Morphine-induced receptor endocytosis in a novel knockin mouse reduces tolerance and dependence

Abstract Opioid drugs, such as morphine, are among the most effective analgesics available. However, their utility for the treatment of chronic pain is limited by side effects including tolerance and dependence. Morphine acts primarily through the mu-opioid receptor (MOP-R) , which is also a target of endogenous opioids. However, unlike endogenous ligands, morphine fails to promote substantial receptor endocytosis both in vitro, and in vivo. Receptor endocytosis serves at least two important functions in signal transduction. First, desensitization and endocytosis act as an "off" switch by uncoupling receptors from G protein. Second, endocytosis functions as an "on" switch, resensitizing receptors by recycling them to the plasma membrane. Thus, both the off and on function of the MOP-R are altered in response to morphine compared to endogenous ligands. To examine whether the low degree of endocytosis induced by morphine contributes to tolerance and dependence, we generated a knockin mouse that expresses a mutant MOP-R that undergoes morphine-induced endocytosis. Morphine remains an excellent antinociceptive agent in these mice. Importantly, these mice display substantially reduced antinociceptive tolerance and physical dependence. These data suggest that opioid drugs with a pharmacological profile similar to morphine but the ability to promote endocytosis could provide analgesia while having a reduced liability for promoting tolerance and dependence

Absence of opioid stress-induced analgesia in mice lacking beta-endorphin by site-directed mutagenesis.

A physiological role for beta-endorphin in endogenous pain inhibition was investigated by targeted mutagenesis of the proopiomelanocortin gene in mouse embryonic stem cells. The tyrosine codon at position 179 of the proopiomelanocortin gene was converted to a premature translational stop codon. The resulting transgenic mice display no overt developmental or behavioral alterations and have a normally functioning hypothalamic-pituitary-adrenal axis. Homozygous transgenic mice with a selective deficiency of beta-endorphin exhibit normal analgesia in response to morphine, indicating the presence of functional mu-opiate receptors. However, these mice lack the opioid (naloxone reversible) analgesia induced by mild swim stress. Mutant mice also display significantly greater nonopioid analgesia in response to cold water swim stress compared with controls and display paradoxical naloxone-induced analgesia. These changes may reflect compensatory upregulation of alternative pain inhibitory mechanisms.

Effects of extremely low-frequency electromagnetic fields on morphine-induced conditioned place preferences in rats

In the present study, we examined the effects of extremely low-frequency (ELF) electromagnetic fields on morphine-induced conditioned place preferences in rats. During the conditioning phase (12 days), three groups of rats were placed in a sensory cue-defined environment paired with morphine (10 mg/kg, i.p.) following exposure to either 20 Hz (1.80 mT) or 50 Hz (2.20 mT) or sham electromagnetic fields for 60 min/day, respectively, and were placed in another sensory cue-defined environment paired with physiological saline (1 ml/kg, i.p.) without exposure to electromagnetic fields. After finishing 12 days of conditioning, preference tests for the morphine-paired place were performed during a 10-day withdrawal period. The exposure to electromagnetic fields substantially potentiated morphine-induced place preferences in rodents, suggesting that ELF electromagnetic fields can increase the propensity for morphine-induced conditioned behaviors. (C) 2005 Elsevier Ireland Ltd. All rights reserved.

Effects of scopolamine on morphine-induced conditioned place preference in mice

It is well known that the cholinergic system plays a crucial role in learning and memory. Psychopharmacological studies in humans and animals have shown that a systemic cholinergic blockade may induce deficits in learning and memory. Accumulated studies h

Pentylenetetrazole-induced status epilepticus following training does not impair expression of morphine-induced conditioned place preference

Learning and memory play an important role in morphine addiction. Status epilepticus (SE) can impair the spatial and emotional learning and memory. However, little is known about the effects of SE on morphine-induced conditioned place preference (CPP). Th

Differential mechanisms of morphine antinociceptive tolerance revealed in (beta)arrestin-2 knock-out mice.

