Multiomics Analyses Identify Proline Endopeptidase-Like Protein As a Key Regulator of Protein Trafficking, a Pathway Underlying Alzheimer's Disease Pathogenesis.

Current treatments for Alzheimer's disease (AD) help reduce symptoms for a limited time but do not treat the underlying pathology. To identify potential therapeutic targets for AD, an integrative network analysis was previously carried out using 364 human postmortem control, mild cognitive impairment, and AD brains. This analysis identified proline endopeptidase-like protein (PREPL), an understudied protein, as a downregulated protein in late-onset AD patients. In this study we investigate the role of PREPL. Analyses of data from human postmortem samples and PREPL knockdown (KD) cells suggest that PREPL expression modulates pathways associated with protein trafficking, synaptic activities, and lipid metabolism. Furthermore, PREPL KD impairs cell proliferation and modulates the structure of vesicles, levels of neuropeptide-processing enzymes, and secretion of neuropeptides. In addition, decrease in PREPL levels leads to changes in the levels of a number of synaptic proteins as well as changes in the levels of secreted amyloid beta (Aß) 42 peptide and Tau phosphorylation. Finally, we report that local decrease in PREPL levels in mouse hippocampus attenuates long-term potentiation, suggesting a role in synaptic plasticity. Together, our results indicate that PREPL affects neuronal function by modulating protein trafficking and synaptic function, an important mechanism of AD pathogenesis. SIGNIFICANCE STATEMENT: Integrative network analysis reveals proline endopeptidase-like protein (PREPL) to be downregulated in human sporadic late-onset Alzheimer's disease brains. Down regulation of PREPL leads to increases in amyloid beta secretion, Tau phosphorylation, and decreases in protein trafficking and long-term potentiation.

A Regional and Projection-Specific Role of RGSz1 in the Ventrolateral Periaqueductal Grey in the Modulation of Morphine Reward.

Opioid analgesics exert their therapeutic and adverse effects by activating µ opioid receptors (MOPR); however, functional responses to MOPR activation are modulated by distinct signal transduction complexes within the brain. The ventrolateral periaqueductal gray (vlPAG) plays a critical role in modulation of nociception and analgesia, but the exact intracellular pathways associated with opioid responses in this region are not fully understood. We previously showed that knockout of the signal transduction modulator Regulator of G protein Signaling z1 (RGSz1) enhanced analgesic responses to opioids, whereas it decreased the rewarding efficacy of morphine. Here, we applied viral mediated gene transfer methodology and delivered adeno-associated virus (AAV) expressing Cre recombinase to the vlPAG of RGSz1fl\fl mice to demonstrate that downregulation of RGSz1 in this region decreases sensitivity to morphine in the place preference paradigm, under pain-free as well as neuropathic pain states. We also used retrograde viral vectors along with flippase-dependent Cre vectors to conditionally downregulate RGSz1 in vlPAG projections to the ventral tegmental area (VTA) and show that downregulation of RGSz1 prevents the development of place conditioning to low morphine doses. Consistent with the role for RGSz1 as a negative modulator of MOPR activity, RGSz1KO enhances opioid-induced cAMP inhibition in periaqueductal gray (PAG) membranes. Furthermore, using a new generation of bioluminescence resonance energy transfer (BRET) sensors, we demonstrate that RGSz1 modulates Gαz but not other Gαi family subunits and selectively impedes MOPR-mediated Gαz signaling events invoked by morphine and other opioids. Our work highlights a regional and circuit-specific role of the G protein-signaling modulator RGSz1 in morphine reward, providing insights on midbrain intracellular pathways that control addiction-related behaviors. SIGNIFICANCE STATEMENT: This study used advanced genetic mouse models to highlight the role of the signal transduction modulator named RGSz1 in responses to clinically used opioid analgesics. We show that RGSz1 controls the rewarding efficacy of opioids by actions in ventrolateral periaqueductal gray projections to the ventral tegmental area, a key component of the midbrain dopamine pathway. These studies highlight novel mechanisms by which pain-modulating structures control the rewarding efficacy of opioids.

Biased signaling by endogenous opioid peptides.

