Pentazocine Binding Research Articles

Lessons on the Sigma-1 Receptor in TNBS-Induced Rat Colitis: Modulation of the UCHL-1, IL-6 Pathway.

Inflammatory Bowel Disease (IBD) is an autoimmune ailment of the gastrointestinal (GI) tract, which is characterized by enhanced activation of proinflammatory cytokines. It is suggested that the sigma-1 receptor (σ1R) confers anti-inflammatory effects. As the exact pathogenesis of IBD is still unknown and treatment options are limited, we aimed to investigate the effects of σ1R in 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced experimental colitis. To this end, male Wistar–Harlan rats were used to model colitic inflammation through the administration of TNBS. To investigate the effects of σ1R, Fluvoxamine (FLV, σ1R agonist) and BD1063 (σ1R antagonist) were applied via intracolonic administration to the animals once a day for three days. Our radioligand binding studies indicated the existence of σ1Rs as [3H](+)-pentazocine binding sites, and FLV treatment increased the reduced σ1R maximum binding capacity in TNBS-induced colitis. Furthermore, FLV significantly attenuated the colonic damage, the effect of which was abolished by the administration of BD1063. Additionally, FLV potentially increased the expression of ubiquitin C-terminal hydrolase ligase-1 (UCHL-1) and the levels of endothelial nitric oxide synthase (eNOS), and decreased the levels of interleukin-6 (IL-6) and inducible NOS (iNOS) expression. In summary, our study offers evidence for the anti-inflammatory potential of FLV and σ1R in experimental colitis, and our results present a promising approach to the development of new σ1R-targeted treatment options against IBD.

Distinct Regulation of σ 1 Receptor Multimerization by Its Agonists and Antagonists in Transfected Cells and Rat Liver Membranes.

Extensive studies have shown that the σ 1 receptor (σ 1R) interacts with and modulates the activity of multiple proteins with important biological functions. Recent crystal structures of σ 1R as a homotrimer differ from a dimer-tetramer model postulated earlier. It remains inconclusive whether ligand binding regulates σ 1R oligomerization. Here, novel nondenaturing gel methods and mutational analysis were used to examine σ 1R oligomerization. In transfected cells, σ 1R exhibited as multimers, dimers, and monomers. Overall, σ 1R agonists decreased, whereas σ 1R antagonists increased σ 1R multimers, suggesting that agonists and antagonists differentially affect the stability of σ 1R multimers. Endogenous σ 1R in rat liver membranes also showed similar regulation of oligomerization as in cells. Mutations at key residues lining the trimerization interface (Arg119, Asp195, Phe191, Trp136, and Gly91) abolished multimerization without disrupting dimerization. Intriguingly, truncation of the N terminus reduced σ 1R to apparent monomer. These results demonstrate that multiple domains play crucial roles in coordinating high-order quaternary organization of σ 1R. The E102Q σ 1R mutant implicated in juvenile amyotrophic lateral sclerosis formed dimers only, suggesting that dysregulation of σ 1R multimeric assembly may impair its function. Interestingly, oligomerization of σ 1R was pH-dependent and correlated with changes in [3H](+)-pentazocine binding affinity and Bmax Combined with mutational analysis, it is reasoned that σ 1R multimers possess high-affinity and high-capacity [3H](+)-pentazocine binding, whereas monomers likely lack binding. These results suggest that σ 1R may exist in interconvertible oligomeric states in a dynamic equilibrium. Further exploration of ligand-regulated σ 1R multimerization may provide novel approaches to modulate the function of σ 1R and its interacting proteins. SIGNIFICANCE STATEMENT: The σ 1 receptor (σ 1R) modulates the activities of various partner proteins. Recently, crystal structures of σ 1R were elucidated as homotrimers. This study used novel nondenaturing gel methods to examine σ1R oligomerization in transfected cells and rat liver membranes. Overall, agonist binding decreased, whereas antagonist binding increased σ 1R multimers, which comprised trimers and larger units. σ 1R multimers were shown to bind [3H](+)-pentazocine with high affinity and high capacity. Furthermore, mutational analysis revealed a crucial role of its N-terminal domain in σ 1R multimerization.

