Cortical Synaptic Membranes Research Articles

Early developmental deletion of forebrain Ank2 causes seizure-related phenotypes by reshaping the synaptic proteome

Rare genetic variants in ANK2, which encodes ankyrin-B, are associated with neurodevelopmental disorders (NDDs); however, their pathogenesis is poorly understood. We find that mice with prenatal deletion in cortical excitatory neurons and oligodendrocytes (Ank2-/-:Emx1-Cre), but not with adolescent deletion in forebrain excitatory neurons (Ank2-/-:CaMKIIα-Cre), display severe spontaneous seizures, increased mortality, hyperactivity, and social deficits. Calcium imaging of cortical slices from Ank2-/-:Emx1-Cre mice shows increased neuronal calcium event amplitude and frequency, along with network hyperexcitability and hypersynchrony. Quantitative proteomic analysis of cortical synaptic membranes reveals upregulation of dendritic spine plasticity-regulatory proteins and downregulation of intermediate filaments. Characterization of the ankyrin-B interactome identifies interactors associated with autism and epilepsy risk factors and synaptic proteins. The AMPA receptor antagonist, perampanel, restores cortical neuronal activity and partially rescues survival in Ank2-/-:Emx1-Cre mice. Our findings suggest that synaptic proteome alterations resulting from Ank2 deletion impair neuronal activity and synchrony, leading to NDDs-related behavioral impairments.

Effects of Zinc, Mercury, or Lead on [3H]MK-801 and [3H]Fluorowillardiine Binding to Rat Synaptic Membranes.

Glutamate (Glu) is considered the most important excitatory amino acid neurotransmitter in the mammalian Central Nervous System. Zinc (Zn) is co-released with Glu during synaptic transmission and interacts with Glutamate receptors and transporters. We performed binding experiments using [3H]MK-801 (NMDA), and [3H]Fluorowillardine (AMPA) as ligands to study Zn-Glutamate interactions in rat cortical synaptic membranes. We also examined the effects of mercury and lead on NMDA or AMPA receptors. Zinc at 1nM, significantly potentiates [3H]MK-801 binding. Lead inhibits [3H]MK-801 binding at micromolar concentrations. At millimolar concentrations, Hg also has a significant inhibitory effect. These effects are not reversed by Zn (1nM). Zinc displaces the [3H]FW binding curve to the right. Lead (nM) and Hg (μM) inhibit [3H]FW binding. At certain concentrations, Zn reverses the effects of these metals on [3H]FW binding. These specific interactions serve to clarify the role of Zn, Hg, and Pb in physiological and pathological conditions.

Dynamics of the mouse brain cortical synaptic proteome during postnatal brain development.

Development of the brain involves the formation and maturation of numerous synapses. This process requires prominent changes of the synaptic proteome and potentially involves thousands of different proteins at every synapse. To date the proteome analysis of synapse development has been studied sparsely. Here, we analyzed the cortical synaptic membrane proteome of juvenile postnatal days 9 (P9), P15, P21, P27, adolescent (P35) and different adult ages P70, P140 and P280 of C57Bl6/J mice. Using a quantitative proteomics workflow we quantified 1560 proteins of which 696 showed statistically significant differences over time. Synaptic proteins generally showed increased levels during maturation, whereas proteins involved in protein synthesis generally decreased in abundance. In several cases, proteins from a single functional molecular entity, e.g., subunits of the NMDA receptor, showed differences in their temporal regulation, which may reflect specific synaptic development features of connectivity, strength and plasticity. SNARE proteins, Snap 29/47 and Stx 7/8/12, showed higher expression in immature animals. Finally, we evaluated the function of Cxadr that showed high expression levels at P9 and a fast decline in expression during neuronal development. Knock down of the expression of Cxadr in cultured primary mouse neurons revealed a significant decrease in synapse density.

