The sigma-1 receptor curtails endogenous opioid analgesia during sensitization of TRPV1 nociceptors.

BACKGROUND AND PURPOSE: Peripheral sensitization contributes to pathological pain. While prostaglandin E2 (PGE2) and nerve growth factor (NGF) sensitize peptidergic C-nociceptors (TRPV1+), glial cell line-derived neurotrophic factor (GDNF) sensitizes non-peptidergic C-neurons (IB4+). The sigma-1 receptor (sigma-1R) is a Ca2+ -sensing chaperone known to modulate opoid analgesia. This receptor binds both to TRPV1 and the µ opioid receptor, although the functional repercussions of these physical interactions in peripheral sensitization are unknown. EXPERIMENTAL APPROACH: We tested the effects of sigma-1 antagonism on PGE2-, NGF-, and GDNF-induced mechanical and heat hyperalgesia in mice. We used immunohistochemistry to determine the presence of endomorphin-2, an endogenous µ receptor agonist, on dorsal root ganglion (DRG) neurons. Recombinant proteins were used to study the interactions between sigma-1R, µ- receptor, and TRPV1. We used calcium imaging to study the effects of sigma-1 antagonism on PGE2-induced sensitization of TRPV1+ nociceptors. KEY RESULTS: Sigma1 antagonists reversed PGE2- and NGF-induced hyperalgesia but not GDNF-induced hyperalgesia. Endomorphin-2 was detected on TRPV1+ but not on IB4+ neurons. Peripheral opioid receptor antagonism by naloxone methiodide or administration of an anti-endomorphin-2 antibody to a sensitized paw reversed the antihyperalgesia induced by sigma-1 antagonists. Sigma-1 antagonism transfers sigma-1R from TRPV1 to µ receptors, suggesting that sigma-1R participate in TRPV1-µ receptor crosstalk. Moreover, sigma-1 antagonism reversed, in a naloxone-sensitive manner, PGE2-induced sensitization of DRG neurons to the calcium flux elicited by capsaicin, the prototypic TRPV1 agonist. CONCLUSION AND IMPLICATIONS: Sigma-1 antagonism harnesses endogenous opioids produced by TRPV1+ neurons to reduce hyperalgesia by increasing µ receptor activity.

αN-Acetyl ß-Endorphin Is an Endogenous Ligand of σ1Rs That Regulates Mu-Opioid Receptor Signaling by Exchanging G Proteins for σ2Rs in σ1R Oligomers.

The opioid peptide ß-endorphin coexists in the pituitary and brain in its αN-acetylated form, which does not bind to opioid receptors. We now report that these neuropeptides exhibited opposite effects in in vivo paradigms, in which ligands of the sigma type 1 receptor (σ1R) displayed positive effects. Thus, αN-acetyl ß-Endorphin reduced vascular infarct caused by permanent unilateral middle cerebral artery occlusion and diminished the incidence of N-methyl-D-aspartate acid-promoted convulsive syndrome and mechanical allodynia caused by unilateral chronic constriction of the sciatic nerve. Moreover, αN-acetyl ß-Endorphin reduced the analgesia of morphine, ß-Endorphin and clonidine but enhanced that of DAMGO. All these effects were counteracted by ß-Endorphin and absent in σ1R-/- mice. We observed that σ1Rs negatively regulate mu-opioid receptor (MOR)-mediated morphine analgesia by binding and sequestering G proteins. In this scenario, ß-Endorphin promoted the exchange of σ2Rs by G proteins at σ1R oligomers and increased the regulation of G proteins by MORs. The opposite was observed for the αN-acetyl derivative, as σ1R oligomerization decreased and σ2R binding was favored, which displaced G proteins; thus, MOR-regulated transduction was reduced. Our findings suggest that the pharmacological ß-Endorphin-specific epsilon receptor is a σ1R-regulated MOR and that ß-Endorphin and αN-acetyl ß-Endorphin are endogenous ligands of σ1R.

The σ1 Receptor and the HINT1 Protein Control α2δ1 Binding to Glutamate NMDA Receptors: Implications in Neuropathic Pain.

