MPTP lesions of the nigrostriatal dopaminergic projection decrease [3H]1-[1-(2-thienyl)cyclohexyl]-piperidine binding to PCP site 2: Further evidence that PCP site 2 is associated with the biogenic amine reuptake complex

Our previous studies have demonstrated that, using membranes of guinea pig brain, [³H]1-[1-(2-thienyl)cyclohexyl]piperidine ([³H]TCP) labels not only the phencyclidine binding site associated with the NMDA receptor (PCP site 1), but also a second high affinity binding site which is associated with the biogenic amine reuptake carrier (termed PCP site 2). To test this hypothesis, the binding of [³H]GBR12935 to the dopamine transporter, and [³H]TCP binding to PCP sites 1 and 2 were measured in caudates harvested from control MPTP-treated and reserpine-treated dogs. MPTP treatment decreased dopamine levels by over 99%, decreased [³H]GBR12935 binding by over 90%, decreased [³H]TCP binding to PCP site 2 by about 50%, and had no significant effect on [³H]TCP binding to PCP site 1. These data are consistent with hypothesis that a portion of PCP site 2 is associated with dopaminergic nerve, terminals in dog caudate.     

Differential Interaction of Phencyclidine-Like Drugs with the Dopamine Uptake Complex In Vivo

The phencyclidine (PCP) derivative, [3H]N-[1-(2-benzo[b]thiophenyl)cyclohexyl]piperidine ([3H]BTCP), was used to label in vivo the dopamine uptake complex in mouse brain. The striatum accumulated the highest level of total and specific binding. Drugs which bind to the dopamine uptake site inhibited [3H]BTCP binding on an order similar to their in vitro affinities for the high-affinity [3H]BTCP site. Drugs which label selectively other monoamine uptake complexes. PCP, or sigma recognition sites were ineffective at doses up to 40 mg/kg. PCP bound to and dissociated from the dopamine uptake complex very rapidly. N-[1-(2-Thienyl)cyclohexyl]pideridine (TCP) and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) had no effect at any time or at any dose. These results imply that the pharmacological effects of PCP are due to its simultaneous interaction with the dopamine uptake complex and the PCP receptor. Conversely, TCP and MK-801, which have the same behavioral properties as PCP, exert their action only through the interaction with the PCP receptor.     

N-methyl-D-aspartate/phencyclidine receptor complex of rat forebrain: purification and biochemical characterization

The N-methyl-D-aspartate (NMDA)/phencyclidine (PCP) receptor from rat forebrain was solubilized with sodium cholate and purified by affinity chromatography on amino-PCP-agarose. A 3700-fold purification was achieved. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol revealed four major bands of Mr 67,000, 57,000, 46,000, and 33,000. [3H]Azido-PCP was irreversibly incorporated into each of these bands after UV irradiation. The dissociation constant (Kd) of [1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) binding to the purified NMDA/PCP receptor was 120 nM. The maximum specific binding (Bmax) for [3H]TCP binding was 3.3 nmol/mg of protein. The pharmacological profile of the purified receptor complex was similar to that of the membranal and soluble receptors. The binding of [3H]TCP to the purified receptor was modulated by the NMDA receptor ligands glutamate, glycine, and NMDA.     

Kinetic characterization of the phencyclidine-N-methyl-D-aspartate receptor interaction: evidence for a steric blockade of the channel

The nature of the interactions between the N-methyl-D-aspartate (NMDA) and the phencyclidine (PCP) receptors was studied in membranes obtained from rat cerebral cortex and washed repeatedly to remove endogenous excitatory amino acids. Binding of [3H]-N-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) to its receptor sites in these membranes proceeded slowly and did not reach equilibrium even after incubation for 4 h at 25 degrees C. The dissociation rate of [3H]TCP-receptor complexes was also slow (t1/2 = 128-165 min). Both association and dissociation followed first-order reaction kinetics, with similar time constants (0.0054 min-1). Addition of glutamate and glycine to the washed membranes was immediately followed by a marked increase in the rates of both association of [3H]TCP with the receptors and its dissociation from them (t1/2 = 8 min). Association now followed second-order reaction kinetics. Accelerated association of [3H]TCP with its binding sites could also be induced by NMDA or by glutamate alone, and glycine enhanced the effect. All effects of glutamate and glycine on [3H]TCP binding kinetics were blocked by the competitive NMDA receptor antagonist AP-5 [D-(-)-2-amino-5-phosphovaleric acid]. [3H]TCP-receptor interactions at equilibrium were not altered by AP-5 or by glutamate and glycine. The binding data were fitted to a model in which interactions of [3H]TCP with the receptor involve a two-step process: the outside ligand must cross a barrier (presumably a closed NMDA receptor channel in the absence of agonists). Once agonists are added, this limitation is removed (presumably because the channel is open).(ABSTRACT TRUNCATED AT 250 WORDS)     

