Di-o-tolylguanidine Research Articles

Synthesis of tolyl guanidine as copper corrosion inhibitor with a complementary study on electrochemical and in silico evaluation

A rapid and new synthetic route for N,N′-di-o-tolyl guanidine (DTG) synthesis from cheap materials is reported. The performance of DTG as an excellent inhibitor for delaying copper (Cu) corrosion with an efficiency higher than 98% at 20 × 10−6 M in an acidic solution was investigated via electrochemical measurements. These measurements included PDP, EFM, and EIS spectroscopy. The experimental data indicated that DTG has an efficient inhibiting effect on the corrosion of Cu in acidic media.The DTG was adsorbed on to the Cu surface via chemical adsorption and followed the Langmuir route. The PDP measurements revealed that DTG acted as a mixed inhibitor. Furthermore, EIS data showed that the DTG adsorbed through the metal/electrolyte interface. This resulted in forming a DTG protective layer on the Cu surface, thereby impeding the dissolution of Cu in the acidic solution. The corrosive solution containing the DTG inhibitor after immersion of the Cu specimen for 48 h, which promoted the formation of a complex between the Cu cation and DTG, was investigated via ultraviolet/visible spectroscopy. In addition, the formation of a DTG protective layer on the Cu surface was confirmed via scanning electron microscopy and atomic force microscopy analysis of the Cu surface morphology. Moreover, the active centers for interaction with the Cu surface in an acidic solution were investigated via in silico evaluation of DTG.

Hazards of Using Masking Protocols When Performing Ligand Binding Assays: Lessons From the Sigma-1 and Sigma-2 Receptors.

Sigma-1 and sigma-2 receptors are emerging therapeutic targets. Although the molecular identity of the sigma-2 receptor has recently been determined, receptor quantitation has used, and continues to use, the sigma-1 selective agents (+) pentazocine or dextrallorphan to mask the sigma-1 receptor in radioligand binding assays. Here, we have assessed the suitability of currently established saturation and competition binding isotherm assays that are used to quantify parameters of the sigma-2 receptor. We show that whilst the sigma-1 receptor mask (+) pentazocine has low affinity for the sigma-2 receptor (Ki 406 nM), it can effectively compete at this site with [³H] di-O-tolyl guanidine (DTG) at the concentrations frequently used to mask the sigma-1 receptor (100 nM and 1 µM). This competition influences the apparent affinity of DTG and other ligands tested in this system. A more troublesome issue is that DTG can displace (+) pentazocine from the sigma-1 receptor, rendering it partly unmasked. Indeed, commonly used concentrations of (+) pentazocine, 100 nM and 1 µM, allowed 37 and 11% respectively of sigma-1 receptors to be bound by DTG (300 nM), which could result in an overestimation of sigma-2 receptor numbers in assays where sigma-1 receptors are also present. Similarly, modelled data for 1 µM dextrallorphan show that only 71–86% of sigma-1 receptors would be masked in the presence of 300 nM DTG. Therefore, the use of dextrallorphan as a masking agent would also lead to the overestimation of sigma-2 receptors in systems where sigma-1 receptors are present. These data highlight the dangers of using masking agents in radioligand binding studies and we strongly recommend that currently used masking protocols are not used in the study of sigma-2 receptors. In order to overcome these problems, we recommend the use of a cell line apparently devoid of sigma-1 receptors [e.g., MCF7 (ATCC HTB-22)] in the absence of any masking agent when determining the affinity of agents for the sigma-2 receptor. In addition, assessing the relative levels of sigma-1 and sigma-2 receptors can be achieved using [³H] DTG saturation binding followed by two-site analysis of (+) pentazocine competition binding with [³H] DTG.

