Receptor Transporter Protein 4 (RTP4) in the Hypothalamus Is Involved in the Development of Antinociceptive Tolerance to Morphine.

Receptor transporter protein 4 (RTP4), one of the receptor chaperone proteins, contributes to the maturation and membrane trafficking of opioid receptor heteromers consisting of mu (MOPr) and delta (DOPr) opioid receptors (MOPr-DOPr). Although MOPr-DOPr is known to mediate the development of morphine tolerance, the extent to which RTP4 plays a role in this process has not been elucidated. Given that RTP4 can be upregulated by repeated administration of morphine, especially in the hypothalamus, here we investigated the effect of hypothalamus-selective ablation of RTP4 on the development of antinociceptive tolerance to morphine. In this study, we generated RTP4flox mice and selectively knocked-out RTP4 using local injection of adeno-associated virus expressing Cre recombinase (AAV-Cre) into the hypothalamus. The AAV-Cre injection partially, but significantly, decreased the level of RTP4 expression, and suppressed the development of antinociceptive tolerance to morphine. Next, we examined the mechanism of regulation of RTP4 and found that, in neuronal cells, Rtp4 induction is via Gi and MAPK activation, while, in microglial cells, the induction is via Toll-like receptor 4. Together, these studies highlight the role of MOR activity in regulating RTP4, which, in turn, plays an important role in modulating morphine effects in vivo.

Endogenous oxytocin levels in extracted saliva elevates during breastfeeding correlated with lower postpartum anxiety in primiparous mothers.

BACKGROUND: Breastfeeding in the early postpartum period is expected to have mental benefits for mothers; however, the underlying psychobiological mechanisms remain unclear. Previously, we hypothesized that the release of oxytocin in response to the suckling stimuli during breastfeeding would mediate a calming effect on primiparous mothers, and we examined salivary oxytocin measurements in primiparous mothers at postpartum day 4 using saliva samples without extraction, which was erroneous. Thus, further confirmation of this hypothesis with a precise methodology was needed. METHODS: We collected saliva samples at three time points (baseline, feeding, and post-feeding) to measure oxytocin in 24 primiparous mothers on postpartum day 2 (PD2) and 4 (PD4) across the breastfeeding cycle. Salivary oxytocin levels using both extracted and unextracted methods were measured and compared to determine the qualitative differences. State and trait anxiety and clinical demographics were evaluated to determine their association with oxytocin changes. RESULTS: Breastfeeding elevated salivary oxytocin levels; however, it was not detected to a significant increase in the extraction method at PD4. We found a weak but significant positive correlation between changes in extracted and unextracted oxytocin levels during breastfeeding (feeding minus baseline); there were no other significant positive correlations. Therefore, we used the extracted measurement index for subsequent analysis. We showed that the greater the increase in oxytocin during breastfeeding, the lower the state anxiety, but not trait anxiety. Mothers who exclusively breastfed at the 1-month follow-up tended to be associated with slightly higher oxytocin change at PD2 than those who did not. CONCLUSIONS: Breastfeeding in early postpartum days could be accompanied by the frequent release of oxytocin and lower state anxiety, potentially contributing to exclusive breastfeeding.

Slitrk2 deficiency causes hyperactivity with altered vestibular function and serotonergic dysregulation.

SLITRK2 encodes a transmembrane protein that modulates neurite outgrowth and synaptic activities and is implicated in bipolar disorder. Here, we addressed its physiological roles in mice. In the brain, the Slitrk2 protein was strongly detected in the hippocampus, vestibulocerebellum, and precerebellar nuclei-the vestibular-cerebellar-brainstem neural network including pontine gray and tegmental reticular nucleus. Slitrk2 knockout (KO) mice exhibited increased locomotor activity in novel environments, antidepressant-like behaviors, enhanced vestibular function, and increased plasticity at mossy fiber-CA3 synapses with reduced sensitivity to serotonin. A serotonin metabolite was increased in the hippocampus and amygdala, and serotonergic neurons in the raphe nuclei were decreased in Slitrk2 KO mice. When KO mice were treated with methylphenidate, lithium, or fluoxetine, the mood stabilizer lithium showed a genotype-dependent effect. Taken together, Slitrk2 deficiency causes aberrant neural network activity, synaptic integrity, vestibular function, and serotonergic function, providing molecular-neurophysiological insight into the brain dysregulation in bipolar disorders.

[MOPr-DOPr heteromer: the meaning and possibility as novel therapeutic target for pain control].

