Wayne D. Bowen

Rat liver and kidney contain high densities of σ1 and σ2 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 σ1 probe, bound to sites in liver membranes with Kd = 7.5 nM and Bmax = 2929 fmol/mg protein. [3H](+)-Pentazocine binding sites in kidney had Kd = 23.3 nM and Bmax = 229fmol/mg protein. [3H1,3- Di-o-tolylguanidine ([3H]DTG) and [3H](+)-3-(3-hydroxyphenyl-N-(1-propyl)piperidine ([3H](+)-3-PPP) label both σ1 and σ2 receptors. Parameters for [3H]DTG in the liver were Kd = 17.9 nM and Bmax - 11 895 fmol.mg protein. Similar parameters were observed for [3H](+)-3-PPP, Kd = 51.9 nM and Bmax = 11 070 fmol/mg protein. [3H]DTG bound to rat kidney with Kd = 45.8 nM and Bmax = 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 σ1 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 σ2 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 1S, 2R-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-5H-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 σ2 and σ2 receptors, respectively. Thus, rat liver and kidney contain high densities of σ1 and σ2 receptors, with σ2 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.

A sigma-like binding site in rat pheochromocytoma (PC12) cells: decreased affinity for (+)-benzomorphans and lower molecular weight suggest a different sigma receptor form from that of guinea pig brain

Two highly selective radiolabeled probes for sigma receptors were found to bind with high affinity and capacity to membranes from undifferentiated PC12 cells. [3H]1,3-di-o-tolylguanidine [( 3H]DTG) bound with Kd = 23.7 +/- 4.6 nM and Bmax = 2025 +/- 660 fmol/mg protein. The Kd and Bmax for [3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ([3H](+)-3-PPP) were 86.3 +/- 21.6 nM and 1539 +/- 242 fmol/mg protein, respectively. These binding parameters were comparable to those observed in guinea pig brain, although the Kd for [3H](+)-3-PPP was 3-fold higher in the PC12 membranes. Both the PC12 and guinea pig brain sites exhibited high affinity for haloperidol, moderate affinity for phencyclidine (PCP), and negligible affinity for MK-801, apomorphine, and (-)-sulpiride. These data suggest a relationship of the PC12 site to sigma receptors. However, all (+)-opiates [+)-benzomorphans and (+)-morphinans) tested bound with markedly lower affinity to the PC12 site compared to guinea pig brain. These include (+)-N-allylnormetazocine [+)-SKF 10,047), (+)-pentazocine, and dextrallorphan. In fact, PC12 sites exhibited preference for (-)-benzomorphans, the reverse stereoselectivity of guinea pig brain sites. Binding of [3H]N-[1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) could not be detected, demonstrating absence of PCP receptors on this cell line. Differentiation of cells by treatment with nerve growth factor had no effect on sigma binding parameters. Membranes from guinea pig brain and PC12 cells were photoaffinity-labeled using [3H]azido-di-o-tolylguanidine. In guinea pig brain, a polypeptide of 25 kDa was specifically labeled. However, label was incorporated into polypeptides of 18 kDa and 21 kDa in membranes from PC12 cells. In view of the otherwise similar binding characteristics, the marked differences in affinity for (+)-benzomorphans and molecular weight suggest that PC12 cells contain a molecular form of sigma receptor distinct from that predominant in guinea pig brain. This raises the possibility of multiple sigma receptor types.

Characterization of two novel σ receptor ligands : antidystonic effects in rats suggest σ receptor antagonism

The novel σ receptor ligands, N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino)ethylamine (BD1047) and 1-[2-(3,4-dichlorophenyl)ethyl]-4-methylpiperazine (BD1063), were characterized in rats using binding assays and behavioral studies. In radioligand binding studies, the novel ligands showed marked selectivity for σ binding sites, generally having a 100-fold or better affinity for σ sites compared to nine other tested receptors (opiate, phencyclidine, muscarinic, dopamine, α1-, α2-, β-adrenoceptor, 5-HT1, 5-HT2); the only exception was the affinity of BD1047 for β-adrenoceptors. Competition assays further revealed that the drugs interacted with both σ1 and σ2 binding sites. Although both drugs had preferential affinities for σ1 sites, BD1047 exhibited a higher affinity for σ2 sites than BD1063. In behavioral studies, BD1047 and BD1063 had no effects on their own when unilaterally microinjected into the red nucleus of rats, but both compounds attenuated the dystonia produced by the high affinity σ ligands, di-o-tolylguanidine (DTG) and haloperidol. BD1047 and BD1063 dose-dependently attenuated the dystonia produced by DTG, suggesting a receptor-mediated mechanism, and the dose curve for DTG was shifted to the right in the presence of the novel ligands. BD1047 and BD1063 appear to act as antagonists at σ sites and may represent promising new tools for probing other functional effects associated with σ binding sites.

