Mu-opioid Receptor Desensitization Research Articles

Estradiol and Mu opioid-mediated reward: The role of estrogen receptors in opioid use

Opioid use and opioid use disorder are characterized by sex and gender differences, and some of these differences may be mediated by differences in the hormonal milieu within and across individuals. This review focuses on the role of ovarian hormones, and particularly estradiol, on the endogenous mu opioid receptor system. There is an abundance of data indicating that estradiol influences the activity of endogenous mu opioid peptides, the activation of mu opioid receptors, and the internalization and desensitization of mu opioid receptors. These effects have functional consequences on behaviors mediated by endogenous mu opioid receptor activity and on sensitivity to mu opioid agonists and antagonists. Recent behavioral data suggest these consequences extend to mu opioid reward, and preclinical studies report that estradiol decreases self-administration of mu opioid receptor agonists across a range of experimental conditions. Data collected in human laboratory studies suggest that estradiol may have functionally similar effects in clinical populations, and thus estrogen receptors may be a potential target in the development of novel therapeutics. This review summarizes data from cellular assays to clinical trials to explore how estradiol influences mu opioid receptor activity, as well as potential ways in which estrogen receptors may be targeted to address the problems of opioid use.

Anxiety enhances pain in a model of osteoarthritis and is associated with altered endogenous opioid function and reduced opioid analgesia.

Negative affect, including anxiety and depression, is prevalent in chronic pain states such as osteoarthritis (OA) and associated with greater use of opioid analgesics, potentially contributing to present and future opioid crises. We tested the hypothesis that the interaction between anxiety, chronic pain, and opioid use results from altered endogenous opioid function. A genetic model of negative affect, the Wistar-Kyoto (WKY) rat, was combined with intra-articular injection of monosodium iodoacetate (MIA; 1 mg) to mimic clinical presentation. Effects of systemic morphine (0.5-3.5 mg·kg-1) on pain behaviour and spinal nociceptive neuronal activity were compared in WKY and normo-anxiety Wistar rats 3 weeks after MIA injection. Endogenous opioid function was probed by the blockade of opioid receptors (0.1-1 mg·kg-1 systemic naloxone), quantification of plasma β-endorphin, and expression and phosphorylation of spinal mu-opioid receptor (MOR). Monosodium iodoacetate-treated WKY rats had enhanced OA-like pain, blunted morphine-induced analgesia, and greater mechanical hypersensitivity following systemic naloxone, compared with Wistar rats, and elevated plasma β-endorphin levels compared with saline-treated WKY controls. Increased MOR phosphorylation at the master site (serine residue 375) in the spinal cord dorsal horn of WKY rats with OA-like pain (P = 0.0312) indicated greater MOR desensitization. Reduced clinical analgesic efficacy of morphine was recapitulated in a model of high anxiety and OA-like pain, in which endogenous opioid tone was altered, and MOR function attenuated, in the absence of previous exogenous opioid ligand exposure. These findings shed new light on the mechanisms underlying the increased opioid analgesic use in high anxiety patients with chronic pain.

In vivo and in vitro Characterization of a Partial Mu Opioid Receptor Agonist, NKTR-181, Supports Future Therapeutic Development.

Mu opioid receptor (MOPr) agonists are well-known and frequently used clinical analgesics but are also rewarding due to their highly addictive and often abusive properties. This may lead to opioid use disorder (OUD) a disorder that effects millions of people worldwide. Therefore, novel compounds are urgently needed to treat OUD. As opioids are effective analgesics and OUD often occurs in conjunction with chronic pain, these novel compounds may be opioids, but they must have a low abuse liability. This could be mediated by diminishing or slowing blood-brain barrier transport, slowing target receptor binding kinetics, and showing a long half-life. NKTR-181 is a PEGylated oxycodol and a MOPr agonist that has slowed blood-brain barrier transport, a long half-life, and diminished likeability in clinical trials. In this study, we examined the signaling and behavioral profile of NKTR-181 in comparison with oxycodone to determine whether further therapeutic development of this compound may be warranted. For this preclinical study, we used a number of in vitro and in vivo assays. The signaling profile of NKTR-181 was determined by the electrophysiological assessment of MOPr-Ca2+ channel inhibition in the nociceptive neurons of rodent dorsal root ganglia. Heterologous cell-based assays were used to assess biased agonism and receptor trafficking. Different rodent behavioral models were used to define the NKTR-181-induced relief of effective and reflexive nociception and drug-seeking behavior as assessed by an intravenous self-administration (IVSA) of NKTR-181. We found that NKTR-181 and oxycodone are partial agonists in G-protein signaling and Ca2+ channel inhibition assays and promote limited MOPr desensitization. However, NKTR-181 inhibits Ca2+ channels by a different mechanism than oxycodone and induces a different pattern of arrestin recruitment. In addition, NKTR-181 has a slower receptor on-rate and a slower rate of Ca2+ channel coupling than oxycodone. This signaling profile is coupled with a slower onset of antinociception and limited drug-seeking behavior in comparison with oxycodone. Together with its known long half-life and slow blood-brain barrier transport, these data suggest that NKTR-181 could be further studied as a pharmacotherapeutic treatment modality for OUD.