Many studies suggest opioid receptor (OPr) dimerization modulates the pharmacological properties of opiates. Specifically, heteromerization between OPr types has been reported to lead to changes in intracellular signaling. Thus, ligands targeting heteromers are expected to be novel therapeutic targets with reduced side effects. The heteromers of mu (MOPr) and delta (DOPr) are detected in brain regions involved in pain processing. The bivalent ligand or small molecule were identified as a MOPr-DOPr targeting ligand. These ligands exhibit antinociceptive properties similar to that of morphine with lesser antinociceptive tolerance as compared to morphine. Studies exploring the in vivo regulation of MOPr-DOPr heteromers, showed chronic morphine administration leads to an upregulation of these heteromers in select brain regions. Exploration of mechanisms underlying this phenomenon led us to the G protein-coupled receptor chaperone, RTP4, that is induced by chronic morphine and facilitates the heteromerization of MOPr and DOPr. In this review, I will introduce the simulated structure or property of MOPr-DOPr heteromer, its targeting ligands, and its intracellular regulatory mechanism that include a key molecule like RTP4 that could serve as a scaffold for the development of novel therapeutic drugs with reduced adverse effects, and hence may take place of the conventional clinical opioids.

The impact of elevated blood lead levels in children on maternal health-related quality of life.

Kabwe is a mining town in Zambia that has been ranked among "the ten most polluted places in the world" with previous findings of serious lead (Pb) pollution. In this study, we aim to examine the impact of childhood Pb poisoning on the health-related quality of life (HRQoL) of mothers in Kabwe. The HRQoL was assessed using the Short-Form 36 survey for 404 mothers coming from residences in 40 randomly selected standard enumeration areas (SEAs). Blood lead levels (BLLs) of the household members including the mothers themselves were measured. We found a significant positive correlation between the BLLs of the mothers and their children (R = 0.6385, p < 0.0001), while the BLLs of preschool-aged and school-aged children were significantly higher than those of their mothers and fathers. Using the data sets containing the BLLs of the household members, the age of the mothers, the household income, and the household SEA, we performed stepwise multiple linear regression analyses. The results showed significant negative associations between the representative BLL of household children and the BLL of preschool-aged children with the vitality and mental health scores of their mothers. Additionally, the BLL of school-aged children was only significantly associated with the mental health score of their mothers. By contrast, there was a significant negative association between the BLLs of the mothers with the social role functioning score. This suggests that elevated BLLs in children have a negative impact on the mental health conditions of their mothers regardless of the mothers' BLL.

Factors associated with self-rated health among mineworkers in Zambia: a cross-sectional study.

BACKGROUND: This study aims to examine miners' working conditions and self-rated health status in copper mines in Zambia and to identify the conditions and factors necessary to improve the safety and health of mineworkers. METHODS: A cross-sectional study using a self-administered questionnaire was conducted anonymously among copper mineworkers in Zambia in 2015 and 2016. Five targeted mining companies among 33 were introduced by the Mineworkers' Union of Zambia. Study participants were recruited at the waiting space for underground work, waiting rooms of company clinics/hospitals, and/or at training sessions, which were places permitted by the target companies to perform data collection via convenience sampling. Bivariate analyses (e.g., t tests, Kruskal-Wallis tests, chi-square tests, or Cochran-Armitage tests) and logistic regression analysis were used to analyze differences in demographic characteristics and to compare their working conditions, health conditions, safety management at the workplace, and training opportunities by employment status. RESULTS: In total, 338 responses were analyzed. Regular employees had better working conditions, including higher incomes (P = 0.001), more likely to be guaranteed sickness insurance by the company (P < 0.001), paid holidays (P = 0.094), and sick leave (P = 0.064), although the difference was not statistically significant. Mineworkers' decreased self-rated health was determined by job category (adjusted odds ratio [AOR], 0.41; 95% confidence interval [CI], 0.21, 0.82; P = 0.012). Having experienced violence from the boss/manager (AOR, 0.54; 95% CI, 0.32, 0.91; P = 0.020) was negatively associated with better self-rated health in the crude odds ratio. CONCLUSIONS: Among mineworkers in Zambia, nonunderground work and not having experienced violence from their boss/manager contributed to increased self-rated health. From the perspective of psychological safety and human security, the management of safety and the working environment, including human resource management and preventing harassment/violence, should be assured, especially for underground mineworkers.

The Possible Role of MOPr-DOPr Heteromers and Its Regulatory Protein RTP4 at Sensory Neurons in Relation to Pain Perception.