Distinct regulation of sigma‐1 receptor multimerization by its agonists and antagonists

Since its initial proposal four decades ago, extensive studies have shown that the sigma‐1 receptor (σ1R) interacts with and modulates the activity of multiple proteins with important functions. Recent crystal structures of σ1R as a homo‐trimer differ from a dimer‐tetramer model postulated by early work. Further it is not clear whether ligand binding regulates σ1R multimerization. Here I conducted mutational analyses and examined ligands' effects on σ1R oligomerization using novel non‐denaturing gels. In transfected cells σ1R exhibited as monomer, dimer, and mainly as high‐order multimers. Agonists ((+)pentazocine, (+)SKF10,047, DTG, PRE‐084) decreased, whereas antagonists (BD1008, BD1047, BD1063, haloperidol, NE‐100, progesterone) increased σ1R multimers, suggesting that agonists and antagonists differentially affect the stability of σ1R multimers. Mutations at key residues lining the trimerization interface abolished multimerization but preserved dimerization. Intriguingly, deletion of the transmembrane domain (TM) reduced σ1R to monomer. These results demonstrate that multiple domains play crucial roles in coordinating high‐order quaternary organization of σ1R, which may comprise interconvertible oligomeric states in a dynamic equilibrium. σ1R multimers exhibited high‐affinity and high‐capacity [3H](+)pentazocine binding, whereas monomers lacked binding. In competition binding the antagonist haloperidol appeared to show an apparent 10‐fold higher potency in wild‐type σ1R than in mutants with only dimers, which might explain why haloperidol binding increased σ1R multimers. Further, a σ1R mutant (E102Q) implicated in early‐onset amyotrophic lateral sclerosis exhibited as dimer only, suggesting that dysregulation of σ1R quaternary structure impairs its physiological function. Further exploration of ligand‐regulated σ1R multimerization may provide novel approaches to modulate the function of σ1R and its interacting proteins.Support or Funding InformationButler University Faculty Startup FundThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Oligomeric States of the Purified Sigma-1 Receptor Are Stabilized by Ligands

Sigma-1 receptor (S1R) is a mammalian member of the ERG2 and sigma-1 receptor-like protein family (pfam04622). It has been implicated in drug addiction and many human neurological disorders, including Alzheimer and Parkinson diseases and amyotrophic lateral sclerosis. A broad range of synthetic small molecules, including cocaine, (+)-pentazocine, haloperidol, and small endogenous molecules such as N,N-dimethyltryptamine, sphingosine, and steroids, have been identified as regulators of S1R. However, the mechanism of activation of S1R remains obscure. Here, we provide evidence in vitro that S1R has ligand binding activity only in an oligomeric state. The oligomeric state is prone to decay into an apparent monomeric form when exposed to elevated temperature, with loss of ligand binding activity. This decay is suppressed in the presence of the known S1R ligands such as haloperidol, BD-1047, and sphingosine. S1R has a GXXXG motif in its second transmembrane region, and these motifs are often involved in oligomerization of membrane proteins. Disrupting mutations within the GXXXG motif shifted the fraction of the higher oligomeric states toward smaller states and resulted in a significant decrease in specific (+)-[(3)H]pentazocine binding. Results presented here support the proposal that S1R function may be regulated by its oligomeric state. Possible mechanisms of molecular regulation of interacting protein partners by S1R in the presence of small molecule ligands are discussed.

Involvement of sigma-1 receptors in the antidepressant-like effects of dextromethorphan.

Dextromethorphan is an antitussive with a high margin of safety that has been hypothesized to display rapid-acting antidepressant activity based on pharmacodynamic similarities to the N-methyl-D-aspartate (NMDA) receptor antagonist ketamine. In addition to binding to NMDA receptors, dextromethorphan binds to sigma-1 (σ1) receptors, which are believed to be protein targets for a potential new class of antidepressant medications. The purpose of this study was to determine whether dextromethorphan elicits antidepressant-like effects and the involvement of σ1 receptors in mediating its antidepressant-like actions. The antidepressant-like effects of dextromethorphan were assessed in male, Swiss Webster mice using the forced swim test. Next, σ1 receptor antagonists (BD1063 and BD1047) were evaluated in conjunction with dextromethorphan to determine the involvement of σ receptors in its antidepressant-like effects. Quinidine, a cytochrome P450 (CYP) 2D6 inhibitor, was also evaluated in conjunction with dextromethorphan to increase the bioavailability of dextromethorphan and reduce exposure to additional metabolites. Finally, saturation binding assays were performed to assess the manner in which dextromethorphan interacts at the σ1 receptor. Our results revealed dextromethorphan displays antidepressant-like effects in the forced swim test that can be attenuated by pretreatment with σ1 receptor antagonists, with BD1063 causing a shift to the right in the dextromethorphan dose response curve. Concomitant administration of quinidine potentiated the antidepressant-like effects of dextromethorphan. Saturation binding assays revealed that a Ki concentration of dextromethorphan reduces both the Kd and the Bmax of [3H](+)-pentazocine binding to σ1 receptors. Taken together, these data suggest that dextromethorphan exerts some of its antidepressant actions through σ1 receptors.