Selective Interactions of Valeriana officinalis Extracts and Valerenic Acid with [3H]Glutamate Binding to Rat Synaptic Membranes

Although GABA neurotransmission has been suggested as a mechanism for Valeriana officinalis effects, CNS depression can also be evoked by inhibition of ionotropic (iGluR) and metabotropic glutamate receptors (mGluR). In this study, we examined if aqueous valerian extract interacted with glutamatergic receptors. Freshly prepared aqueous valerian extract was incubated with rat cortical synaptic membranes in presence of 20 nM [3H]Glutamate. Aqueous valerian extract increased [3H]Glutamate binding from 1 × 10−7 to 1 × 10−3 mg/mL. In the presence of (2S,1′S,2′S)-2-(Carboxycyclopropyl)glycine (LCCG-I) and (2S,2′R,3′R)-2-(2′,3′-Dicarboxycyclopropyl)glycine (DCG-IV), Group II mGluR agents, valerian extract markedly decreased [3H]Glutamate binding, while (2S)-2-amino-3-(3,5-dioxo-1,2,4-oxadiazolidin-2-yl) propanoic acid) (quisqualic acid, QA), Group I mGluR agonist, increased [3H]Glutamate binding. At 0.05 mg/mL aqueous valerian extract specifically interacted with kainic acid NMDA and AMPA receptors. Valerenic acid, a marker compound for Valeriana officinalis, increased the [3H]Glutamate binding after 1.6 × 10−2 mg/mL, and at 0.008 mg/mL it interacted only with QA (Group I mGluR). The selective interactions of valerian extract and valerenic acid with Group I and Group II mGluR may represent an alternative explanation for the anxiolytic properties of this plant.

Syntheses and binding affinities of 6-nitroquipazine analogues for serotonin transporter. part 2: 4-Substituted 6-nitroquipazines

Eleven 4-substituted derivatives of 6-nitroquipazine were synthesized and evaluated for their abilities to displace [ 3H]citalopram binding to the rat cortical synaptic membranes. Among them, 4-chloro-6-nitroquipazine was shown to possess the highest binding affinity ( K i=0.03 nM) which was approximately 6 times higher than that of 6-nitroquipazine ( K i=0.17 nM) itself. In this paper, we describe the syntheses of 4-substituted 6-nitroquipazine derivatives, the results of corresponding biological evaluation and the SAR study.

Involvement of amino-acid side chains of membrane proteins in the binding of glutathione to pig cerebral cortical membranes.

Glutathione (GSH), a general antioxidant and detoxifying compound, is the most abundant thiol-containing peptide in the central nervous system. It has been earlier shown to regulate the functions of glutamate receptors and to possess specific binding sites in both neurons and glial cells. The possible involvement of disulfide bonds, cysteinyl, arginyl, lysyl, glutamyl, and aspartyl residues in the binding of tritiated GSH to specific sites in pig cerebral cortical synaptic membranes was now studied after covalent modification of membrane proteins. Treatment of synaptic membranes with the thiol-modifying reagents 5,5'-dithio-bis(2-nitrobenzoate) (DTNB) and 4,4'-dithiodipyridine (DDP) dramatically enhanced the binding of [3H]GSH in a dose-dependent manner. Dithiothreitol (DTT) alone reduced the binding, but pretreatment of the membranes with DTT potentiated the enhancing effect of DTNB. On the other hand, when the modification with DTNB was followed by treatment with DTT, the enhancement by DTNB was completely reversed. N-ethylmaleimide, a thiol alkylating agent, and phenylisothiocyanate, a thiol- and amino-group modifying compound, reduced the binding, and their effects were additive. The guanidino-modifying agent phenylglyoxal reduced the binding but the carboxyl-modifying reagent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide had no significant effect. The results indicate that cysteinyl side chains and disulfide bonds are essential in the binding of GSH to membrane proteins and that arginyl and lysyl side chains may also be directly involved in this process.

Pharmacological characterization of (10bS)-1,2,3,5,6,10b-hexahydropyrrolo[2,1-a]isoquinoline oxalate (YSL-3S) as a new alpha2-adrenoceptor antagonist.