Effect of Sigma agonist 1, 3 di-o-tolyl-guanidine (DTG) on Squizophrenia and Immobility responses

Recently in our laboratory we found that DTG (sigma receptor agonist 1,3-di-o-tolyl-guanidine) sub-chronic administration reduced the hyperlocomotor activity and reversed the neuronal hypotrophy in the prefrontal cortex, amygdala and nucleus accumbens, generated in rats with neonatal ventral hippocampus lesion (nVHL). We also observed that DTG reversed some of the behavioral and neuromorphological effects of nVHL rats, which supports the possibility that DTG has beneficial effects in the management of symptoms of schizophrenia. We also found that DTG had effects on immobility responses, in the unlesioned rats increased the duration of the dorsal immobility but it did not have effect on the duration of immobility elicited by clamping. However, the nVHL increased the duration of immobility elicited by clamping, but did not have effect on the duration of the dorsal immobility. It should be noted that DTG counteracted the increase in the duration of the immobility by clamping produced by nVHL. The increase in the duration of the dorsal immobility produced by DTG was counteracted by nVHL. We suggested that the differential effect on these two immobility responses, is due to they are different varieties of immobility mediated by different mechanisms. Therefore, we believe that this evidence could help us to connect, schizophrenia with immobility reaction, which may be associated or somehow present in catatonic schizophrenia Key words; immobility response, schizophrenia, neonatal ventral hippocampus lesion, sigma agonist.

Pharmacological evaluation of SN79, a sigma (σ) receptor ligand, against methamphetamine-induced neurotoxicity in vivo

Methamphetamine is a highly addictive psychostimulant drug of abuse, causing hyperthermia and neurotoxicity at high doses. Currently, there is no clinically proven pharmacotherapy to treat these effects of methamphetamine, necessitating identification of potential novel therapeutic targets. Earlier studies showed that methamphetamine binds to sigma (σ) receptors in the brain at physiologically relevant concentrations, where it “acts in part as an agonist.” SN79 (6-acetyl-3-(4-(4-(4-florophenyl)piperazin-1-yl)butyl)benzo[d]oxazol-2(3H)-one) was synthesized as a putative σ receptor antagonist with nanomolar affinity and selectivity for σ receptors over 57 other binding sites. SN79 pretreatment afforded protection against methamphetamine-induced hyperthermia and striatal dopaminergic and serotonergic neurotoxicity in male, Swiss Webster mice (measured as depletions in striatal dopamine and serotonin levels, and reductions in striatal dopamine and serotonin transporter expression levels). In contrast, di-o-tolylguanidine (DTG), a well established σ receptor agonist, increased the lethal effects of methamphetamine, although it did not further exacerbate methamphetamine-induced hyperthermia. Together, the data implicate σ receptors in the direct modulation of some effects of methamphetamine such as lethality, while having a modulatory role which can mitigate other methamphetamine-induced effects such as hyperthermia and neurotoxicity.

Expression of σ receptors of human urinary bladder tumor cells (RT-4 cells) and development of a competitive receptor binding assay for the determination of ligand affinity to human σ 2 receptors

A selective competitive binding assay for the determination of the affinity of compounds to the human σ 2 receptor using 96-well multiplates and a solid state scintillator was developed. In the assay system, [ 3H]ditolylguanidine (DTG) was used as radioligand and membrane homogenates from human RT-4 cells physiologically expressing σ 2 receptors served as receptor material. In order to block the interaction of the unselective radioligand [ 3H]DTG with σ 1 receptors, all experiments were performed in the presence of the σ 1 selective ligand (+)-pentazocine. The density of σ 2 receptors of the cells was analyzed by a saturation experiment with [ 3H]DTG. The radioligand [ 3H]DTG was bound to a single, saturable site on human σ 2 receptors, resulting in a B max value of 2108 ± 162 fmol/mg protein and K d-value of 8.3 ± 2.0 nM. The expression of competing σ 1 receptors was evaluated by performing a saturation experiment using the σ 1 selective radioligand [ 3H](+)-pentazocine, which resulted in a B max value of 279 ± 40 fmol/mg protein and K d value of 13.4 ± 1.6 nM. For validation of the σ 2 binding assay, the K i-values of four σ 2 ligands (ditolylguanidine, haloperidol, rimczole and BMY-14802) were determined with RT-4 cell membrane preparations. The K i values obtained from these experiments are in good accordance with the K i-values obtained with rat liver membrane preparations as receptor material and with K i values given in the literature.

