Designer Benzodiazepines' Activity on Opioid Receptors: A Docking Study.

BACKGROUND: Previous studies have reported that benzodiazepines (BZDs) seem to enhance euphoric and reinforcing properties of opioids in opioid users so that a direct effect on opioid receptors has been postulated, together with a possible synergistic induction of severe side effects due to co use of BDZs and opioids. This is particularly worrisome given the appearance on the market of designer benzodiazepines (DBZDs), whose activity/toxicity profiles are scarcely known. OBJECTIVES: This study aimed to evaluate, through computational studies, the binding affinity (or lack thereof) of 101 DBZDs identified online on the kappa, mu, and delta opioid receptors (K, M, DOR); and to assess whether their mechanism of action could include activation of the latter. METHODS: MOE® was used for the computational studies. Pharmacophore mapping based on strong opioids agonist binders' 3D chemical features was used to filter the DBZDs. Resultant DBZDs were docked into the crystallised 3D active conformation of KOR (PDB6B73), DOR (PDB6PT3) and MOR (PDB5C1M). Co-crystallised ligands and four strong agonists were used as reference compounds. A score (S, Kcal/mol) representative of the predicted binding affinity, and a description of ligand interactions were obtained from MOE®. RESULTS: The docking results, filtered for S < -8.0 and the interaction with the Asp residue, identified five DBZDs as putative binders of the three ORs : ciclotizolam, fluloprazolam, JQ1, Ro 48-6791, and Ro 48-8684. CONCLUSION: It may be inferred that at least some DBZDs may have the potential to activate opioid receptors. This could mediate/increase their anxiolytic, analgesic, and addiction potentials, as well as worsen the side effects associated with opioid co-use.

Positive allosteric modulation of endogenous delta opioid receptor signaling in the enteric nervous system is a potential treatment for gastrointestinal motility disorders.

Allosteric modulators (AMs) are molecules that can fine-tune signaling by G protein-coupled receptors (GPCRs). Although they are a promising therapeutic approach for treating a range of disorders, allosteric modulation of GPCRs in the context of the enteric nervous system (ENS) and digestive dysfunction remains largely unexplored. This study examined allosteric modulation of the delta opioid receptor (DOR) in the ENS and assessed the suitability of DOR AMs for the treatment of irritable bowel syndrome (IBS) symptoms using mouse models. The effects of the positive allosteric modulator (PAM) of DOR, BMS-986187, on neurogenic contractions of the mouse colon and on DOR internalization in enteric neurons were quantified. The ability of BMS-986187 to influence colonic motility was assessed both in vitro and in vivo. BMS-986187 displayed DOR-selective PAM-agonist activity and orthosteric agonist probe dependence in the mouse colon. BMS-986187 augmented the inhibitory effects of DOR agonists on neurogenic contractions and enhanced reflex-evoked DOR internalization in myenteric neurons. BMS-986187 significantly increased DOR endocytosis in myenteric neurons in response to the weakly internalizing agonist ARM390. BMS-986187 reduced the generation of complex motor patterns in the isolated intact colon. BMS-986187 reduced fecal output and diarrhea onset in the novel environment stress and castor oil models of IBS symptoms, respectively. DOR PAMs enhance DOR-mediated signaling in the ENS and have potential benefit for the treatment of dysmotility. This study provides proof of concept to support the use of GPCR AMs for the treatment of gastrointestinal motility disorders.NEW & NOTEWORTHY This study assesses the use of positive allosteric modulation as a pharmacological approach to enhance opioid receptor signaling in the enteric nervous system. We demonstrate that selective modulation of endogenous delta opioid receptor signaling can suppress colonic motility without causing constipation. We propose that allosteric modulation of opioid receptor signaling may be a therapeutic strategy to normalize gastrointestinal motility in conditions such as irritable bowel syndrome.

LP1 and LP2: Dual-Target MOPr/DOPr Ligands as Drug Candidates for Persistent Pain Relief.

