Pentazocine Binding Research Articles

(-)1-(Benzofuran-2-yl)-2-propylaminopentane shows survival effect on cortical neurons under serum-free condition through sigma receptors.

1. The rapid cell death of cortical neurons in serum-free culture was rescued by the condition medium from the high-density culture, but not by brain-derived neurotrophic factor or basic fibroblast growth factor. 2. Similar rescue was observed by the addition of (-)BPAP, an impulse enhancer, and (+)-pentazocine, a sigma receptor agonist. These actions were blocked by BD1063, a sigma receptor antagonist. 3. (-)BPAP showed a weak displacement activity in the [3H]pentazocine binding to synaptic membranes from rat cerebral cortex. 4. These findings suggest that (-)BPAP and (+)-pentazocine have unique survival activity on cortical neurons through sigma receptors.

Cloning and characterization of a mouse sigma1 receptor.

A cDNA clone (S2-1a) isolated from a mouse brain cDNA library, using a guinea pig sigma1 cDNA as probe, has high homology to the predicted protein sequence of the guinea pig (88%) and human (90%) sigma1 receptors. Northern analysis revealed a major mRNA of approximately 1.8 kb in a wide range of mouse tissues, with highest levels in brain, liver, kidney, and thymus. Southern analysis and chromosomal mapping in the mouse suggested a single-copy gene in region A5-B2 of chromosome 4. Expression of the clone in MCF-7 and CHO cells led to a pronounced increase in (+)-[3H]pentazocine binding with a selectivity profile consistent with sigma1 receptors. In vitro translation yielded a protein of approximately 28 kDa, as did transfection of a probe containing the hemagglutinin (HA) epitope (S2-1a.HA) into CHO cells, as determined by western analysis using an antibody directed against HA. (+)-[3H]-Pentazocine binding to immunopurified HA-tagged receptor demonstrated conclusively that S2-1a.HA encodes a high-affinity (+)-[3H]pentazocine binding site with characteristics of a murine sigma1 receptor. An antisense oligodeoxynucleotide designed from S2-1a potentiated opioid analgesia in vivo.

Up-regulation of [formula omitted]DTG but not [formula omitted](+)-pentazocine labeled σ sites in mouse spinal cord by chronic morphine treatment

To monitor the possible effect of morphine on σ sites, binding characteristics of [ 3 H] (+)-pentazocine and [ 3 H] 1,3-di-(2-tolyl)guanidine (DTG) to brain and spinal cord membranes of morphine-treated and control mice were compared. For morphine treatment, a single injection (100 mg/kg, s.c.) of morphine was followed 4 h later by pellet implantation (75 mg morphine free base). Animals were sacrificed 24, 72 h or 7 days later. The equilibrium dissociation value ( K d) and the density ( B max) of [ 3 H] (+)-pentazocine binding remained unaffected by morphine treatment. Also, no change was found in K d and B max values of [ 3 H] DTG labeled σ 2 subtypes after any morphine treatment schedule when measured in the presence of 100 nM (+)-pentazocine. However, the B max of [ 3 H] DTG binding in the spinal cord in the absence of 100 nM (+)-pentazocine, was significantly elevated 72 h after implantation of the morphine pellet and recovered by 7 days, a time when the antinociceptive effect produced by the morphine pellet had dissipated. These data suggest that one population of σ sites, that has a high affinity for [ 3 H] DTG, but is not equivalent with the [ 3 H] (+)-pentozocine labeled σ 1 subtype or the [ 3 H] DTG labeled σ 2 subtype, is upregulated by morphine and, therefore, may play a role in the development of tolerance to or dependence on the effects of morphine.

