Search for novel ligands selective at a polyamine recognition domain on theN-methyl-d-aspartate receptor complex using membrane binding techniques

Among over 60 polyamine derivatives tested, onlyN-(3-aminopropyl)octanediamine and bis-(3-aminopropyl)nonanediamine (TE393) markedly inhibited [³H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) binding at equilibrium in the presence of added spermidine (SPD) in “non-washed” rat brain synaptic membranes, without affecting that in the absence of added SPD. Although TE393 significantly potentiated [³H]MK-801 binding before equilibrium in the presence ofl-glutamic acid (Glu) alone or both Glu and glycine (Gly) added in “Triton-treated” membranes, the putative polyamine antagonists 1,10-decanediamine (DA10) and arcaine invariably inhibited binding irrespective of the addition of agonists. In the absence of added SPD, in addition, TE393 markedly enhanced abilities of both Glu and Gly to potentiate [³H]MK-801 binding before equilibrium. However, TE393 induced a rightward shift of the concentration-response curve of SPD for [³H]MK-801 binding before equilibrium. Moreover, TE393 was effective in potentiating binding of an antagonist but not an agonist radioligand to the NMDA domain and in inhibiting binding of an antagonist but not an agonist radioligand to the Gly domain. The potentiation of NMDA antagonist binding by TE393 occurred in a manner sensitive to prevention by arcaine but not by DA10. These results suggest that TE393 may be a novel ligand at the polyamine domain with an ability to interact with both the NMDA and Gly recognition domains in antagonist-preferring forms.     

Radioligand binding studies reveal agmatine is a more selective antagonist for a polyamine-site on the NMDA receptor than arcaine or ifenprodil

Ifenprodil, arcaine and agmatine have all been reported to inhibit the NMDA receptor by actions at polyamine-sites, however the specific sites with which these compounds interact is unknown. Here we used radioligand binding of [3H]MK-801 to a membrane preparation from rat cerebral cortex to investigate the interactions of these compounds with the NMDA receptor complex. In the absence of exogenous polyamines, agmatine reduced [3H]MK-801 binding only at concentrations over 500 micro M, as opposed to the putative polyamine-site antagonists arcaine and ifenprodil which directly reduce ligand binding at much lower concentrations (5 micro M) in the absence of polyamines. In our studies, all three compounds significantly reduced spermidine-potentiated [3H]MK-801 binding, however agmatine was the only compound effective at concentrations below those that produced direct inhibition of [3H]MK-801 binding. Under these conditions, agmatine had a K(i)=14.8 micro M for spermidine-potentiated [3H]MK-801 binding and displayed characteristics of a competitive antagonist. Agmatine, as well as ifenprodil and arcaine, also displaced [3H]spermidine from rat cortical membranes at concentrations similar to those that were effective at reducing spermidine-potentiated [3H]MK-801 binding. In conclusion, these data suggest that agmatine reduces the potentiating effects of polyamines by competitive antagonism at a specific site on the NMDA receptor complex, and that these actions of agmatine differ from those of ifenprodil and arcaine.     

Glycine modulation of the phencyclidine binding site in mammalian brain

Neurophysiological studies have shown that glycine potentiates the NMDA response in cultured neurons by a strychnine-insensitive mechanism. Autoradiographic data have demonstrated a correspondence between strychnine-insensitive [3H]glycine binding sites and NMDA-sensitive [3H]glutamate binding sites. Here we report that in synaptic plasma membranes from rat brain, the binding of a PCP analog, [3H]TCP, was enhanced more than 5-fold by 1 microM glycine. This glycine stimulation of binding of [3H]TCP was blocked by the competitive NMDA-receptor antagonist, D-AP7. These data provide support for the hypothesis that a unique amino acid recognition site is associated with the proposed NMDA/PCP receptor complex in brain.     

Binding of [3H]desglycinyl remacemide to rat brain membranes: association with the benzomorphan attachment site of the N-methyl-D-aspartic acid receptor channel.