Relevance of Mu-Opioid Receptor Splice Variants and Plasticity of Their Signaling Sequelae to Opioid Analgesic Tolerance.

Opioid dose escalation to effectively control pain is often linked to the current prescription opioid abuse epidemic. This creates social as well as medical imperatives to better understand the mechanistic underpinnings of opioid tolerance to develop interventions that minimize it, thereby maximizing the analgesic effectiveness of opioids. Profound opioid analgesic tolerance can be observed in the absence of mu-opioid receptor (MOR) downregulation, aggregate MOR G protein uncoupling, and MOR desensitization, in the absence of impaired G protein coupled receptor kinase phosphorylation, arrestin binding, or endocytosis. Thus, we have explored alternative biochemical sequelae that might better account for opioid analgesic tolerance. Our findings indicate that substantial plasticity among upstream and downstream components of opioid receptor signaling and the emergence of alternative signaling pathways are major contributors to opioid analgesic tolerance. An exemplar of this plasticity is our findings that chronic morphine upregulates the MOR variants MOR-1B2 and MOR-1C1 and phosphorylation of their C-terminal sites not present in MOR-1, events causally associated with the chronic morphine-induced shift in MOR G protein coupling from predominantly Gi/Go inhibitory to Gs-stimulatory adenylyl cyclase signaling. The unique feature(s) of these variants that underlies their susceptibility to adapting to chronic morphine by altering the nature of their G protein coupling reveals the richness and pliability of MOR signaling that is enabled by generating a wide diversity of MOR variants. Furthermore, given differential anatomical expression patterns of MOR variants, MOR splice variant-dependent adaptations to chronic morphine could enable mechanistic underpinnings of tolerance and dependence that are CNS region- and cell-specific.

Activation of MORs in the VTA induces changes on cFos expression in different projecting regions: Effect of inflammatory pain

Chronic pain is a worldwide major health problem and many pain-suffering patients are under opioid based therapy. Epidemiological data show that pain intensity correlates with the risk of misuse of prescription opioids, and other drugs of abuse including alcohol. This increased vulnerability to suffer Substance Use Disorders could be, in part, caused by functional changes that occur over the mesocorticolimbic system, a brain pathway involved in reward processing and addiction. Previous data in rats revealed that inflammatory pain desensitizes mu opioid receptors (MORs) in the ventral tegmental area (VTA). As a consequence, pain alters dopamine release in the nucleus accumbens (NAc) derived from MOR activation in the VTA and also increases intake of high doses of heroine. Given that the VTA neurons target different brain regions, in the present study we first analyzed changes induced by inflammatory pain in the MOR dependent activation pattern of the main VTA projecting areas. To do that, we administered two doses (7 or 14 ng) of DAMGO (MORs agonist) or artificial cerebrospinal fluid (aCSF) focally into the VTA of rats and measured the activation in projection areas by cFos immunohistochemistry. Our results show that focal injections of DAMGO in the VTA increases cFos expression in the majority of its projecting areas, namely NAc, basolateral amygdala (BLA), cingulate cortex (ACC) and bed nucleus of the stria terminalis (BNST), as compared to aCSF. Second, we analyzed whether inflammatory pain would affect to cFos expression using a group of rats injected with CFA in the hind paw. In this case, we found that cFos expression was not significantly different between DAMGO and aCSF administered rats in BLA, ACC and BNST. Our results confirm that inflammatory pain induces desensitization of VTA MORs in a region dependent manner which can be very relevant for addictive behaviours.

