The flexible termini of conantokin G define its interactions with NMDA receptors

Converging lines of evidence suggest that the N-methyl-D-aspartic acid (NMDA) antagonistproperties of conantokin G (ConG) are mediated through a novel polyamine-associated site.Moreover, structural modification of the heptadecapeptide yields peptides that can eithermimic the NMDA antagonist properties of the parent peptide or produce polyamine-likeactions at NMDA receptors. We synthesized a panel of ConG fragments and evaluated theireffects using a neurochemical assay that predicts pharmacological actions at NMDA receptors.While the C-terminal tetrapeptide elicited a polyamine-like activation of [3H]MK-801 bindingwith a potency comparable to spermine, the N-terminal pentapeptide produced a marginalinhibition of spermine-enhanced [3H]MK-801 binding. These observations suggest that theparent peptide interacts with two distinct sites on NMDA receptors. In contrast, amino acidreplacements in the middle region of ConG resulted in analogues that were of comparable orgreater potency than the parent peptide. The Ala7,Tyr10 derivative is of particular interestsince it is a potent inhibitor (IC50 80 nM) of spermine-enhanced [3H]MK-801 binding, andmay thus serve as a precursor for studies designed to 125I-label putative ConG binding sites.Our observations are also consistent with the hypothesis that the termini of ConG are essentialfor an interaction with NMDA receptors, while the middle region of this peptide serves as aspacer unit. This hypothesis is consonant with spectroscopic evidence that ConG possessesa central rigid helical backbone with flexible N- and C-terminal regions. Nonetheless, ConGvariants in which the termini were connected with conformationally stabilized- or 310-helical spacers grew progressively less potent as NMDA antagonists as the structural stabilityof these peptides increased. Thus, the middle region of ConG appears to possess functionsother than providing conformational stability. These newly synthesized ConG derivatives mayserve as a basis for the design of novel peptidic or peptidomimetic agents.     

Pharmacological characterization of the N-methyl-d-aspartate (NMDA) receptor-channel in rodent and dog brain and rat spinal cord using [3H]MK-801 binding

The biochemical and pharmacological properties of [³H]MK-801 binding to the N-methyl-d-aspartate (NMDA) receptor-channel in homogenates of mouse, guinea pig and dog brain, dog cerebral cortex and rat spinal cord were determined using radioligand binding techniques. Specific [³H]MK-801 binding increased linearily with increasing tissue concentration and in general represented 80–93% of the total binding at 6–8 nM radioligand concentration. [³H]MK-801 interacted with brain and spinal homogenates with high affinity. The dissociation constants (K _d ) for all tissues studied were similar ranging between 7.9 and 11.9 nM, whereas the maximum number of binding sites (B_max) showed a wide, tissue-dependent range (0.1–6.75 pmol/mg protein). The rank order of tissue enrichment was found to be as follows: mouse brain>>dog cerebral cortex>>dog brain>> guinea pig brain>>rat spinal cord. Specific [³H]MK-801 binding in rodent and dog brain, dog cerebral cortex and rat spinal cord exhibited a similar pharmacological profile 9correlation coefficients=0.93–0.99). The rank order of potency of unlabelled compounds competing for [³H]MK-801 binding was: (+)MK-801>(–)MK-801>phencyclidine>(–)cyclazocine>>(+)cyclazocine ketamine>(+)N-allyl-N-normetazocine>(–)N-allyl-N-normetazocine>(–)pentazocine>(+)pentazocine. NMDA, Kainate, quisqualate and several other compounds failed to inhibit [³H]MK-801 binding at 100 M. In modulation studies conducted on extensively washed dog cortex membranes, Mg²⁺ ions stimulated [³H]MK-801 binding at 10 M-1 mM (EC₅₀=91.5 M) and then inhibited the binding from 1 mM to 10 mM (IC₅₀=3.1 mM). Glycine stimulated [³H]MK-801 binding at 30 nM-1 mM (EC₅₀=256 nM). In contrast, Zn²⁺ ions inhibited the binding of [³H]MK-801 binding site exhibited similar pharmacological and biochemical properties. These data appear to suggest that the pharmacological profile of the NMDA-receptor-channel is species and tissue independent.     

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.     

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.     