Desglycinyl remacemide (DGR), a biologically active metabolite of remacemide, was radiolabeled in an attempt to develop a ligand binding assay to identify its site of action. Incubation of the radioligand with membranes obtained from P2 fractions of whole rat brain revealed a single population of specific [3H]-DGR binding sites having a Kd of 290 nM and a Bmax of 1.3 pmole/mg protein. The specific binding of [3H]-DGR is most enriched in the P2 subcellular fraction and is heterogeneously distributed throughout the brain. The binding of [3H]-DGR to rat brain membranes was inhibited most potently by MK-801 and SKF-10,047. In contrast, haloperidol, and other sigma receptor-active agents, were relatively inactive at this site. These data suggest that DGR interacts with a channel blocking site on the NMDA receptor.     

Differential inhibition by ferrous ions of [3H]MK-801 binding to native N-methyl-D-aspartate channel in neonatal and adult rat brains

In vitro addition or pretreatment with >/=1 microM ferrous chloride markedly inhibited in a concentration-dependent manner [3H]dizocilpine (MK-801) binding to an open ion channel associated with the N-methyl-D-aspartate (NMDA) receptor in rat brain synaptic membranes. The addition of NMDA agonists invariably attenuated the inhibition of [3H]MK-801 binding in hippocampal synaptic membranes previously treated with ferrous chloride, without significantly affecting that in cerebellar synaptic membranes. In the absence of spermidine, ferrous chloride was more potent in inhibiting binding in the cerebral cortex and hippocampus in adult rats than in those in rats at 3 days after birth, while in the striatum [3H]MK-801 binding was 10 times more sensitive to inhibition by added ferrous chloride in neonatal rats than in adult rats. Addition of spermidine significantly attenuated the potency of ferrous chloride to inhibit binding in the cerebral cortex of adult rats, with facilitation of the inhibition in newborn rats. Moreover, spermidine significantly reduced the inhibitory potency of ferrous chloride in neonatal rat striatum, without markedly affecting that in adult rat striatum. These results suggest that ferrous ions may interfere with opening processes of the native NMDA channel through molecular mechanisms peculiar to neuronal development in a manner associated with the polyamine recognition domain.     

Supporting evidence for negative modulation by protons of an ion channel associated with theN-methyl-D-aspartate receptor complex in rat brain using ligand binding techniques

The addition ofL-glutamic acid (Glu) alone, both Glu and glycine (Gly) or Glu/Gly/spermidine (SPD) was effective in potentiating[³H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) binding before equilibrium to an ion channel associated with theN-methyl-D-aspartate (NMDA) receptor complex in brain synaptic membranes extensively washed and treated with Triton X-100. The binding dependent on Glu almost linearly increased in proportion to decreasing proton concentrations at a pH range of 6.0 to 9.0 in external incubation medium, while a Gly-dependent portion of the binding increased with decreasing proton concentrations up to a pH of 7.5 with a plateau thereafter. In contrast, the SPD-dependent binding increased in proportion to decreasing proton concentrations up to a pH of 7.0 with a gradual decline thereafter. Similar profiles were also obtained with [³H]MK-801 binding at equilibrium, with an exception that significant binding of [³H]MK-801 was detected in the absence of any added agonists. The potency of SPD to potentiate [³H]MK-801 binding before equilibrium increased in proportion to decreasing proton concentrations, with those of both Glu and Gly being unchanged. In contrast, the ability of (+)MK-801 to displace [³H]MK-801 binding at equilibrium was not significantly affected by a decrement of external proton concentrations from pH 7.5 to pH 8.5 in the presence of Glu/Gly and Glu/Gly/SPD added. However, similar changes in external proton concentrations did not similarly affect binding of several radioligands for the NMDA and Gly domains on the receptor complex. Decreasing proton concentrations were effective in exponentially potentiating binding of [³H]SPD at a pH range of 6.0 to 9.0 without virtually altering [³H]D, L-α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid binding. In addition, [³H]kainic acid binding markedly decreased with decreasing proton concentrations only in the presence of Ca²⁺ ions. These results suggest that protons negatively modulate neuronal responses mediated by the NMDA receptor ionophore complex through interference with opening mechanisms of the channel domain without disturbing association processes of the endogenous agonists with the respective recognition domains in rat brain. Moreover, possible modulation by protons of responses mediated by the kainate receptor in the presence of Ca²⁺ ions at concentrations that occur in vivo is also suggested.     

