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Opioids: cellular mechanisms of tolerance and physical dependence
Opioids: cellular mechanisms of tolerance and physical dependence Chris P Bailey1 and Mark Connor2 Morphine and other opioids are used and abused for their analgesic and rewarding properties. Tolerance to these effects develops over hours/days to weeks, as can physical and psychological dependence. Despite much investigation, the precise cellular mechanisms underlying opioid tolerance and dependence remain elusive. Recent studies examining m-opioid receptor desensitization and trafficking have revealed several potential mechanisms for acute receptor regulation. Other studies have reported changes in many other proteins that develop during chronic opioid treatment or withdrawal and such changes may be partly responsible for the cellular and synaptic adaptations to prolonged opioid exposure. While these studies have added to our knowledge of the cellular processes participating in opioid tolerance and dependence, the challenge remains to integrate these observations into a coherent explanation of the complex changes observed in whole animals chronically exposed to opioids. Addresses 1 Department of Pharmacology, University of Bristol, School of Medical Sciences, Bristol BS8 1TD, UK 2 Pain Management Research Institute, Kolling Institute, University of Sydney at Royal North Shore Hospital, St Leonards 2065, New South Wales, Australia Corresponding author: Bailey, CP ([email protected])
Current Opinion in Pharmacology 2005, 5:60–68 This review comes from a themed issue on Neurosciences Edited by Graeme Henderson, Hilary Little and Jenny Morton Available online 19th December 2004 1471-4892/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coph.2004.08.012
Introduction Opioid drugs are used clinically as unsurpassed analgesic agents but are also illegally abused on the street to induce a sense of well-being and euphoria. Tolerance to opioids, defined as a loss of effect following repeated treatments such that a higher dose is required for equivalent effect, limits the analgesic efficacy of these drugs [1] and contributes to the social problems surrounding recreational opioid abuse. In animals, tolerance to the anti-nociceptive effects of opioids can be observed even after a single dose, and continues to develop over many weeks of drug treatment. A complex interplay of events occurring at the single cell level and also in neuronal networks are likely to contribute to whole animal opioid tolerance, with distinct mechanisms being more important at different times during chronic exposure. This review summarizes key recent advances in our understanding of the mechanisms underlying the phenomenon of cellular tolerance, which is the reduced response to opioid agonists by m-opioid receptor (MOR)expressing neurons during chronic agonist exposure. Cellular tolerance following prolonged opioid receptor activation could result from alterations in receptor coupling, receptor number, the amount of effector protein or the capacity of an effector to be regulated by opioid receptors. Recent work has focused largely on the idea that the capacity of agonists to recruit various MOR regulatory events is a major determinant of their propensity to induce both tolerance and dependence. Concurrent work is increasingly promoting the idea that tolerance and dependence/withdrawal are molecularly separable phenomena, and therefore this review also covers recent studies examining possible cellular substrates of physical dependence, including adaptations unmasked on withdrawal from chronic opioid treatment.
Receptor desensitization and trafficking Abbreviations AC adenylyl cyclase CREB cAMP response element binding protein DAMGO [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin DOR d-opioid receptor GABA g-aminobutyric acid GAT-1 GABA transporter GRK G-protein-coupled receptor kinase LC locus coeruleus MAPK mitogen-activated protein kinase MOR m-opioid receptor NK1 neurokinin-1 receptor PAG periaqueductal grey PLD2 phospholipase D2 Current Opinion in Pharmacology 2005, 5:60–68
The analgesic and rewarding properties of opioid drugs occur through activation of MORs [2]. MORs are Gi/ocoupled receptors and, like many other G-proteincoupled receptors, can undergo rapid desensitization and internalization following exposure to agonist [3,4,5]. These acute receptor regulatory processes have assumed a central role in discussions into the development of cellular tolerance to morphine and other MOR agonists [4]. The generally accepted mechanism underlying MOR desensitization and internalization begins with phosphorylation of activated receptors by G-protein-coupled receptor kinases (GRKs), followed by arrestin binding. At this point, the www.sciencedirect.com
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receptor is in a desensitized state at the plasma membrane. Arrestin-bound receptors can then be internalized via a clathrin-dependent pathway, and either recycled to the cell surface or downregulated [3,4,6] (Figure 1). In heterologous expression systems, opioid receptor desensitization can also be modulated by second mes-
senger-linked protein kinases such as protein kinase C, protein kinase A and calcium/calmodulin-dependent kinase II [7]. The relative importance of different kinases in regulating opioid receptor activity in neurons has not yet been resolved, and the potential interactions between second-messenger-linked protein kinases, GRKs and arrestins in MOR desensitization, internalization and