Subchronic administration of N-[2-(3,4-dichlorophenyl)ethyl]- N-methyl-2-(dimethylamino)ethylamine (BD1047) alters σ 1 receptor binding

BD1047 ( N-[2-(3,4-dichlorophenyl)ethyl]- N-methyl-2-(dimethylamino)ethylamine) is known to bind with high affinity and selectivity to σ sites in vitro. In prior in vivo studies, it has been shown to attenuate the dystonic postures and orofacial dyskinesias that are produced by σ receptor ligands, including the neuroleptic haloperidol. Since abnormal movements, such as dystonic postures and orofacial dyskinesias, are side effects that are associated with many σ-active neuroleptics, compounds such as BD1047 may have therapeutic potential for preventing and treating these unwanted movements. A possible limitation to the therapeutic potential of BD1047, however, is that at least in cell culture and albeit weak, it can be cytotoxic. Therefore, the present study analyzed the possible neurotoxic effects of in vivo subchronic intracerebroventricular infusion of BD1047 (10 nmol/h) or artificial cerebrospinal fluid (CSF) into rat brains using osmotic minipumps for 7 or 14 days. Following a 24 h wash-out period, the animals were killed, the brains removed, and P 2 membranes prepared. Membranes from rats treated for 7 or 14 days with BD1047 showed a marked decrease in [ 3H](+)-pentazocine binding as compared to membranes from CSF-treated animals, suggesting a loss of σ 1 receptor binding. Histological examination of brain sections processed for Nissl stains and glial fibrillary acidic protein (GFAP) immunohistochemistry excluded the possibility of a cytotoxically induced down-regulation, suggesting possible receptor internalization or desensitization mediated via σ 1 sites. Under the conditions used in our study, BD1047 does not appear to be neurotoxic, and the data, when taken together with other studies, suggest that BD1047 acts as a partial agonist at σ sites. © 1997 Elsevier Science B.V. All rights reserved.

Sigma binding in a human neuroblastoma cell line.

Behaviorally, sigma1 agents modulate opioid analgesia. To examine possible mechanisms responsible for these interactions, we have identified a cell line containing both sigma1 and opioid receptors. [3H](+)-pentazocine binding in BE(2)-C human neuroblastoma cells is high affinity (KD 3.4 +/- 0.7 nM) and high density (Bmax 2.98 +/- 0.14 pmol/mg protein). Competition studies reveal a selectivity profile similar to that of sigma1 sites in guinea pig brain. (+)-Pentazocine has no effect upon either basal or forskolin-stimulated cyclase in the BE(2)-C cells, but cAMP accumulation is inhibited by the morphine, DPDPE and naloxone benzoylhydrazone. (+)-Pentazocine at concentrations as high as 10 microM does not affect this opioid effect, implying that sigma1/opioid interactions are not mediated at the level of the cell. This suggest that their behavioral interactions result from interacting neural circuits. Although (+)-pentazocine is without effect in the cyclase system, it does block carbachol-stimulated phosphoinositol turnover (IC50 6.5 +/- 1.14 microM). The specificity of the effect is confirmed by the ability of haloperidol (1 microM) to shift the IC50 value of (+)-pentazocine 2-fold to the right.

Solubilization and characterization of binding sites for [3H]NE-100, a novel and potent sigma 1 ligand, from guinea pig brain.

The binding sites for [3H]NE-100, a newly defined sigma 1 ligand, was solubilized from guinea pig brain, using zwitterionic detergent 3-[(3-c holamidopropyl) dimethylamino]-1-propanesulfonate (CHAPS), and the properties of the solubilized binding sites were compared to those for [3H](+)-pentazocine, a selective sigma 1 ligand. The pharmacological selectivity of solubilized sites for both [3H]NE-100 and [3H](+)-pentazocine was identical to that obtained from membrane preparations. Stereoselectivity of benzomorphan such as pentazocine and SKF10,047 was preserved in displacing [3H]NE-100 binding in solubilized preparations as observed in membrane preparations. The inhibitory potencies of several sigma ligands on [3H]NE-100 binding were similar to those on [3H](+)-pentazocine binding, indicating that the pharmacological characteristics of the binding sites for [3H]NE-100 are retained after solubilization. Phenytoin augmented the binding of [3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl) piperidine hydrochloride (3-PPP) to solubilized sigma binding sites while it had no effect on the binding of [3H]NE-100. Furthermore, the inhibitory effect of putative sigma receptor agonists such as (+)-3-PPP and dextromethorphan were enhanced by phenytoin; the effects of haloperidol, a putative sigma receptor antagonist, were unaltered. Molecular weight of [3H]NE-100 binding protein was estimated to be 440KDa by Sepharose CL-6B gel filtration chromatography, and the value was identical to that of [3H](+)-pentazocine binding protein, a putative sigma 1 binding protein. These findings indicate that [3H]NE-100 binding sites are putative sigma 1 binding sites, and that NE-100 may act as an antagonist at sigma 1 binding sites.

