Steroid binding at sigma receptors suggests a link between endocrine, nervous, and immune systems.

Specific sigma binding sites have been identified in the mammalian brain and lymphoid tissue. In this study, certain gonadal and adrenal steroids, particularly progesterone, were found to inhibit sigma receptor binding in homogenates of brain and spleen. The findings suggest that steroids are naturally occurring ligands for sigma receptors and raise the possibility that these sites mediate some aspects of steroid-induced mental disturbances and alterations in immune functions.

Opiate receptor: demonstration in nervous tissue.

Tritiated naloxone, a powerful opiate antagonist, specifically binds to an opiate receptor of mammalian brain and guinea pig intestine. Competition for the opiate receptor by various opiates and their antagonists closely parallels their pharmacological potency. The opiate receptor is confined to nervous tissue.

Biologically active MK-801 and SKF-10,047 binding sites distinct from those in rat brain are expressed on human lung cancer cells.

We have shown previously that cultured human lung cancer cells of different histologic types express multiple opioid receptors that can regulate their growth. In this report, we show that these cells also express specific, saturable, and high-affinity binding sites (Kd approximately 1 nM) for the non-opioid phencyclidine (PCP), [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b]cyclohepten-5,10-imine hydrogen maleate] (MK-801) and sigma N-allylnormetazocine (SKF-10,047) receptor ligands. Characterization of these binding sites showed them to be protein in nature and sensitive to the guanine nucleotide GTP. Pharmacological studies showed that (+) MK-801 and (+) SKF-10,047 competed with each other for their binding sites and also for the methadone binding site present in these cells. However, the mu and delta opioid ligands did not compete for (+) MK-801 and (+) SKF-10,047 binding sites. In addition, these binding sites on lung cancer cells appear to be distinct from the N-methyl D-aspartate/PCP receptor ionophore complex reported to be present in rat brain. MK-801 and SKF-10,047, at nM concentrations, were found to inhibit the growth of these cells in culture within a few hours of exposure, and this effect was irreversible after 24 h. The growth effects of these ligands could not be reversed by the opioid antagonist naloxone, suggesting involvement of nonopioid type receptors in the actions of these ligands. The abundant expression of biologically active MK-801 and SKF-10 047 binding sites in these cell lines, distinct from those in rat brain, suggests that these cell lines may prove to be a valuable source for further characterization and purification of these binding sites.

Receptors and secretory actions of sigma/phencyclidine agonists in anterior pituitary cells.

Opiate receptor subtypes in the adenohypophysis were analyzed by binding studies with tritiated etorphine, phencyclidine (PCP), and N-allylnormetazocine [(+)SKF 10,047] in anterior pituitary cell (AC) cultures and membranes, and in cell populations separated by centrifugal elutriation. In cultured AC, specific binding of [3H]etorphine revealed two sets of saturable sites with Kd values of 5 nM and about 10 microM. The high affinity [3H]etorphine sites were present in low concentration and represent specific opiate receptors that mediate the direct inhibitory actions of etorphine and morphine on LH release in vitro. The more abundant low affinity sites, observed in the presence of higher concentrations of unlabeled opiates, exhibited the properties of sigma/PCP receptors. In intact AC and pituitary membranes, specific [3H]PCP binding was saturable with respect to labeled and unlabeled ligand concentrations, and Scatchard analysis revealed a single class of relatively high affinity [3H]PCP-binding sites (Kd = 98 nM in pituitary membranes). Relative potencies derived from inhibition of [3H]PCP binding in AC by PCP-related drugs were: (-) cyclazocine greater than dexoxadrol greater than N-[1-(2-Thienyl)cyclohexil]piperidine greater than PCP greater than (+)SKF 10,047 greater than levaxodral greater than (+)cyclazocine less than (-)SKF 10,047 greater than (+)ethylketocyclazocine greater than haloperidol greater than (-)ethylketocyclazocine. In elutriated pituitary cells, specific [3H]PCP binding was correlated with the LH content of the individual cell fractions. The binding of (+)-[3H]SKF 10,047 was also specific and saturable in AC and anterior pituitary membranes, which contained two classes of binding sites with Kd values of 87 nM and 3.3 microM. In fractionated pituitary cells, specific binding of (+)-[3H]SKF 10,047 was similar in enriched lactotrophs and gonadotrophs. The high affinity class of (+)-[3H]SKF 10,047-binding sites probably corresponds to sigma-receptors, and the low affinity class to PCP receptors. In contrast to the inhibitory actions of opiates on LH release in vitro, PCP and (+)SKF 10,047 stimulated LH release in cultured AC and enhanced the secretory responses to GnRH as well as KCl. The stimulation of LH release by PCP was dependent on extracellular calcium and is probably related to increased transmembrane calcium influx. The stimulatory sites may correspond to selective sigma/PCP receptors, and could represent a distinct nonopiate receptor subtype with the potential for modulation of gonadotropin secretion.