Nerve injury produces neuropathic pain through the binding of α2δ1 proteins to glutamate N-methyl-D-aspartate receptors (NMDARs). Notably, mice with a targeted deletion of the sigma 1 receptor (σ1R) gene do not develop neuropathy, whereas mice lacking the histidine triad nucleotide-binding protein 1 (Hint1) gene exhibit exacerbated allodynia. σ1R antagonists more effectively diminish neuropathic pain of spinal origin when administered by intracerebroventricular injection than systemically. Thus, in mice subjected to unilateral sciatic nerve chronic constriction injury (CCI), we studied the participation of σ1Rs and HINT1 proteins in the formation of α2δ1-NMDAR complexes within the supraspinal periaqueductal gray (PAG). We found that δ1 peptides required σ1Rs in order to interact with the NMDAR NR1 variant that contains the cytosolic C1 segment. σ1R antagonists or low calcium levels provoke the dissociation of σ1R-NR1 C1 dimers, while they barely affect the integrity of δ1-σ1R-NR1 C1 trimers. However, HINT1 does remove δ1 peptides from the trimer, thereby facilitating the subsequent dissociation of σ1Rs from NMDARs. In σ1R-/- mice, CCI does not promote the formation of NMDAR-α2δ1 complexes and allodynia does not develop. The levels of α2δ1-σ1R-NMDAR complexes increase in HINT1-/- mice and after inducing CCI, degradation of α2δ1 proteins is observed. Notably, σ1R antagonists but not gabapentinoids alleviate neuropathic pain in these mice. During severe neuropathy, the metabolism of α2δ1 proteins may account for the failure of many patients to respond to gabapentinoids. Therefore, σ1Rs promote and HINT1 proteins hinder the formation α2δ1-NMDAR complexes in the PAG, and hence, the appearance of mechanical allodynia depends on the interplay between these proteins.

Effects of Gαi2 and Gαz protein knockdown on alpha2A-adrenergic and cannabinoid CB1 receptor regulation of MEK-ERK and FADD pathways in mouse cerebral cortex.

BACKGROUND: Alpha2A-adrenergic (α2A-AR) and cannabinoid CB1 (CB1-R) receptors exert their functions modulating multiple signaling pathways, including MEK-ERK (extracellular signal-regulated kinases) and FADD (Fas-associated protein with death domain) cascades. These molecules are relevant in finding biased agonists with fewer side effects, but the mechanisms involving their modulations by α2A-AR- and CB1-R in vivo are unclear. This study investigated the roles of Gαi2 and Gαz proteins in mediating α2A-AR- and CB1-R-induced alterations of MEK-ERK and FADD phosphorylation (p-) in mouse brain cortex. METHODS: Gαi2 or Gαz protein knockdown was induced in mice with selective antisense oligodeoxinucleotides (ODNs; 3 nmol/day, 5 days) prior to UK-14,304 (UK or brimonidine; 1 mg/kg) or WIN55212-2 (WIN; 8 mg/kg) acute treatments. Inactivated (p-T286) MEK1, activated (p-S217/221) MEK1/2, activated (p-T202/Y204) ERK1/2, p-S191 FADD, and the corresponding total forms of these proteins were quantified by immunoblotting. RESULTS: Increased (+ 88%) p-T286 MEK1 cortical density, with a concomitant reduction (-43%) of activated ERK was observed in UK-treated mice. Both effects were attenuated by Gαi2 or Gαz antisense ODNs. Contrastingly, WIN induced Gαi2- and Gαz-independent upregulations of p-T286 MEK1 (+ 63%), p-S217/221 MEK1/2 (+ 86%), and activated ERK (+ 111%) in brain. Pro-apoptotic FADD was downregulated (- 34 to 39%) following UK and WIN administration, whereas the neuroprotective p-S191 FADD was increased (+ 74%) in WIN-treated mice only. None of these latter effects required from Gαi2 or Gαz protein integrity. CONCLUSION: The results indicate that α2A-AR (UK), but not CB1-R (WIN), agonists use Gαi2 and Gαz proteins to modulate MEK-ERK, but not FADD, pathway in mouse brain cortex.

Human HINT1 Mutant Proteins that Cause Axonal Motor Neuropathy Exhibit Anomalous Interactions with Partner Proteins.