CPP, a new potent and selective NMDA antagonist. Depression of central neuron responses, affinity for [H]d-AP5 binding sites on brain membranes and anticonvulsant activity

Properties of a new potent antagonist acting selectively at N-methyl-D-aspartate (NMDA) type excitatory amino acid receptors are described. This compound, 3-((+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) is more potent than all previously reported NMDA antagonists in depressing mammalian spinal neuronal responses (cat and immature rat), in its affinity for [3H]D-AP5 (a radiolabelled NMDA antagonist) binding sites on rat brain membranes, and as an anticonvulsant in mice.     

Effect of phosphatidylserine on the binding properties of glutamate receptors in brain sections from adult and neonatal rats

The effects of phosphatidylserine (PS) on the binding properties of the AMPA (-amino-3-hydroxy-5-methylisoxazolepropionic acid) and NMDA ( N-methyl-d-aspartate) subtypes of glutamate receptors were analyzed by quantitative autoradiography of [³H]AMPA, [³H]6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and [³H]glutamate binding on at brain tissue sections. Preincubation of brain sections with PS produced an increase in [³H]AMPA binding without modifying the binding properties of [3H]CNQX, an antagonist of AMPA receptors. This effect of PS appeared to be specific for the AMPA subtype of glutamate receptors as the same treatment did not modify [³H]glutamate binding to the NMDA receptors. Furthermore, the PS-induced increase in [3H]AMPA binding was different in various brain structures, being larger in the molecular layer of the cerebellum and almost absent in the striatum. Preincubation with calcium also augmented [³H]AMPA binding, and the lack of additivity of the effects of calcium and PS on [³H]AMPA binding strongly suggests that both treatments share a common mechanism(s) for producing increased agonist binding. Finally, the effect of PS on AMPA receptor properties was markedly reduced in rat brain sections prepared from neonatal rats at a developmental stage that is normally characterized by the absence of LTP expression in certain brain regions. The present data are consistent with the hypothesis that alteration in the lipid composition of synaptic membranes may be an important mechanism for regulating AMPA receptor properties. which could be involved in producing long-lasting changes in synaptic operation.     

3H]1-[2-(2-thienyl)cyclohexyl]piperidine labels two high-affinity binding sites in human cortex: further evidence for phencyclidine binding sites associated with the biogenic amine reuptake complex

Previous work demonstrated two high-affinity PCP binding sites in guinea pig brain labeled by [3H]TCP (1-(1-[2-thienyl]cyclohexyl)piperidine): site 1 (N-methyl-D-aspartate [NMDA]-associated) and site 2 (dopamine-reuptake complex associated). The present study examined brain membranes prepared from various species, including human, for the presence of site 2, defined as binding in the presence of (+)-5-methyl-10,11-dihydro-5H-dibenzo [a, d]cyclohepten-5,10-imine maleate ((+)-MK801) minus binding in the presence of 10 microM TCP (nonspecific binding). Studies were conducted in absence of sodium which was found to be inhibitory to [3H]TCP binding. The results demonstrated detectable levels of site 2 in brain membranes of guinea pig, rabbit, pig, mouse, sheep, and human but not in the rat or chicken. Using human cortical membranes, site 2 was the predominant binding site. Detailed studies conducted with human cortical tissue showed that high-affinity dopamine (1-[2- [bis(4-fluorophenyl)-methoxy]ethyl]-4-(3-phenylpropyl)piperazine (GBR12909)], [1,2]benzo(b)thiophenylcyclo-hexylpiperidine (BTCP), and serotonin (fluoxetine) uptake inhibitors produced a wash-resistant inhibition of [3H]TCP binding to site 2, but not site 1. Preincubation of guinea pig brain membranes with BTCP was shown to produce an increase in the dissociation rate of [3H]TCP from PCP site 2. Structure activity studies with various uptake inhibitors showed that GBR12909, benztropine, fluoxetine, and BTCP have higher affinity for site 2 than for site 1. (+)-MK801, ketamine, and tiletamine were very selective for site 1, whereas dexoxadrol and TCP were moderately selective for site 1. These results suggest that human cortex possesses high-affinity PCP binding sites associated with biogenic reuptake binding sites, and that guinea pig brain, but not rat brain, may be an appropriate animal model for studying PCP site 2 in human brain.     