Delayed treatments for stroke influence neuronal death in rat organotypic slice cultures subjected to oxygen glucose deprivation

A major limitation of current stroke therapies is the need to treat candidate patients within 3 h of stroke onset. Human umbilical cord blood cell (HUCBC) and the sigma receptor agonist 1,3, di-o-tolylguanidine (DTG) administration both caused significant reductions in brain damage in the rat middle cerebral artery occlusion model of stroke when administered at delayed timepoints. In vivo, these treatments suppress the infiltration of peripheral lymphocytes into the brain in addition to decreasing neurodegeneration. An ex vivo organotypic slice culture (OTC) model was utilized to characterize the efficacy of these treatments in mitigating neurodegeneration in ischemic brain tissue in the absence of the peripheral immune system. Slice cultures subjected to oxygen glucose deprivation (OGD) had significantly elevated levels of degenerating neurons and microglial nitric oxide production when compared to their normoxic counterparts. In cultures subjected to OGD, HUCBC but not DTG treatment reduced the number of degenerating neurons and the production of microglial derived nitric oxide back to levels detected in normoxic controls. These data show that HUCBC treatment can mediate direct neuroprotection and suppress innate inflammation in ischemic brain tissue in the absence of the peripheral immune system, whereas DTG requires peripheral effects to mediate neuroprotection. These experiments yield insight into the mechanisms by which these neuroprotective treatments function at delayed timepoints following stroke.

Female Sex Hormones Decrease Constitutive Endothelin-1 Release via Endothelial Sigma-1/Cocaine Receptors: An Action Independent of the Steroid Hormone Receptors

Cardiovascular disease is rare in premenopausal women compared to men. The authors investigate sex hormone-induced endothelin-1 (ET-1) release and the involvement of classic sex hormone receptors as well as the ability of sigma-1/cocaine receptors to respond to sex hormones. ET-1 release was measured in the supernatant of endothelial cells after treatment with beta-estradiol, progesterone, testosterone, or combined with their antagonists, and with the sigma-1 receptor ligand ditolylguanidine (DTG), or haloperidol, a sigma-1 receptor antagonist. Binding assays were performed using 2.5 x 10(-8) M [3H]DTG. Female sex hormones decreased ET-1 release whereas testosterone increased it, sex hormone antagonists only slightly attenuated or had no effect on the respective hormone's effect. DTG totally blocked the female sex hormone-induced inhibition on ET-1 release, whereas testosterone-induced stimulation was not affected. However, haloperidol blocked both. [3H]DTG binding was displaced by beta -estradiol but not by testosterone. DTG-binding sites account for 513 +/- 114 per cell, KD 8.79 nM. These data suggest that besides classic steroid hormone receptors, sigma-1/cocaine receptors mediate the effects of female sex hormones on ET-1 release, an up to now unknown signalling pathway. Results also suggest that female and male sex hormones may bind to different sites on sigma-1 receptors, exerting opposite pharmacological effects.

Conformationally restricted analogs of BD1008 and an antisense oligodeoxynucleotide targeting σ 1 receptors produce anti-cocaine effects in mice

Cocaine's ability to interact with σ receptors suggests that these proteins mediate some of its behavioral effects. Therefore, three novel σ receptor ligands with antagonist activity were evaluated in Swiss Webster mice: BD1018 (3 S-1-[2-(3,4-dichlorophenyl)ethyl]-1,4-diazabicyclo[4.3.0]nonane), BD1063 (1-[2-(3,4-dichlorophenyl)ethyl]-4-methylpiperazine), and LR132 (1 R,2 S-(+)- cis- N-[2-(3,4-dichlorophenyl)ethyl]-2-(1-pyrrolidinyl)cyclohexylamine). Competition binding assays demonstrated that all three compounds have high affinities for σ 1 receptors. The three compounds vary in their affinities for σ 2 receptors and exhibit negligible affinities for dopamine, opioid, GABA A and NMDA receptors. In behavioral studies, pre-treatment of mice with BD1018, BD1063, or LR132 significantly attenuated cocaine-induced convulsions and lethality. Moreover, post-treatment with LR132 prevented cocaine-induced lethality in a significant proportion of animals. In contrast to the protection provided by the putative antagonists, the well-characterized σ receptor agonist di- o-tolylguanidine (DTG) and the novel σ receptor agonist BD1031 (3 R-1-[2-(3,4-dichlorophenyl)ethyl]-1,4-diazabicyclo[4.3.0]nonane) each worsened the behavioral toxicity of cocaine. At doses where alone, they produced no significant effects on locomotion, BD1018, BD1063 and LR132 significantly attenuated the locomotor stimulatory effects of cocaine. To further validate the hypothesis that the anti-cocaine effects of the novel ligands involved antagonism of σ receptors, an antisense oligodeoxynucleotide against σ 1 receptors was also shown to significantly attenuate the convulsive and locomotor stimulatory effects of cocaine. Together, the data suggests that functional antagonism of σ receptors is capable of attenuating a number of cocaine-induced behaviors.

