Mu-opioid Receptor Signaling Research Articles

Mu-opioid receptor signalling switch to excitatory following chronic morphine persists upon treatment cessation

Mu-opioid receptor signalling switch to excitatory following chronic morphine persists upon treatment cessation

Heteromerization Modulates mu Opioid Receptor Functional Properties in vivo.

Mu opioid receptors modulate a large number of physiological functions. They are in particular involved in the control of pain perception and reward properties. They are also the primary molecular target of opioid drugs and mediate their beneficial analgesic effects, euphoric properties as well as negative side effects such as tolerance and physical dependence. Importantly, mu opioid receptors can physically associate with another receptor to form a novel entity called heteromer that exhibits specific ligand binding, signaling, and trafficking properties. As reviewed here, in vivo physical proximity has now been evidenced for several receptor pairs, subsequent impact of heteromerization on native mu opioid receptor signaling and trafficking identified and a link to behavioral changes established. Selective targeting of heteromers as a tool to modulate mu opioid receptor activity is therefore attracting growing interest and raises hopes for innovative therapeutic strategies.

Oprm1 A112G, a single nucleotide polymorphism, alters expression of stress-responsive genes in multiple brain regions in male and female mice

BackgroundOPRM1 A118G, a functional human mu-opioid receptor (MOR) polymorphism, is associated with drug dependence and altered stress responsivity in humans as well as altered MOR signaling. MOR signaling can regulate many cellular processes, including gene expression, and many of the long-term, stable effects of drugs and stress may stem from changes in gene expression in diverse brain regions. A mouse model bearing an equivalent polymorphism (Oprm1 A112G) was previously generated and studied. Mice homozygous for the G112 allele show differences in opioid- and stress-related phenotypes.ApproachThe current study examines the expression of 24 genes related to drug and stress responsivity in the caudoputamen, nucleus accumbens, hypothalamus, hippocampus, and amygdala of drug-naïve, stress-minimized, male and female mice homozygous for either the G112 variant allele or the wild-type A112 allele.ResultsWe detected nominal genotype-dependent changes in gene expression of multiple genes. We also detected nominal sex-dependent as well as sex-by-genotype interaction effects on gene expression. Of these, four genotype-dependent differences survived correction for multiple testing: Avp and Gal in the hypothalamus and Oprl1 and Cnr1 in the hippocampus.ConclusionsChanges in the regulation of these genes by mu-opioid receptors encoded by the G112 allele may be involved in some of the behavioral and molecular consequences of this polymorphism observed in mice.

Heat Shock Protein 90α, Assisted by Co‐Chaperones p23 and Cdc37, Promotes Opioid Anti‐Nociception in the Brain via Promoting ERK MAPK Signaling

Reducing opioid side effects like constipation and addiction has been a strong focus for improving opioid therapy in treating acute and chronic pain. One such approach includes manipulation of mu opioid receptor (MOR) signaling cascades, such as βarrestin2. However, the MOR signaling complex and mechanisms of signaling regulation are poorly understood. In our earlier work, we identified Heat shock protein 90 (Hsp90) as a novel MOR signaling regulator, which could be a target for improving the therapeutic profile of opioids. We found that intracerebroventricular (icv) 17‐AAG, a well‐established Hsp90 inhibitor, strongly blocked or abolished morphine anti‐nociception in paw incision and HIV neuropathic pain in mice. The current study was designed to identify molecular mechanisms of this regulation, including which isoform(s) and/or co‐chaperone(s) are involved in the regulatory effect of Hsp90 on opioid signaling. Male and female CD‐1 mice were treated with selective inhibitors for different Hsp90 isoforms (KU‐A, KUNB106, KUNG65) and co‐chaperones (KU177, gedunin, celastrol) via icv injection. We found that an Hsp90α selective inhibitor blocked morphine anti‐nociception by ~ 80% in the paw incision model, however, inhibitors for Hsp90β and Grp94 had no impact on morphine anti‐nociception. Knockdown of Hsp90α in the brain by CRISPR‐Cas9 also showed strong inhibition of morphine anti‐nociception in paw incision, confirming the results. Further, inhibition of the co‐chaperones p23 and Cdc37, but not Aha1, showed similar effects as Hsp90α inhibition in the paw incision model. To determine the molecular mechanism of these activities, we found that Hsp90α inhibitor treatment blocked ERK and JNK MAPK activation induced by the selective MOR agonist DAMGO without changing the expression of Hsp70 in mouse periaqueductal grey (PAG). Treatment with the ERK MAPK inhibitor U0126 recapitulated our results of Hsp90 inhibition on paw incision, HIV neuropathic, and tail flick pain, suggesting that ERK MAPK regulation is the means by which Hsp90α regulates morphine anti‐nociception. Identification of the isoforms and co‐chaperones involved in Hsp90 regulation of ERK and opioid anti‐nociception may permit more selective manipulation of the MOR signaling machinery to improve opioid efficacy and reduce side effects.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Phosphatidylethanolamine‐Binding Protein Promotes Opioid Anti‐Nociception in the Brain and Spinal Cord by Reducing βarrestin2 Recruitment to the Mu Opioid Receptor

