Comparison of L-[3H]glutamate, D-[3H]aspartate, DL-[3H]AP5 and [3H]NMDA as ligands for NMDA receptors in crude postsynaptic densities from rat brain

L-[3H]Glutamate, D-[3H]aspartate, DL-[3H]2-amino-5-phosphonovalerate (AP5) and [3H]N-methyl-D-aspartate (NMDA) were evaluated as radioligands using postsynaptic density (PSD) preparations from rat brain. L[3H]Glutamate had the highest affinity and greatest percentage specific binding, followed by D-[3H]aspartate greater than DL-[3H]AP5 greater than [3H]NMDA. The pharmacological specificity of the binding of each radioligand indicated an interaction with NMDA-preferring receptors, the order of potency of displacing compounds tested being L-glutamate greater than D-aspartate greater than D-AP5 greater than DL-AP5 greater than ibotenate greater than NMDLA greater than quisqualate. For L-[3H]glutamate, the data revealed an interaction with two sites, the major one having NMDA receptor characteristics and the minor one resembling the quisqualate-preferring receptor. Against L-[3H]glutamate binding, quisqualate showed a two-component inhibition profile with an affinity of 25 microM at the NMDA site and 0.19 microM at the quisqualate site. Thus, by using several radioligands possessing activity at NMDA receptors, we confirm that an NMDA receptor binding site is present in crude PSDs. Although it is less selective than D-[3H]aspartate, DL-[3H]AP5 and [3H]NMDA, L-[3H]glutamate remains, by virtue of its high affinity, the ligand of choice for the study of NMDA receptors in preparations where such sites predominate.     

Characterization of the binding of [3H]L-689,560, an antagonist for the glycine site on the N-methyl-D-aspartate receptor, to rat brain membranes.

The binding characteristics of [3H]L-689,560 [(+/-)-4-(trans)-2-carboxy-5,7-dichloro-4-phenylaminocarbonylamino -1,2,3,4- tetrahydroquinoline], a selective antagonist for the glycine site on the N-methyl-D-aspartate receptor, have been evaluated using rat cortex/hippocampus P2 membranes. Specific [3H]L-689,560 binding was saturable, having a Kd of 2.97 nM and a Bmax of 4.15 pmol/mg of protein. The Bmax value was not significantly different from that obtained for [3H]glycine in the same membrane preparation, and L-689,560 and glycine were found to be mutually competitive. The specific binding of [3H]L-689,560 (1 nM) represented 96 +/- 0.02% (four experiments) of total binding. Association experiments at 4 degrees revealed that [3H]L-689,560 reached equilibrium in 120 min, with a t1/2 of 40 min. The dissociation of [3H]L-689,560 was slow at 4 degrees (t1/2 = 118 min), allowing the use of filtration to separate free from bound radioactivity. Both association and dissociation curves were best fitted by a double-exponential function, suggesting the presence of two components. Comparison of IC50 values obtained using [3H]glycine and [3H]L-689,560 binding for 21 glycine site ligands (including agonists, partial agonists, and antagonists, with affinities spanning 5 orders of magnitude) showed a 1:1 correlation, with a correlation coefficient of 0.97. This suggests that efficacy does not have a large influence on the affinity of glycine site ligands when an agonist or antagonist radioligand is used. Ligands for other amino acid recognition sites did not directly inhibit [3H]L-689,560 binding. [3H]L-689,560 is an improved radioligand for the glycine site that will facilitate further investigations of its properties.     

Differential binding properties of [3H]dextrorphan and [3H]MK-801 in heterologously expressed NMDA receptors

The N-methyl-D-aspartate receptor (NMDAR) antagonists: MK-801, phencyclidine and ketamine are open-channel blockers with limited clinical value due to psychotomimetic effects. Similarly, the psychotomimetic effects of the dextrorotatory opioids, dextromethorphan and its metabolite dextrorphan, derive from their NMDAR antagonist actions. Differences in the use dependency of blockade, however, suggest that the binding sites for MK-801 and dextrorphan are distinct. In the absence of exogenous glutamate and glycine, the rate of association of [3H]MK-801 with wild-type NR1-1a/NR2A receptors was considerably slower than that for [3H]dextrorphan. Glutamate individually, and in the presence of the co-agonist glycine, had substantial effects on the specific binding of [3H]MK-801, while the binding of [3H]dextrorphan was not affected. Mutation of residues N616 and A627 in the NR1 subunit had a profound effect on [3H]MK-801 binding affinity, while that of [3H]dextrorphan was unaltered. In contrast, NR1 residues, W611 and N812, were critical for specific binding of [3H]dextrorphan to NR1-1a/NR2A complexes with no corresponding influence on that of [3H]MK-801. Thus, [3H]dextrorphan and [3H]MK-801 have distinct molecular determinants for high-affinity binding. The ability of [3H]dextrorphan to bind to a closed channel, moreover, indicates that its recognition site is shallower in the ion channel domain than that of MK-801 and may be associated with the extracellular vestibule of the NMDAR.     