The 14 kDa histidine triad nucleotide-binding protein 1 (HINT1) is critical to maintain the normal function of motor neurons. Thus, a series of human HINT1 mutants cause autosomal recessive axonal neuropathy with neuromyotonia. HINT1 establishes a series of regulatory interactions with signaling proteins, some of which are enriched in motor neurons, such as the type 1 sigma receptor or intracellular domain (ICD) of transmembrane teneurin 1, both of which are also implicated in motor disturbances. In a previous study, we reported the capacity of HINT1 to remove the small ubiquitin-like modifier (SUMO) from a series of substrates and the influence of HINT1 mutants on this activity. We now report how human HINT1 mutations affect the interaction of HINT1 with the regulator of its SUMOylase activity, calcium-activated calmodulin, and its substrate SUMO. Moreover, HINT1 mutants exhibited anomalous interactions with G protein coupled receptors, such as the mu-opioid, and with glutamate N-methyl-D-aspartate receptors as well. Additionally, these HINT1 mutants showed impaired associations with transcriptional regulators such as the regulator of G protein signaling Z2 protein and the cleaved N-terminal ICD of teneurin 1. Thus, the altered enzymatic activity of human HINT1 mutants and their anomalous interactions with partner proteins may disrupt signaling pathways essential to the normal function of human motor neurons.

Calmodulin Supports TRPA1 Channel Association with Opioid Receptors and Glutamate NMDA Receptors in the Nervous Tissue.

Transient receptor potential ankyrin member 1 (TRPA1) belongs to the family of thermo TRP cation channels that detect harmful temperatures, acids and numerous chemical pollutants. TRPA1 is expressed in nervous tissue, where it participates in the genesis of nociceptive signals in response to noxious stimuli and mediates mechanical hyperalgesia and allodynia associated with different neuropathies. The glutamate N-methyl-d-aspartate receptor (NMDAR), which plays a relevant role in allodynia to mechanical stimuli, is connected via histidine triad nucleotide-binding protein 1 (HINT1) and type 1 sigma receptor (σ1R) to mu-opioid receptors (MORs), which mediate the most potent pain relief. Notably, neuropathic pain causes a reduction in MOR antinociceptive efficacy, which can be reversed by blocking spinal NMDARs and TRPA1 channels. Thus, we studied whether TRPA1 channels form complexes with MORs and NMDARs that may be implicated in the aforementioned nociceptive signals. Our data suggest that TRPA1 channels functionally associate with MORs, delta opioid receptors and NMDARs in the dorsal root ganglia, the spinal cord and brain areas. These associations were altered in response to pharmacological interventions and the induction of inflammatory and also neuropathic pain. The MOR-TRPA1 and NMDAR-TRPA1 associations do not require HINT1 or σ1R but appear to be mediated by calcium-activated calmodulin. Thus, TRPA1 channels may associate with NMDARs to promote ascending acute and chronic pain signals and to control MOR antinociception.

The Sigma 2 receptor promotes and the Sigma 1 receptor inhibits mu-opioid receptor-mediated antinociception.

The Sigma-1 receptor (σ1R) has emerged as an interesting pharmacological target because it inhibits analgesia mediated by mu-opioid receptors (MOR), and also facilitates the development of neuropathic pain. Based on these findings, the recent cloning of the Sigma-2 receptor (σ2R) led us to investigate its potential role as a regulator of opioid analgesia and of pain hypersensitivity in σ2R knockout mice. In contrast to σ1R deficient mice, σ2R knockout mice developed mechanical allodynia following establishment of chronic constriction injury-induced neuropathic pain, which was alleviated by the σ1R antagonist S1RA. The analgesic effects of morphine, [D-Ala, N-MePhe, Gly-ol]-encephalin (DAMGO) and ß-endorphin increased in σ1R-/- mice and diminished in σ2R-/- mice. The analgesic effect of morphine was increased in σ2R-/- mice by treatment with S1RA. However, σ2R-/- mice and wild-type mice exhibited comparable antinociceptive responses to the delta receptor agonist [D-Pen2,5]-encephalin (DPDPE), the cannabinoid type 1 receptor agonist WIN55,212-2 and the α2-adrenergic receptor agonist clonidine. Therefore, while σR1 inhibits and σ2R facilitates MOR-mediated analgesia these receptors exchange their roles when regulating neuropathic pain perception. Our study may help identify new pharmacological targets for diminishing pain perception and improving opioid detoxification therapies.

The ALS-related σ1R E102Q Mutant Eludes Ligand Control and Exhibits Anomalous Response to Calcium.