Alpha2-adrenoceptor antagonists, which can enhance synaptic norepinephrine levels by blocking feedback inhibition processes, are potentially useful in the treatment of disease states such as depression, memory impairment, impotence and sexual dysfunction. (10bS)-1,2,3,5,6,10b-Hexahydropyrrolo[2,1-a]isoquinoline oxalate (YSL-3S) was evaluated in several in vitro biological tests to establish its pharmacological profile of activities as an alpha2-adrenoceptor antagonist. Saturation binding assay revealed that [3H]rauwolscine bound to the alpha2-adrenoceptors with a Kd value of 6.3+/-0.5 nM and a Bmax value of 251+/-39 fmol/mg protein in rat cortical synaptic membranes. Competitive binding assay showed that YSL-3S inhibited the binding of [3H]rauwolscine (1 nM) in a concentration-dependent manner with a Ki value of 98.2+/-12.1 nM while it did not inhibit the binding of [3H]cytisine (1.25 nM) to neuronal nicotinic cholinergic receptors. The Ki values of yohimbine, clonidine and norepinephrine for [3H]rauwolscine binding were 15.8+/-1.0, 40.1+/-5.9 and 40.0+/-11.5 nM, respectively. In addition, the binding affinity of YSL-3S for alpha2-adrenoceptors was higher than that of its antipode and the racemic mixture. The functional activity of YSL-3S at the presynaptic alpha2-adrenoceptors was assessed using the prostatic portion of the rat vas deferens. Clonidine inhibited field-stimulated contractions of the vas deference in a dose-dependent manner. The presence of YSL-3S or yohimbine caused a parallel, rightward the dose-response curve of clonidine in a dose-dependent manner, indicating an antagonistic action at the presynaptic alpha2-adrenoceptors. The pA2 values of yohimbine and YSL-3S were 7.66+/-0.13 and 6.64+/-0.18, respectively. The results indicate that YSL-3S acts as a competitive antagonist at presynaptic alpha2-adrenoceptors with a potency approximately ten times lower than yohimbine, but is devoid of binding affinity for neuronal nicotinic cholinergic receptors.

Specific glutathione binding sites in pig cerebral cortical synaptic membranes

Glutathione (γ-glutamylcysteinylglycine) is a neuromodulator at glutamate receptors, but may also act as a neurotransmitter at sites of its own. The Na +-independent binding of [ 3H]glutathione to pig cortical synaptic membranes was characterized here using glycine, cysteine analogs, dipeptides and glutathione derivatives, and ligands selective for known glutamate receptors. l-Glutamate, pyroglutamate, quinolinate, ( S)-5-fluorowillardiine and 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione were weak inhibitors at concentrations of 0.5 or 1 mM. d-Glutamate, l- and d-aspartate, glutamine, quisqualate, kynurenate, other N-methyl- d-aspartate receptor ligands and non- N-methyl- d-aspartate receptor ligands failed to displace [ 3H]glutathione. Except for weak inhibition by d-serine (0.5 mM), glycine and other ligands of the glycine co-activatory site in the N-methyl- d-aspartate receptors had no displacing effect. Similarly, metabotropic glutamate group I, II and III receptor agonists and antagonists and compounds acting at the glutamate uptake sites were generally inactive. Glutathione, oxidized glutathione, S-nitrosoglutathione, γ- l-glutamylcysteine, cysteinylglycine, cysteine, cysteamine and cystamine were the most potent displacers ( ic 50 values in the micromolar range), followed by dithiothreitol, glutathione sulfonate and the S-alkyl derivatives of glutathione ( S-methyl-, -ethyl-, -propyl-, -butyl- and -pentylglutathione). l-Homocysteinate and aminomethanesulfonate exhibited a moderate efficacy. Thiokynurenate, a cysteine analog and an antagonist at the N-methyl- d-aspartate receptor glycine co-activatory site, was a potent activator of glutathione binding. At 1 mM, some dipeptides also slightly activated the binding, γ- l-glutamylleucine and γ- l-glutamyl-GABA being the most effective. The specific binding sites for glutathione in brain synaptic membranes are not identical to any known excitatory amino acid receptor. The cysteinyl moiety is crucial in the binding of glutathione. The oxidation or alkylation of the cysteine thiol group reduces the binding affinity. The strong activation by thiokynurenate may indicate that the glutathione receptor protein contains a modulatory site to which co-agonists may bind and allosterically activate glutathione binding. The novel population of specific binding sites of glutathione gives rise to the possibility that they may have profound effects on synaptic functions in the mammalian central nervous system. The glutathione binding sites may be an important, and for the most part unrecognized, component in signal transduction in the brain.