Opioids have been widely accepted as an effective treatment for chronic pain at a cost of serious side effects, including tolerance and addiction. Side effects are caused in part by desensitization and downregulation of mu opioid receptors (MOR) through recruitment of βarrestin2. Phosphatidylethanolamine‐Binding Protein (PEBP) is known to regulate either Raf Kinase or G Protein Receptor Kinase‐2 (GRK2) through phosphorylation by Protein Kinase C (PKC), and GRK2 is a major player in the recruitment of βarrestin2. However, PEBP has not been investigated for its role in MOR signaling and opioid response. We first tested this using an in vitro model of βarrestin2 recruitment in MOR expressing U2OS cells. We found that by inhibiting PEBP through siRNA knockdown or the small molecule inhibitor locostatin, we could increase the recruitment of βarrestin2 to the MOR in 2 separate assays. Conversely, we found that activating PKC via phorbol myristate acetate (PMA) led to PEBP phosphorylation/activation, and a reduction in βarrestin2 recruitment to the MOR, which could be blocked by PEBP knockdown. We then tested the role of PEBP in vivo by injection of locostatin into the brain or spinal cord, or CRISPR editing of PEBP in the spinal cord. We found that blocking PEBP in vivo led to a strong reduction in morphine‐induced tail flick anti‐nociception in both the brain and spinal cord. Together, these results support the hypothesis that PEBP acts to sequester GRK2 and block βarrestin2 recruitment downstream of the MOR both in vivo and in vitro. These results also suggest that enhanced PKC/PEBP activation by opioid drugs could further reduce opioid side effects and enhance analgesic efficacy.Support or Funding InformationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

(312) - Heat shock protein 90 promotes opioid anti-nociception in the brain and represses opioid anti-nociception in the spinal cord through the differential regulation of ERK MAPK signaling

Opioid drugs are efficacious in treating moderate to severe acute and chronic pain, but are limited by serious side effects. Recent efforts to create biased opioids without these side effects by selectively engaging downstream signal transduction (e.g. βarrestin2) has been limited by our lack of knowledge of the mu opioid receptor (MOR) signaling complex. In our earlier work, we identified Heat shock protein 90 (Hsp90) as a novel MOR signaling regulator. We found that intracerebroventricular (icv) 17-AAG, a well-established Hsp90 inhibitor, strongly blocked or abolished morphine anti-nociception in paw incision and HIV neuropathy pain in mice. In our current work, we've extended from these findings to examine the role of Hsp90 in the spinal cord. In contrast to our findings in the brain, we found that intrathecal (it) .5 nmol 17-AAG for 24 hours in CD-1 mice caused strongly increased morphine anti-nociception in tail flick and paw incision pain (80.7–88.6% increase). This was confirmed using the alternate Hsp90 inhibitor KU-32 as well as Rotarod testing, ruling out off-target effects. We examined ERK MAPK signaling in the brain and spinal cord in response to opioids after 17-AAG inhibition, and found that ERK MAPK activation was lost in the brain, but potentiated in the spinal cord. By using icv and it administration of 5 µg U0126, a selective ERK inhibitor, we demonstrated that the effects of Hsp90 inhibition in the brain and spinal cord could be completely explained by ERK signaling changes. By using selective inhibitors, we further found that the molecular isoform Hsp90α and the co-chaperones p23 and Cdc37 (but not Aha1) were responsible for the signaling and anti-nociception changes observed in the brain. Overall, these results demonstrate different regional effects of Hsp90 and ERK signaling that have a strong impact on opioid anti-nociception, which could be used to improve opioid drug discovery.