Sigma receptor type 1 (σ1R) is a transmembrane protein expressed throughout the central nervous system and in certain peripheral tissues. The human σ1R E102Q mutation causes juvenile amyotrophic lateral sclerosis (ALS), likely by inducing a series of alterations in calcium efflux from the endoplasmic reticulum (ER) to mitochondria that affects calcium homeostasis and cellular survival. Here, we report the influence of calcium on σ1R E102Q associations with glutamate N-methyl-D-aspartate receptors (NMDARs), binding immunoglobulin protein (BiP), and transient receptor potential calcium channels A1, V1, and M8. The mutant protein inhibited the binding of calmodulin to these calcium channels and interacted less with BiP than wild-type σ1R, thereby contributing to calcium homeostasis dysfunction. Mutant σ1R, but not wild-type σ1R, strongly bound to histidine triad nucleotide binding protein 1, which regulates neuromuscular synaptic organization and target selection through teneurin 1. While ligands regulated the association of σ1R wild-type with NMDARs and BiP, they failed to modulate the interaction between these proteins and the σ1R E102Q mutant. Thus, the σ1R E102Q mutant exhibited an anomalous response to cytosolic calcium levels, altered affinity for target proteins, and a loss of response to regulatory ligands. We believe that these modifications may contribute to the onset of juvenile ALS.

Ligands Exert Biased Activity to Regulate Sigma 1 Receptor Interactions With Cationic TRPA1, TRPV1, and TRPM8 Channels.

The sigma 1 receptor (σ1R) and the mu-opioid receptor (MOR) regulate the transient receptor potential (TRP) V1 calcium channel. A series of proteins are involved in the cross-regulation between MORs and calcium channels like the glutamate N-methyl-D-aspartate receptor (NMDAR), including the histidine triad nucleotide-binding protein 1 (HINT1), calmodulin (CaM), and the σ1R. Thus, we assessed whether similar mechanisms also apply to the neural TRP ankyrin member 1 (TRPA1), TRP vanilloid member 1 (TRPV1), and TRP melastatin member 8 (TRPM8). Our results indicate that σ1R and CaM bound directly to cytosolic regions of these TRPs, and this binding increased in the presence of calcium. By contrast, the association of HINT1 with these TRPs was moderately dependent on calcium. The σ1R always competed with CaM for binding to the TRPs, except for its binding to the TRPA1 C-terminal where σ1R binding cooperated with that of CaM. However, σ1R dampened HINT1 binding to the TRPA1 N-terminal. When the effect of σ1R ligands was addressed, the σ1R agonists PRE084 and pregnenolone sulfate enhanced the association of the σ1R with the TRPM8 N-terminal and TRPV1 C-terminal in the presence of physiological calcium, as seen for the σ1R-NMDAR interactions. However, these agonists dampened σ1R binding to the TRPA1 and TRPV1 N-terminal domains, and also to the TRPA1 C-terminal, as seen for σ1R-binding immunoglobulin protein (BiP) interactions in the endoplasmic reticulum (ER). By contrast, the σ1R antagonists progesterone and S1RA reduced the association of σ1R with TRPA1 and TRPV1 C-terminal regions, as seen for the σ1R-NMDAR interactions. Conversely, they enhanced the σ1R interaction with the TRPA1 N-terminal, as seen for σ1R-BiP interactions, whereas they barely affected the association of σ1R with the TRPV1 N-terminal. Thus, depending on the calcium channel and the cytosolic region examined, the σ1R agonists pregnenolone sulfate and PRE084 opposed or collaborated with the σ1R antagonists progesterone and S1RA to disrupt or promote such interactions. Through the use of cloned cytosolic regions of selected TRP calcium channels, we were able to demonstrate that σ1R ligands exhibit biased activity to regulate particular σ1R interactions with other proteins. Since σ1Rs are implicated in essential physiological processes, exploiting such ligand biases may represent a means to develop more selective and efficacious pharmacological interventions.

The Axonal Motor Neuropathy-Related HINT1 Protein Is a Zinc- and Calmodulin-Regulated Cysteine SUMO Protease.