Interaction of new antidepressants with sigma-1 receptor chaperones and their potentiation of neurite outgrowth in PC12 cells

The sigma-1 receptor chaperone located in the endoplasmic reticulum (ER) may be implicated in the mechanistic action of some antidepressants. The present study was undertaken to examine whether new antidepressant drugs interact with the sigma-1 receptor chaperone. First, we examined the effects of selective serotonin reuptake inhibitors (SSRIs) (fluvoxamine, paroxetine, sertraline, citalopram and escitalopram), serotonin and noradrenaline reuptake inhibitors (SNRIs) (duloxetine, venlafaxine, milnacipran), and mirtazapine, a noradrenaline and specific serotonergic antidepressant (NaSSA), on [(3)H](+)-pentazocine binding to rat brain membranes. Then, we examined the effects of these drugs on nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. The order of potency for drugs at the sigma-1 receptor chaperone was as follows: fluvoxamine>sertraline>fluoxetine>escitalopram>citalopram>paroxetine>duoxetine. Venlafaxine, milnacipran, and mirtazapine showed very weak affinity for this chaperone. Furthermore, fluvoxamine, fluoxetine, escitalopram, and mirtazapine significantly potentiated NGF-induced neurite outgrowth in cell assays, and the effects of all these drugs, excluding mirtazapine, were antagonized by NE-100, a selective antagonist of the sigma-1 receptor chaperone. Moreover, the effects of fluvoxamine and fluoxetine on neurite outgrowth were also antagonized by sertraline, indicating that sertraline may be an antagonist at the sigma-1 receptor chaperone. The effect of mirtazapine on neurite outgrowth was antagonized by the selective 5-hydroxytryptamine1A receptor antagonist WAY-100635. These findings suggest that activation at the sigma-1 receptor chaperone may be involved in the action of some SSRIs, such as fluvoxamine, fluoxetine and escitalopram. In contrast, mirtazapine independently potentiated neurite outgrowth in PC12 cells, indicating that this beneficial effect may mediate its pharmacological effect.

3D-QSAR and docking studies of pentacycloundecylamines at the sigma-1 (σ1) receptor

Pentacycloundecylamine (PCU) derived compounds have been shown to be promising lead structures for the development of novel drug candidates aimed at a variety of neurodegenerative and psychiatric diseases. Here we show for the first time a 3D quantitative structure–activity relationship (3D-QSAR) for a series of aza-PCU-derived compounds with activity at the sigma-1 (σ1) receptor. A comparative molecular field analysis (CoMFA) model was developed with a partial least squares cross validated (q2) regression value of 0.6, and a non-cross validated r2 of 0.9. The CoMFA model was effective at predicting the sigma-1 activities of a test set with an r2 >0.7. We also describe here the docking of the PCU-derived compounds into a homology model of the sigma-1 (σ1) receptor, which was developed to gain insight into binding of these cage compounds to the receptor. Based on docking studies we evaluated in a [3H]pentazocine binding assay an oxa-PCU, NGP1-01 (IC50=1.78μM) and its phenethyl derivative (IC50=1.54μM). Results from these studies can be used to develop new compounds with specific affinity for the sigma-1(σ1) receptor.

Sigma-1 receptors are essential for capsaicin-induced mechanical hypersensitivity: Studies with selective sigma-1 ligands and sigma-1 knockout mice

We evaluated the role of σ1 receptors on capsaicin-induced mechanical hypersensitivity and on nociceptive pain induced by punctate mechanical stimuli, using wild-type and σ1 receptor knockout (σ1-KO) mice and selective σ1 receptor-acting drugs. Mutation in σ1-KO mice was confirmed by PCR analysis of genomic DNA and, at the protein level, by [3H](+)-pentazocine binding assays. Both wild-type and σ1-KO mice not treated with capsaicin showed similar responses to different intensities of mechanical stimuli (0.05–8g force), ranging from innocuous to noxious, applied to the hind paw. This indicates that σ1 gene inactivation does not modify the perception of punctate mechanical stimuli. The intraplantar (i.pl.) administration of capsaicin induced dose-dependent mechanical allodynia in wild-type mice (markedly reducing both the threshold force necessary to induce paw withdrawal and the latency to paw withdrawal induced by a given force). In contrast, capsaicin was completely unable to induce mechanical hypersensitivity in σ1-KO mice. The high-affinity and selective σ1 antagonists BD-1063, BD-1047 and NE-100, administered subcutaneously (s.c.), dose-dependently inhibited mechanical allodynia induced by capsaicin (1μg,i.pl.), yielding ED50 (mg/kg) values of 15.80±0.93, 29.31±1.65 and 40.74±7.20, respectively. The effects of the σ1 antagonists were reversed by the σ1 agonist PRE-084 (32mg/kg, s.c.). None of the drugs tested modified the responses induced by a painful mechanical punctate stimulus (4g force) in nonsensitized animals. These results suggest that σ1 receptors are essential for capsaicin-induced mechanical hypersensitivity, but are not involved in mechanical nociceptive pain.