Morphine induces antinociception by activating mu opioid receptors (muORs) in spinal and supraspinal regions of the CNS. (Beta)arrestin-2 (beta)arr2), a G-protein-coupled receptor-regulating protein, regulates the muOR in vivo. We have shown previously that mice lacking (beta)arr2 experience enhanced morphine-induced analgesia and do not become tolerant to morphine as determined in the hot-plate test, a paradigm that primarily assesses supraspinal pain responsiveness. To determine the general applicability of the (beta)arr2-muOR interaction in other neuronal systems, we have, in the present study, tested (beta)arr2 knock-out ((beta)arr2-KO) mice using the warm water tail-immersion paradigm, which primarily assesses spinal reflexes to painful thermal stimuli. In this test, the (beta)arr2-KO mice have greater basal nociceptive thresholds and markedly enhanced sensitivity to morphine. Interestingly, however, after a delayed onset, they do ultimately develop morphine tolerance, although to a lesser degree than the wild-type (WT) controls. In the (beta)arr2-KO but not WT mice, morphine tolerance can be completely reversed with a low dose of the classical protein kinase C (PKC) inhibitor chelerythrine. These findings provide in vivo evidence that the muOR is differentially regulated in diverse regions of the CNS. Furthermore, although (beta)arr2 appears to be the most prominent and proximal determinant of muOR desensitization and morphine tolerance, in the absence of this mechanism, the contributions of a PKC-dependent regulatory system become readily apparent.

Morphine peripheral analgesia depends on activation of the PI3K gamma/AKT/nNOS/NO/K(ATP) signaling pathway

Morphine is one of the most prescribed and effective drugs used for the treatment of acute and chronic pain conditions. In addition to its central effects, morphine can also produce peripheral analgesia. However, the mechanisms underlying this peripheral action of morphine have not yet been fully elucidated. Here, we show that the peripheral antinociceptive effect of morphine is lost in neuronal nitric-oxide synthase null mice and that morphine induces the production of nitric oxide in primary nociceptive neurons. The activation of the nitric-oxide pathway by morphine was dependent on an initial stimulation of PI3K gamma/AKT protein kinase B (AKT) and culminated in increasedactivation of K(ATP) channels. In the latter, this intracellular signaling pathway might cause a hyperpolarization of nociceptive neurons, and it is fundamental for the direct blockade of inflammatory pain by morphine. This understanding offers new targets for analgesic drug development.

Involvement of the midbrain periaqueductal gray 5-HT1A receptors in social conflict induced analgesia in mice

Recent results from our laboratory have shown that 30-bites social conflict in mice produces a high-intensity, short-term analgesia which is attenuated by systemically injected 5-HT1A receptor agonists, such as BAY R 1531 (6-methoxy-4-(di-n-propylamino)-1,3,4,5-tetrahydrobenz(c,d)indole hydrochloride) and gepirone. The present study investigated the effects of these drugs, as well as the 5-HT1A receptor antagonist WAY 100135 (N-tert-butyl-3-(4-(2-methoxyphenyl)piperazine-1-yl)-2-phenylpropanamide) injected into the midbrain periaqueductal gray matter of mice on 30-bites analgesia. Four to five days after guide-cannula implantation, each mouse received microinjection of gepirone (30 nmol/0.2 mu l), BAY R 1531 (10 nmol/0.2 mu l), WAY 100135 (10 nmol/0.2 mu l), saline (0.9% NaCl) or vehicle (saline + 4% Tween 80) 5 min before either an aggressive (30 bites) or a non-aggressive interaction. Nociception was assessed by the tail-flick test made before as well as 1, 5, 10 and 20 min after social interaction. The full 5-HT1A receptor agonist BAY R 1531 blocked, whereas, WAY 100135 and gepirone intensified 30-bites analgesia, Neither non-aggressive interaction, per se, nor the three compounds given after this type of social interaction significantly changed nociception. These results indicate that 5-HT1A receptors in the periaqueductal gray inhibit analgesia induced by social conflict in mice. (C) 1998 Elsevier B.V. B.V.