Differential inhibition by ferrous ions of [H]MK-801 binding to native N-methyl- -aspartate channel in neonatal and adult rat brains

In vitro addition or pretreatment with ≥1 μM ferrous chloride markedly inhibited in a concentration-dependent manner [³H]dizocilpine (MK-801) binding to an open ion channel associated with the N-methyl- -aspartate (NMDA) receptor in rat brain synaptic membranes. The addition of NMDA agonists invariably attenuated the inhibition of [³H]MK-801 binding in hippocampal synaptic membranes previously treated with ferrous chloride, without significantly affecting that in cerebellar synaptic membranes. In the absence of spermidine, ferrous chloride was more potent in inhibiting binding in the cerebral cortex and hippocampus in adult rats than in those in rats at 3 days after birth, while in the striatum [³H]MK-801 binding was 10 times more sensitive to inhibition by added ferrous chloride in neonatal rats than in adult rats. Addition of spermidine significantly attenuated the potency of ferrous chloride to inhibit binding in the cerebral cortex of adult rats, with facilitation of the inhibition in newborn rats. Moreover, spermidine significantly reduced the inhibitory potency of ferrous chloride in neonatal rat striatum, without markedly affecting that in adult rat striatum. These results suggest that ferrous ions may interfere with opening processes of the native NMDA channel through molecular mechanisms peculiar to neuronal development in a manner associated with the polyamine recognition domain.     

Preparation of rat brain membranes highly enriched with opiate kappa binding sites using site-directed acylating agents: Optimization of assay conditions

The goal of this study was to determine optimal conditions with which to measure opiate kappa binding sites in rat brain. Membranes were pretreated with mu-selective (BIT) and delta-selective (FIT) site-directed acylating agents (Rice et al., Science 220, 314-316), and the binding of [3H]bremazocine to the residual binding sites was defined as the kappa binding site. The binding of [3H]bremazocine to BIT/FIT-treated membranes was greatly increased by conducting the assay at 0 degrees C in the presence of 0.4 M NaCl. Using this 0 degrees C/NaCl assay condition, the binding of [3H]bremazocine was best described by a one-site binding model with a KD of 0.45 nM and a Bmax of 378 fmol/mg protein. Autoradiographic studies demonstrated that, using this assay condition, [3H]bremazocine densely labeled the deep layers of guinea pig cortex, an area known to be enriched with kappa binding sites. These and additional data suggest that the binding of [3H]bremazocine to the kappa binding site of rat brain is optimally assayed at 0 degrees C in the presence of 0.4 M NaCl using BIT/FIT-treated membranes and that rat brain is endowed with a high level of kappa binding sites.     

Interaction of insecticides of the pyrethroid family with specific binding sites on the voltage-dependent sodium channel from mammalian brain

Measurement of neurotoxin binding in rat brain membranes and neurotoxin-activated 22Na+ influx in neuroblastoma cells were used to define the site and mechanism of action of pyrethroids and DDT on sodium channels. A highly potent pyrethroid, RU 39568, alone enhanced the binding of [3H]batrachotoxinin A 20-alpha-benzoate up to 30 times. This effect was amplified by the action of neurotoxins such as sea anemone toxins and brevetoxin acting at different sites of the sodium channel protein in brain membranes. The ability of various pyrethroids and DDT to enhance batrachotoxin binding was related to their capacity to activate tetrodotoxin sensitive 22Na+ uptake. These results point to an allosteric mechanism of pyrethroids and DDT action involving preferential binding to active states of sodium channels which have high affinity for neurotoxins, causing persistent activation of sodium channels. Pyrethroids do not block [3H]tetrodotoxin binding, 125I-Anemonia sulcata toxin 2 binding, 125I-Tityus serrulatus toxin gamma binding at neurotoxin receptor sites 1, 3 and 4 respectively. Pyrethroids appear to act at a new neurotoxin receptor site on the sodium channel. The distribution of pyrethroid binding sites in rat brain was determined by quantitative autoradiographic procedures using the property of pyrethroids to reveal binding sites for [3H]batrachotoxinin A 20-alpha-benzoate.     