Solving the Opioid Crisis: Respiratory Depression by Opioids as Critical End Point

Solving the Opioid Crisis: Respiratory Depression by Opioids as Critical End Point

Opioid Activity in the Locus Coeruleus Is Modulated by Chronic Neuropathic Pain.

Pain affects both sensory and emotional aversive responses, often provoking depression and anxiety-related conditions when it becomes chronic. As the opioid receptors in the locus coeruleus (LC) have been implicated in pain, stress responses, and opioid drug effects, we explored the modifications to LC opioid neurotransmission in a chronic constriction injury (CCI) model of short- and long-term neuropathic pain (7 and 30days after nerve injury). No significant changes were found after short-term CCI, yet after 30days, CCI provoked an up-regulation of cAMP (cyclic 5'-adenosine monophosphate), pCREB (phosphorylated cAMP response element binding protein), protein kinase A, tyrosine hydroxylase, and electrical activity in the LC, as well as enhanced c-Fos expression. Acute mu opioid receptor desensitization was more intense in these animals, measured as the decline of the peak current caused by [Met5]-enkephalin and the reduction of forskolin-stimulated cAMP produced in response to DAMGO. Sustained morphine treatment did not markedly modify certain LC parameters in CCI-30d animals, such as [Met5]-enkephalin-induced potassium outward currents or burst activity and c-Fos rebound after naloxone precipitation, which may limit the development of some typical opioid drug-related adaptations. However, other phenomena were impaired by long-term CCI, including the reduction in forskolin-stimulated cAMP accumulation by DAMGO after naloxone precipitation in morphine dependent animals. Overall, this study suggests that long-term CCI leads to changes at the LC level that may contribute to the anxiodepressive phenotype that develops in these animals. Furthermore, opioid drugs produce complex adaptations in the LC in this model of chronic neuropathic pain.

Phosphatidylethanolamine‐Binding Protein Promotes Opioid Anti‐Nociception in the Brain and Spinal Cord by Reducing βarrestin2 Recruitment to the Mu Opioid Receptor

Opioids have been widely accepted as an effective treatment for chronic pain at a cost of serious side effects, including tolerance and addiction. Side effects are caused in part by desensitization and downregulation of mu opioid receptors (MOR) through recruitment of βarrestin2. Phosphatidylethanolamine‐Binding Protein (PEBP) is known to regulate either Raf Kinase or G Protein Receptor Kinase‐2 (GRK2) through phosphorylation by Protein Kinase C (PKC), and GRK2 is a major player in the recruitment of βarrestin2. However, PEBP has not been investigated for its role in MOR signaling and opioid response. We first tested this using an in vitro model of βarrestin2 recruitment in MOR expressing U2OS cells. We found that by inhibiting PEBP through siRNA knockdown or the small molecule inhibitor locostatin, we could increase the recruitment of βarrestin2 to the MOR in 2 separate assays. Conversely, we found that activating PKC via phorbol myristate acetate (PMA) led to PEBP phosphorylation/activation, and a reduction in βarrestin2 recruitment to the MOR, which could be blocked by PEBP knockdown. We then tested the role of PEBP in vivo by injection of locostatin into the brain or spinal cord, or CRISPR editing of PEBP in the spinal cord. We found that blocking PEBP in vivo led to a strong reduction in morphine‐induced tail flick anti‐nociception in both the brain and spinal cord. Together, these results support the hypothesis that PEBP acts to sequester GRK2 and block βarrestin2 recruitment downstream of the MOR both in vivo and in vitro. These results also suggest that enhanced PKC/PEBP activation by opioid drugs could further reduce opioid side effects and enhance analgesic efficacy.Support or Funding InformationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Desensitization and Tolerance of Mu Opioid Receptors on Pontine Kölliker-Fuse Neurons