Differentiation of [3H]phencyclidine and (+)-[3H]SKF-10,047 binding sites in rat cerebral cortex.

The potency of a series of opioid and non-opioid psychotomimetic drugs to inhibit the specific binding of [3H]PCP and (+)-[3H]SKF-10,047 to rat cerebral cortical membranes was examined. (+)-PCMP, the 3-methylpiperidino analog of PCP, was a potent inhibitor of the specific binding of both ligands. All of the other 12 compounds examined, however, displayed a 3-277-fold selectivity for either [3H]PCP or (+)-[3H]SKF-10,047 binding. These results suggest that although these opioid and non-opioid psychotomimetics bind to both sites, most have significantly different affinities. The binding sites for [3H]PCP appear to be distinct from the 'sigma' binding sites labeled with (+)-[3H]SKF-10,047.

Quantitative characterization of multiple binding sites for phencyclidine and N-allylnormetazocine in membranes from rat and guinea pig brain.

Quantitative ligand binding studies have been used to characterize binding sites for N-allylnormetazocine ((+)SKF10,047) (SKF), 1-(1-phenylcyclohexyl) piperidine (PCP), N-[1-(2-thienyl)cyclohexyl]piperidine (TCP) and haloperidol in membranes from the brain of rat and guinea pig under conditions which permitted simultaneous analysis of the binding of both PCP and SKF. Using four labelled ligands (SKF, TCP, PCP and haloperidol), each displaced by the corresponding four unlabelled ligands, four classes of binding sites were observed in membranes from the brain of the rat, corresponding to sigma (sigma), two classes of PCP sites (PCP1, PCP2) and dopamine (D2) sites. The sigma site was suppressed by 50 nM haloperidol, while the PCP1 and PCP2 sites were not. These results were confirmed by studies employing a self- and cross-displacement design and dose-response surfaces for SKF and TCP, with and without blockade by haloperidol of the sigma site. Using mathematical modelling, employing the program LIGAND, it was possible to reject simpler models involving a common "PCP/sigma" site or a model involving only two classes of sites (sigma and PCP). Similar methods were used to identify two classes of sigma binding sites and two classes of PCP binding sites, in membranes prepared from the brain of the guinea pig. The relative potencies of 18 ligands for displacement of (+)[3H]SKF10,047 and [3H]TCP were compared: there were significant qualitative and quantitative differences in the "sigma" binding sites in the brain of rat and guinea pig, while the PCP binding sites were very similar in the two species.

Novel nonnarcotic analgesics with an improved therapeutic ratio. Structure-activity relationships of 8-(methylthio)- and 8-(acylthio)-1,2,3,4,5,6-hexahydro-2,6-methano-3-benzazocines.