3H]dihydroergocryptine binding in rat brain.

[3H]dihydroergocryptine (DHE) appears to bind to alpha-adrenergic receptor sites in rabbit uterine membranes. We have characterized the binding of [3H]DHE to membranes prepared from rat cerebral cortex. alpha-Adrenergic agents were less potent and dopamine and serotonin, more potent, in displacing brain DHE binding than in uterus. Furthermore brain DHE binding sites demonstrated less stereospecificity for catecholamines than sites in uterus. Dopamine displaced DHE binding with about the same potency in cerebellar and cerebral cortical membranes, but was 10 times as potent in displacing DHE binding in the striatum. The binding of [3H]DHE in brain is complex and differs significantly from the rabbit uterus. There are two possible explanations for this discrepancy. [3H]DHE may bind a single site in brain with properties differing from known peripheral adrenergic receptors or DHE may bind to multiple sites in brain, sites which may or may not represent other neurotransmitter receptors.     

Presence of the binding of a variety of ligands related to ionotropic excitatory amino acid receptors in rat retina.

The binding of [3H]L-glutamic (Glu), [3H](+/-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic and [3H]D,L-(E)-2-amino-4-propyl-5-phosphono-3-pentenoic acids was detected in rat retinal membranes extensively washed and treated with a low concentration of Triton X-100, in addition to the binding of both [3H]glycine (Gly) and [3H]5,7-dichlorokynurenic acid. Furthermore, retinal membranes exhibited the binding of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne in the presence of Glu, Gly and spermidine irrespective of the incubation period employed. Rat retina also contained the binding of [3H]kainic acid as well as the binding of [3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid which was sensitive to potentiation by potassium thiocyanate. In addition, the binding of both [3H](+)-3-(hydroxyphenyl)-N-(l-propyl)piperidine and [3H]1,3-di-o-tolyl-guanidine was found in rat retinal membranes extensively washed but not treated with Triton X-100. These results give support for the proposal that the rodent retina contains subclasses of ionotropic brain excitatory amino acid receptors including the N-methyl-D-aspartate receptor ionophore complex as well as sigma sites.     

Characterization of NK-1 receptors in guinea pig and rat brain membranes with NK-1 peptides and a non-peptide antagonist.

The major finding of the present investigation is the demonstration of different NK-1 receptors in rat and guinea pig brain membranes with CP 96345 (non-peptide NK-1 antagonist) and R-544 (NK-1 peptide antagonist). We used [3H][Sar9,Met(O2)11]SP, the highly selective ligand for NK-1 receptor to compare NK-1 binding sites in rat and guinea pig brain membranes. Scatchard analysis revealed the existence of a single population of [3H][Sar9,Met(O2)11]SP binding sites in both preparations. The affinity and the maximal number of binding sites were found closely similar in rat (Kd 2 nM, Bmax = 37 fmol/mg protein) and guinea pig brain membranes (Kd = 3 nM, Bmax = 25 fmol/mg of protein). The order of potency of neurokinins to inhibit [3H][Sar9,Met(O2)11]SP binding from rat brain (SP > NKA > NKB) was found different of that observed on guinea pig brain (SP > NKB > NKA). Results obtained with [Sar9,Met(O2)11]SP, [beta Ala8]NKA(4-10) and [MePhe7]NKB suggest that selective agonists cannot discriminate between NK-1 receptors of different species. Using the non-peptide antagonist CP 96345 and the tripeptide R-544, we found that these two NK-1 antagonists discriminate between rat and guinea pig [3H][Sar9,Met(O2)11]SP binding sites.     

A novel GABAA antagonist [3H]SR 95531: microscopic analysis of binding in the rat brain and allosteric modulation by several benzodiazepine and barbiturate receptor ligands