Aims: Histidine triad nucleotide-binding protein 1 (HINT1) exhibits proapoptotic and tumor-suppressive activity. HINT1 binds to transcription factors such as teneurin1 and to the regulator of G protein signaling 17 (RGS) (Z2) protein, which incorporates the small ubiquitin-like modifier (SUMO), and is implicated in several types of cancer. HINT1 interacts with proteins such as PKCγ and Raf-1 through zinc ions provided by the cysteine-rich domain of RGSZ2 and the coupled neural nitric oxide synthase (nNOS). Recently, a series of HINT1 mutants have been reported to cause human autosomal recessive axonal neuropathy with neuromyotonia (ARAN-NM). However, the specific alteration in the function of HINT1 induced by these mutants remains to be elucidated. Because sumoylation modifies protein association and transcriptional regulation, we investigated whether HINT1 exhibits zinc- and redox-regulated sumoylase activity, which may be altered in those mutants. Results: HINT1 exhibits cysteine protease activity to remove SUMO from a variety of signaling proteins. HINT1 sumoylase activity is blocked by zinc, and it is released by nitric oxide or calcium-activated calmodulin (CaM). HINT1 contains a SUMO-interacting motif (110-116 HIHLHVL) and the catalytic triad Cys84-Asp87-His114 in the C-terminal region. Thus, zinc probably provided by the RGSZ2-nNOS complex may bind to Cys84 to block HINT1 isopeptidase activity. Innovation: To date, HINT1 is the only sumoylase that is regulated by two alternate pathways, redox- and calcium-activated CaM. Conclusion: The 15 human HINT1 mutants reported to cause ARAN-NM exhibited altered sumoylase activity, which may contribute to the onset of this human motor disease.

Cannabidiol enhances morphine antinociception, diminishes NMDA-mediated seizures and reduces stroke damage via the sigma 1 receptor.

Cannabidiol (CBD), the major non-psychotomimetic compound present in the Cannabis sativa plant, exhibits therapeutic potential for various human diseases, including chronic neurodegenerative diseases, such as Alzheimer's and Parkinson's, ischemic stroke, epilepsy and other convulsive syndromes, neuropsychiatric disorders, neuropathic allodynia and certain types of cancer. CBD does not bind directly to endocannabinoid receptors 1 and 2, and despite research efforts, its specific targets remain to be fully identified. Notably, sigma 1 receptor (σ1R) antagonists inhibit glutamate N-methyl-D-aspartate acid receptor (NMDAR) activity and display positive effects on most of the aforesaid diseases. Thus, we investigated the effects of CBD on three animal models in which NMDAR overactivity plays a critical role: opioid analgesia attenuation, NMDA-induced convulsive syndrome and ischemic stroke. In an in vitro assay, CBD disrupted the regulatory association of σ1R with the NR1 subunit of NMDAR, an effect shared by σ1R antagonists, such as BD1063 and progesterone, and prevented by σ1R agonists, such as 4-IBP, PPCC and PRE084. The in vivo administration of CBD or BD1063 enhanced morphine-evoked supraspinal antinociception, alleviated NMDA-induced convulsive syndrome, and reduced the infarct size caused by permanent unilateral middle cerebral artery occlusion. These positive effects of CBD were reduced by the σ1R agonists PRE084 and PPCC, and absent in σ1R-/- mice. Thus, CBD displays antagonist-like activity toward σ1R to reduce the negative effects of NMDAR overactivity in the abovementioned experimental situations.

Sigma 1 Receptor Antagonists Inhibit Manic-Like Behaviors in Two Congenital Strains of Mice.

Background: Several currently available animal models reproduce select behavioral facets of human mania as well as the abnormal glutamatergic neurotransmission and dysregulation of glycogen synthase kinase 3ß that accompanies this disease. Methods: In this study, we addressed the therapeutic potential of ligands of sigma receptor type 1 (σ1R) in 2 putative models of mania: the "manic" Black Swiss outbred mice from Taconic farms (BStac) and mice with the 129 genetic background and histidine triad nucleotide-binding protein 1 (HINT1) deletion (HINT1-/- mice) that exhibit bipolar-like behaviors. Results: The activity of control mice, which do not exhibit manic-like behaviors in the forced swim test, was significantly enhanced by MK801, an inhibitor of glutamate N-methyl-D-aspartate receptor activity, an effect that was not or barely observed in manic-like mice. Typical mood stabilizers, such as glycogen synthase kinase 3ß inhibitors, but not σ1R ligands, reduced the N-methyl-D-aspartate receptor-mediated behaviors in control mice. Notably, σ1R antagonists S1RA, PD144418, BD1047, and BD1063, but not σ1R agonists PRE084 and PPCC, attenuated the manic-like behaviors of BStac and HINT1-/- mice by increasing antiactivity behaviors. The antimanic effects of a single administration of σ1R antagonists persisted for at least 24 hours, and these drugs did not alter the behavior of the "bipolar" HINT1-/- mice during pro-depressive episodes. Conclusions: σ1R antagonists exhibit a selective normalizing effect on specific behavioral domains of mania without altering control (normal) or depressive-like behaviors.