Although persistent pain is estimated to affect about 20% of the adult population, current treatments have poor results. Polypharmacology, which is the administration of more than one drug targeting on two or more different sites of action, represents a prominent therapeutic approach for the clinical management of persistent pain. Thus, in the drug discovery process the "one-molecule-multiple targets" strategy nowadays is highly recognized. Indeed, multitarget ligands displaying a better antinociceptive activity with fewer side effects, combined with favorable pharmacokinetic and pharmacodynamic characteristics, have already been shown. Multitarget ligands possessing non-opioid/opioid and opioid/opioid mechanisms of action are considered as potential drug candidates for the management of various pain conditions. In particular, dual-target MOPr (mu opioid peptide receptor)/DOPr (delta opioid peptide receptor) ligands exhibit an improved antinociceptive profile associated with a reduced tolerance-inducing capability. The benzomorphan-based compounds LP1 and LP2 belong to this class of dual-target MOPr/DOPr ligands. In the present manuscript, the structure-activity relationships and the pharmacological fingerprint of LP1 and LP2 compounds as suitable drug candidates for persistent pain relief is described.

Endogenous opioid and cannabinoid systems modulate the muscle pain: A pharmacological study into the peripheral site.

The participation of the peripheral opioid and cannabinoid endogenous systems in modulating muscle pain and inflammation has not been fully explored. Thus, the aim of this study was to investigate the involvement of these endogenous systems during muscular-tissue hyperalgesia induced by inflammation. Hyperalgesia was induced by carrageenan injection into the tibialis anterior muscles of male Wistar rats. We padronized an available Randal-Sellito test adaptation to evaluate nociceptive behavior elicited by mechanical insult in muscles. Western blot analysis was performed to evaluate the expression levels of opioid and cannabinoid receptors in the dorsal root ganglia. The non-selective opioid peptide receptor antagonist (naloxone) and the selective mu opioid receptor MOP (clocinnamox) and kappa opioid receptor KOP (nor-binaltorphimine) antagonists were able to intensify carrageenan-induced muscular hyperalgesia. On the other hand, the selective delta opioid receptor (DOP) antagonist (naltrindole) did not present any effect on nociceptive behavior. Moreover, the selective inhibitor of aminopeptidases (Bestatin) provoked considerable dose-dependent analgesia when intramuscularly injected into the hyperalgesic muscle. The CB1 receptor antagonist (AM251), but not the CB2 receptor antagonist (AM630), intensified muscle hyperalgesia. All irreversible inhibitors of anandamide hydrolase (MAFP), the inhibitor for monoacylglycerol lipase (JZL184) and the anandamide reuptake inhibitor (VDM11) decreased carrageenan-induced hyperalgesia in muscular tissue. Lastly, MOP, KOP and CB1 expression levels in DRG were baseline even after muscular injection with carrageenan. The endogenous opioid and cannabinoid systems participate in peripheral muscle pain control through the activation of MOP, KOP and CB1 receptors.

A selective delta opioid receptor agonist SNC80, but not KNT-127, induced tremor-like behaviors via hippocampal glutamatergic system in mice.

Although delta opioid receptors (DOP) are now known to play a major role in modulating chronic pain and controlling emotional processes, unfortunately, some DOP agonists, such as SNC80, reportedly produced convulsive-like behaviors manifesting as tremor-like behaviors in a preclinical study. Therefore, these induced convulsions limit the progress of the clinical development of DOP agonists. However, mechanisms underlying DOP-induced convulsant activity remain unclarified. Thus, the study aimed to elucidate mechanisms that could cause tremor-like behaviors of SNC80. These drugs were microinjected into the ventral hippocampus CA3 (vCA3), amygdala (AMY), and insular cortex (IC) of mice. In addition, we examined the extracellular glutamate levels after DOP agonist local treatment. Microinjection of SNC80 into the vCA3 increased the number of tremor-like behaviors and extracellular glutamate levels but did not cause tremor-like behaviors in mice when microinjected into IC and AMY. Pretreatment with α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainite receptor antagonist CNQX into vCA3 totally inhibited the SNC80-induced increases in tremor-like behaviors. In contrast, another DOP agonist, KNT-127, did not cause tremor-like behaviors in any of the tested brain areas. Further, the extracellular glutamate levels in the hippocampus were significantly lower in the KNT-127-treated mice than in the SNC80-treated mice. Our results showed that the administration of SNC80, but not KNT-127, into vCA3 induced tremor-like behaviors by activating glutamatergic neurons in mice. We propose that KNT-127 should be further studied clinically as a DOP agonist that is expected to have a low risk for convulsions than those resulting in antinociceptive and antidepressant effects.

Shifting motivational states: The effects of nucleus accumbens dopamine and opioid receptor activation on a modified effort-based choice task.