Heteromers between mu opioid receptor (MOPr) and delta opioid receptor (DOPr) (i.e., MOPr-DOPr heteromer) have been found to be expressed in different brain regions, in the spinal cord, and in dorsal root ganglia. Recent studies on this heteromer reveal its important pathophysiological function in pain regulation including neuropathic pain; this suggests a role as a novel therapeutic target in chronic pain management. In addition, receptor transporter protein 4 (RTP4) has been shown to be involved in the intracellular maturation of the MOPr-DOPr heteromers. RTP4 appears to have unique distribution in vivo being highly expressed in sensory neurons and also macrophages; the latter are effector cells of the innate immune system that phagocytose foreign substances and secrete both pro-inflammatory and antimicrobial mediators; this suggests a possible contribution of RTP4 to neuronal immune-related pathological conditions such as neuropathic pain. Although RTP4 could be considered as an important therapeutic target in the management of pain via MOPr-DOPr heteromer, a few reports have supported this. This review will summarize the possible role or functions of the MOPr-DOPr heteromer and its regulatory molecule RTP4 in pain modulation at sensory neurons.

Aiming at Ideal Therapeutics-MOPr/DOPr or MOPr-DOPr Heteromertargeting Ligand.

BACKGROUND AND OBJECTIVE: The recent alarming reports related to "opioid crisis" necessitate the development of safer and effective analgesics without unwanted side effects. Thus, there needs to be an alternative target or strategy for the development of drugs for the treatment of opioid use/abuse. As one of the novel targets, in these two decades, ligands targeting opioid receptor "heteromerization" including mu-opioid receptor (MOPr)-delta opioid receptor (DOPr) heteromer have been proposed and the pharmacological advancement of reduced side effects has been broadly accepted and well recognized. In this review, some of the ligands targeting both MOPr and DOPr or MOPr-DOPr heteromers are introduced especially focusing on their pharmacological effects in vivo. CONCLUSION: It has been found that most of those ligands possess potent antinociceptive activity (as much as or higher than that of morphine) with reduced side effects such as tolerance. In addition, some of them are also able to reduce or prevent physiological withdrawal symptoms observed under chronic opioid use. Importantly, there are an increasing number of evidence that show changes in heteromer expression in various pathological animal models and these strongly argue for targeting heteromers for the development of the next generation of pain medication in the near future.

Activation of µ-δ opioid receptor heteromers inhibits neuropathic pain behavior in rodents.

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.

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.

Two delta opioid receptor subtypes are functional in single ventral tegmental area neurons, and can interact with the mu opioid receptor.

The mu and delta opioid receptors (MOR and DOR) are highly homologous members of the opioid family of GPCRs. There is evidence that MOR and DOR interact, however the extent to which these interactions occur in vivo and affect synaptic function is unknown. There are two stable DOR subtypes: DPDPE sensitive (DOR1) and deltorphin II sensitive (DOR2); both agonists are blocked by DOR selective antagonists. Robust motivational effects are produced by local actions of both MOR and DOR ligands in the ventral tegmental area (VTA). Here we demonstrate that a majority of both dopaminergic and non-dopaminergic VTA neurons express combinations of functional DOR1, DOR2, and/or MOR, and that within a single VTA neuron, DOR1, DOR2, and MOR agonists can differentially couple to downstream signaling pathways. As reported for the MOR agonist DAMGO, DPDPE and deltorphin II produced either a predominant K+ dependent hyperpolarization or a Cav2.1 mediated depolarization in different neurons. In some neurons DPDPE and deltorphin II produced opposite responses. Excitation, inhibition, or no effect by DAMGO did not predict the response to DPDPE or deltorphin II, arguing against a MOR-DOR interaction generating DOR subtypes. However, in a subset of VTA neurons the DOR antagonist TIPP-Ψ augmented DAMGO responses; we also observed DPDPE or deltorphin II responses augmented by the MOR selective antagonist CTAP. These findings directly support the existence of two independent, stable forms of the DOR, and show that MOR and DOR can interact in some neurons to alter downstream signaling.

Neuroprotective DAMPs member prothymosin alpha has additional beneficial actions against cerebral ischemia-induced vascular damages.

Prothymosin alpha (ProTα) suppresses stress-induced necrosis of cultured cortical neurons. As neuroprotection alone could not explain the long-lasting protective actions against cerebral ischemia by ProTα, we further examined whether ProTα, in addition to neuroprotective effects, has other anti-ischemic activities. When recombinant mouse ProTα (rmProTα) at 0.3 mg/kg was intravenously (i.v.) given 2 h after the start of tMCAO, all mice survived for more than 14 days. In evaluation of CD31- and tomato lectin-labeling as well as IgG and Evans blue leakage, rmProTα treatment (0.1 mg/kg) largely blocked ischemia-induced vascular damages. Therefore, rmProTα has novel beneficial effects against ischemia-induced brain damage through vascular mechanisms.