Developmental regulation and effect of early undernutrition on phosphorylation of rat cortical synaptic membrane proteins

Undernutrition during early postnatal life was employed in rats by restricting the feeding time. The synaptic membrane fraction from cerebral cortex of normal and undernourished rats of various ages was prepared and endogenous protein phosphorylation studied. Many of the synaptic membrane proteins were found to be phosphorylated in an age-dependent manner. Early undernutrition affects the phosphorylation of various proteins in a complex way; most affected were 48-, 52-, 61- and 74-kDa proteins. These proteins were found to have phosphorylations mainly at tyrosine residues. This finding indicates that tyrosine phosphorylations may be affected most by early undernutrition. Adequate nutrition after early undernutrition removes most of the effects of undernutrition on synaptic protein phosphorylation. To address the question of how undernutrition may affect protein phosphorylation, we studied the lipid content of synaptic membrane fraction as it can affect membrane properties, including the fluidity. We found that undernutrition affects phosphorylation of most of the synaptic membrane proteins in the same manner in which it affects the cholesterol–phospholipid ratio of synaptic membrane and, hence, the fluidity of the membrane. This indicates that lipid biosynthesis is one of the ways by which undernutrition can affect synaptic membrane protein phosphorylation.

Characterization of [ [formula omitted]]thiocolchicoside binding sites in rat spinal cord and cerebral cortex

Thiocolchicoside, a semi-synthetic derivative of the naturally occurring compound colchicoside with a relaxant effect on skeletal muscle, has been found to displace both [ 3 H ]γ-aminobutyric acid ([ 3 H ]GABA) and [ 3 H ]strychnine binding, suggesting an interaction with both GABA and strychnine-sensitive glycine receptors. In order to gain further insight into the interaction of thiocolchicoside with these receptors, the binding of [ 3 H ]thiocolchicoside in rat spinal cord–brainstem and cortical synaptic membranes was characterized. [ 3 H ]Thiocolchicoside binding was saturable in both tissues examined. In spinal cord–brainstem membranes, we found a K D of 254±47 nM and a B max of 2.39±0.36 pmol/mg protein, whereas in cortical membranes, a K D of 176 nM and a B max of 4.20 pmol/mg protein was observed. A similar K D value was found in kinetic experiments performed in spinal cord–brainstem membranes. Heterologous displacement experiments showed that GABA and strychnine displaced the binding in a dose-dependent manner, whereas glycine was ineffective. [ 3 H ]Thiocolchicoside binding was also displaced by several GABA A receptor agonists and antagonists, but not by baclofen, flunitrazepam, guvacine, picrotoxin or by other drugs unrelated to GABA transmission. In spinal cord–brainstem, and to a lower extent, in cortical membranes, GABA and its analogs were not able to completely displace [ 3 H ]thiocolchicoside specific binding indicating that, besides GABA A receptors, thiocolchicoside can bind to another unidentified site. Unlabelled thiocolchicoside, however, completely displaced [ 3 H ]muscimol binding both in cortical and in spinal cord–brainstem synaptic membranes with an IC 50 in the low μM range. Neurosteroids were found to modulate the binding in cortical but not in spinal cord–brainstem synaptic membranes. We conclude that [ 3 H ]thiocolchicoside binding shows a pharmacological profile indicating an interaction with the GABA A receptor. The different affinities for the GABA A receptor agonists and antagonists and sensitivity to neurosteroids obtained in the cerebral cortex and in the spinal cord may indicate a preferential interaction of the compound with a subtype of the GABA A receptor. The data also indicate that [ 3 H ]thiocolchicoside binds to another site(s), whose nature remains to be elucidated.

Distribution and Fluidizing Action of Soluble and Aggregated Amyloid β-Peptide in Rat Synaptic Plasma Membranes

The effects of soluble and aggregated amyloid beta-peptide (Abeta) on cortical synaptic plasma membrane (SPM) structure were examined using small angle x-ray diffraction and fluorescence spectroscopy approaches. Electron density profiles generated from the x-ray diffraction data demonstrated that soluble and aggregated Abeta1-40 peptides associated with distinct regions of the SPM. The width of the SPM samples, including surface hydration, was 84 A at 10 degrees C. Following addition of soluble Abeta1-40, there was a broad increase in electron density in the SPM hydrocarbon core +/-0-15 A from the membrane center, and a reduction in hydrocarbon core width by 6 A. By contrast, aggregated Abeta1-40 contributed electron density to the phospholipid headgroup/hydrated surface of the SPM +/-24-37 A from the membrane center, concomitant with an increase in molecular volume in the hydrocarbon core. The SPM interactions observed for Abeta1-40 were reproduced in a brain lipid membrane system. In contrast to Abeta1-40, aggregated Abeta1-42 intercalated into the lipid bilayer hydrocarbon core +/-0-12 A from the membrane center. Fluorescence experiments showed that both soluble and aggregated Abeta1-40 significantly increased SPM bulk and protein annular fluidity. Physico-chemical interactions of Abeta with the neuronal membrane may contribute to mechanisms of neurotoxicity, independent of specific receptor binding.