The nucleus accumbens (NAc) is critical for regulating the appetitive and consummatory phases of motivated behavior. These experiments examined the effects of dopamine and opioid receptor manipulations within the NAc during an effort-based choice task that allowed for simultaneous assessment of both phases of motivation. Male Sprague-Dawley rats received bilateral guide cannulas targeting the NAc core and were tested in 1-hr sessions with free access to rat chow and the choice to work for sugar pellets on a progressive ratio 2 (PR2) reinforcement schedule. Individual groups of rats were tested following stimulation or blockade of NAc D1-like or D2-like receptors, stimulation of µ-, δ-, or κ-opioid receptors, or antagonism of opioid receptors. Behavior was examined under ad libitum conditions and following 23-h food restriction. NAc blockade of the D1-like receptors or stimulation of the D2 receptor reduced break point for earning sugar pellets; D2 receptor stimulation also modestly lowered chow intake. NAc µ-opioid receptor stimulation increased intake of the freely-available chow while simultaneously reducing break point for the sugar pellets. In non-restricted conditions, δ-opioid receptor stimulation increased both food intake and breakpoint. There were no effects of stimulating NAc D1 or κ receptors, nor did blocking D2 or opioid receptors affect task behavior. These data support prior literature linking dopamine to appetitive motivational processes, and suggest that µ- and δ-opioid receptors affect food-directed motivation differentially. Specifically, µ-opioid receptors shifted behavior towards consumption, and δ-opioid receptor enhanced both sugar-seeking and consumption of the pabulum chow when animals were not food restricted.

Delta-opioid receptor-mediated modulation of excitability of individual hippocampal neurons: mechanisms involved.

BACKGROUND: Delta-opioid receptor (DOR)-mediated modulation of hippocampal neural networks is involved in emotions, cognition, and in pathophysiology and treatment of mood disorders. In this study, we examined the effects of DOR agonist (SNC80) and antagonist (naltrindole) on the excitability of individual hippocampal neurons. METHODS: Primary neuronal cultures were prepared from hippocampi of newborn rats and cultivated in vitro for 8-14 days (DIV8-14). The effects of SNC80 naltrindole on evoked and spontaneous action potentials (APs) were measured at DIV8-9 and DIV13-14, respectively. RESULTS: SNC80 (100 µM) potentiated spontaneous AP firing and stimulated sodium current; naltrindole had opposite effects. The stimulatory effect of 100 µM of SNC80 was revoked by pre-administration of 1 µM of naltrindole. SNC80 and naltrindole induced similar inhibitory effects on the evoked AP firing and on the calcium current. Further, SNC80 inhibited both peak and sustained potassium currents. Naltrindole had no effect on potassium currents. CONCLUSION: We suggest that the effects of naltrindole and high concentration of SNC80 on the sodium currents are mediated via DORs and underlying the changes in spontaneous activity. The inhibitory effects of SNC80 on calcium and potassium currents might also be DOR-dependent; these currents might mediate SNC80 effect on the evoked AP firing. The inhibitory effects of naltrindole on calcium and of low doses of SNC80 on sodium currents might be however DOR independent. The behavioral effects of SNC80 and naltrindole, observed in previous studies, might be mediated, at least in part, via the modulatory effect of these ligands on the excitability of hippocampal neurons.

Exploiting the Power of Stereochemistry in Drug Action: 3-[(2S,6S,11S)-8-Hydroxy-6,11-dimethyl-1,4,5,6-tetrahydro-2,6-methano-3-benzazocin-3(2H)-yl]-N-phenylpropanamide as Potent Sigma-1 Receptor Antagonist.

(+)-(2S,6S,11S)- and (-)-(2R,6R,11R)-Benzomorphan derivatives have a different binding affinity for sigma-1 (σ1R) and opioid receptors, respectively. In this study, we describe the synthesis of the (+)-enantiomer [(+)-LP1] of the benzomorphan MOR agonist/DOR antagonist LP1 [(-)-LP1]. The binding affinity of both (+)-LP1 and (-)-LP1 for σ1R and sigma-2 receptor (σ2R) was tested. Moreover, (+)-LP1 opioid receptor binding affinity was also investigated. Finally, (+)-LP1 was tested in a mouse model of inflammatory pain. Our results showed a nanomolar σ1R and binding affinity for (+)-LP1. Both (+)-LP1 and (-)-LP1 elicited a significant analgesic effect in a formalin test. Differently from (-)-LP1, the analgesic effect of (+)-LP1 was not reversed by naloxone, suggesting a σ1R antagonist profile. Furthermore, σ1R agonist PRE-084 was able to unmask the σ1R antagonistic component of the benzomorphan compound. (+)-LP1 could constitute an useful lead compound to develop new analgesics based on mechanisms of action alternative to opioid receptor activation.

