Search

We previously showed that prolonged morphine treatment and chronic inflammation both enhanced delta opioid receptor (deltaOR) cell surface density in lumbar spinal cord neurons. Here, we sought to determine whether administration of morphine to rats with chronic inflammation would further increase the bio-availability of deltaOR, and thereby the analgesic properties of the deltaOR agonist deltorphin, over that produced by inflammation alone. We found that chronic inflammation produced by injection of complete Freund's adjuvant (CFA) into the hind paw resulted in a bilateral increase in the binding and internalization of fluorescent deltorphin in neurons of the lumbar spinal cord as did prolonged morphine treatment [Morinville A, Cahill CM, Aibak H, Rymar VV, Pradhan A, Hoffert C, Mennicken F, Stroh T, Sadikot AF, O'Donnell D, Clarke PB, Collier B, Henry JL, Vincent JP, Beaudet A (2004a) Morphine-induced changes in delta opioid receptor trafficking are linked to somatosensory processing in the rat spinal cord. J Neurosci 24:5549-5559]. This effect was accompanied by an increase in the antinociceptive efficacy of intrathecal deltorphin as measured using the tail-flick test. Treatment of CFA-injected rats with morphine decreased the cell surface availability of deltaOR in neurons of the dorsal horn of the lumbar spinal cord as compared with treatment with CFA alone. Behaviorally, it significantly enhanced the antihyperalgesic effects of deltorphin (plantar test; % maximum possible antihyperalgesic effect (MPAHE)=113.5%+/-32.4% versus 26.1%+/-11.6% in rats injected with CFA alone) but strongly reduced the antinociceptive efficacy of the drug (tail-flick test; % maximum possible antinociceptive effect (MPE)=29.6%+/-3.6% versus 66.6%+/-6.3% in rats injected with CFA alone) suggesting that the latter, but not the former, is linked to the deltaOR trafficking events observed neuroanatomically. These results demonstrate that in chronic inflammation, the antihyperalgesic effects of deltaOR agonists may be enhanced by morphine pre-treatment. They also reveal a dichotomy between mechanisms underlying antihyperalgesic and antinociceptive effects of deltaOR agonists.

Zoning-based organic farming has become increasingly attractive in Indonesia for producing healthy food, improving the quality of land and the environment, and increasing the welfare of farmers. The problem is that organic farming areas should be certified by a legal agency called the National Standardization Agency and the cost should be paid by farmers in those areas. Zoning allows the farmers to jointly manage farming areas, including payment of certification costs. However, the success of zoning-based organic farming implementation depends on farmers’ acceptability. This study aims to analyse perceptions and knowledge regarding zoning-based organic farming plans, measure the level of farmers’ willingness to accept (WTA) zoning-based organic farming plans and determine the effect of economic, ecological, and social support factors, as well as other factors towards WTA the zoning-based organic farming system. This study found that the majority of farmers perceive zoning-based organic farming as economically viable and ecologically and socially supported compared to conventional farming since most of the farmers have good knowledge regarding the importance of zoning. Although the farmers have long been experiencing conventional farming, the number of WTA farmers in zoning-based organic farming is very high. The study confirmed that the ecological aspect is the most influential in accepting zoning-based organic farming. Economic factors have a very strong influence on increasing farmers’ WTA zoning-based organic farming. This study also suggests that the implementation of zoning-based organic farming needs to be sustainably supported by the community.

Inflammatory bowel disease (IBD) is a multifactorial disease, which is thought to be an interplay between genetic, environment, microbiota, and immune-mediated factors. Dysbiosis in the gut microbial composition, caused by antibiotics and diet, is closely related to the initiation and progression of IBD. Differences in gut microbiota composition between IBD patients and healthy individuals have been found, with reduced biodiversity of commensal microbes and colonization of opportunistic microbes in IBD patients. Gut microbiota can, therefore, potentially be used for diagnosing and prognosticating IBD, and predicting its treatment response. Currently, there are no curative therapies for IBD. Microbiota-based interventions, including probiotics, prebiotics, synbiotics, and fecal microbiota transplantation, have been recognized as promising therapeutic strategies. Clinical studies and studies done in animal models have provided sufficient evidence that microbiota-based interventions may improve inflammation, the remission rate, and microscopic aspects of IBD. Further studies are required to better understand the mechanisms of action of such interventions. This will help in enhancing their effectiveness and developing personalized therapies. The present review summarizes the relationship between gut microbiota and IBD immunopathogenesis. It also discusses the use of gut microbiota as a noninvasive biomarker and potential therapeutic option.

Konflik sosial merupakan bentuk dasar interaksi yang terjadi secara alamiyah. Konflik sosial adalah entitas dan realitas dalam kehidupan bermasyarakat. George Simmel mengkonsepkan gagasan konflik sosial sebagai tindakan permusuhan timbal balik, yang dipicu oleh benturan sifat-sifat manusia konfliktis dan sifat alamiah manusia feeling of hostility. Penelitian ini bertujuan untuk (1) mendeskripsikan bentuk dan penyebab konflik sosial dalam novel Aib dan Nasib karya Minanto berdasarkan perspektif George Simmel; (2) mendeskripsikan penyelesaian konflik sosial dalam novel Aib dan Nasib karya Minanto berdasarkan perspektif George Simmel. Jenis penelitian ini adalah kualitatif deskriptif dan studi pustaka (library research). Teknik pengumpulan data menggunakan teknik baca dan teknik catat. Analisis data mengacu pada model Miles Huberman, yaitu melalui tahapan reduksi data, pemaparan data, dan penarikan kesimpulan. Hasil penelitian menunjukkan bahwa konflik soial dalam novel Aib dan Nasib karya Minanto berdasarkan perspektif George Simmel terdiri dari: (1) tiga bentuk konflik sosial yaitu: a. konflik kepentingan dengan satu wujud konflik sosial, yaitu adu mulut; b. konflik antar pribadi dengan empat wujud konflik sosial: adu mulut, sindiran, pengeroyokan, dan ketegangan; c. konflik dalam hubungan intim dengan dua wujud konflik sosial: adu mulut dan ketegangan; (2) empat penyelesaian konflik yaitu koersi, mediasi, toleransi, dan konversi.