Fenfluramine diminishes NMDA receptor-mediated seizures via its mixed activity at serotonin 5HT2A and type 1 sigma receptors.

Fenfluramine exhibits antiepileptic properties and thus diminishes epileptiform discharges in experimental animal models of Dravet syndrome. Fenfluramine is metabolized into norfenfluramine in vivo, which shows greater affinity and agonist activity at serotonin 5HT2 receptors (5HT2R) than fenfluramine. In this study, we found that fenfluramine and norfenfluramine disrupted the regulatory association of the sigma 1 receptor (σ1R) with NR1 subunits of glutamate N-methyl-D-aspartate receptors (NMDAR), an effect that was also produced by σ1R antagonists such as S1RA and prevented by σ1R agonists such as PPCC. The antagonists removed σ1R bound to NMDAR NR1 subunits enabling calcium-regulated calmodulin (CaM) to bind to those subunits. As a result, CaM may inhibit calcium permeation through NMDARs. The serotoninergic activity of fenfluramine at 5HT2AR, and likely also at 5HT2CR, collaborated with its activity at σ1Rs to prevent the convulsive syndrome promoted by NMDAR overactivation. Notably, fenfluramine enhanced the inhibitory coupling of G protein-coupled receptors such as 5HT1AR and cannabinoid type 1 receptor with NMDARs, thus allowing the more effective restrain of NMDAR activity. Thus, fenfluramine circumvents the negative side effects of direct NMDAR antagonists and may improve the quality of life of subjects affected by such proconvulsant dysfunctions.

The Sigma-1 Receptor Antagonist, S1RA, Reduces Stroke Damage, Ameliorates Post-Stroke Neurological Deficits and Suppresses the Overexpression of MMP-9.

The glutamate N-methyl-D-aspartate receptor (NMDAR) plays an essential role in the excitotoxic neural damage that follows ischaemic stroke. Because the sigma-1 receptor (σ1R) can regulate NMDAR transmission, exogenous and putative endogenous regulators of σ1R have been investigated using animal models of ischaemic stroke. As both agonists and antagonists provide some neural protection, the selective involvement of σ1Rs in these effects has been questioned. The availability of S1RA (E-52862/MR309), a highly selective σ1R antagonist, prompted us to explore its therapeutic potential in an animal model of focal cerebral ischaemia. Mice were subjected to right middle cerebral artery occlusion (MCAO), and post-ischaemic infarct volume and neurological deficits were determined across a range of intervals after the stroke-inducing surgery. Intracerebroventricular or intravenous treatment with S1RA significantly reduced the cerebral infarct size and neurological deficits caused by permanent MCAO (pMCAO). Compared with the control/sham-operated mice, the neuroprotective effects of S1RA were observed when delivered up to 5 h prior to surgery and 3 h after ischaemic onset. Interestingly, neither mice with the genetic deletion of σ1R nor wild-type mice that were pre-treated with the σ1R agonist PRE084 showed beneficial effects after S1RA administration with regard to stroke infarction. S1RA-treated mice showed faster behavioural recovery from stroke; this finding complements the significant decreases in matrix metalloproteinase-9 (MMP-9) expression and reactive astrogliosis surrounding the infarcted cortex. Our data indicate that S1RA, via σ1R, holds promising potential for clinical application as a therapeutic agent for ischaemic stroke.

Schizophrenia and depression, two poles of endocannabinoid system deregulation.

