ArticleLiterature Review

Role of Receptor Internalization in Opioid Tolerance and Dependence

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Abstract

Agonist-induced mu-opioid receptor (MOPr) internalization has long been suggested to contribute directly to functional receptor desensitization and opioid tolerance. In contrast, recent evidence suggests that opioid receptor internalization could in fact reduce opioid tolerance in vivo, but the mechanisms that are responsible for the internalization-mediated protection against opioid tolerance are controversely discussed. One prevailing hypothesis is, that receptor internalization leads to decreased receptor signaling and therefore to reduced associated compensatory changes in downstream signaling systems that are involved in the development of opioid tolerance. However, numerous studies have demonstrated that desensitized and internalized mu-opioid receptors are rapidly recycled to the cell surface in a reactivated state, thus counteracting receptor desensitization and opioid tolerance. Further studies revealed agonist-selective differences in the ability to induce opioid receptor internalization. Recently it has been demonstrated that the endocytotic efficacies of opioids are negatively correlated to the induced opioid tolerance. Thus, clearer understanding of the role of opioid receptor trafficking in the regulation of opioid tolerance and dependence will help in the treatment of patients suffering from chronic pain or drug dependence.

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... Chronic opioid exposure can also alter function of remaining MOPR by producing a switch in MOPR G-protein coupling from Gi/o to Gs, leading to activation of adenylyl cyclase rather than inhibition (Wang et al., 2005). MOPR activation and subsequent phosphorylation by GPCR kinases can also lead to the recruitment of β-arrestins, which-in conjunction with many other effectors-leads to MOPR receptor desensitization and internalization (Koch and Höllt, 2008;Roeckel et al., 2016;Corder et al., 2017;Derouiche et al., 2020;Massaly et al., 2021). MOPR phosphorylation at sites involved in receptor desensitization and internalization are observed in mice seven days after partial sciatic nerve ligation, a manipulation that produces tolerance to both the analgesic and conditioned reinforcing properties of morphine (Petraschka et al., 2007). ...
... The development of tolerance stems from desensitization of the opioid system and inflammatory immune responses within peripheral and central nervous systems (Zhu et al., 1999;Dumas and Pollack, 2008;Koch and Höllt, 2008;Matsui et al., 2014;Corder et al., 2017;Lueptow et al., 2018;Eidson and Murphy, 2019). Following activation, opioid receptors can be phosphorylated by GPCR kinases, which triggers G-protein uncoupling and binding of β-arrestins (Dumas and Pollack, 2008;Zhou et al., 2021). ...
... β-arrestin pathway signaling causes desensitized receptors to remain inactive at the plasma membrane, facilitates their endocytosis and subsequent degradation or recycling. As such, these cellular mechanisms represent a critical component in facilitating the development of tolerance at multiple levels (Hutchings et al., 1997;Bohn et al., 2000;Koch and Höllt, 2008;Zhou et al., 2021). Biased agonists, that preferentially activate G-protein signaling cascades with minimal β-arrestin pathway activity, have received great interest as therapeutic alternatives with the thought that such ligands may minimize the development of tolerance and other unwanted side-effects (Ballantyne and Chavkin, 2020). ...
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Decades of research advances have established a central role for endogenous opioid systems in regulating reward processing, mood, motivation, learning and memory, gastrointestinal function, and pain relief. Endogenous opioid systems are present ubiquitously throughout the central and peripheral nervous system. They are composed of four families, namely the μ (MOPR), κ (KOPR), δ (DOPR), and nociceptin/orphanin FQ (NOPR) opioid receptors systems. These receptors signal through the action of their endogenous opioid peptides β-endorphins, dynorphins, enkephalins, and nociceptins, respectfully, to maintain homeostasis under normal physiological states. Due to their prominent role in pain regulation, exogenous opioids—primarily targeting the MOPR, have been historically used in medicine as analgesics, but their ability to produce euphoric effects also present high risks for abuse. The ability of pain and opioid use to perturb endogenous opioid system function, particularly within the central nervous system, may increase the likelihood of developing opioid use disorder (OUD). Today, the opioid crisis represents a major social, economic, and public health concern. In this review, we summarize the current state of the literature on the function, expression, pharmacology, and regulation of endogenous opioid systems in pain. Additionally, we discuss the adaptations in the endogenous opioid systems upon use of exogenous opioids which contribute to the development of OUD. Finally, we describe the intricate relationship between pain, endogenous opioid systems, and the proclivity for opioid misuse, as well as potential advances in generating safer and more efficient pain therapies.
... These receptors are expressed by primary sensory neurons including the small myelinated Aδ-fibers or unmyelinated C fibers as well as neurons at supra-spinal sites of the pain pathway (7,8). A growing body of evidence shows that desensitization of mu-opioid receptor and the progressive loss of receptor function under continued exposure to the opioid may underlie morphine tolerance (5,9,10). However, other molecular changes in other neurotransmitter receptors such as N-methyl-D-aspartate (NMDA) receptors and signaling molecules including protein kinases are proposed to be involved in morphine-induced analgesic tolerance (9,(11)(12)(13). ...
... A growing body of evidence shows that desensitization of mu-opioid receptor and the progressive loss of receptor function under continued exposure to the opioid may underlie morphine tolerance (5,9,10). However, other molecular changes in other neurotransmitter receptors such as N-methyl-D-aspartate (NMDA) receptors and signaling molecules including protein kinases are proposed to be involved in morphine-induced analgesic tolerance (9,(11)(12)(13). ...
... Receptor phosphorylation is thought to be a key initial event for acute mu-opioid receptor desensitization (9). It is shown that several kinases can phosphorylate mu-opioid receptors in response to agonist activation and modulate their activity (21). ...
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Backgrounds: Calcium/calmodulin-dependent protein kinase IIα (CaMKIIα) may modulate function of mu-opioid receptors by phosphorylation and therefore, be involved in development of morphine-induced analgesic tolerance. Objective: We aimed to examine changes in gene expression of CamKIIα in the lumbosacral cord and midbrain during induction of morphine analgesic tolerance. Materials and Methods: Male Wistar rats weighing 250-300g were used. Two groups of rats (n=6 per group) received saline (1 ml/kg) or morphine (10 mg/ml/kg) twice-daily for eight days, and induction of morphine analgesic tolerance was assessed using a hotplate test on days 1, 4 and 8 of the injections. The lumbosacral spinal cord and midbrain were also dissected in six independent groups (n=4 per group) on days 1, 4 and 8 of saline or morphine injections to examine changes in gene expression of CamKIIα with a semi-quantitative RT-PCR method. Results: The result of the hotplate test showed that those rats receiving repeated injections of morphine developed tolerance and exhibited significant decreases in antinociception on days 4 and 8 of the injections compared to that of day one (P<0.001). The result of gene expression in the lumbosacral cord showed that there were no significant changes in the CamKIIα gene expression on days 1 and 8 but its expression was significantly increased by 102 % on day 4 of the injections (P<0.01). In addition, the CamKIIα gene expression in the midbrain showed no significant changes on days 1 and 4 but it was significantly decreased by 67 % on day 8 of morphine injections (P<0.01). Conclusion: It can be concluded that changes in the CamKIIα gene expression in the lumbosacral cord and midbrain during repeated injections of morphine may differently be associated with induction of morphine tolerance. Keywords: Gene expression, Drug tolerance, Analgesia, Spinal cord, Mesencephalon
... The exact underlying molecular mechanisms responsible for tolerance has remained to be completely understood. A growing body of evidence shows that µ-opioid receptor desensitization and progressive loss of receptor function under continued exposure to the opioid may underlie morphine tolerance (Martini and Whistler, 2007;Koch and Hollt, 2008 phosphorylation is thought to be a key initial event for acute µ-opioid receptor desensitization (Koch and Hollt, 2008). In addition, tolerance is associated with the development of abnormal pain sensitivity such as hyperalgesia (Chen et al., 2007;Chen et al., 2008). ...
... The exact underlying molecular mechanisms responsible for tolerance has remained to be completely understood. A growing body of evidence shows that µ-opioid receptor desensitization and progressive loss of receptor function under continued exposure to the opioid may underlie morphine tolerance (Martini and Whistler, 2007;Koch and Hollt, 2008 phosphorylation is thought to be a key initial event for acute µ-opioid receptor desensitization (Koch and Hollt, 2008). In addition, tolerance is associated with the development of abnormal pain sensitivity such as hyperalgesia (Chen et al., 2007;Chen et al., 2008). ...
... The exact underlying molecular mechanisms responsible for tolerance has remained to be completely understood. A growing body of evidence shows that µ-opioid receptor desensitization and progressive loss of receptor function under continued exposure to the opioid may underlie morphine tolerance (Martini and Whistler, 2007;Koch and Hollt, 2008;Williams et al., 2013). Receptor phosphorylation is thought to be a key initial event for acute µ-opioid receptor desensitization (Koch and Hollt, 2008). ...
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Transient receptor potential vanilloid type 1 (TRPV1) and protein kinase γ (PKCγ) are involved in sensitization/desensitization to noxious stimuli. We aimed to examine gene expression of TRPV1 and PKCγ in rat lumbosacral cord and midbrain on days 1, 4 and 8 of induction of morphine analgesic tolerance. Two groups of male Wistar rats received twice daily saline (1 ml/kg) or morphine (10 mg/kg) for eight days and were monitored for analgesic tolerance with a hotplate test on days 1, 4 and 8 of the injections. Six independent groups in three sets were also treated with saline or morphine, decapitated on days 1, 4 or 8 of the schedule, respectively and their lumbosacral cord and midbrain were dissected. The result of the hotplate test revealed induction of analgesic tolerance on days 4 and 8 of morphine injections. The TRPV1 gene expression in the lumbosacral cord was significantly increased only on day 4 of morphine injections but the PKCγ gene expression remained with no significant changes on days 1, 4 and 8 of the injections. In the midbrain, the TRPV1 gene expression was significantly increased only on day 1 of morphine injections; however, the PKCγ gene expression was significantly increased on days 4 and 8 of morphine injections. It can be concluded that the TRPV1 gene expression in the lumbosacral cord and midbrain is associated with early phase of morphine-induced analgesic tolerance but the PKCγ gene expression is altered only in midbrain at later phase of the process.
... The exact underlying molecular mechanisms responsible for tolerance has remained to be completely understood. A growing body of evidence shows that µ-opioid receptor desensitization and progressive loss of receptor function under continued exposure to the opioid may underlie morphine tolerance (Martini and Whistler, 2007;Koch and Hollt, 2008 phosphorylation is thought to be a key initial event for acute µ-opioid receptor desensitization (Koch and Hollt, 2008). In addition, tolerance is associated with the development of abnormal pain sensitivity such as hyperalgesia (Chen et al., 2007;Chen et al., 2008). ...
... The exact underlying molecular mechanisms responsible for tolerance has remained to be completely understood. A growing body of evidence shows that µ-opioid receptor desensitization and progressive loss of receptor function under continued exposure to the opioid may underlie morphine tolerance (Martini and Whistler, 2007;Koch and Hollt, 2008 phosphorylation is thought to be a key initial event for acute µ-opioid receptor desensitization (Koch and Hollt, 2008). In addition, tolerance is associated with the development of abnormal pain sensitivity such as hyperalgesia (Chen et al., 2007;Chen et al., 2008). ...
... The exact underlying molecular mechanisms responsible for tolerance has remained to be completely understood. A growing body of evidence shows that µ-opioid receptor desensitization and progressive loss of receptor function under continued exposure to the opioid may underlie morphine tolerance (Martini and Whistler, 2007;Koch and Hollt, 2008;Williams et al., 2013). Receptor phosphorylation is thought to be a key initial event for acute µ-opioid receptor desensitization (Koch and Hollt, 2008). ...
Article
Introduction: Transient receptor potential vanilloid type 1 (TRPV1) and protein kinase Cγ (PKCγ) are involved in sensitization/desensitization to noxious stimuli. We aimed to examine the gene expression levels of TRPV1 and PKCγ in rat lumbosacral cord and midbrain on days 1, 4 and 8 of induction of morphine analgesic tolerance. Methods: Two groups of male Wistar rats received twice daily saline (1 ml/kg) or morphine (10 mg/kg) for eight days and were monitored for analgesic tolerance with a hotplate test on days 1, 4 and 8 of the injections. Six independent groups in three sets were also treated with saline or morphine, decapitated on days 1, 4 or 8 of the schedule, respectively and their lumbosacral cord and midbrain were dissected. Results: The result of the hotplate test revealed induction of analgesic tolerance on days 4 and 8 of morphine injections. The TRPV1 gene expression in the lumbosacral cord was significantly increased only on day 4 of morphine injections, but the PKCγ gene expression remained with no significant changes on days 1, 4 and 8. In the midbrain, the TRPV1 gene expression was significantly increased only on day 1; however, the PKCγ gene expression was significantly increased on days 4 and 8 of morphine injections. Conclusion: It can be concluded that the TRPV1 gene expression changes in the lumbosacral cord and midbrain is associated with early phase of morphine-induced analgesic tolerance but the PKCγ gene expression is altered only in midbrain at the later phase of process. © 2016, Iranian Society of Physiology and Pharmacology. All rights reserved.