Opioids, such as morphine and fentanyl, are widely used for the treatment of severe pain; however, prolonged treatment with these drugs leads to the development of tolerance and can lead to opioid use disorder. The "Opioid Epidemic" has generated a drive for a deeper understanding of the fundamental signaling mechanisms of opioid receptors. It is generally thought that the three types of opioid receptors (µ, δ, κ) are activated by endogenous peptides derived from three different precursors: Proopiomelanocortin, proenkephalin, and prodynorphin. Posttranslational processing of these precursors generates >20 peptides with opioid receptor activity, leading to a long-standing question of the significance of this repertoire of peptides. Here, we address some aspects of this question using a technical tour de force approach to systematically evaluate ligand binding and signaling properties ([35S]GTPγS binding and ß-arrestin recruitment) of 22 peptides at each of the three opioid receptors. We show that nearly all tested peptides are able to activate the three opioid receptors, and many of them exhibit agonist-directed receptor signaling (functional selectivity). Our data also challenge the dogma that shorter forms of ß-endorphin do not exhibit receptor activity; we show that they exhibit robust signaling in cultured cells and in an acute brain slice preparation. Collectively, this information lays the groundwork for improved understanding of the endogenous opioid system that will help in developing more effective treatments for pain and addiction.

GPR83 engages endogenous peptides from two distinct precursors to elicit differential signaling.

PEN is an abundant neuropeptide that activates GPR83, a G protein-coupled receptor that is considered a novel therapeutic target due to its roles in regulation of feeding, reward, and anxiety-related behaviors. The major form of PEN in the brain is 22 residues in length. Previous studies have identified shorter forms of PEN in mouse brain and neuroendocrine cells; these shorter forms were named PEN18, PEN19 and PEN20, with the number reflecting the length of the peptide. The C-terminal five residues of PEN20 are identical to the C-terminus of a procholecystokinin (proCCK)-derived peptide, named proCCK56-62, that is present in mouse brain. ProCCK56-62 is highly conserved across species although it has no homology to the bioactive cholecystokinin domain. ProCCK56-62 and a longer form, proCCK56-63 were tested for their ability to engage GPR83. Both peptides bind GPR83 with high affinity, activate second messenger pathways, and induce ligand-mediated receptor endocytosis. Interestingly, the shorter PEN peptides, ProCC56-62, and ProCCK56-63 differentially activate signal transduction pathways. Whereas PEN22 and PEN20 facilitate receptor coupling to Gai, PEN18, PEN19 and ProCCK peptides facilitate coupling to Gas. Furthermore, the ProCCK peptides exhibit dose dependent Ga subtype selectivity in that they faciliate coupling to Gas at low concentrations and Gai at high concentrations. These data demonstrate that peptides derived from two distinct peptide precursors can differentially activate GPR83, and that GPR83 exhibits Ga subtype preference depending on the nature and concentration of the peptide. These results are consistent with the emerging idea that endogenous neuropeptides function as biased ligands. Significance Statement We found that peptides derived from proCCK bind and activate GPR83, a G protein-coupled receptor that is known to bind peptides derived from proSAAS. Different forms of the proCCK- and proSAAS-derived peptides show biased agonism, activating Gas or Gai depending on the length of the peptide and/or its concentration.

Autoantibodies Blocking M3 Muscarinic Receptors Cause Postganglionic Cholinergic Dysautonomia.

A 10-year-old girl presented with ileus, urinary retention, dry mouth, lack of tears, fixed dilated pupils, and diffuse anhidrosis 7 days after a febrile illness. We hypothesized that her syndrome was due to autoimmunity against muscarinic acetylcholine receptors, blocking their activation. Using an indirect enzyme-linked immunosorbent assay for all 5 muscarinic receptors (M1 -M5 ), we identified in the patient's serum antibodies that selectively bound to M3 receptors. In vitro functional studies confirmed that these autoantibodies selectively blocked M3 receptor activation. Thus, autoantibodies against M3 acetylcholine receptors cause acute postganglionic cholinergic dysautonomia. ANN NEUROL 2020;88:1237-1243.

Regulation of Opioid Receptors by Their Endogenous Opioid Peptides.