Binding properties of SA4503, a novel and selective σ1 receptor agonist

The binding profiles of SA4503 (1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)piperazine dihydrochloride), a novel σ receptor ligand, to σ 1 and σ 2 receptor subtypes in guinea pig and rat brain membranes were evaluated. SA4503 showed a high affinity for the σ 1 receptor subtype labeled by (+)-[ 3H]pentazocine (IC 50 = 17.4 ± 1.9 nM), while it had about 100-fold less affinity for the σ 2 receptor subtype labeled by [ 3H]1,3-di(2-tolyl)guanidine ([ 3H]DTG) in the presence of 200 nM (+)-pentazocine. SA4503 showed little affinity for 36 other receptors, ion channels and second messenger systems. The inhibition curves of SA4503 for (+)-[ 3H]pentazocine binding were shifted to the right in the presence of guanosine 5′- o-(3-thiotriphosphate) (GTPγS), as similar to those of (+)-3-(3-hydroxyphenyl)- N-(1-propyl)piperidine ((+)-3-PPP) and (+)-pentazocine, σ 1 receptor agonists. SA4503 significantly increased the K D value, but did not affect the B max value for specific (+)-[ 3H]pentazocine binding. These results indicated that SA4503 is a potent and selective agonist for the σ 1 receptor subtype in the brain. In addition, SA4503 inhibited specific (+)-[ 3H]pentazocine binding in a competitive manner.

Interactions of selective serotonin reuptake inhibitors with subtypes of σ receptors in rat brain

The interactions of selective serotonin reuptake inhibitors and tricyclic antidepressants with subtypes of σ receptors were investigated. The rank order of affinity ( K i values) from competition experiments of [ 3H](+)-pentazocine binding to σ 1 sites was: fluvoxamine > sertraline > S(+)-fluoxetine > (±)-fluoxetine > citalopram > imipramine > paroxetine > desipramine > R(−)-fluoxetine > (±)-norfluoxetine. The K i values of all drugs for σ 2 sites were more than 1000 nM. Furthermore, all drugs were more potent at σ 1, sites than at σ 2 sites. These findings suggest that σ receptors ( σ 1, site) may play, in some way, a role in the actions of selective serotonin reuptake inhibitors.

The sigma ligand JO 1784 prevents trimethyltin-induced behavioural and sigma-receptor dysfunction in the rat.

Recently much research interest has focused on the possible therapeutic uses of sigma-receptor ligands in psychiatric and neurodegenerative disorders. In the present study, the potential neuroprotective effects of chronic (52 days) administration of (+) cinnamyl-l-phenyl-l-N-methyl-N-cyclo propylene (JO 1784) (1 and 3 mg/kg subcutaneously), a potent and selective sigma receptor ligand, were assessed in the trimethyltin (8 mg/kg intraperitoneally) model of memory dysfunction. JO 1784 (3 mg/kg subcutaneously) prevented the trimethyltin-induced deficits in locomotor activity, passive avoidance and radial maze performance, while the lower dose of JO 1784 had little or no effect. Trimethyltin was also shown to produce a marked reduction in the binding of [3H] (+)-pentazocine to sigma-receptor sites in limbic brain structures, as detected by quantitative autoradiography, which was particularly evident in the hippocampal pyramidal cells. JO 1784 (3 mg/kg subcutaneously) successfully attenuated this loss of [3H] (+)-pentazocine binding sites in the hippocampus (CA1, CA3 and CA4 regions) and in the substantia innominata. This neuroprotective effect of JO 1784 in the trimethyltin model would seem to be related to the modulatory effects of this sigma ligand on trimethyltin-induced glutamate neurotoxicity.