Fentanyl Induces Rapid Onset Hyperalgesic Priming: Type I at Peripheral and Type II at Central Nociceptor Terminals.

Systemic fentanyl induces hyperalgesic priming, long-lasting neuroplasticity in nociceptor function characterized by prolongation of inflammatory mediator hyperalgesia. To evaluate priming at both nociceptor terminals, we studied, in male Sprague Dawley rats, the effect of local administration of agents that reverse type I (protein translation) or type II [combination of Src and mitogen-activated protein kinase (MAPK)] priming. At the central terminal, priming induced by systemic, intradermal, or intrathecal fentanyl was reversed by the combination of Src and MAPK inhibitors, but at the peripheral terminal, it was reversed by the protein translation inhibitor. Mu-opioid receptor (MOR) antisense prevented fentanyl hyperalgesia and priming. To determine whether type I and II priming occur in the same population of neurons, we used isolectin B4-saporin or [Sar9, Met(O2)11]-substance P-saporin to deplete nonpeptidergic or peptidergic nociceptors, respectively. Following intrathecal fentanyl, central terminal priming was prevented by both saporins, whereas that in peripheral terminal was not attenuated even by their combination. However, after intradermal fentanyl, priming in the peripheral terminal requires both peptidergic and nonpeptidergic nociceptors, whereas that in the central terminal is dependent only on peptidergic nociceptors. Pretreatment with dantrolene at either terminal prevented fentanyl-induced priming in both terminals, suggesting communication between central and peripheral terminals mediated by intracellular Ca2+ signaling. In vitro application of fentanyl increased cytoplasmic Ca2+ concentration in dorsal root ganglion neurons, which was prevented by pretreatment with dantrolene and naloxone. Therefore, acting at MOR in the nociceptor, fentanyl induces hyperalgesia and priming rapidly at both the central (type II) and peripheral (type I) terminal and this is mediated by Ca2+ signaling.SIGNIFICANCE STATEMENT Fentanyl, acting at the μ-opioid receptor (MOR), induces hyperalgesia and hyperalgesic priming at both the central and peripheral terminal of nociceptors and this is mediated by endoplasmic reticulum Ca2+ signaling. Priming in the central terminal is type II, whereas that in the peripheral terminal is type I. Our findings may provide useful information for the design of drugs with improved therapeutic profiles, selectively disrupting individual MOR signaling pathways, to maintain an adequate long-lasting control of pain.

Role of GPCR (mu-opioid)-receptor tyrosine kinase (epidermal growth factor) crosstalk in opioid-induced hyperalgesic priming (type II).

Repeated stimulation of mu-opioid receptors (MORs), by an MOR-selective agonist DAMGO induces type II priming, a form of nociceptor neuroplasticity, which has 2 components: opioid-induced hyperalgesia (OIH) and prolongation of prostaglandin-E2 (PGE2)-induced hyperalgesia. We report that intrathecal antisense knockdown of the MOR in nociceptors, prevented the induction of both components of type II priming. Type II priming was also eliminated by SSP-saporin, which destroys the peptidergic class of nociceptors. Because the epidermal growth factor receptor (EGFR) participates in MOR signaling, we tested its role in type II priming. The EGFR inhibitor, tyrphostin AG 1478, prevented the induction of prolonged PGE2-induced hyperalgesia, but not OIH, when tested out to 30 days after DAMGO. However, even when repeatedly injected, an EGFR agonist did not induce hyperalgesia or priming. A phosphopeptide, which blocks the interaction of Src, focal adhesion kinase (FAK), and EGFR, also prevented DAMGO-induced prolongation of PGE2 hyperalgesia, but only partially attenuated the induction of OIH. Inhibitors of Src and mitogen-activated protein kinase (MAPK) also only attenuated OIH. Inhibitors of matrix metalloproteinase, which cleaves EGF from membrane protein, markedly attenuated the expression, but did not prevent the induction, of prolongation of PGE2 hyperalgesia. Thus, although the induction of prolongation of PGE2-induced hyperalgesia at the peripheral terminal of peptidergic nociceptor is dependent on Src, FAK, EGFR, and MAPK signaling, Src, FAK, and MAPK signaling is only partially involved in the induction of OIH.

Mu opioid receptor signaling in the nucleus accumbens shell increases responsiveness of satiety-modulated lateral hypothalamus neurons.