Relationship between modulation of the cerebellorubrospinal system in the in vitro turtle brain and changes in motor behavior in rats: effects of novel sigma ligands

Saturation and competition binding studies showed that the turtle brain contains σ sites labeled by both [ 3H]di-o-tolylguanidine (DTG) and [ 3H](+)-pentazocine. There was a significant correlation between the IC 50 values of σ ligands for [ 3H]DTG sites in the turtle vs. rat brain, suggesting that the sites are comparable in the two species. In contrast, [ 3H](+)-pentazocine, which primarily labels σ1 sites in the rodent brain, labels a heterogeneity of sites in the turtle brain. In extracellular recordings from the in vitro turtle brainstem, some σ ligands enhanced the burst responses of red nucleus (RN) neurons (DTG, haloperidol, BD1031, BD1052, BD1069) while other σ ligands decreased the burst responses (BD1047, BD1063). Control compounds (turtle Ringer vehicle control, opiate antagonist naloxone, atypical neuroleptic sulpiride) had no significant effects on the RN burst responses recorded from the in vitro turtle brain. The ED 50s of the ligands for altering the burst responses in RN neurons from the turtle brain were correlated with their IC 50s for turtle brain sites labeled with [ 3H]DTG, but not [ 3H](+)-pentazocine; this pattern is identical to that previously reported in rats, where there is a correlation between the potencies of σ ligands for producing dystonic postures after microinjection into the rat RN and their binding to rat brain sites labeled with [ 3H]DTG, but not [ 3H](+)-pentazocine. When the novel σ ligands were microinjected into the rat RN, dystonic postures were produced by ligands that increased the burst duration of RN neurons in the turtle brain. Novel σ ligands that reduced the burst responses in the in vitro turtle brain have previously been reported to have no effects on their own when microinjected into the rat RN, but to block the dystonic postures produced by other σ ligands. Taken together, the data suggest that the opposite effects of the novel ligands in the turtle electrophysiological studies represent the actions of agonists vs. antagonists, and that the directionality of the effects has predictive value for the expected motor effects of the drugs.

Involvement of σ receptors in the modulation of the glutamatergic/NMDA neurotransmission in the dopaminergic systems

Extracellular single-unit recordings and iontophoresis were used to examine the effects of different selective σ receptor ligands on dopaminergic and glutamatergic N-methyl- d-aspartate (NMDA) neurotransmissions both in origin (A10 and A9 areas) and terminal (nucleus accumbens and caudate nucleus) regions of the rat mesolimbic and nigrostriatal dopaminergic systems. The selective σ 1 receptor ligands 2-[4-(4-methoxy-benzyl)piperazin-1-yl-methyl]4-oxo[4 H]-benzo-thiazolin-2-one (S-21377), systemically administered (1.2 mg/kg, i.v., cumulative dose), and 2[(4-benzyl piperazin-1-yl) mothyl] naphthalene, dichiorydrate (S-21378), iontophoretically applied, slightly increased the spontaneous firing rate and potentiated the NMDA-induced neuronal activation of dopaminergic neurons in the A9 and A10 regions. (+) N-cyclopropylmethyl- N-methyl-1,4-diphenyl-1-ethyl-butyl-2- N (JO-1784), another selective σ 1 receptor ligand produced no or little effect in these areas. The systemic administration of the selective σ 2 receptor ligand 1,4-bis-spiro[isobenzofuran-1(3 H), 4′-piperidin-1′yl]butane (Lu 29–252) (2 mg/kg, i.v., cumulative dose) did not modify the firing activity of A9 and A10 dopaminergic neurons, but significantly potentiated the NMDA-induced increase in firing activity of A10 dopaminergic neurons. None of the σ receptor ligands tested had any effects on the dopamine-induced suppression of firing. In the nucleus accumbens, the systemic administration of (JO-1784), (40 μg/kg, i.v.), (+)-pentazocine (30 μg/kg, i.v.), another selective σ 1 receptor ligand, and of the non selective σ 1 receptor ligand di-tolyl-guanidine (DTG) (20 μg/i.v.) produced a significant increase of NMDA-induced neuronal activation. Microiontophoretic applications of JO-1784 also potentiated the NMDA response. They also increased significantly the suppressant effect of dopamine on NMDA and kainate-induced activations of accumbens neurons. In the caudate nucleus, (+)-pentazocine, but not JO-1784, potentiated slightly the neuronal response to NMDA. None of the σ receptor ligands tested did modify significantly the responses of caudate and accumbens neurons to kainate. These findings suggest that at least two subtypes of σ 1 receptors may affect differentially the glutamate NMDA neurotransmission in the terminal and origin regions of the mesolimbic and nigrostriatal dopaminergic systems. These results also demonstrate the existence of a functional interaction between σ 2 and NMDA receptors in the A10 region.