Identification of GPR83 as the receptor for the neuroendocrine peptide PEN.

PEN is an abundant peptide in the brain that has been implicated in the regulation of feeding. We identified a receptor for PEN in mouse hypothalamus and Neuro2A cells. PEN bound to and activated GPR83, a G protein (heterotrimeric guanine nucleotide)-binding protein)-coupled receptor (GPCR). Reduction of GPR83 expression in mouse brain and Neuro2A cells reduced PEN binding and signaling, consistent with GPR83 functioning as the major receptor for PEN. In some brain regions, GPR83 colocalized with GPR171, a GPCR that binds the neuropeptide bigLEN, another neuropeptide that is involved in feeding and is generated from the same precursor protein as is PEN. Coexpression of these two receptors in cell lines altered the signaling properties of each receptor, suggesting a functional interaction. Our data established PEN as a neuropeptide that binds GPR83 and suggested that these two ligand-receptor systems-PEN-GPR83 and bigLEN-GPR171-may be functionally coupled in the regulation of feeding.

P-glycoprotein inhibitors improve effective dose and time of pregabalin to inhibit intermittent cold stress-induced central pain.

Pregabalin (PGB) is a valuable therapeutic drug against chronic pain. Here we attempted to perform the combinatorial drug therapy with P-glycoprotein (P-gp) inhibitors to lower therapeutic dosage of PGB in the intermittent cold stress-induced fibromyalgia-like pain model. Single intracerebroventricular (i.c.v.) PGB injection exerted long-lasting anti-hyperalgesic effects for 72 h, while the effect of PGB given intraperitoneally (i.p.) disappeared within 3 h. Importantly, the pretreatment with P-gp inhibitors markedly prolonged the PGB (i.p.) effects, which lasted for 72 h. These results suggest that the combinatorial treatment with P-gp inhibitor enables the prolongation of dose-interval for PGB.

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.

Quality of partogram monitoring at a primary health centre in Zambia.

BACKGROUND: The World Health Organization (WHO) recommends using a partogram to reduce maternal and neonatal mortality, especially in developing countries. Some previous studies conducted in African countries suggested that appropriate use of a partogram with standardised monitoring was associated with good labour outcomes. However, the compliance rates of recording differed among the monitoring items on the partogram and the quality of monitoring has not been examined adequately. OBJECTIVE: to examine the compliance for each monitoring item on the WHO partogram and the quality of the monitoring. DESIGN: a retrospective and observation study. METHODS: a retrospective review of partograms (n=200) was undertaken in a health centre in Lusaka, Zambia. We excluded referral cases, admission with full dilatation, birth before arrival, childbirth within 30 minutes, and false labour. Finally, 125 partograms were examined to assess the recording compliance for each monitoring item. An observation study in the delivery room and interviews with midwives were also conducted to examine the quality of monitoring for labour. The research ethics committee of the Division of Health Sciences, Osaka University Graduate School of Medicine and the Biomedical Research Ethics Committee of the University of Zambia approved the study. FINDINGS: the lowest recording rate of the frequency of uterine contractions at the time of admission was 69.6%. The highest compliance rates in the active phase were found for the descent of the fetal head and cervix dilatation at 97.6% and 97.3%, respectively. The lowest rate was found for the mother's pulse rate at 25.5%, whereas 27.1% of the women admitted in the latent phase were diagnosed as entering the active phase in the acceleration phase. In addition, the methods of abdominal palpation for assessing uterine contractions and intermittent fetal heart rate monitoring were not appropriate. KEY CONCLUSIONS AND IMPLICATIONS: Zambian midwives have acquired sufficient understanding regarding the usefulness of the WHO partogram. However, there were differences in the compliance rates for each monitoring item due to a lack of medical devices and inappropriate monitoring skill. To improve labour outcomes with the WHO partogram, it is necessary to improve the recording and compliance rates for each monitoring item, as well as to upgrade the quality of monitoring.

Heteromers of µ-δ opioid receptors: new pharmacology and novel therapeutic possibilities.