Figure 1
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Pathways for acute m-opioid receptor regulation. The phenomena of opioid receptor activation, uncoupling and internalization are well described, but the precise mechanisms underlying the experimental observations are largely undefined. The pink shaded area represents states of the receptor that are likely to be uncoupled from signalling. Although it is assumed that once MOR is phosphorylated and bound by arrestin it is functionally desensitized, alternative mechanisms of desensitization might also occur. Opioid receptors are basally phosphorylated, but the kinases responsible for this are unknown. A role for serine/threonine protein kinases such as protein kinase A, calmodulin-dependent protein kinase and protein kinase C in receptor desensitization and/or the recruitment of GRK-dependent receptor trafficking have been suggested, but they have yet to be defined in detail. Similarly, specific tyrosine kinases that phosphorylate the MOR have not been identified. Approximately 80% of MORs that undergo endocytosis are recycled; the remaining 20% are degraded in lysosomes. AP-2, adaptor protein 2; Arr3, arrestin-3; P, phosphate. www.sciencedirect.com
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recycling are also ill-defined. Nonetheless, kinasemediated rapid receptor desensitization is likely to be an obligatory event preceding the development of cellular tolerance. Indirect evidence that arrestin-dependent MOR desensitization contributes to morphine tolerance in vivo comes from studies in arrestin-3 and GRK3 knockout mice. In arrestin-3 knockout mice, the acute antinociceptive response to morphine or heroin was enhanced and both acute and chronic tolerance to the antinociceptive effects of morphine was significantly attenuated [8,9]. However, arrestin-3 deletion had no effect on the acute antinociceptive potency of etorphine, fentanyl or methadone [8–10]. By contrast, deletion of GRK3 produced a modest inhibition of the development of tolerance to fentanyl, but not to morphine, in antinociceptive tests. Desensitization of fentanyl actions on population responses in hippocampal slices was also abolished [11], whereas desensitization of morphine actions was only slightly attenuated. Significantly, morphine withdrawal was not affected by deletion of arrestin-3, and loss of GRK3 did not affect withdrawal following chronic fentanyl treatment, suggesting that the development of tolerance and dependence are separable phenomena. In neurons, the molecular details of MOR desensitization have not been defined, although a single study in nucleus raphe magnus neurons reported that desensitization following short (<15 s) applications of DAMGO ([D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin; a MOR agonist) or morphine could be reduced by intracellular application of a GRK2 inhibitory peptide [12]. Agonist-induced rapid MOR desensitization and internalization
In isolated cells, high efficacy MOR agonists such as DAMGO have consistently been shown to cause MOR desensitization over seconds to minutes [13,14,15, 16,17,18]. However, this is not always the case for the less efficacious agonist morphine. In locus coeruleus (LC) neurons, an agonist’s ability to produce rapid desensitization of potassium channel activation was found to correlate with its effectiveness at promoting receptor internalization in HEK-293 cells [13,14]. Notably, morphine produced little desensitization in LC neurons or MOR internalization in HEK-293 cells, whereas DAMGO, methadone and met-enkephalin produced both substantial rapid desensitization in LC neurons and receptor internalization in HEK-293 cells. In contrast to effects observed in LC neurons, morphine produced a similar level of rapid desensitization in pituitary-derived AtT-20 cells as did DAMGO and methadone. However, morphine caused minimal MOR internalization in AtT20 cells, as in HEK-293 cells, in direct contrast to the robust internalization seen with DAMGO and methadone [16]. Further, a point mutation in MOR (T180A) Current Opinion in Pharmacology 2005, 5:60–68
resulted in a receptor that internalized normally following DAMGO exposure, but which did not exhibit DAMGOinduced desensitization when expressed in AtT-20 cells [17]. Thus rapid MOR desensitization does not always correlate with receptor internalization, most notably in cells challenged with morphine. The specific role of MOR internalization in MOR desensitization is not clear. Receptors are likely to be desensitized when still in the plasma membrane following receptor phosphorylation and/or arrestin binding, and internalization of the desensitized receptor might be fundamentally important for receptor resensitization, rather than being a significant component of desensitization. It is not known why morphine causes rapid MOR desensitization in some cell systems but not in others. It is possible that this is a result of the varying intracellular environment of different cells, raising the possibility that opioid receptor desensitization is mediated by different processes, and that different protein kinases are differentially activated or expressed in different cell types. For example, a recent study has shown that morphine can be converted into a rapidly desensitizing MOR agonist in LC neurons when protein kinase C is activated by phorbol esters or concomitant stimulation of Gq-coupled receptors [18]. Why does morphine cause minimal rapid MOR internalization?