Effect of intravenous alizapride on spinal morphine-induced pruritus

Background. This double-blind study was undertaken to determine whether alizapride inhibits spinal morphine-induced pruritus.Methods. Eighty-four patients undergoing Caesarean section under spinal anaesthesia (100 mg of hyperbaric lidocaine 5% plus morphine 0.2 mg) were randomly allocated to one of two groups. just after birth, alizapride-50 mg (alizapride group) or metoclopramide 10 mg (metoclopramide group) were injected i.v. Patients were assessed after surgery for pruritus (absent, mild, moderate or severe) or other untoward symptoms.Results. In the metoclopramide group, pruritus was absent in 5 (12%) patients, mild in 23 (55%), moderate in 11 (26%), and severe in 3 (7%), while in the alizapride group, these incidences were, respectively, 5 (12%), 33 (79%), 4 (10%), and 0 (P=0.045, chi(2)-test). There was no difference in the incidence of side-effects, which were all minor.Conclusions. Alizapride reduced the severity of morphine-induced pruritus.

Comparison of droperidol and ondansetron prophylactic effect on subarachnoid morphine-induced pruritus

The prophylactic effect of ondansetron on subarachnoid morphine-induced pruritus is controversial, while evidence suggests that droperidol prevents pruritus. The aim of this study is to compare the effects of droperidol and ondansetron on subarachnoid morphine-induced pruritus. 180 ASA I or II patients scheduled to undergo cesarean sections under subarachnoid anesthesia combined with morphine 0.2mg were randomized to receive, after the child's birth, metoclopramide 10mg (Group I - control), droperidol 2.5mg (Group II) or ondansetron 8mg (Group III). Postoperatively, the patients were assessed for pruritus (absent, mild, moderate or severe) or other side effects by blinded investigators. Patients were also blinded to their group allocation. The tendency to present more severe forms of pruritus was compared between groups. NNT was also determined. Patients assigned to receive droperidol [Proportional odds ratio: 0.45 (95% confidence interval 0.23-0.88)] reported less pruritus than those who received metoclopramide. Ondansetron effect was similar to metoclopramide [Proportional odds ratio: 0.95 (95% confidence interval 0.49-1.83)]. The NNT for droperidol and ondansetron was 4.0 and 14.7, respectively. Ondansetron does not inhibit subarachnoid morphine-induced pruritus.

The role of morphine-6-glucuronide (M6G) in pain control

Morphine-6beta-D-glucuronide (M6G) is an analgesically active metabolite of morphine, accounting for approximate to10% of the morphine dose when administered by systemic routes to humans. Although M6G is more hydrophilic than morphine, it crosses the blood-brain barrier, albeit relatively slowly. For this reason, it is generally thought that, after chronic dosing, M6G contributes significantly to the analgesic effects of systemically administered morphine. Owing to its polar nature, M6G is cleared from the systemic circulation primarily via renal elimination. As M6G accumulates in patients with renal impairment, there is an increased risk of M6G-induced respiratory depression in renal failure patients who are being dosed chronically with systemic morphine. Consistent with its analgesic and respiratory depressant properties, M6G binds to the p-opioid receptor in a naloxone-reversible manner. Although the affinity of M6G for the mu-opioid receptor is similar to or slightly less than that of morphine, preclinical studies in rodents show that M6G is one to two orders of magnitude more potent than morphine when administered by central routes. This major discrepancy between the markedly higher intrinsic antinociceptive potency of M6G relative to morphine, despite their similar p-opioid receptor binding affinities, is difficult to reconcile. It has been proposed that M6G mediates its pain-relieving effects through a novel 'M6G opioid receptor', while others have argued that M6G may have higher efficacy than morphine for transduction of intracellular events. When administered by parenteral routes to rodents, M6G's antinociceptive potency is no more than twofold higher than morphine. In humans, the analgesic efficacy and respiratory depressant potency of M6G relative to morphine have been assessed in a number of short-term studies involving the intrathecal or intravenous routes of administration. For example, in hip replacement patients, intrathecal M6G provided excellent postoperative analgesia but the occurrence of late respiratory depression in 10% of these patients raised serious concern about safety. In postoperative patients, intravenous M6G administered by means of patient-controlled analgesia (PCA), or bolus plus PCA, produced no analgesia in one study and limited analgesia in another. Similarly, there was a lack of significant analgesia in healthy volunteers who received intravenous M6G for the alleviation of experimental pain (carbon dioxide applied to the nasal mucosa). In contrast, satisfactory analgesia was produced by bolus doses of intravenous M6G administered to patients with cancer pain, and to healthy volunteers with experimentally-induced ischaemic, electrical or thermal (ice water) pain. Studies to date in healthy volunteers suggest that intravenous M6G may be a less potent respiratory depressant and have a lower propensity for producing nausea and vomiting than morphine. However, it is unclear whether equi-analgesic doses of M6G and morphine were compared. Clearly, more extensive short-term trials, together with studies involving chronic M6G administration, are necessary before the potential clinical utility of M6G as an analgesic drug in its own right can be determined.