... With long term use, however, physiological dependence and tolerance increase due to internalization of receptors and other adaptive processes [6]. Efficacy of clinical pain control decreases requiring increasing doses of analgesic agents, corresponding to increases in side effect profiles [6]. ...
... With long term use, however, physiological dependence and tolerance increase due to internalization of receptors and other adaptive processes [6]. Efficacy of clinical pain control decreases requiring increasing doses of analgesic agents, corresponding to increases in side effect profiles [6]. The range of side effects include respiratory depression, constipation, cognitive dysfunction, and psychiatric comorbidities such as anxiety and depression [7][8][9][10]. ...
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As awareness increases about the side effects of opioids and risks of misuse, opioid use and appropriate weaning of opioid therapies have become topics of significant clinical relevance among pediatric populations. Critically ill hospitalized neonates, children, and adolescents routinely receive opioids for analgesia and sedation as part of their hospitalization, for both acute and chronic illnesses. Opioids are frequently administered to manage pain symptoms, reduce anxiety and agitation, and diminish physiological stress responses. Opioids are also regularly prescribed to youth with chronic pain. These medications may be prescribed during the initial phase of a diagnostic workup, during an emergency room visit; as an inpatient, or on an outpatient basis. Following treatment for underlying pain conditions, it can be challenging to appropriately wean and discontinue opioid therapies. Weaning opioid therapy requires special expertise and care to avoid symptoms of increased pain, withdrawal, and agitation. To address this challenge, there have been enhanced efforts to implement opioid-reduction during pharmacological therapies for pediatric pain management. Effective pain management therapies and their outcomes in pediatrics are outside the scope of this paper. The aims of this paper were to: 1) Review the current practice of opioid-reduction during pharmacological therapies; and 2) highlight concrete opioid weaning strategies and management of opioid withdrawal.
... The favorable profile of methadone in terms of reduced tolerance in comparison with other opioids is another key point for the administration of methadone for chronic pain therapy. Some preclinical evidence suggests it has an optimal profile regarding the ability to induce opioid receptor internalization that may explain this clinical phenomenon [18]. Despite its proven efficacy, methadone has a relevant potential for drug interactions and may be associated with serious ADRs, among which is the dose-independent prolongation of the QT interval [19] leading to rare but potentially fatal arrhythmias. ...
... The median VAS (IQR) was 8.5 (8)(9) at T 0 , 5 (3-7) at T 1 , 5 (4-7) at T 2 , 7 (4-8) at T 3 , and 3 (2-6) at T 4 . The median number of pills of rescue medications (IQR) was 95 (34-240) at T 0 , 5 at T 1 , 15 (5-60) at T 2 , 8 (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) at T 3 , and 10 (3-16) at T 4 as tapering of methadone was quick and easy. Altogether, our observations suggest that the optimization of the treatment, including either the association with or a formulation containing methylnaltrexone, which has been shown to be able to counteract the constipation [21], would relevantly increase the persistence on treatment, thus increasing the proportion of patients that might benefit from LDM. ...
Article
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Objectives: A refractory chronic migraine (RCM) accompanied by medication-overuse headache (MOH) is an extremely disabling disease. Evidence suggests that in selected patients, chronic opioids may be a valuable therapeutic option for RCM. The aim of the present study was to evaluate the effectiveness and safety of prophylaxis with low-dose methadone (LDM) in patients affected by RCM with continuous headache and MOH. Methods: A prospective cohort study was performed between May 2012 and November 2015 at the Headache Center and Toxicology Unit of the Careggi University Hospital. Eligible patients were treated with prophylactic LDM and followed up for 12 months. Headache exacerbations, pain intensity, use of rescue medications, and occurrence of adverse drug reactions (ADRs) were recorded. Results: Thirty patients (24 females, median age 48 years) were enrolled. Nineteen (63%) patients dropped out, mainly because of early ADRs (n = 10), including nausea, vomiting, and constipation. At last available follow-up, LDM was associated with a significant decrease in the number of headache attacks/month (from a median of 45 (interquartile range 30-150) to 16 (5-30), p < 0.001), in pain intensity (from 8.5 (8-9) to 5 (3-6), p < 0.001), and in the number of rescue medications consumed per month (from 95 (34-240) to 15 (3-28), p < 0.001). No misuse or diversion cases were observed. Conclusion: LDM could represent a valuable and effective option in selected patients affected by RCM with continuous headache and MOH, although the frequency of early ADRs poses major safety concerns. Randomized controlled trials are needed to confirm the efficacy and safety of LDM prophylaxis.
... Thus, MOR downregulation represents one of many mechanisms contributing to opioid tolerance. In multiple studies, MOR mRNA expression remains unaltered by opioid agonists, which suggests that agonist-selective MOR downregulation occurs post-transcriptionally [129][130][131][132][133]. Let-7 miRNAs can downregulate MOR without affecting its mRNA expression through a mechanism that interferes with translation initiation [134]. Translational repression occurs when cytosolic let-7 miRNAs get incorporated into RISC and its interaction with MOR 3 UTR leads to MOR mRNA recruitment to P-bodies where it becomes degraded by de-capping enzymes and exonucleases [110,134]. ...
... In multiple studies, MOR mRNA expression remains unaltered by opioid agonists, which suggests that agonist-selective MOR downregulation occurs post-transcriptionally [129][130][131][132][133]. Let-7 miRNAs can downregulate MOR without affecting its mRNA expression through a mechanism that interferes with translation initiation [134]. Translational repression occurs when cytosolic let-7 miRNAs get incorporated into RISC and its interaction with MOR 3 UTR leads to MOR mRNA recruitment to P-bodies where it becomes degraded by de-capping enzymes and exonucleases [110,134]. Other mechanisms are likely involved in opioid tolerance besides let-7 downregulation of MOR since knocking down let-7 in the brain only partially reduces morphine antinociceptive tolerance. ...
Article
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Cardiovascular disease (CVD) is a significant cause of morbidity and mortality across the world. A large proportion of CVD deaths are secondary to coronary artery disease (CAD) and myocardial infarction (MI). Even though prevention is the best strategy to reduce risk factors associated with MI, the use of cardioprotective interventions aimed at improving patient outcomes is of great interest. Opioid conditioning has been shown to be effective in reducing myocardial ischemia-reperfusion injury (IRI) and cardiomyocyte death. However, the molecular mechanisms behind these effects are under investigation and could provide the basis for the development of novel therapeutic approaches in the treatment of CVD. Non-coding RNAs (ncRNAs), which are functional RNA molecules that do not translate into proteins, are critical modulators of cardiac gene expression during heart development and disease. Moreover, ncRNAs such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are known to be induced by opioid receptor activation and regulate opioid signaling pathways. Recent advances in experimental and computational tools have accelerated the discovery and functional characterization of ncRNAs. In this study, we review the current understanding of the role of ncRNAs in opioid signaling and opioid-induced cardioprotection.
... Opioids are still the last resort for moderate to severe pain but rapid development of analgesic tolerance (a profound loss of drug potency) remains as a frustrating problem (Ren et al., 2015). There are many hypotheses about how opioid tolerance develops, including, functional decoupling of opioid receptors from G-proteins which leads to receptor desensitization, phosphorylation of opioid receptors, internalization and/or down-regulation of -opioid receptor, upregulation of the cAMP (cyclic adenosine monophosphate) and PKC (protein kinase C) pathways, neuroimmune activation and neuroinflammation, glutamate homeostasis, nitric oxide production, modulatory role of ion channels or neurotrophic factors and activation of glial cells (astrocytes and microglia) at the level of the spinal cord (Ben-Eliyahu et al., 1992;Bian et al., 2012;Dang and Christie, 2010;DeLeo et al., 2004;Fukagawa et al., 2013;Koch and Höllt, 2008;Kolesnikov et al., 1993;Martini and Whistler, 2007;Ossipov et al., 2004;Vacca et al., 2013). ...
Article
The development of morphine-induced antinociceptive tolerance limits its therapeutic efficacy in pain management. Atorvastatin, or competitive inhibitor of 3-hydroxy-methyl-glutaryl coenzyme A (HMG-CoA) reductase, is mainstay agent in hypercholesterolemia treatment. Beyond the cholesterol-lowering activity, exploration of neuroprotective properties of this statin indicates its potential benefit in central nervous disorders. The aim of the present study was to assess the effects of atorvastatin in development and expression of morphine-induced analgesic tolerance in male mice and probable involvement of nitric oxide. Chronic and acute treatment with atorvastatin 10 and 20 mg/kg, respectively, could alleviate morphine tolerance in development and expression phases. Chronic co-administration of nitric oxide synthase (NOS) inhibitors including L-NAME (non selective NOS inhibitor; 2 mg/kg), aminoguanidine (selective inducible NOS inhibitor; 50 mg/kg) and 7-NI (selective neuronal NOS inhibitor; 15 mg/kg) with atorvastatin blocked the protective effect of atorvastatin in tolerance reversal. Moreover, reversing the atorvastatin effect was also observed in acute simultaneous treatment of L-NAME (5 mg/kg) and aminoguanidine (100 mg/kg) with atorvastatin. Co-treatment of guanylyl cyclase inhibitor, ODQ (chronic dose: 10 mg/kg and acute dose: 20 mg/kg) was associated with prevention of atorvastatin anti-tolerance properties. Our results revealed that the atorvastatin modulating role in morphine antinociceptive tolerance is mediated at least in part via nitric oxide in animal pain models of hot plate and tail flick.
... Scale bar: 100 μm phosphorylation of intracellular receptor domains resulting in an uncoupling of G protein signaling and a recruitment of the endocytotic machinery leading to receptor internalization [1,52]. Several studies display a role of different protein kinases, such as PKC or MAPKs, in MOR phosphorylation [27]. Tolerant mice showed increased expression and phosphorylation of total PKC, and of PKCγ and PKCε, the isoforms mainly involved in morphine analgesic tolerance [45], that was attenuated by treatment with DAPT. ...
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The development of analgesic tolerance to opioids is an important limitation in the management of chronic pain. Spinal cord glial cell activation appears to play a pivotal role in the development and maintenance of opioid tolerance, indicating the presence of an opioid-induced neuronal–glial interaction; however, how opioids drive this cross-talk is still elusive. In search of treatments to attenuate morphine analgesic tolerance, our research focused on the role of Notch signaling pathway, one of the most important mechanisms of cell-to-cell interactions, in the spinal dorsal horn after morphine repeated exposure and whether Notch inhibition attenuates morphine analgesic tolerance. Double immunofluorescence experiments on spinal sections from morphine-tolerant mice showed a neuronal localization of Notch-1 receptor whereas the Notch ligand Jagged was localized on neighboring astrocytes. Morphine-induced μ opioid receptor (MOR) stimulation triggered Notch-1 signaling activation and this event was mediated by astrocyte JNK activation. Notch-1 activation selectively reduced the expression of histone deacetylase (HDAC)-1, resulting in an overphosphorylation of PKC and ERK, kinases involved in MOR phosphorylation and internalization after repeated morphine exposure. Notch-1 signaling inhibition, through intrathecal administration of the γ-secretase inhibitor, DAPT, counteracted PKC and ERK overphosphorylation, MOR internalization, and analgesic tolerance. Conversely, the HDAC-1 inhibitor, LG325, further aggravated MOR internalization, PKC overphosphorylation, and analgesic tolerance. Our findings implicate the MOR-triggered Notch-1 signaling in promoting MOR internalization and morphine analgesic tolerance by epigenetic regulation mechanisms. These data suggest that Notch-1 inhibitors could represent an innovative therapeutic perspective for the management of opioid tolerance in chronic pain therapy.
... Cocaine dependence is positively correlated with MOR expression in the cingulate, frontal and temporal cortex, caudate, and thalamic areas of the reward circuit [17], and alcohol dependence is positively correlated with MOR expression in the ventral striatum and nucleus accumbens [18]. The effects of opioid use on MOR expression is inconclusive, with some studies showing downregulation and others upregulation in these reward areas [19]. Moreover, the MOR is present in nearly all brain areas associated with feeding, including those involved in both the energy regulation and reward aspects of food consumption [20]. ...