Conversion of the 8-phenolic 1,2,3,4,5,6-hexahydro-2,6-methano-3-benzazocines to the corresponding 8-thiophenolic analogues was achieved by three different routes. Diazotization of 8-amino-2,6-methano-3-benzazocine (2) followed by the reaction with CH3SNa afforded 8-(methylthio)-1,2,3,4,5,6-hexahydro-2,6-methano-3-benzazocine (3). Another route using Grewe cyclization was also examined for the synthesis of 3. As the most effective route, Newman-Kwart rearrangement of benzazocines was selected and closely investigated. 8-(N,N-Dimethylthiocarbamoyl)oxy derivatives (6a-e) rearranged to 8-(N,N-dimethylcarbamoyl)thio derivatives (7a-e) in good yields. Reductive cleavage of 7a-e and subsequent methylation or acylations gave the title compounds (3, 8-24). Although analgesic activities of sulfur-containing benzazocines decreased compared to the corresponding hydroxy compounds, the N-methyl derivative (S-metazocine, 8) showed potent analgesic activity.

Enantiomeric N-substituted N-normetazocines: a comparative study of affinities at sigma, PCP, and mu opioid receptors.

The optical antipodes of N-allyl-N-normetazocine (2; SKF 10047, NANM) were the original compounds used for the classification of the sigma receptor as distinct from other receptors such as the PCP (NMDA), opioid, and dopamine receptors. Later studies showed that (+)-N-(dimethylallyl)-N-normetazocine [(+)-4, (+)-pentazocine] was more potent and selective for the sigma receptor. In order to gain additional structure-activity relationship information, several N-substituted N-normetazocine analogs were prepared and evaluated for their sigma-1 ([3H]-(+)-3-PPP or [3H]-(+)-pentazocine), PCP ([3H]TCP), and mu opioid ([3H]DAMGO) receptor binding affinities. (+)-N-Benzyl-N-normetazocine [(+)-10)] possessed subnanomolar affinities for the sigma site, Ki = 0.67. The analog (+)-10 showed greater than 14,000- and 2400-fold selectivity, respectively, for the sigma receptor relative to the PCP and mu opioid receptors. The N-substituted N-normetazocines were enantioselective for the sigma site. The (+)-N-benzyl analog, (+)-10, showed a 55-fold selectivity relative to (-)-10. Analysis of the data also revealed that (+)-normetazocine [(+)-1] [Ki = 30 nM] possessed the highest affinity for the PCP receptor. However, (+)-metazocine [(+)-5] (Ki = 41 nM) was the most selective compound for the PCP receptor relative to the sigma (51-fold) and mu opioid (greater than 200-fold) sites.

Phencyclidine-like effects of tetrahydroisoquinolines and related compounds.

A series of 1,2,3,4-tetrahydroisoquinolines, tetrahydrothieno[2,3-c]pyridines, and related compounds were evaluated for their ability to inhibit binding of [3H]-1-[1-(2-thienyl)piperidine and [3H]-N-allylnormetazocine to phencyclidine (PCP) and sigma receptors, respectively. A representative series of compounds was evaluated in behavioral assays to determine the ability of the compounds to induce PCP-like stereotyped behavior and ataxia. All of the compounds caused stereotyped behavior and ataxia, indicating their agonist actions at the PCP site.

A comparison of the binding of sigma opioids and phencyclidine, and the interaction with antipsychotic drugs in rat brain membranes.

The present study investigates the relationship between binding at the sigma site labelled by the prototypic sigma ligand (+)-[3H]-N-allylnormetazocine [+)-[3H]-SKF10,047) and binding at the phencyclidine (PCP) site labelled by [3H]-phencyclidine in rat whole brain membranes. (+)-[3H]-SKF10,047 bound with a KD of 251 +/- 66 nM. [3H]-PCP bound with a KD of 180 +/- 35 nM (KD +/- asymptotic s.e.). The potencies of a range of compounds to displace these ligands were only poorly correlated (r = 0.3). Furthermore selective displacement of (+)-[3H]-SKF10,047 but not of [3H]-PCP was demonstrated using the non-selective dopamine ligand haloperidol and the dopamine2-selective ligand 3-(3-hydroxyphenyl)N-n-propylpiperidine (3PPP). These results indicate that the sigma and PCP sites are different entities. The relationship between binding at the sigma site and dopamine receptors was investigated in rat whole brain membranes and in striatal membranes. (+/-)-SKF10,047 displaced [3H]-haloperidol bound to whole brain membranes with a greater potency than it displaced [3H]-haloperidol bound to striatal membranes. The opposite was true for the dopamine antagonist, clozapine, which showed greater potency in striatal membranes. Comparison of [3H]-haloperidol binding in whole brain and striatum gave only a poor correlation (r = 0.6). Hence, different binding sites would appear to exist in these brain regions, the binding of [3H]-haloperidol to whole brain being predominantly to sigma sites and the binding to striatum being predominantly to dopamine receptors.