Dissociation of the motor effects of (+)-pentazocine from binding to σ1 sites

Radioligand binding and behavioral studies were conducted to determine whether a relationship existed between the motor effects produced by (+)-pentazocine and its binding to σ sites. Scatchard analyses revealed decreased [ 3H](+)-pentazocine binding in middle aged rats (5–6 months old) compared to young adult rats (2–3 months old). However, there was no difference between the extent of circling behavior or dystonia produced by microinjection of (+)-pentazocine into the substantia nigra or red nucleus in the older animals compared to the young adult rats. There was also a significant decrease in [ 3H](+)-pentazocine binding in rats chronically treated with haloperidol. Again, however, despite the reduction in [ 3H](+)-pentazocine binding, there was no difference between the extent of dystonia produced by unilateral intrarubral microinjection of (+)-pentazocine into animals chronically treated with haloperidol vs. saline. The postural changes produced by (+)-pentazocine could not be attenuated with coadministration of the putative σ receptor antagonist BD1047 ( N-[2-(3,4-dichlorophenyl)ethyl]- N-methyl-2-(dimethylamino)ethylamine), or the opiate receptor antagonist naloxone. However, the (+)-opiate, (+)-nordihydrocodeinone, partially attenuated the postural effects of (+)-pentazocine, despite its very low affinity for σ 1, σ 2, or opiate receptors. Taken together with previous studies, the results suggest that [ 3H](+)-pentazocine is a potent and selective probe for σ 1 binding sites, but the in vivo effects of (+)-pentazocine cannot be fully attributed to actions through these sites. Some of the in vivo effects of (+)-pentazocine appear to involve other binding sites that are not detected under the conditions normally used in in vitro assays.

Novel potent sigma 1 ligands: N-[omega-(tetralin-1-yl)alkyl]piperidine derivatives.

A series of substituted N-[(tetralin-1-yl)alkyl]piperidines and a number of related N-di-n-propyl-[(tetralin-1-yl)alkyl]amines were prepared. Structural modifications such as piperidine substitutions, intermediate chain lengthening, and the nature of the aromatic ring were explored in order to identify structural requirements for selective sigma 1 affinity. They were tested in radioligand binding assays on sigma 1, 5-HT1A and 5-HT2 serotonergic, PCP (phencyclidine), and D-2 dopaminergic receptors. Almost all the compounds reported here showed a high to superpotent sigma 1 affinity, and some compounds also demonstrated a widespread selectivity over the other receptors. In [3H]-(+)-pentazocine binding, 3,3-dimethyl-1-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)-n- propyl] piperidine (24) and 3,3-dimethyl-1-[4-(1,2,3,4-tetrahydronaphthalen-1-yl)-n- butyl]piperidine (26) reached the lowest Ki values (0.4 and 0.8 nM, respectively); compound 24 also demonstrated a considerable PCP affinity (Ki = 34.2 nM), whereas compound 26 was suitably selective. Furthermore the presence of a 4-benzyl substituent on the piperidine ring (compound 16, Ki = 3.9 nM on sigma 1 sites) caused an increase in 5-HT1A affinity (Ki < 0.14 nM).

Characterization of [3H]pentazocine binding sites in post-mortem human frontal cortex.

We investigated binding characteristics of [3H](+)-pentazocine in homogenates of post-mortem human frontal cortex. At equilibrium, specific binding was linear with protein concentration, was saturable, reversible, stereoselective, heat-labile and was nearly absent in the white matter. Saturation experiments revealed a KD of 3.68 +/- 0.46nM and a B(max) of 0.636 +/- 0.107 pmol/mg protein. The rank order of Ki values of competing substances was: haloperidol < N,N'-di(o-tolyl)guanidine (DTG) < (+)-SKF 10,047 < (-)-SKF 10,047. We also examined the influence of age, gender, hemisphere, post-mortem time and storage time of brain tissue at -80 degrees C on [3H](+)-pentazocine binding sites. Of these variables, only age was significantly related to [3H](+)-pentazocine binding (diminished binding with increasing age). Together, our results demonstrate the presence of specific [3H](+)-pentazocine binding sites in post-mortem human brain tissue. Furthermore, the binding sites decrease with increasing age and are apparently independent of gender, hemisphere, post-mortem time and storage time of brain tissue.