Opioid signaling in the nucleus accumbens shell (sNAcc) has been implicated in hedonic feeding and binge eating behavior. The sNAcc projects to the lateral hypothalamus (LH), and this pathway has been suggested to modulate palatability-driven feeding behavior. In this study, we investigated the effects of sNAcc mu opioid receptor (MOR) stimulation on firing rates of LH neurons in previously sated rats. Neural firing in the LH was recorded while food-deprived rats performed an operant task to obtain sweetened Intralipid (a 4% fat emulsion containing 5% sucrose) before and after bilateral sNAcc infusion of either a MOR agonist [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) or a saline control solution. During sessions in which saline was infused into the sNAcc, the number of trials completed after infusion were significantly lower than the number completed before infusion, likely reflecting animals' increased satiety state. During sessions in which DAMGO was infused into the sNAcc, the decrease in the number of trials completed (comparing post- vs. pre-infusion trials) was significantly attenuated. Electrophysiological recording showed that the percentage of LH neurons showing an excitatory response due to behavioral events (cue presentation, lever press, lever retraction, and consumption) was reduced in post vs. pre-saline infusion period. However, the percentage of LH neurons showing excitatory responses to the same behavioral events was similar in pre- and post-DAMGO infusion periods. These findings suggest that MOR stimulation in sNAcc leads to an increase in stimulus-evoked excitatory signaling in LH neurons which could contribute to preventing satiety-induced decline in palatable food intake.

Delta opioid receptor antagonism/inverse agonism and its effect on mu opioid receptor signaling in SH‐SY5Y cells

Opioid analgesics are prescribed with success to combat chronic and severe pain, yet there are many clinical and public health problems associated with the use of these medications that include constipation, respiratory depression and physical dependence. Academic scientists to pharmaceutical companies are developing opioid drugs, G‐protein biased agonists and allosteric modulators to name a few, in an effort to identify safer and more effective analgesics. Previous studies have reported that the co‐administration of naltrindole (a delta opioid receptor antagonist) with morphine (mu opioid receptor agonist) in vivo maintains analgesia yet reduces the development of morphine tolerance and dependence. Following this observation, in collaboration with the Mosberg lab we have discovered and characterized several mu agonist/delta antagonist “bifunctional compounds” that lack tolerance and dependence liabilities in vivo. The goal of my studies is to use these bifunctional compounds, as well as selective mu and delta opioid receptor ligands, in an attempt to explain this phenomenon on a molecular level. The SH‐SY5Y human neuroblastoma cell line is an ideal system in which to assess mu and delta receptor cross‐talk because it endogenously expresses both receptor types. My first objective was to demonstrate mu opioid receptor tolerance at the level of the G protein (a decrease in GTPγ35S binding) using the selective agonist DAMGO. Compared to untreated cells, DAMGO (1μM, 6 hours) treated cells showed a 50% reduction in DAMGO‐mediated GTPγ35S binding and a slight decrease in basal GTPγ35S binding. I then found that the addition of the delta inverse agonist ICI 174,864 (1μM, 6 hours) prevented DAMGO‐mediated tolerance. To further study this response I used another delta antagonist TIPPΨ and three bifunctional compounds. Overall the results suggest that delta opioid receptor inhibition is advantageous for sustained mu opioid receptor function in a cellular model of opioid tolerance. Experiments are ongoing to decipher how delta blockade alters mu‐opioid receptor signaling.Support or Funding InformationDA‐03910 and NIGMS‐GM007767

Hyperalgesic priming (type II) induced by repeated opioid exposure: maintenance mechanisms.