Rôle et implication du système cannabinoïde dans la modulation périphérique de la douleur inflammatoire et neuropathique

Les dérivés de l’opium (opioïdes) et du cannabis (cannabinoïdes) présentent de nombreuses propriétés intéressantes. Suite à l’identification de leurs récepteurs respectifs, diverses stratégies pharmacologiques ont tenté d’exploiter leurs propriétés analgésiques. Le clonage des récepteurs cannabinoïdes CB1 et CB2 a favorisé la découverte de composés endogènes pour ces récepteurs, les endocannabinoïdes, dont les deux plus étudiés sont l’anandamide et le 2-arachidonyl glycérol (2-AG). Cette découverte a également mené à l’identification d’enzymes qui catalysent l’inactivation de ces cannabinoïdes endogènes : une amidohydrolase des acides gras ou FAAH ainsi qu’une monoacylglycérol lipase ou MAGL. Le système cannabinoïde endogène est régulé à la hausse dans une variété de processus pathologiques, tels que les douleurs inflammatoire et neuropathique. Cette augmentation est habituellement interprétée comme une réaction physiologique visant à rétablir l’homéostasie et elle a notamment été observée en périphérie. Les endocannabinoïdes semblent donc agir de façon spécifique à des moments clés dans certains tissus ciblés afin de minimiser les conséquences reliées au déclenchement de ces douleurs. Cette observation est très intéressante d’un point de vue thérapeutique puisqu’elle suggère la possibilité de cibler les enzymes de dégradation des endocannabinoïdes dans le but d’augmenter leurs concentrations locales et d’ainsi prolonger leur action neuromodulatrice. En périphérie, l’activation des récepteurs cannabinoïdes induit des effets antinociceptifs bénéfiques tout en minimisant les effets indésirables souvent associés à leur activation centrale. Nous avons orienté nos travaux vers la modulation périphérique de ce système endogène à l’aide d’inhibiteurs des enzymes de dégradation des endocannabinoïdes afin d’évaluer leur potentiel thérapeutique et d’élucider les mécanismes d’action qui sous-tendent leurs effets dans des modèles animaux de douleurs inflammatoire et neuropathique. Nous avons démontré que cette approche permet de soulager les symptômes associés à ces deux types de douleurs, et ce via les récepteurs CB1 et CB2. Les systèmes cannabinoïde et opioïde présentent des similitudes, dont des localisations similaires le long des voies de la douleur, des mécanismes d’action relayés par des récepteurs couplés aux protéines G et des propriétés pharmacologiques communes telles que l’analgésie. Le système opioïde est impliqué dans les effets antinociceptifs induits par les cannabinoïdes. À l’inverse, le rôle joué par le système cannabinoïde dans ceux induits par la morphine demeure incertain. Nous avons démontré que les effets antinociceptifs périphériques et spinaux produits par la morphine sont diminués chez les souris génétiquement modifiées chez lesquelles l’expression des récepteurs CB1 ou CB2 a été éliminée, laissant supposer un rôle pour ces récepteurs dans les effets de la morphine. Nous avons de plus démontré que la diminution de l'analgésie produite par la morphine dans ces souris n'est pas causée par un dysfonctionnement des récepteurs opioïdes mu (MOP) ni par une régulation à la baisse de ces récepteurs. Nos résultats confirment l'existence d'interactions fonctionnelles entre les systèmes cannabinoïde et opioïde au niveau périphérique et spinal. Ces observations sont prometteuses d’un point de vue thérapeutique puisqu’une modulation périphérique ciblée des niveaux d’endocannabinoïdes et d’opioïdes endogènes permettrait de produire des effets analgésiques bénéfiques potentiellement synergiques tout en minimisant les effets indésirables associés à l’activation centrale de ces systèmes.