Morphine and other opiates are among the most widely prescribed and clinically useful medications for the treatment of chronic pain. However, the applicability of these compounds has been severely hampered by the rapid development of tolerance and physical dependence that typically accompanies their repeated use. A growing body of evidence has implicated the regulated functioning of μ-δ opioid receptor heteromers in both the modulation of morphine-mediated antinociception, and in the limitation of undesirable side effects resulting from chronic opiate exposure. Moreover, μ-δ heteromers exhibit unique ligand binding characteristics and signaling properties, indicating that pharmacological targeting of the μ-δ heteromer may represent a novel therapeutic approach for the management of chronic pain and addiction disorders. Therefore, the present review will attempt to summarize the latest relevant findings regarding the regulation and functional characteristics of the μ-δ heteromer both in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Intestinal type sinonasal adenocarcinomas are a rare malignancy of the nasal cavity and paranasal sinuses ascending in the nasal cavity are particularly unusual. We report an infrequent case of an Intestinal sinonasal adenocarcinoma from the left lateral nasal wall. A 60-year-old Hispanic male complained of complete progressive nasal obstruction and epistaxis for 8months. A nasal endoscopic exam displayed a prominent enlargement, which remained in connection with the anterior part of left inferior turbinate. Computed tomography revealed a medium-defined, soft tissue dense lesion in the left anterior nasal cavity. Mass biopsy histology confirmed a low grade differentiate Intestinal type sinonasal adenocarcinomas. The patient underwent 8cycles of chemotherapy including paclitaxel, platinol and fluorouracil. According to the medical records, the patient responded well to the chemotherapy treatment until about six cycles. When a follow up CT scan showed tumor growth, the patient was placed on Doxorubicin but developed toxicity. The main treatment modality is surgical resection with histological clear margins but concerning the low differentiation, low response of chemotherapy, and surgical risk we offered an option with gamma knife radiosurgery. In our case, the patient demonstrated satisfactory relief of symptoms and reported the ability to breathe through the nose. 80% of tumor resolution was observed in 1month following the CT scan. Keywords: Intestinal type sinonasal adenocarcinomas, Nasal tumor, Gamma knife radiosurgery, Chemotherapy, Nasal cancer

This paper presents the characterization of the microbial community responsible for the in-situ bioremediation of hexachlorocyclohexane (HCH). Microbial community structure and function was analyzed using 16S rRNA amplicon and shotgun metagenomic sequencing methods for three sets of soil samples. The three samples were collected from a HCH-dumpsite (450 mg HCH/g soil) and comprised of a HCH/soil ratio of 0.45, 0.0007, and 0.00003, respectively. Certain bacterial; (Chromohalobacter, Marinimicrobium, Idiomarina, Salinosphaera, Halomonas, Sphingopyxis, Novosphingobium, Sphingomonas and Pseudomonas), archaeal; (Halobacterium, Haloarcula and Halorhabdus) and fungal (Fusarium) genera were found to be more abundant in the soil sample from the HCH-dumpsite. Consistent with the phylogenetic shift, the dumpsite also exhibited a relatively higher abundance of genes coding for chemotaxis/motility, chloroaromatic and HCH degradation (lin genes). Reassembly of a draft pangenome of Chromohalobacter salaxigenes sp. (∼8X coverage) and 3 plasmids (pISP3, pISP4 and pLB1; 13X coverage) containing lin genes/clusters also provides an evidence for the horizontal transfer of HCH catabolism genes.

Abstract Background It is hypothesized that complex interactions between multiple environmental factors and genetic factors are implicated in sporadic Alzheimer's disease (AD); however, the underlying mechanisms are poorly understood. Importantly, recent evidence reveals that expression and activity levels of the β-site APP cleaving enzyme 1 (BACE1), which initiates amyloid-β (Aβ) production, are elevated in AD brains. In this study, we investigated a molecular mechanism by which sex and stress interactions may accelerate β-amyloidogenesis and contribute to sporadic AD. Results We applied 5-day restraint stress (6 h/day) to the male and female 5XFAD transgenic mouse model of AD at the pre-pathological stage of disease, which showed little amyloid deposition under non-stressed control conditions. Exposure to the relatively brief behavioral stress increased levels of neurotoxic Aβ42 peptides, the β-secretase-cleaved C-terminal fragment (C99) and plaque burden in the hippocampus of female 5XFAD mice but not in that of male 5XFAD mice. In contrast, significant changes in the parameters of β-amyloidosis were not observed in the cerebral cortex of stressed male or female 5XFAD mice. We found that this sex- and brain region-specific acceleration of β-amyloidosis was accounted for by elevations in BACE1 and APP levels in response to adverse stress. Furthermore, not only BACE1 mRNA but also phosphorylation of the translation initiation factor eIF2α (a proposed mediator of the post-transcriptional upregulation of BACE1) was elevated in the hippocampus of stressed female 5XFAD mice. Conclusions Our results suggest that the higher prevalence of sporadic AD in women may be attributable to the vulnerability of female brains (especially, the hippocampus) to stressful events, which alter APP processing to favor the β-amyloidogenesis through the transcriptional and translational upregulation of BACE1 combined with elevations in its substrate APP.