An electrophilic affinity ligand based on (+)-MK801 distinguishes PCP site 1 from PCP site 2

The electrophilic affinity ligand, (+)-3-isothiocyanato-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine hydrochloride {(+)-MK801-NCS} was characterized for its ability to acylate phencyclidine (PCP) and sigma binding sites in vivo. Initial studies, conducted with mouse brain membranes, characterized the binding sites labeled by [³H]1-[1-(2-thienyl)cyclohexyl]piperidine ([³H]TCP). The Kd values of [³H]TCP for PCP site 1 (MK801-sensitive) and PCP site 2 (MK801-insensitive) were 12 nM and 68 nM, with Bmax values of 1442 and 734 fmol/mg protein, respectively. Mice were sacrificed 18–24 hours following intracerebroventricular administration of the acylator. The administration of (+)-MK801-NCS increased [³H]TCP binding to site 2, but not to site. 1. Although (+)-MK801-NCS decreased [³H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d; cbcyclohepten-5,10-imine maleate ([³H](+)-MK801) binding to site 1, it had no effect on [³H]TCP binding to site 1. Viewed collectively with other published data, these data support the hypothesis that PCP sites 1 and 2 are distinct binding sites, and that [³H]TCP and [³H](+)-MK801 label different domains of the PCP binding site associated with the NMDA receptor.Abbreviations ((+)-MK801) (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine - ((+)-MK801-NCS) (+)-3-isothiocyanato-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine hydrochloride - (PCP) 1-(1-phencyclohexyl)piperidine - (TCP) 1-{1-(2-thienyl)cyclohexyl}piperidine - (DTG) (2-(tllyl)guanidine - (metaphit) (1-(1-(3-isothiocyanatophenyl)-cyclohexyl)piperidine) - (NMDA) N-methyl-D-aspartate - (HEPPSO) (N-[2-hydroxyethyl]piperazine-N-[2-hydroxypropanesulfoni c acid] - ((+)-MK801-NCS) (+)-5-methyl(3-isothiocyanatophenyl)-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine - (NMDA) N-methyl-D-aspartate Address reprint requests to Dr. Rothman, Phone (410)550-1487.FAX 410-550-2997     

Multiple mode of binding of phencyclidines: high affinity association between phencyclidine receptors in rat brain and a monovalent ion-sensitive polypeptide

Two populations of phencyclidine (PCP) binding sites are shown to exist in the rat brain: a high-affinity monovalent ion-sensitive site (Kd of 10-14 nM for [3H]TCP, [3H]N-[1-(2-thienyl)cyclohexyl]piperidine), which exists in both the frontal cortex and the hippocampus, and a lower affinity site (Kd of 80-130 nM for [3H]TCP) which is found in the hippocampus but not in the frontal cortex. The nature of the interactions between the ion-binding sites and the high affinity PCP receptors depend on both ligand structure (PCP or TCP) and the ion involved (K' or Na'). The high-affinity sites are associated with an Mr 90,000 polypeptide whose labeling by [3H]azido phencyclidine is selectively inhibited by monovalent ions.     