Quantitative autoradiographic localization of NMDA receptors in rat brain using [3H]CPP: comparison with [3H]TCP binding sites

The regional distribution of N-methyl-D-aspartate (NMDA) receptors in rat brain using the selective NMDA receptor ligand [3H]3-[+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) has been quantitated by in vitro autoradiography. [3H]CCP binding was highest in the CA1 region of the hippocampus. Relatively high levels of binding were also observed in the cerebral cortex, while moderate binding was obtained in the thalamus, striatum and granule cell layer of the cerebellum. Concurrent studies examining the phencyclidine receptor ligand [3H]1-[1-(2-thienyl)cyclohexyl]-piperidine (TCP), revealed a similar pattern of binding that correlated well with the localization of [3H]CPP-labeled NMDA receptors (r = 0.88, P less than 0.01).     

3H]CGP 61594, the first photoaffinity ligand for the glycine site of NMDA receptors

Activation of NMDA receptors requires the presence of glycine as a coagonist which binds to a site that is allosterically linked to the glutamate binding site. To identify the protein constituents of the glycine binding site in situ the photoaffinity label [3H]CGP 61594 was synthesized. In reversible binding assays using crude rat brain membranes, [3H]CGP 61594 labeled with high affinity (K(D) = 23 nM) the glycine site of the NMDA receptor. This was evident from the Scatchard analysis, the displacing potencies of various glycine site ligands and the allosteric modulation of [3H]CGP 61594 binding by ligands of the glutamate and polyamine sites. Electrophysiological experiments in a neocortical slice preparation identified CGP 61594 as a glycine antagonist. Upon UV-irradiation, a protein band of 115 kDa was specifically photolabeled by [3H]CGP 61594 in brain membrane preparations. The photolabeled protein was identified as the NR1 subunit of the NMDA receptor by NR1 subunit-specific immunoaffinity chromatography. Thus, [3H]CGP 61594 is the first photoaffinity label for the glycine site of NMDA receptors. It will serve as a tool for the identification of structural elements that are involved in the formation of the glycine binding domain of NMDA receptors in situ and will thereby complement the mutational analysis of recombinant receptors.     

Differential modulation of [3H]TCP binding to the NMDA receptor by L-glutamate and glycine

At equilibrium (4 h incubation), [3H]TCP (N-(1-[2-thienyl]-cyclohexyl)-3,4-[3H]piperidine) binding to well-washed rat forebrain membranes was enhanced in a concentration-dependent and 2-APV (2-amino-5-phosphonovaleric acid)-sensitive fashion by L-glutamate (EC50 = 0.2 microM; maximal effect +280%). L-glutamate (10 microM) increased the affinity of [3H]TCP from 78 to 28 nM, but was without effect on the maximal binding capacity. The enhancing effect of L-glutamate on [3H]TCP binding was potentiated by glycine in a concentration-dependent manner (EC50 = 50 nM, maximal effect +30% in the presence of 10 microM L-glutamate; EC50 = 2 microM, maximal effect +29% in the presence of 0.1 microM L-glutamate). This effect was strychnine-insensitive. Glycine failed to enhance [3H]TCP binding in the presence of 10 microM 2-APV. The glycine effect was due to an increase in affinity (Kd = 21 nM in the presence of 10 microM glycine and 10 microM L-glutamate); glycine did not affect the maximal binding capacity. The glycine enhancement of L-glutamate-stimulated [3H]TCP binding was not antagonised by 1 microM strychnine and was mimicked by L-serine and L-alanine but not by GABA, taurine or beta-alanine. Kinetic analysis of the glycine and L-glutamate enhancement of [3H]TCP binding indicated that the L-glutamate effect was related to a decrease in the [3H]TCP dissociation rate while the glycine effect was due to an increase in the rate of [3H]TCP association in the presence of L-glutamate.     