The reason why morphine is less effective at producing MOR internalization remains obscure, although there is increasing evidence that morphine is not as effective as agonists such as DAMGO or etorphine at initiating specific steps in the GRK/arrestin pathway [7,10,19]. Indeed, morphine has been shown to induce substantially less phosphorylation of a putative GRK phosphorylation site (Ser375) in heterologously expressed MORs [19]. Furthermore, experiments in embryonic fibroblasts taken from arrestin-2/3 knockout mice, which completely lack native arrestin, confirmed that morphine activation of heterologously expressed MORs resulted in less translocation of fluorescent arrestin constructs to the plasma membrane than did other opioid agonists [10]. An alternative explanation for the differences between morphine and DAMGO emerged following reports that DAMGO, but not morphine, activated phospholipase D2 (PLD2) [20,21]. Activation of recombinant PLD2 in HEK-293 cells facilitated MOR internalization, whereas inhibition of this enzyme blocked it. Native MORs have not yet been shown to couple to PLD2 in neurons, but this pathway is the first reported to be activated by DAMGO but not morphine. Interestingly, a study modelling ligand/receptor/effector interactions suggested that the failure of morphine to recruit receptor regulatory mechanisms, including internalization, could be explained largely on kinetic grounds; this intriguing theory deserves more experimental attention [22]. www.sciencedirect.com
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Does morphine cause greater tolerance because of less MOR internalization?
Equianalgesic doses of opioid drugs do not produce equivalent amounts of tolerance in the whole animal. In particular, tolerance to the antinociceptive effects of morphine is greater than that seen with higher efficacy agonists such as etorphine or fentanyl [23]. Although this could be attributable to the fact that morphine would occupy more receptors than etorphine or fentanyl at equianalgesic doses, this finding, coupled with the repeated observation that morphine produces little MOR internalization, has generated two contrasting ideas concerning the role of MOR internalization in the development of cellular tolerance and dependence in vivo. If MOR resensitization is dependent upon internalization and dephosphorylation in endosomes (see Figure 1), then morphine might produce greater cellular tolerance because desensitized morphine-bound MORs cannot be internalized and resensitized; the other hypothesis suggests that, because morphine does not promote receptor internalization, receptors remain at the plasma membrane and continue to signal, thus disproportionately recruiting adaptations that induce cellular tolerance [24]. The idea that MOR resensitization following morphine treatment is comparatively less than with other opioid drugs is most strongly supported by evidence from cells heterologously expressing high densities of MORs. In these cells, resensitization of the MOR response following morphine-induced desensitization was significantly slower than resensitization following DAMGO treatment [19,25,26]. This has been attributed to relatively slower dephosphorylation of morphine-activated receptors compared with DAMGO-activated receptors [19], although direct evidence for MOR dephosphorylation in endosomes is still lacking. Unfortunately, the interpretation of these studies is significantly hampered by a lack of correlation between the time-course of MOR desensitization (maximal in approximately 4 h) and receptor internalization (maximal in less than 30 min), as well as by the failure to demonstrate any correlation between receptor re-insertion and recovery of agonist responses [3]. The notion that morphine produces more profound cellular adaptations than do other agonists because of its reduced capacity to initiate receptor internalization remains controversial [14,15,24,27]. One cellular consequence of chronic morphine is ‘superactivation’ of adenylyl cyclase (AC); it is claimed that this occurs as an adaptive response to prolonged MOR activation by morphine [24]. However, acute ‘superactivation’ of AC is an adaptation produced by all MOR agonists, including those that cause rapid MOR desensitization and internalization. Superactivation might be a correlate of the upregulated AC activity reported in chronically opioidtreated animals, but this in vivo phenomenon is also likely to involve increased AC protein [28]. Morphine produced www.sciencedirect.com
greater AC superactivation in HEK-293 cells transfected with MORs than it did in cells expressing mutant MORs that internalized in response to morphine; however, the differences in AC superactivation produced by methadone acting at these internalizing and non-internalizing MOR variants was modest [24]. Furthermore, the observation that a low concentration of DAMGO reduced morphine-induced AC superactivation by ‘dragging’ MORs into the cell via activation of one half of a putative MOR dimer [27] could not be reproduced using other measurements of receptor desensitization in neurons or receptor internalization in HEK-293 cells [14,15]. The relationship between processes involved in MOR desensitization, internalization and trafficking and the development of cellular tolerance in neurons remains to be established. Studies in neurons have not yet directly correlated MOR desensitization with receptor internalization, and as the vast majority of desensitization and internalization studies have focused on the first hour of agonist treatment we do not know how the processes underlying rapid desensitization relate to loss of function following longer-term opioid agonist treatment. There are, however, hints that the mechanisms of acute receptor desensitization and resensitization are altered by chronic morphine treatment of animals, because the recovery of MOR responses in LC neurons following an acute desensitizing stimulus is slower and less complete in neurons from morphine-treated animals than in controls [29]. Further studies in neurons are essential, and the observation that morphine produces redistribution and aggregation of MORs in the dendrites but not soma of cultured nucleus accumbens neurons [30] reinforces the idea that commonly utilized non-neuronal cell models might not be appropriate for studying opioid receptor regulation owing to their lack of crucial components of regulatory pathways that exist in neurons.