Solubilization of Rat Brain Phencyclidine Receptors in an Active Binding Form That Is Sensitive to N-Methyl-d-Aspartate Receptor Ligands

Phencyclidine (PCP) receptors were successfully solubilized from rat forebrain membranes with 1% sodium cholate. Approximately 58% of the initial protein and 20-30% of the high-affinity PCP binding sites were solubilized. The high affinity toward PCP-like drugs, the stereo-selectivity of the sites, and the sensitivity to N-methyl-D-aspartate (NMDA) receptor ligands were preserved. Binding of the potent PCP receptor ligand N-[3H][1-(2-thienyl)cyclohexyl] piperidine ([3H]TCP) to the soluble receptors was saturable (KD = 35 nM), and PCP-like drugs inhibited [3H]TCP binding in a rank order of potency close to that observed for the membrane-bound receptors; the most potent inhibitors were TCP (Ki = 31 nM) and the anticonvulsant MK-801 (Ki = 50 nM). The NMDA receptor antagonist 2-amino-5-phosphonovaleric acid inhibited binding of [3H]TCP to the soluble receptors; glutamate or NMDA diminished this inhibition in a dose-dependent manner. Taken together, the results indicate that the soluble PCP receptor preparation contains the glutamate recognition sites and may represent a single receptor complex for PCP and NMDA, as suggested by electrophysiological data. The successful solubilization of the PCP receptors in an active binding form should now facilitate their purification.     

Differential regulation of sigma and PCP receptors after chronic administration of haloperidol and phencyclidine in mice

The existence of multiple receptor sites for the psychotomimetic agents phencyclidine (PCP) and some opiate-benzomorphans such as (+)N-allylnormetazocine ([+]SKF 10,047) in the mammalian central nervous system is well documented. These are: 1) sigma/PCP (sigma p) site, which binds both PCP and psychotomimetic opiates but not antipsychotics such as haloperidol, 2) PCP site, which selectively binds PCP analogs, and 3) sigma/haloperidol (sigma h) site, for which certain antipsychotics and (+)SKF 10,047, but not PCP analogs, display high affinity. In this study we examined the regulation of these receptor sites after chronic treatment of mice with either PCP or haloperidol. The following radiolabeled ligands were used to assess binding to the various receptor subtypes: [3H]-1-[1-[3-hydroxyphenyl)cyclohexyl]piperidine ([3H]PCP-3-OH; sigma p and PCP sites), [3H]thienyl-phencyclidine ([3H]TCP; PCP site), (+)-[3H]SKF 10,047 (sigma p and sigma h sites), and [3H]haloperidol (sigma h and D-2 dopamine receptors). Treatment of mice for 1, 7, 14, and 21 days with PCP (10 mg.kg-1.day-1) failed to induce variations in sigma p, sigma h, and PCP receptor binding. However, similar treatment with haloperidol (4 mg.kg-1.day-1) induced: 1) complete elimination of the binding to sigma h sites, 2) up-regulation of D-2 dopamine receptors, and 3) no change in sigma p and PCP receptor binding after 14 or 21 days of treatment. However, a single day of haloperidol treatment or in vitro incubation of mouse brain membranes with haloperidol failed to alter receptor binding. This study suggests that prolonged treatment of mice with haloperidol induces a loss in sigma h receptors that are presumably associated with certain psychotomimetic effects. This phenomenon is accompanied by an up-regulation of D-2 dopamine receptors.     

Characterization of [3H]desmethylimipramine binding in bovine adrenal medulla: interactions with sigma- and (or) phencyclidine-receptor ligands.

High-affinity binding sites (apparent KD 2.87 nM) for [3H]desmethylimipramine ([3H]DMI), have been demonstrated and characterized in membrane preparations of bovine adrenal medulla. The binding of [3H]DMI improved upon pretreatment of the membrane with KCl and was saturable, sodium dependent, and potently inhibited by nisoxetine and imipramine. [3H]DMI binding was also inhibited by various phencyclidine (PCP)- and (or) sigma-receptor ligands, with the following order of potency: haloperidol > rimcazole > (-)-butaclamol > dextromethorphan > MK-801 > (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ((+)-3-PPP) > PCP > N-(2-thienyl)cyclohexyl-3,4-piperidine (TCP) > (+)-SKF-10047 > (-)-SKF-10047. The inhibition produced by sigma ligands was not attributed to stimulation of either sigma 1- or sigma 2-receptors, owing to inactivity of the selective sigma-receptor ligands (+)-pentazocine and 1,3-di(2-tolyl)guanidine (DTG). The inhibition of [3H]DMI binding by sigma- and PCP-receptor ligands was not attributed to PCP1- or PCP2-receptor stimulation, owing to the decreased potency (100-fold) of these ligands in [3H]DMI assays compared with the affinity for brain PCP1 sites, and the ineffectiveness of the PCP2-ligand N-(1-(2-benzo(b)thiophenyl)cyclohexyl)piperidine (BTCP). Scatchard analysis of the inhibition by the sigma-ligands haloperidol and (+)-3-PPP, as well as the PCP1 receptor ligand MK-801, demonstrated noncompetitive interaction with the site bound by [3H]DMI. These studies indicate that bovine adrenomedullary membranes possess a specific receptor for the noradrenaline uptake inhibitor [3H]DMI, which is sensitive to allosteric modulation produced by PCP and sigma-ligands.     