Regional Heterogeneity of Polyamine Effects on the N-Methyl-D-Aspartate Receptor in Rat Brain

Abstract: Polyamines have pronounced effects on N-methyl-D-aspartate (NMDA) receptors in vitro and may be important modulators of NMDA receptor activity in vivo. There is considerable regional heterogeneity in the effects of polyamines on [³H]MK-801 binding in rat brain sections. For example, spermidine enhances the binding of [³H]MK-801 to a much greater extent in the striatum than in the cortex. To further explore the basis for this regional heterogeneity, the effects of polyamines on [³H]MK-801 binding were measured in well-washed membranes prepared from frontal cortex and striatum. There was no difference in the concentration-response relationship for spermidine or the K_D for [³H]MK-801 in the presence of 75 μM spermidine, suggesting that the regional difference seen in tissue sections is due to an endogenous factor that is either removed or inactivated during the preparation of membranes. Comparison of spermidine concentration-response curves in washed and unwashed tissue sections revealed that washing selectively enhanced the E_max value in the ventromedial caudate putamen without changing the EC₅₀. This is consistent with the possibility that a noncompetitive polyamine antagonist is being removed from this region during washing. There was no regional variability in the effects of the putative inverse agonist 1, 10-diaminodecane, consistent with recent suggestions that this polyamine inhibits the NMDA receptor at a site distinct from the one at which polyamines act to enhance NMDA receptor function. Agents that modulate the redox state of the NMDA receptor did not eliminate the regional heterogeneity of polyamine effects. Furthermore, the stimulatory effect of glycine in these regions did not correlate with that of spermidine. These results suggest the existence of one or more endogenous factors that noncompetitively influence the effects of polyamines in a regionspecific manner.     

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 [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.     

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.     

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.     

Changes of [<Superscript>3</Superscript>H]Muscimol, [<Superscript>3</Superscript>H]Flunitrazepam and [<Superscript>3</Superscript>H]MK-801 Binding in Rat Brain by Prolonged Ventricular Infusion of 7-Nitroindazole

In the present study, we have investigated the effects of prolonged inhibition of nitric oxide synthase (NOS) by infusion of neuronal NOS (nNOS) inhibitor, 7-nitroindazole (7-NI), to examine modulation of NMDA and GABA_A receptor binding in rat brain. The duration of sleeping time was significantly increased by the pre-treatment with 7-NI (100 mg/kg) 30 min before pentobarbital (40 mg/kg) treatment in rats. However, the duration of pentobarbital-induced sleep was shortened by the prolonged infusion of 7-NI into cerebroventricle for 7 days. We have investigated the effect of NOS inhibitor on NMDA and GABA_A receptor binding characteristics in discrete areas of brain regions by using autoradiographic techniques. The GABA_A receptors were analyzed by quantitative autoradiography using [³H]muscimol and [³H]flunitrazepam binding, and NMDA receptor binding was analyzed by using [³H]MK-801 binding in rat brain slices. Rats were infused with 7-NI (500 pmol/10 l/ h, i.c.v.) for 7 days, through pre-implanted cannula by osmotic minipumps. The levels of [³H]muscimol were markedly elevated in cortex, caudate putamen, and thalamus while the levels of [³H]flunitrazepam binding were only elevated in cerebellum by NOS inhibitor. However, there was no change in the level of [³H]MK-801 binding except decreasing in the thalamus. These results show that the prolonged inhibition of NOS by 7-NI-infusion highly elevates [³H]muscimol binding in a region-specific manner and decreases the pentobarbital-induced sleep.     

Pathologically Activated Neuroprotection via Uncompetitive Blockade of N-Methyl-d-aspartate Receptors with Fast Off-rate by Novel Multifunctional Dimer Bis(propyl)-cognitin

