Ropizine concurrently enhances and inhibits [3H]dextromethorphan binding to different structures of the guinea pig brain: autoradiographic evidence for multiple binding sites

Ropizine (10 microM) produces a simultaneous enhancement and inhibition of [3H]dextromethorphan (DM) high-affinity binding to different areas of the guinea pig brain. These results imply that there are two distinct types of high-affinity [3H]DM binding sites, which are present in variable proportions in different brain structures. The ropizine-enhanced [3H]DM binding type was preferentially inhibited by (+)-pentazocine. This is consistent with the presumption that the (+)-pentazocine-sensitive site is identical with the common site for DM and 3-(-3-Hydroxyphenyl)-N-(1-propyl)piperidine ((+)-3-PPP). The second binding type, which is inhibited by ropizine and is not so sensitive to (+)-pentazocine, has not been fully characterized. This study demonstrates that the biphasic effects of ropizine are due, at least in part, to the effects of ropizine on two different types of [3H]DM binding sites. However, this study does not rule out that common DM/(+)-3-PPP site also might be inhibited by higher concentrations of ropizine.     

Computer-assisted analysis of dextromethorphan and (+)-3-(-3-hydroxyphenyl)-N-(1-propyl)piperidine binding sites in rat brain. Allosteric effects of ropizine

Computer-assisted analysis of self- and cross-displacement studies between dextromethorphan (DM) and (+)-3-(3-hydroxyphenyl)-N-(1-propyl) piperidine ((+)-3-PPP) demonstrated in the rat brain the existence of two high-affinity and one low-affinity binding site for each ligand. One high-affinity site is the common DM1/sigma 1 site, the affinity of which is allosterically increased 4 to 5-fold by 10 microM ropizine. The Kd values of the DM1/sigma 1 for DM and (+)-3-PPP are 17 and 11 nM respectively. DM binds to the second high-affinity site (R2) with a Kd of 15 nM; this site has low affinity for (+)-3-PPP. Conversely, (+)-3-PPP binds with high affinity (Kd 53 nM) to another site (R3), that has low-affinity for DM. The Bmax of the common DM1/sigma 1 site in the rat is about ten times smaller than that in the guinea pig. Thus, extreme caution should be exercised in extrapolating from one species to another. Since DM and most sigma ligands bind to more than one site, not all of which are shared, it is important not to attribute the complex pharmacological effects of these ligands to a single hypothetical receptor.     

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.     

Pharmacological specificity of some psychotomimetic and antipsychotic agents for the sigma and PCP binding sites

The pharmacological specificity of representative psychotomimetic agents such as phencyclidine (PCP) analogs, opiate benzomorphans and several antipsychotic agents was assessed for the sigma and PCP binding sites. In a series of binding experiments, in rat brain membranes, sigma and PCP binding sites were labeled with [3H]-1-[1-(3-hydroxyphenyl)cyclohexyl]piperidine [( 3H]PCP-3-OH), (+) [3H]-N-allylnormetazocine [(+) [3H]SKF 10047] and (+) [3H]-3-[3-hydroxy-phenyl]-N-(1-propyl)piperidine [(+) [3H]-3-PPP]. PCP analogs inhibit potently high affinity [3H]PCP-3-OH binding and (+) [3H]SKF 10047 binding, moderately the low affinity binding component of [3H]PCP-3-OH and very weakly (+) [3H]-3-PPP binding. (+)SKF 10047 and cyclazocine are potent to moderate inhibitors of (+) [3H]SKF 10047, high affinity [3H]PCP-3-OH and (+) [3H]-3-PPP binding, but extremely weak inhibitors of low affinity [3H]PCP-3-OH binding. The antipsychotic agents display high affinity for (+) [3H]-3-PPP binding sites, moderate affinity for (+) [3H]SKF 10047 sites and have no effect on either the high or low affinity [3H]PCP-3-OH binding. The present data further support the existence of multiple sigma and PCP binding sites.     