Effects of monovalent cations on neostriatal dopamine D2 receptors labeled with [3H]raclopride

Specific [3H]raclopride binding to dopamine D2 receptors in the rabbit neostriatum was investigated in the presence of the monovalent cations sodium, lithium and potassium. NaCl and LiCl produced concentration-dependent elevations in specific [3H]raclopride binding with sodium inducing approximately 50% more binding than lithium. Inhibition of [3H]raclopride binding by the antagonist (+)-butaclamol was unaffected by the presence of sodium or lithium in the incubation medium. In contrast, the potency of dopamine to compete with [3H]raclopride was decreased by these two ions. This effect was more pronounced in the presence of sodium than lithium and was observed for both the high- and low-affinity states of the D2 receptor. The guanine nucleotide derivative 5'-guanylylimidodiphosphate (Gpp(NH)p) reduced the potency of dopamine to compete with [3H]raclopride binding in both the presence and absence of cations; however, this effect of Gpp(NH)p was a shift of the D2 receptors from a high to a lower affinity state. Saturation binding curves in the presence of sodium or lithium were compared with experiments carried out in the absence of monovalent cations (sucrose) and demonstrated that these ions increased the affinity (judged by the equilibrium dissociation constant Kd) of the neostriatal [3H]raclopride binding sites. While NaCl produced a significantly greater change in the Kd of [3H]raclopride binding as compared to LiCl, no differences were apparent in the maximum binding capacity (Bmax) values determined in the presence of these two cations. In conclusion, the results indicate that [3H]raclopride binding to rabbit neostriatal membranes exhibits a sensitivity to monovalent cations that is consistent with the ionic regulatory properties of the D2 receptor. Moreover, although lithium and sodium influence specific [3H]raclopride binding in a similar manner, there appear to be quantitative differences between these two ions.     

[3H]5,7-Dichlorokynurenic acid,a novel radioligand labels NMDA receptor-associated glycine binding sites

A strychnine-insensitive glycine binding site is located on the N-methyl-d-aspartate (NMDA)-preferring glutamate receptor complex. Kynurenic acid analogs are antagonists at this binding site. A derivative of kynurenic acid, 5,7-dichlorokynurenic acid (5,7-DCKA) was radiolabeled with ³H and used to study antagonist binding to the glycine recognition site. This ligand ([³H]5,7-DCKA) showed high affinity (K_d = 69 nM), saturable (B_max = 14.5 pmol/mg protein) binding to rat brain membranes. A variety of agonists and antagonists inhibited the binding of [³H]5,7-DCKA and [³H]glycine in a similar fashion (r = 0.93). In addition, glutamate site agonists and antagonists exerted opposite allosteric effects on [³H]5,7-DCKA binding suggesting that [³H]5,7-DCKA preferentially binds to the agonist-activated conformation of the receptor.     

ACTH binds to [3H]MK-801-labelled rat hippocampal NMDA receptors.

The molecular mechanism of the clinical antiepileptic/antimyoclonic action of adrenocorticotrophic hormone (ACTH) is unknown. To explore the possible role of excitatory amino acid receptors, we studied the influence of ACTH and ACTH fragments in vitro on the binding of [3H]MK-801 to rat hippocampus, a region relevant to epilepsy. ACTH-(1-39), ACTH-(1-24), and ACTH-(1-17) displayed micromolar affinities compared to the nanomolar affinity of MK-801, whereas ACTH-(4-10) and four clinically used anticonvulsants were inactive. These findings are not specific for NMDA receptors but conform to the rank order of potency of ACTH fragments at other receptor binding sites.     