Abstract Introduction Heterotopic pregnancy is diagnosed as the presence of two gestations simultaneously. This is a rare situation with a reported prevalence of 0.08% in normal conception. Case presentation We report a case of a 24-year-old primigravida of Indian origin who was seen in the emergency department with a diagnosis of a ruptured ectopic pregnancy. A careful ultrasound assessment led to the diagnosis of a heterotopic pregnancy. Immediate surgical intervention with supportive measures resulted in a successful outcome. Conclusion An obstetrician should keep in mind the occurrence of a heterotopic pregnancy while dealing with pregnant females. The ectopic gestation invariably ruptures over a period of time leaving the patient in an emergency situation. A quick assessment and careful handling of the normal gestation can lead the patient to term with gratifying results.

Abstract Background G Protein-Coupled Receptors (GPCRs) are a large and diverse family of membrane proteins whose members participate in the regulation of most cellular and physiological processes and therefore represent key pharmacological targets. Although several bioinformatics resources support research on GPCRs, most of these have been designed based on the traditional assumption that monomeric GPCRs constitute the functional receptor unit. The increase in the frequency and number of reports about GPCR dimerization/oligomerization and the implication of oligomerization in receptor function makes necessary the ability to store and access information about GPCR dimers/oligomers electronically. Results We present here the requirements and ontology (the information scheme to describe oligomers and associated concepts and their relationships) for an information system that can manage the elements of information needed to describe comprehensively the phenomena of both homo- and hetero-oligomerization of GPCRs. The comprehensive information management scheme that we plan to use for the development of an intuitive and user-friendly GPCR-Oligomerization Knowledge Base (GPCR-OKB) is the result of a community dialog involving experimental and computational colleagues working on GPCRs. Conclusion Our long term goal is to disseminate to the scientific community organized, curated, and detailed information about GPCR dimerization/oligomerization and its related structural context. This information will be reported as close to the data as possible so the user can make his own judgment on the conclusions drawn for a particular study. The requirements and ontology described here will facilitate the development of future information systems for GPCR oligomers that contain both computational and experimental information about GPCR oligomerization. This information is freely accessible at http://www.gpcr-okb.org.

Cannabinoid and opioid receptor activities can be modulated by a variety of post-translational mechanisms including the formation of interacting complexes. This study examines the involvement of endogenous and exogenous chaperones in modulating the abundance and activity of cannabinoid CB 1 receptor (CB 1 R), δ opioid receptor (DOR), and CB 1 R-DOR interacting complexes. Focusing on endogenous protein chaperones, namely receptor transporter proteins (RTPs), we examined relative mRNA expression in the mouse spinal cord and found RTP4 to be expressed at higher levels compared with other RTPs. Next, we assessed the effect of RTP4 on receptor abundance by manipulating RTP4 expression in cell lines. Overexpression of RTP4 causes an increase and knock-down causes a decrease in the levels of CB 1 R, DOR, and CB 1 R-DOR interacting complexes; this is accompanied by parallel changes in signaling. The ability of small molecule lipophilic ligands to function as exogenous chaperones was examined using receptor-selective antagonists. Long-term treatment leads to increases in receptor abundance and activity with no changes in mRNA supporting a role as pharmacological chaperones. Finally, the effect of cannabidiol (CBD), a small molecule ligand and a major active component of cannabis, on receptor abundance and activity in mice was examined. We find that CBD administration leads to increases in receptor abundance and activity in mouse spinal cord. Together, these results highlight a role for chaperones (proteins and small molecules) in modulating levels and activity of CB 1 R, DOR, and their interacting complexes potentially through mechanisms including receptor maturation and trafficking. SIGNIFICANCE STATEMENT: This study highlights a role for chaperones (endogenous and small membrane-permeable molecules) in modulating levels of cannabinoid CB 1 receptor, delta opioid receptor, and their interacting complexes. These chaperones could be developed as therapeutics for pathologies involving these receptors., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

Ketamine is a glutamate receptor antagonist that was developed over 50 years ago as an anesthetic agent. At subanesthetic doses, ketamine and some metabolites are analgesics and fast-acting antidepressants, presumably through targets other than glutamate receptors. We tested ketamine and its metabolites for activity as allosteric modulators of opioid receptors expressed as recombinant receptors in heterologous systems and with native receptors in rodent brain; signaling was examined by measuring GTP binding, β -arrestin recruitment, MAPK activation, and neurotransmitter release. Although micromolar concentrations of ketamine alone had weak agonist activity at μ opioid receptors, the combination of submicromolar concentrations of ketamine with endogenous opioid peptides produced robust synergistic responses with statistically significant increases in efficacies. All three opioid receptors ( μ , δ , and κ ) showed synergism with submicromolar concentrations of ketamine and either methionine-enkephalin (Met-enk), leucine-enkephalin (Leu-enk), and/or dynorphin A17 (Dyn A17), albeit the extent of synergy was variable between receptors and peptides. S -ketamine exhibited higher modulatory effects compared with R -ketamine or racemic ketamine, with ∼100% increase in efficacy. Importantly, the ketamine metabolite 6-hydroxynorketamine showed robust allosteric modulatory activity at μ opioid receptors; this metabolite is known to have analgesic and antidepressant activity but does not bind to glutamate receptors. Ketamine enhanced potency and efficacy of Met-enkephalin signaling both in mouse midbrain membranes and in rat ventral tegmental area neurons as determined by electrophysiology recordings in brain slices. Taken together, these findings support the hypothesis that some of the therapeutic effects of ketamine and its metabolites are mediated by directly engaging the endogenous opioid system. SIGNIFICANCE STATEMENT: This study found that ketamine and its major biologically active metabolites function as potent allosteric modulators of μ , δ , and κ opioid receptors, with submicromolar concentrations of these compounds synergizing with endogenous opioid peptides, such as enkephalin and dynorphin. This allosteric activity may contribute to ketamine's therapeutic effectiveness for treating acute and chronic pain and as a fast-acting antidepressant drug., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