Acute desensitization of mu opioid receptors is thought to be an initial step in the development of tolerance to opioids. Given the resistance of the respiratory system to develop tolerance, desensitization of neurons in the Kölliker-Fuse (KF), a key area in the respiratory circuit, was examined. The activation of G protein-coupled inwardly rectifying potassium current was measured using whole-cell voltage-clamp recordings from KF and locus coeruleus (LC) neurons contained in acute rat brain slices. A saturating concentration of the opioid agonist [Met5]-enkephalin (ME) caused significantly less desensitization in KF neurons compared with LC neurons. In contrast to LC, desensitization in KF neurons was not enhanced by activation of protein kinase C or in slices from morphine-treated rats. Cellular tolerance to ME and morphine was also lacking in KF neurons from morphine-treated rats. The lack of cellular tolerance in KF neurons correlates with the relative lack of tolerance to the respiratory depressant effect of opioids.

A biased mu‐opioid receptor positive allosteric modulator that does not alter receptor regulation

Currently used opioid analgesics compete with endogenous opioid peptide ligands by binding to the orthosteric site of the mu opioid receptor (MOPr). Activation of this G protein‐coupled receptor (GPCR) affords analgesia but also causes respiratory depression, sedation, constipation, and euphoria leading to a high addictive liability. Our lab is currently pursuing the idea that positive allosteric modulators of MOPr (mu‐PAMs) may be a way to treat pain with a better therapeutic profile. Such compounds, including the lead ligand BMS‐986122, enhance the affinity, potency, and efficacy of opioid agonists to bind MOPr and activate G protein. On the other hand, BMS‐986122 does not stimulate G protein activation, as measured by [35S]GTPγS binding in the absence of an orthosteric ligand. Here, we sought to determine if BMS‐986122 activates non‐G protein mediated pathways downstream of MOPr and to investigate the long‐term consequences of BMS‐986122 exposure on the regulation of MOPr in vitro using C6 rat glioma cells expressing rat MOPr. The results demonstrate that BMS‐986122 initiates phosphorylation of ERK1/2 in a pertussis‐toxin insensitive manner supporting a G protein‐independent mechanism. BMS‐986122 alone did not cause any long‐term adaptations in MOPr signaling. Moreover, in contrast to its ability to enhance agonist‐mediated activation of G protein, BMS‐986122 failed to enhance agonist‐induced desensitization of MOPr as measured by receptor downregulation, receptor phosphorylation, and loss of receptor‐mediated signaling following long‐term agonist exposure. Development of biased allosteric ligands at GPCRs represents a novel avenue for analgesic drug development. Future work will determine the physiological consequences of this biased PAM in mouse models of pain.Support or Funding InformationFunded by DA039997

Protein kinase C-mediated mu-opioid receptor phosphorylation and desensitization in rats, and its prevention during early diabetes.

Painful diabetic neuropathy is associated with impaired opioid analgesia; however, the precise mechanism in sensory neurons remains unclear. This study aimed to identify putative mechanisms involved in modified opioid responsiveness during early streptozotocin-induced diabetes in rats. In this study, we demonstrate that in diabetic animals, impaired peripheral opioid analgesia is associated with a reduction in functional mu-opioid receptor (MOR) G protein coupling. Mu-opioid receptor immunoreactive neurons colocalized with activated forms of protein kinase C (PKC) and with the receptor for advanced glycation end products (RAGE) during streptozotocin-induced diabetes. Moreover, MOR phosphorylation at Thr370 in sensory neurons of diabetic rats, and thus desensitization, was due to RAGE-dependent PKC activation. Importantly, blocking PKC activation using PKC selective inhibitor, silencing RAGE with intrathecal RAGE siRNA, or inhibiting advanced glycation end product (AGE) formation prevented sensory neuron MOR phosphorylation and, consequently, restored MOR G protein coupling and analgesic efficacy. Thus, our findings give the first in vivo evidence of a RAGE-dependent PKC-mediated heterologous MOR phosphorylation and desensitization in sensory neurons under pathological conditions such as diabetic neuropathy. This may unravel putative mechanisms and suggest possible prevention strategies of impaired opioid responsiveness.

Inflammatory Pain Promotes Increased Opioid Self-Administration: Role of Dysregulated Ventral Tegmental Area μ Opioid Receptors.