Modulation of [3H]MK-801 binding to NMDA receptors in vivo and in vitro

[3H]MK-801 binding in vivo was used to determine the occupancy of NMDA receptor ligands shown to allosterically modulate binding in vitro. ED(50) values (mg/kg) were obtained for the channel blockers (+)-5-methyl-10,11-dihydro-5,4-dibenzo[a,d]cyclohepten-5,10-imine maleate ((+)-MK-801, 0.2), 1-(1-phenylcyclohexyl)piperidine (phencyclidine, PCP, 1.7) and ketamine (4.4). Antagonists at the glutamate (DL-(2-carboxypiperazine-4-yl)propyl-1-phosphonate (DL-CPP, 5.7)) and glycine site (7-Chloro-4-hydroxy-3-(3-phenoxy)-phenyl-2(H)quinolinone (L-701,324, 14.1), 3R(+)cis-4-methyl-pyrrollid-2-one (L-687,414, 15.1)) inhibited [3H]MK-801 binding in vivo to varying maximum levels (69%, 103% and 45%, respectively). NR2B subunit-selective compounds acting at the ifenprodil site inhibited [3H]MK-801 in vivo by a maximum of 52-72% and gave ED(50) values (mg/kg) of: (+/-)-(1S*, 2S*)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol ((+/-)CP-101,606), 1.9; (+/-)-(3R, 4S)-3-[4-(4-fluorophenyl)-4-hydroxypiperidin-1-yl]chroman-4,7-diol ((+/-)CP-283,097), 1.8; (+/-)-(R*, S*)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperidine propanol ((+/-)Ro 25-6981), 1.0; ifenprodil, 6.0. The glycine site agonist D-serine stimulated binding to 151% of control with an ED(50) of 1.7 mg/kg. Results show that [3H]MK-801 binding in vivo may be used to measure receptor occupancy of ligands acting not only within the ion channel but also at modulatory sites on the NMDA receptor complex.     

Cocaine receptors labeled by [3H]2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane

The potent cocaine analog 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)-tropane (CFT, also designated WIN 35,428) was tritiated and evaluated as a molecular probe for cocaine receptors in caudate putamen membranes of cynomolgus monkeys. Kinetic, saturation, and competition experiments indicated that [3H]CFT, like [3H]cocaine, bound to at least two components. Association and dissociation of the radioligand at 0-4 degrees occurred in two phases; the t 1/2 for dissociation of the fast and slow components was 2.5 and 23 min, respectively. Saturation analysis revealed high and low affinity binding components with affinities (Kd) of 4.7 +/- 1.2 and 60 +/- 12 nM (means +/- SE) and densities (Bmax) of 50 +/- 18 and 290 +/- 20 pmol/g of tissue, respectively. [3H]CFT was displaced stereoselectively by the enantiomers of cocaine and by the diastereoisomers of the phenyltropane analog of cocaine. Most congeners displaced [3H]CFT fully, with shallow competition curves (nH, 0.69-0.81). In contrast, several monoamine uptake inhibitors structurally unrelated to cocaine (GBR 12909, Lu 19-005, and mazindol) displaced a maximum of about 90% specifically bound [3H]CFT, with steeper competition curves (nH, 0.89-1.3), suggesting that these drugs bind to a subpopulation of [3H]CFT-labeled sites. The rank order of potency observed in the present study is identical to the rank order of potency at binding sites labeled by [3H]cocaine: Lu 19-005 greater than mazindol greater than CFT greater than GBR 12909 greater than (-)-cocaine greater than bupropion greater than WIN 35,140 greater than (+)-cocaine. Moreover, there is a high positive correlation (r, 0.99, p less than 0.001) between the affinities of drugs at sites labeled by [3H]CFT and [3H]cocaine. The results show that [3H]CFT and [3H]cocaine bind to a similar spectrum of sites in monkey caudate putamen. Because of its higher affinity and slower dissociation rate, [3H]CFT appears to be a superior radioligand probe for these sites.     

Light microscopic autoradiographic localisation of [3H]glycine and [3H]strychnine binding sites in rat brain

Receptor autoradiography has been employed to determine the distribution of strychnine-insensitive glycine binding sites in rat brain using [3H]glycine as a ligand. The location was significantly different from and more widespread than glycine sensitive [3H]strychnine binding sites. Highest binding densities were observed in hippocampus, cortex, subiculum and amygdala followed by striatum, cerebellum and olfactory areas. Characterisation of the binding indicated that it was saturable, of high affinity, stereoselective and displaced by structurally related amino acids. The results support the existence of two glycine receptor subtypes: strychnine-sensitive and strychnine-insensitive.     

Comparison of the uptake of [3H]-gabapentin with the uptake of L-[3H]-leucine into rat brain synaptosomes.