UNLABELLED: Several studies suggest that heteromerization between µ (MOP) and δ (DOP) opioid receptors modulates the signalling properties of the individual receptors. For example, whereas activation of MOP receptors by an agonist induces G protein-mediated signalling, the same agonist induces ß-arrestin-mediated signalling in the context of the MOP-DOP receptor heteromer. Moreover, heteromer-mediated signalling is allosterically modulated by a combination of MOP and DOP receptor ligands. This has implications in analgesia given that morphine-induced antinociception can be potentiated by DOP receptor ligands. Recently reagents selectively targeting the MOP-DOP receptor heteromer such as bivalent ligands, antibodies or membrane permeable peptides have been generated; these reagents are enabling studies to elucidate the contribution of endogenously expressed heteromers to analgesia as well as to the development of side-effects associated with chronic opioid use. Recent advances in drug screening technology have led to the identification of a MOP-DOP receptor heteromer-biased agonist that activates both G protein-mediated and ß-arrestin-mediated signalling. Moreover, this heteromer-biased agonist exhibits potent antinociceptive activity but with reduced side-effects, suggesting that ligands targeting the MOP-DOP receptor heteromer form a basis for the development of novel therapeutics for the treatment of pain. In this review, we summarize findings regarding the biological and functional characteristics of the MOP-DOP receptor heteromer and the in vitro and in vivo properties of heteromer-selective ligands. LINKED ARTICLES: This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.

Direct bidirectional µ-opioid control of midbrain dopamine neurons.

The ventral tegmental area (VTA) is required for the rewarding and motivational actions of opioids and activation of dopamine neurons has been implicated in these effects. The canonical model posits that opioid activation of VTA dopamine neurons is indirect, through inhibition of GABAergic inputs. However, VTA dopamine neurons also express postsynaptic µ-opioid peptide (MOP) receptors. We report here that in Sprague Dawley rat, the MOP receptor-selective agonist DAMGO (0.5-3 µM) depolarized or increased the firing rate of 87 of 451 VTA neurons (including 22 of 110 dopamine neurons). This DAMGO excitation occurs in the presence of GABAA receptor blockade and its EC50 value is two orders of magnitude lower than for presynaptic inhibition of GABA release on to VTA neurons. Consistent with a postsynaptic channel opening, excitations were accompanied by a decrease in input resistance. Excitations were blocked by CdCl2 (100 µM, n = 5) and ω-agatoxin-IVA (100 nM, n = 3), nonselective and Cav2.1 Ca(2+) channel blockers, respectively. DAMGO also produced a postsynaptic inhibition in 233 of 451 VTA neurons, including 45 of 110 dopamine neurons. The mean reversal potential of the inhibitory current was -78 ± 7 mV and inhibitions were blocked by the K(+) channel blocker BaCl2 (100 µM, n = 7). Blockade of either excitation or inhibition unmasked the opposite effect, suggesting that MOP receptors activate concurrent postsynaptic excitatory and inhibitory processes in most VTA neurons. These results provide a novel direct mechanism for MOP receptor control of VTA dopamine neurons.

Molecular characterization of eluxadoline as a potential ligand targeting mu-delta opioid receptor heteromers.

Eluxadoline, an orally active mixed µ opioid receptor (µOR) agonist δ opioid receptor (δOR) antagonist developed for the treatment of diarrhea-predominant irritable bowel syndrome, normalizes gastrointestinal (GI) transit and defecation under conditions of novel environment stress or post-inflammatory altered GI function. Furthermore, compared to loperamide, which is used to treat non-specific diarrhea, the effects of eluxadoline on GI transit occur over a wider dosage range. However, the mechanisms of action of eluxadoline are unclear. In this study, we compared the ability of eluxadoline and loperamide to activate G-protein- and ß-arrestin-mediated signaling at µOR homomers or µOR-δOR heteromers in heterologous cells. We also examined the ability of both compounds to reduce castor oil induced diarrhea in wild type (WT) and mice lacking δOR. We find that eluxadoline is more potent than loperamide in eliciting G-protein activity and ß-arrestin recruitment in µOR expressing cells. However, in cells expressing µOR-δOR heteromers, the potency of eluxadoline is higher, but its maximal effect is lower than that of loperamide. Moreover, in these cells the signaling mediated by eluxadoline but not loperamide is reduced by µOR-δOR heteromer-selective antibodies. We find that in castor oil-induced diarrhea eluxadoline is more efficacious compared to loperamide in WT mice, and δOR appears to play a role in this process. Taken together these results indicate that eluxadoline behaves as a potent µOR agonist in the absence of δOR, while in the presence of δOR eluxadoline's effects are mediated through the µOR-δOR heteromer.