CGP-39653) in rat brain synaptic membranes, with less potent displacement of binding of dl -α-amino-3-hydroxy-5-[³H]-methylisoxazole-4-propionic and [³H]kainic acids. Liquid chromatographic analysis revealed that both GSH and GSSG were contaminated with l -Glu by <1%. Both GSH and GSSG potentiated (+)-5-[³H]methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine ([³H]MK-801) binding in a manner similar to that found with l -Glu. Pre-treatment with glutamate dehydrogenase (GDH) induced a marked rightward shift of the concentration-response curve for l -Glu in the presence of NAD without affecting that in its absence, whereas GDH was ineffective in affecting the potentiation by both GSH and GSSG even in the presence of NAD. In the presence of GSH at a maximally effective concentration, both glycine (Gly) and spermidine potentiated [³H]MK-801 binding to a somewhat smaller extent than that found in the presence of l -Glu at a maximally effective concentration. The potentiation of [³H]MK-801 binding by GSH was invariably attenuated by addition of CGP-39653, d -2-amino-5-phosphonovaleric acid (d -AP5), and 5,7-dichlorokynurenic acid (DCKA), whereas GSH was effective in diminishing potencies of CGP-39653, d -AP5, DCKA, and 6,7-dichloroquinoxaline-2,3-dione to inhibit [³H]MK-801 binding when determined in the presence of both l -Glu and Gly. These results suggest that glutathione may be an endogenous agonist selective for the N-methyl-d -aspartate (NMDA) recognition domain on the NMDA receptor ionophore complex.     

Aromatic analogs of arcaine inhibit MK-801 binding to the NMDA receptor

Aromatic analogs of arcaine were shown to have inhibitory effects on the binding of the channel blocking drug [³H]MK-801 to the NMDA receptor complex. The most potent compound of the series was an N,N′-bis(propyl)guanidinium which inhibited [³H]MK-801 binding with an IC₅₀ of 0.58 μM and an IC₅₀ of 12.17 μM upon addition of 100 μM spermidine. The increase in IC₅₀ upon addition of spermidine suggests competitive antagonism between the inhibitor and spermidine at the arcaine-sensitive polyamine site of the NMDA receptor complex.     

Regulation of the Phosphoinositide Cascade by Polyamines in Brain

Abstract: The endogenous polyamines spermidine and spermine enhanced guanosine 5′-O-(3-thiotriphosphate) (GTP-γ-S)-stimulated phosphoinositide turnover with EC₅₀ values of 100 ± 30 and 50 ± 15 µM, respectively, whereas the synthetic polyamines N,N′-bis(3-aminopropyl)-1,3-propanediamine and -ethylenediamine inhibited GTP-γ-S-stimulated phosphoinositide turnover, with maximal inhibition at 1 mM. Kinetic analysis of GTP-γ-S-stimulated phosphoinositide turnover in the absence and presence of spermidine showed that the K_m for GTP-γ-S was not changed (1,303 ± 270 and 1,069 ± 214 nM, respectively), whereas the V_max was increased by 206% (1,566 ± 141 and 4,792 ± 84 cpm, respectively), indicating that spermidine and GTP-γ-S acted at different sites. Spermidine also enhanced Ca²⁺-stimulated phosphoinositide turnover in the absence of GTP-γ-S by decreasing the Ca²⁺ requirement of the phosphoinositide-specific phospholipase C. Arcaine and agmatine, polyamine antagonists at the NMDA receptor complex, did not block the effects of spermidine on GTP-γ-S- and Ca²⁺-induced phosphoinositide turnover, suggesting that the spermidine effects are not mediated through these specific polyamine sites. Furthermore, spermidine increased the level of [³H]phosphatidylinositol 4-phosphate (EC₅₀ = 120 ± 10 µM), without affecting significantly the levels of [³H]phosphatidylinositol and [³H]phosphatidylinositol 4,5-bisphosphate. Collectively these data indicate that the enhanced phosphoinositide turnover induced by spermidine in the presence of GTP-γ-S or Ca²⁺ is mediated through multiple levels of the phosphoinositide turnover cascade.     