Presence of sigma and phencyclidine (PCP)-like receptors in membrane preparations of bovine adrenal medulla.

Receptor binding studies with [3H]-(+)SKF-10047 were carried out to characterize the putative sigma (sigma) and phencyclidine (PCP) receptors in membrane preparations of bovine adrenal medulla. Specific binding of the radiolabelled compound was observed after incubation with the membrane preparation at 37 degrees, the equilibrium being reached at 20 min and the maximal binding being observed with 0.6 mg/ml protein. Saturation binding studies were performed at equilibrium (30 min at 37 degrees with 0.5 mg/ml of membrane protein) in the presence of haloperidol (1 microM) or 1-[1-(2-thienyl) cyclohexyl] piperidine (TCP; 0.2 microM) to block sigma or PCP receptors, respectively. The binding of [3H]-(+)SKF-10047 was characterized by two distinct components. A high affinity binding site (haloperidol sensitive) had an apparent KD of 8.3 nM and a Bmax of 67 pmol/g protein. A lower affinity binding site (TCP sensitive) had an apparent KD of 32.7 nM and a Bmax of 83 pmol/g protein. The drug specificity of the high affinity binding site resembled that of the putative sigma receptor, being potently inhibited by haloperidol and pentazocine. The binding pharmacology of the low affinity site resembled that of the phencyclidine receptor, being potently displaced by TCP and PCP. The binding of [3H]-(+)SKF-10047 to both receptors showed marked stereoselectivity for the dextrorotatory (+) isomer of SKF-10047 and was insensitive to the receptor specific opioid ligands DAGO (mu), DSLET (delta) and U-69593 (kappa). These data indicate that bovine adrenal medulla contains sigma and PCP-like receptors.

In vivo binding of [3H]d-N-allylnormetazocine and [3H]haloperidol to sigma receptors in the mouse brain.

Specific binding to sigma sites has been demonstrated and characterized in vitro using [3H]d-N-allylnormetazocine ([3H]d-NANM) and [3H]haloperidol ([3H]HAL) as ligands. As an extension of these experiments, we examined the regional in vivo specific binding of [3H]d-NANM and [3H]HAL in the mouse brain. Specific in vivo sigma binding was seen with both ligands; average estimates of specific binding across brain regions were 54 per cent and 56 per cent of total brain radioactivity, using [3H]d-NANM and [3H]HAL, respectively. Both ligands showed high levels of specific binding in the cerebellum, medulla-pons and midbrain, and lowest levels in the hippocampus. Estimated average [3H]d-NANM binding to phencyclidine (PCP) receptors across seven brain regions was only 13 per cent of total brain radioactivity, and showed a more uniform regional distribution than sigma binding. While the distributions of in vivo specific binding of [3H]d-NANM and [3H]HAL to sigma sites were comparable to findings obtained in vitro, the present estimates of in vivo [3H]d-NANM binding to PCP sites did not resemble the distribution of PCP receptors found in vitro. The results suggest that radiolabelled d-NANM and HAL may be useful for imaging sigma binding sites in vivo.

Discriminative stimulus properties of (+)-N-allylnormetazocine in the rat: correlations with (+)-N-allylnormetazocine and phencyclidine receptor binding.