We previously developed a model of opioid-induced neuroplasticity in the peripheral terminal of the nociceptor that could contribute to opioid-induced hyperalgesia, type II hyperalgesic priming. Repeated administration of mu-opioid receptor (MOR) agonists, such as DAMGO, at the peripheral terminal of the nociceptor, induces long-lasting plasticity expressed, prototypically as opioid-induced hyperalgesia and prolongation of prostaglandin E2-induced hyperalgesia. In this study, we evaluated the mechanisms involved in the maintenance of type II priming. Opioid receptor antagonist, naloxone, induced hyperalgesia in DAMGO-primed paws. When repeatedly injected, naloxone-induced hyperalgesia, and hyperalgesic priming, supporting the suggestion that maintenance of priming involves changes in MOR signaling. However, the knockdown of MOR with oligodeoxynucleotide antisense did not reverse priming. Mitogen-activated protein kinase and focal adhesion kinase, which are involved in the Src signaling pathway, previously implicated in type II priming, also inhibited the expression, but not maintenance of priming. However, when Src and mitogen-activated protein kinase inhibitors were coadministered, type II priming was reversed, in male rats. A second model of priming, latent sensitization, induced by complete Freund's adjuvant was also reversed, in males. In females, the inhibitor combination was only able to inhibit the expression and maintenance of DAMGO-induced priming when knockdown of G-protein-coupled estrogen receptor 30 (GPR30) in the nociceptor was performed. These findings demonstrate that the maintenance of DAMGO-induced type II priming, and latent sensitization is mediated by an interaction between, Src and MAP kinases, which in females is GPR30 dependent.

Endogenous central amygdala mu-opioid receptor signaling promotes sodium appetite in mice

Due to the importance of dietary sodium and its paucity within many inland environments, terrestrial animals have evolved an instinctive sodium appetite that is commensurate with sodium deficiency. Despite a well-established role for central opioid signaling in sodium appetite, the endogenous influence of specific opioid receptor subtypes within distinct brain regions remains to be elucidated. Using selective pharmacological antagonists of opioid receptor subtypes, we reveal that endogenous mu-opioid receptor (MOR) signaling strongly drives sodium appetite in sodium-depleted mice, whereas a role for kappa (KOR) and delta (DOR) opioid receptor signaling was not detected, at least in sodium-depleted mice. Fos immunohistochemistry revealed discrete regions of the mouse brain displaying an increased number of activated neurons during sodium gratification: the rostral portion of the nucleus of the solitary tract (rNTS), the lateral parabrachial nucleus (LPB), and the central amygdala (CeA). The CeA was subsequently targeted with bilateral infusions of the MOR antagonist naloxonazine, which significantly reduced sodium appetite in mice. The CeA is therefore identified as a key node in the circuit that contributes to sodium appetite. Moreover, endogenous opioids, acting via MOR, within the CeA promote this form of appetitive behavior.

Decreased CB receptor binding and cannabinoid signaling in three brain regions of a rat model of schizophrenia

Schizophrenia is a serious mental health disorder characterized by several behavioral and biochemicel abnormalities. In a previous study we have shown that mu-opioid (MOP) receptor signaling is impaired in specific brain regions of our three-hit animal model of schizophrenia. Since the cannabinoid system is significantly influenced in schizophrenic patients, in the present work we investigated cannabinoid (CB) receptor binding and G-protein activation in cortical, subcortical and cerebellar regions of control and ‘schizophrenic’ rats. Cannabinoid agonist (WIN-55,212-2 mesylate) mediated G-protein activation was consistently decreased in all areas tested, and the difference was extremely significant in membranes prepared from the cerebellum. Interestingly, the cerebellar activity of WIN-55,212-2 stimulated G-proteins was substantially higher than those of cerebral cortex and subcortical region in control animals, indicating a primordial role of the cannabinoid system in the cerebellum. At the level of radioligand binding, the affinities of the CB receptors were also markedly decreased in the model animals. Capacity of the [3H]WIN-55,212-2 binding was only higher in the cerebellum of ‘schizophrenic’ model rats. Taken together, in all three brain areas of model rats both cannabinoid receptor binding and cannabinoid agonist-mediated G-protein activation were regularly decreased. Our results revealed that besides the opioids, the endocannabinoid – cannabis receptor system also shows impairment in our rat model, increasing its face validity and translational utility.

A biased mu‐opioid receptor positive allosteric modulator that does not alter receptor regulation