Ketamine: The final frontier or another depressing end?

Two decades ago, the observation of a rapid and sustained antidepressant response after ketamine administration provided an exciting new avenue in the search for more effective therapeutics for the treatment of clinical depression. Research elucidating the mechanism(s) underlying ketamine's antidepressant properties has led to the development of several hypotheses, including that of disinhibition of excitatory glutamate neurons via blockade of N-methyl-d-aspartate (NMDA) receptors. Although the prominent understanding has been that ketamine's mode of action is mediated solely via the NMDA receptor, this view has been challenged by reports implicating other glutamate receptors such as AMPA, and other neurotransmitter systems such as serotonin and opioids in the antidepressant response. The recent approval of esketamine (Spravato™) for the treatment of depression has sparked a resurgence of interest for a deeper understanding of the mechanism(s) underlying ketamine's actions and safe therapeutic use. This review aims to present our current knowledge on both NMDA and non-NMDA mechanisms implicated in ketamine's response, and addresses the controversy surrounding the antidepressant role and potency of its stereoisomers and metabolites. There is much that remains to be known about our understanding of ketamine's antidepressant properties; and although the arrival of esketamine has been received with great enthusiasm, it is now more important than ever that its mechanisms of action be fully delineated, and both the short- and long-term neurobiological/functional consequences of its treatment be thoroughly characterized.

Delta- and mu-opioid pathways are involved in the analgesic effect of Ocimum basilicum L in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Ocimum basilicum L. is a perennial herb that has been used in traditional Asian Indian medicine for thousands of years as a natural anti-inflammatory, antibiotic, diuretic, and analgesic. AIM OF THE STUDY: The present study was conducted to investigate the analgesic effects of basil essential oil (BEO) in inflammatory pain models and identify underlying mechanisms. We further investigated whether BEO affects physiological pain and motor coordination. MATERIALS AND METHODS: The analgesic effects of BEO were assessed in various mouse experimental pain models using formalin, acetic acid, heat, and carrageenan as stimuli. BEO was administered by intraperitoneal injection or inhalation. The involvement of various pathways in the analgesic effect of BEO was assessed by pretreating mice with selective pharmacological inhibitors, administered intraperitoneally. Opioid pathways were tested using the κ-opioid antagonist 5'-guanidinonaltrindole (GNTI; 0.3 mg/kg), δ-opioid antagonist naltrindole (NTD; 5 mg/kg) and µ-opioid antagonist naloxone (NAL; 8 mg/kg); nitric oxide (NO) pathways were tested using the NO synthase inhibitor N-nitro l-arginine methyl ester (L-NAME; 37.5 mg/kg) and NO precursor L-arginine (L-Arg; 600 mg/kg); and KATP channel pathways were tested using the ATP-sensitive K+ channel blocker, glibenclamide-hippuric acid (GHA, 2 mg/kg). Potential effects of BEO on motor coordination were assessed using a rotarod test. RESULTS: BEO exerted analgesic effects in all pain models. Notably, pretreatment with naltrindole, naloxone, or L-arginine significantly reduced the analgesic effects of BEO in the formalin test. BEO increased mean withdrawal latencies in a thermal plantar test at a high dose, but not at lower doses. BEO had no effect on motor coordination. CONCLUSIONS: Our findings indicate that the analgesic effects of BEO are primarily mediated by delta- and mu-opioid pathways and further suggest that BEO has potential for development as an analgesic agent for the relief of inflammatory pain.

Oxycodone suppresses the apoptosis of hippocampal neurons induced by oxygen-glucose deprivation/recovery through caspase-dependent and caspase-independent pathways via κ- and δ-opioid receptors in rats.