Over the past decade, boldine, a naturally occurring alkaloid found in several plant species including the Chilean Boldo tree, has garnered attention for its efficacy in rodent models of human disease. Some of the properties that have been attributed to boldine include antioxidant activities, neuroprotective and analgesic actions, hepatoprotective effects, anti-inflammatory actions, cardioprotective effects and anticancer potential. Compelling data now indicates that boldine blocks connexin (Cx) hemichannels (HCs) and that many if not all of its effects in rodent models of injury and disease are due to CxHC blockade. Here we provide an overview of boldine's pharmacological properties, including its efficacy in rodent models of common human injuries and diseases, and of its absorption, distribution, pharmacokinetics, and metabolism., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Sáez, Burrell, Cahill, Cullen, Devi, Gilbert, Graham, Gurvich, Havton, Iyengar, Khanna, Palermo, Siddiq, Toro, Vasquez, Zhao and Cardozo.)

Neuropeptides are ubiquitous in the nervous system. Research into neuropeptides has been limited by a lack of experimental tools that allow for the precise dissection of their complex and diverse dynamics in a circuit-specific manner. Opioid peptides modulate pain, reward and aversion and as such have high clinical relevance. To illuminate the spatiotemporal dynamics of endogenous opioid signaling in the brain, we developed a class of genetically encoded fluorescence sensors based on kappa, delta and mu opioid receptors: κLight, δLight and µLight, respectively. We characterized the pharmacological profiles of these sensors in mammalian cells and in dissociated neurons. We used κLight to identify electrical stimulation parameters that trigger endogenous opioid release and the spatiotemporal scale of dynorphin volume transmission in brain slices. Using in vivo fiber photometry in mice, we demonstrated the utility of these sensors in detecting optogenetically driven opioid release and observed differential opioid release dynamics in response to fearful and rewarding conditions., (© 2024. The Author(s).)

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 (A β ) 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., Competing Interests: The authors do not have any conflicts of interest to disclose., (U.S. Government work not protected by U.S. copyright.)

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 RGSz1 fl\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 G α z but not other G α i family subunits and selectively impedes MOPR-mediated G α z 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., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)

Carboxypeptidase E (CPE) is an essential enzyme that contributes to the biosynthesis of the vast majority of neuropeptides and peptide hormones. There are several reports claiming that small decreases in CPE activity cause physiological changes in animals and/or cultured cells, but these studies did not provide evidence that neuropeptide levels were affected by decreased CPE activity. In the present study, we tested if CPE is a rate-limiting enzyme in neuropeptide production using CpeNeo mice, which contain a neomycin cassette within the Cpe gene that eliminates enzyme expression. Homozygous CpeNeo/Neo mice show defects found in Cpe fat/fat and/or Cpe global knockout (KO) mice, including greatly decreased levels of most neuropeptides, severely impaired fertility, depressive-like behavior, adult-onset obesity, and anxiety-like behavior. Removal of the neomycin cassette with Flp recombinase under a germline promoter restored expression of CPE activity and resulted in normal behavioral and physiological properties, including levels of neuropeptides. Mice heterozygous for the CpeNeo allele have greatly reduced levels of Cpe mRNA and CPE-like enzymatic activity. Despite the decreased levels of Cpe expression, heterozygous CpeNeo mice are behaviorally and physiologically identical to wild-type mice, with normal levels of most neuropeptides. These results indicate that CPE is not a rate-limiting enzyme in the production of most neuropeptides, casting doubt upon studies claiming small decreases in CPE activity contribute to obesity or other physiological effects., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Abstract: Diabetic neuropathy, often associated with diabetes mellitus, is a painful condition with no known effective treatment except glycemic control. Studies with neuropathic pain models report alterations in cannabinoid and opioid receptor expression levels; receptors whose activation induces analgesia. We examined whether interactions between CB1R and opioid receptors could be targeted for the treatment of diabetic neuropathy. For this, we generated antibodies that selectively recognize native CB1R-MOR and CB1R-DOR heteromers using a subtractive immunization strategy. We assessed the levels of CB1R, MOR, DOR, and interacting complexes using a model of streptozotocin-induced diabetic neuropathy and detected increased levels of CB1R, MOR, DOR, and CB1R-MOR complexes compared with those in controls. An examination of G-protein signaling revealed that activity induced by the MOR, but not the DOR agonist, was potentiated by low nanomolar doses of CB1R ligands, including antagonists, suggesting an allosteric modulation of MOR signaling by CB1R ligands within CB1R-MOR complexes. Because the peptide endocannabinoid, hemopressin, caused a significant potentiation of MOR activity, we examined its effect on mechanical allodynia and found that it blocked allodynia in wild-type mice and mice with diabetic neuropathy lacking DOR (but have CB1R-MOR complexes). However, hemopressin does not alter the levels of CB1R-MOR complexes in diabetic mice lacking DOR but increases the levels of CB1R-DOR complexes in diabetic mice lacking MOR. Together, these results suggest the involvement of CB1R-MOR and CB1R-DOR complexes in diabetic neuropathy and that hemopressin could be developed as a potential therapeutic for the treatment of this painful condition., (Copyright © 2021 International Association for the Study of Pain.)