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Roux-en-Y gastric bypass surgery (RYGB) is the most common and effective weight loss procedure for severe obesity. However, a significant increase in addictive behaviors and new-onset substance use disorder (SUD) are sometimes observed post-surgery. The endogenous opioid system is known to play a major role in motivated behavior and reward, as well as the abuse of substances, including alcohol, tobacco, opioids and highly palatable foods. Here, we examined the effects of RYGB on mu-opioid receptor levels in the brain. Male Sprague-Dawley rats were assigned to one of four groups: standard diet with sham surgery (control), ad libitum high-energy high-fat (HF) diet with sham surgery, calorie restricted HF diet with sham surgery (Sham-FR), or HF diet with RYGB surgery. Control and HF groups were fed their respective diets for 8 weeks, with surgery performed on the eighth week. After 9 weeks on their respective diets post-surgery, animals were sacrificed for mu-opioid receptor autoradiography using the [³H] [D-Ala2,N-Me-Phe4-Gly5-ol]- enkephalin (DAMGO) ligand. Rats with RYGB showed reduced DAMGO binding in the central amygdala compared to sham-operated HF diet controls, and in the hypothalamus compared to high-fat fed Sham-FR. Diet alone did not change [³H] DAMGO binding in any region. These findings show that RYGB surgery, independent of diet or caloric restriction, decreases mu opioid signaling in specific regions important for stress and energy regulation. Thus, RYGB surgery may lead to greater stress sensitivity via downregulated mu opioid signaling in the central amygdala, which may contribute to the observed increased risk in some subjects for addictive behavior.
... Micro-opioid receptors (MOPr) are a class of opioid receptors belonging to superfamily of seven transmembrane helix receptors. Activation of opioid receptors causes neuronal inhibition via multiple downstream effectors (Koch and Höllt, 2008). It has been shown that the agonist D-Ala2, Me Phe4, Glyol5-enkephalin (DAMGO) induced activation of MOPr also causes activation of PLD2 in an ARF dependent manner (Haberstock-Debic et al., 2003;Koch et al., 2003;Rankovic et al., 2009). ...
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Phosphatidic acid (PA) is a simple glycerophospholipid with a well-established role as an intermediate in phospholipid biosynthesis. In addition to its role in lipid biosynthesis, PA has been proposed to act as a signaling molecule that modulates several aspects of cell biology including membrane transport. PA can be generated in eukaryotic cells by several enzymes whose activity is regulated in the context of signal transduction and enzymes that can metabolize PA thus terminating its signaling activity have also been described. Further, several studies have identified PA binding proteins and changes in their activity are proposed to be mediators of the signaling activity of this lipid. Together these enzymes and proteins constitute a PA signaling toolkit that mediates the signaling functions of PA in cells. Recently, a number of novel genetic models for the analysis of PA function in vivo and analytical methods to quantify PA levels in cells have been developed and promise to enhance our understanding of PA functions. Studies of several elements of the PA signaling toolkit in a single cell type have been performed and are presented to provide a perspective on our understanding of the biochemical and functional organization of pools of PA in a eukaryotic cell. Finally, we also provide a perspective on the potential role of PA in human disease, synthesizing studies from model organisms, human disease genetics and analysis using recently developed PLD inhibitors.
... When delivered acutely, opioid agonists inhibit adenylyl cyclase (AC) activity [47], as observed here for Adcy5. Conversely, a chronic opioid treatment increases AC levels, inducing a superactivation of cAMP which leads to opioid tolerance and dependence [48]. After binding of the agonists, opioid receptors go through a phosphorylation process triggered by G protein-coupled receptor kinases (GRK) and second messenger-regulated kinases (PKC, PKA, CaMK-protein kinases C and A and Ca 2+ /calmodulin-dependent protein kinases). ...
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The impact of vitamin D on sensory function, including pain processing, has been receiving increasing attention. Indeed, vitamin D deficiency is associated with various chronic pain conditions, and several lines of evidence indicate that vitamin D supplementation may trigger pain relief. However, the underlying mechanisms of action remain poorly understood. We used inflammatory and non-inflammatory rat models of chronic pain to evaluate the benefits of vitamin D3 (cholecalciferol) on pain symptoms. We found that cholecalciferol supplementation improved mechanical nociceptive thresholds in monoarthritic animals and reduced mechanical hyperalgesia and cold allodynia in a model of mononeuropathy. Transcriptomic analysis of cerebrum, dorsal root ganglia, and spinal cord tissues indicate that cholecalciferol supplementation induces a massive gene dysregulation which, in the cerebrum, is associated with opioid signaling (23 genes), nociception (14), and allodynia (8), and, in the dorsal root ganglia, with axonal guidance (37 genes) and nociception (17). Among the identified cerebral dysregulated nociception-, allodynia-, and opioid-associated genes, 21 can be associated with vitamin D metabolism. However, it appears that their expression is modulated by intermediate regulators such as diverse protein kinases and not, as expected, by the vitamin D receptor. Overall, several genes—Oxt, Pdyn, Penk, Pomc, Pth, Tac1, and Tgfb1—encoding for peptides/hormones stand out as top candidates to explain the therapeutic benefit of vitamin D3 supplementation. Further studies are now warranted to detail the precise mechanisms of action but also the most favorable doses and time windows for pain relief.
... But prolonged exposure to these drugs is limited due to the rapid development of tolerance and dependence. Numerous molecular mechanisms have been suggested for these undesirable effects of morphine, including desensitization of μopioid receptor via receptor internalization, uncoupling from G-protein, up-regulation of cAMP pathway, activation of N-methyl D-aspartate (NMDA) system, central neuroimmune activation and NOS activation (Bailey and Connor, 2005;Ben-Eliyahu et al., 1992;Bian et al., 2012;Dang and Christie, 2012;Deleo et al., 2004;DuPen et al., 2007;Koch and Höllt, 2008;Kolesnikov et al., 1993;Koppert, 2007;Martini and Whistler, 2007;Ossipov et al., 2004). A great body of evidence suggests the NO pathway as a proven mechanism involved in morphine-induced tolerance/dependence phenomena. ...
... De même, l'inhibition de la ß-arrestine 2 augmente l'effet analgésique de la morphine tout en réduisant la dépression respiratoire et la constipation qu'elle produit (Raehal and Bohn 2005). Au niveau du récepteur, un défaut de couplage avec la protéine G, une accélération de la phosphorylation par différentes kinases (GRK, PKC, PKA, PI3K) ou par des protéines régulatrices comme les RGS (regulators of G protein signaling), un défaut d'internalisation ou un défaut de recyclage impliquant le recrutement de de la ß-arrestine 2, ont été évoqués même s'il n'y a pas toujours de parallélisme évident entre défaut d'internalisation et tolérance (Jim and Wang 2006;Koch and Höllt 2008;Simonnet and Laulin 2001). L'utilisation d'un agoniste qui favorise l'internalisation a permis de réduire la tolérance induite par des injections répétées de morphine chez la souris (Posa et al. 2016). ...
Thesis
L’utilisation des opioïdes est à l’origine de phénomènes de tolérance et d’hyperalgésie induite (HIO) aussi bien chez l’animal qu’en utilisation clinique. Ces phénomènes surviennent avec tous les opioïdes de manière dose-dépendante. Les mécanismes qui les sous-tendent sont complexes et imparfaitement connus. Le rémifentanil et le sufentanil sont les opioïdes les plus utilisés en France en anesthésie-réanimation. Leur utilisation s’accompagne d’une HIO qui majore la douleur postopératoire et peut être responsable de la persistance de la douleur à long terme. La perception des stimuli nociceptifs chez un patient sous anesthésie générale n’est pas aisée et repose encore sur des signes cliniques indirects d’activation du système sympathique. Ces signes peu sensibles et peu spécifiques conduisent à sous doser ou sur-doser les patients en opioïdes. Récemment, un nouvel outil de monitorage de la nociception est apparu, l’analgesia nociception index (ANI). L’ANI reflète le tonus parasympathique et de ce fait permettrait aux anesthésistes de mieux adapter le dosage des opioïdes. Dans cette thèse, nous avons d’abord évalué la sensibilité et la spécificité de l’ANI à détecter les stimuli nociceptifs, puis montré qu’elles étaient supérieures à celles des signes cliniques, et supérieures à d’autres indices de monitorage proposés. Ensuite nous avons validé la capacité de l’ANI à guider l’analgésie peropératoire du rémifentanil dans différentes situations.Sur le plan expérimental, nous avons exploré, après une exposition courte ou prolongée à différentes doses de rémifentanil et de sufentanil, les mécanismes associés à l’hyperalgésie thermique in vivo, chez la souris, et ex vivo, sur la voie des MAP kinases ERK1/2 et sur le trafic membranaire des récepteurs opioïdes de type µ (MOR) dans différentes cultures cellulaires. Chez la souris, nous avons mis en évidence une hyperalgésie précoce au saut sur plaque chaude, après exposition aux doses les plus élevées de rémifentanil, mais pas avec le sufentanil. De plus, nous n’avons pas observé d’HIO sur le léchage des pattes.Sur les cultures cellulaires, le rémifentanil comme le sufentanil activent la voie des MAPK ERK1/2 lors d’une exposition courte, avec apparition d’une désensibilisation lorsque l’exposition se prolonge. Le rémifentanil comme le sufentanil induisent une internalisation précoce et progressive des récepteurs MOR.
... The molecular mechanisms underlying opioid tolerance are not fully understood. A well-accepted view is that opioid tolerance is associated with opioid receptor phosphorylation, desensitization, and endocytosis [52][53][54]. Since mGluR2 deletion produced enhanced opioid analgesia, it is unlikely that mGluR2 regulates mu-opioid receptor internalization processes that lead to tolerance to opioid analgesia. ...
Article
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Opioid abuse is a rapidly growing public health crisis in the USA. Despite extensive research in the past decades, little is known about the etiology of opioid addiction or the neurobiological risk factors that increase vulnerability to opioid use and abuse. Recent studies suggest that the type 2 metabotropic glutamate receptor (mGluR2) is critically involved in substance abuse and addiction. In the present study, we evaluated whether low-mGluR2 expression may represent a risk factor for the development of opioid abuse and addiction using transgenic mGluR2-knockout (mGluR2-KO) rats. Compared to wild-type controls, mGluR2-KO rats exhibited higher nucleus accumbens (NAc) dopamine (DA) and locomotor responses to heroin, higher heroin self-administration and heroin intake, more potent morphine-induced analgesia and more severe naloxone-precipitated withdrawal symptoms. In contrast, mGluR2-KO rats displayed lower motivation for heroin self-administration under high price progressive-ratio (PR) reinforcement conditions. Taken together, these findings suggest that mGluR2 may play an inhibitory role in opioid action, such that deletion of this receptor results in an increase in brain DA responses to heroin and in acute opioid reward and analgesia. Low-mGluR2 expression in the brain may therefore be a risk factor for the initial development of opioid abuse and addiction.
... In recent years, there have been substantial efforts to understand the molecular regulation of desensitization and internalization of MOR [4,[59][60][61][62][63]. Studies have shown that the MOR agonists fentanyl and DAMGO are more efficacious at recruiting β-arrestin to the receptor [64,65], subsequently causing internalization of the receptor, when compared to morphine, which shows delayed recruitment of β-arrestin and a slower internalization [66][67][68]. ...
... But prolonged exposure to these drugs is limited due to the rapid development of tolerance and dependence. Numerous molecular mechanisms have been suggested for these undesirable effects of morphine, including desensitization of μopioid receptor via receptor internalization, uncoupling from G-protein, up-regulation of cAMP pathway, activation of N-methyl D-aspartate (NMDA) system, central neuroimmune activation and NOS activation (Bailey and Connor, 2005;Ben-Eliyahu et al., 1992;Bian et al., 2012;Dang and Christie, 2012;Deleo et al., 2004;DuPen et al., 2007;Koch and Höllt, 2008;Kolesnikov et al., 1993;Koppert, 2007;Martini and Whistler, 2007;Ossipov et al., 2004). A great body of evidence suggests the NO pathway as a proven mechanism involved in morphine-induced tolerance/dependence phenomena. ...
... Tolerance to opioids and other drugs is defined as a state of adaptation where the effect of drug is diminished with time after prolong exposure. Different mechanisms are held responsible such as m-opioids receptor internalization and desensitization, upregulation of cAMP, protein kinase C (PKC), activation of glutamate, neuroimmune or nitric oxide system, activation of neurotropic factors or glial cells and or ion channels at the central or peripheral level of nervous system [32][33][34][35][36][37][38][39][40][41][42]. Several researcher recommend that those drugs which interact with proinflammatory cytokine's and their respective pathway attenuate the development of morphine tolerance [43,44]. ...