Displacement of [ 3H]glutathione by cysteine and glutathione derivatives in pig cerebral cortical synaptic membranes

The pharmacology and structure-function relationships of the binding of glutathione (y-glutamylcysteinylglycine, GSI-I) to its specific receptors in the central nervous system have not been characterised. We have tested the effects of cysteine analogues, y-glutamyl dipeptides and glutathione derivatives on the Nd-independent binding of [3H]GSH to pig cortical synaptic membranes. GSH, oxidized glutathione (GSSG), S-nitrosoglutathione, y-Lglutamylcysteine, cyste-inylglycine, cysteine and cystamine were the most potent displacers (IC50 values in the micromolar range), ghttathione sulfonate and the Salkyl derivatives of glutathione (S-methyl-, -ethyl-, propyl-, -butyl-, penthylglutathione), Lhomocysteate and aminomethanesulfonate exhibited moderate efficacy, some y-glutamyl dipeptides were very weak displacers and Lcysteate, taurine and homotaurine inactive. At 1 mM y-Lglutamylleucine, y-L-glutamyl-GABA and y-Dglutamyltaurine slightly activated the binding. We conclude that the cysteinyl moiety and the free thiol group of glutathione is crucial in the binding.

Sedative and hypothermic effects induced by β‐asarone, a main component of Acorus calamus

In the present study we investigate the behavioural effects of β-asarone, the main compound of the essential oil obtained from Acorus calamus. β-asarone, when administered intraperitoneally in rats, exerts sedative and hypothermic but not analgesic effects. β-asarone, however, when administered in association with the cannabinomimetic drug WIN 55,212-2, was shown to potentiate some of the typical behavioural activities induced in animals by cannabinoids. Binding assays, performed on cortical synaptic membrane preparations using a specific cannabinoid radioligand ([3H]CP-55,940), excluded the ability of β-asarone to exert a direct agonistic activity on CB1 receptors. Hence, β-asarone cannot be considered a pure cannabinomimetic agent but it can be envisaged at least as an allosteric modulatory agent. © 1998 John Wiley & Sons, Ltd.

Ligand binding to porcine ionotropic glutamate receptors with chemically modified arginyl residues

The involvement of arginyl residues in the binding of ligands to different ionotropic glutamate receptors, to the modulatory glycine site in the N-methyl- d-aspartate (NMDA) receptor and to the NMDA-governed ionophore was assessed with porcine cortical synaptic membranes. The arginyl residues were covalently modified with phenylglyoxal. The binding and protection experiments showed that arginine residue(s) are directly involved in the binding of ligands to the agonist sites of all ionotropic glutamate receptors and to the modulatory glycine site but not to the ion channel in the NMDA receptor complex.

Role of histidyl residues in the binding of ligands to the porcine N-methyl- d-aspartate receptor

Possible involvement of histidyl residues in the binding of ligands to ionotropic glutamate receptors and to modulatory sites on the N-methyl- d-aspartate (NMDA) receptor was assessed in porcine cortical synaptic plasma membranes after covalent modification with diethyl pyrocarbonate (DEPC). Binding of [ 3H]glutamate to the NMDA sites was enhanced but to the 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and kainate receptors unaffected by 1 and 5 mM DEPC. Binding of 3-[(R)-carboxypiperazin-4-yl]-[1,2– 3H]propyl-1-phosphonate ([ 3H]CPP) was reduced in a dose-dependent manner by DEPC and the activation of binding by 1-hydroxy-3-amino-2-pyrrolidone (HA-966) blocked by 10 mM DEPC. DEPC reduced the strychnine-insensitive binding of [ 3H]glycine and the glycine- and glutamate-activated binding of [ 3H]dizocilpine. Protection experiments indicated that histidyl residues are directly involved in the binding of glycine (but not HA-966) and allosterically modulate the binding of glutamate, CPP and dizocilpine. The results corroborate the existence of agonist- and antagonist-preferring sites or conformational states of the NMDA receptors.