Other proteins that may affect cellular tolerance The rewarding, dependence-producing and analgesic effects of opioids result from activation of MORs in several brain regions simultaneously. Functional and gene expression studies have suggested an ever-growing number of target proteins that are likely to be involved in in vivo adaptations to chronic opioid treatment. These proteins and their impact on opioid tolerance have been studied by diverse techniques, including inhibition with pharmacological antagonists, knockdown of expression with antisense oligonucleotides and genomic deletion in mice. d-opioid receptors
Although morphine is a preferential MOR agonist, evidence suggests that other opioid receptors and endogenous ligands, particularly those of the d-opioid receptor (DOR) system, are intimately involved in Current Opinion in Pharmacology 2005, 5:60–68
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responses to acute and chronic morphine. Deletion of the genes for DORs and preproenkephalin, the precursor of both m and d endogenous peptide agonists, inhibits the development of morphine tolerance in antinociceptive tests without affecting the development of adaptations expressed during morphine withdrawal [31,32]. Although the potency of morphine is unchanged in DOR knockout mice [31], morphine analgesia in wildtype animals is made more effective by co-administration of either DOR agonists or antagonists [33]. MORs and DORs exist on overlapping populations of neurons in pain-modulating regions of the CNS, and an intriguing explanation for MOR/DOR synergy is that MOR and DOR can exist as heterodimers [33,34]. More evidence for a close association between MORs and DORs comes from studies showing that treatment of rats or mice with MOR agonists for several days leads to translocation of DORs to neuronal plasma membranes [35,36]. In sensory neurons, activation of DORs leads to translocation of intracellular DORs to the cell surface via membrane fusion of vesicles that also contain the pro-nociceptive peptide calcitonin gene-related peptide, the release of which might be expected to oppose the antinociceptive effect of morphine and thus could contribute to morphine tolerance [37]. Neurokinins and other neurotransmitters
Genetic deletion of an ever-growing number of neurotransmitters and/or their receptors also modifies the development of tolerance to the antinociceptive effects of morphine, the rewarding effects of morphine and the expression of opioid withdrawal [38], although in most cases the anatomical substrates and molecular mechanisms of these effects are not established. The most intriguing studies implicate recently identified neurotransmitters such as orexin, as well as well-known transmitters such as neurokinins [39,40]. Neurokinin-1 receptor (NK1) knockout mice do not self-administer morphine or exhibit conditioned place preference to morphine administration, although their withdrawal signs are largely preserved [40,41]. Possible target sites for NK1-mediated effects include the nucleus accumbens and amygdala [42]. Putative heterodimerization between NK1 receptors and MORs affects receptor trafficking in HEK-293 cells, but the role of heterodimerization with NK1 receptors in normal MOR function remains to be established [43]. G protein regulator and receptor trafficking proteins
Deletion of genes encoding proteins involved in regulating G protein function (e.g. RGS-9) [44] or receptor desensitization and trafficking (e.g. arrestin-3) [8–10,45] have profound effects on the potency of morphine to produce reward or analgesia, as well as the development of tolerance. There is considerable evidence that chronic opioid treatment can affect neuronal mRNA and protein levels of RGS [44,46,47] and GRKs [48–50], as well as Current Opinion in Pharmacology 2005, 5:60–68
the protein level and phosphorylation state of arrestin-3 [45,48,51,52]. Evidence from heterologous systems indicates that RGS proteins are also involved in opioidinduced AC supersensitization [53,54]. However, although it is tempting to speculate that disruption of the direct interaction between MORs and these proteins abrogates adaptations to chronic opioid treatment, deletion of these proteins will affect the signalling of multiple G-protein-coupled receptors both on morphinesensitive neurons and on other neurons in MOR-related circuits; currently, there are no data from RGS-9, GRK3 or arrestin-3 knockout animals to demonstrate that cellular or synaptic adaptations to chronic morphine treatment are affected. Direct evidence that an RGS protein in MOR-containing neurons contributes to morphine tolerance comes from a study that demonstrated increased RGS4 proteins levels following chronic morphine treatment; intracellular application of this protein attenuated the acute response to morphine in these neurons [46]. Synaptic transmission and plasticity
It is perhaps not surprising that deletion of genes encoding proteins involved in synaptic transmission and synaptic plasticity, such as the NR2A subunit of the N-methyl-D-aspartate receptor [55,56], also affects the development and expression of morphine tolerance and dependence. Clever experiments have extended the utility of gene knockout mice by re-introducing the deleted gene into specific brain regions using either virally mediated transfer (e.g. for RGS-9) or electroporation (e.g. for NR2A). NR2A re-introduction in the ventral tegmental area and periaqueductal grey (PAG) regions partially restored the expression of tolerance to the antinociceptive effects of morphine, whereas reintroduction into the nucleus accumbens partially restored morphine withdrawal signs [55]. In addition to the potential role of RGS proteins in tolerance, selective expression in the nucleus accumbens of an RGS-9 construct also abolished the exaggerated sensitivity to the rewarding properties of morphine present in RGS-9 knockout mice [44].