Transcriptional regulation of human mu-opioid receptor gene: functional characterization of activating and inhibitory transcription factors

The organization of the nervous and immune systems is characterized by obvious differences and striking parallels. Both systems need to relay information across very short and very long distances. The nervous system communicates over both long and short ranges primarily by means of more or less hardwired intercellular connections, consisting of axons, dendrites, and synapses. Longrange communication in the immune system occurs mainly via the ordered and guided migration of immune cells and systemically acting soluble factors such as antibodies, cytokines, and chemokines. Its short-range communication either is mediated by locally acting soluble factors or transpires during direct cell–cell contact across specialized areas called “immunological synapses” (Kirschensteiner et al., 2003). These parallels in intercellular communication are complemented by a complex array of factors that induce cell growth and differentiation: these factors in the immune system are called cytokines; in the nervous system, they are called neurotrophic factors. Neither the cytokines nor the neurotrophic factors appear to be completely exclusive to either system (Neumann et al., 2002). In particular, mounting evidence indicates that some of the most potent members of the neurotrophin family, for example, nerve growth factor (NGF) and brainderived neurotrophic factor (BDNF), act on or are produced by immune cells (Kerschensteiner et al., 1999) There are, however, other neurotrophic factors, for example the insulin-like growth factor-1 (IGF-1), that can behave similarly (Kermer et al., 2000). These factors may allow the two systems to “cross-talk” and eventually may provide a molecular explanation for the reports that inflammation after central nervous system (CNS) injury has beneficial effects (Moalem et al., 1999). In order to shed some more light on such a cross-talk, therefore, transcription factors modulating mu-opioid receptor (MOPr) expression in neurons and immune cells are here investigated. More precisely, I focused my attention on IGF-I modulation of MOPr in neurons and T-cell receptor induction of MOPr expression in T-lymphocytes. Three different opioid receptors [mu (MOPr), delta (DOPr), and kappa (KOPr)] belonging to the G-protein coupled receptor super-family have been cloned. They are activated by structurallyrelated exogenous opioids or endogenous opioid peptides, and contribute to the regulation of several functions including pain transmission, respiration, cardiac and gastrointestinal functions, and immune response (Zollner and Stein 2007). MOPr is expressed mainly in the central nervous system where it regulates morphine-induced analgesia, tolerance and dependence (Mayer and Hollt 2006). Recently, induction of MOPr expression in different immune cells induced by cytokines has been reported (Kraus et al., 2001; Kraus et al., 2003). The human mu-opioid receptor gene (OPRM1) promoter is of the TATA-less type and has clusters of potential binding sites for different transcription factors (Law et al. 2004). Several studies, primarily focused on the upstream region of the OPRM1 promoter, have investigated transcriptional regulation of MOPr expression. Presently, however, it is still not completely clear how positive and negative transcription regulators cooperatively coordinate cellor tissue-specific transcription of the OPRM1 gene, and how specific growth factors influence its expression. IGF-I and its receptors are widely distributed throughout the nervous system during development, and their involvement in neurogenesis has been extensively investigated (Arsenijevic et al. 1998; van Golen and Feldman 2000). As previously mentioned, such neurotrophic factors can be also produced and/or act on immune cells (Kerschenseteiner et al., 2003). Most of the physiologic effects of IGF-I are mediated by the type I IGF surface receptor which, after ligand binding-induced autophosphorylation, associates with specific adaptor proteins and activates different second messengers (Bondy and Cheng 2004). These include: phosphatidylinositol 3-kinase, mitogen-activated protein kinase (Vincent and Feldman 2002; Di Toro et al. 2005) and members of the Janus kinase (JAK)/STAT3 signalling pathway (Zong et al. 2000; Yadav et al. 2005). REST plays a complex role in neuronal cells by differentially repressing target gene expression (Lunyak et al. 2004; Coulson 2005; Ballas and Mandel 2005). REST expression decreases during neurogenesis, but has been detected in the adult rat brain (Palm et al. 1998) and is up-regulated in response to global ischemia (Calderone et al. 2003) and induction of epilepsy (Spencer et al. 2006). Thus, the REST concentration seems to influence its function and the expression of neuronal genes, and may have different effects in embryonic and differentiated neurons (Su et al. 2004; Sun et al. 2005). In a previous study, REST was elevated during the early stages of neural induction by IGF-I in neuroblastoma cells. REST may contribute to the down-regulation of genes not yet required by the differentiation program, but its expression decreases after five days of treatment to allow for the acquisition of neural phenotypes. Di Toro et al. proposed a model in which the extent of neurite outgrowth in differentiating neuroblastoma cells was affected by the disappearance of REST (Di Toro et al. 2005). The human mu-opioid receptor gene (OPRM1) promoter contains a DNA sequence binding the repressor element 1 silencing transcription factor (REST) that is implicated in transcriptional repression. Therefore, in the fist part of this thesis, I investigated whether insulin-like growth factor I (IGF-I), which affects various aspects of neuronal induction and maturation, regulates OPRM1 transcription in neuronal cells in the context of the potential influence of REST. A series of OPRM1-luciferase promoter/reporter constructs were transfected into two neuronal cell models, neuroblastoma-derived SH-SY5Y cells and PC12 cells. In the former, endogenous levels of human mu-opioid receptor (hMOPr) mRNA were evaluated by real-time PCR. IGF-I upregulated OPRM1 transcription in: PC12 cells lacking REST, in SH-SY5Y cells transfected with constructs deficient in the REST DNA binding element, or when REST was down-regulated in retinoic acid-differentiated cells. IGF-I activates the signal transducer and activator of transcription-3 (STAT3) signaling pathway and this transcription factor, binding to the STAT1/3 DNA element located in the promoter, increases OPRM1 transcription. T-cell receptor (TCR) recognizes peptide antigens displayed in the context of the major histocompatibility complex (MHC) and gives rise to a potent as well as branched intracellular signalling that convert naïve T-cells in mature effectors, thus significantly contributing to the genesis of a specific immune response. In the second part of my work I exposed wild type Jurkat CD4+ T-cells to a mixture of CD3 and CD28 antigens in order to fully activate TCR and study whether its signalling influence OPRM1 expression. Results were that TCR engagement determined a significant induction of OPRM1 expression through the activation of transcription factors AP-1, NF-kB and NFAT. Eventually, I investigated MOPr turnover once it has been expressed on T-cells outer membrane. It turned out that DAMGO induced MOPr internalisation and recycling, whereas morphine did not. Overall, from the data collected in this thesis we can conclude that that a reduction in REST is a critical switch enabling IGF-I to up-regulate human MOPr, helping these findings clarify how human MOPr expression is regulated in neuronal cells, and that TCR engagement up-regulates OPRM1 transcription in T-cells. My results that neurotrophic factors a and TCR engagement, as well as it is reported for cytokines, seem to up-regulate OPRM1 in both neurons and immune cells suggest an important role for MOPr as a molecular bridge between neurons and immune cells; therefore, MOPr could play a key role in the cross-talk between immune system and nervous system and in particular in the balance between pro-inflammatory and pro-nociceptive stimuli and analgesic and neuroprotective effects.

Why does low dose morphine augment fentanyl analgesia?