Activation of µ, δ, and κ opioid receptors by endogenous opioid peptides leads to the regulation of many emotional and physiological responses. The three major endogenous opioid peptides, ß-endorphin, enkephalins, and dynorphins result from the processing of three main precursors: proopiomelanocortin, proenkephalin, and prodynorphin. Using a knockout approach, we sought to determine whether the absence of endogenous opioid peptides would affect the expression or activity of opioid receptors in mice lacking either proenkephalin, ß-endorphin, or both. Since gene knockout can lead to changes in the levels of peptides generated from related precursors by compensatory mechanisms, we directly measured the levels of Leu-enkephalin and dynorphin-derived peptides in the brain of animals lacking proenkephalin, ß-endorphin, or both. We find that whereas the levels of dynorphin-derived peptides were relatively unaltered, the levels of Leu-enkephalin were substantially decreased compared to wild-type mice suggesting that preproenkephalin is the major source of Leu-enkephalin. This data also suggests that the lack of ß-endorphin and/or proenkephalin does not lead to a compensatory change in prodynorphin processing. Next, we examined the effect of loss of the endogenous peptides on the regulation of opioid receptor levels and activity in specific regions of the brain. We also compared the receptor levels and activity in males and females and show that the lack of ß-endorphin and/or proenkephalin leads to differential modulation of the three opioid receptors in a region- and gender-specific manner. These results suggest that endogenous opioid peptides are important modulators of the expression and activity of opioid receptors in the brain.

Five Decades of Research on Opioid Peptides: Current Knowledge and Unanswered Questions.

In the mid-1970s, an intense race to identify endogenous substances that activated the same receptors as opiates resulted in the identification of the first endogenous opioid peptides. Since then, >20 peptides with opioid receptor activity have been discovered, all of which are generated from three precursors, proenkephalin, prodynorphin, and proopiomelanocortin, by sequential proteolytic processing by prohormone convertases and carboxypeptidase E. Each of these peptides binds to all three of the opioid receptor types (µ, δ, or κ), albeit with differing affinities. Peptides derived from proenkephalin and prodynorphin are broadly distributed in the brain, and mRNA encoding all three precursors are highly expressed in some peripheral tissues. Various approaches have been used to explore the functions of the opioid peptides in specific behaviors and brain circuits. These methods include directly administering the peptides ex vivo (i.e., to excised tissue) or in vivo (in animals), using antagonists of opioid receptors to infer endogenous peptide activity, and genetic knockout of opioid peptide precursors. Collectively, these studies add to our current understanding of the function of endogenous opioids, especially when similar results are found using different approaches. We briefly review the history of identification of opioid peptides, highlight the major findings, address several myths that are widely accepted but not supported by recent data, and discuss unanswered questions and future directions for research. SIGNIFICANCE STATEMENT: Activation of the opioid receptors by opiates and synthetic drugs leads to central and peripheral biological effects, including analgesia and respiratory depression, but these may not be the primary functions of the endogenous opioid peptides. Instead, the opioid peptides play complex and overlapping roles in a variety of systems, including reward pathways, and an important direction for research is the delineation of the role of individual peptides.

Regulation of an Opioid Receptor Chaperone Protein, RTP4, by Morphine.

Signaling by classic analgesics, such as morphine, is governed primarily by the relative abundance of opioid receptors at the cell surface, and this is regulated by receptor delivery to, and retrieval from, the plasma membrane. Although retrieval mechanisms, such as receptor endocytosis, have been extensively investigated, fewer studies have explored mechanisms of receptor maturation and delivery to the plasma membrane. A previous study implicated receptor transporter proteins (RTPs) in the latter process. Since not much is known about regulation of RTP expression, we initiated studies examining the effect of chronic morphine administration on the levels of RTPs in the brain. Among the four RTPs, we detected selective and region-specific changes in RTP4 expression; RTP4 mRNA is significantly upregulated in the hypothalamus compared with other brain regions. We examined whether increased RTP4 expression impacted receptor protein levels and found a significant increase in the abundance of mu opioid receptors (MOPrs) but not other related G protein-coupled receptors (GPCRs, such as delta opioid, CB1 cannabinoid, or D2 dopamine receptors) in hypothalamic membranes from animals chronically treated with morphine. Next, we used a cell culture system to show that RTP4 expression is necessary and sufficient for regulating opioid receptor abundance at the cell surface. Interestingly, selective MOPr-mediated increase in RTP4 expression leads to increases in cell surface levels of MOPr-delta opioid receptor heteromers, and this increase is significantly attenuated by RTP4 small interfering RNA. Together, these results suggest that RTP4 expression is regulated by chronic morphine administration, and this, in turn, regulates opioid receptor cell surface levels and function.

G Protein-Coupled Receptor Heteromers.