Interaction of the N-Methyl—aspartate Receptor Complex with a Novel Synapse-associated Protein, SAP102

Ionotropic glutamate receptors are known to cluster at high concentration on the postsynaptic membrane of excitatory synapses, but the mechanism by which this occurs is poorly understood. Studies on the neuromuscular junction and central inhibitory synapses suggest that clustering of neurotransmitter receptors requires its interaction with a cytoplasmic protein. Recently, in vitro studies have shown that members of the N-methyl--aspartate (NMDA) class of glutamate receptors interact with a synapse-associated protein, SAP90 (PSD-95). However, evidence for the in vivo interaction of NMDA receptors with SAPs is still lacking. In the present study, we demonstrate the specific interaction between SAP102, a novel synapse-associated protein, and the NMDA receptor complex from the rat cortical synaptic plasma membranes using co-immunoprecipitation techniques. No association was observed between SAP102 and GluR1, a member of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate class of glutamate receptors. To identify the domain on the NMDA receptor responsible for this interaction, we constructed hexahistidine fusion proteins from different regions of the NR1a and NR2 subunits of the NMDA receptor. Immunoblot overlay experiments showed that while the C-terminal domain of the NR2 subunit displayed strong binding, the NR1a intracellular C-terminal tail did not interact with SAP102. The site of interaction was more precisely located to the last 20 amino acids of the NR2 subunit as indicated by the interaction of the synthetic peptide with SAP102. In summary, we demonstrate here for the first time an in vivo interaction between the native NMDA receptor complex and a synapse-associated protein. These results suggest that SAP102 may play an important role in NMDA receptor clustering and immobilization at excitatory synapses.

Zinc Deficiency Decreases the Concentration of N-Methyl-D-Aspartate Receptors in Guinea Pig Cortical Synaptic Membranes

Zinc deficiency in guinea pigs decreases glutamate-stimulated calcium uptake in cortical synaptosomes. Glutamate not only stimulates calcium uptake but also potentiates the binding of the drug dizocilpine (MK-801) to an internal site of the N-methyl-D-aspartate receptor/calcium channel, a subtype of the glutamate receptor. The purpose of this study was to determine whether the effect of zinc deficiency on calcium uptake by glutamate-stimulated synaptosomes is related to N-methyl-D-aspartate receptor number or function, as measured by MK-801 binding. Immature guinea pigs consumed a low zinc (<1 mg/kg) diet ad libitum or an adequate zinc (100 mg/kg) diet, either ad libitum or restricted to maintain weight similar to that of the low zinc animals. Binding of MK-801 to cortical membranes was measured first in the presence of saturating concentrations of glutamate or N-methyl-D-aspartate in combination with glycine. Zinc deficiency significantly reduced the concentration of MK-801 binding sites (20%) regardless of the potentiating agonist used, but had no effect on binding affinity. The binding of MK-801 in response to 1, 10 and 100 µmol/L glycine, in the presence of 100 nmol/L glutamate, was then measured and found to be significantly reduced (12%). The results suggest that zinc deficiency decreases the number of functional N-methyl-D-aspartate receptor/channels in cortical membranes, probably because of impaired channel opening.

Effect of Anions on [3H]CGP 39653 Binding to NMDA Receptors in Brain

We previously demonstrated biphasic effects of salts on the binding of [3H]CGP 39653, a ligand for NMDA recognition sites, in well-washed membranes from rat cerebral cortex. Low concentrations of the salts increased binding, and higher concentrations produced inhibition. The increase in binding was due to conversion of NMDA recognition sites from a low- to a high-affinity state by cations (Soc. Neurosci. Abstr. 19:1784). In the present study, we evaluated the effects of sodium salts of different anions on [3H]CGP 39653 binding to NMDA recognition sites under conditions in which all binding sites were transformed from the low- to the high-affinity state. In well-washed rat cortical synaptic membranes, all anions studied inhibited [3H]CGP 39653 binding with different potencies. The range of IC50 values was from 2 mM to 300 mM. The order of potencies was as follows: citrate > EDTA > phosphate > carbonate > iodinate > nitrate > bromide > chloride > acetate > fluoride. Of the anions tested, HEPES had the lowest potency (IC50 > 400 mM). These findings have implications for the design of in vitro binding assays. They also suggest that in vivo conditions are complicated by the presence of endogenous anions which can alter interactions of ligands with NMDA recognition sites.