Recent reports have demonstrated that the synthetic gamma-aminobutyric acid (GABA)-derivative, SR 95531 [2-(3'-carbethoxy-2'-propyl)-3-amino-6-paramethoxy-phenyl-pyrid azinium bromide], possesses selective GABAA antagonistic properties. Because of its potency for recognition of GABAA sites, this agent has been used to identify GABAA receptors. In the present investigation, we studied the binding of [3H]SR 95531 to tissue sections of rat brain using microscopic analysis of receptor localization. The appropriate binding conditions for defining GABAA receptors with this radioligand were obtained by determining the dissociation and association kinetics, and performing saturation and displacement studies. Using membrane preparations from whole rat brain (or brain regions representing cortex, striatum, hippocampus, midbrain-thalamus, medulla-pons and cerebellum), saturation and displacement studies were analyzed, and allosteric modulation of [3H]SR 95531 binding was examined by including several benzodiazepine and barbiturate receptor ligands in the incubation media. To assess the stereoselective properties of [3H]SR 95531 binding in rat membranes, numerous barbiturates were added during the incubation. The binding of [3H]SR 95531 was demonstrated to be saturable, specific and to bind with relatively high affinity to low-affinity GABAA sites. Scatchard analysis performed on saturation data of binding to tissue sections showed a dissociation constant (KD) of 42.4 nM and a maximum number of binding sites (Bmax) of 105.8 fmol/mg tissue. Microscopic analysis showed that intermediate to high densities of [3H]SR 95531 binding occurred in brain regions containing intermediate to high densities of low-affinity GABAA receptor sites. The binding of [3H]SR 95531 to membranes also appeared to occur at low-affinity GABAA sites. Results from competition studies demonstrated that [3H]SR 95531 is displaceable by GABAA agents and displaced preferentially by GABAA antagonists. Scatchard analysis of saturation experiments from membrane preparations indicated that the KD and Bmax from the centrifugation assay was 53.0 nM and 4.26 pmol/mg protein, respectively. Using the filtration assay, binding to membranes yielded a KD value of 45.6 nM and a Bmax of 0.77 pmol/mg protein. The allosteric modulation data demonstrated that numerous benzodiazepine and barbiturate agents inhibited [3H]SR 95531 binding and this varied according to brain region. Several barbiturates included in the incubation media exhibited a stereoselective inhibition of [3H]SR 95531 binding to whole rat brain membranes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Developmental maturation of the N-methyl- -aspartic acid receptor channel complex in postnatal rat brain

The N-methyl- -aspartate (NMDA) receptor plays an important role in developmental plasticity. Previous studies have reported differences between the NMDA receptor-channel complex in the rat pup brain and the adult brain. In the present study, modulation of the NMDA channel complex as a function of age was measured to determine when the temporal switching of the NMDA receptor from the immature form to the adult mature form takes place. [³H]MK-801 binding was measured in the rat forebrain from postnatal day 1 to day 21. Our data suggest the presence of two types of NMDA receptors — an immature type and a mature type. The immature NMDA receptor, seen during the early postnatal period (day 1–day 14) is highly sensitive to spermidine, -glutamate alone potentiates [³H]MK-801 binding, and glycine failed to potentiate an -glutamate-induced increase in [³H]MK-801 binding. During the late postnatal period (after day 14) spermidine alone did not increase [³H]MK-801 binding as potently as it did during the early postnatal period, high-affinity [³H]MK-801 binding was not seen in the presence of -glutamate alone, and -glutamate and glycine or -glutamate and spermidine or -glutamate, glycine and spermidine together, significantly increased [³H]MK-801 binding in a manner similar to that reported in the adult brain. Together, the pharmacology of the NMDA receptor during the early postnatal period differs from the adult-like receptor seen during the late postnatal period, and that in rats the apparent switching of the NMDA receptor from the immature type to the mature type takes place after the second postnatal week.     

Age-related alterations of NMDA-receptor properties in the mouse forebrain: partial restoration by chronic phosphatidylserine treatment.

The effect of aging on the properties of N-methyl-D-aspartate (NMDA) receptors in the forebrain of female NMRI mice was investigated using the antagonist [3H]MK-801 as radioligand. Compared to young (3 months) mice, aged (20 months) mice showed changes of the properties of the NMDA receptor at three different levels: (1) the density was reduced by about 35%; (2) the efficacy of L-glutamate and glycine for stimulating specific [3H]MK-801 binding was enhanced, probably because more NMDA receptor-associated ion channels are closed under baseline conditions in the aged brain; (3) the affinity of L-glutamate and glycine to its binding sites at the NMDA receptor complex was also enhanced. Chronic treatment of aged mice with phosphatidylserine (20 mg/kg, i.p., once daily) for three weeks completely normalized enhanced efficacy and affinity of L-glutamate and glycine and elevated NMDA receptor density by approximately 25%. These findings are consistent with the assumptions that deficits of the NMDA receptor are one of the mechanisms of age-related cognitive impairment and that the beneficial effects of phosphatidylserine treatment on cognitive deficits of aged individuals might be partially due to the effects of this drug on age-related NMDA receptor deficits.     