Adaptive changes following chronic opioid treatment that might underlie physical dependence Adaptive changes in neurons and neuronal communication are a hallmark of chronic morphine treatment; such adaptations must underlie altered behaviour associated with morphine dependence and withdrawal syndrome, as well as drug-induced craving and relapse to drug use (see also review by Weiss in this issue). Adaptations affecting neuronal excitability and synaptic transmission
The most well-described consequence of morphine withdrawal is a rebound increase in cAMP levels, probably www.sciencedirect.com
Opioids: cellular mechanisms of tolerance and physical dependence Bailey and Connor 65
produced by ‘superactivation’ of AC and upregulation of the amount of enzyme. There is also evidence that activation of TrkB receptors by brain-derived neurotrophic factor can contribute to cAMP superactivation in LC neurons [57]. Earlier studies are reviewed by Williams, Christie and Manzoni [28], and the strong evidence for enhanced g-aminobutyric acid (GABA)mediated synaptic transmission during morphine withdrawal in brain regions such as the PAG, ventral tegmental area, nucleus accumbens and dorsal raphe is discussed in detail. AC-mediated effects on GABA release are largely mediated by protein kinase A, but some effects are attributable to the conversion of cAMP to adenosine (Figure 2), [58]. For example, in mouse (but not rat) PAG, there is a protein kinase A-dependent increase in GABA release and a concomitant elevation of extracellular adenosine, which acts on inhibitory A1 receptors to reduce excess GABA release [59]. Inhibitors of cAMP metabolism and nucleoside transport block adenosine-dependent inhibition of GABA release, implying that the withdrawal-induced adenosine tone results
from excessive AC activation. Another study has demonstrated enhanced adenosine inhibition of synaptic inputs to nucleus accumbens neurons [60], an effect attributed to a reduction in adenosine transport. Although there is considerable evidence for adaptations to chronic morphine treatment at the level of nerve terminals, little progress has been made in elucidating the mechanisms that underlie changes in neuronal cell body firing rates that occur during opioid withdrawal [28]. The identity of ‘cation channels’ proposed to underlie withdrawal excitation of opioid-sensitive PAG or LC neurons has not been established; however, the recent observation that currents mediated by the dopamine transporter can profoundly affect the firing rate of dopaminergic neurons [61] indicates that the activity of similar transporters (e.g. those for noradrenaline or GABA) during opioid withdrawal is worth examining, particularly in light of the dramatic changes in neurotransmitter release during this process. There is evidence for alterations of glutamate transporter expression following
Figure 2
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Hyperexcitability and enhanced transmitter release during opioid withdrawal. Under normal conditions, MOR activation can result in inhibition of AC, inhibition of Ca2+ channels and opening of voltage-gated K+ channels. Following withdrawal from chronic opioid treatment, many neurons exhibit hyperexcitability at the soma, a process thought to be mediated, at least in part, by induction of a cation current through protein kinase A (PKA) activation following rebound cAMP superactivation or by increased levels of AC. Additionally, transmitter release at the nerve terminal can be enhanced during opioid withdrawal. The mechanisms underlying this process include rebound elevated cAMP levels or increased amounts of AC protein, causing enhanced transmitter release through PKA activation. Upregulated AC is, in turn, more sensitive to inhibition by opioids. In addition, enhanced cAMP production can also enhance extracellular levels of adenosine, causing inhibition of transmitter release through adenosine A1 receptors. Figure adapted from [28]. www.sciencedirect.com
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chronic morphine treatment in glia [62], spinal cord neurons [63] and hippocampal neurons [64], although it is not known whether these changes in glutamate transporters occur in opioid-sensitive neurons. Transcription factors underlying adaptations to opioids
Synaptic plasticity is thought to be important in mediating certain behavioural effects of chronic opioid treatment (see review by Jones and Bonci in this issue). Activation of the cAMP-activated transcription factor — cAMP response element binding protein (CREB) — is heavily implicated in various forms of synaptic plasticity and, because elevations in cAMP have long been considered central to the expression of opioid withdrawal, CREB is thought to be important for withdrawal-dependent synaptic plasticity [65]. Region-specific increases in phosphorylated CREB have been demonstrated in the rat brain following morphine withdrawal, notably in the nucleus accumbens, amygdala and hypothalamus [66]; manipulating CREB activity in the nucleus accumbens affects behavioural responses to rewarding stimuli, including morphine [67]. Few reports have linked changes in CREB activity to any specific cellular adaptation to acute or chronic opioid treatment, with the exception of one study that demonstrated attenuated hyperexcitability in LC neurons following morphine withdrawal in CREB1 knockout mice, or in mice with a CREB1 mutant that showed decreased transcriptional activity [68].