Comparison of [3H] phencyclidine ([3H] PCP) and [3H] N-[1-(2-thienyl) cyclohexyl] piperidine ([3H] TCP) binding properties to rat and human brain membranes

The investigation of [3H] PCP and [3H] TCP binding properties to rat cerebrum and cerebellum resulted in the demonstration of multiple binding sites for the two drugs. In the two tissue preparations PCP had a lower affinity than TCP. In membranes from the cerebrum an equal number of high affinity binding sites were present for [3H] PCP and [3H] TCP. However, low affinity binding sites were two times more numerous for [3H] PCP than for [3H] TCP. In the cerebellum, the number of high and low affinity sites labeled by the two radioligands was identical, but the number of high affinity sites was about 7 fold lower than in the cerebrum. Taken together these results may indicate that in the cerebrum [3H] PCP labels other sites than NMDA/PCP receptor(s), maybe sigma receptors and/or the dopamine uptake complex. In human cerebral cortex samples [3H] TCP also bound to two different sites. The number of high and low affinity sites were 12 and 3 times, respectively, less abundant than in the rat cerebrum. Low affinity sites were of higher affinity (5 times) than corresponding sites in the rat brain. In the human cerebellum [3H] TCP binding parameters were identical to those measured in the same region in the rat.     

Binding studies and photoaffinity labeling identify two classes of phencyclidine receptors in rat brain

Binding and photoaffinity labeling experiments were employed in order to differentiate 1-(1-phenylcyclohexyl)piperidine (PCP) receptor sites in rat brain. Two classes of PCP receptors were characterized and localized: one class binds [3H]-N-[1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) with high affinity (Kd = 10-15 nM) and the other binds the ligand with a relatively low affinity (Kd = 80-100 nM). The two classes of sites have different patterns of distribution. Forebrain regions are characterized by high-affinity sites (hippocampus greater than frontal cortex greater than thalamus greater than olfactory bulb greater than hypothalamus), but some parts (e.g., hippocampus, hypothalamus) contain low-affinity sites as well. In the cerebellum only low-affinity sites were detected. Binding sites for [3H]PCP and for its photolabile analogue [3H]azido-PCP showed a regional distribution similar to that of the [3H]TCP sites. The neuroleptic drug haloperidol did not block binding to either the high- or the low-affinity [3H]TCP sites, whereas Ca2+ inhibited binding to both. Photoaffinity labeling of the PCP receptors with [3H]AZ-PCP indicated that five specifically labeled polypeptides of these receptors (Mr 90,000, 62,000, 49,000, 40,000, and 33,000) are unevenly distributed in the rat brain. Two of the stereoselectively labeled polypeptides (Mr 90,000 and 33,000) appear to be associated with the high- and low-affinity [3H]TCP-binding sites; the density of the Mr 90,000 polypeptide in various brain regions correlates well with the localization of the high-affinity sites, whereas the density of the Mr 33,000 polypeptide correlates best with the distribution of the low-affinity sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycine-Like Modulation of N-Methyl-d-Aspartate Receptors by a Monoclonal Antibody That Enhances Long-Term Potentiation