Article
Morphine is a μ-opioid analgesic drug which is used in the treatment and management of chronic pain. However, due to development of antinociceptive tolerance its clinical use is limited. Thalidomide is an old glutamic acid derivative which recently reemerged because of its potential to counteract a number of disorders including neurodegenerative disorders. The potential underlying mechanisms and effects of thalidomide on morphine-induced antinociceptive tolerance is still elusive. Hence, the present study was designed to explore the effect of thalidomide on the development and expression of morphine antinociceptive tolerance targeting l-arginine-nitric oxide (NO) pathway in mice and T98G human glioblastoma cell line. When thalidomide was administered in a dose of 17.5 mg/kg before each dose of morphine chronically for 5 days it prevented the development of antinociceptive tolerance. Also, a single dose of thalidomide 20 mg/kg attenuated the expression phase of antinociceptive tolerance. The protective effect of thalidomide was augmented in development phase when co-administration with NOS inhibitors like L-NAME (non- selective NOS inhibitor; 2 mg/kg) or aminoguanidine (selective inducible NOS inhibitor; 50 mg/kg). Also, the reversal effect of thalidomide in expression phase was potentiated when concomitantly administrated with L-NAME (5 mg/kg) or aminoguanidine (100 mg/kg). Co-administration of ODQ (a guanylyl cyclase inhibitor) 10 mg/kg in developmental phase or 20 mg/kg in expression phase also progressively increased the pain threshold. In addition, thalidomide (20 μM) also significantly inhibited the overexpression of iNOS gene induced by morphine (2.5 μM) in T98G cell line. Hence, our findings suggest that thalidomide has protective effect both in the development and expression phases of morphine antinociceptive tolerance. It is also evident that this effect of thalidomide is induced by the inhibition of NOS enzyme predominantly iNOS.
... Since physical symptoms of tolerance and withdrawal are an expected outcome of long-term opioid therapy (Hojsted et al., 2010;Koch & Hollt, 2008;O'Brien, Volkow, & Li, 2006;Portenoy, 1990) and since items addressing this issue are unique to AUDADIS-IV based criteria, we simulated omission of these physical-based criteria and calculated rates of problematic use with and without them. Following this criteria alternation we repeated the evaluations of agreement using kappa statistic between the three questionnaires and the altered AUDADIS-IV based diagnostic criteria. ...
Article
Prescription opioid medications are commonly used for the treatment of chronic pain. Assessments of problematic opioid use among pain patients are inconsistent across studies, partially due to differences between various measures. Therefore, the most appropriate measure to use is often unclear. In this study we assessed problematic opioid use in a sample of 551 individuals receiving treatment for chronic pain, using three questionnaires: the Alcohol Use Disorder and Associated Disabilities Interview Schedule – Fourth Edition (AUDADIS-IV), the Current Opioid Misuse Measure (COMM) questionnaire and Portenoy's criteria (PC). These questionnaires yielded discordant positive rates of problematic use: 52.6%, 28.7%, and 17.1%, respectively, which did not change substantially when excluding AUDADIS-IV criteria of physical symptoms of tolerance and withdrawal. Although these three questionnaires share some statistically correlated content-based congruent questions, positive response rates to them were significantly different based on construction features, including questionnaires' referred time-frame, wording of questions and response alternatives. The findings of the present study illustrate strengths and limitations of the AUDADIS-IV, COMM and PC in diagnosing problematic opioid use in a population of adults suffering from chronic pain, and highlight the importance of recognizing and addressing specific questionnaire and question-related differences when identifying problematic opioid use in this population.
... Opioid receptors have been classified into three subtypes; m, δ and κ based upon a characteristic pharmacology using highly selective ligands (Yu et al., 2003;Al-Hasani and Bruchas, 2011). Mu-opioid receptor (MOR) is one of the opioid receptor subtypes that belong to the G protein-coupled receptor super family (Law et al., 2000;Koch and Hollt, 2008;Gretton and Droney, 2014;). It has high affinity of binding with morphine and other opioid peptides and is critical for the rewarding effects of heroin and morphine (Kosten and George, 2002;Yu et al., 2003, McDonald andLambert, 2005). ...
Article
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Morphine and Thymoquinone (TQ) are both opioid receptor stimulating compounds although they have different pharmacological origins. Morphine is a natural opioid derivative whereas TQ is one of the main pharmacologically active compounds from Nigella sativa oils. Recently, a great deal of attention had been focused on the potential roles of TQ in opioid dependence therapy by focusing on mu-opioid receptor, a primary site of action for morphine's effects. This study was carried out to study the effects of TQ on protein expression of mu-opioid receptors mediated by chronic morphine treatment in opioid receptor expressing cell line (U87 glioblastoma cells). U87 cells was grown in tissue culture flasks with RPMI 1640 medium containing 1 mmol/L L-glutamine, supplemented with 10% (v/v) fetal bovine serum (FBS), and 1% (w/v) penicillin/streptomycin. The cell viability was assessed by the trypan blue dye and manually counted using a haemocytometer. The MTT assay was used to determine the cytotoxic effects of Morphine and TQ. The protein concentration of human mu-opioid receptor (MOR) level in the cells was determined using the Cusabio® ELISA Kit. Data obtained from this assay indicated that 35 µM morphine and 60.9 µM TQ gives maximum MOR protein concentration. Co-treatment of morphine with TQ and methadone increased the MOR protein concentration (*P < 0.05). These finding suggest that TQ could possibly reduce the tolerance and dependence at cellular level by increasing MOR protein concentration. However, it needs to be further confirmed at molecular level and in-vivo study.. KEYWORDS: Morphine, thymoquinone, mu-opioid receptors protein concentration, opioid receptor expressing cell (U87 MG). ABBREVIATIONS: TQ = thymoquinone, MOR = mu-opioid receptor, MG = monoglioma, ELISA = Enzyme-linked immunosorbent assay.
... In recent years, there have been substantial efforts to understand the molecular regulation of desensitization and internalization of MOR [4,[59][60][61][62][63]. Studies have shown that the MOR agonists fentanyl and DAMGO are more efficacious at recruiting β-arrestin to the receptor [64,65], subsequently causing internalization of the receptor, when compared to morphine, which shows delayed recruitment of β-arrestin and a slower internalization [66][67][68]. ...
Article
Full-text available
The interplay between the dopamine (DA) and opioid systems in the brain is known to modulate the additive effects of substances of abuse. On one hand, opioids serve mankind by their analgesic properties, which are mediated via the mu opioid receptor (MOR), a Class A G protein-coupled receptor (GPCR), but on the other hand, they pose a potential threat by causing undesired side effects such as tolerance and dependence, for which the exact molecular mechanism is still unknown. Using human embryonic kidney 293T (HEK 293T) and HeLa cells transfected with MOR and the dopamine D2 receptor (D2R), we demonstrate that these receptors heterodimerize, using an array of biochemical and biophysical techniques such as coimmunoprecipitation (co-IP), bioluminescence resonance energy transfer (BRET1), Fӧrster resonance energy transfer (FRET), and functional complementation of a split luciferase. Furthermore, live cell imaging revealed that D2LR, when coexpressed with MOR, slowed down internalization of MOR, following activation with the MOR agonist [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO).
... However, the currently accepted idea that G protein-biased ligands should not produce tolerance has little evidence to support it and our data suggest that even opioids biased toward G protein might cause tolerance, limiting their utility as analgesics. Previous studies suggest that the development of tolerance may depend not only on long-term adaptations connected with βarr2 function but should be considered as an attribute of a particular ligand (Koch and Höllt, 2008). For example, according to some reports, tolerance to antinociceptive properties of morphine is mediated by c-Jun N-terminal kinase rather than by the action of βarr2 (Kuhar et al., 2015;Marcus et al., 2015;Yuill et al., 2016). ...
Article
Background and purpose: The concept of opioid ligands biased toward the G protein pathway with minimal recruitment of β-arrestin-2 has become a promising approach for the development of novel, efficient and potentially nonaddictive opioid therapeutics. A recently discovered biased μ-opioid receptor agonist, PZM21, was reported to be analgesic and possess reduced side effects. Here, we aimed to further investigate the behavioural and biochemical properties of PZM21. Experimental approach: We evaluated antinociceptive effects of systemic and intrathecal PZM21 administration. Its addiction-like properties were determined using several behavioural approaches: conditioned place preference, locomotor sensitization, precipitated withdrawal and self-administration. Further, we assessed the influence of PZM21 on morphine-induced antinociception, tolerance and reward. Effects of PZM21 on striatal release of monoamines were evaluated using brain microdialysis. Key results: PZM21 caused long-lasting dose-dependent antinociception. It did not induce reward- and reinforcement-related behaviour, however, its repeated administration led to antinociceptive tolerance and naloxone-precipitated withdrawal symptoms. Pretreatment with PZM21 enhanced morphine-induced antinociception and attenuated the expression of morphine reward. In comparison to morphine, PZM21 administration led to moderate release of dopamine and robust release of serotonin in the striatum. Conclusion and implications: PZM21 presents antinociceptive efficacy and does not possess rewarding or reinforcing properties. However, its clinical application may be restricted, as it induces tolerance and withdrawal symptoms. Notably, its ability to diminish morphine reward implicates that PZM21 may be useful in opioid use disorder therapy.
... The pathological mechanisms of tolerance are quite complex and largely unknown and involve the interplay of multiple regulatory mechanisms occurring both at the level of individual opioid-responsive cells and at the level of neuronal circuits [26,27]. Tolerance involves degrees of desensitization and downregulation, depending on the characteristic of the opioid agonist, although receptor downregulation is determinant but not necessary for opioid tolerance [28,29]. The mu-opioid receptors' (MORs) tolerance is not only agonist-dependent, but it also depends on the opioid dose, the route of administration, the duration of the receptors' exposure and the cellular environment in which receptors are expressed [28,30]. ...
Article
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Chronic low back pain (CLBP) due to osteoarthritis represents a therapeutic challenge worldwide. Opioids are extensively used to treat such pain, but the development of tolerance, i.e., less susceptibility to the effects of the opioid, which can result in a need for higher doses to achieve the same analgesic effect, may limit their use. Animal models suggest that ultra-low doses of opioid antagonists combined with opioid agonists can decrease or block the development of opioid tolerance. In this retrospective study, we tested this hypothesis in humans. In 2019, 53 patients suffering from CLBP were treated with either Oxycodone and Naloxone Prolonged Release (27 patients, OXN patients) or Oxycodone Controlled Release (26 patients, OXY patients). The follow-up period lasted 2 years, during which 10 patients discontinued the treatment, 5 out of each group. The remaining 43 patients reached and maintained the targeted pain relief, but at 18 and 24 months, the OXY patients showed a significantly higher oxycodone consumption than OXN patients to reach the same level of pain relief. No cases of respiratory depression or opioid abuse were reported. There were no significant differences in the incidence of adverse effects between the two treatments, except for constipation, more common in OXY patients. From our results, we can affirm that a long-term opioid treatment with oxycodone-naloxone combination, when compared with oxycodone only, may significantly hinder the development of opioid tolerance. We were also able to confirm, in our cohort, the well known positive effect of naloxone in terms of opioid-induced bowel dysfunction incidence reduction.
... An emerging literature identifies a role for NAc D2Rexpressing MSNs in opioid withdrawal [66][67][68]. Morphine tolerance is regulated by reductions in mu-opioid receptor availability at the cell surface, either by transcriptional regulation or internalization of the receptor [69,70]. Studies have reported downregulation of Oprm1 mRNA following chronic experimenterdelivered morphine due to either promoter hypermethylation [71] or an increase in miRNAs that cause degradation of Oprm1 transcript [72]. ...
Article
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Opioid exposure is known to cause transcriptomic changes in the nucleus accumbens (NAc). However, no studies to date have investigated cell type-specific transcriptomic changes associated with volitional opioid taking. Here, we use single nucleus RNA sequencing (snRNAseq) to comprehensively characterize cell type-specific alterations of the NAc transcriptome in rats self-administering morphine. One cohort of male Brown Norway rats was injected with acute morphine (10 mg/kg, i.p.) or saline. A second cohort of rats was allowed to self-administer intravenous morphine (1.0 mg/kg/infusion) for 10 consecutive days. Each morphine-experienced rat was paired with a yoked saline control rat. snRNAseq libraries were generated from NAc punches and used to identify cell type-specific gene expression changes associated with volitional morphine taking. We identified 1106 differentially expressed genes (DEGs) in the acute morphine group, compared to 2453 DEGs in the morphine self-administration group, across 27 distinct cell clusters. Importantly, we identified 1329 DEGs that were specific to morphine self-administration. DEGs were identified in novel clusters of astrocytes, oligodendrocytes, and D1R- and D2R-expressing medium spiny neurons in the NAc. Cell type-specific DEGs included Rgs9 , Celf5 , Oprm1 , and Pde10a . Upregulation of Rgs9 and Celf5 in D2R-expressing neurons was validated by RNAscope. Approximately 85% of all oligodendrocyte DEGs, nearly all of which were associated with morphine taking, were identified in two subtypes. Bioinformatic analyses identified cell type-specific upstream regulatory mechanisms of the observed transcriptome alterations and downstream signaling pathways, including both novel and previously identified molecular pathways. These findings show that volitional morphine taking is associated with distinct cell type-specific transcriptomic changes in the rat NAc and highlight specific striatal cell populations and novel molecular substrates that could be targeted to reduce compulsive opioid taking.