phine, and which of these adaptations give rise to the persistently altered behaviours found in animals and humans exposed to chronic opioids. The next few years should yield further progress towards defining the cellular molecular processes underlying the behavioural adaptations to opioid drugs.
Update The mechanism linking upregulated AC activity and morphine-withdrawal-induced hyperexcitability of PAG neurons has been established in a recent paper [72], which demonstrated that most PAG neurons in brain slices from morphine-dependent mice exhibit a novel opioid-sensitive cation conductance following morphine withdrawal. This conductance was produced by a PKAdependent increase in the activity of the GABA transporter (GAT-1), and inhibitors of GAT-1 blocked the withdrawal-induced hyperexcitability of PAG neurons. GAT-1 inhibitors are currently used to treat epilepsy; this study suggests they may also have efficacy in treating opioid withdrawal symptoms.
Acknowledgements This work was funded in part by the Wellcome Trust (CPB) and the National Health and Medical Research Council of Australia Project Grant #302002 (MC).
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
Mitogen-activated protein kinases
Mitogen-activated protein kinases (MAPKs) are activated by many G-protein-coupled receptors, including opioid receptors, and their activation has been implicated in synaptic plasticity [69]. Acute morphine treatment increases levels of phosphorylated (activated) MAPK in some brain regions including the anterior cingulate, somatosensory and association cortices and LC, but decreases activated MAPK levels in the nucleus accumbens and amygdala [70,71]. Interestingly, with the exception of the LC, the increases in activated MAPKs were seen in neurons adjacent to those expressing MORs, suggesting that opioid modulation of intercellular communication is important in modulating MAPK activity.
Conclusions The cellular consequences of MOR activation are well understood, and significant progress has been made in understanding the acute regulation of MORs heterologously expressed in cell lines. Furthermore, the involvement of diverse neurotransmitter systems and intracellular signalling proteins in acute and chronic opioid actions has been revealed by the use of knockout mice. However, the challenge for biologists interested in understanding the consequences of chronic morphine exposure is to establish which of the defined actions of morphine at the molecular level are important for producing cellular tolerance and synaptic adaptation to morCurrent Opinion in Pharmacology 2005, 5:60–68
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(beta)arrestin-2 knock-out mice. J Neurosci 2002, 22:10494-10500. 10. Bohn LM, Dykstra LA, Lefkowitz RJ, Caron MG, Barak LS: Relative opioid efficacy is determined by the components of the G protein-coupled receptor desensitization machinery. Mol Pharmacol 2004, 66:106-112. 11. Terman GW, Jin W, Cheong Y-P, Lowe J, Caron M, Lefkowitz RJ, Chavkin C: G-protein receptor kinase 3 (GRK3) influences opioid analgesic tolerance but not opioid withdrawal. Br J Pharmacol 2004, 141:55-64. 12. Li AH, Wang HL: G protein-coupled receptor kinase 2 mediated mu-opioid receptor desensitization in GABAergic neurons of the nucleus raphe magnus. J Neurochem 2001, 77:435-444. 13. Alvarez VA, Arttamangkul S, Dang V, Salem A, Whistler J, von Zastow M, Grandy DK, Williams JT: m-Opioid receptors: ligand-dependent activation of potassium conductance, desensitization and internalization. J Neurosci 2002, 22:5769-5776. This study demonstrates a strong correlation between the capacity of an opioid ligand to produce MOR desensitization in LC neurons and MOR internalization in HEK-293 cells. This is the best evidence to date for a role of processes leading to receptor internalization being involved in termination of MOR signalling in a neuron. 14. Bailey CP, Couch D, Johnson E, Griffiths K, Kelly E, Henderson G: m-Opioid receptor desensitization in mature rat neurons: lack of interaction between DAMGO and morphine. J Neurosci 2003, 23:10515-10520. An important study that directly tests the hypothesis that MOR desensitization produced by morphine can be rescued by concurrent application of an agonist that produces MOR internalization. The study finds no evidence to support this hypothesis in either neurons or cell lines heterologously expressing MORs. 15. Blanchet C, Sollini M, Luscher C: Two distinct forms of desensitization of G-protein coupled inwardly rectifying potassium currents evoked by alkaloid and peptide m-opioid receptor agonists. Mol Cell Neurosci 2003, 24:517-523. 16. Borgland SL, Connor M, Osborne PB, Furness JB, Christie MJ: Opioid agonists have different efficacy profiles for G protein activation, rapid desensitization, and endocytosis of mu-opioid receptors. J Biol Chem 2003, 278:18776-18784. This is the first study to directly correlate the abilities of different opioid ligands to cause rapid MOR desensitization and internalization in the same cell system (AtT20 cells). They found that the efficacies of ligands to activate MOR correlated well with desensitization, but were distinct from their abilities to cause endocytosis.