Cerebral ischemia/reperfusion injury (CIRI) can lead to perioperative neurocognitive disorders (PND) during clinical recanalization procedures in cerebral vessels, principally due to neuronal apoptosis in the hippocampus. Oxycodone appears to be a multiple opioid receptor agonist and exerts intrinsic antinociception activity via κ-opioid receptor (KOR). Recent evidence has revealed that activation of both δ-opioid receptor (DOR) and KOR can provide neuroprotection against CIRI in vivo and in vitro. In our study, we established an oxygen-glucose deprivation/recovery (OGD/R) model with fetal hippocampal neurons and found that oxycodone could induce CIRI tolerance in these neurons, primarily through KOR and DOR. Possible mechanisms might involve the regulatory effect of oxycodone on the MAPK-Bcl2/Bax-caspase-9-caspase-3 pathway, as well as its inhibitory effect on cellular reactive oxygen species (ROS) production and mitochondrial membrane potential activation. Taken together, our findings may indicate a potential method for the prevention and treatment of PND associated with CIRI.

Emerging Treatment Targets for Migraine and Other Headaches.

Migraine is a complex disorder that is characterized by an assortment of neurological and systemic effects. While headache is the most prominent feature of migraine, a host of symptoms affecting many physiological functions are also observed before, during, and after an attack. Furthermore, migraineurs are heterogeneous and have a wide range of responses to migraine therapies. The recent approval of calcitonin gene-related-peptide based therapies has opened up the treatment of migraine and generated a renewed interest in migraine research and discovery. Ongoing advances in migraine research have identified a number of other promising therapeutic targets for this disorder. In this review, we highlight emergent treatments within the following biological systems: pituitary adenylate cyclase activating peptdie, 2 non-mu opioid receptors that have low abuse liability - the delta and kappa opioid receptors, orexin, and nitric oxide-based therapies. Multiple mechanisms have been identified in the induction and maintenance of migraine symptoms; and this divergent set of targets have highly distinct biological effects. Increasing the mechanistic diversity of the migraine tool box will lead to more treatment options and better patient care.

Stimulation of spinal δ-opioid receptors attenuates the exercise pressor reflex in decerebrate rats.

A reflex arising from contracting hindlimb muscle is responsible in part for the increases in arterial pressure and heart rate evoked by exercise. The afferent arm of this reflex comprises group III and IV afferents. δ-Opioid receptors are expressed predominately on the spinal endings of group III afferents, whereas µ-opioid receptors are expressed predominately on the spinal endings of group IV afferents. Using stimuli that activated group III afferents, namely static contraction, calcaneal tendon stretch, and lactic acid injection into the superficial epigastric artery, we tested the hypothesis that, in rats with either patent or ligated femoral arteries, activation of pre- and postsynaptic δ-opioid receptors in the dorsal horn attenuated pressor reflex responses to these stimuli. In rats with patent arteries or ligated femoral arteries, [d-Pen2,5]enkephalin (DPDPE), a δ-opioid agonist injected intrathecally (10 µg in 10 µl), significantly attenuated the pressor responses to contraction, stretch, and lactic acid (all P < 0.05). Naltrindole, a δ-opioid receptor antagonist, prevented the attenuation. In contrast, DPDPE did not attenuate the pressor response to capsaicin injection into the superficial epigastric artery in either group of rats (both P > 0.05). Intrathecal injection of saline (10 µl), the vehicle for DPDPE, had no effect on the pressor responses in either group of rats. We conclude that activation of spinal δ-opioid receptors attenuates reflexes evoked by group III afferents in both freely perfused and ligated rats.

Sex Differences in the Rat Hippocampal Opioid System After Oxycodone Conditioned Place Preference.