Role of Sigma-1 Receptor C-terminal Segment in Inositol 1,4,5-Trisphosphate Receptor Activation: CONSTITUTIVE ENHANCEMENT OF CALCIUM SIGNALING IN MCF-7 TUMOR CELLS

Sigma-1 receptor (sigma-1R) agonists enhance inositol 1,4,5-trisphosphate (IP3)-dependent calcium release from endoplasmic reticulum by inducing dissociation of ankyrin B 220 (ANK 220) from the IP3 receptor (IP3R-3), releasing it from inhibition. MCF-7 breast tumor cells express little or no sigma-1R and were used here to investigate the effect of receptor overexpression and the role of its N- and C-terminal segments in function. We stably expressed intact sigma-1R (amino acids (aa) 1-223; lines 11 and 41), N-fragment (aa 1-100; line K3), or C-fragment (aa 102-223; line sg101). C-fragment expressed as a peripheral membrane-bound protein that was removable from the endoplasmic reticulum membrane by chaotropic salt wash, consistent with lack of a putative transmembrane domain. The expressed sigma-1R, N-fragment, and C-fragment exhibited normal, low affinity, and no [3H](+)-pentazocine binding activity, respectively. All transfected lines showed constitutive enhancement of bradykinin (BDK)-induced calcium release, because of a decrease in BDK ED50 values. Interestingly, sigma-1R and C-fragment had high activities, whereas the N-fragment was much less active. The antagonist BD1063 behaved as an inverse agonist in sigma-1R cells, whereas C-fragment was insensitive to ligand regulation. Like BDK, vasopressin- and ATP-induced calcium release was enhanced with the same pattern in cell lines. Anti-IP3R-3 immunoprecipitates from cells expressing sigma-1R or C-fragment contained significantly less ANK 220 compared with untransfected or N-fragment cells, indicating a higher amount of ankyrin-free IP3R-3. Anti-ankyrin B immunoprecipitates contained sigma-1R or C-fragment, with markedly lower levels of N-fragment present. These results suggest that sigma-1R overexpression drives sigma agonist-independent dissociation of ANK 220 from IP3R-3, resulting in activation. The C-terminal segment plays a key role in the interaction.

Irreversible blockade of sigma‐1 receptors by haloperidol and its metabolites in guinea pig brain and SH‐SY5Y human neuroblastoma cells

We evaluated the effect of haloperidol (HP) and its metabolites on [(3)H](+)-pentazocine binding to sigma(1) receptors in SH-SY5Y human neuroblastoma cells and guinea pig brain P(1), P(2) and P(3) subcellular fractions. Three days after a single i.p. injection in guinea pigs of HP (but not of other sigma(1) antagonists or (-)-sulpiride), [(3)H](+)-pentazocine binding to brain membranes was markedly decreased. Recovery of sigma(1) receptor density to steady state after HP-induced inactivation required more than 30 days. HP-metabolite II (reduced HP, 4-(4-chlorophenyl)-alpha-(4-fluorophenyl)-4-hydroxy-1-piperidinebutanol), but not HP-metabolite I (4-(4-chlorophenyl)-4-hydroxypiperidine), irreversibly blocked sigma(1) receptors in guinea pig brain homogenate and P(2) fraction in vitro. We found similar results in SH-SY5Y cells, which suggests that this process may also take place in humans. HP irreversibly inactivated sigma(1) receptors when it was incubated with brain homogenate and SH-SY5Y cells, but not when incubated with P(2) fraction membranes, which suggests that HP is metabolized to inactivate sigma(1) receptors. Menadione, an inhibitor of the ketone reductase activity that leads to the production of HP-metabolite II, completely prevented HP-induced inactivation of sigma(1) receptors in brain homogenates. These results suggest that HP may irreversibly inactivate sigma(1) receptors in guinea pig and human cells, probably after metabolism to reduced HP.