Chronic postnatal phencyclidine treatment on [ 3HI](+) pentazocine binding in juvenile rat brain

Chronic phencyclidine (PCP) administrations in postnatal rats produce long-term behavioral changes. Here we report the effects of repeated postnatal PCP treatment on [3H](+)pentazocine binding in juvenile male rats. Saturation analyses of the binding data showed no significant difference in any of the binding characteristics between chronic PCP-treated rats and saline-treated controls suggesting that mechanisms other than alterations in or, binding underlie the behavioral effects of repeated postnatal PCP administration in immature rats.

Anticonvulsant activity of caramiphen analogs

Caramiphen potently blocks maximal electroshock (MES)-induced seizures in mice and rats. The anticonvulsant mechanism has been hypothesized to be due to high-affinity binding to σ recognition sites in brain. To study the structureactivity relationship for anticonvulsant activity of caramiphen we evaluated 8 analogs in MES-induced seizures in rats and also determined whether a correlation exists between anticonvulsant potency and σ binding affinity. Some of the analogs potently inhibited σ binding but were devoid of anticonvulsant activity. Aminocaramiphen 2 (ED 50 = 3.4 mg/kg) and N-methyl-4-piperidinyl 1-phenylcyclopentanecarboxylate 9 (ED 50 = 4.8 mg/kg) showed anticonvulsant activity comparable to caramiphen (ED 50 = 3.1 mg/kg), although in σ binding assays the affinities were 3-and 30-fold less than caramiphen, respectively. In the presence of 250 μM of phenytoin, caramiphen and p-aminocaramiphen showed 3- to 5-fold increases in affinity for [ 3H](+)pentazocine binding, whereas piodocaramiphen, which was inactive as an anticonvulsant, showed no change in affinity for σ binding. These results indicate that anticonvulsant activity of the caramiphen analogs is not due to interaction with o binding sites.

Rat liver and kidney contain high densities of σ 1 and σ 2 receptors: characterization by ligand binding and photoaffinity labeling