Regulation of σ-Receptors: High- and Low-Affinity Agonist States, GTP Shifts, and Up-Regulation by Rimcazole and 1,3-Di(2-Tolyl)guanidine

The regulation of the central sigma-binding site was investigated using both in vitro and in vivo manipulations in conjunction with radioligand binding. The displacement of the binding of R(+)-[3H]3-[3-hydroxyphenyl]-N-(1-propyl)piperidine [R(+)-[3H]3-PPP] to cortical homogenates by a range of drugs was consistent with the site labelled being a sigma-receptor. (+)-SKF 10,047, (-)-SKF 10,047, (+/-)-cyclazocine, phencyclidine, and dexoxadrol displaced R(+)-[3H]3-PPP with pseudo-Hill coefficients of less than 1. Further analysis employing nonlinear curve fitting techniques demonstrated that displacement data for these compounds were described better by a model whereby R(+)-[3H]3-PPP was displaced from two discrete sites; approximately 65% of the total sites were in the high-affinity state. In the presence of 10 mM Mg2+ and 0.3 mM GTP, displacement curves for (+)-SKF 10,047 and (+/-)-cyclazocine were shifted to the right. These findings were due to the shift of some 15% of the high-affinity binding sites to a low-affinity state. Saturation experiments revealed that 0.3 mM GTP acted competitively to decrease the affinity of R(+)-[3H]3-PPP for the sigma sites. The sigma-binding site was thus likely to be linked to a guanine nucleotide regulatory (G) protein. Thus sigma drugs could be subdivided on the basis of their GTP sensitivity and pseudo-Hill coefficients, and by analogy with other receptors R(+)-3-PPP, (+)-SKF 10,047, and (+/-)-cyclazocine, may be putative sigma-agonists. 1,3-Di(2-tolyl)guanidine (DTG), rimcazole, and haloperidol displaced R(+)-[3H]3-PPP with pseudo-Hill coefficients of approximately unity and thus may be sigma-antagonists. Subchronic treatment with rimcazole was characterized by slight sedation and a concomitant up-regulation, with a decrease in the affinity, of sigma-binding sites. The schedule of rimcazole also increased dopamine turnover in the nucleus accumbens; both the concentration of 3,4-dihydroxyphenylacetic acid (DOPAC) and the DOPAC/dopamine ratio were elevated. DTG produced similar alterations to the binding parameters of the sigma-binding site; however, changes were not observed in general behavior or accumbal dopamine turnover. sigma-Receptors are likely to be linked to a G protein and are functionally involved in the CNS.     

Discrimination of Multiple [3H]5-Hydroxytryptamine Binding Sites by the Neuroleptic Spiperone in Rat Brain

Certain neuroleptic drugs, such as spiperone and (+) butaclamol, can discriminate between two populations of [³H]5-hydroxytryptamine ([³H]5-HT) binding sites in rat brain. The butyrophenone neuroleptic spiperone shows the greatest selectivity for these two binding sites, having at least a 3000-fold difference between its dissociation constants (2-12 nM versus 35,000 nM) for the high- and low-affinity sites, respectively. Inhibition of [³H]5-HT binding by spiperone in rat frontal cortex and corpus striatum yields distinctly biphasic inhibition curves with Hill slopes significantly less than unity. Results from nonlinear regression analysis of these inhibition studies were consistent with a two-site model in each brain region. In the frontal cortex the high-affinity neuroleptic sites comprised about 60% of the total [^3/H]5-HT binding sites whereas in the corpus striatum they accounted for only 20% of the sites. Furthermore, saturation studies of [³H]5-HT binding assayed in the absence or presence of 1 μM-spiperone (a concentration that completely blocks the high-affinity site while having minimal activity at the low-affinity site) reveal a parallel shift in the Scatchard plot with no change in the dissociation constant of [³H]5-HT, but a significant decrease (64% in frontal cortex or 28% in corpus striatum) in the number of specific binding sites. These observations are consistent with the existence of at least two populations of [³H]5-HT binding sites having a differential regional distribution in rat brain.     