Expression, Subcellular Localization, and Regulation of Sigma Receptor in Retinal Müller Cells

Sigma receptors (sigmaRs) are nonopioid, nonphencyclidine binding sites with robust neuroprotective properties. Type 1 sigmaR1 (sigmaR1) is expressed in brain oligodendrocytes, but its expression and binding capacity have not been analyzed in retinal glial cells. This study examined the expression, subcellular localization, binding activity, and regulation of sigmaR1 in retinal Müller cells. Primary mouse Müller cells (MCs) were analyzed by RT-PCR, immunoblotting, and immunocytochemistry for the expression of sigmaR1, and data were compared with those of the rat Müller cell line (rMC-1) and the rat ganglion cell line (RGC-5). Confocal microscopy was used to determine the subcellular sigmaR1 location in primary mouse MCs. Membranes prepared from these cells were used for binding assays with [3H]-pentazocine (PTZ). The kinetics of binding, the ability of various sigmaR1 ligands to compete with sigmaR1 binding, and the effects of donated nitric oxide (NO) and reactive oxygen species (ROS) on binding were examined. sigmaR1 is expressed in primary mouse MCs and is localized to the nuclear and endoplasmic reticulum membranes. Binding assays showed that in primary mouse MCs, rMC-1, and RGC-5, the binding of PTZ was saturable. [3H]-PTZ bound with high affinity in RGC-5 and rMC-1 cells, and the binding was similarly robust in primary mouse MCs. Competition studies showed marked inhibition of [3H]-PTZ binding in the presence of sigmaR1-specific ligands. Incubation of cells with NO and ROS donors markedly increased sigmaR1 binding activity. MCs express sigmaR1 and demonstrate robust sigmaR1 binding activity, which is inhibited by sigmaR1 ligands and is stimulated during oxidative stress. The potential of Müller cells to bind sigmaR1 ligands may prove beneficial in retinal degenerative diseases such as diabetic retinopathy.

C.14.03 The long-term - maximising potential for rehabilitation

The sigma-1 receptor chaperone located in the endoplasmic reticulum (ER) may be implicated in the mechanistic action of some antidepressants. The present study was undertaken to examine whether new antidepressant drugs interact with the sigma-1 receptor chaperone. First, we examined the effects of selective serotonin reuptake inhibitors (SSRIs) (fluvoxamine, paroxetine, sertraline, citalopram and escitalopram), serotonin and noradrenaline reuptake inhibitors (SNRIs) (duloxetine, venlafaxine, milnacipran), and mirtazapine, a noradrenaline and specific serotonergic antidepressant (NaSSA), on [3H](+)-pentazocine binding to rat brain membranes. Then, we examined the effects of these drugs on nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. The order of potency for drugs at the sigma-1 receptor chaperone was as follows: fluvoxamine>sertraline>fluoxetine>escitalopram>citalopram>paroxetine>duoxetine. Venlafaxine, milnacipran, and mirtazapine showed very weak affinity for this chaperone. Furthermore, fluvoxamine, fluoxetine, escitalopram, and mirtazapine significantly potentiated NGF-induced neurite outgrowth in cell assays, and the effects of all these drugs, excluding mirtazapine, were antagonized by NE-100, a selective antagonist of the sigma-1 receptor chaperone. Moreover, the effects of fluvoxamine and fluoxetine on neurite outgrowth were also antagonized by sertraline, indicating that sertraline may be an antagonist at the sigma-1 receptor chaperone. The effect of mirtazapine on neurite outgrowth was antagonized by the selective 5-hydroxytryptamine1A receptor antagonist WAY-100635. These findings suggest that activation at the sigma-1 receptor chaperone may be involved in the action of some SSRIs, such as fluvoxamine, fluoxetine and escitalopram. In contrast, mirtazapine independently potentiated neurite outgrowth in PC12 cells, indicating that this beneficial effect may mediate its pharmacological effect.