We have identified a monoclonal antibody, B6B21, that significantly elevates long-term potentiation when applied to CA1 pyramidal cell apical dendrites in rat hippocampal slices and characterized its binding to N-methyl-D-aspartate-receptor complexes using extensively washed hippocampal membranes. Five micrograms of affinity-purified B6B21 per 100 micrograms of membranes gave a two- to threefold elevation in N-[1-(2-thienyl)cyclohexyl]-3,4-[3H]piperidine ([3H]TCP) binding. When [3H]TCP binding was stimulated by the combined addition of maximal concentrations of glutamate, glycine, and magnesium, B6B21 no longer stimulated [3H]TCP binding. Like glycine, B6B21 enhanced the effect of N-methyl-D-aspartate and glutamate in stimulating [3H]TCP binding. Moreover, B6B21 reversed 7-chlorokynurenic acid inhibition of [3H]TCP binding, but it had no effect on the inhibition of [3H]TCP binding by D-(-)-2-amino-5-phosphonovaleric acid. B6B21 increased the rate of association and dissociation of [3H]TCP, but had no effect on equilibrium binding. Glutamate, but not glycine, however, increased B6B21-enhancement of [3H]TCP association and dissociation. B6B21 binding at strychnine-insensitive glycine sites was confirmed by direct measurement of [3H]glycine binding. These results suggest that B6B21 binds directly to N-methyl-D-aspartate receptors and displays properties similar to glycine.     

Solubilization of phencyclidine receptors from rat cerebral cortex in an active ligand binding state

A phencyclidine (PCP) receptor binding site has been solubilized in an active ligand-binding state from rat cerebral cortical membranes with sodium deoxycholate. Optimal receptor solubilization occurs at a detergent/protein ratio of 0.5 (w/w); for 5 mg protein/ml solubilized with 0.25% sodium deoxycholate, about 60% of the protein and 25% of the receptor is solubilized. Specific binding of either [3H]-N-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) or [3H]MK-801 is measurable by filtration through Sephadex G-50 columns or glass fiber filters; more than 60% of the binding activity is stable after 48 h at 4 degrees C. In the presence of detergent, [3H]TCP binding exhibits a Kd of 250 nM, a Bmax of 0.56 pmol/mg protein, and a pharmacological profile consistent with that of the membrane-bound PCP receptor, although most drugs bind with affinities 2 to 8 fold lower than in membranes. Upon reduction of detergent concentration, binding parameters approximate those for the membrane-bound receptor ([3H]TCP binding: Kd = 48 nM, Bmax = 1.13 pmol/mg protein).     

Phencyclidine and related compounds evoked [3H]dopamine release from rat mesencephalic cell cultures by a mechanism independent of the phencyclidine receptor, sigma binding site, or dopamine uptake site

At concentrations greater than or equal to 100 microM, phencyclidine (PCP), N-(1-(2-thienyl)-cyclohexyl)piperidine (TCP), and MK-801 induced [3H]dopamine release from dissociated cell cultures of rat mesencephalon. This release was Ca2+ independent and tetrodotoxin insensitive. Tetrodotoxin (2 microM) itself had no effect on spontaneous release of [3H]dopamine. [3H]Dopamine release was induced by 1,3-di(2-tolyl)guanidine, a sigma ligand, and by 4-aminopyridine (1-3 mM), a K+ channel blocker. No stereoselectivity was observed for [3H]dopamine release evoked by the dioxadrol enantiomers, dexoxadrol, and levoxadrol, or by enantiomers of N-allylnormetazocine (SKF 10,047). The selective dopamine uptake inhibitor 1-(2-[bis(4-fluorophenyl)methoxy]ethyl)-4-(3-phenylpropyl)piperazine dihydrochloride (GBR 12909) did not affect spontaneous or TCP-evoked [3H]dopamine release. Together, these data suggest that the dopamine-releasing effects of PCP-like compounds on the mesencephalic cells were not mediated by actions at the PCP receptor or sigma binding site, Ca2+, or Na+ channels, or at the high affinity dopamine uptake site. It remains conceivable that blocking actions of PCP-like compounds at voltage-regulated K+ channels may at least partly explain the response. These results are discussed in comparison with findings in intact brain.     