This study provides critical new insights that show that inflammatory pain alters heroin intake through a desensitization of MORs located within the VTA. These findings expand our knowledge of the interactions between inflammatory pain and opioid abuse liability, and should help to facilitate the development of novel and safer opioid-based strategies for treating chronic pain.

Desensitization of mu opioid receptors on Kölliker‐Fuse neurons

Tolerance to the respiratory depressant effects of opioids develops less readily than tolerance to analgesia, creating a dosing limit in chronic pain patients. Desensitization of mu opioid receptors is considered an initial step in the development of tolerance. Currently, most information about desensitization has been obtained in locus coeruleus (LC) neurons. Given the lack of respiratory tolerance, this study examined desensitization of mu opioid receptors on respiratory‐controlling Kölliker‐Fuse (KF) neurons. In voltage‐clamp recordings from KF neurons in rat brain slice, the opioid receptor agonist [Met5]enkephalin (ME) caused an outward current in a population of KF neurons due to activation of GIRK conductance. Compared to ME, the partial opioid agonist morphine produced a current that was proportionally larger than the morphine‐mediated current in LC brain slice preparations, indicating that KF neurons may have a large receptor reserve and thus be resistant to desensitization. Desensitization was induced by prolonged (10 min) perfusion of a high concentration of ME (30 µM). Desensitization of the ME‐mediated GIRK current was significantly less in KF neurons than in LC neurons. In contrast to LC, desensitization was not enhanced by activation of PKC or in slices from morphine‐treated rats. Thus, a lack of desensitization of mu opioid receptors on KF neurons may contribute to the relative lack of tolerance to the respiratory depressant effect of opioids. This will be tested using in situ and in vivo preparations of morphine‐treated rats. Supported by DA038069 (ESL) and DA08163 (JTW).

Direct Association of Mu-Opioid and NMDA Glutamate Receptors Supports their Cross-Regulation: Molecular Implications for Opioid Tolerance

In the nervous system, the interaction of opioids like morphine and its derivatives, with the G protein-coupled Mu-opioid receptor (MOR) provokes the development of analgesic tolerance, as well as physical dependence. Tolerance implies that increasing doses of the drug are required to achieve the same effect, a phenomenon that contributes significantly to the social problems surrounding recreational opioid abuse. In recent years, our understanding of the mechanisms that control MOR function in the nervous system, and that eventually produce opioid tolerance, has increased greatly. Pharmacological studies have identified a number of signaling proteins involved in morphine-induced tolerance, including the N-methyl-D-aspartate acid glutamate receptor (NMDAR), nitric oxide synthase (NOS), protein kinase C (PKC), protein kinase A (PKA), calcium (Ca²⁺)/calmodulin (CaM)-dependent kinase II (CaMKII), delta-opioid receptor (DOR) and the regulators of G-protein signaling (RGS) proteins. There is general agreement on the critical role of the NMDAR/nNOS/CaMKII pathway in this process, which is supported by the recent demonstration of a physical association between MORs and NMDARs in post-synaptic structures. Indeed, it is feasible that treatments that diminish morphine tolerance may target distinct elements within the same regulatory MOR-NMDAR pathway. Accordingly, we propose a model that incorporates the most relevant signaling components implicated in opioid tolerance in which, certain signals originating from the activated MOR are perceived by the associated NMDAR, which in turn exerts a negative feedback effect on MOR signaling. MOR- and NMDAR-mediated signals work together in a sequential and interconnected manner to ultimately induce MOR desensitization. Future studies of these phenomena should focus on adding further components to this signaling pathway in order to better define the mechanism underlying MOR desensitization in neural cells.

How to design an opioid drug that causes reduced tolerance and dependence

Mu opioid receptor (MOR) agonists such as morphine are extremely effective treatments for acute pain. In the setting of chronic pain, however, their long-term utility is limited by the development of tolerance and physical dependence. Drug companies have tried to overcome these problems by simply "dialing up" signal transduction at the receptor, designing more potent and efficacious agonists and more long-lasting formulations. Neither of these strategies has proven to be successful, however, because the net amount of signal transduction, particularly over extended periods of drug use, is a product of much more than the pharmacokinetic properties of potency, efficacy, half-life, and bioavailability, the mainstays of traditional pharmaceutical screening. Both the quantity and quality of signal transduction are influenced by many regulated processes, including receptor desensitization, trafficking, and oligomerization. Importantly, the efficiency with which an agonist first stimulates signal transduction is not necessarily related to the efficiency with which it stimulates these other processes. Here we describe recent findings that suggest MOR agonists with diminished propensity to cause tolerance and dependence can be identified by screening drugs for the ability to induce MOR desensitization, endocytosis, and recycling. We also discuss preliminary evidence that heteromers of the delta opioid receptor and the MOR are pronociceptive, and that drugs that spare such heteromers may also induce reduced tolerance.