Rat liver and kidney were investigated for the presence of sigma (σ) receptor subtypes by radioligand binding with three highly selective σ probes and by photoaffinity labeling using [H]azido-di- o-tolyguanidine ([ 3H]azido-DTG). [ 3(+)- Pentazocine, a highly selective σ 1 probe, bound to sites in liver membranes with K d = 7.5 nM and B max = 2929 fmol/mg protein. [ 3H](+)-Pentazocine binding sites in kidney had K d = 23.3 nM and B max = 229fmol/mg protein. [ 3 H1,3- Di-o- tolylguanidine ([ 3H]DTG) and [ 3 H](+)-3-(3- hydroxyphenyl-N-(1- propyl)piperidine ([ 3H](+)-3-PPP) label both σ 1 and σ 2 receptors. Parameters for [ 3H]DTG in the liver were K d = 17.9 nM and B max - 11 895 fmol.mg protein. Similar parameters were observed for [ 3H](+)-3-PPP, K d = 51.9 nM and B max = 11 070 fmol/mg protein. [ 3H]DTG bound to rat kidney with K d = 45.8 nM and B max = 1190 fmol/mg protein. The observation that either [ 3H]DTG or [ 3H](+)-3-PPP labeled a higher number of sites relative to [ 3H](+)-pentazocine suggested that liver and kidney contain both subtypes of σ receptor. This was confirmed by competition studies vs. [ 3H](+)-pentazocine and [ 3H]DTG (in the presence of dextrallorphan to mask σ 1 sites). In both tissues, [ 3H](+)-pentazocine labeled sites with high affinity for haloperidol and enantioselectivity for (+)-benzomorphans over (−)-benzomorphans. [ 3H]DTG + dextrallorphan labeled sites in both tissues which also high affinity for haloperidol, but which had the characteristic σ 2 property of low affinity for (+)-benzomorphans and enantioselectivity for (−)-benzomorphans over the corresponding (+)-isomer. Similar results were obtained with [ 3H](+)-3-PPP + dextrallorphan. Several novel aryl diamines, such as 1 S, 2 R- cis- N-[2-(3,4-dichlorophenylethyl]- N-methyl-2-(1-pyrrolidinyl)cyclohexylamine BD737) and N-2- (3,4-dichlorophenyl)ethyl]- N-methyl-2-(1-pyrrolidinyl)ethylamine (BD1008), bound to both sites with high affinity. Photoaffinity labeling with 10 nM [ 3H]azido-DTG resulted in specific labeling of polypeptides of 25 kDa and 21.5 kDa and 21.5 kDa. Dextrallorphan (100 nM or 500 nM) completely blocked labeling of the 25 kDa polypeptide, but had no effect on labeling of the lower molecular weight protein. (+)-10,11-Dihydro-5-methyl-5 H-dibenzol[ a, d]cyclohepten-5,10-imine((+)_-MK-801) had no effect on labeling of either polypeptide. These data are consistent with the notion that the 25 kDa and 21.5 proteins represent σ 2 and σ 2 receptors, respectively. Thus, rat liver and kidney contain high densities of σ 1 and σ 2 receptors, with σ 2 sites comprising 75–80% of the total σ population. The high density of σ receptor subtypes may indicate important functions in hepatic and renal tissues.

Multiple [ 3H]DTG binding sites in guinea pig cerebellum: evidence for the presence of non-specific binding

The characteristics of the low affinity component of 1,3-di(2- 3H]tolyl)guanidine binding to the guinea pig cerebellum were investigated. Saturation binding assays where σ 1 receptors were masked with dextrallorphan indicated that 1,3-di(2-[5- 3H]tolyl)guanidine bound to cerebellar membranes in a fashion best described by a 1 site + non-specific binding model with a low density of specific binding sites ( B max ≈ 200 fmol/mg protein). Boiling the cerebellar membranes before addition to the saturation assay had no effect on the density of 1,3-di(2-[5- 3H]tolyl)guanidine binding. In contrast, both the K d and B max for 1,3-di(2-[5- 3H]tolyl)guanidine binding to liver membranes was significantly reduced by boiling, as was the density of [ 3H](+)-pentazocine binding to cerebellum and liver. Thus, a substantial component of 1,3-di(2-[5- 3H]tolyl)guanidine binding in the guinea pig cerebellum is to non-specific, proteinaceous binding sites with some of the pharmacological characteristics of the σ 2 binding site.

3H](+)-Pentazocine binding to rat brain σ1 receptors

[ 3H](+)-Pentazocine binding has been characterized in the rat brain. It binds to a single population of binding sites with affinity of about 7 nM and density of 280 fmol/mg protein. [ 3H](+)-Pentazocine binding is not enriched in the crude synaptic membrane, being about 1 6 of what we found in the crude membrane preparation. The binding, like that for other σ ligands, was enriched in the microsomal and nuclear fractions. The inhibition by haloperidol, proadifen and d-fenfluramine was the same in the crude synaptic membrane, nuclear and microsomal fractions, suggesting that [ 3H](+)-pentazocine binds to a homogeneous protein in the different subcellular fractions. Our pharmacological characterization using 45 different drugs suggests that the [ 3H](+)-pentazocine binding site in rat brain differs from other σ ligands, like N-propyl-3-(3-hydroxyphenyl)piperidine ([ 3H](+)-3PPP), N, N′-di( o-tolyl)guanidine ([ 3H]DTG) and (+)- N-allylnormetazocine ([ 3H](+)-SKF10,047). [ 3H](+)-Pentazocine binding in rat brain is inhibited by σ compounds and some cytochrome P450 ligands, like proadifen and 1-[2-[bis(4-fluorophenyl) methoxy]ethyl]-4-[3-phenylpropyl] piperazine (GBR 12909), although with considerably lower potency than reported for other σ ligands. Other inhibitors are some serotonin uptake blockers or their metabolites and phenylalkylamines.