The functional sensitisation of sigma receptors following chronic selective serotonin reuptake inhibitor treatment.

The purpose of the present study was to investigate the potential impairment of normal motor function following chronic selective serotonin reuptake inhibitor treatment that may result from sensitisation of sigma receptors. Rats were chronically treated with either sertraline, citalopram, paroxetine or fluvoxamine and a selective sigma receptor ligand, di-o-tolylguanidine (DTG), for 28 days. All animals then received an acute intra-rubral injection of either DTG or saline. Following the direct injection of DTG into the red nucleus, rats chronically treated with DTG exhibit a maximal behavioural response characterised as a pronounced dystonia. Animals chronically treated with sertraline and citalopram elicited a response similar to that of control animals following the acute DTG challenge, whereas chronic treatment with paroxetine and fluvoxamine significantly decreased and increased the dystonic response, respectively. Facial spasticity and vacuous chewing movements were associated with, and reflected the extent of, the DTG-induced dystonia. Changes in regional biogenic amine concentrations were also determined. The concentrations of serotonin and noradrenaline were determined in the brain stem and cerebellum following the intra-rubral injection of either saline or DTG in animals that had been chronically treated with a selective serotonin reuptake inhibitor or DTG. There was a significant increase in serotonin concentration in the brain stem as a result of chronic DTG and fluvoxamine treatments. The increase in serotonin correlated with the reported potentiation of dystonia in animals that received 28 days treatment with these drugs. The potentiation of dystonia following chronic DTG and fluvoxamine treatments suggests that these drugs sensitise the sigma2 receptors, an effect that does not appear to be shared by citalopram, sertraline or paroxetine.

Potentiation by dehydroepiandrosterone of the neuronal response to N-methyl-D-aspartate in the CA3 region of the rat dorsal hippocampus: an effect mediated via sigma receptors.

We have previously shown in vivo that low doses of selective sigma (sigma) receptor ligands potentiate selectively and dose-dependently the excitatory response of pyramidal neurons to microiontophoretic applications of N-methyl-D-aspartate (NMDA) in the CA3 region of the rat dorsal hippocampus. As several neuroactive steroids such as progesterone and testosterone have a high affinity for sigma receptors, the effects of some neuroactive steroids on the NMDA-induced neuronal response were assessed using extracellular unitary recordings of CA3 dorsal hippocampus pyramidal neurons obtained in anesthetized Sprague-Dawley rats. Low doses of dehydroepiandrosterone (DHEA) potentiated selectively and dose-dependently the NMDA response without affecting those to acetylcholine or quisqualate. This potentiating effect of DHEA was suppressed by the selective sigma 1 antagonist NE-100 and by the non-selective sigma antagonist haloperidol. Low doses of progesterone and of testosterone did not modify the NMDA response, but reversed the potentiating effects of DHEA as well as those of the non-steroidal sigma ligands di-tolylguanidine (DTG), (+)pentazocine and JO-1784. The two neuroactive steroids with a low affinity for sigma receptors, pregnenolone and pregnenolone sulfate, had no effect on the NMDA response, and did not modify the potentiation of the NMDA response induced by DHEA and by non-steroidal sigma ligands. The potentiation of the NMDA response by DTG (1 microgram/kg i.v.) was significantly greater in ovariectomized rats than in males and non-ovariectomized females on either day one or three of the estrous cycle. These results suggest that some neuroactive steroids such as DHEA, progesterone and testosterone modulate the NMDA response via sigma receptors. Furthermore, they also indicate that endogenous progesterone and testosterone, by acting as non-selective sigma antagonists, may produce a tonic dampening of the function of sigma receptors and consequently a decrease in the NMDA receptor function.