Effect of some potent adenosine uptake inhibitors on benzodiazepine binding in the CNS

A series of nucleoside transport inhibitors has been tested for their ability to displace [³H]diazepam binding to CNS membranes. No correlation between their potency as [³H]adenosine uptake blockers and as inhibitors of [³H]diazepam binding was found, either in rat or guinea-pig brain tissue. Dipyridamole, a potent adenosine transport inhibitor interacted strongly (K_i = 54 nM) with peripheral-type benzodiazepine binding sites (“acceptor sites”) and was 4–5 fold weaker in displacing [³H]methylclonazepam and [³H]Ro15-1788, ligands selective for the specific central benzodiazepine “receptor”. Unlike the benzodiazepines, dipyridamole had no anticonvulsant action against metrazole-induced convulsions in mice. Ro5-4864, a benzodiazepine which selectively interacts with the peripheral-type benzodiazepine binding site, was approximately equipotent with diazepam in inhibiting [³H]adenosine uptake in brain tissue. These results do not support the idea of a very close link between high-affinity central binding sites for clinically-active benzodiazepines and the adenosine uptake site. The possibility of a connection between benzodiazepine “acceptor” sites and the membrane nucleoside transporter is discussed.     

Dextromethorphan binding sites in the guinea pig brain

1. Dextromethorphan (DM), a dextrorotatory nonopioid antitussive, binds to specific high-affinity sites in the central nervous system. These sites are distinct from the opioid and other known neurotransmitter receptor sites. Antitussives such as carbetapentane and caramiphen also bind to DM sites with a nanomolar affinity. 2. The anticonvulsant drugs phenytoin and ropizine produce an allosteric enhancement of the binding of [3H]DM to guinea pig brain. DM, carbetapentane, and caramiphen also are efficacious anticonvulsant agents in the rat maximal electroshock seizures test, and DM enhances the anticonvulsant effects of phenytoin (PHT). 3. These results suggest that drugs that bind to the DM sites could be used alone as anticonvulsants or in combination with PHT to lower its effective dose and reduce its side effects. 4. The investigation of the DM binding sites may help to open new approaches for the treatment of convulsive disorders and to explain further some of the molecular mechanisms of neutronal excitability.     

Solubilization of benzodiazepine binding site from rat cortex.

The high-affinity binding site for [³H] diazepam has been solubilized from rat brain using 0.5% Lubrol-PX. Using a polyethylene glycol (PEG)-γ-globulin assay, it has been possible to demonstrate solubilization of about 60% of the binding sites in a single step. The solubilized binding site possesses a K_D of 11 nM for [³H] diazepam compared to approximately 4 nM for the membrane-bound form, and binding is to a single class of sites. The order of potency of benzodiazepines is identical for the solubilized receptor and the membrane-bound form. Binding of [³H] diazepam is temperature dependent and higher at 4° than 37°C. Both urea and guanidine-HC1 were capable of totally inhibiting binding, and this inhibition was partly reversible; neither sulfhydryl groups nor carbohydrate moieties seem to be important for binding. γ-Aminobutyric acid which enhanced [³H] diazepam binding to membrane fractions was without effect on the solubilized binding site.     

Autoradiographic characterization of binding sites for [3H]NE-100 in guinea pig brain