... 88 Opioid tolerance could be developed by opioid receptor phosphorylation and uncoupling upregulation of the cyclic adenosine monophosphate pathway, agonist-selective m-opioid receptor internalization or the activation of NMDA receptors. 77,89,90 Low-dose ketamine, a noncompetitive antagonist of NMDA receptors, may be appropriate in SCD patients with VOC not responsive to standard opioid analgesics. 91 The adjuvants include antihistamines, antidepressants, benzodiazepines, anticonvulsants and so on. ...
Article
Objectives The hallmark of sickle cell disease (SCD) is acute and chronic pain, and the pain dominates the clinical characteristics of SCD patients. Although pharmacological treatments of SCD targeting the disease mechanisms have been improved, many SCD patients suffer from pain. To overcome the pain of the disease, there have been renewed requirements to understand the novel molecular mechanisms of the pain in SCD. Methods We concisely summarized the molecular mechanisms of SCD-related acute and chronic pain, focusing on potential drug targets to treat pain. Results Acute pain of SCD is caused by vaso-occulusive crisis (VOC), impaired oxygen supply or infarction-reperfusion tissue injuries. In VOC, inflammatory cytokines include tryptase activate nociceptors and transient receptor potential vanilloid type 1. In tissue injury, the secondary inflammatory response is triggered and causes further tissue injuries. Tissue injury generates cytokines and pain mediators including bradykinin, and they activate nociceptive afferent nerves and trigger pain. The main causes of chronic pain are from extended hyperalgesia after a VOC and central sensitization. Neuropathic pain could be due to central or peripheral nerve injury, and protein kinase C might be associated with the pain. In central sensitization, neuroplasticity in the brain and the activation of glial cells may be related with the pain. Discussion In this review, we summarized the molecular mechanisms of SCD-related acute and chronic pain. The novel treatments targeting the disease mechanisms would interrupt complications of SCD and reduce the pain of the SCD patients.
... La désensibilisation des récepteurs et la diminution de leur expression pourraient être responsables de cette tolérance cellulaire. Néanmoins, la littérature suggère que le traitement chronique de morphine ne modifie pas l'expression des MOR (Koch & Hollt, 2008). C'est pourquoi de nombreuses études se sont concentrées sur la désensibilisation de ces récepteurs. ...
Thesis
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Les effets de la morphine sont influencés par le sexe. Chez le rongeur, son métabolisme implique sa glucuronidation en morphine-3-glucuronide (M3G). La M3G provoque une hyperalgésie qui pourrait s’opposer aux effets antinociceptifs de la morphine. Nous avons constaté que l’antinociception induite par la morphine est plus forte chez la souris mâle et que la tolérance antinociceptive se développe plus rapidement chez la souris femelle. La M3G possède un effet pronociceptif qui ne semble pas dépendre du sexe. Nous avons quantifié la morphine et la M3G dans le sang et dans des régions cérébrales impliquées dans le contrôle de la douleur après une injection de morphine, et nous avons constaté que les ratios métaboliques M3G/morphine étaient largement supérieurs chez les femelles. Nous avons également observé un métabolisme central de la morphine in vivo. Nous avons conclu que le métabolisme périphérique et central était influencé par le sexe et que le métabolisme central de la morphine pourrait participer aux différences d’antinociception liées au sexe. Cependant, son implication dans la tolérance antinociceptive semble limitée.
... Studies in animal models suggest that the endogenous opioid system has an important role in controlling appetite in animal models [50][51][52][53]. Low MOR levels were detected in the dorsal striatum of patients affected with schizophrenia [12] and in long-term opiate drug users [54,55]. In contrast, high MOR levels in the ventral striatum are associated with alcohol dependence [56,57]; and higher MOR availability in the anterior cingulate and frontal cortex are associated with cocaine dependence [58]. ...
Article
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Ultrastructural, neurochemical, and molecular alterations within the striatum are associated with the onset and progression of Parkinson’s disease (PD). In PD, the dopamine-containing neurons in the substantia nigra pars compacta (SNc) degenerate and reduce dopamine-containing innervations to the striatum. The loss of striatal dopamine is associated with enhanced corticostriatal glutamatergic plasticity at the early stages of PD. However, with disease progression, the glutamatergic corticostriatal white matter tracts (WMTs) also degenerate. We analyzed the levels of Mu opioid receptors (MORs) in the corticostriatal WMTs, as a function of α-Synuclein (α-Syn) toxicity in transgenic mouse brains. Our data show an age-dependent loss of MOR expression levels in the striatum and specifically, within the caudal striatal WMTs in α-Syn tg mouse brains. The loss of MOR expression is associated with degeneration of the myelinated axons that are localized within the corticostriatal WMTs. In brains affected with late stages of PD, we detect evidence confirming the degeneration of myelinated axons within the corticostriatal WMTs. We conclude that loss of corticostriatal MOR expression is associated with degeneration of corticostriatal WMT in α-Syn tg mice, modeling PD.
... Morphine receptors are expressed in both myelinated Aδ-fibres and unmyelinated C fibres and neurons at supra-spinal sites of the pain pathway (Liu et al., 2012). Classical morphine tolerance is due to desensitization of the mu-opioid receptor under continued exposure to the opioid (Koch and Hollt, 2008;Williams et al., 2013). Furthermore, the N-methyl-D-aspartate (NMDA) receptors and signaling molecules including protein kinases are involved in morphine-induced analgesic tolerance (Ahmadi and Rashdi, 2016). ...
Article
Haloperidol is an antipsychotic agent recently described as an antinociceptive drug able to mediate the antagonism of sigma-1 receptors while morphine is an opioid used in the treatment of neuropathic pain. The objectives of this work were to determine the type of interaction generated by the combination of morphine and haloperidol in neuropathic pain induced by chronic constriction injury and to evaluate morphine tolerance and side effects. The antiallodynic and anti-hyperalgesic effects of morphine (0.01-3.16 mg/kg, s.c.) and haloperidol (0.0178-0.1778 mg/kg, s.c.) were determined after single-doses, in monotherapy and combined, using the acetone and von Frey tests, respectively. Evaluations were performed until 10-days postsurgery. Data were processed using “Surface of Synergic Interaction analysis”. The rotarod test was used to evaluate motor coordination, and the constipation test was performed using 5% charcoal. The effects of haloperidol and BD-1063, sigma-1 receptor antagonists, naloxone and PRE-084 (sigma-1 agonist) were determined using the morphine-tolerance model. Morphine (0.0316 mg/kg)+haloperidol (0.0178 mg/kg) was determined to be the optimal combination. Morphine-tolerance was observed on day 5 after 11 administrations, although in animals that received the combination, tolerance was delayed until day 8. PRE-084 and naloxone administered on day 5 in animals treated with the combination resulted in a blockade of its antiallodynic effects. Adverse effects of constipation or motor incoordination were not shown in animals treated with morphine+haloperidol. In conclusion, haloperidol enhances the antinociceptive effects of morphine without significant adverse effects, as it is able to disrupt or delay the morphine-tolerance in neuropathic pain.
... Several mechanisms, including internalization of the m-opioid receptor 7,19 and abnormal activation of the Nmethyl-D-aspartate (NMDA) receptor, leading to central sensitization to innoxious stimuli, have been suggested to underlie OIH development. 20,50 Moreover, opioids are known to induce the release of serotonin (5-hydroxytryptamine ) in the spinal dorsal horn, which contributes to opioid analgesia. ...
Article
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Opioid usage for pain therapy is limited by its undesirable clinical effects, including paradoxical hyperalgesia, also known as opioid-induced hyperalgesia, (OIH). However, the mechanisms associated with the development and maintenance of OIH remain unclear. Here, we investigated the effect of serotonin inhibition by the 5-HT3 receptor antagonist, ondansetron (OND), as well as serotonin deprivation via its synthesis inhibitor para-chlorophenylalanine (PCPA), on mouse OIH models, with particular focus on astrocyte activation. Co-administering of OND and morphine, in combination with serotonin depletion, inhibited mechanical hyperalgesia and astrocyte activation in the spinal dorsal horn of mouse OIH models. Although previous studies have suggested that activation of astrocytes in the spinal dorsal horn is essential for the development and maintenance of OIH, herein, treatment with carbenoxolone (CBX), a gap junction inhibitor that suppresses astrocyte activation, did not ameliorate mechanical hyperalgesia in mouse OIH models. These results indicate that serotonin in the spinal dorsal horn, and activation of the 5-HT3 receptor play essential roles in OIH induced by chronic morphine, while astrocyte activation in the spinal dorsal horn serves as a secondary effect of OIH. Our findings further suggest that serotonergic regulation in the spinal dorsal horn may be a therapeutic target of OIH. Perspective The current study revealed that the descending serotonergic pain-facilitatory system in the spinal dorsal horn is crucial in OIH, and that activation of astrocytes is a secondary phenotype of OIH. Our study offers new therapeutic targets for OIH and may help reduce inappropriate opioid use.
... GPCR endocytosis starts a cascade of intricate signaling events such as activating a second wave of cAMP signaling (51) and is implicated in pathological processes such as opioid addiction (52). While the kinetics of bulk cAMP accumulation during GPCR endocytosis have been characterized (53), the dynamics of cAMP accumulation within the specific microdomains important in this process on May 24, 2021 http://advances.sciencemag.org/ ...
Article
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Growing evidence suggests that many essential intracellular signaling events are compartmentalized within kinetically distinct microdomains in cells. Genetically encoded fluorescent biosensors are powerful tools to dissect compartmentalized signaling, but current approaches to probe these microdomains typically rely on biosensor fusion and overexpression of critical regulatory elements. Here, we present a novel class of biosensors named FluoSTEPs (fluorescent sensors targeted to endogenous proteins) that combine self-complementing split green fluorescent protein, CRISPR-mediated knock-in, and fluorescence resonance energy transfer biosensor technology to probe compartmentalized signaling dynamics in situ. We designed FluoSTEPs for simultaneously highlighting endogenous microdomains and reporting domain-specific, real-time signaling events including kinase activities, guanosine triphosphatase activation, and second messenger dynamics in live cells. A FluoSTEP for 3',5'-cyclic adenosine monophosphate (cAMP) revealed distinct cAMP dynamics within clathrin microdomains in response to stimulation of G protein-coupled receptors, showcasing the utility of FluoSTEPs in probing spatiotemporal regulation within endogenous signaling architectures.
... In contrast, opioid-induced acute analgesic tolerance could be prevented by PKC inhibition (Ueda et al., 2001) or knockdown of cellular FK506 binding protein 12 (FKBP12) (Yan et al., 2014), which leads to MOR1 internalization. One plausible theory underlying these processes is that MOR1 internalization followed by rapid recycling contributes to its functional re-sensitization and counteracts opioid tolerance (Koch and Höllt, 2008). However, MOR1 internalization without rapid recycling would lead to reduced MOR1 cell surface availability and contribute to opioid tolerance. ...
Article
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•ANXA2 is a novel MOR1-interacting protein regulating MOR1 sub-cellular localization.•ANXA2 retains MOR1 in late recycling endosomes after remifentanil exposure.
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Emotions modulate behavioural priorities at the central and peripheral nervous system. Understanding emotions from the perspective of specific neurotransmitter systems is critical, because the central role of affect in multiple psychopathologies, and the role of specific neuroreceptor systems as corresponding drug targets. Here we provide an integrative overview of molecular imaging studies that have targeted the human emotion circuit at the level of specific neuroreceptors and transmitters. We focus specifically on opioid, dopamine and serotonin system given their key role in modulating motivation and emotions, and discuss how they contribute to both healthy and pathological emotions.
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Evidence has shown that downstream signaling by mu opioid receptor (MOR) agonists that recruit β-arrestin2 may lead to the development of tolerance. Also, it has been suggested that opioid receptor desensitization and cyclic AMP overshoot contributes to the development of tolerance and occurrence of withdrawal, respectively. Therefore, studies were conducted with 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(isoquinoline-3-carboxamido)morphinan (NAQ), a MOR selective partial agonist discovered in our laboratory, to characterize its effect on β-arrestin2 recruitment and precipitation of a cyclic AMP overshoot. DAMGO, a MOR full agonist dose-dependently increased β-arrestin2 association with the MOR, whereas NAQ did not. Moreover, NAQ displayed significant, concentration-dependent antagonism of DAMGO-induced β-arrestin2 recruitment. After prolonged morphine treatment of mMOR-CHO cells, there was a significant overshoot of cAMP upon exposure to naloxone, but not NAQ. Moreover, prolonged incubation of mMOR-CHO cells with NAQ did not result in desensitization nor downregulation of the MOR. In functional studies comparing NAQ with nalbuphine in the cAMP inhibition, Ca2+flux and [35S]GTPγS binding assays, NAQ did not show agonism in the Ca2+flux assay but showed partial agonism in the cAMP and [35S]GTPγS assays. Also, NAQ significantly antagonized DAMGO-induced intracellular Ca2+increase. In conclusion, NAQ is a low efficacy MOR modulator that lacks β-arrestin2 recruitment function and does not induce cellular hallmarks of MOR adaptation and fails to precipitate a cellular manifestation of withdrawal in cells pretreated with morphine. These characteristics are desirable if NAQ is pursued for opioid abuse treatment development.