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.

σ1 Receptors in rat striatum regulate NMDA-stimulated [ 3H]dopamine release via a presynaptic mechanism

The role of the σ 1 receptor in the regulation of N-methyl- d-aspartate (NMDA)-stimulated [ 3H]dopamine release from rat striatal slices was examined. The σ receptor agonist 1 S,2 R-(−)- N-[2-(3,4-dichlorophenyl)ethyl]- N-methyl-2-(1-pyrrolidinyl)cyclohexylamine (BD737) inhibited stimulated release in a concentration-dependent manner. The σ 1 receptor antagonist, 1-(cyclopropylmethyl)-4-(2′-(4″-fluorophenyl)-2′-oxoethyl)piperidine HBr (DuP 734), reversed inhibition of release by BD737. Haloperidol, di- o-tolylguanidine (DTG) and N-[2-(3,4-dichlorophenyl)ethyl]- N-methyl-2-(1-pyrrolidinyl)ethylamine (BD1008) reversed the BD737-mediated inhibition of release. Haloperidol and DTG also antagonized inhibition of stimulated release by (+)-pentazocine. Furthermore, BD737 and (+)-pentazocine inhibited stimulated release in the presence of tetrodotoxin, suggesting that σ 1 receptors regulating dopamine release are located on dopaminergic nerve terminals. These data suggest that σ 1 receptors may be important in the regulation of glutamate-stimulated dopamine release.

1,3-di- o-Tolylguanidine (DTG) differentially affects acute and tonic formalin pain: Antagonism by rimcazole

The role of the sigma receptor in prolonged pain was examined by assessing the effects of 1,3, di- o-tolylguanidine (DTG), a selective sigma receptor ligand, on the formalin test in mice. Formalin injected subcutaneously into the hindpaw produces a biphasic pain response: an acute phase of short duration followed by a longer-lasting tonic phase. DTG (10 mg/kg, IP) potently reduced pain behavior in the acute phase but increased pain behavior in the tonic phase. Rimcazole (5 and 10 mg/kg, IP), a selective sigma receptor antagonist, blocked both the DTG-induced decrease and increase in pain behavior observed in the acute and tonic phases, respectively. These data support previous findings indicating a modulatory role for the sigma receptor in nociceptive processes, and suggest that this receptor differentially modulates acute vs. tonic pain.

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, α 1-, α 2-, β-adrenoceptor, 5-HT 1, 5-HT 2); the only exception was the affinity of BD1047 for β-adrenoceptors. Competition assays further revealed that the drugs interacted with both σ 1 and σ 2 binding sites. Although both drugs had preferential affinities for σ 1 sites, BD1047 exhibited a higher affinity for σ 2 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.

DTG-induced circling behaviour in rats may involve the interaction between σ sites and nigro-striatal dopaminergic pathways

The distribution of σ sites in brain areas enriched in dopamine, and the finding that circling behaviour can be elicited by specific σ ligands such as DTG (di- o-tolylguanidine) suggest a modulatory role of these sites in the dopaminergic system. The present study was carried out to investigate further this hypothesis. Circling behaviour induced in rats by unilateral intranigral injection of DTG (10 nmol/rat) was decreased by haloperidol (0.1 mg/kg, i.p.), clebopride (0.25 mg/kg, i.p.) and SCH 23390 (0.03 mg/kg, s.c.) indicating that an interaction between σ sites and the midbrain dopaminergic system may be involved in this rotational behaviour. Microdialysis experiments in freely moving rats showed that unilateral intranigral injection of DTG (5, 10, 20 nmol/rat) produced increases in extracellular levels of dopamine metabolites (DOPAC, HVA) in the ipsilateral striatum which correlated with the number of rotations. In addition intranigral injection of DTG (10 nmol/rat) produced increases in tissular dopamine metabolite levels in the ipsilateral striatum without affecting dopamine metabolite levels in limbic structures. These results indicate that σ sites may be involved in the modulation of the dopaminergic motor system.