Sigma receptors: recent advances and new clinical potentials

Several recent advances are leading to a better understanding of sigma receptors. Here we focus on our recent findings regarding cellular functions of sigma-2 receptors and discuss their possible clinical implications. Agonists at sigma-2 receptors induced changes in cell morphology and apoptosis in various cell types. Sigma-2 receptor activation produced both transient and sustained increases in [Ca++]i, derived from different intracellular stores. These changes in [Ca++]i and cytotoxic effects are mediated by intracellular sigma-2 receptors. Sigma-2 agonists induced apoptosis in drug-resistant cancer cells, enhanced the potency of DNA damaging agents, and down-regulated expression of p-glycoprotein mRNA. Thus, sigma-2 receptor agonists may be useful in treatment of drug-resistant cancers. Sigma radioligands have been used in tumor imaging. We also discuss how sigma-2 antagonists might prevent the irreversible motor side effects of typical neuroleptics. Sigma-2 receptors may subserve a novel signalling pathway to apoptosis, involved in regulation of cell proliferation and/or viability.

Cytotoxic effects of sigma ligands: sigma receptor-mediated alterations in cellular morphology and viability

The morphological effects of several neuroleptics as well as other novel and prototypic sigma ligands were examined by addition to cultures of C6 glioma cells. Sigma ligands caused loss of processes, assumption of spherical shape, and cessation of cell division. The time course and magnitude of this effect were dependent on the concentration of sigma ligand. Continued exposure to sigma compounds ultimately resulted in cell death. However, the morphological effect was reversible when sigma ligand was removed shortly after rounding. The potency of compounds to produce these effects generally correlated with binding affinity at sigma receptors of C6 glioma cell membranes labeled with [3H](+)-pentazocine. At a concentration of 100 microM, haloperidol, reduced haloperidol, fluphenazine, perphenazine, trifluoperazine, BD737, LR172, BD1008, and SH344 produced significant effects in 3–6 hr of exposure. Other compounds, such as trifluperidol, thioridazine, and (-)-butaclamol, produced significant effects by 24 hr of exposure. Despite the requirement of micromolar concentrations of ligand (some compounds were effective at 30 microM), the effect showed a remarkable specificity for compounds exhibiting sigma receptor binding affinity. Neuroleptics lacking potent sigma affinity [e.g., (-)- sulpiride, (+)-butaclamol, and clozapine] and other compounds that lack significant sigma affinity but that are agonists or antagonists at dopamine, serotonin, adrenergic, glutamate, phencyclidine, GABA, opiate, or muscarinic cholinergic receptors were without effect on cellular morphology at concentrations up to 300 microM over a period of 72 hr. Likewise, blockers and activators of Na+, K+, and Ca2+ channels and a monoamine oxidase inhibitor devoid of sigma affinity were without effect. Interestingly, 1,3-di-o-tolylguanidine (DTG), (+)-3-(3- hydroxyphenyl)-N-(1-propyl)piperidine [(+)-3-PPP], (+)-pentazocine, (+)- cyclazocine, and other sigma-active benzomorphans and morphinans appeared inactive in up to 72 hr of culture. However, these compounds interacted synergistically with a subeffective dose of BD737 (30 microM) to produce effects usually in 6 hr or less. Also, the pH of the culture medium had a profound effect on the activity of sigma compounds. Increasing the pH from the normal range of 7.2–7.4 to pH 8.3– 8.5 shifted the dose curves (30, 100, 300 microM) for all sigma compounds to the left. Under these conditions, DTG, (+)-3-PPP, and benzomorphans produced effects in 24 hr or less.(ABSTRACT TRUNCATED AT 400 WORDS)

Metabolites of haloperidol display preferential activity at σ receptors compared to dopamine D-2 receptors

Haloperidol bound with equal affinity to sigma and dopamine D-2 receptors (KI = 2.8 nM). Compared to haloperidol, its carbonyl-reduced metabolite bound to sigma receptors with nearly equal affinity. However, reduced haloperidol bound to dopamine receptors with 85-fold lower affinity compared to haloperidol (KI = 239 nM). The chlorophenyl-hydroxy-piperidine metabolite of haloperidol lacked affinity for dopamine receptors, but bound with moderate affinity to sigma receptors (KI = 326 nM). The carboxylic acid metabolite lacked affinity for either receptor. Like haloperidol, (+)-pentazocine, and 1,3-di-o-tolylguanidine, reduced haloperidol potently inhibited the phosphoinositide response to muscarinic agonists in rat brain synaptoneurosomes, an assay which monitors sigma agonist activity. This metabolite also produced a dystonic alteration of head position in rats when microinjected into the red nucleus. However, unlike observations with haloperidol and other sigma ligands, this effect was associated with pathological changes in the red nucleus. Therefore, it cannot be attributed to sigma receptor interactions with certainty. These findings suggest that administration of haloperidol results initially in effects mediated through both dopamine and sigma receptors, but as metabolism proceeds the sigma actions would be expected to decline at a significantly slower rate than the dopaminergic actions.