Neomycin Is an Agonist at a Polyamine Site on the N-Methyl-D-Aspartate Receptor

Neomycin appears as a full agonist and spermidine as a partial agonist at the site where polyamines enhance 1-[1-(2-thienyl)cyclohexyl][3H]piperidine ([3H]TCP) binding on the N-methyl-D-aspartate (NMDA) receptor. Other aminoglycosides also enhance [3H]TCP binding with efficacies roughly proportional to the number of primary amine groups. The polyamine antagonists ifenprodil and arcaine inhibit enhancement of [3H]TCP binding by spermidine or neomycin. The inhibition of [3H]TCP binding by arcaine is apparently competitively reduced by neomycin and spermidine, supporting a common site. Diethylenetriamine (previously described as a polyamine antagonist) may be a partial agonist. Enhancement by neomycin or spermidine is not additive to that of Mg2+, consistent with competition of Mg2+ and spermidine or neomycin at the site where these compounds enhance [3H]TCP binding. Polyamines also enhance the binding of the competitive antagonist 2-(2-carboxypiperazin-4-yl)[3H]propyl-1-phosphonic acid ([3H]CPP). Neomycin, which does not enhance [3H]CPP binding, inhibits the enhancement by spermidine. That this site is distinct from the site where spermidine and neomycin increase [3H]TCP binding is supported by different pharmacology. Arcaine and diethylenetriamine do not inhibit spermidine enhancement of [3H]CPP binding. Mg2+ also does not compete with the spermidine enhancement of [3H]CPP binding. Ifenprodil inhibits the spermidine enhancement of [3H]CPP binding. The data suggest two or more polyamine sites, with arcaine selective for the site that enhances [3H]TCP binding. Neomycin is an agonist at one polyamine site and antagonist to the second.     

[3H]N-[1-(2-Thienyl)cyclohexyl]-3,4-Piperidine ([3H]TCP) Binding in Human Frontal Cortex: Decreases in Alzheimer-Type Dementia

We studied [3H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine [( 3H]TCP) binding to human frontal cortex obtained at autopsy from 10 histologically normal controls and eight histopathologically verified cases with Alzheimer-type dementia (ATD). Extensively washed membrane preparations were used to minimize the effects of endogenous substances. In ATD frontal cortex, the total concentration (Bmax) of [3H]TCP binding sites was significantly reduced by 40-50%. The apparent dissociation constant (KD) values showed no significant change. The reduction in binding capacity was also apparent in Triton X-100-treated membrane preparations, and there was a linear correlation between the number of [3H]TCP binding sites and that of N-methyl-D-aspartate (NMDA)-sensitive [3H]glutamate binding sites. [3H]TCP binding sites spared in ATD brains retained the affinity for the ligand and the reactivity to NMDA, L-glutamate, and glycine. These results suggest that the primary change in NMDA receptor-ion channel complex in ATD brains is the reduction of its number, possibly reflecting the loss of neurons bearing these receptor complexes, and that the functional linkage within the receptor complexes spared in ATD brains remains normal.     

[3H]cocaine labels a binding site associated with the serotonin transporter in guinea pig brain: Allosteric modulation by paroxetine

We studied the characteristics of [³H]cocaine binding to membranes prepared from whole guinea pig brain. Cocaine binding was specific and saturable. A one-site binding model fit the data adequately: the Kd value of [³H]cocaine was 44 nM with a Bmax value of 280 fmol/mg protein. The rank order of potency for the [³H]cocaine binding site was paroxetine > clomipramine > (–)-cocaine > fluoxetine > mazindol > desipramine > GBR12909 > phencyclidine > benztropine > GBR12935 > (+)-cocaine. The IC50 values of these drugs for inhibition of [³H]cocaine binding were highly correlated with their IC50 values for inhibition of [³H]5-HT uptake into synaptosomes prepared from whole guinea pig brain. High affinity 5-HT uptake inhibitors produced dose-dependent wash-resistant (pseudoirreversible) inhibition of [³H]cocaine binding. The wash-resistant inhibition produced by paroxetine was due to an increase in the Kd of [³H]cocaine binding sites, and was accompanied by an increase in the dissociation rate, consistent with an allosteric mechanism. These studies suggest that, using membranes prepared from whole guinea pig brain, [³H]cocaine labels a binding site associated with serotonin transporter and that paroxetine and cocaine bind to different sites on the serotonin transporter.Abbreviations GBR12909 1-(2-{bis(4-fluorophenyl)methoxy}ethyl)-4-{3-phenylpropyl}piperazine - TCP 1-{1-(2-thienyl)cyclohexyl}piperidine - BTCP N-{1-(2-benzo(b)thiophenyl)cyclohexyl}piperidine - PCP 1-(1-phenylcyclohexyl)piperidine - GBR12935 (1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine) - CMI clomipramine     