Characterization of [3H]MK-801 binding to N-methyl-D-aspartate receptors in cultured rat cerebellar granule neurons and involvement in glutamate-mediated toxicity

The conditions required for growth and survival of cerebellar granule neurons in vitro are known to alter the developmental regulation of NMDA receptor subunit mRNA. In the present report, we have examined the functional and pharmacological characteristics of NMDA receptors on cerebellar granule neurons at 12 days in culture (12 DIC). Under open-channel conditions in extensively washed membranes, [³H]MK-801 labeled a uniform population of sites (K_d = 3.2 ± 0.3 nM) in a saturable manner (B_max = 416 ± 18 fmol/mgl); however, biexponential association and dissociation kinetics indicated the possible existence of at least two NMDA receptor populations that differ in pharmacological properties. The kinetically derived equilibrium dissociation constants for the high- and low-affinity binding components were 0.56 and 771 nM, respectively. The equilibrium competition analysis of MK-801 and other channel-blocking compounds as displacers of [³H]MK-801 revealed the presence of high- and low-affinity binding sites with relative apportionments of 70% and 30%, respectively. The rank-order potency profile of competitor binding at the high-affinity site was (+)-MK-801 > TCP > dextrorphan > dextromethorphan > (+)-ketamine. When tested for the ability to protect 12 DIC cerebellar granule neurons from acute glutamate-induced toxicity, the neuroprotective rank-order potency of these compounds was MK-801 > TCP > dextrorphan > (+)-ketamine > dextromethorphan, which correlated significantly with the high-affinity competition binding profile and thus established the role of NMDA receptors in glutamate toxicity. The findings of these experiments indicate that NMDA receptors on 12 DIC cerebellar granule neurons are a heterogenous population that functionally mediate glutamate-induced neurotoxicity. The heterogenous [³H]MK-801 binding sites may represent NMDA receptor channels composed of different subunits. © 1997 John Wiley & Sons, Inc.     

Effects of ifenprodil on the N-methyl-D-aspartate receptor ionophore complex in rat brain.

The effects of a cerebral anti-ischemic drug ifenprodil on the receptor ionophore complex of an N-methyl-D-aspartate (NMDA)-sensitive subclass of central excitatory amino acid receptors were examined using [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10- imine (MK-801) binding in rat brain synaptic membrane preparations as a biochemical measure. The binding in membrane preparations not extensively washed was markedly inhibited not only by competitive NMDA antagonists such as (+/-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic, D-2-amino-5-phosphonovaleric and D-2-amino-7-phosphonoheptanoic acids, but also by competitive antagonists at the strychnine-insensitive glycine (Gly) site including 7-chlorokynurenic acid and 6,7-dichloroquinoxaline-2,3-dione. Among several proposed ligands for alpha-adrenergic receptors tested, ifenprodil most potently inhibited the binding in these membrane preparations due to a decrease in the density of the binding sites without significantly affecting the affinity. Ifenprodil also inhibited the binding of [3H]N-[1-(2-thienyl)cyclohexyl]piperidine as well as of [3H]MK-801 to open NMDA channels in a concentration-dependent manner at concentrations above 10 nM in membrane preparations extensively washed but not treated by a detergent, with a Hill coefficient of less than unity. Further treatment of extensively washed membrane preparations with a low concentration of Triton X-100 resulted in an almost complete abolition of [3H]MK-801 binding, and the binding was restored to the level found in membrane preparations not extensively washed following the addition of both L-glutamic acid (Glu) and Gly. Ifenprodil was effective in inhibiting [3H]MK-801 binding via reducing both initial association and dissociation rates in Triton-treated membrane preparations, irrespective of the presence of Glu and Gly added. The binding in Triton-treated membrane preparations was additionally potentiated by the polyamine spermidine in a concentration-dependent manner at concentrations above 10 microM in the presence of both Glu and Gly at maximally effective concentrations. Ifenprodil invariably diminished the abilities of these three stimulants to potentiate [3H]MK-801 binding at concentrations over 1 microM in a manner that the maximal responses each were reduced. These results suggest that ifenprodil does not interfere with the NMDA receptor complex as a specific isosteric antagonist at the polyamine domain in contrast to the prevailing view.     

Allosteric Regulation of the N-Methyl-d-Aspartate Receptor-Linked Ion Channel Complex and Effects of Ethanol in Ethanol-Withdrawal Seizure-Prone and -Resistant Mice