Uncompetitive N-methyl-d-aspartate (NMDA) receptor antagonists with fast off-rate (UFO) may represent promising drug candidates for various neurodegenerative disorders. In this study, we report that bis(propyl)-cognitin, a novel dimeric acetylcholinesterase inhibitor and γ-aminobutyric acid subtype A receptor antagonist, is such an antagonist of NMDA receptors. In cultured rat hippocampal neurons, we demonstrated that bis(propyl)-cognitin voltage-dependently, selectively, and moderately inhibited NMDA-activated currents. The inhibitory effects of bis(propyl)-cognitin increased with the rise in NMDA and glycine concentrations. Kinetics analysis showed that the inhibition was of fast onset and offset with an off-rate time constant of 1.9 s. Molecular docking simulations showed moderate hydrophobic interaction between bis(propyl)-cognitin and the MK-801 binding region in the ion channel pore of the NMDA receptor. Bis(propyl)-cognitin was further found to compete with [³H]MK-801 with a K_i value of 0.27 μm, and the mutation of NR1(N616R) significantly reduced its inhibitory potency. Under glutamate-mediated pathological conditions, bis(propyl)-cognitin, in contrast to bis(heptyl)-cognitin, prevented excitotoxicity with increasing effectiveness against escalating levels of glutamate and much more effectively protected against middle cerebral artery occlusion-induced brain damage than did memantine. More interestingly, under NMDA receptor-mediated physiological conditions, bis(propyl)-cognitin enhanced long-term potentiation in hippocampal slices, whereas MK-801 reduced and memantine did not alter this process. These results suggest that bis(propyl)-cognitin is a UFO antagonist of NMDA receptors with moderate affinity, which may provide a pathologically activated therapy for various neurodegenerative disorders associated with NMDA receptor dysregulation.     

Role of NMDA Receptors in Pentobarbital Tolerance/Dependence

Effects of continuous pentobarbital administration on binding characteristics of [³H]MK-801 in the rat brain were examined by autoradiography. Animals were rendered tolerant to pentobarbital using i.c.v. infusion of pentobarbital (300g/10l/hr for 7 days) by osmotic minipumps and dependent by abrupt withdrawal from pentobarbital. The levels of [³H]MK-801 binding were elevated in rats 24-hr after withdrawal from pentobarbital while there were no changes except in septum and anterior ventral nuclei in tolerant rats. For assessing the role of NMDA receptor in barbiturate action, an NMDA receptor antagonist (MK-801, 2.7 femto g/10l/hr) was co-infused with pentobarbital. The pentobarbital-infused group had a shorter duration of pentobarbital-induced loss of righting reflex (sleeping time) than that of the control group, and MK-801 alone did not affect the righting reflex. However, co-infusion of MK-801 blocked hyperthermia, and prolonged the onset of convulsions induced by t-butylbicyclophosphorothionate (TBPS) in pentobarbital withdrawal rats. In addition, elevated [³⁵S]TBPS binding was significantly attenuated by co-infusion with MK-801. These results suggest the involvement of NMDA receptor up-regulation in pentobarbital withdrawal and that the development of dependence can be attenuated by the treatment of subtoxic dose of MK-801.     

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.     

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.     

Phorbol Ester Differentiates the Levels of [<Superscript>3</Superscript>H]MK-801 Binding in Rats Lines Selected for Differential Sensitivity to the Hypnotic Effects of Ethanol

These studies addressed the possible involvement between sensitivity to the hypnotic action of ethanol and function of the NMDA receptor. The studies were carried out using high-alcohol sensitive (HAS) and low-alcohol sensitive (LAS) rats, two rats having differential sensitivity to the acute hypnotic action of ethanol. The animal models were developed by a selective breeding experiment. Using a quantitative autoradiograph technique, it was demonstrated that [³H]MK-801 binding to the NMDA receptor was highest in hippocampus in both HAS and LAS rats, but significant [³H]MK-801 binding was also detected in cortex, caudate-putamen, and thalamus of HAS and LAS rats. The density of [³H]MK-801 binding was lower only in cerebellar granule layers of untreated HAS rats as compared to the same brain area in untreated LAS rats. Activation of protein kinase C (PKC) by 100 nM PDBu, increased [³H]MK-801 binding in cortex, caudate-putamen, thalamus, central gray, and cerebellum of HAS rats but activation of PKC did not influence [³H]MK-801 binding in LAS rats. These activation of PKC differentiates between [³H]MK-801 binding of HAS and LAS rats in frontal cortex (layer II-IV and cingulate), caudate-putamen, and ventral lateral thalamic nuclei. The basal level of PKC- mRNA was higher in HAS rats than that of LAS rats. These results suggest that the activation of PKC potentiates NMDA receptor function of the rat line which is more sensitive to alcohol (HAS) but does not affect [³H]MK-801 binding of alcohol resistant (LAS) rats.     