G protein-coupled receptors (GPCRs) compose one of the largest families of membrane proteins involved in intracellular signaling. They are involved in numerous physiological and pathological processes and are prime candidates for drug development. Over the past decade, an increasing number of studies have reported heteromerization between GPCRs. Many investigations in heterologous systems have provided important indications of potential novel pharmacology; however, the physiological relevance of these findings has yet to be established with endogenous receptors in native tissues. In this review, we focus on family A GPCRs and describe the techniques and criteria to assess their heteromerization. We conclude that advances in approaches to study receptor complex functionality in heterologous systems, coupled with techniques that enable specific examination of native receptor heteromers in vivo, are likely to establish GPCR heteromers as novel therapeutic targets.

Opioid-Induced Signaling and Antinociception Are Modulated by the Recently Deorphanized Receptor, GPR171.

ProSAAS is one of the most widely expressed proteins throughout the brain and was recently found to be upregulated in chronic fibromyalgia patients. BigLEN is a neuropeptide that is derived from ProSAAS and was recently discovered to be the endogenous ligand for the orphan G protein-coupled receptor GPR171. Although BigLEN-GPR171 has been found to play a role in feeding and anxiety behaviors, it has not yet been explored in pain and opioid modulation. The purpose of this study was to evaluate this novel neuropeptide-receptor system in opioid-induced antinociception. We found that GPR171 is expressed in GABAergic neurons within the periaqueductal gray, which is a key brain area involved in pain modulation and opioid functions. We also found that, although the GPR171 agonist and antagonist do not have nociceptive effects on their own, they oppositely regulate morphine-induced antinociception with the agonist enhancing and antagonist reducing antinociception. Lastly, we showed that the GPR171 antagonist or receptor knockdown decreases signaling by the mu-opioid receptor, but not the delta-opioid receptor. Taken together, these results suggest that antagonism of the GPR171 receptor reduces mu opioid receptor signaling and morphine-induced antinociception, whereas the GPR171 agonist enhances morphine antinociception, suggesting that GPR171 may be a novel target toward the development of pain therapeutics. SIGNIFICANCE STATEMENT: GPR171 is a recently deorphanized receptor that is expressed within the periaqueductal gray and can regulate mu opioid receptor signaling and antinociception. This research may contribute to the development of new therapeutics to treat pain.

Collybolide is a novel biased agonist of κ-opioid receptors with potent antipruritic activity.

Among the opioid receptors, the κ-opioid receptor (κOR) has been gaining considerable attention as a potential therapeutic target for the treatment of complex CNS disorders including depression, visceral pain, and cocaine addiction. With an interest in discovering novel ligands targeting κOR, we searched natural products for unusual scaffolds and identified collybolide (Colly), a nonnitrogenous sesquiterpene from the mushroom Collybia maculata. This compound has a furyl-δ-lactone core similar to that of Salvinorin A (Sal A), another natural product from the plant Salvia divinorum Characterization of the molecular pharmacological properties reveals that Colly, like Sal A, is a highly potent and selective κOR agonist. However, the two compounds differ in certain signaling and behavioral properties. Colly exhibits 10- to 50-fold higher potency in activating the mitogen-activated protein kinase pathway compared with Sal A. Taken with the fact that the two compounds are equipotent for inhibiting adenylyl cyclase activity, these results suggest that Colly behaves as a biased agonist of κOR. Behavioral studies also support the biased agonistic activity of Colly in that it exhibits ∼10-fold higher potency in blocking non-histamine-mediated itch compared with Sal A, and this difference is not seen in pain attenuation by these two compounds. These results represent a rare example of functional selectivity by two natural products that act on the same receptor. The biased agonistic activity, along with an easily modifiable structure compared with Sal A, makes Colly an ideal candidate for the development of novel therapeutics targeting κOR with reduced side effects.

Morphine Regulated Synaptic Networks Revealed by Integrated Proteomics and Network Analysis.