The Effect of Protein Kinase C and G Protein-Coupled Receptor Kinase Inhibition on Tolerance Induced by μ-Opioid Agonists of Different Efficacy

Differences in the mechanisms underlying tolerance and mu-opioid receptor desensitization resulting from exposure to opioid agonists of different efficacy have been suggested previously. The objective of this study was to determine the effects of protein kinase C (PKC) and G protein-coupled receptor kinase (GRK) inhibition on antinociceptive tolerance in vivo to opioid agonists of different efficacy. A rapid (8-h) tolerance-induction model was used where each opioid was repeatedly administered to naive mice. Animals were then challenged with the opioid after injection of a kinase inhibitor to determine its effects on the level of tolerance. Tolerance to meperidine, morphine, or fentanyl was fully reversed by the PKC inhibitor 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)carbazole (Gö6976). However, in vivo tolerance to [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) was not reversed by PKC inhibition. The novel small-molecule GRK inhibitors beta-adrenergic receptor kinase 1 inhibitor and 2-(8-[(dimethylamino) methyl]-6,7,8,9-tetrahydropyridol[1,2-a]indol-3-yl)-3-(1-methylindol-3-yl)maleimide (Ro 32-0432) did not reverse the tolerance to meperidine, fentanyl, or morphine but did reverse the tolerance to DAMGO. To correlate GRK-dependent DAMGO-induced tolerance with mu-opioid receptor desensitization, we used in vitro whole-cell patch-clamp recording from mouse locus coeruleus neurons and observed that the GRK inhibitors reduced DAMGO-induced desensitization of mu-opioid receptors, whereas the PKC inhibitor had no effect. These results suggest that tolerance induced by low- and moderate-efficacy mu-opioid receptor agonists is dependent on PKC, whereas tolerance induced by the high-efficacy agonist DAMGO is dependent on GRK.

Involvement of PKCα and G‐protein‐coupled receptor kinase 2 in agonist‐selective desensitization of µ‐opioid receptors in mature brain neurons

The ability of an agonist to induce desensitization of the mu-opioid receptor (MOR) depends upon the agonist used. Furthermore, previous data suggest that the intracellular mechanisms underlying desensitization may be agonist-specific. We investigated the mechanisms underlying MOR desensitization, in adult mammalian neurons, caused by morphine (a partial agonist in this system) and DAMGO (a high-efficacy agonist). MOR function was measured in locus coeruleus neurons, by using whole-cell patch-clamp electrophysiology, in rat and mouse brain slices (both wild-type and protein kinase C (PKC)alpha knockout mice). Specific isoforms of PKC were inhibited by using inhibitors of the receptors for activated C-kinase (RACK), and in vivo viral-mediated gene-transfer was used to transfect neurons with dominant negative mutants (DNMs) of specific G-protein-coupled receptor kinases (GRKs). Morphine-induced desensitization was attenuated by using RACK inhibitors that inhibit PKCalpha, but not by other isoform-specific inhibitors. Further, the PKC component of morphine-induced desensitization was absent in locus coeruleus neurons from PKCalpha knockout mice. The PKC-enhanced morphine-induced desensitization was not affected by over-expression of a GRK2 dominant negative mutant (GRK2 DNM). In contrast, DAMGO-induced MOR desensitization was independent of PKC activity but was reduced by over-expression of the GRK2 DNM but not by that of a GRK6 DNM. In mature mammalian neurons, different MOR agonists can induce MOR desensitization by different mechanisms, morphine by a PKCalpha-mediated, heterologous mechanism and DAMGO by a GRK-mediated, homologous mechanism. These data represent functional selectivity at the level of receptor desensitization.