The receptor binding specificity and neuroanatomical distribution of [³H]NE-100 (N, N- dipropyl-2- [4- methoxy-3- (2- phenylethoxy) phenyl] ethylamine monohydrochloride)-labeled sigma receptor in guinea pig brain were examined using quantitative autoradiography. NE-100 potently inhibited [³H]NE-100 binding to slide-mounted sections of guinea pig brain with the IC₅₀ value of 1.09 nM, therefore, NE-100 apparently has high affinity binding sites. Competition studies, under conditions similar to those used to visualize the receptor, yielded the following rank order of potency: NE-100 > haloperidol > DuP734 > (+)pentazocine ⪢ (−)pentazocine. Non-sigma ligands such as phencyclidine (PCP), MK-801 and (−)sulpiride had negligible affinities for [³H]NE-100 binding sites. High densities of [³H]NE-100 binding sites displaceable by haloperidol were present in the granule layer of the cerebellum, the cingulate cortex, the CA3 region of the hippocampus, the hypothalamus and the pons. The distribution of [³H]NE-100 binding sites was consistent with that of [³H](+)pentazocine, a sigma₁ ligand. These sigma sites may possibly be related to various aspects of schizophrenia.     

Methyllycaconitine and (+)-anatoxin-a differentiate between nicotinic receptors in vertebrate and invertebrate nervous systems

Specific high-affinity binding sites for 125I-alpha-bungarotoxin and (-)-[3H]nicotine have been measured in rat brain and locust (Schistocerca gregaria) ganglia. The binding sites for 125I-alpha-bungarotoxin had similar Kd values of 1.5 x 10(-9) and 0.8 x 10(-9) M for rat and locust preparations, respectively; the corresponding values for the (-)-[3H]nicotine-binding site were 9.3 x 10(-9) and 1.7 x 10(-7) M. Methyllycaconitine (MLA) potently inhibited 125I-alpha-bungarotoxin binding in both rat and locust. MLA was a less effective inhibitor of (-)-[3H]nicotine binding whereas (+)-anatoxin-a was a very potent inhibitor at this site in the rat but not in the locust. These data suggest that (+)-anatoxin-a is a useful probe for the high-affinity nicotine-binding receptor in vertebrate brain, whereas MLA is a preferential probe for the subclass of receptor that binds alpha-bungarotoxin.     

Specific calcium antagonist binding sites in brain

The binding of the calcium antagonist [³H] nitrendipine ([³H] NDP) to brain and heart is described and the brain site is characterized. The binding is saturable, specific and of very high affinity with K_D values of 0.16 nM in brain and 0.21 nM in heart. Our kinetic results are similar to those recently reported by two other groups (1,2), indicating a saturable, high affinity binding site in brain. In brain the binding sites are enriched in crude nuclear and synaptosomal fractions. The highest levels of binding are seen in the hippocampus, caudate and cerebral cortex with much lower levels in the cerebellum and pons. Calcium has a marked stimulatory effect on [³H] NDP binding at 10^?4 M. Addition of 0.5 mM CaCl₂ to EDTA treated membranes nearly doubles the number of binding sites. Of the many drugs and neurotransmitters tested only other calcium antagonists, i.e., verapamil, inhibit binding (IC₅₀ = 250 nM). The inhibition of [³H] NDP binding by verapamil is apparently non-competitive and not complete, suggesting that [³H] NDP binds to several sites, only some of which are inhibited by verapamil. The [³H] NDP binding site is probably a protein since it is very sensitive to trypsin, heat and sulfhydryl reagents.     

Interactions Between the Glutamate and Glycine Recognition Sites of the N-Methyl-d-Aspartate Receptor from Rat Brain, as Revealed from Radioligand Binding Studies