The activity of certain G protein-coupled receptors (GPCRs) and of glutamate N-Methyl-D-aspartate receptors (NMDARs) is altered in both schizophrenia and depression. Using postmortem prefrontal cortex samples from subjects with schizophrenia or depression, we observed a series of opposite changes in the expression of signaling proteins that have been implicated in the cross-talk between GPCRs and NMDARs. Thus, the levels of HINT1 proteins and NMDAR NR1 subunits carrying the C1 cytosolic segment were increased in depressives and decreased in schizophrenics, respect to matched controls. The differences in NR1 C1 subunits were compensated for via altered expression of NR1 subunits lacking the C1 segment; thus, the total number of NR1 subunits was comparable among the three groups. GPCRs influence the function of NR1 C1-containing NMDARs via PKC/Src, and thus, the association of mu-opioid and dopamine 2 receptors with NR1 C1 subunits was augmented in depressives and decreased in schizophrenics. However, the association of cannabinoid 1 receptors (CB1Rs) with NR1 C1 remained nearly constant. Endocannabinoids, via CB1Rs, control the presence of NR1 C1 subunits in the neural membrane. Thus, an altered endocannabinoid system may contribute to the pathophysiology of schizophrenia and depression by modifying the HINT1-NR1 C1/GPCR ratio, thereby altering GPCR-NMDAR cross-regulation.

Increased PKC activity and altered GSK3ß/NMDAR function drive behavior cycling in HINT1-deficient mice: bipolarity or opposing forces.

Mice with histidine triad nucleotide-binding protein 1 (HINT1) deletion exhibit manic-like symptoms that evolve into depressive-like behavior in response to stressful paradigms. Molecular and electrophysiological studies have indicated that HINT1-/- mice exhibit increased PKC, PKA, and GSK3ß activities, as well as glutamate N-methyl-D-aspartate receptor (NMDAR)/α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptor (AMPAR) and NR2B/NR2A subunit ratios. Pharmacological interventions stabilized their behavior but through different mechanisms. GSK3ß inhibitors and valproate directly attenuated the expression of the manic-like symptoms, whereas PKC inhibition, lamotrigine, or risperidone promoted NMDAR-mediated depressive-like behaviors that counterbalanced the preexisting manic-like symptoms. Naïve HINT1-/- mice exposed to stressful paradigms rapidly manifested depressive-like behaviors in subsequent stressful situations, a capacity that persisted for a couple of weeks thereafter. During the depressive-like phase, citalopram, amitriptyline and MK801 precipitated manic-like behaviors in stressed HINT1-/- mice. Notably, the antagonism of NMDARs prevented HINT1-/- mice from alternating behaviors in response to stress. A comparison with "manic" Black Swiss mice indicated that in HINT1-/- mice, PKC supports manic-like symptoms and reduces the expression of depressive-like behaviors via activation of GSK3ß and regulation of NR2B-enriched NMDARs. HINT1-/- mice represent a suitable model for studying human BPD and may facilitate the identification of novel targets and drugs to treat this mental disorder.

Endocannabinoid control of glutamate NMDA receptors: the therapeutic potential and consequences of dysfunction.

Glutamate is probably the most important excitatory neurotransmitter in the brain. The glutamate N-methyl-D-aspartate receptor (NMDAR) is a calcium-gated channel that coordinates with G protein-coupled receptors (GPCRs) to establish the efficiency of the synaptic transmission. Cross-regulation between these receptors requires the concerted activity of the histidine triad nucleotide-binding protein 1 (HINT1) and of the sigma receptor type 1 (σ1R). Essential brain functions like learning, memory formation and consolidation, mood and behavioral responses to exogenous stimuli depend on the activity of NMDARs. In this biological context, endocannabinoids are released to retain NMDAR activity within physiological limits. The efficacy of such control depends on HINT1/σ1R assisting in the physical coupling between cannabinoid type 1 receptors (CB1Rs) and NMDARs to dampen their activity. Subsequently, the calcium-regulated HINT1/σ1R protein tandem uncouples CB1Rs to prevent NMDAR hypofunction. Thus, early recruitment or a disproportionate cannabinoid induced response can bring about excess dampening of NMDAR activity, impeding its adequate integration with GPCR signaling. Alternatively, this control circuit can apparently be overridden in situations where bursts of NMDAR overactivity provoke convulsive syndromes. In this review we will discuss the possible relevance of the HINT1/σ1R tandem and its use by endocannabinoids to diminish NMDAR activity and their implications in psychosis/schizophrenia, as well as in NMDAR-mediated convulsive episodes.