Role of protein kinase C in functional selectivity for desensitization at the µ‐opioid receptor: from pharmacological curiosity to therapeutic potential

Opioid agonists are the best therapy for moderate to severe pain, but clinical use is limited due to the development of tolerance and dependence. For the first time, Bailey and co-workers have demonstrated functional selectivity for agonist-induced desensitization of mu-opioid receptors (MOR) in mature rat locus coeruleus neurons. Native MORs are differentially desensitized through separate, agonist-dependent signalling pathways; desensitization of the morphine-occupied receptor occurs via a protein kinase C alpha-dependent pathway while [D-Ala(2), N-MePhe(4), Gly-ol]enkephalin-mediated desensitization is via a G protein receptor kinase subtype 2-dependent mechanism. These results suggest that MORs adopt separate conformational states that either result in different efficiencies of G protein activation or access to phosphorylation by desensitization machinery (e.g. protein kinase C alpha or G protein receptor kinase subtype 2). Further study of the interaction of protein kinase C with MORs in native neurons will enhance our understanding of agonist-induced functional selectivity for desensitization at MORs and provide important insights into how to selectively modulate agonist efficacy to enhance therapeutic capabilities of opioid drugs.

ADP-ribosylation factor 6 regulates mu-opioid receptor trafficking and signaling via activation of phospholipase D2

Endocytosis of the mu-opioid receptor (MOPr) has been shown to play a protective role against the development of tolerance to opioid drugs by facilitating receptor reactivation and recycling. It has been further demonstrated, that the opioid-mediated and ADP-ribosylation factor (ARF)-dependent activation of phospholipase D2 (PLD2) is a prerequisite for MOPr endocytosis. In this study, we investigated which particular ARF protein is involved in opioid-mediated PLD2 activation and what are the mechanisms of ARF function in MOPr trafficking and signaling. By coexpressing the MOPr and dominant negative or constitutively active ARF mutants in human embryonic kidney (HEK) 293 cells and primary cultured cortical neurons as well as by using siRNA technology, we identified the ARF6 protein to be involved in the regulation of MOPr endocytosis. We also found that expression of an effector domain mutant of ARF6, which is incapable of activating PLD, blocked agonist-induced endocytosis suggesting that ARF6 function in MOPr trafficking is PLD2-mediated. Analogously, opioid-mediated activation of PLD2 is blocked in the presence of dominant negative ARF6 mutants. Finally, we also showed that ARF6 protein influences the recycling/reactivation of internalized MOPr and thus modulates agonist-induced MOPr desensitization. Together, these results provide evidence that ARF6 protein regulates MOPr trafficking and signaling via PLD2 activation and hence affects the development of opioid receptor desensitization and tolerance.

Brain-specific Gαz interacts with Src tyrosine kinase to regulate Mu-opioid receptor-NMDAR signaling pathway

There is a certain cross-talk in the nervous system between N-methyl-D-aspartate receptors (NMDARs) and Mu-opioid receptors (MORs). While NMDARs participate in the desensitization of MORs, these in turn modulate NMDAR-mediated glutamate responses. The G protein coupled receptors (GPCRs) activate NMDARs via Src although the role of Gα subunits in this process is not well defined. We have found that in the absence of MOR activation, the brain specific Gαz subunit binds to and stabilizes Src in its inactive form. The administration of morphine provokes the phosphorylation of specific cytosolic tyrosine residues in NMDAR2A subunits. This was achieved by PKCγ disrupting this Gαz–Src complex, enabling Src to be activated (pTyr416) by binding to GαiGTP proteins. These changes increased the activation of the calcium/calmodulin-dependent protein kinase II (CaMKII), thereby promoting MOR desensitization. This regulatory pathway is disrupted by inhibiting PKC, preventing MOR-activated Gαi2 subunits from gaining control over Src. Thus, in neural cells the Gαz subunits exert a negative control on Src function reducing the activating influence of MORs on this tyrosine kinase. This MOR-triggered signaling pathway recruits PKCγ and Gαi subunits to activate Src tyrosine kinase, resulting in the potentiation of NMDAR function. Most relevant, this mechanism which operates in neural cells is essential for the development of tolerance to the analgesic effects of morphine.