Abstract: The effects of ethanol, glycine, and spermidine on the specific binding of [³H]MK-801 were characterized in Triton-treated membranes prepared from the hippocampus and cortex of ethanol-withdrawal seizure-prone (WSP) and -resistant (WSR) mice. Glycine, an allosteric agonist at the NMDA receptor-linked ion channel complex, caused an increase in specific [³H]MK-801 binding to hippocampal membrane preparations. There were no significant differences in EC₅₀ values between the selected lines for the effect of glycine (WSP, 391.7 ± 48.4 nM; WSR, 313.4 ± 77 nM) in the presence of 10 µM NMDA or in the maximal response to the agonist (WSP, 1.75 ± 0.26 pmol/mg of protein; WSR, 1.67 ± 0.22 pmol/mg of protein). The EC₅₀ values for the spermidine-induced increase in [³H]MK-801 binding in membranes from hippocampus in the absence (WSP, 11.7 ± 0.83 µM; WSR, 9.98 ± 1.29 µM) or in the presence of 10 µM glycine and 10 µM NMDA (WSP, 2.1 ± 0.35 µM; WSR, 2.37 ± 0.42 µM) also did not differ. Similar results were obtained in cortical membranes. Saturation isotherms indicated that there was no difference in the density of [³H]MK-801 binding sites, or in their affinity for the radioligand, between the mouse lines. In addition, administration of ethanol by inhalation (24 h) to WSP and WSR mice did not cause an increase in the density of [³H]MK-801 binding sites, and there was no difference in the density or affinity of binding sites between the mouse lines. Withdrawal from ethanol (6 h), which causes an increase in the severity of handling-induced convulsions in WSP mice, also did not alter the binding site density or affinity for radioligand. The results suggest that the characteristics of the NMDA receptor-linked ion channel complex in the tissue preparations described here do not differ in WSP and WSR mice. Thus, genetic differences in seizure susceptibility during ethanol withdrawal can be dissociated from the total density of hippocampal or cortex NMDA receptors under activating conditions.     

NMDA and AMPA Receptors Evoke Transmitter Release from Noradrenergic Axon Terminals in the Rat Spinal Cord

N-methyl-D-aspartate (NMDA) stimulated release of [³H]noradrenaline (NA) from prelabelled rat spinal cord slices. The release was partially insensitive to tetrodotoxin (TTX) and was inhibited by the NMDA antagonist MK-801. Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) also evoked release of [³H]NA, which was enhanced by blocking AMPA receptor desensitization with cyclothiazide. AMPA-evoked release was inhibited by the non-NMDA antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)-quinoxaline (NBQX) but was not affected by TTX. NMDA and AMPA showed synergistic effects, indicating co-existence of NMDA and AMPA receptors on noradrenergic terminals. Kainate evoked [³H]NA release only at high concentrations and the release was not potentiated by blocking kainate receptor desensitization with concanavalin A. Thus, the results indicate that there are stimulatory presynaptic NMDA and AMPA receptors on noradrenergic axon terminals in the spinal cord and that they interact synergistically to evoke release of [³H]NA.     

Selective Release of Spermine and Spermidine from the Rat Striatum by N-Methyl-d-Aspartate Receptor Activation In Vivo

The intrastriatal infusion of N-methyl-D-aspartate (NMDA; 250-1,000 microM) via a dialysis cannula in anesthetized rats resulted in a marked and rapid increase in the concentrations of spermine and spermidine recovered in the dialysate. Extracellular concentrations of NMDA-released spermine and spermidine were calculated to be in the low micromolar range. Putrescine levels were not significantly affected by NMDA. The effects of NMDA (500 microM) were blocked by the previous systemic injection of MK-801 (3 mg/kg, i.p.) but were insensitive to the intrastriatal infusion of tetrodotoxin (1 microM). Intrastriatally infused kainate or quisqualate (1,000 microM) did not increase polyamine levels in the dialysate. Spermine and spermidine dialysate levels were also significantly increased by the infusion of high concentrations of K+ (greater than 100 mM), although the effects of K+ were considerably less marked than those of NMDA. Striatal polyamines are released into the extracellular space specifically by NMDA receptor activation. Because of their multiple effects on receptor- and voltage-operated cation channels, polyamines that are released by NMDA receptor activation may play an important role in phenomena already attributed to NMDA receptor stimulation, such as long-term potentiation, synaptic plasticity, and neurotoxicity.     

NMDA Receptors with Different Sensitivities to Magnesium and Ifenprodil Control the Release of [14C]Acetylcholine and [3H]Spermidine from Rat Striatal Slices in Vitro

Abstract: KCI (20–100 mM) and W-methyl-D-aspartate (NMDA, 100–1,000 μM) produce concomitant concentration-dependent increases in the release of previously captured [¹⁴C]acetylcholine and [³H]spermidine from rat striatal slices in vitro. The effects of NMDA (300μM) on striatal [¹⁴C]acetylcholine and [³H]spermidine release were blocked with equal potencies by the competitive NMDA antagonist CGP 37849, the glycine site antagonist L-689,560, and the NMDA channel blocker dizocilpine. In contrast, although NMDA-evoked [¹⁴C]acetylcholine release was antagonized by ifenprodil (IC₅₀= 5.3 μM) and MgCl₂, (IC₅₀= 200 μM), neither compound antagonized the NMDA-evoked release of [³H]spermidine at concentrations up to 100 μM (ifenprodil) or 1 mM (MgCl₂). Distinct NMDA receptor subtypes with different sensitivities to magnesium and ifenprodil therefore exist in the rat striaturn.     