1. Gabapentin is a novel anticonvulsant with an unknown mechanism of action. Homogenate binding studies described elsewhere have suggested that [3H]-gabapentin binds to a site in brain similar to the large neutral amino acid (LNAA) uptake site, termed system-L. 2. This study describes an investigation into the uptake of [3H]-gabapentin into a crude synaptosomal preparation from cerebral cortex of rat brain. Characterization studies showed that [3H]-gabapentin is taken up into synaptosomes by a system that is similar to that responsible for the uptake of L-[3H]-leucine. This system is sodium-independent, temperature-sensitive and requires ATP for function. 3. Kinetic studies of [3H]-gabapentin uptake produced a Michaelis constant (KM = 160 microM) similar to that observed for L-[3H]-leucine (KM = 110.3 microM). Vmax values were 837.1 pmol mg-1 protein min-1 and 2.192 nmol mg-1 protein min-1 respectively. 4. Gabapentin and L-leucine mutually inhibit their uptake. Lineweaver-Burke plots of these data demonstrate that inhibition occurs by a competitive mechanism. Further to this the Dixon transformation of the data illustrates that these two substrates share a common uptake site by the similarity between their calculated Ki and KM values (gabapentin inhibition of L-[3H]-leucine uptake: Ki = 160 microM; L-leucine inhibition of [3H]-gabapentin uptake: Ki = 262 microM). 5. Studies into the effect of gabapentin, the system-L-specific ligand 2-(-)-endoamino-bicycloheptane-2-carboxylic acid (BCH), and the system-A-specific ligand alpha-(methyl-amino)-isobutyric acid (MeAIB), on the initial rate of uptake of [3H]-glycine, L-[3H]-glutamate, L-[3H]-glutamine, and L-[3H]-leucine were performed. At 100 microM, gabapentin significantly inhibited initial rate of uptake of [3H]-glycine (29%), L-[3H]-glutamate (22%) and L-[3H]-leucine (40%). 6. Gabapentin is taken up into synaptosomes by a system similar to system-L, responsible for the uptake of large neutral amino acids. Gabapentin will also inhibit the uptake of certain excitatory amino acids in this synaptosomal preparation. The implications of these findings for the mechanism of action for gabapentin are unclear. The data presented here may suggest an intracellular site for mechanism of action for this compound. Similarly changes in levels of amino acid pools may be involved in the mechanism of gabapentin's anticonvulsant action.     

3H]ifenprodil binding to NMDA receptors in porcine hippocampal brain membranes

The present study characterized the behavioral effects of the selective alpha(2)-adrenoceptor antagonist, atipamezole, in a rat model of focal cerebral ischemia. Atipamezole (1 mg/kg, s.c.) or desipramine (5 mg/kg, i.p.), a noradrenaline reuptake blocker, was administered either as a single injection 2 days after ischemia induction or for 10 days thereafter (subacute administration). A subacute atipamezole treatment given 30 min before behavioral assessment improved performance in the limb-placing test (days 5, 7, 9, and 11) and in the foot-slip test (days 3 and 7), but not in the beam-walking test. There was no difference between experimental groups in behavioral performance following a single administration of atipamezole or following single or subacute administration of desipramine. The drug treatments did not attenuate the impairment of spatial cognitive performance of ischemic rats in the Morris water-maze test. These results suggest that repeated use-dependent release of noradrenaline by atipamezole facilitates the sensorimotor recovery following focal cerebral ischemia in rats.     

Cellular localization of [3H]-diazepam receptors

Isolated fractions enriched in bovine cortical astroglial cells or nerve endings were used to determine the receptor site for [³H]-diazepam binding. The results suggest strong enrichment in glial cells for the benzodiazepine receptor. Both the inhibition of diazepam binding by other benzodiazepines and the affinity of the glial receptor for diazepam suggest that this receptor is similar to that previously reported in crude CNS membrane fractions.     

Sigma receptors on NCB-20 hybrid neurotumor cells labeled with (+)[3H]SKF 10,047 and (+)[3H]3-PPP

(+)[3H]SKF 10,047 and (+)[3H]3-PPP label a homogeneous population of sites in NCB-20 cell membranes that apparently represent benzomorphan specific binding sites previously reported for this cell line. Their drug specificity indicates that these sites are very similar to sigma receptor binding sites labeled in brain tissues by these ligands and do not represent PCP receptors.