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Over 90% of Chronic pain (CP) patients receive opioids-based treatments which led to a public health crisis with lasting impacts on social and economic wellbeing based on opioid addiction. Opioids act through activation of μ (MOR), δ (DOR) and κ (KOR) opioid receptors which are broadly and differentially distributed throughout the brain. Chronic opioid consumption leads to brain changes such as alterations on neurotransmission, dendritic branching and spine density, as well as an increase in apoptosis. To overcome opioid-related issues, extensive efforts have been made to search for an alternative treatment. Bioactive molecules secreted by Stem Cells, collectively known as secretome, have shown a positive impact in different pain models. However, there is a lack of studies on the role of secretome in modulating opioid receptors. By using cerebral organoids (CeO), a self-organized, functional and multicellular 3D structure that resemble the brain, we were able to identify MOR, DOR and KOR at different stages of maturation. Treatment with secretome increased MOR expression highlighting a possible role in pain signaling mechanisms. Opioid treatments did not impact the expression of neuronal maturation markers but together with secretome, they increased astrogliogenesis. Opioid treated organoids presented higher dopamine secretion recapitulating an important physiological event after opioid exposure. This work demonstrates that CeO is an important model system for the study of opioid signaling with potential implications to the understanding of basic mechanisms related to pain physiology.
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Significance statement: The mechanisms underlying opioid tolerance and susceptibility to opioid addiction remain unclear. The present studies demonstrate that a single-point mutation at the T394 phosphorylation site in the C-terminal of mu opioid receptor (MOR) results in loss of opioid tolerance and enhanced vulnerability to heroin self-administration. These findings suggest that modulation of the MOR-T394 phosphorylation or dephosphorylation status may have therapeutic potential in management of pain, opioid tolerance, and opioid abuse and addiction. Accordingly, MOR-T394 mutation or polymorphisms could be a risk factor in developing opioid abuse and addiction and therefore be used as a new biomarker in prediction and prevention of opioid abuse and addiction.
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The use of opioids in pain management is hampered by the emergence of analgesic tolerance, which leads to increased dosing and side effects, both of which have contributed to the opioid epidemic. One promising potential approach to limit opioid analgesic tolerance is activating the CNS endocannabinoid system, via activation of CB1 receptors (CB1Rs) in the descending pain inhibitory pathway. In this review, we first discuss preclinical and clinical evidence revealing the potential of pharmacological activation of CB1Rs in modulating opioid tolerance, including activation by phytocannabinoids, synthetic CB1R agonists, endocannabinoid degradation enzyme inhibitors, and recently discovered CB1R positive allosteric modulators. On the other hand, as nonpharmacological pain relief is advocated by the US‐NIH to combat the opioid epidemic, we also discuss contributions of peripheral neuromodulation, involving the electrostimulation of peripheral nerves, in addressing chronic pain and opioid tolerance. The involvement of supraspinal endocannabinoid systems in peripheral neuromodulation‐induced analgesia is also discussed.
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Functional selectivity at the μ opioid receptor (μR), a prototypical GPCR that is a physiologically relevant target for endogenous opioid neurotransmitters and analgesics, has been a major focus for drug discovery in the recent past. The cellular mechanisms that mediate functional selectivity, however, are still being fully elucidated. The present work tested the hypothesis that lifetimes of agonist-induced receptor-arrestin clusters at the cell surface controls the magnitude of arrestin signaling, and therefore functional selectivity, on μR. We show that endomorphin-2 (EM2), an arrestin-biased ligand for μR, lengthens surface lifetimes of receptor-arrestin clusters significantly compared to morphine. The lengthening of lifetimes required two specific leucines on the C-terminal tail of μR. Mutation of these leucines to alanines decreased arrestin-mediated signaling by EM2 without affecting G-protein signaling, suggesting that lengthened endocytic lifetimes were required for arrestin-biased signaling by EM2. Lengthening surface lifetime by pharmacologically slowing endocytosis was sufficient to increase arrestin-mediated signaling by both EM2 and the clinically relevant agonist morphine. Our findings show that distinct ligands can leverage specific sequence elements on the μR to regulate receptor endocytic lifetimes and the magnitude of arrestin-mediated signaling, and implicate these sequences as important determinants of functional selectivity in the opioid system.
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Symptomatic neuromas and chronic neuropathic pain are significant problems affecting patients' quality of life and independence that are challenging to treat. These symptoms are due to structural and functional changes that occur peripherally within neuromas, as well as alterations that occur centrally within the brain and spinal cord. A multimodal approach is most effective, with goals to minimize opioid use, to capitalize on the synergistic effects of nonopioid medications and to explore potential benefits of novel adjunctive treatments.
Opioid receptors are divided into the three classical types: MOP(μ:mu), DOP(δ:delta) and KOP(κ:kappa) that are naloxone-sensitive and an additional naloxone-insensitive nociceptin/orphanin FQ(N/OFQ) peptide receptor(NOP). Studies to determine opioid receptor location and turnover variably rely on; (i) measuring receptor mRNA, (ii) genetically tagging receptors, (iii) labelling receptors with radioligands, (iv) use of antibodies in immunohistochemistry/Western Blotting or (v) measuring receptor function coupled with the use of selective antagonists. All have their drawbacks with significant issues relating to mRNA not necessarily predicting protein, poor antibody selectivity and utility of radiolabels in low expression systems. In this minireview we discuss use of fluorescently labelled opioid receptor ligands. To maintain the pharmacological properties of the corresponding parent ligand fluorescently labelled ligands must take into account fluorophore (brightness and propensity to bleach), linker length and chemistry, and site to which the linker (and hence probe) will be attached. Use of donor and acceptor fluorophores with spectral overlap facilitates use in FRET type assays to determine proximity of ligand or tagged receptor pairs. There is a wide range of probes of agonist and antagonist nature for all four opioid receptor types; caution is needed with agonist probes due to the possibility for internalization. We have produced two novel ATTO based probes; DermorphinATTO488 (MOP) and N/OFQATTO594 (NOP). These probes label MOP and NOP in a range of preparations and using N/OFQATTO594 we demonstrate internalization and ligand-receptor interaction by FRET. Fluorescent opioid probes offer potential methodological advantages over more traditional use of antibodies and radiolabels.
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For thousands of years opioids have been the first-line treatment option for pain management. However, the tolerance and addiction potential of opioids limit their applications in clinic. NFP, a MOR/KOR dual-selective opioid antagonist, was identified as a ligand that significantly antagonized the antinociceptive effects of morphine with lesser withdrawal effects than naloxone at similar doses. To validate the potential application of NFP in opioid addiction treatment, a series of in vitro and in vivo assays were conducted to further characterize its pharmacological profile. In calcium mobilization assays and MOR internalization studies, NFP showed the apparent capacity to antagonize DAMGO-induced calcium flux and etorphine-induced MOR internalization. In contrast to the opioid agonists DAMGO and morphine, cells pretreated with NFP did not show apparent desensitization and down regulation of the MOR. Though in vitro bidirectional transport studies showed that NFP might be a P-gp substrate, in warm-water tail-withdrawal assays it was able to antagonize the antinociceptive effects of morphine indicating its potential central nervous system activity. Overall these results suggest that NFP is a promising dual selective opioid antagonist that may have the potential to be used therapeutically in opioid use disorder treatment.
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Opioid overdoses recently became the leading cause of accidental death in the US, marking an increase in the severity of the opioid use disorder (OUD) epidemic that is impacting global health. Current treatment protocols for OUD are limited to opioid medications, including methadone, buprenorphine, and naltrexone. While these medications are effective in many cases, new treatments are required to more effectively address the rising societal and interpersonal costs associated with OUD. In this article, we review the opioid and cholinergic systems, and examine the potential of acetylcholine (ACh) as a treatment target for OUD. The cholinergic system includes enzymes that synthesize and degrade ACh and receptors that mediate the effects of ACh. ACh is involved in many central nervous system functions that are critical to the development and maintenance of OUD, such as reward and cognition. Medications that target the cholinergic system have been approved for the treatment of Alzheimer’s disease, tobacco use disorder, and nausea. Clinical and preclinical studies suggest that medications such as cholinesterase inhibitors and scopolamine, which target components of the cholinergic system, show promise for the treatment of OUD and further investigations are warranted.
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Pain management is the specialized medical practice of modulating pain perception and thus easing the suffering and improving the life quality of individuals suffering from painful conditions. Since this requires the modulation of the activity of endogenous systems involved in pain perception, and given the large role that the opioidergic system plays in pain perception, opioids are currently the most effective pain treatment available and are likely to remain relevant for the foreseeable future. This contributes to the rise in opioid use, misuse, and overdose death, which is currently characterized by public health officials in the United States as an epidemic. Historically, the majority of preclinical rodent studies were focused on morphine. This has resulted in our understanding of opioids in general being highly biased by our knowledge of morphine specifically. However, recent in vitro studies suggest that direct extrapolation of research findings from morphine to other opioids is likely to be flawed. Notably, these studies suggest that different opioid analgesics (opioid agonists) engage different downstream signaling effects within the cell, despite binding to and activating the same receptors. This recognition implies that, in contrast to the historical status quo, different opioids cannot be made equivalent by merely dose adjustment. Notably, even at equianalgesic doses, different opioids could result in different beneficial and risk outcomes. In order to foster further translational research regarding drug-specific differences among opioids, here we review basic research elucidating differences among opioids in pharmacokinetics, pharmacodynamics, their capacity for second messenger pathway activation, and their interactions with the immune system and the dopamine D2 receptors.
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The biological process of opioid analgesic tolerance remains nowadays elusive. In particular the mechanism by which opioid receptor desensitization occurs has not been completely elucidated to date. One possible hypothesis involves the internalization of MOR. Here, we describe a simple in vitro protocol to investigate the localization of MOR-1 after repeated morphine administration in the spinal cord of morphine-tolerant mice, using western blotting and immunofluorescence techniques. © 2021, Springer Science+Business Media, LLC, part of Springer Nature.
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Opioids are some of the most potent analgesics available. However, their effectiveness is limited by the development of analgesic tolerance. Traditionally, tolerance was thought to occur by termination of m-opioid receptor (MOR) signaling via desensitization and internalization. Contradictory findings led to a more recent proposal that sustained MOR signaling caused analgesic tolerance. However, this view has also been called into question. We recently discovered that the plateletderived growth factor receptor(PDGFR)-b signaling system is both necessary and sufficient to cause opioid tolerance. We therefore propose a completely new hypothesis: That opioid tolerance is mediated by selective cellular signals and is independent of MOR internalization. To test this hypothesis, we developed an automated software-based method to perform unbiased analyses of opioid-induced MOR internalization in the rat substantia gelatinosa. We induced tolerance with either morphine, which did not cause MOR internalization, or fentanyl, which did. We also blocked tolerance by administering morphine or fentanyl with the PDGFR-b inhibitor imatinib.Wefound that imatinib blocked tolerance without altering receptor internalization induced by either morphine or fentanyl. We also showed that imatinib blocked tolerance to other clinically used opioids. Our findings indicate that opioid tolerance is not dependent upon MOR internalization and support the novel hypothesis that opioid tolerance is mediated by intracellular signaling that can be selectively targeted. This suggests the exciting possibility that undesirable opioid side effects can be selectively eliminated, dramatically improving the safety and efficacy of opioids. © 2020 by The American Society for Pharmacology and Experimental Therapeutics.
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Converging lines of evidence from epidemiological, preclinical, and experimental studies indicate that the endocannabinoid system may be involved in the pathophysiology of schizophrenia and suggest that the cannabinoid receptor 1 may be a potential therapeutic target. In view of this, we first provide an overview of the endocannabinoid system and systematically review the evidence for cannabinoid 1 receptor alterations in animal models of schizophrenia and clinical studies in schizophrenia. MEDLINE, EMBASE, Psych Articles and Psych INFO were systematically searched from inception until January 7th, 2020. Of 1187 articles, 24 articles were included in the systematic review, including 8 preclinical studies measuring the cannabinoid 1 receptor in the context of an established animal model of schizophrenia and 16 clinical studies investigating the cannabinoid 1 receptor in schizophrenia. The majority of preclinical studies (6/8) have shown that the cannabinoid 1 receptor is reduced in the context of animal models of schizophrenia. Moreover, the majority of in vivo clinical imaging studies (3/4) that used arterial blood sampling to quantify the radiotracer kinetics have shown reduced cannabinoid 1 receptor availability in schizophrenia. However, mixed findings have been reported in ex vivo literature, including reports of no change (5/11), increased (4/11), and decreased (2/11) receptor levels. We review methodological reasons for these discrepancies and review how cannabinoid 1 receptor dysfunction may contribute to the pathophysiology of schizophrenia, drawing on the role of the receptor in regulating synaptic transmission and synaptic plasticity. We also discuss how the cannabinoid 1 receptor may be a potential therapeutic target.