Opioids dominate the field of pain management because of their ability to provide analgesia in many medical circumstances. However, side effects including respiratory depression, constipation, tolerance, physical dependence, and the risk of addiction limit their clinical utility. Fear of these side effects results in the under-treatment of acute pain. For many years, research has focused on ways to improve the therapeutic index (the ratio of desirable analgesic effects to undesirable side effects) of opioids. One strategy, combining opioid agonists that bind to different opioid receptor types, may prove successful.^ We discovered that subcutaneous co-administration of a moderately analgesic dose of the mu-opioid receptor (MOR) selective agonist fentanyl (20μg/kg) with subanalgesic doses of the less MOR-specific agonist morphine (100ng/kg-100μg/kg), augmented acute fentanyl analgesia in rats. Parallel [35S]GTPγS binding studies using naïve rat substantia gelatinosa membrane treated with fentanyl (4μM) and morphine (1nM-1pM) demonstrated a 2-fold increase in total G-protein activation. This correlation between morphine-induced augmentation of fentanyl analgesia and G-protein activation led to our proposal that interactions between MORs and DORs underlie opioid-induced augmentation. We discovered that morphine-induced augmentation of fentanyl analgesia and G-protein activity was mediated by DORs. Adding the DOR-selective antagonist naltrindole (200ng/kg, 40nM) at doses that did not alter the analgesic or G-protein activation of fentanyl, blocked increases in analgesia and G-protein activation induced by fentanyl/morphine combinations. Equivalent doses of the MOR-selective antagonist cyprodime (20ng/kg, 4nM) did not block augmentation. Substitution of the DOR-selective agonist SNC80 for morphine yielded similar results, further supporting our conclusion that interactions between MORs and DORs are responsible for morphine-induced augmentation of fentanyl analgesia and G-protein activation. Confocal microscopy of rat substantia gelatinosa showed that changes in the rate of opioid receptor internalization did not account for these effects.^ In conclusion, fentanyl analgesia augmentation by subanalgesic morphine is mediated by increased G-protein activation resulting from functional interactions between MORs and DORs, not changes in MOR internalization. Additional animal and clinical studies are needed to determine whether side effect incidence changes following opioid co-administration. If side effect incidence decreases or remains unchanged, these findings could have important implications for clinical pain treatment. ^

Postoperative analgesic effects of epidural administration of neostigmine alone or in combination with morphine in ovariohysterectomized dogs

Objective-To evaluate analgesic effects of epidurally administered neostigmine alone or in combination with morphine in dogs after ovariohysterectomy.Animals-40 healthy bitches.Procedures-After acepromazine premedication, anesthesia was induced. Dogs randomly received 1 of the following 4 epidural treatments 30 minutes before ovariohysterectomy (n = 10/group): saline (0.9% NaCl) solution (control), morphine (0.1 mg/kg), neostigmine (10 pg/kg), or morphine-neostigmine (0.1 mg/kg and 10 pg/kg, respectively). Analgesia was assessed for 24 hours after surgery by use of a visual analogue.scale (VAS; scale of 0 to 10) or numeric descriptive scale (NDS; scale of 0 to 24) and by the need for supplemental analgesia (morphine [0.5 mg/kg, IM] administered when VAS was >= 4 or NDS was >= 8).Results-Significantly more control dogs (n = 8) received supplemental analgesia, compared with the number of neostigmine-treated dogs (1); no dogs in the remaining groups received supplemental analgesia. Compared with values for the control dogs, the NDS scores were lower for morphine-neostigmine-treated dogs (from 2 to 6 hours and at 12 hours) and for morphine-treated dogs (all time points). The NDS scores were lower for morphine-treated dogs at 3, 12, and 24 hours, compared with values for neostigmine-treated dogs. The VAS was less sensitive than the NDS for detecting differences among groups.Conclusions and Clinical Relevance-Epidurally administered neostigmine reduced the use of supplemental analgesia after ovariohysterectorny in dogs. However, analgesic effects were less pronounced than for epidurally administered morphine or morphine-neostigmine. Adding neostigmine to epidurally administered morphine did not potentiate opioid-induced analgesia.