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 G as or G ai depending on the length of the peptide and/or its concentration., (Copyright © 2020 American Society for Pharmacology and Experimental Therapeutics.)

Chronic pain is a major health issue, and the search for new analgesics has become increasingly important because of the addictive properties and unwanted side effects of opioids. To explore potentially new drug targets, we investigated mutations in the NTRK1 gene found in individuals with congenital insensitivity to pain with anhidrosis (CIPA). NTRK1 encodes tropomyosin receptor kinase A (TrkA), the receptor for nerve growth factor (NGF) and that contributes to nociception. Molecular modeling and biochemical analysis identified mutations that decreased the interaction between TrkA and one of its substrates and signaling effectors, phospholipase Cγ (PLCγ). We developed a cell-permeable phosphopeptide derived from TrkA (TAT-pQYP) that bound the Src homology domain 2 (SH2) of PLCγ. In HEK-293T cells, TAT-pQYP inhibited the binding of heterologously expressed TrkA to PLCγ and decreased NGF-induced, TrkA-mediated PLCγ activation and signaling. In mice, intraplantar administration of TAT-pQYP decreased mechanical sensitivity in an inflammatory pain model, suggesting that targeting this interaction may be analgesic. The findings demonstrate a strategy to identify new targets for pain relief by analyzing the signaling pathways that are perturbed in CIPA.

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., (© 2021 The British Pharmacological Society.)

Anxiety disorders are prevalent across the United States and result in a large personal and societal burden. Currently, numerous therapeutic and pharmaceutical treatment options exist. However, drugs to classical receptor targets have shown limited efficacy and often come with unpleasant side effects, highlighting the need to identify novel targets involved in the etiology and treatment of anxiety disorders. GPR83, a recently deorphanized receptor activated by the abundant neuropeptide PEN, has also been identified as a glucocorticoid regulated receptor (and named GIR) suggesting that this receptor may be involved in stress-responses that underlie anxiety. Consistent with this, GPR83 null mice have been found to be resistant to stress-induced anxiety. However, studies examining the role of GPR83 within specific brain regions or potential sex differences have been lacking. In this study, we investigate anxiety-related behaviors in male and female mice with global knockout and following local GPR83 knockdown in female mice. We find that a global knockdown of GPR83 has minimal impact on anxiety-like behaviors in female mice and a decrease in anxiety-related behaviors in male mice. In contrast, a local GPR83 knockdown in the basolateral amygdala leads to more anxiety-related behaviors in female mice. Local GPR83 knockdown in the central amygdala or nucleus accumbens (NAc) showed no significant effect on anxiety-related behaviors. Finally, dexamethasone administration leads to a significant decrease in receptor expression in the amygdala and NAc of female mice. Together, our studies uncover a significant, but divergent role for GPR83 in different brain regions in the regulation of anxiety-related behaviors, which is furthermore dependent on sex., Competing Interests: NT is employed by Regeneron Pharmaceuticals, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Fakira, Lueptow, Trimbake and Devi.)

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, β-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, β-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, β-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 β-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 β-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.

Many signal transduction systems have an apparent redundancy built into them, where multiple physiological agonists activate the same receptors. Whether this is true redundancy, or whether this provides an as-yet unrecognized specificity in downstream signaling, is not well understood. We address this question using the kappa opioid receptor (KOR), a physiologically relevant G protein-coupled receptor (GPCR) that is activated by multiple members of the Dynorphin family of opioid peptides. We show that two related peptides, Dynorphin A and Dynorphin B, bind and activate KOR to similar extents in mammalian neuroendocrine cells and rat striatal neurons, but localize KOR to distinct intracellular compartments and drive different post-endocytic fates of the receptor. Strikingly, localization of KOR to the degradative pathway by Dynorphin A induces sustained KOR signaling from these compartments. Our results suggest that seemingly redundant endogenous peptides can fine-tune signaling by regulating the spatiotemporal profile of KOR signaling., Competing Interests: JK, AG, IG, LD, MP No competing interests declared, (© 2021, Kunselman et al.)

Antibodies represent powerful tools to examine signal transduction pathways. Here, we present a strategy integrating multiple state-of-the-art methods to produce, validate, and utilize antibodies. Focusing on understudied synaptic proteins, we generated 137 recombinant antibodies. We used yeast display antibody libraries from the B cells of immunized rabbits, followed by FACS sorting under stringent conditions to identify high affinity antibodies. The antibodies were validated by high-throughput functional screening, and genome editing. Next, we explored the temporal dynamics of signaling in single cells. A subset of antibodies targeting opioid receptors were used to examine the effect of treatment with opiates that have played central roles in the worsening of the 'opioid epidemic.' We show that morphine and fentanyl exhibit differential temporal dynamics of receptor phosphorylation. In summary, high-throughput approaches can lead to the identification of antibody-based tools required for an in-depth understanding of the temporal dynamics of opioid signaling.