3H]-(+)-pentazocine binding to sigma recognition sites in human cerebellum

The binding characteristics of the sigma-1 selective benzomorphan [ 3H]-(+)-pentazocine were determined in human cerebellar membranes. Saturation binding analysis revealed two affinity sites with a K DH of 1.4 ± 0.7 nM and a K DL of 33.6 ± 11.9 nM. Kinetic studies performed at 25°C demonstrated reversible binding with association with association and dissociation rate constants determined for two classes of sites. In saturation binding studies, the addition of (+)-SKF 10,047 occluded binding of [ 3H]-(+)-pentazocine to high affinity sigma binding sites. The affinity profile of ligands displacing [ 3H]-(+)-pentazocine was consistent with the labeling of sigma-1 recognition sites with haloperidol > (+)-pentazocine > (+)-SKF 10,047 > (+)-3-PPP > DTG > (−)-pentazocine > (−)-SKF 10,047. The potency of the putative D 3 receptor-selective ligand (±)-7-OH-DPAT was close to that measured for (+)-pentazocine in displacement experiments. These data suggest that [ 3H]-(+)-pentazocine labels sigma-1 sites in human cerebellum under appropriate assay conditions.

NE-100, a novel potent σ ligand, preferentially binds to σ 1 binding sites in guinea pig brain

The selectivity of N, N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]-ehthylamine monohydrochloride (NE-100) for σ 1 and σ 2 binding sites was studied by means of binding of [ 3H](+)-pentazocine and [ 3 H]1,3- di-o- tolylguanidine ([ 3H]HTG) in guinea pig brain. NE-100 inhibited [ 3H](+)-pentazocine binding to σ 1 binding sites potently with an IC 50 value of 1.54±0.26 nM while it had a weak effect on [ 3H]DTG binding to σ 2 binding sites. The inhibitory effect of NE-100 on [ 3H](+)-pentazocine was 55 times more potent than that on [ 3H]DTG binding. These results suggest that NE-100 is a potent and selective ligand for σ 1 binding sites.

Characterization of the binding of [ 3H](+)-pentazocine to σ recognition sites in guinea pig brain

The selective σ compound (+)-pentazocine was radiolabeled and its binding characteristics in guinea pig brain membranes were investigated. [ 3H](+)-Pentazocine bound to a single high-affinity site with a K D of 2.9 nM and a B max of 1998 fmol/mg protein. Saturation was achieved at a ligand concentration of 15 nM. Maximal specific binding was observed at 37°C and was greater than 90% of total binding. Equilibrium was reached by 120 min and dissociation was complete by 420 min, with a t 1,2 of 121 min. Li +, Ca 2+ and Mg 2+ inhibited binding at high concentrations, and binding was insensitive to adenyl and guanyl nucleotides. Stereoselectivity was observed for the inhibition of binding by benzomorphans, 3-(3-hydroxyphenyl)-N-propylpiperidine and butaclamol, and the (+) enantiomers and α diastereomers of pentazocine and cyclazocine were more potent than their corresponding (−) enantiomers and β diastereomers. The rank order of potency for the σ reference agents to displace [ 3H](+)-pentazocine binding was similar to that reported using the [ 3H]σ ligands dextromethorphan, 1,3-di(2-tolyl)guuanidine and (+)-3-(3-hydroxyphenyl)-N-propylpiperidine. Haloperidol, (+)-pentazocine, (+)-3-(3-hydroxyphenyl)-N-propylpiperidine and rimcazole were competitive inhibitors of binding to the [ 3H](+)-pentazocine-defined σ recognition site, suggesting that these different structural classes of compounds all bind to a single molecular entity.