Evidence for a multi-site model of the rat brain σ 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 σ 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 irradiotion, whereas irradiation had little effect on the potency of unlabeled (+)-SKF 10,047 and (+)-pentazocine. These data suggest that σ-related (+)-benzomorphans and non-benzomorphans interact either with distinct, allosterically coupled sites on the same σ receptor macromolecule or with different populations of σ receptor types.

Synthesis and evaluation of optically pure [3H]-(+)-pentazocine, a highly potent and selective radioligand for σ receptors

Tritium‐labeled (+)‐pentazocine ([3H]‐1b) of specific activity 26.6 Ci/mmol was synthesized in 3 steps starting with (+)‐normetazocine (2) of defined optical purity. [3H]‐1b has been characterized as a highly selective ligand for labeling of σ receptors. Competition data revealed that [3H]‐1b could be displaced from guinea pig brain membrane preparations with a number of commonly used σ receptor ligands. [3H]‐1b exhibited saturable, enantioselective binding with a K d of 5.13±0.97 nM and a B max of 1146±122 fmol/mg protein. Phencyclidine (PCP) displaced [3H]‐1b with low affinity while MK‐801 was inactive, thus indicating insignificant activity at the PCP‐binding site; apomorphine failed to displace [3H]‐1b indicating lack of dopamine receptor cross‐reactivity. Since the affinity of [3H]‐1b is about 6 times that of the two commonly employed σ ligands ((+)‐3‐[3H]PPP and [3H]DTG) and since it is more selective for σ receptors than the benzomorphan [3H]SKF‐10,047, it represents the first example of a highly selective benzomorphan based σ receptor ligand. [3H]‐1b should prove useful for further study of the structure and function of σ receptors.

Two novel σ receptor ligands, BD1047 and lr172, attenuate cocaine-induced toxicity and locomotor activity

Abstract The ability of cocaine to interact with σ receptors indicates that these sites may mediate the negative properties associated with cocaine use, such as toxicity and addiction. Previous studies have shown that the novel σ receptor ligand, BD1008 (N-[2-(3,4-dicholophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)ethylamine), effectively protects against cocaine-induced convulsions and locomotor activity in mice. Therefore, BD1047 ([2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(diamino)ethylamine) and LR172 (N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-homopiperidinyl)ethylamine), two analogs of BD1008, were tested to determine if they also have anti-cocaine properties. Receptor binding assays showed that BD1047 and LR172 both have high affinities for σ receptors, but low to negligible affinities for dopamine, opioid, phencyclidine, and 5-HT2 sites. In behavioral studies, pretreatment of mice with BD1047 or LR172 reduced the convulsions, lethality, and locomotor activity produced by cocaine. The data indicates a possible role for σ receptor ligands in the treatment of cocaine overdose and addiction.

Involvement of sigma receptors in the behavioral effects of cocaine: evidence from novel ligands and antisense oligodeoxynucleotides

Pharmacological and molecular biological tools were used to validate the involvement of sigma receptors in the actions of cocaine. Radioligand binding studies demonstrated significant levels of sigma receptors in the brain and heart, where cocaine interacts preferentially with the sigma(1) subtype. In behavioral pharmacological studies using mice, nine novel sigma receptor antagonists significantly attenuated cocaine-induced convulsions, while structural analogs with weak interactions with sigma receptors were ineffective. In contrast to the protection provided by the antagonists, a classical sigma receptor agonist exacerbated the convulsive effects of cocaine. The antagonists also attenuated cocaine-induced lethality, with the best compound protecting against death even when administered as a post-treatment. At doses where the antagonists had no effect on baseline locomotor activity, they significantly attenuated the locomotor stimulatory effects of cocaine, suggesting their ability to block the psychomotor as well as the toxic effects of cocaine. To further validate that the anti-cocaine effects were achieved by interfering with cocaines access to sigma receptors, antisense oligodeoxynucleotides against sigma(1) receptors were shown to attenuate the convulsive and locomotor stimulatory effects of cocaine. Together, the studies support the involvement of sigma receptors, particularly the sigma(1) subtype, in the behavioral effects of cocaine.