Neuropeptides and peptide hormones are important cell-cell signaling molecules that mediate many physiological processes. Unlike classic neurotransmitters, peptides undergo cell-type-specific post-translational modifications that affect their biological activity. To enable the identification of the peptide repertoire of a genetically defined cell type, we generated mice with a conditional disruption of the gene for carboxypeptidase E (Cpe), an essential neuropeptide-processing enzyme. The loss of Cpe leads to accumulation of neuropeptide precursors containing C-terminal basic residues, which serve as tags for affinity purification. The purified peptides are subsequently identified using quantitative peptidomics, thereby revealing the specific forms of neuropeptides in cells with the disrupted Cpe gene. To validate the method, we used mice expressing Cre recombinase under the proopiomelanocortin (Pomc) promoter and analyzed hypothalamic and pituitary extracts, detecting peptides derived from proopiomelanocortin (as expected) and also proSAAS in POMC neurons. This technique enables the analyses of specific forms of peptides in any Cre-expressing cell type., Competing Interests: Declaration of Interests The authors declare that they have no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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 (M 1 -M 5 ), we identified in the patient's serum antibodies that selectively bound to M 3 receptors. In vitro functional studies confirmed that these autoantibodies selectively blocked M 3 receptor activation. Thus, autoantibodies against M 3 acetylcholine receptors cause acute postganglionic cholinergic dysautonomia. ANN NEUROL 2020;88:1237-1243., (© 2020 American Neurological Association.)

Cannabidiol (CBD) is the second most abundant component of the Cannabis plant and is known to have effects distinct from Δ 9 -tetrahydrocannabinol (THC). Many studies that examined the behavioral effects of CBD concluded that it lacks the psychotomimetic effects attributed to THC. However, CBD was shown to have a broad spectrum of effects on several conditions such as anxiety, inflammation, neuropathic pain, and epilepsy. It is currently thought that CBD engages different targets and hence CBD's effects are thought to be due to multiple molecular mechanisms of action. A well-accepted set of targets include GPCRs and ion channels, with the serotonin 5-HT 1A receptor and the transient receptor potential cation channel TRPV1 channel being the two main targets. CBD has also been thought to target G protein-coupled receptors (GPCRs) such as cannabinoid and opioid receptors. Other studies have suggested a role for additional GPCRs and ion channels as targets of CBD. Currently, the clinical efficacy of CBD is not completely understood. Evidence derived from randomized clinical trials, in vitro and in vivo models and real-world observations support the use of CBD as a drug treatment option for anxiety, neuropathy, and many other conditions. Hence an understanding of the current status of the field as it relates to the targets for CBD is of great interest so, in this review, we include findings from recent studies that highlight these main targets., (© 2020 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.)

In this work, we studied a series of carfentanyl amide-based opioid derivatives targeting the mu opioid receptor (μOR) and the delta opioid receptor (δOR) heteromer as a credible novel target in pain management therapy. We identified a lead compound named MP135 that exhibits high G-protein activity at μ-δ heteromers compared to the homomeric δOR or μOR and low β-arrestin2 recruitment activity at all three. Furthermore, MP135 exhibits distinct signaling profile, as compared to the previously identified agonist targeting μ-δ heteromers, CYM51010. Pharmacological characterization of MP135 supports the utility of this compound as a molecule that could be developed as an antinociceptive agent similar to morphine in rodents. In vivo characterization reveals that MP135 maintains untoward side effects such as respiratory depression and reward behavior; together, these results suggest that optimization of MP135 is necessary for the development of therapeutics that suppress the classical side effects associated with conventional clinical opioids.

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., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Post-translational modifications (PTMs) are key events in signal transduction since they affect protein function by regulating their abundance and/or activity. PTMs involve the covalent attachment of functional groups to specific amino acids. Since they tend to be generally reversible, PTMs serve as regulators of signal transduction pathways. G-protein-coupled receptors (GPCRs) are major signaling proteins that undergo multiple types of PTMs. In this Review, we focus on the opioid receptors, members of GPCR family A, and highlight recent advances in the field that have underscored the importance of PTMs in the functional regulation of these receptors. Since opioid receptor activity plays a central role in the development of tolerance and addiction to morphine and other drugs of abuse, understanding the molecular mechanisms regulating receptor activity is of fundamental importance., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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 ([ 35 S]GTPγS binding and β-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 β-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., Competing Interests: The authors declare no competing interests.

Several reports support the idea that µ- and δ-opioid receptors (ORs) may exist as heterodimers in brain regions involved in pain signaling. The unique pharmacology of these heteromers may present a novel analgesic target. However, the role of µ-δ heteromers in sensory neurons involved in pain and opioid analgesia remains unclear, particularly during neuropathic pain. We examined the effects of spinal nerve injury on µ-δ heteromer expression in dorsal root ganglion (DRG) neurons and the effects of a µ-δ heteromer-targeting agonist, CYM51010, on neuropathic pain behavior in rats and mice. An L5 spinal nerve ligation (SNL) in rats significantly decreased µ-δ heteromer expression in L5 DRG but increased heteromer levels in uninjured L4 DRG. Importantly, in SNL rats, subcutaneous injection of CYM51010 inhibited mechanical hypersensitivity in a dose-related manner (EC50: 1.09 mg/kg) and also reversed heat hyperalgesia and attenuated ongoing pain (2 mg/kg, subcutaneously). HEK-293T cell surface-labeled with µ- and δ-ORs internalized both receptors after exposure to CYM51010. By contrast, in cells transfected with µ-OR alone, CYM51010 was significantly less effective at inducing receptor internalization. Electrophysiologic studies showed that CYM51010 inhibited the C-component and windup phenomenon in spinal wide dynamic range neurons of SNL rats. The pain inhibitory effects of CYM51010 persisted in morphine-tolerant rats but was markedly attenuated in µ-OR knockout mice. Our studies show that spinal nerve injury may increase µ-δ heterodimerization in uninjured DRG neurons, and that µ-δ heteromers may be a potential therapeutic target for relieving neuropathic pain, even under conditions of morphine tolerance.