Despite its efficacy, the use of morphine for the treatment of chronic pain remains limited because of the rapid development of tolerance, dependence and ultimately addiction. These undesired effects are thought to be because of alterations in synaptic transmission and neuroplasticity within the reward circuitry including the striatum. In this study we used subcellular fractionation and quantitative proteomics combined with computational approaches to investigate the morphine-induced protein profile changes at the striatal postsynaptic density. Over 2,600 proteins were identified by mass spectrometry analysis of subcellular fractions enriched in postsynaptic density associated proteins from saline or morphine-treated striata. Among these, the levels of 34 proteins were differentially altered in response to morphine. These include proteins involved in G-protein coupled receptor signaling, regulation of transcription and translation, chaperones, and protein degradation pathways. The altered expression levels of several of these proteins was validated by Western blotting analysis. Using Genes2Fans software suite we connected the differentially expressed proteins with proteins identified within the known background protein-protein interaction network. This led to the generation of a network consisting of 116 proteins with 40 significant intermediates. To validate this, we confirmed the presence of three proteins predicted to be significant intermediates: caspase-3, receptor-interacting serine/threonine protein kinase 3 and NEDD4 (an E3-ubiquitin ligase identified as a neural precursor cell expressed developmentally down-regulated protein 4). Because this morphine-regulated network predicted alterations in proteasomal degradation, we examined the global ubiquitination state of postsynaptic density proteins and found it to be substantially altered. Together, these findings suggest a role for protein degradation and for the ubiquitin/proteasomal system in the etiology of opiate dependence and addiction.

GPR171 is a hypothalamic G protein-coupled receptor for BigLEN, a neuropeptide involved in feeding.

Multiple peptide systems, including neuropeptide Y, leptin, ghrelin, and others, are involved with the control of food intake and body weight. The peptide LENSSPQAPARRLLPP (BigLEN) has been proposed to act through an unknown receptor to regulate body weight. In the present study, we used a combination of ligand-binding and receptor-activity assays to characterize a Gαi/o protein-coupled receptor activated by BigLEN in the mouse hypothalamus and Neuro2A cells. We then selected orphan G protein-coupled receptors expressed in the hypothalamus and Neuro2A cells and tested each for activation by BigLEN. G protein-coupled receptor 171 (GPR171) is activated by BigLEN, but not by the C terminally truncated peptide LittleLEN. The four C-terminal amino acids of BigLEN are sufficient to bind and activate GPR171. Overexpression of GPR171 leads to an increase, and knockdown leads to a decrease, in binding and signaling by BigLEN and the C-terminal peptide. In the hypothalamus GPR171 expression complements the expression of BigLEN, and its level and activity are elevated in mice lacking BigLEN. In mice, shRNA-mediated knockdown of hypothalamic GPR171 leads to a decrease in BigLEN signaling and results in changes in food intake and metabolism. The combination of GPR171 shRNA together with neutralization of BigLEN peptide by antibody absorption nearly eliminates acute feeding in food-deprived mice. Taken together, these results demonstrate that GPR171 is the BigLEN receptor and that the BigLEN-GPR171 system plays an important role in regulating responses associated with feeding and metabolism in mice.

Identification of a µ-δ opioid receptor heteromer-biased agonist with antinociceptive activity.

G protein-coupled receptors play a pivotal role in many physiological signaling pathways. Mounting evidence suggests that G protein-coupled receptors, including opioid receptors, form dimers, and dimerization is necessary for receptor maturation, signaling, and trafficking. However, the physiological role of dimerization in vivo has not been well-explored because of the lack of tools to study these dimers in endogenous systems. To address this problem, we previously generated antibodies to µ-δ opioid receptor (µOR-δOR) dimers and used them to study the pharmacology and signaling by this heteromer. We also showed that the heteromer exhibits restricted distribution in the brain and that its abundance is increased in response to chronic morphine administration. Thus, the µOR-δOR heteromer represents a potentially unique target for the development of therapeutics to treat pain. Here, we report the identification of compounds targeting µOR-δOR heteromers through high-throughput screening of a small-molecule library. These compounds exhibit activity in µOR-δOR cells but not µOR or δOR cells alone. Among them, CYM51010 was found to be a µOR-δOR-biased ligand, because its activity is blocked by the µOR-δOR heteromer antibody. Notably, systemic administration of CYM51010 induced antinociceptive activity similar to morphine, and chronic administration of CYM51010 resulted in lesser antinociceptive tolerance compared with morphine. Taken together, these results suggest that CYM51010, a µOR-δOR-biased ligand, could serve as a scaffold for the development of a unique type (heteromer-biased) of drug that is more potent and without the severe side effects associated with conventional clinical opioids.

Opioid receptor function is regulated by post-endocytic peptide processing.