Currently used opioid analgesics compete with endogenous opioid peptide ligands by binding to the orthosteric site of the mu opioid receptor (MOPr). Activation of this G protein‐coupled receptor (GPCR) affords analgesia but also causes respiratory depression, sedation, constipation, and euphoria leading to a high addictive liability. Our lab is currently pursuing the idea that positive allosteric modulators of MOPr (mu‐PAMs) may be a way to treat pain with a better therapeutic profile. Such compounds, including the lead ligand BMS‐986122, enhance the affinity, potency, and efficacy of opioid agonists to bind MOPr and activate G protein. On the other hand, BMS‐986122 does not stimulate G protein activation, as measured by [35S]GTPγS binding in the absence of an orthosteric ligand. Here, we sought to determine if BMS‐986122 activates non‐G protein mediated pathways downstream of MOPr and to investigate the long‐term consequences of BMS‐986122 exposure on the regulation of MOPr in vitro using C6 rat glioma cells expressing rat MOPr. The results demonstrate that BMS‐986122 initiates phosphorylation of ERK1/2 in a pertussis‐toxin insensitive manner supporting a G protein‐independent mechanism. BMS‐986122 alone did not cause any long‐term adaptations in MOPr signaling. Moreover, in contrast to its ability to enhance agonist‐mediated activation of G protein, BMS‐986122 failed to enhance agonist‐induced desensitization of MOPr as measured by receptor downregulation, receptor phosphorylation, and loss of receptor‐mediated signaling following long‐term agonist exposure. Development of biased allosteric ligands at GPCRs represents a novel avenue for analgesic drug development. Future work will determine the physiological consequences of this biased PAM in mouse models of pain.Support or Funding InformationFunded by DA039997

Impedance-based analysis of mu opioid receptor signaling and underlying mechanisms

The mu opioid receptor is a G-protein coupled receptor able to signal through the Gαi/o class of G-protein and β-arrestin pathways, stimulating down-stream effector pathways. Signaling bias occurs when different receptor agonists lead to different signaling outcomes. Traditionally these have been studied using end-point assays. Real-time cellular analysis platforms allow for the analysis of the holistic effects of receptor activation as an integrated output. While this allows for different ligands to be compared rapidly, the cellular mechanisms underlying the signal are not well described. Using an impedance based system, the impedance responses for two opioid ligands, morphine and DAMGO were examined.The impedance responses for these two agonists, while showing similar features, were distinct from each other. Some of the mechanisms underlying the mu opioid receptor coupled impedance changes were investigated. It was found that the response is a result of discrete cellular processes, including G-protein signaling and protein kinase phosphorylation.

DNA Methylation Modulates Nociceptive Sensitization after Incision.

DNA methylation is a key epigenetic mechanism controlling DNA accessibility and gene expression. Blockade of DNA methylation can significantly affect pain behaviors implicated in neuropathic and inflammatory pain. However, the role of DNA methylation with regard to postoperative pain has not yet been explored. In this study we sought to investigate the role of DNA methylation in modulating incisional pain and identify possible targets under DNA methylation and contributing to incisional pain. DNA methyltranferase (DNMT) inhibitor 5-Aza-2′-deoxycytidine significantly reduced incision-induced mechanical allodynia and thermal sensitivity. Aza-2′-deoxycytidine also reduced hindpaw swelling after incision, suggesting an anti-inflammatory effect. Global DNA methylation and DNMT3b expression were increased in skin after incision, but none of DNMT1, DNMT3a or DNMT3b was altered in spinal cord or DRG. The expression of proopiomelanocortin Pomc encoding β-endorphin and Oprm1 encoding the mu-opioid receptor were upregulated peripherally after incision; moreover, Oprm1 expression was further increased under DNMT inhibitor treatment. Finally, local peripheral injection of the opioid receptor antagonist naloxone significantly exacerbated incision-induced mechanical hypersensitivity. These results suggest that DNA methylation is functionally relevant to incisional nociceptive sensitization, and that mu-opioid receptor signaling might be one methylation regulated pathway controlling sensitization after incision.

Mu opioid receptor modulation in the nucleus accumbens lowers voluntary wheel running in rats bred for high running motivation

The exact role of opioid receptor signaling in mediating voluntary wheel running is unclear. To provide additional understanding, female rats selectively bred for motivation of low (LVR) versus high voluntary running (HVR) behaviors were used. Aims of this study were 1) to identify intrinsic differences in nucleus accumbens (NAc) mRNA expression of opioid-related transcripts and 2) to determine if nightly wheel running is differently influenced by bilateral NAc injections of either the mu-opioid receptor agonist D-Ala2, NMe-Phe4, Glyo5-enkephalin (DAMGO) (0.25, 2.5 μg/side), or its antagonist, naltrexone (5, 10, 20 μg/side). In Experiment 1, intrinsic expression of Oprm1 and Pdyn mRNAs were higher in HVR compared to LVR. Thus, the data imply that line differences in opioidergic mRNA in the NAc could partially contribute to differences in wheel running behavior. In Experiment 2, a significant decrease in running distance was present in HVR rats treated with 2.5 μg DAMGO, or with 10 μg and 20 μg naltrexone between hours 0–1 of the dark cycle. Neither DAMGO nor naltrexone had a significant effect on running distance in LVR rats. Taken together, the data suggest that the high nightly voluntary running distance expressed by HVR rats is mediated by increased endogenous mu-opioid receptor signaling in the NAc, that is disturbed by either agonism or antagonism. In summary, our findings on NAc opioidergic mRNA expression and mu-opioid receptor modulations suggest HVR rats, compared to LVR rats, express higher running levels mediated by an increase in motivation driven, in part, by elevated NAc opioidergic signaling.