On the role of NMDA receptors in blood pressure regulation in spontaneously hypertensive rats (SHR)

Summary In the present study the binding of [³H]MK-801 to glutamatergic receptors of the NMDA type was compared in spontaneously hypertensive (SHR) and normotensive (WKY) rats in various brain structures (including nucleus tractus solitarii) by quantitative receptor autoradiography. Additionally, blood pressure changes after treatment with the NMDA antagonist MK-801 were studied in both strains. There were no differences between SHR and WKY rats either in the level of [³H]MK-801 binding or in the hypertensive reaction to MK-801.     

Early isolation from conspecific song does not affect the normal developmental decline of N-methyl-D-aspartate receptor binding in an avian song nucleus

Early effects of experience on synaptic reorganization and behavior often involve activation of N-methyl-D -aspartate (NMDA) receptors. We have begun to explore the role of this glutamate-receptor subtype in the development of learned birdsong. Song learning in zebra finches occurs during a restricted period that coincides with extensive synaptic reorganization within neural regions controlling song behavior. In one brain region necessary for song learning, the lateral magnocellular nucleus of the anterior neostriatum (lMAN), NMDA receptor binding is twice as high at the onset of song learning as in adulthood. In the present study, we used quantitative autoradiography with the noncompetitive NMDA antagonist [³H]MK-801 to examine more closely the developmental decline in NMDA receptor binding within lMAN and found that it occurred gradually over the period of song learning and was not associated with a particular stage of the learning process. In addition, early isolation from conspecific song did not affect [³H]MK-801 binding in lMAN at 30, 60, or 80 days. Since behavioral studies confirmed that our isolate rearing conditions extended the sensitive period for song learning, we conclude that the normal developmental decline in overall NMDA receptor binding within lMAN does not terminate the capacity for song learning. Finally, early deafening, which prevents both stages of song learning, also did not affect [³H]MK-801 binding in lMAN at 80 days, indicating that the decline in NMDA receptor binding occurs in the absence of auditory experiences associated with song development. © 1995 John Wiley & Sons, Inc.     

Synthetic Analogues of Conantokin-G: NMDA Antagonists Acting Through a Novel Polyamine-Coupled Site

Abstract: Conantokin-G (con-G) is a 17-amino-acid polypeptide that acts as an N-methyl-d -aspartate (NMDA) antagonist. This action has been attributed to a specific but noncompetitive inhibition of the positive modulatory effects of polyamines at NMDA receptors. Con-G possesses several unusual structural features, including five γ-carboxyglutamate (Gla) residues and a high degree of helicity in aqueous media. Previous structure-activity studies indicated that one or more Gla residues are necessary for NMDA antagonist activity. Con-G analogues were synthesized with alanine (Ala), serine (Ser), and phosphoserine substituted for Gla to assess the contribution of individual Gla residues to biological activity and secondary structure. Replacement of Gla in positions 3 and 4 resulted in polypeptides with markedly reduced and no NMDA antagonist actions, respectively. In contrast, Gla residues in positions 7, 10, and 14 are not required for NMDA antagonist actions because the potencies of con-G analogues containing Ser⁷, Ser¹⁰, Ala¹⁴, and Ser¹⁴ to inhibit spermine-stimulated [³H]MK-801 binding are similar to the parent peptide. Moreover, the Ala⁷ derivative of con-G was about fourfold more potent than the parent peptide both as an inhibitor of spermine-stimulated increases in [³H]MK-801 binding (IC₅₀ of ～45 nM) and in reducing NMDA-stimulated increases in cyclic GMP levels (IC₅₀ of ～77 nM) in cerebellar granule cell cultures. Although con-G and its analogues assumed mixtures of 3₁₀ and α-helices, no clear-cut relationship was evinced between the NMDA antagonist properties of these peptides and the degree of helicity they assumed in aqueous solutions. Together with the inability of con-G to affect 5,7-dichloro[³H]kynurenic acid, [³H]CGP-39653, and [³H]ifenprodil binding, these data are consistent with the hypothesis that this polypeptide acts at a unique, polyamine-associated site on NMDA receptors.