The ON:OFF switch, σ1R-HINT1 protein, controls GPCR-NMDA receptor cross-regulation: implications in neurological disorders.

In the brain, the histidine triad nucleotide-binding protein 1 (HINT1) and sigma 1 receptors (σ1Rs) coordinate the activity of certain G-protein coupled receptors (GPCRs) with that of glutamate N-methyl-D-aspartate receptors (NMDARs). To determine the role of HINT1-σ1R in the plasticity of GPCR-NMDAR interactions, substances acting at MOR, cannabinoid CB1 receptor, NMDAR and σ1R were injected into mice, and their effects were evaluated through in vivo, ex vivo, and in vitro assays. It was observed that HINT1 protein binds to GPCRs and NMDAR NR1 subunits in a calcium-independent manner, whereas σ1R binding to these proteins increases in the presence of calcium. In this scenario, σ1R agonists keep HINT1 at the GPCR and stimulate GPCR-NMDAR interaction, whereas σ1R antagonists transfer HINT1 to NR1 subunits and disengage both receptors. This regulation is lost in σ1R-/- mice, where HINT1 proteins mostly associate with NMDARs, and GPCRs are physically and functionally disconnected from NMDARs. In HINT1-/- mice, ischemia produces low NMDAR-mediated brain damage, suggesting that several different GPCRs enhance glutamate excitotoxicity via HINT1-σ1R. Thus, several GPCRs associate with NMDARs by a dynamic process under the physiological control of HINT1 proteins and σ1Rs. The NMDAR-HINT1-σ1R complex deserves attention because it offers new therapeutic opportunities.

The σ1 receptor engages the redox-regulated HINT1 protein to bring opioid analgesia under NMDA receptor negative control.

AIMS: The in vivo pharmacology of the sigma 1 receptor (σ1R) is certainly complex; however, σ1R antagonists are of therapeutic interest, because they enhance mu-opioid receptor (MOR)-mediated antinociception and reduce neuropathic pain. Thus, we investigated whether the σ1R is involved in the negative control that glutamate N-methyl-d-aspartate acid receptors (NMDARs) exert on opioid antinociception. RESULTS: The MOR C terminus carries the histidine triad nucleotide-binding protein 1 (HINT1) coupled to the regulator of G-protein signaling RGSZ2-neural nitric oxide synthase assembly. Activated MORs stimulate the production of nitric oxide (NO), and the redox zinc switch RGSZ2 converts this signal into free zinc ions that are required to recruit the redox sensor PKCγ to HINT1 proteins. Then, PKCγ impairs HINT1-RGSZ2 association and enables σ1R-NR1 interaction with MOR-HINT1 complexes to restrain opioid signaling. The inhibition of NOS or the absence of σ1Rs prevents HINT1-PKCγ interaction, and MOR-NMDAR cross-regulation fails. The σ1R antagonists transitorily remove the binding of σ1Rs to NR1 subunits, facilitate the entrance of negative regulators of NMDARs, likely Ca(2+)-CaM, and prevent NR1 interaction with HINT1, thereby impairing the negative feedback of glutamate on opioid analgesia. INNOVATION: A redox-regulated process situates MOR signaling under NMDAR control, and in this context, the σ1R binds to the cytosolic C terminal region of the NMDAR NR1 subunit. CONCLUSION: The σ1R antagonists enhance opioid analgesia in naïve mice by releasing MORs from the negative influence of NMDARs, and they also reset antinociception in morphine tolerant animals. Moreover, σ1R antagonists alleviate neuropathic pain, probably by driving the inhibition of up-regulated NMDARs.

Detecting zinc release induced by mu-opioid receptor agonists in brain slices.

After iron, zinc is the second most abundant transition metal in living organisms and it is known to be a contributory factor in a series of neurological disorders. In biological systems zinc exists as either bound Zn(2+), representing the majority of the total zinc in tissues, or free (chelatable) Zn(2+). Several fluorescents dyes have been developed to detect free zinc when it is released from zinc-binding proteins, which occurs via redox mechanisms in response to the stimulation of a number of neurotransmitter receptors, including the µ opioid receptor. Here we describe a detailed protocol to detect drug stimulated intracellular zinc release in rodent brain slices using time-lapse microscopy and fluorescence imaging.