m-opioid receptor desensitization and resensitization. J Neurochem 2004, 88:680-688. 22. Woolf PJ, Linderman JJ: Untangling ligand induced activation and desensitization of G protein coupled receptors. Biophys J 2003, 84:3-13. 23. Duttaroy A, Yoburn BC: The effect of intrinsic efficacy on opioid tolerance. Anesthesiology 1995, 82:1226-1236. 24. Finn AK, Whistler JL: Endocytosis of the mu opioid receptor reduces tolerance and a cellular hallmark of withdrawal. Neuron 2001, 32:829-839. 25. Koch T, Schulz S, Pfeiffer M, Klutzny M, Schroder H, Kahl E, Hollt V: C-terminal splice variants of the mouse mu-opioid receptor differ in morphine-induced internalization and receptor resensitization. J Biol Chem 2001, 276:31408-31414. 26. Beyer A, Koch T, Schroder H, Schulz S, Hollt V: Effect of the A118G polymorphism on binding affinity, potency and agonist-mediated endocytosis, desensitization, and resensitization of the human mu-opioid receptor. J Neurochem 2004, 89:553-560. 27. He L, Fong J, von Zastrow M, Whistler JL: Regulation of opioid receptor trafficking and morphine tolerance by receptor oligomerization. Cell 2002, 108:271-282. 28. Williams JT, Christie MJ, Manzoni O: Cellular and synaptic adaptations mediating opioid dependence. Physiol Rev 2001, 81:299-343. 29. Dang VC, Williams JT: Chronic morphine treatment reduces recovery from opioid desensitization. J Neurosci 2004, 24:7699-7706. 30. Haberstock-Debic H, Wein M, Barot M, Colago EEO, Rahman Z, Neve RL, Pickel VM, Nestler EJ, von Zastrow M, Svingos AL: Morphine acutely regulates opioid receptor trafficking selectively in dendrites of nucleus accumbens neurons. J Neurosci 2003, 23:4324-4332. This study describes redistribution of MORs by morphine specifically in dendrites of nucleus accumbens neurons, suggesting that morphineinduced MOR internalization may occur in a compartent-selective manner in neurons. 31. Zhu Y, King MA, Schuller AP, Nitsche JF, Reidl M, Elde RP, Unterwald E, Pasternak GW, Pinat JE: Retention of supraspinal delta-like analgesia and loss of morphine tolerance in d opioid receptor knockout mice. Neuron 1999, 24:243-252.
17. Celver J, Xu M, Jin W, Lowe J, Chavkin C: Distinct domains on the m-opioid receptor control uncoupling and internalization. Mol Pharmacol 2004, 65:528-537.
32. Nitsche JF, Schuller AGP, King MA, Zengh M, Pasternak GW, Pintar JE: Genetic dissociation of opiate tolerance and physical dependence in d-opioid receptor-1 and Preproenkephalin knock-out mice. J Neurosci 2002, 22:10906-10913.
18. Bailey CP, Kelly E, Henderson G: Protein kinase C activation enhances morphine-induced rapid desensitization of m-opioid receptors in mature rat locus coeruleus neurons. Mol Pharmacol 2004, in press.
33. Gomes I, Gupta A, Filipovska J, Szeto HH, Pintar JE, Devi LA: A role for heterodimerization of m and d opiate receptors in enhancing morphine analgesia. Proc Natl Acad Sci USA 2004, 101:5135-5139.
19. Schulz S, Mayer D, Pfeiffer M, Stumm R, Koch T, Hollt V: Morphine induces terminal micro-opioid receptor desensitization by sustained phosphorylation of serine-375. EMBO J 2004, 23:3282-3289. The first study to identify the phosphorylation of a specific residue in MOR (Ser375) using a phosphospecific antibody. In HEK-293 cells, agonistinduced Ser375 phosphorylation correlated with receptor desensitization and internalization.