Binding characteristics of [3H]opioid ligands to active opioid binding sites solubilized from rat brain membranes by glycodeoxycholate and NaCl: the recovery of binding activity by dilution

This paper describes the binding properties of [3H]peptidergic opioid ligands to binding sites solubilized from rat brain membranes by the treatment with 0.125% sodium glycodeoxycholate and 1 M NaCl. The highest amount of the specific binding of [3H]-[D-Ala2-, Met5]enkephalinamide was obtainable when 10-fold diluted solubilized preparations were incubated in the presence of 0.1 mM MnCl2 and 100 mM NaCl at 0 degree C (on ice) for 3 h. With this assay condition, the significant binding of following [3H]opioid ligands, which have been thought to be selective for receptor types, was also observed: [3H]-[D-Ala2, MePhe4, Gly-ol5]enkephalin (mu-type), [3H]-[D-Ala2, D-Leu5]enkephalin (delta-type) and [3H]dynorphin1-9 (kappa-type). The number of binding sites in solubilized preparations for each [3H]ligand corresponded to 40-50% recovery of original membrane-bound binding sites. The Scatchard plot of the concentration-saturation binding curve showed only one class of binding sites, with a high affinity for each [3H]ligand. Apparent dissociation constants between solubilized receptors and [3H]ligands were the same as membrane-bound ones, but the ligand specificity for each receptor-type, which was examined by binding inhibition tests with unlabeled ligands, decreased. Present results indicate that heterogeneous opioid receptors in rat brain membranes seem to be transformed into less heterogeneous forms through the treatment with glycodeoxycholate and NaCl and the dilution process.     

Polyamine effects uponN-methyl-D-aspartate receptor functioning: differential alteration by glutamate and glycine site antagonists

Polyamines such as spermidine potentiate activation of theN-methyl-D-aspartate (NMDA)-type excitatory amino acid receptor. The goal of the present study was to investigate interactions between the putative polyamine binding site and previously described sites for glutamate and glycine. Binding of the high-potency PCP receptor ligand [³H]MK-801 to well-washed rat brain membranes was used as an in vitro probe of NMDA receptor activation. Spermidine concentration-response studies were performed in the absence and presence of both glutamate and glycine, with and withoutD-(−)-2-amino-5-phosphonovaleric acid (D(−)AP-5) or 7-chlorokynurenic acid (7Cl-KYN). Incubation in the presence of spermidine alone induced a 20.4-fold increase in [³H]MK-801 binding with an EC₅₀ value of 13.3 μM. The mean concentration of spermidine which induced maximal stimulation of binding was 130 μM (n = 10,S.E.M.= 24.66,range= 25–250 μM). Glutamate (10 μM) decreased the EC₅₀ value for spermidine-induced stimulation of [³H]MK-801 binding to 3.4 μM. Glycine (10 μM) did not significantly alter either maximum spermidine-induced [³H]MK-801 binding or the EC₅₀ value for spermidine-induced stimulation of [³H]MK-801 binding. Incubation in the presence of the specific glutamate antagonistD(−)AP-5 attenuated [³H]MK-801 binding in a glutamate-reversible fashion. The competitive glycine antagonist 7Cl-KYN decreased maximum spermidine-induced [³H]MK-801 binding in a glycine-reversible fashion. In addition, 7Cl-KYN increased the EC₅₀ value for spermidine-induced stimulation of [³H]MK-801 binding whileD(−)AP-5 was without effect. These findings suggest that glutamate and glycine regulate the polyamine binding site differentially. PCP-like agents induce a psychotomimetic state closely resembling schizophrenia by inhibiting NMDA receptor-mediated neurotransmission. The ability of polyamines to modulate NMDA receptor functioning suggests a potential site for pharmacological intervention.