Abstract: The N-methyl-d -aspartate (NMDA) receptor possesses two distinct amino acid recognition sites, one for glutamate and one for glycine, which appear to be allosterically linked. Using rat cortex/hippocampus P₂ membranes we have investigated the effect of glutamate recognition site ligands on [³H]glycine (agonist) and (±)4-trans-2-car-boxy-5,7-dichloro-4-[³H]phenylaminocarbonylamino-1,2,3,4-tetrahydroquinoline ([³H]l -689,560; antagonist) binding to the glycine site and the effect of glycine recognition site ligands on l -[³H]glutamate (agonist), dl -3-(2-carboxypiperazin-4-yl)-[³H]propyl-1 -phosphonate ([³H]-CPP; “C-7” antagonist), and cis-4-phosphonomethyl-2-[³H]piperidine carboxylate ([³H]CGS-19755; “C-5” antagonist) binding to the glutamate site. “C-7” glutamate site antagonists partially inhibited [³H]l -689,560 binding but had no effect on [³H]glycine binding, whereas “C-5” antagonists partially inhibited the binding of both radioligands. Glycine, d -serine, and d -cycloserine partially inhibited [³H]CGS-19755 binding but had little effect on l -[³H]-glutamate or [³H]CPP binding, whereas the partial agonists (+)-3-amino-1-hydroxypyrrolid-2-one [(+)-HA-966], 3R-(+)cis-4-methyl-HA-966 (l -687,414), and 1-amino-1-carboxycyclobutane all enhanced [³H]CPP binding but had no effect on [³H]CGS-19755 binding, and (+)-HA-966 and l -687,414 inhibited l -[³H]glutamate binding. The association and dissociation rates of [³H]l -689,560 binding were decreased by CPP and d -2-amino-5-phosphonopentanoic acid (“C-5”). Saturation analysis of [³H]l -689,560 binding carried out at equilibrium showed that CPP had little effect on the affinity or number of [³H]l -689,560 binding sites. These results indicate that complex interactions occur between the glutamate and glycine recognition sites on the NMDA receptor. In addition, mechanisms other than allosterism may underlie some effects, and the possibility of a steric interaction between CPP and [³H]l -689,560 is discussed.     

SKF 525-A and cytochrome P-450 ligands inhibit with high affinity the binding of [3H]dextromethorphan and sigma ligands to guinea pig brain.

The DM1/sigma 1 site binds dextromethorphan (DM) and sigma receptor ligands. The broad binding specificity of this site and its peculiar subcellular distribution prompted us to explore the possibility that this site is a member of the cytochrome P-450 superfamily of enzymes. We tested the effects of the liver microsomal monooxygenase inhibitor SKF 525-A (Proadifen), and other P-450 substrates on the binding of [3H]dextromethorphan, [3H]3-(-3-Hydroxyphenyl)-N-(1-propyl)piperidine and (+)-[3H]1,3-Di-o-tolyl-guanidine ([3H]DTG) to the guinea pig brain. SKF 525-A, l-lobeline and GBR-12909 inhibited the binding of the three labeled ligands with nM affinity. Each drug has identical nM Ki values for the high-affinity site labeled by the three ligands. This indicated that they displaced the labeled ligands from the common DM1/sigma 1 site. Debrisoquine and sparteine, prototypical substrates for liver debrisoquine 4-hydroxylase, displayed Ki values of 9-13 and 3-4 microM respectively against the three labeled ligands. These results, the broad specificity of the DM1/sigma 1 binding site, and its peculiar subcellular distribution, raises the possibility that this binding site is a member of the cytochrome P-450 superfamily of isozymes, rather than a neurotransmitter receptor. These findings may have important implications for the understanding of the therapeutic, side effects and toxicity of several neurotropic drugs.     

Solubilization and Characterization of σ-Receptors from Guinea Pig Brain Membranes

The sigma-receptor, a distinct binding site in brain tissue that may mediate some of the psychotomimetic properties of benzomorphan opiates and phencyclidine, has been solubilized using the ionic detergent sodium cholate. Binding assays were performed with the solubilized receptor using vacuum filtration over polyethyleneimine-treated glass fiber filters. The pharmacological specificity of the solubilized binding site for sigma-receptor ligands is nearly identical to the membrane-bound form of the receptor, with the order of potencies for displacement of the selective sigma-ligand [3H]di-o-tolylguanidine ([3H]DTG) closely correlated. The stereoselectivity for (+)-benzomorphan opiate enantiomers was retained by the solubilized receptor. The soluble receptor retained high affinity for binding of [3H]DTG (KD = 28 +/- 0.5 nM) and (+)-[3H]3-(3-hydroxyphenyl)-N-(1-propyl)piperidine [(+)-[3H]3-PPP] (KD = 36 +/- 2 nM). Photoaffinity labeling of the solubilized receptor by [3H]p-azido-DTG, a sigma-selective photoaffinity label, resulted in labeling of a 29-kilodalton polypeptide identical in size to that labeled in intact membranes. Estimation of the Stokes radius of the [3H]DTG binding site was obtained by Sepharose CL-6B chromatography in the presence of 20 mM cholate and calculated to be 8.7 nm. This value was identical to the molecular size found for the binding sites of the sigma-selective ligands (+)-[3H]3-PPP and (+)-[3H]SKF-10,047, supporting the hypothesis that all three ligands bind to the same macromolecular complex.     