Compensatory effect by sigma 1 (σ 1) receptor stimulation during alcohol withdrawal in mice performing an object recognition task

Chronic alcohol consumption (CAC) provokes intense neurobiological alterations, which lead, notably, to an important abstinence syndrome upon withdrawal with deleterious cognitive consequences. We here examined the effect of activation or inactivation of the σ 1 receptor during CAC withdrawal on the cognitive abilities of Swiss mice. Animals consumed an alcohol 10%/sucrose 30 g/l solution during 4 months. Control groups consumed only the sucrose vehicle solution. Then, animals experienced a progressive, 16 days long, CAC withdrawal, during which they were administered once daily with saline, igmesine (10 mg/kg i.p.), a σ 1 receptor agonist, or BD1047 (10 mg/kg i.p.), a σ 1 antagonist. Mice were then tested using an object exploration task, to evaluate their locomotor and exploratory activities and reactions to object habituation, spatial change or novel object presentation. CAC-treated animals showed augmentation of locomotion, anxiety and object exploration, which impeded correct reaction to object habituation, spatial change or novelty. Treatment with the σ 1 ligands, ineffective in control groups, resulted in decrease of the hyper-responsiveness and restored habituation. However, correct reactions to spatial change and novelty were only produced by the σ 1 agonist treatment. Moreover, the σ 1 receptor hippocampal expression was increased in CAC-treated mice. Treatments with both σ 1 ligands regulated its expression, but subcellular fractionation experiments revealed that the agonist treatment increased [ 3H](+)-pentazocine binding to σ 1 sites in the plasma membrane fraction, while the antagonist maintained it only in the microsomal, putatively endoplasmic reticulum, fraction. In conclusion, CAC increased the σ 1 receptor expression in the hippocampus of mice. Regulation of its expression during withdrawal, notably using a selective agonist, allowed not only to attenuate the CAC-induced hyper-responsiveness, but also to restore correct cognitive abilities.

Modulation of bradykinin‐induced calcium changes in SH‐SY5Y cells by neurosteroids and sigma receptor ligands via a shared mechanism

In this study we investigated the effects of sigma receptor ligands and neurosteroids on bradykinin-induced intracellular calcium concentration ([Ca2+]i) changes in SH-SY5Y neuroblastoma cells. [Ca2+]i levels in cells loaded with fura-2 were monitored with dual-wavelength ratiometric fluorescence measurement. Submicromolar concentrations of bradykinin elicited [Ca2+]i responses with a fast rise followed by a slow decline in these cells. Preincubation of low micromolar concentrations of the neurosteroids pregnenolone, dehydroepiandrosterone (DHEA), or the prototypic sigma (sigma) receptor agonist (+)pentazocine potentiated bradykinin-induced [Ca2+]i changes in SH-SY5Y cells. The sigma receptor antagonist haloperidol blocked the enhancing effects on [Ca2+]i by (+)pentazocine or pregnenolone. Progesterone did not significantly affect the basal [Ca2+]i level or bradykinin-induced [Ca2+]i changes in these cells. However, coincubation of progesterone with (+)pentazocine, pregnenolone, or DHEA reversed their potentiating effects. The antagonistic effects of haloperidol and progesterone on the potentiating effects of (+)pentazocine and pregnenolone suggested that these ligands might act through a common mechanism. We further showed that progesterone, pregnenolone, and DHEA competed for [3H]+pentazocine binding in SH-SY5Y cells with Ki values of 0.13 +/- 0.03 microM, 0.98 +/- 0.34 microM, and 5.2 +/- 1.4 microM, respectively. Thus, the modulation of bradykinin-induced [Ca2+]i changes by neurosteroids in these cells is likely due to their actions on sigma receptors.

3H]BHDP as a novel and selective ligand for σ1 receptors in liver mitochondria and brain synaptosomes of the rat

The binding profile of [ 3H]BHDP ([ 3H] N-benzyl- N′-(2-hydroxy-3,4-dimethoxybenzyl)-piperazine) was evaluated. [ 3H]BHDP labelled a single class of binding sites with high affinity ( K d=2–3 nM) in rat liver mitochondria and synaptic membranes. The pharmacological characterization of these sites using σ reference compounds revealed that these sites are σ receptors and, more particularly, σ1 receptors. Indeed, BHDP inhibited [ 3H]pentazocine binding, a marker for σ1 receptors, with high affinity in a competitive manner. BHDP is selective for σ1 receptors since it did not show any relevant affinity for most of the other receptors, ion channels or transporters tested. Moreover, in an in vitro model of cellular hypoxia, BHDP prevented the fall in adenosine triphosphate (ATP) levels caused by 24 h hypoxia in cultured astrocytes. Taken together, these results demonstrate that [ 3H]BHDP is a potent and selective ligand for σ1 receptors showing cytoprotective effects in astrocytes.

Evidence for sigma-1-like receptors in isolated rat liver mitochondrial membranes.