Differential up-regulation of voltage-dependent na + channels induced by phenytoin in brains of genetically seizure-susceptible (el) and control (ddy) mic

We investigated the effect of in vivo administration of an antiepileptic drug, phenytoin, on the saxitoxin binding capacity of receptor site 1 of the Na + channel α-subunit, and the expression activity of the channel messenger RNA in epileptic El mouse brains, as compared with parental ddY mice. Subchronic treatment with phenytoin (25 mg/kg per day) for 14 days increased the [ 3H]saxitoxin binding to brain-derived synaptic membranes of both El and control ddY mice in a time dependent manner. This increase plateaued at 21 ± 4% in El mice and 28 ± 3% in ddY control mice after administration of phenytoin for seven days. After cessation of treatment with phenytoin, [ 3H]saxitoxin binding capacity returned to the basal level within two weeks in both ddY and El brains. Scatchard plot analysis revealed that the phenytoin treatment caused a 20–30% increase in maximum binding capacity of [ 3H]saxitoxin binding without any change in equilibrium dissociation constant in the brain cortical synaptic membranes of both epileptic El and control ddY mice. A single injection of phenytoin (25 mg/kg) elevated the level of Na + channel messenger RNA within l h in ddY mouse brains. The increase in Na + channel messenger RNA reached a peak (about 80% increase) after 5 h of phenytoin administration in a concentration-dependent manner (6.25–50 mg/kg). On the other hand, in El mouse brains, Na + channel messenger RNA was not elevated until more than 5 h after phenytoin injection, and was increased by only about 33%. These results indicate that subchronic administration of phenytoin enhanced the expression of voltage-dependent Na + channel messenger RNA, which was followed by an increase in the number of Na + channels in both El and ddY mice. However, a single injection of phenytoin revealed a delayed expression of Na + channel messenger RNA in El mouse brain, compared to ddY brains, suggesting that the molecular basis for up-regulation of Na + channels in epileptic El brains is abnormal.

125I‐Ifenprodil: Synthesis and Characterization of Binding to a Polyamine‐Sensitive Site in Cerebral Cortical Membranes

The characteristics of binding sites in rat cerebral cortical synaptic membranes labeled by 125I-ifenprodil, a noncompetitive NMDA receptor antagonist, are described. 125I-ifenprodil was synthesized using Na125I in the presence of chloramine-T and purified by paper chromatography. Binding of the 125I-ligand was optimal at pH 7.7 in 5 mM Tris.HCl buffer. Equilibrium binding of 125I-ifenprodil was displaced by spermine (1 mM) but not by ifenprodil or its analogue, SL 82.0715 (both 16.7 microM). Zn2+, Ca2+, and Mg2+ inhibited specific binding of 125I-ifenprodil in a concentration-dependent manner, with IC50 values of 0.11, 1.1, and 1.7 mM, respectively. The dissociation constant (KD) for unlabeled ifenprodil determined by saturation binding was 205 nM. Scatchard plots of saturation data appeared curvilinear but were best described by a single-binding-site model (Hill coefficient = 0.95), with a density of binding sites (Bmax) of 141 pmol/mg of protein. Binding of 125I-ifenprodil was inhibited by polyamines, with a rank potency order of spermine > spermidine > putrescine = 1,3-diaminopropane. The pattern of inhibition produced by spermidine was apparently competitive. Ifenprodil congeners also fully inhibited polyamine-sensitive binding of 125I-ifenprodil, with a rank potency order of ifenprodil > SL 82.0715 = tibalosine > nylidrin = isoxsuprine. It was found that sigma/antitussive agents partially inhibited specific binding, but inclusion of the sigma drug GBR 12909 had little effect on the binding of 125I-ifenprodil, suggesting this site was not involved. The binding site labeled by 125I-ifenprodil is polyamine sensitive, has a discrete pharmacological profile, and apparently is unrelated to the sigma site.