Effects of Excitatory Amino Acids on Cerebral Oxygen Consumption and Blood Flow in Rat

This investigation tested the importance of excitatory amino acids' effects on regional cerebral O₂ consumption and the concomitant changes in cerebral blood flow (rCBF) in isoflurane anesthetized rats. In the glutamate or N-methyl-D-aspartate (NMDA) groups, 10^–2 M glutamate or NMDA was topically applied to the right cortex and the left cortex was used as a control. One mg/kg dizocilpine maleate (MK-801), a non-competitive NMDA receptor antagonist, was administered (iv) to the MK-801 group and saline was given to the control group. Cortical rCBF was determined using ¹⁴C-iodoantipyrine and regional O₂ extraction was measured microspectrophotometrically. Cerebral O₂ consumption increased 77% after glutamate (contralateral cortex: 9.0 ± 1.1 ml O₂/min/100 g, glutamate treated cortex: 15.9 ± 3.9), while a 46% increase was observed with the same concentration of NMDA (contralateral cortex: 9.8 ± 2.0, NMDA treated cortex: 14.3 ± 5.5). After MK-801, the O₂ consumption decreased to 37% of the control value (control cortex: 7.0 ± 1.3, MK-801 treated cortex: 2.6 ± 3.9). MK-801 significantly decreased cerebral O₂ extraction from 7.1 ± 1.3 ml O₂/100 ml (control cortex) to 5.3 ± 0.6 (MK-801 treated cortex). However, there was no significant difference in cerebral O₂ extraction between treated and contralateral cortex in either the glutamate or NMDA groups. The increase in O₂ consumption caused by glutamate or NMDA was coupled with increased rCBF. Glutamate increased rCBF from 95 ± 5 ml/min/100 g (contralateral cortex) to 165 ± 31 (treated cortex), while NMDA increased rCBF from 114 ± 12 (contralateral cortex) to 178 ± 60 (treated cortex). MK-801 decreased O₂ consumption with a lesser decrease of rCBF. The rCBF was 48 ± 9 in the MK-801 treated cortex and 99 ± 22 in the control cortex. Some substances produced by the activation of NMDA receptors may be related to the coupling of cerebral metabolism and blood flow, since after blockade of NMDA receptors with MK-801, this relationship is uncoupled. These findings suggest that glutamatergic processes have a major effect on cerebral O₂ consumption and that this is at least partly due to NMDA receptors.     

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.     

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.     

Changes of NMDA receptor binding in spontaneously epileptic rat and parent strains

We measured the binding of [³H]3-[(±)2-carboxypiperazin-4-yl]propyl-1-phosphonic acid ([³H]CPP), a competitive ligand forN-methyl-d-aspartate (NMDA) receptors, in double mutant spontaneously epileptic rats (SER:zi/zi, tm/tm) and their parent strains, zitter rats, and tremor rats, and WTC rats (control rats from tremor rats derived from Kyoto:Wistar rats) before and after the onset of seizures in tremor rats and SER. Significantly lower [³H]CPP binding receptor density (B_max) was found in the cortex of SER and zitter rats at 12–15 weeks of age than in that of WTC rats and tremor rats, and at 4 weeks of age the Bmax in zitter rats was lower than that in the other strains. The reduction of Bmax in SER at 12–15 weeks of age may reflect a down regulation of NMDA receptors due to repetitive tonic seizures in SER.     

Long-Lasting Ibogaine Protection against NMDA-Induced Convulsions in Mice

Ibogaine, a putative antiaddictive drug, is remarkable in its apparent ability to downgrade withdrawal symptoms and drug craving for extended periods of time after a single dose. Ibogaine acts as a non-competitive NMDA receptor antagonist, while NMDA has been implicated in long lasting changes in neuronal function and in the physiological basis of drug addiction. The purpose of this study was to verify if persistent changes in NMDA receptors could be shown in vivo and in vitro after a single administration of ibogaine. The time course of ibogaine effects were examined on NMDA-induced seizures and [³H] MK-801 binding to cortical membranes in mice 30min, 24, 48, and 72h post treatment. Ibogaine (80 mg/kg, ip) was effective in inhibiting convulsions induced by NMDA at 24 and 72 hours post administration. Likewise, [³H] MK-801 binding was significantly decreased at 24 and 72 h post ibogaine. No significant differences from controls were found at 30min or 48h post ibogaine. This long lasting and complex pattern of modulation of NMDA receptors prompted by a single dose of ibogaine may be associated to its antiaddictive properties.