Phosphatidylethanolamine‐Binding Protein Regulates Mu Opioid Receptor Induced βarrestin2 Recruitment and Downstream Signaling

The signaling regulators and pathways of the mu‐opioid receptor (MOR) are being studied to develop functionally selective MOR ligands. Such novel analgesic drugs may alleviate pain without the side effects and drawbacks of typical opioids. We have analyzed proteomics databases available in the literature to find candidate proteins that are regulated in the brain by chronic opioid treatment. One such candidate, phosphatidylethanolamine‐binding protein (PEBP), is a cytoplasmic protein with a highly conserved structure that has been shown to regulate GPCR signaling through both the Raf/MEK/ERK pathway and G protein‐coupled receptor kinase 2 (GRK2), a major feedback inhibitor of GPCRs which has been implicated in a number of neurological disorders including chronic pain. The expression of PEBP was also found to be regulated by chronic morphine treatment in a proteomic study of rodent brain, suggesting that PEBP may regulate MOR signaling, however PEBP has not yet been associated with MOR signaling. We have knocked down PEBP in MOR‐ expressing HEK, U2OS, N2a and SH‐SY5Y cells using specific siRNA to determine changes in either MOR‐induced ERK signaling or βarrestin2 (βarr2) recruitment and receptor internalization, with and without GRK2 overexpression. PEBP knockdown does not change MOR agonist induced ERK signaling efficacy or potency, but βarr2 recruitment, MOR internalization, and chronic morphine desensitization potency is increased. This suggests a role for PEBP in regulating MOR induced βarr2 recruitment and attendant opioid induced side effects. We will continue to study the role of PEBP in pain and opioid response in vivo, and are developing in vitro drug discovery screens with PEBP as a target.

Dual κ-agonist/μ-antagonist opioid receptor modulation reduces levodopa-induced dyskinesia and corrects dysregulated striatal changes in the nonhuman primate model of Parkinson disease.

Effective medical management of levodopa-induced dyskinesia (LID) remains an unmet need for patients with Parkinson disease (PD). Changes in opioid transmission in the basal ganglia associated with LID suggest a therapeutic opportunity. Here we determined the impact of modulating both mu and kappa opioid receptor signaling using the mixed agonist/antagonist analgesic nalbuphine in reducing LID and its molecular markers in the nonhuman primate model. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated macaques with advanced parkinsonism and reproducible LID received a range of nalbuphine doses or saline subcutaneously as: (1) monotherapy, (2) acute coadministration with levodopa, and (3) chronic coadministration for 1 month. Animals were assessed by blinded examiners for motor disability and LID severity using standardized rating scales. Plasma levodopa levels were determined with and without nalbuphine, and postmortem brain samples were subjected to Western blot analyses. Nalbuphine reduced LID in a dose-dependent manner by 48% (p < 0.001) without compromising the anti-PD effect of levodopa or changing plasma levodopa levels. There was no tolerance to the anti-LID effect of nalbuphine given chronically. Nalbuphine coadministered with levodopa was well tolerated and did not cause sedation. Nalbuphine monotherapy had no effect on motor disability. Striatal tissue analyses showed that nalbuphine cotherapy blocks several molecular correlates of LID, including overexpression of ΔFosB, prodynorphin, dynorphin A, cyclin-dependent kinase 5, and increased phosphorylation of DARPP-32 at threonine-34. Nalbuphine reverses the molecular milieu in the striatum associated with LID and is a safe and effective anti-LID agent in the primate model of PD. These findings support repurposing this analgesic for the treatment of LID.

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 Ca2+-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. Antioxid. Redox Signal. 22, 799–818.