Competitive interactions at [3H]1,3-di(2-tolyl)guanidine (DTG)-defined sigma recognition sites in guinea pig brain.

In saturation binding experiments, (+)pentazocine, (+)3-(3-hydroxyphenyl)-N-propylpiperidine (3-PPP), haloperidol and rimcazole did not inhibit the binding of [3H]DTG in a purely competitive fashion. Although Scatchard analysis indicated that [3H]DTG bound to a single site, the inhibition curves of some, but not all, reference compounds exhibited Hill coefficients of less than 0.8. The Scatchard data were consistent with a model of hyperbolic competitive inhibition of binding to the [3H]DTG-defined sigma site, although other possibilities such as negative cooperativity or binding to two sites cannot be definitively excluded. Compounds from numerous pharmacological and structural classes inhibited the binding of [3H]DTG, suggesting that interactions of [3H]DTG with other receptors may have confounded the Scatchard analysis of the binding of [3H]DTG to sigma recognition sites.     

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     

[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     

Effect of sodium on [3H]ethylketocyclazocine binding to opioid receptors in frog brain membranes

The specific binding of (³H)ethylketocyclazocine to frog brain membrane preparation was enhanced in the presence of sodium ions administered as NaCl, both at 0 °C and at room temperature. The optimal NaCl concentration was 25 mM at 0 °C and 50 mM at 24 °C. MgCl₂ inhibited the [³H]ethylketocyclazocine binding. Two binding sites (high and low affinity) were established with [³H]ethylketocyclazocine as ligand by equilibrium binding studies. Addition of NaCl increased the B_max of the low-affinity site more than that of the high-affinity site at both temperatures. Affinities were higher at 0 °C than at 24 °C. TheK _D values were not significantly influenced by sodium ions. The dissimilarities between the rat and frog brain opioid receptors in [³H]ethylketocyclazocine binding are attributed to the different lipid composition of the two membranes.Abbreviations used DAGO D-Ala²-(Me)Phe⁴-Gly-ol⁵-enkephalin - DALE d-Ala²-l-Leu⁵-enkephalin - DADLE d-Ala²-d-Leu⁵-enkephalin - EKC Ethylketocyclazocine - DHM Dihydromorphine - BIT 2-(p-ethoxybenzyl)1-diethylaminoethyl-5-isothiocyanobenzimidazole isothiocyanate - FIT Fentanyl isothiocyanate     

Opioid binding properties in bovine retina

Saturable binding sites for tritiated dihydromorphine ([³H]DHM), D-Ala²-D-Leu⁵-enkephalin ([³H]DADL) and etorphine were found in a crude synaptosomal preparation of bovine retina. Scatchard analysis of saturation binding curves of each ligand was curvilinear and the presence of two independent binding sites inferred. The density of binding sites of [³H]etorphine was similar to that reported in brain crude synaptosomal preparations, and the affinity for the high affinity binding site to each ligand was similar to values determined in brain. Moreover, the regulation of the binding sites by GTP and sodium was also similar to that observed in brain. Selective binding sites for [³H]DADL (δ-sites) were not detectable, although binding sites similar in nature to μ-binding sites were detected.