Recent studies have identified a stereospecific (+)-NANM binding site that binds psychotomimetic opioids and phencyclidine (PCP) but has a distribution in brain different from the PCP binding site. Since (+)-NANM has no opioid activity and (-)-NANM has opioid activity, rats were trained to discriminate (+)-NANM from saline in order to develop an ability to distinguish the (+)-NANM cues from other opioid agonist and antagonist activities. Cyclazocine, PCP, and ketamine all produced (+)-NANM-like stimuli in a dose-dependent manner. Behaviorally, cyclazocine and PCP are equipotent to (+)-NANM whereas ketamine is 6.7 times less potent than (+)-NANM. Pentazocine had the highest affinity for the (+)-[3H]NANM binding site, yet did not produce (+)-NANM-like discriminative stimuli. By contrast, ketamine had the lowest binding affinity for the (+)-[3H]NANM binding site and did produce (+)-NANM-like discriminative stimuli. Drug discrimination potencies relative to (+)-NANM were not predictive of relative binding affinities at (+)-NANM or PCP binding sites, although there was a trend toward a stronger correlation with the PCP binding site. Therefore, the discriminative stimulus properties of (+)-NANM cannot be explained by pharmacologic actions at either (+)-NANM or PCP binding sites alone, and may involve concurrent actions at both sites.

3H-SKF10047 receptor binding studies. Attempts to define the opioid sigma receptor.

The results of experiments are described in which attempts have been made to use the multi-site ligand (d,l)3H-SKF10047 to define a binding site that may be the opioid sigma site. This was attempted by using highly selective blocking agents to eliminate binding at the other opioid sites. The remaining bound 3H-SKF10047, approximately 20% of total specific bound, was then characterized using competitive binding studies with different types of opioids. Under these conditions, a binding site was identified which is highly selective for the benzmorphans and certain morphinans known to cause dysphoria. The kappa selective agent (l)U-50488 did not demonstrate high activity for this site and neither did several mu opioids, PCP or the (d) enantiomers of two opioid benzmorphans. The carefully defined site may be the putative opioid sigma receptor.

Opiate and opioid peptide binding in rat goldfish: further evidence for opiate receptor heterogeneity.

The binding of a series of 3H-labeled mu, kappa, sigma and delta opioid agonists and an antagonist has been examined in rat and goldfish brain membranes. A variety of treatments, including NaCl, N-ethylmaleimide, trypsin and chymotrypsin show dramatic differences both between the two species and between the different 3H-ligands.

The anti-amnesic effects of sigma1 (sigma1) receptor agonists confirmed by in vivo antisense strategy in the mouse.

The sigma1 (sigma1) receptor cDNA was recently cloned in several animal species, including the mouse. In order to firmly establish the implication of sigma1 receptors in memory, a phosphorothioate-modified antisense oligodeoxynucleotide (aODN) targeting the sigma1 receptor mRNA and a mismatched analog (mODN) were administered intracerebroventricularly for 3 days in mice. Scatchard analyses of in vitro (+)-[3H]SKF-10,047 binding to sigma1 sites showed that Bmax values were significantly decreased in the hippocampus (-58.5%) and cortex (-38.1%), but not in the cerebellum, of aODN treated mice, as compared to saline- or mODN-treated animals. In vivo binding levels were also significantly decreased after aODN treatment in the hippocampus and cortex but not in the cerebellum. The anti-amnesic effects of the selective sigma1 agonists PRE-084 or SA4503 were evaluated against the learning impairments induced by dizocilpine or scopolamine, respectively, using spontaneous alternation behavior and passive avoidance task. The anti-amnesic effects of PRE-084 or SA4503, observed after saline- or mODN-treatment, were blocked after aODN administration. These observations bring a molecular basis to the modulatory role of sigma1 receptors in memory processes.

Beneficial effects of sigma agonists on the age-related learning impairment in the senescence-accelerated mouse (SAM).