Most neuroendocrine peptides are generated in the secretory compartment by proteolysis of the precursors at classical cleavage sites consisting of basic residues by well studied endopeptidases belonging to the subtilisin superfamily. In contrast, a subset of bioactive peptides is generated by processing at non-classical cleavage sites that do not contain basic residues. Neither the peptidases responsible for non-classical cleavages nor the compartment involved in such processing has been well established. Members of the endothelin-converting enzyme (ECE) family are considered good candidate enzymes because they exhibit functional properties that are consistent with such a role. In this study we have explored a role for ECE2 in endocytic processing of δ opioid peptides and its effect on modulating δ opioid receptor function by using selective inhibitors of ECE2 that we had identified previously by homology modeling and virtual screening of a library of small molecules. We found that agonist treatment led to intracellular co-localization of ECE2 with δ opioid receptors. Furthermore, selective inhibitors of ECE2 and reagents that increase the pH of the acidic compartment impaired receptor recycling by protecting the endocytosed peptide from degradation. This, in turn, led to a substantial decrease in surface receptor signaling. Finally, we showed that treatment of primary neurons with the ECE2 inhibitor during recycling led to increased intracellular co-localization of the receptors and ECE2, which in turn led to decreased receptor recycling and signaling by the surface receptors. Together, these results support a role for differential modulation of opioid receptor signaling by post-endocytic processing of peptide agonists by ECE2.

AT1R-CB1R heteromerization reveals a new mechanism for the pathogenic properties of angiotensin II.

The mechanism of G protein-coupled receptor (GPCR) signal integration is controversial. While GPCR assembly into hetero-oligomers facilitates signal integration of different receptor types, cross-talk between Gαi- and Gαq-coupled receptors is often thought to be oligomerization independent. In this study, we examined the mechanism of signal integration between the Gαi-coupled type I cannabinoid receptor (CB(1)R) and the Gαq-coupled AT1R. We find that these two receptors functionally interact, resulting in the potentiation of AT1R signalling and coupling of AT1R to multiple G proteins. Importantly, using several methods, that is, co-immunoprecipitation and resonance energy transfer assays, as well as receptor- and heteromer-selective antibodies, we show that AT1R and CB(1)R form receptor heteromers. We examined the physiological relevance of this interaction in hepatic stellate cells from ethanol-administered rats in which CB(1)R is upregulated. We found a significant upregulation of AT1R-CB(1)R heteromers and enhancement of angiotensin II-mediated signalling, as compared with cells from control animals. Moreover, blocking CB(1)R activity prevented angiotensin II-mediated mitogenic signalling and profibrogenic gene expression. These results provide a molecular basis for the pivotal role of heteromer-dependent signal integration in pathology.

Environmental enrichment enhances ethanol preference over social reward in male swiss mice: Involvement of oxytocin-dopamine interactions.

The impact of environmental enrichment (EE) on natural rewards, including social and appetitive rewards, was investigated in male Swiss mice. EE, known for providing animals with various stimuli, was assessed for its effects on conditioned place preference (CPP) associated with ethanol and social stimuli. We previously demonstrated that EE increased the levels of the prosocial neuropeptide oxytocin (OT) in the hypothalamus and enhanced ethanol rewarding effects via an oxytocinergic mechanism. This study also investigated the impact of EE on social dominance and motivation for rewards, measured OT-mediated phospholipase C (PLC) activity in striatal membranes, and assessed OT expression in the hypothalamus. The role of dopamine in motivating rewards was considered, along with the interaction between OT and D1 receptors (DR) in the nucleus accumbens (NAc). Results showed that EE mice exhibited a preference for ethanol reward over social reward, a pattern replicated by the OT analogue Carbetocin. EE mice demonstrated increased social dominance and reduced motivation for appetitive taste stimuli. Higher OT mRNA levels in the hypothalamus were followed by diminished OT receptor (OTR) signaling activity in the striatum of EE mice. Additionally, EE mice displayed elevated D1R expression, which was attenuated by the OTR antagonist (L-368-889). The findings underscore the reinforcing effect of EE on ethanol and social rewards through an oxytocinergic mechanism. Nonetheless, they suggest that mechanisms other than the prosocial effect of EE may contribute to the ethanol pro-rewarding effect of EE and Carbetocin. They also point towards an OT-dopamine interaction potentially underlying some of these effects.