GPR83, the receptor for the neuropeptide PEN, exhibits high expression in the nucleus accumbens of the human and rodent brain, suggesting that it plays a role in modulating the mesolimbic reward pathway. However, the cell-type specific expression of GPR83, its functional impact in the reward pathway, and in drug reward-learning has not been fully explored. Using GPR83/eGFP mice, we show high GPR83 expression on cholinergic interneurons in the nucleus accumbens and moderate expression on ventral tegmental area dopamine neurons. In GPR83 knockout mice, baseline dopamine release in the nucleus accumbens is enhanced which disrupts the ratio of tonic vs phasic release. Additionally, GPR83 knockout leads to changes in the expression of dopamine-related genes. Using the morphine conditioned place preference model, we identify sex differences in morphine reward-learning, show that GPR83 is upregulated in the nucleus accumbens following morphine conditioned place preference, and show that shRNA-mediated knockdown of GPR83 in the nucleus accumbens leads to attenuation morphine reward. Together, these findings detect GPR83 expression in the reward-pathway, and show its involvement in dopamine release and morphine reward-learning., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Cannabinoid 1 (CB 1 R) and delta opioid receptors (DOR) associate to form heteromers that exhibit distinct pharmacological properties. Not much is known about CB 1 R-DOR heteromer location or signaling along the pain circuit in either animal models or patients with chemotherapy-induced peripheral neuropathy (CIPN). Here, we use paclitaxel to induce CIPN in mice and confirm the development of mechanical allodynia. Under these conditions, we find significant increases in CB 1 R-DOR heteromers in the dorsal spinal cord of mice with CIPN as well as in postmortem spinal cords from human subjects with CIPN compared to controls. Next, we investigated receptor signaling in spinal cords of mice with CIPN and found that treatment with a combination of low signaling doses of CB 1 R and DOR ligands leads to significant enhancement in G-protein activity that could be selectively blocked by the CB 1 R-DOR antibody. Consistent with this, administration of subthreshold doses of a combination of ligands (CB 1 R agonist, Hu-210, and DOR agonist, SNC80) leads to significant attenuation of allodynia in mice with CIPN that is not seen with the administration of individual ligands, and this could be blocked by the CB 1 R-DOR antibody. Together, these results imply that CB 1 R-DOR heteromers upregulated during CIPN-associated mechanical allodynia could serve as a potential target for treatment of neuropathic pain including CIPN., Competing Interests: The authors declare no competing financial interest.

Neuropeptides are chemical messengers that act to regulate a number of physiological processes, including feeding, reward, pain, and memory, among others. PEN is one of the most abundant hypothalamic neuropeptides; however, until recently, its target receptor remained unknown. In this Review, we summarize recent developments in research focusing on PEN and its receptor GPR83. We describe the studies leading to the deorphanization of GPR83 as the receptor for PEN. We also describe the signaling mediated by the PEN-GPR83 system, as well as the physiological roles in which PEN-GPR83 has been implicated. As studies have suggested a role for the PEN-GPR83 system in food intake and body weight regulation, as well as in drug addiction and reward disorders, a thorough understanding of this novel neuropeptide-receptor system will help identify novel therapeutic targets to treat pathophysiological conditions involving PEN-GPR83.

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, CB 1 cannabinoid, or D 2 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., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

It is well accepted that treatment of chronic pain with morphine leads to μ opioid receptor (MOR) desensitization and the development of morphine tolerance. MOR activation by the selective peptide agonist, D-Ala2, N-MePhe4, Gly-ol]-enkephalin(DAMGO), leads to robust G protein receptor kinase activation, β-arrestin recruitment, and subsequent receptor endocytosis, which does not occur in an activation by morphine. However, MOR activation by morphine induces receptor desensitization, in a Protein kinase C (PKC) dependent manner. PKC inhibitors have been reported to decrease receptor desensitization, reduce opiate tolerance, and increase analgesia. However, the exact role of PKC in these processes is not clearly delineated. The difficulties in establishing a particular role for PKC have been, in part, due to the lack of reagents that allow the selective identification of PKC targets. Recently, we generated a conformation state-specific anti-PKC antibody that preferentially recognizes the active state of this kinase. Using this antibody to selectively isolate PKC substrates and a proteomics strategy to establish the identity of the proteins, we examined the effect of morphine treatment on the PKC targets. We found an enhanced interaction of a number of proteins with active PKC, in the presence of morphine. In this article, we discuss the role of these proteins in PKC-mediated MOR desensitization and analgesia. In addition, we posit a role for some of these proteins in mediating pain by TrKA activation, via the activation of transient receptor potential cation channel subfamily V member 1 (TRPV1). Finally, we discuss how these new PKC interacting proteins and pathways could be targeted for the treatment of pain., Competing Interests: The authors declare no conflict of interest.

Neuropeptides are the largest class of intercellular signaling molecules, contributing to a wide variety of physiological processes. Neuropeptide receptors are therapeutic targets for a broad range of drugs, including medications to treat pain, addiction, sleep disorders, and nausea. In addition to >100 peptides with known functions, many peptides have been identified in mammalian brain for which the cognate receptors have not been identified. Similarly, dozens of "orphan" G protein-coupled receptors have been identified in the mammalian genome. While it would seem straightforward to match the orphan peptides and receptors, this is not always easily accomplished. In this review we focus on peptides named PEN and big LEN, which are among the most abundant neuropeptides in mouse brain, and their recently identified receptors: GPR83 and GPR171. These receptors are co-expressed in some brain regions and are able to interact. Because PEN and big LEN are produced from the same precursor protein and co-secreted, the interaction of GPR83 and GPR171 is physiologically relevant. In addition to interactions of these two peptides/receptors, PEN and LEN are co-localized with neuropeptide Y and Agouti-related peptide in neurons that regulate feeding. In this review, using these peptide receptors as an example, we highlight the multiple modes of regulation of receptors and present the emerging view that neuropeptides function combinatorially to generate a network of signaling messages. The complexity of neuropeptides, receptors, and their signaling pathways is important to consider both in the initial deorphanization of peptides and receptors, and in the subsequent development of therapeutic applications., (Copyright © 2017 Elsevier Inc. All rights reserved.)