Although opioid addiction has risen dramatically, the role of gender in addiction has been difficult to elucidate. We previously found sex-dependent differences in the hippocampal opioid system of Sprague-Dawley rats that may promote associative learning relevant to drug abuse. The present studies show that although female and male rats acquired conditioned place preference (CPP) to the mu-opioid receptor (MOR) agonist oxycodone (3 mg/kg, I.P.), hippocampal opioid circuits were differentially altered. In CA3, Leu-Enkephalin-containing mossy fibers had elevated levels in oxycodone CPP (Oxy) males comparable to those in females and sprouted in Oxy-females, suggesting different mechanisms for enhancing opioid sensitivity. Electron microscopy revealed that in Oxy-males delta opioid receptors (DORs) redistributed to mossy fiber-CA3 synapses in a manner resembling females that we previously showed is important for opioid-mediated long-term potentiation. Moreover, in Oxy-females DORs redistributed to CA3 pyramidal cell spines, suggesting the potential for enhanced plasticity processes. In Saline-injected (Sal) females, dentate hilar parvalbumin-containing basket interneuron dendrites had fewer MORs, however plasmalemmal and total MORs increased in Oxy-females. In dentate hilar GABAergic dendrites that contain neuropeptide Y, Sal-females compared to Sal-males had higher plasmalemmal DORs, and near-plasmalemmal DORs increased in Oxy-females. This redistribution of MORs and DORs within hilar interneurons in Oxy-females would potentially enhance disinhibition of granule cells via two different circuits. Together, these results indicate that oxycodone CPP induces sex-dependent redistributions of opioid receptors in hippocampal circuits in a manner facilitating opioid-associative learning processes and may help explain the increased susceptibility of females to opioid addiction acquisition and relapse.

Met-enkephalin improves metabolic syndrome in high fat diet challenged mice through promotion of adipose tissue browning.

Obesity and its related metabolic disorders including insulin resistance and fatty liver become major public health concerns in both developed and developing countries. Brown adipose tissue (BAT), a critical organ of energy expenditure due to thermogenesis, has been considered as an attractive target for prevention or treatment of obesity and obesity related diseases. Previous studies indicate Met-enkephalin (MetEnk) has the potential on adipocyte browning, however, whether MetEnk displays the impact on adipocyte browning in vivo to improve obesity associated morbidities is still unclear. In the present study, we showed that MetEnk effectively prevented high fat diet (HFD) induced C57BL/6J mice weight gain, clearly enhanced glucose tolerance and insulin sensitivity, and dramatically reduced hepatic steatosis in HFD fed mice. Mechanically, MetEnk restored protein kinase A (PKA) signaling pathway in HFD challenged mice and promoted subcutaneous white adipose tissue (WAT) browning. Our study suggests that MetEnk can be considered as a potential therapeutic peptide for diet-induced obesity and metabolic disorders.

Methylation Products of 6ß- N-Heterocyclic Substituted Naltrexamine Derivatives as Potential Peripheral Opioid Receptor Modulators.

Two 6ß- N-heterocyclic naltrexamine derivatives, NAP and NMP, have been identified as peripherally selective mu opioid receptor (MOR) antagonists. To further enhance the peripheral selectivity of both compounds, the 17-amino group and the nitrogen atom of the pyridine ring in both NAP and NMP were methylated to obtain dMNAP and dMNMP, respectively. Compared with NAP and NMP, the binding affinities of dMNAP and dMNMP shifted to MOR and KOR (kappa opioid receptor) dual selective and they acted as moderate efficacy partial agonists. The results from radioligand binding studies were further confirmed by molecular docking studies. In vivo studies demonstrated that dMNAP and dMNMP did not produce antinociception nor did they antagonize morphine's antinociceptive activity, indicating that these compounds did not act on the central nervous system. Meanwhile, both dMNAP and dMNMP significantly slowed down fecal excretion, which indicated that they were peripherally acting opioid receptor agonists. All together, these results suggested that dMNAP and dMNMP acted as peripheral mu/kappa opioid receptor modulators and may be applicable in the treatment of diarrhea in patients with bowel dysfunction.

Peripheral delta opioid receptors mediate duloxetine antiallodynic effect in a mouse model of neuropathic pain.

Peripheral delta opioid (DOP) receptors are essential for the antiallodynic effect of the tricyclic antidepressant nortriptyline. However, the population of DOP-expressing cells affected in neuropathic conditions or underlying the antiallodynic activity of antidepressants remains unknown. Using a mouse line in which DOP receptors were selectively ablated in cells expressing Nav1.8 sodium channels (DOP cKO), we established that these DOP peripheral receptors were mandatory for duloxetine to alleviate mechanical allodynia in a neuropathic pain model based on sciatic nerve cuffing. We then examined the impact of nerve cuffing and duloxetine treatment on DOP-positive populations using a knock-in mouse line expressing a fluorescent version of the DOP receptor fused with the enhanced green fluorescent protein (DOPeGFP). Eight weeks postsurgery, we observed a reduced proportion of DOPeGFP-positive small peptidergic sensory neurons (calcitonin gene-related peptide (CGRP) positive) in dorsal root ganglia and a lower density of DOPeGFP-positive free nerve endings in the skin. These changes were not present in nerve-injured mice chronically treated with oral duloxetine. In addition, increased DOPeGFP translocation to the plasma membrane was observed in neuropathic conditions but not in duloxetine-treated neuropathic mice, which may represent an additional level of control of the neuronal activity by DOP receptors. Our results therefore established a parallel between changes in the expression profile of peripheral DOP receptors and mechanical allodynia induced by sciatic nerve cuffing.