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The chimeric peptide EN-9 was reported as a κ-opioid/neuropeptide FF receptors bifunctional agonist that modulated chronic pain with no tolerance. Many lines of evidence have shown that the effect of the κ-opioid receptor is mediated by not only its specific activation but also downstream events participation, especially interaction with the μ-opioid receptor pathway in antinociception and tolerance on most occasions. The present study investigated the acute and chronic cross-tolerance of EN-9 with μ-opioid receptor agonist EM-2, DAMGO, and morphine after intracerebroventricularly (i.c.v) injection in the mouse tail-flick test. In the acute tolerance test, EN-9 showed symmetrical acute cross-tolerance to DAMGO but no cross-tolerance to EM2. In the chronic tolerance test, EN-9 had no tolerance after eight days of repeated administration. However, EN-9 illustrated complete cross-tolerance to morphine and symmetrical cross-tolerance to EM2. In addition, inhibition of NPFF receptor could induce the tolerance development of EN-9. These findings indicated that supraspinal EN-9-induced antinociception contains additional components, which are mediated by the downstream μ-opioid receptor pathway both in acute and chronic treatment, whereas the subtypes of μ-opioid receptor or NPFF system pathway involved in antinociceptive effects induced by EN-9 are complex. Identifying the receptor mechanism could help design preferable bifunctional opioid compounds.
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This study investigated the effect of DAMGO-induced μ opioid receptor (MOR) internalization on morphine tolerance. Male Sprague-Dawley rats (200–250 g) aged 6–8 weeks were administered morphine via intrathecal (i.t.) injection (15 μg/10 μl twice daily for 5 days) to induce antinociceptive tolerance, which was evaluated using the tail-flick and paw-withdrawal tests. Response latency was calculated as the percentage of maximum possible effect (%MPE). A bolus of DAMGO was administered by i.t. injection on day 6, and the tail-flick and paw-withdrawal tests were carried out 24, 48, and 72 h later. Membrane and cytosolic MOR expression was assessed by western blotting. HEK293 cells were transfected with MOR-FLAG plasmid and after 6 days of morphine treatment (10 μM), the cells were treated with 1 μM DAMGO, and MOR localization was examined by immunofluorescence analysis 30 and 60 min later. Repeated morphine treatment induced tolerance after 5 days; however, i.t. DAMGO administration restored morphine sensitivity and enhanced acute morphine-induced antinociception after 24, 48, and 72 h. In HEK293 cells, DAMGO treatment stimulated MOR internalization after 30 min and MOR recycling to the membrane after 1 h. Membrane and cytoplasmic MOR expression in vivo was unchanged 24, 48, and 72 h after i.t. DAMGO injection. Morphine does not cause significant MOR internalization or downregulation, and can readily induce tolerance. DAMGO counters this effect by enhancing receptor endocytosis, thereby reversing morphine-induced antinociceptive tolerance and restoring its analgesic efficacy.
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Emotions modulate behavioral priorities via central and peripheral nervous systems. Understanding emotions from the perspective of specific neurotransmitter systems is critical, because of the central role of affect in multiple psychopathologies and the role of specific neuroreceptor systems as corresponding drug targets. Here, we provide an integrative overview of molecular imaging studies that have targeted the human emotion circuit at the level of specific neuroreceptors and transmitters. We focus specifically on opioid, dopamine, and serotonin systems, given their key role in modulating motivation and emotions, and discuss how they contribute to both healthy and pathological emotions.Keywords Molecular imaging Human emotions Dopamine systemSerotonin systemOpioid system
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μ opiate receptors, the principal sites for opiate analgesia and reward, can display compensatory responses to opiate agonist drug administration. Agonist-induced K channel responses mediated by these receptors desensitize when examined in Xenopus oocyte expression systems. Mechanisms underlying such processes could include phosphorylation events similar to those reported to desensitize other G-protein-linked receptors. We used C-terminally directed anti-μ receptor antibodies to immunoprecipitate a phosphoprotein with size appropriate for the μ receptor from stably expressing Chinese hamster ovary cells. Phosphorylation of this μ opiate receptor protein was enhanced approximately 5-fold by treatment with the μ agonist morphine. The time course and dose-response relationships between μ receptor phosphorylation and agonist-induced desensitization display interesting parallels. Phosphorylation of μ opiate receptor protein is also enhanced 5-fold by treatment with the protein kinase C activator phorbol 12-myristate 13-acetate. The protein kinase inhibitor staurosporine blocked the effect of phorbol 12-myristate 13-acetate on μ receptor phosphorylation. However, staurosporine failed to block morphine-induced phosphorylation. These observations suggest that several biochemical pathways can lead to μ receptor phosphorylation events that may include mechanisms involved in μ receptor desensitization.
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It has been known for some time that chronic treatment of neuronal cells and tissues with opioids, contrary to their acute effect, leads to an increase in cAMP accumulation. This phenomenon, defined as adenylyl cyclase superactivation, has been implicated in opiate addiction, yet the mechanism by which it is induced remains unclear. Here, we show that this phenomenon can be reproduced and studied in COS-7 cells cotransfected with adenylyl cyclase type V and μ-opioid receptor cDNAs. These cells display acute opioid inhibition of adenylyl cyclase activity, whereas prolonged exposure to the μ-agonist morphine or [-Ala², N-methyl-Phe⁴, Gly-ol⁵]enkephalin leads to a time-dependent superactivation of adenylyl cyclase. This superactivated state is reversible, because it is gradually lost following agonist withdrawal. Adenylyl cyclase superactivation can be prevented by pertussis toxin pretreatment, indicating the involvement of Gi/o proteins, or by cotransfection with the carboxyl terminus of β-adrenergic receptor kinase or with α-transducin (scavengers of Gβγ dimers), indicating a role for the G protein βγ dimers in adenylyl cyclase superactivation. However, contrary to several other Gβγ-dependent signal transduction mechanisms (e.g. the extracellular signal-regulated kinase 2/MAP kinase pathway), adenylyl cyclase superactivation is not affected by the Ras dominant negative mutant N17-Ras.
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Morphine is a powerful pain reliever, but also a potent inducer of tolerance and dependence. The development of opiate tolerance occurs on continued use of the drug such that the amount of drug required to elicit pain relief must be increased to compensate for diminished responsiveness. In many systems, decreased responsiveness to agonists has been correlated with the desensitization of G-protein-coupled receptors. In vitro evidence indicates that this process involves phosphorylation of G-protein-coupled receptors and subsequent binding of regulatory proteins called beta-arrestins. Using a knockout mouse lacking beta-arrestin-2 (beta arr2-/-), we have assessed the contribution of desensitization of the mu-opioid receptor to the development of morphine antinociceptive tolerance and the subsequent onset of physical dependence. Here we show that in mice lacking beta-arrestin-2, desensitization of the mu-opioid receptor does not occur after chronic morphine treatment, and that these animals fail to develop antinociceptive tolerance. However, the deletion of beta-arrestin-2 does not prevent the chronic morphine-induced up-regulation of adenylyl cyclase activity, a cellular marker of dependence, and the mutant mice still become physically dependent on the drug.
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The ability of a system to regulate its responsiveness in the presence of a continuous stimulus, often termed desensitization, has been extensively characterized for the beta2-adrenergic receptor (beta2AR). beta2AR signalling is rapidly attenuated through receptor phosphorylation and subsequent binding of the protein beta-arrestin. Ultimately the receptor undergoes internalization, and although the molecular mechanism is unclear, receptor phosphorylation and beta-arrestin binding have been implicated in this processs. Here we report that beta-arrestin and arrestin-3, but not visual arrestin, promote beta2AR internalization and bind with high affinity directly and stoichiometrically to clathrin, the major structural protein of coated pits. Moreover, beta-arrestin/arrestin chimaeras that are defective in either beta2AR or clathrin binding show a reduced ability to promote beta2AR endocytosis. Immunofluorescence microscopy of intact cells indicates an agonist-dependent colocalization of the beta2AR and beta-arrestin with clathrin. These results show that beta-arrestin functions as an adaptor in the receptor-mediated endocytosis pathway, and suggest a general mechanism for regulating the trafficking of G-protein-coupled receptors.
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The sterile insect technique (SIT) is used as an environment-friendly means of suppressing Mediterranean fruit fly (Ceratitis capitata; ‘medfly’) populations in the Arava valley of Israel. The technique depends on released sterile males effectively wresting the reproductive potential away from wild, fertile males. Studies carried out in other countries have indicated that sterile males may sometimes be of inferior sexual competitiveness in comparison with their wild counterparts and that this may inhibit SIT efficacy. In the present study, field-cage experiments were conducted to investigate the sexual competitiveness of sterile male medflies (genetic sexing strain Vienna 4/Tol-94) produced in and shipped from Guatemala, in the presence of wild males in Israel. In addition, we checked whether pre-release chilling affects their sexual success. Sterile and wild males were found to be similar in mating frequency, latency until mating, insemination probability, and duration of copulations during which no sperm were stored. There was, however, weak evidence that copulations involving sperm storage were shorter for sterile males. Chilling did not influence any element of male sexual performance. In both experiments, copulations culminating in sperm storage by females were longer than those that failed, suggesting that processes occurring early on in copulation may sometimes be the source of sexual failure. Overall, these results indicate a high standard of vigor in the sterile male medfiies used in the SIT program presently followed in Israel.
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The mu-opioid receptor mediates not only the beneficial painkilling effects of opiates like morphine but also the detrimental effects of chronic exposure such as tolerance and dependence. Different studies have linked tolerance to opioid receptor desensitization. Agonist activation of the mu-opioid receptor stimulates a mitogen-activated protein kinase (MAPK) activity, but the functional significance of this pathway remains unclear. We have focused on the MAPK signaling cascade to study mu-opioid receptor desensitization. We report that inhibition of the MAPK pathway blocks desensitization of mu-opioid receptor signaling as well as the loss of receptor density due to internalization. Our results suggest that a feedback signal emanating from the MAPK cascade is required for mu-opioid receptor desensitization.
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Opiate receptor down-regulation in neuroblastoma X glioma NG108-15 hybrid cells possibly involved the internalization of ligand-receptor complexes during chronic treatment. However, receptor internalization was not supported by the observed decrease in [3H] enkephalin(D-Ala2,D-Leu5) ( [3H]DADLE) associated with the hybrid cells during prolonged incubation with 10 nM [3H]DADLE at 37 degrees C. This decrease in [3H]DADLE bound was determined to be due to degradation of the ligand-receptor complexes, for a time-dependent increase in [3H]DADLE bound was observed when the incubations were carried out in the presence of 0.1 mM chloroquine. The increase did not exceed the amount of down-regulated receptor, could be blocked by naloxone, and was not observed at 24 degrees C. The [3H]DADLE bound in the presence of chloroquine was not sensitive to trypsin or to 20 microM diprenorphine. The accumulated [3H]DADLE was demonstrated to be intracellularly located by the fractionation of the homogenates in self-generating Percoll gradients. In the presence of chloroquine, a time-dependent translocation of [3H]DADLE from the plasma membrane-enriched fractions to the lysosome-enriched fractions was observed. The translocation was not observed at 24 degrees C in the presence of chloroquine or at 37 degrees C in the absence of chloroquine. The [3H]DADLE in the lysosome-enriched fractions was not sensitive to trypsin and remained bound in the presence of chloroquine. With the removal of chloroquine, an increase in the release of [3H]DADLE into the medium was observed. Sephadex G-50 column chromatography of the sodium deoxycholate extracts of the lysosome-enriched fractions suggested that the [3H]DADLE was bound to macromolecules intracellularly. Thus, chronic [3H]DADLE treatment of the hybrid cells resulted in an internalization of ligand-receptor complexes which were degraded in the lysosomes. Subsequently, the [3H]DADLE was regurgitated by the hybrid cells.
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Caveolae are 50-100-nm membrane microdomains that represent a subcompartment of the plasma membrane. Previous morphological studies have implicated caveolae in (a) the transcytosis of macromolecules (including LDL and modified LDLs) across capillary endothelial cells, (b) the uptake of small molecules via a process termed potocytosis involving GPI-linked receptor molecules and an unknown anion transport protein, (c) interactions with the actin-based cytoskeleton, and (d) the compartmentalization of certain signaling molecules, including G-protein coupled receptors. Caveolin, a 22-kD integral membrane protein, is an important structural component of caveolae that was first identified as a major v-Src substrate in Rous sarcoma virus transformed cells. This finding initially suggested a relationship between caveolin, transmembrane signaling, and cellular transformation. We have recently developed a procedure for isolating caveolin-rich membrane domains from cultured cells. To facilitate biochemical manipulations, we have applied this procedure to lung tissue--an endothelial and caveolin-rich source-allowing large scale preparation of these complexes. These membrane domains retain approximately 85% of caveolin and approximately 55% of a GPI-linked marker protein, while they exclude > or = 98% of integral plasma membrane protein markers and > or = 99.6% of other organelle-specific membrane markers tested. Characterization of these complexes by micro-sequencing and immuno-blotting reveals known receptors for modified forms of LDL (scavenger receptors: CD 36 and RAGE), multiple GPI-linked proteins, an anion transporter (plasma membrane porin), cytoskeletal elements, and cytoplasmic signaling molecules--including Src-like kinases, hetero-trimeric G-proteins, and three members of the Rap family of small GTPases (Rap 1--the Ras tumor suppressor protein, Rap 2, and TC21). At least a fraction of the actin in these complexes appeared monomeric (G-actin), suggesting that these domains could represent membrane bound sites for microfilament nucleation/assembly during signaling. Given that the majority of these proteins are known molecules, our current studies provide a systematic basis for evaluating these interactions in vivo.