Sigma receptor regulation of norepinephrine release from rat hippocampal slices

Multiple sigma receptor subtypes have been identified in the hippocampus, yet their physiological role remains largely undefined. In the current study, we examined the role of sigma receptors in the regulation of N-methyl- d-aspartate (NMDA)-stimulated [ 3H]norepinephrine ([ 3H]NE) release from rat hippocampal slices. Both sigma agonists (+)pentazocine and BD737 inhibited stimulated norepinephrine release in a concentration-dependent manner. The sigma 1 antagonist DuP 734 completely antagonized the inhibition of release by all concentrations of BD737 tested. However, DuP 734 only partially reversed inhibition of release by (+)pentazocine concentrations above 100 nM. 1,3 Di- o-tolylguanidine (DTG), but not haloperidol, antagonized BD737-mediated inhibition of release. DTG also completely antagonized inhibition of release by 100 nM (+)pentazocine yet haloperidol produced only a partial reversal. A combination of DuP 734 and haloperidol produced complete reversal of (+)pentazocine-mediated inhibition, suggesting potential involvement of multiple sigma receptor subtypes in the regulation of norepinephrine release. Both (+)pentazocine and BD737 failed to inhibit stimulated release in the presence of tetrodotoxin, suggesting that sigma receptors regulating NE release are not located on noradrenergic nerve terminals. These results suggest that sigma receptors may be a therapeutic target for disorders resulting from noradrenergic imbalance in hippocampus.

Pharmacological evidence for the involvement of sigma sites in DTG-induced contralateral circling in rats

The central distribution of sigma sites labelled by di- o -tolylguanidine (DTG), a compound which has specific affinity for sigma sites, and its ability to produce postural movements, are consistent with the hypothesis that sigma sites may play a functional role in the regulation of movement. The aim of the present study was to evaluate the specificity of the circling behaviour induced by unilateral intranigral injection of DTG in rats. As previously described, DTG produced dose-dependent unilateral rotations (2.5–20 nmol/rat). A similar dose-dependent circling behaviour was observed with DMTG and (+) NANM (3–40 nmol/rat), compounds which bind to both sigma and PCP sites, and with haloperidol (3–20 nmol/rat) whereas raclopride and d,l-sulpiride did not elicit any circling (10 nmol/rat). DTG-induced circling after intranigral injection (10 nmol/rat) was decreased in a dose-dependent manner by rimcazole (20–40 mg/kg, i.p.), a selective ligand for sigma sites, and by BMY 14802 (3, 10, 30mg/kg, i.p.), ifenprodil and eliprodil (1, 3, 10 mg/kg, i.p.), non-selective sigma ligands. In contrast, naloxone (1 mg/kg, s.c.) and CGS 19755 (1, 3, 10 mg/kg, i.p.) did not change the DTG-induced circling. Eliprodil failed to inhibit circling produced by compounds devoid of any affinity for sigma sites such as APV, dizocilpine or muscimol, indicating the specificity of the inhibition observed with eliprodil on the DTG-induced circling. These results suggest that circling behaviour produced by unilateral intranigral injection of DTG may be a useful model for studying the interaction of new drugs with sigma sites and might also be used to investigate further the potential involvement of these sites in the regulation of the activity of the nigro-striatal dopaminergic pathways.

Oocytes from Xenopus laevis contain an intrinsic σ2-like binding site

In preparation for expression studies for rat brain σ-binding sites, Xenopus oocytes were tested for the presence of [ 3 H]di-o- tolylguanidine (DTG)-binding sites. Native oocytes were found to contain two intrinsic [ 3H]DTG-binding sites, a high-affinity site ( K d = 32 ± 6 nM, B max of 45.7 ± 19 pmol/mg protein) and a low-affinity binding site ( K d = 1.3 ± 0.7 μM, B max of 3.2 ± 0.7 nmol/mg protein). In a series of radioligand-binding-displacement studies, the high-affinity binding sites were found to have a binding profile which has a similar K d to that of the mammalian σ 2-binding site (32 vs. 38 nM). Comparison of the IC 50 values for inhibition of [ 3H]DTG binding in rat liver and oocytes for DTG, haloperidol (HAL), (−)-pentazocine, (+)-3-(3-hydroxyphenyl)- N-propylpiperidine hydrochloride ((+)-3-PPP), (+(-pentazocine and Zn 2+, showed similarity in rank (r 2 = 0.913) but a 7-fold lower potency in oocytes. These results suggest that the high-affinity [ 3H]DTG-binding site in oocytes represents a σ 2-like binding site.