1. Sigma (sigma) receptors have generated a great deal of interest on the basis of their possible roles in various pathologies, including cytoprotection. Although the exact function of sigma-1 (sigma(1)) receptors is not yet known, their role in the regulation of intracellular Ca(2+) levels and sterol biosynthesis, functions that could be assigned to mitochondria, are the only mechanisms described. 2. Using preparations of purified rat liver and brain mitochondria we demonstrate herein the presence of sigma-like binding sites. [(3)H](+)-pentazocine, a sigma(1) radioligand was used to label these sites. 3. In the liver, [(3)H](+)-pentazocine labelled one class of binding sites with high affinity (K(d)=3 nM), similar to that observed in liver microsomes and synaptic membranes. These sites were located on the outer mitochondrial membranes and displayed high affinity for other sigma(1) ligands namely, haloperidol, ifenprodil, carbetapentane or 1,3-di(2-tolyl)guanidine (DTG). 4. The presence of sigma(1) receptors on liver mitochondria was confirmed using double fluorescence immunostaining. 5. [(3)H](+)-pentazocine binding sites were also found on brain mitochondria but they appeared pharmacologically distinct to the liver ones as [(3)H](+)-pentazocine and typical sigma(1) ligands displayed lower affinities for these sites. Nevertheless, [(3)H](+)-pentazocine binding on both liver and brain mitochondria was modulated by progesterone, a putative endogenous ligand for sigma receptors. 6. Our data demonstrates the presence of [(3)H](+)-pentazocine binding sites with pharmacological characteristics identical to sigma(1) receptors on rat liver mitochondrial membranes. The pharmacological significance of these sites and their role on mitochondrial function remain unknown.

Molecular cloning and pharmacological characterization of the rat sigma 1 receptor

In an effort to further understand the pharmacology of sigma receptors, we have cloned the rat homolog of the sigma 1 receptor. We isolated a cDNA clone (rs2-2) from rat brain tissue using reverse transcriptase-polymerase chain reaction (RT-PCR) and 5′ and 3′ rapid amplification of cDNA ends (RACE) that encoded a full-length sequence of 223 amino acids. The predicted protein sequence of the clone has high homology with that of the murine (93.3%), guinea pig (93.7%), and human (96%) sigma 1 receptors. Northern analysis showed a major mRNA band of approximately 1.8 kb. RT-PCR revealed the presence of the mRNA in all the tissues tested, with high levels in the brain, spinal cord, liver, thymus, adrenal glands, and kidneys. When expressed in Chinese hamster ovary (CHO) cells, the level of sigma 1 binding increased markedly, and the binding profile was consistent with sigma 1 sites. However, measurable levels of sigma 1 binding present in the cell lines before transfection made the interpretation of these results difficult. To ensure that the binding reflected the transfected protein, we tagged the receptors with a hemagglutinin (HA) epitope at the amino terminus and examined binding in immunoprecipitated receptors. Western analysis using an antisera against the HA epitope revealed a molecular weight of ∼28 kDa, close to the predicted value. The receptor binding profile of the immunopurified receptor was consistent with that seen with traditional sigma 1 binding sites. Thus, rs2-2.HA encodes a high-affinity [ 3H](+)-pentazocine binding site with characteristics of a rat sigma 1 receptor.

Expression of the purported sigma 1 (σ 1) receptor in the mammalian brain and its possible relevance in deficits induced by antagonism of the NMDA receptor complex as revealed using an antisense strategy