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In Xenopus oocytes expressing the rat mu receptor and the G protein-gated, inwardly rectifying K channel (known as KGA or GIRK1), application of [DAla2,MePhe4,Glyol5]enkephalin), a mu opioid agonist,evoked a dose-dependent increase in K conductance. With sustained agonist exposure, the amplitude of the response decayed with a t of 8 ± 2 min. In oocytes coexpressing the mu and 5HT1A receptors with GIRK1, stimulation of either receptor resulted in heterologous desensitization of the subsequent response to the other. Injection of guanosine 5′-O-(thiotriphosphate) (1 mM) increased the basal GIRK1 activity and the total response to the application of agonist, but did not affect the rate of desensitization. Basal channel activity in the absence of agonist also desensitized at the same rate when the oocytes were exposed to high K (96 mM) solution. The above results indicate that the desensitization of the response occurred at a site downstream of the receptor, possibly at the channel. The rate of desensitization was not significantly altered by any of the following treatments: removal of external Ca, preloading the oocytes with 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-tetra-(acetoxymethyl)-ester (0.5-1 mM), elevation of cAMP levels, treatment with phorbol esters (1 μM), staurosporine (0.5 μM), okadaic acid (1 μM), or cytochalasin B (0.5 μM). These results suggest that desensitization may not involve a calcium or phosphorylation-dependent mechanism.
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Opioids are some of the most efficacious analgesics used in humans. Prolonged administration of opioids, however, often causes the development of drug tolerance, thus limiting their effectiveness. To explore the molecular basis of those mechanisms that may contribute to opioid tolerance, we have isolated a cDNA for the human mu opioid receptor, the target of such opioid narcotics as morphine, codeine, methadone, and fentanyl. The receptor encoded by this cDNA is 400 amino acids long with 94% sequence similarity to the rat mu opioid receptor. Transient expression of this cDNA in COS-7 cells produced high-affinity binding sites to mu-selective agonists and antagonists. This receptor displays functional coupling to a recently cloned G-protein-activated K+ channel. When both proteins were expressed in Xenopus oocytes, functional desensitization developed upon repeated stimulation of the mu opioid receptor, as observed by a reduction in K+ current induced by the second mu receptor activation relative to that induced by the first. The extent of desensitization was potentiated by both the multifunctional calcium/calmodulin-dependent protein kinase and protein kinase C. These results demonstrate that kinase modulation is a molecular mechanism by which the desensitization of mu receptor signaling may be regulated at the cellular level, suggesting that this cellular mechanism may contribute to opioid tolerance in humans.
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A mu opioid receptor and a G protein-activated K+ channel were coexpressed in Xenopus oocytes. Stimulation of the mu opioid receptor induced an inwardly rectifying current that was blocked by opioid receptor antagonist naloxone, indicating that the mu opioid receptor is functionally coupled to the K+ channel. The coupling is mediated by G proteins, since pertussis toxin treatment reduced the K+ current and injection of GTP gamma S (guanosine 5'-O-(thiotriphosphate)) enhanced it. Repeated stimulation of the mu receptor leads to desensitization, as the K+ current from the second stimulation was reduced to 70% of that from the first one. Both cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) regulate this process, but in opposite direction. Activation of PKC by treatment of the oocyte with phorbol ester potentiated the desensitization of the mu receptor-induced current. However, incubation of the cell with a membrane-permeable cAMP analog, 8-chlorophenylthio-cAMP, completely abolished the desensitization. The cAMP effect appears to be mediated by PKA, since injection of a PKA catalytic subunit showed the same effect as cAMP incubation. These results suggest that PKA and PKC differentially regulate the mu opioid receptor coupling to the G protein-activated K+ channel.
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The molecular basis of opioid tolerance/dependence has long eluded researchers, but recent advances in receptor regulation have suggested a useful conceptual approach to the problem. In NG108-15 neuroblastoma × glioma hybrid (NG) cells, opioid agonists inhibit adenylate cyclase in a dose-dependent, naloxone-antagonizable fashion. Chronic treatment with opioid agonists results in a series of molecular processes that, in a tolerance-like fashion, counteract this inhibition. These processes include desensitization and down-regulation of receptors and an increase in adenylate cyclase activity.Opioid inhibition of adenylate cyclase and opioid receptor down-regulation also have been observed in the brain. However, most studies have found that the receptors coupled to adenylate cyclase are not of the mu type, which a thought to be the primary mediators of opioid analgesia. Down-regulation has been observed for both mu and delta opioid receptors in the brain. However, in most case, the time course of down-regulation is not correlated with that for tolerance development, and chronic morphine treatment does not result in down-regulation. Thus, opioid receptors in the brain, like those in NG cells, are subject to dynamic regulation by agonists, which probably has an important role in their function. However, it remains to be established that opioid receptor regulation is the basis of opioid tolerance and dependence.
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Agonist exposure of many G protein-coupled receptors stimulates an activation of extracellular signal-regulated protein kinases (ERKs) 1 and 2, members of the mitogen-activated protein kinase (MAPK) family. Here, we show that treatment of human embryonic kidney (HEK) 293 cells stably transfected to express the rat μ-opioid receptor (MOR1) with [D-Ala2,MePhe4,Gly5-ol]enkephalin (DAMGO) stimulated a rapid and transient (3-5-min) activation and nuclear translocation of MAPK. Exposure of these cells to the MAPK kinase 1 inhibitor PD98059 not only prevented MAPK activation but also inhibited homologous desensitization of the μ-opioid receptor. We have therefore determined the effect of PD98059 on agonist-induced μ-receptor phosphorylation. DAMGO stimulated a threefold increase in MOR1 phosphorylation within 20 min that could be reversed by the antagonist naloxone. PD98059 produced a dose-dependent inhibition of agonist-promoted μ-receptor phosphorylation with an IC50 of 20 μM. DAMGO also induced MOR1 internalization that peaked at 30 min. Confocal microscopy revealed that DAMGO-induced MOR1 internalization was also largely inhibited in the presence of PD98059. U0126, another chemically unrelated inhibitor of the MAPK cascade, mimicked the effect of PD98059 on μ-receptor phosphorylation and desensitization. MOR1 itself, however, appears to be a poor substrate for MAPK because μ-receptors immunoprecipitated from stably transfected HEK 293 cells were not phosphorylated by exogenous ERK 2 in vitro. The fact that morphine also triggered MAPK activation but did not induce MOR1 internalization indicates that receptor internalization was not required for MOR1-mediated mitogenic signaling. We conclude that MOR1 stimulates a rapid and internalization-independent MAPK activation. Activation of the MAPK cascade in turn may not only relay mitogenic signals to the nucleus but also trigger initial events leading to phosphorylation and desensitization of the μ-opioid receptor.
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By metabolically labeling tissue slices from striatum and thalamus with [32P]orthophosphoric acid and immunoprecipitating the receptor with μ receptor-specific antiserum, we found that the endogenous μ receptor in the brain tissue did undergo phosphorylation. The phosphorylation occurred at basal level (no drug treatment) and was enhanced with DAMGO-treatment. The enhancement of the phosphorylation was blocked by naloxone. Morphine stimulation also increased the phosphorylation, but the amount of enhancement was less than that caused by DAMGO-treatment. μ receptor phosphorylation in the thalamus was much greater than the striatum, while no phosphorylation of the μ receptor in the cerebellum was detected, even with DAMGO treatment. The extent of μ receptor phosphorylation identified in the thalamus, striatum and cerebellum is consistent with the previous studies of μ receptor distribution. The time course and dose–response studies demonstrated that μ receptor phosphorylation was a rapid event, exhibited a positive dose-dependent response, and was similar to that observed in the cloned μ receptor in CHO cells. Furthermore, we correlated the change of μ receptor phosphorylation with the desensitization of the μ receptor function, specifically, inhibition of adenylyl cyclase activity in the thalamus of morphine-tolerant rats. We found that in the thalamus of rats chronically treated with morphine, the enhancement of μ receptor phosphorylation in basal and DAMGO-treated samples paralleled the desensitization of DAMGO-mediated inhibition of adenylyl cyclase. Our results suggest that μ receptor phosphorylation in vivo may play an important role in the modulation of mu receptor function following both acute exposure to morphine and during the development of morphine tolerance.
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Narcotics affect adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] in two opposing ways, both mediated by the opiate receptor. The first process is the readily reversible inhibition of the enzyme by narcotics; the second is a compensatory increase in enzyme activity which is delayed in onset and relatively stable. Late positive regulation of the enzyme counteracts the inhibitory influence of morphine and is responsible for narcotic dependence and tolerance. The coupled inhibitory and positive regulatory mechanisms for adenylate cyclase provide a means of activating and deactivating neural circuits hours after the initial event and thus may play a role in a memory process.
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In vivo and in vitro approaches were used to investigate a possible change in the opiate receptors during the development of tolerance/ depende. With the pAx method no significant change in the apparent pA2 of naloxone in tolerant rats in vivo could be found, indicating that no substantial change in the affinity for the receptors takes place. Comparison of receptor binding of 3H-etorphine and 3H-naloxone to rat brain homogenate in vitro showed no difference in binding between naive and tolerant rats. The displacement of small amounts of high labeled antagonist or agonist by increasing amounts of unlabeled antagonist in mouse brain in vivo offered the possibility of characterizing properties of receptors in the intact animal. This technique revealed no indication of a change in the number of receptor sites in tolerant animals. An apparently lower affinity in the tolerant animals could be explained by the morphine present in these animals. Displacement of 3H-etorphine from receptors by a high amount of unlabeled naltrexone in vivo could also be demonstrated by autoradiography.
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The effect of continuous intrathecal infusions of opioids was studied in rats. Chronic intrathecal infusion of the highly selective mu agonist, [NMPhe3, D-Pro4]morphiceptin produced a rapid onset of tolerance to the drug in the analgesic test. However, membrane prepared from the spinal cords of the rats chronically infused with a low dose of the drug showed no statistically significant change in the number of mu or delta receptor binding sites. In addition, membrane prepared from rats challenged with a single high-dose bolus injection of [NMPhe3, D-Pro4]morphiceptin did not produce alterations in the receptor binding number. If the chronically treated rats were challenged with an acute bolus dose of [NMPhe3, D-Pro4]morphiceptin, there was a significant decrease in the number of binding sites. The reduced binding site number was observed for the mu ligand but not for the delta ligand. A similar decrease of receptor binding can also be achieved by chronic infusion of the drug at high doses. Scatchard plot showed a decrease of maximum mu binding sites in the membranes prepared from the combined chronic infusion-acute injection treated rats. Brain tissue from the same rats showed no change in the number of mu and delta receptor binding sites, indicating that the down-regulation of mu receptors was confined to the spinal cord only. Morphine did not induce receptor down-regulation by acute, chronic or combined treatments. These results suggest that in the rat spinal cord, tolerance can be induced without apparent receptor down-regulation.(ABSTRACT TRUNCATED AT 250 WORDS)
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The effects of phosphorylation of a mu-opioid receptor on signal transduction to G-protein were studied. The mu-opioid receptor purified from rat whole brains was reconstituted with purified Gi1 in phosphatidylcholine vesicles. DAGO, a mu-opioid agonist at 1 microM-1 mM increased GTPase activity by 10-110% of control, in a concentration-dependent manner. When the mu-opioid receptor was phosphorylated by cyclic AMP-dependent protein kinase prior to reconstitution with Gi1, the DAGO-stimulation was markedly reduced (20% increase at 1 mM DAGO).
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Quantitative autoradiography was used to show the locations of mu-opiate receptor binding sites which are upregulated following chronic morphine treatment in rats. A saturating concentration of the mu-specific ligand [3H]D-ala2-N-methyl-Phe4,Gly-ol5-enkephalin was used to label sites in slide-mounted sections through one level of the thalamus in rats implanted subcutaneously with morphine pellets for 5 days. In vitro binding and autoradiography showed the largest increase in binding in the hypothalamus, especially the ventromedial nucleus (155%), with smaller increases in the basolateral and medial amygdaloid nuclei and the striatum. The set of structures showing the upre