Interaction of 18-methoxycoronaridine with nicotinic acetylcholine receptors in different conformational states

The interaction of 18-methoxycoronaridine (18-MC) with nicotinic acetylcholine receptors (AChRs) was compared with that for ibogaine and phencyclidine (PCP). The results established that 18-MC: (a) is more potent than ibogaine and PCP inhibiting (±)-epibatidine-induced AChR Ca²⁺ influx. The potency of 18-MC is increased after longer pre-incubation periods, which is in agreement with the enhancement of [³H]cytisine binding to resting but activatable Torpedo AChRs, (b) binds to a single site in the Torpedo AChR with high affinity and inhibits [³H]TCP binding to desensitized AChRs in a steric fashion, suggesting the existence of overlapping sites. This is supported by our docking results indicating that 18-MC interacts with a domain located between the serine (position 6′) and valine (position 13′) rings, and (c) inhibits [³H]TCP, [³H]ibogaine, and [³H]18-MC binding to desensitized AChRs with higher affinity compared to resting AChRs. This can be partially attributed to a slower dissociation rate from the desensitized AChR compared to that from the resting AChR. The enthalpic contribution is more important than the entropic contribution when 18-MC binds to the desensitized AChR compared to that for the resting AChR, and vice versa. Ibogaine analogs inhibit the AChR by interacting with a luminal domain that is shared with PCP, and by inducing desensitization.     

Modulation of glutamate-induced uncompetitive blocker binding to the NMDA receptor by temperature and by glycine

The effect of temperature on the binding of [3H]-N-[1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) to the ion channel of the N-methyl-D-aspartate (NMDA) receptors was studied in washed rat brain-cortex membranes. Raising the temperature from 5 to 33 degrees C resulted in a significant increase in the association rates of [3H]TCP binding measured in the presence of 1 microM glutamate and 1 microM glycine, but was less effective in the absence of the added agonists. No such effects of temperature on the dissociation rates of [3H]TCP-receptor complexes were observed. In the absence of agonists, neither the association nor the dissociation binding components varied with temperature, suggesting a diffusion-controlled limitation of access of the ligand to its site within the nonactivated NMDA receptor. No evidence was found for a temperature-dependent change in the density of [3H]TCP binding sites or for heterogeneity of [3H]TCP binding sites associated with the NMDA receptor, even though when approaching equilibrium the binding kinetics in the presence of glutamate and glycine deviated from an ordinary bimolecular reaction scheme. The data were fitted instead to a two-exponent binding function, comprising the sum of a fast and a slow binding component. Their corresponding time constants exhibited an increase with temperature, and the increase of each one was correlated significantly with the corresponding decrease in the equilibrium binding constant; however, there was no temperature-related change in the relative proportions of the two components, with the fast binding component (alpha) accounting for 50-70% of the site population.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization df a [3H]Glycine Recognition Site as a Modulatory Site of the N-Methyl-D-Aspartate Receptor Complex

A [3H]glycine recognition site in rat brain synaptic plasma membranes (SPM) has been identified, having characteristics expected of a modulatory component of the N-methyl-D-aspartate receptor complex. Incubation of SPM with [3H]glycine for 10 min at 2 degrees C results in saturable, reversible binding with a KD of 0.234 microM and a Bmax of 9.18 pmol/mg. A pharmacological analysis of this binding site indicates that D-serine (Ki = 0.27 microM), D-alanine (Ki = 1.02 microM), and D-cycloserine (Ki = 2.33 microM) are potent inhibitors of binding, whereas the corresponding L isomers have significantly less activity (Ki = 25.4 microM, 15.9 microM, and greater than 100 microM, respectively). Inactive at concentrations of up to 100 microM were strychnine, L-valine, N,N-dimethylglycine, aminomethylphosphonate, and aminomethylsulfonate. The active compounds were analyzed further for their ability to stimulate [3H]1-[1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) binding to Triton X-100-washed SPM. Results indicate that the affinity of the compounds for the [3H]glycine recognition site correlates with the ability of these analogues to stimulate [3H]TCP binding.