[This corrects the article DOI: 10.1371/journal.pone.0187306.].

G-protein coupled receptors (GPCRs) are a superfamily of receptors responsible for initiation of a myriad of intracellular signaling cascades. Currently, GPCRs represent approximately 34% of marketed pharmaceuticals, a large portion of which have no known endogenous ligand. These orphan GPCRs represent a large pool of novel targets for drug development. Very recently, the neuropeptide PEN, derived from the proteolytic processing of the precursor proSAAS, has been identified as a selective, high-affinity endogenous ligand for the orphan receptor, GPR83. GPR83 is highly expressed in the brain, spleen and thymus, indicating that this receptor may be a target to treat neurological and immune disorders. In the brain GPR83 is expressed in regions involved in the reward pathway, stress/anxiety responses, learning and memory and metabolism. However, the cell type specific expression of GPR83 in these regions has only recently begun to be characterized. In the immune system, GPR83 expression is regulated by Foxp3 in T-regulatory cells that are involved in autoimmune responses. Moreover, in the brain this receptor is regulated by interactions with other GPCRs, such as the recently deorphanized receptor, GPR171, and other hypothalamic receptors such as MC4R and GHSR. The following review will summarize the properties of GPR83 and highlight its known and potential significance in health and disease, as well as its promise as a novel target for drug development., (© 2018 Elsevier Inc. All rights reserved.)

Studies show that neuropeptide-receptor systems in the basolateral amygdala (BLA) play an important role in the pathology of anxiety and other mood disorders. Since GPR171, a recently deorphanized receptor for the abundant neuropeptide BigLEN, is expressed in the BLA, we investigated its role in fear and anxiety-like behaviors. To carry out these studies we identified small molecule ligands using a homology model of GPR171 to virtually screen a library of compounds. One of the hits, MS0021570_1, was identified as a GPR171 antagonist based on its ability to block (i) BigLEN-mediated activation of GPR171 in heterologous cells, (ii) BigLEN-mediated hyperpolarization of BLA pyramidal neurons, and (iii) feeding induced by DREADD-mediated activation of BigLEN containing AgRP neurons in the arcuate nucleus. The role of GPR171 in anxiety-like behavior or fear conditioning was evaluated following systemic or intra-BLA administration of MS0021570_1, as well as following lentiviral-mediated knockdown of GPR171 in the BLA. We find that systemic administration of MS0021570_1 attenuates anxiety-like behavior while intra-BLA administration or knockdown of GPR171 in the BLA reduces anxiety-like behavior and fear conditioning. These results indicate that the BigLEN-GPR171 system plays an important role in these behaviors and could be a novel target to develop therapeutics to treat psychiatric disorders.

Intracellular peptides generated by limited proteolysis are likely to function inside and outside cells and could represent new possibilities for drug development. Here, we used several conformational-sensitive antibodies targeting G-protein coupled receptors to screen for novel pharmacological active peptides. We find that one of these peptides, DITADDEPLT activates cannabinoid type 1 receptors. Single amino acid modifications identified a novel peptide, DIIADDEPLT (Pep19), with slightly better inverse agonist activity at cannabinoid type 1 receptors. Pep19 induced uncoupling protein 1 expression in both white adipose tissue and 3T3-L1 differentiated adipocytes; in the latter, Pep19 activates pERK1/2 and AKT signaling pathways. Uncoupling protein 1 expression induced by Pep19 in 3T3-L1 differentiated adipocytes is blocked by AM251, a cannabinoid type 1 receptors antagonist. Oral administration of Pep19 into diet-induced obese Wistar rats significantly reduces adiposity index, whole body weight, glucose, triacylglycerol, cholesterol and blood pressure, without altering heart rate; changes in the number and size of adipocytes were also observed. Pep19 has no central nervous system effects as suggested by the lack of brain c-Fos expression, cell toxicity, induction of the cannabinoid tetrad, depressive- and anxiety-like behaviors. Therefore, Pep19 has several advantages over previously identified peripherally active cannabinoid compounds, and could have clinical applications.

The N-terminal region of G protein-coupled receptors can be efficiently targeted for the generation of receptor-selective antibodies. These antibodies are useful for the biochemical characterization of the receptors. In this study, we developed a set of criteria to select the optimal epitope and applied them to generate antibodies to the N-terminal region of 34 different G protein-coupled receptors. The antibody characterization revealed that a subset of antibodies exhibited increased recognition of the receptor following agonist treatment and this increase could be blocked by treatment with the receptor antagonist. An analysis of the epitopes showed that those antibodies that exhibit increased recognition are on average twelve residues long, have an overall net negative charge and are enriched in aspartic and glutamic acids. These antibodies are useful since they facilitate studies examining dose dependent increases in recognition of receptors in heterologous cells as well as in native tissue. Another interesting use of these antibodies is that they facilitate measuring changes in receptor recognition in brain following peripheral drug administration; for example, systemic administration of cocaine, a blocker of dopamine transporter that increases local dopamine levels at the synapse, was found to lead to increases in antibody recognition of dopamine receptors in the brain. Taken together these studies, in addition to describing novel tools to study native receptors, provide a framework for the generation of antibodies to G protein-coupled receptors that can detect ligand-induced conformational changes.