Amniotic-fluid ingestion enhances central δ-opioid-induced hypoalgesia in rats in the cold-water tail-flick assay in a repeated-measures design.

Placental Opioid Enhancing Factor (POEF) is found in amniotic fluid (AF) and placenta. When ingested, it enhances opioid-mediated pain relief. Our laboratory has shown that ingestion of AF specifically enhances the hypoalgesia associated with δ-opioid receptor activation in the brain. The specific biochemical compound in AF responsible for the enhancement of δ-opioid activity is of great interest as an analgesic adjunct for pain but is unknown at this time. Research efforts to isolate and characterize this biochemical compound are hampered by the lack of an algesiometric assay that allows repeated measurement of pain threshold and repeated exposure to δ-opioid receptor activation. The cold water tail-flick assay (CWTF) may be a sensitive and reliable pain threshold test of (a) all species of opioids that is (b) not subject to repeated-testing effects. Therefore the CWTF test is potentially ideal for the study of δ opioid systems in a repeated measures design. Here, we confirm these attributes of the CWTF test, and determined that (a) there are no repeated-exposure effects associated with the CWTF assay; (b) there are no repeated-exposure effects associated with repeated central injections of DPDPE ([D-Pen2,D-Pen5]-Enkephalin, a selective δ-opioid agonist) as measured by the CWTF assay; and (c) ingestion of AF in conjunction with a central injection of DPDPE produced the same hypoalgesic enhancement as previously found using another assay.

Up-regulation of µ-, δ- and κ-opioid receptors in concanavalin A-stimulated rat spleen lymphocytes.

Regulation of µ-, δ- and κ-opioid receptor protein level in spleen lymphocytes when stimulated by mitogen is not known. To answer the question whether these cells do express opioid receptor (OR) proteins, primary, fresh rat spleen lymphocytes were prepared and stimulated for 48 h with mitogenic dose of Con A. The unstimulated lymphocytes did not express µ- and δ-OR proteins in detectable amounts, however, stimulation with Con A resulted in appearance of clearly detectable immunoblot signals of both µ-OR and δ-OR. κ-OR were detected already in primary cells and increased 2.4-fold in Con A-stimulated cells. These results were supported by data obtained by flow cytometry analysis indicating a dramatic increase in number of µ-, δ- and κ-OR expressing cells after mitogen stimulation. The newly synthesized µ-, δ- and κ-OR in Con A-stimulated spleen lymphocytes were present in the cells interior and not functionally mature, at least in terms of their ability to enhance activity of trimeric G proteins determined by three different protocols of agonist-stimulated, high-affinity [35S]GTPγS binding assay. The up-regulation of µ-, δ- and κ-OR was associated with specific decrease of their cognate trimeric G proteins, Gi1α/Gi2α; the other Gα and Gß subunits were unchanged. The level of ß-arrestin-1/2 was also decreased in Con A-stimulated splenocytes. We conclude that up-regulation of OR expression level in spleen lymphocytes by Con A proceeds in conjunction with down-regulation of their intracellular signaling partners, Gi1α/Gi2α proteins and ß-arrestin-1/2. These regulatory proteins are expressed in high amounts already in unstimulated cells and decreased by mitogen stimulation.

Evidence and Function Relevance of Native DOR-MOR Heteromers.

Opioid receptors are the sites of action for morphine and most other clinically used opioid drugs. Abundant evidence now demonstrates that different opioid receptor types can physically associate to form heteromers. Owing to their constituent monomers' involvement in analgesia, mu/delta opioid receptor (M/DOR) heteromers have been a particular focus of attention. Understandings of the physiological relevance and indisputable proof of M/DOR formation in vivo are still evolving. This aspect of the field has been slow to progress in large part by the limitations of most available experimental models; recently however, promising progress is being made. As a result, the long-repeated promise of opioid receptor heteromers as selective therapeutic targets is now being realized.