Sigma (σ) receptors have generated a great deal of interest on the basis of their possible role in psychosis, neuroprotection and various other behaviors including learning processes. The existence of at least two classes of σ receptor binding sites (σ 1 and σ 2) is now well established. The recent cloning of the mouse, guinea pig and human σ 1 receptors has allowed the study of the discrete distribution of the σ 1 receptor mRNA in rodent and human brain tissues using in situ hybridization. Overall, the sites of expression of specific σ 1 receptor mRNA signals were in accordance to the anatomical distribution of σ 1 receptor protein first established by quantitative receptor autoradiography. Specific σ 1 receptor hybridization signals were found to be widely, but discretely distributed, in mouse and guinea pig brain tissues. The highest levels of transcripts were seen in various cranial nerve nuclei. Lower, but still high hybridization signals were observed in mesencephalic structures such as the red nucleus, periaqueductal gray matter and substantia nigra, as well as in some diencephalic structures including such as the habenula and the arcuate, paraventricular and ventromedial hypothalamic nuclei. Superficial (I–II) and deeper (IV–VI) cortical laminae were moderately labeled in the mouse brain. Moderate levels of σ 1 receptor mRNA were also found in the pyramidal cell layer and the dentate gyrus of the hippocampal formation. Other structures such as the thalamus and amygdaloid body also expressed the σ 1 receptor mRNA although to a lesser extent. In murine peripheral tissues, strong hybridization signals were observed in the liver, white pulp of the spleen and the adrenal gland. In the postmortem human brain, moderate levels of σ 1 receptor mRNA, distributed in a laminar fashion, were detected in the temporal cortex with the deeper laminae (IV–VI) being particularly enriched. In the hippocampal formation, the strongest hybridization signals were observed in the dentate gyrus while all other subfields of the human hippocampal formation expressed lower levels of the σ 1 receptor mRNA. Antisense oligodeoxynucleotides against the purported σ 1 receptor were used next to investigate the possible role of this receptor in dizocilpine (MK-801)/NMDA receptor blockade-induced amnesia. Following a continuous intracerebroventricular infusion of a specific σ 1 receptor antisense into the third ventricle (0.4 nmol/h for 5 days), σ 1/[ 3H](+)pentazocine binding was significantly reduced in mouse brain membrane homogenates while a scrambled antisense control was without effect. Moreover, the σ 1 receptor antisense treatments (5 nmol/injection, every 12 h×3 or 0.4 nmol/h for 5 days) attenuated (+)MK-801/NMDA receptor blockade-induced cognitive deficits in the treated mice while a scrambled antisense control had no effect. Taken together, these results demonstrate the widespread, but discrete, distribution of the σ 1 receptor mRNA in the mammalian central nervous system. Moreover, antisense treatments against the purported σ 1 receptor gene reduced specific σ 1/[ 3H](+)pentazocine binding and modulated cognitive behaviors associated with NMDA receptor blockade providing further evidence for the functional relevance of the cloned gene.

Repeated haloperidol treatment decreases σ 1 receptor binding but does not affect its mRNA levels in the guinea pig or rat brain

The effects of chronic treatment with haloperidol on sigma (σ) receptors were investigated across brain regions and species. The regional distribution of [ 3H](+)-pentazocine binding to σ 1 receptor was similar between the guinea pig and rat brains. The highest level of binding was detected in the brain stem and lowest in the striatum and hippocampus. The regional distribution of [ 3H]1,3-di (2-tolyl) guanidine ([ 3H]DTG) binding in the presence of 100 nM (+)-pentazocine to σ 2 receptor was similar to that of the [ 3H](+)-pentazocine binding in the guinea pig brain, while in the rat brain high levels of [ 3H]DTG binding were detected in the cortex, frontal cortex and cerebellum. The intraperitoneal administration of 2 mg/kg of haloperidol to guinea pig and rats once a day for 21 days produced inhibition of [ 3H](+)-pentazocine binding but did not affect [ 3H]DTG binding to σ 2 receptors in any brain region examined. The effects of haloperidol on [ 3H](+)-penazocine binding in the rat were much weaker than those in the guinea pig. The regional distribution of the level of σ 1 receptor mRNA determined by the ribonuclease protection assay was similar to that of the [ 3H](+)-pentazocine binding activity, except in the cortex and cerebellum where the levels of σ 1 receptor mRNA were low in guinea pig and rat. Treatment with haloperidol did not affect the levels of σ 1 receptor mRNA in any brain region in either species. These findings suggested that the σ receptors differentially distributed in brain regions are down regulated by treatment with haloperidol across σ receptor subtypes and animal species without changing the transcriptional activity of the σ 1 receptor. The mechanisms by which σ receptors could be differently regulated in vivo by chronic treatment with haloperidol in different species may contribute to the therapeutic efficacy of haloperidol.

Binding of [ 35S]GTPγS stimulated by (+)-pentazocine, sigma receptor agonist, is abundant in the guinea pig spleen

Here we measured sigma receptor agonist, [ 3H](+)-pentazocine binding and (+)-pentazocine-stimulated [ 35S]GTPγS binding throughout brain regions and peripheral organs of mice and guinea pigs to investigate the distribution of G protein-coupled sigma receptors. There was no significant correlationship between both distributions, in which the [ 3H](+)-pentazocine binding is highest in the liver of each species, while the [ 35S]GTPγS binding is highest in the guinea pig spleen. The agonist-stimulated [ 35S]GTPγS binding in the spleen was also confirmed by in situ autoradiography using sections. Thus it is suggested that there are at least two subtypes, metabotropic and nonmetabotropic sigma receptors, and the former ones are abundant in the guinea pig spleen.