A beneficial effect of sigma (sigma) agonists was previously described on several pharmacological models of learning impairments. We examined this effect in senescence-accelerated mice (SAM), which has been developed as a murine model of aging and cognitive dysfunction. SAMP8/Ta (P8, senescence-prone substrain), 10-12 months of age, showed significant impairments in mnemonic capacities, as compared to age-matched SAMR1/Ta controls (R1, senescence-resistant substrain). Tests included open-field behavior, spontaneous alternation performances in the Y-maze, step-down passive avoidance and place learning after repetitive training in a water-maze. Pretreatment with the sigma agonists JO-1784 (igmesine) or PRE-084, at 0.1-3 mg/kg, s.c., significantly improved spontaneous alternation and passive avoidance performances in P8. JO-1784 or PRE-084, at 1 mg/kg, also improved place learning in the water-maze, and retention, in term of escape latency. The implication of sigma sites was indicated by the lack of significant effect of JO-1783, the inactive enantiomer of JO-1784, and by the ability of BMY-14802 (5 mg/kg, i.p.) to antagonize the effects on passive avoidance of JO-1784 (0.5 mg/kg) or PRE-084 (1 mg/kg). Subchronic treatments with JO-1784 (0.5 mg/kg/day) or PRE-084 (1 mg/kg/day) during 10 days, allowed a significant improvement of learning during training in the water-maze, but retention was not significantly ameliorated. These results confirmed the interest of the SAM substrains as an experimental model for senile memory impairment and showed that sigma agonists could improve the quality of learning, although they seem less effective on long-term memory retrieval upon chronic administration.

(+)-[3H]SK&F 10,047 binding sites in rat liver.

Stereospecific binding sites for (+)-[3H]SKF 10,047 have been demonstrated in rat liver. These binding sites were shown to be opioid receptors of the sigma type, and this was confirmed by their binding properties: reversibility, saturability, stereospecificity, effects of ions and various pharmacological drugs on (+)-[3H]SK sigma F 10,047 binding. The authors suggest that (+)-[3H]SKF 10,047 binding sites in liver and brain are a part of novel 'sigmergic' regulatory system.

Evidence for a multi-site model of the rat brain sigma receptor.

Irradiation of rat brain membranes with light of 254 nm, a treatment which modifies ultra-violet absorbing residues in proteins, decreased binding of both [3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ([ 3H](+)-3-PPP) and [3H]1,3-di-o-tolylguanidine ([3H]DTG) to sigma receptors. For [3H](+)-3-PPP, this was due to a decreased Bmax. In contrast, irradiation markedly increased binding of [3H](+)-N-allylnormetazocine ([3H](+)-SKF 10,047) due to a decrease in the Kd. Both unlabeled DTG and haloperidol were competitive inhibitors of [3H](+)-3-PPP binding to untreated membranes, causing an increase in the Kd and no change in the Bmax. The benzomorphans, (+)-SKF 10,047 and (+)-pentazocine, were uncompetitive inhibitors, causing a decrease in both the Kd and Bmax for [3H](+)-3-PPP. Finally, the ability of DTG and (+)-3-PPP to inhibit binding of [3H](+)-SKF 10,047 was markedly reduced by ultra-violet irradiation, whereas irradiation had little effect on the potency of unlabeled (+)-SKF 10,047 and (+)-pentazocine. These data suggest that sigma-related (+)-benzomorphans and non-benzomorphans interact either with distinct, allosterically coupled sites on the same sigma receptor macromolecule or with different populations of sigma receptor types.

[3H]PCP-3-OH and (+)[3H]SKF 10047 binding sites in rat brain membranes: evidence of multiplicity.

Specific binding of one of the most potent analogs of phencyclidine (PCP), [3H]PCP-3-OH, in rat brain membranes revealed the labeling of high (Kd = 0.5 nM) and low (Kd = 16 nM) affinity binding sites. (+)SKF 10047 potently inhibited high, but not low, affinity [3H]PCP-3-OH binding. (+)[3H]SKF 10047 apparently labeled the high affinity PCP-3-OH binding site and also an additional site, sensitive to haloperidol, which is distinct from the two sites labeled by [3H]PCP-3-OH.