Allosteric interactions between δ and κ opioid receptors in peripheral sensory neurons.

The peripheral δ opioid receptor (DOR) is an attractive target for analgesic drug development. There is evidence that DOR can form heteromers with the κ-opioid receptor (KOR). As drug targets, heteromeric receptors offer an additional level of selectivity and, because of allosteric interactions between protomers, functionality. Here we report that selective KOR antagonists differentially altered the potency and/or efficacy of DOR agonists in primary cultures of adult rat peripheral sensory neurons and in a rat behavioral model of thermal allodynia. In vitro, the KOR antagonist nor-binaltorphimine (nor-BNI) enhanced the potency of [D-Pen(2,5)]-enkephalin (DPDPE), decreased the potency of [D-Ala(2),D-Leu(5)]-enkephalin (DADLE), and decreased the potency and efficacy of 4-[(R)-[(2S,5R)-4-allyl-2,5-dimethylpiperazin-1-yl](3-methoxyphenyl)methyl]-N,N-diethylbenzamide (SNC80) to inhibit prostaglandin E(2) (PGE(2))-stimulated adenylyl cyclase activity. In vivo, nor-BNI enhanced the effect of DPDPE and decreased the effect of SNC80 to inhibit PGE(2)-stimulated thermal allodynia. In contrast to nor-BNI, the KOR antagonist 5'-guanidinonaltrindole (5'-GNTI) reduced the response of DPDPE both in cultured neurons and in vivo. Evidence for DOR-KOR heteromers in peripheral sensory neurons included coimmunoprecipitation of DOR with KOR, a DOR-KOR heteromer selective antibody augmented the antinociceptive effect of DPDPE in vivo, and the DOR-KOR heteromer agonist 6'-GNTI inhibited adenylyl cyclase activity in vitro as well as PGE(2)-stimulated thermal allodynia in vivo. Taken together, these data suggest that DOR-KOR heteromers exist in rat primary sensory neurons and that KOR antagonists can act as modulators of DOR agonist responses most likely through allosteric interactions between the protomers of the DOR-KOR heteromer.

Opioid receptor heteromers in analgesia.

Opiates such as morphine and fentanyl, a major class of analgesics used in the clinical management of pain, exert their effects through the activation of opioid receptors. Opioids are among the most commonly prescribed and frequently abused drugs in the USA; however, the prolonged use of opiates often leads to the development of tolerance and addiction. Although blockade of opioid receptors with antagonists such as naltrexone and naloxone can lessen addictive impulses and facilitate recovery from overdose, systemic disruption of endogenous opioid receptor signalling through the use of these antagonistic drugs can have severe side effects. In the light of these challenges, current efforts have focused on identifying new therapeutic targets that selectively and specifically modulate opioid receptor signalling and function so as to achieve analgesia without the adverse effects associated with chronic opiate use. We have previously reported that opioid receptors interact with each other to form heteromeric complexes and that these interactions affect morphine signalling. Since chronic morphine administration leads to an enhanced level of these heteromers, these opioid receptor heteromeric complexes represent novel therapeutic targets for the treatment of pain and opiate addiction. In this review, we discuss the role of heteromeric opioid receptor complexes with a focus on mu opioid receptor (MOR) and delta opioid receptor (DOR) heteromers. We also highlight the evidence for altered pharmacological properties of opioid ligands and changes in ligand function resulting from the heteromer formation.

Oxytocin and vasopressin: Signalling, behavioural modulation and potential therapeutic effects.

Oxytocin (OT) and vasopressin (AVP) are endogenous ligands for OT and AVP receptors in the brain and in the peripheral system. Several studies demonstrate that OT and AVP have opposite roles in modulating stress, anxiety and social behaviours. Interestingly, both peptides and their receptors exhibit high sequence homology which could account for the biased signalling interaction of the peptides with OT and AVP receptors. However, how and under which conditions this crosstalk occurs in vivo remains unclear. In this review we shed light on the complexity of the roles of OT and AVP, by focusing on their signalling and behavioural differences and exploring the crosstalk between the receptor systems. Moreover, we discuss the potential of OT and AVP receptors as therapeutic targets to treat human disorders, such as autism, schizophrenia and drug abuse. LINKED ARTICLES: This article is part of a themed issue on Building Bridges in Neuropharmacology. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.8/issuetoc.