34. Gomes I, Jordan BA, Gupta A, Trapaidze N, Nagy V, Devi LA: Heterodimerization of mu and delta opioid receptors. J Neurosci 2000, 20:RC110.
20. Koch T, Brandenberg L-O, Schulz S, Liang Y, Klein J, Hollt V: ADP-ribosylation factor-dependent phospolipase D2 activation is required for agonist-induced m-opioid receptor endocytosis. J Biol Chem 2003, 278:9979-9986. This paper showed that DAMGO stimulation of heterologously expressed MOR activated heterologously expressed PLD2, and that this interaction was essential for MOR endocytosis. Intriguingly, morphine did not stimulate PLD2, providing the first demonstration of a second messenger pathway differentially activated by MOR agonists, thereby providing a potential explanation for the relative inability of morphine to promote MOR endocytosis.
36. Morinville A, Cahill CM, Esdaile MJ, Aibak H, Collier B, Kieffer BL, Beaudet A: Regulation of d-opioid receptor trafficking via m-opioid receptor stimulation: evidence from m-opioid receptor knock-out mice. J Neurosci 2003, 23:4888-4898.
21. Koch T, Brandenberg L-O, Liang Y, Schulz S, Beyer A, Schroder H, Hollt V: Phospholipase D2 modulates agonist-induced
39. Georgescu D, Zachariou V, Barrot M, Mieda M, Willie JT, Eisch AJ, Yanagisawa M, Nestler EJ, DiLeone RJ: Involvement of lateral
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35. Cahill CM, Morinville A, Lee M-C, Vincent J-P, Collier B, Beaudet A: Prolonged morphine treatment targets d opioid receptors to neuronal plasma membranes and enhances d-mediated antinociception. J Neurosci 2001, 21:7598-7607.
37. Bao L, Jin S-X, Zhang C, Wang L-H, Xu Z-Z, Zhang F-X, Wang L-C, Ning F-S, Cai H-J, Guan J-S et al.: Activation of delta opioid receptors induces receptor insertion and neuropeptide secretion. Neuron 2003, 37:121-133. 38. Contet C, Kieffer BL, Befort K: Mu opioid receptor; a gateway to drug addiction. Curr Opin Neurobiol 2004, 14:370-378.
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hypothalamic peptide orexin in morphine dependence and withdrawal. J Neurosci 2003, 23:3106-3111. 40. Murtra P, Sheasby AM, Hunt SP, De Felipe C: Rewarding effects of opiates are absent in mice lacking the receptor for substance P. Nature 2000, 405:180-183. 41. Ripley TL, Gadd CA, De Felipe C, Hunt SP, Stephens DN: Lack of self-administration and behavioural sensitization to morphine, but not cocaine, in mice lacking NK1 receptors. Neuropharmacology 2002, 43:1258-1268. 42. Gadd CA, Murtra P, De Felipe C, Hunt SP: Neurokinin-1 receptorexpressing neurons in the amygdala modulate morphine reward and anxiety behaviours in the mouse. J Neurosci 2003, 23:8271-8280. 43. Pfeiffer M, Kirscht S, Stumm R, Koch T, Wu D, Laugsch M, Schroder H, Hollt V, Schulz S: Heterodimerization of substance P and m-opioid receptors regulates receptor trafficking and resensitization. J Biol Chem 2003, 278:51630-51637. 44. Zachariou V, Georgescu D, Sanchez N, Rahman Z, DiLeone R, Berton O, Neve RL, Sim-Selley LJ, Selley DE, Gold SJ, Nestler EJ: Essential role for RGS9 in opiate action. Proc Natl Acad Sci USA 2003, 100:13656-13661. This study describes how mice lacking the gene for RGS-9 display enhanced morphine reward and analgesia, and delayed tolerance and withdrawal. Furthermore, by specifically reinserting the RGS9 gene into the nucleus accumbens, the enhancement in morphine reward is reversed.
An elegant study whereby GluRe1 N-methl-D-aspartate receptors were re-inserted into specific brain regions of GluRe1-null mice, demonstrating the relative importance of discrete brain regions in distinct behavioural effects of chronic morphine treatments. 56. Miyamoto Y, Yamada K, Nagai T, Mori H, Mishina M, Furukawa H, Noda Y, Nabeshima T: Behavioural adaptations to addictive drugs in mice lacking the NMDA receptor e1 subunit. Eur J Neurosci 2004, 19:151-158. 57. Akbarian S, Rios M, Liu RJ, Gold SJ, Fong HF, Zeiler S, Coppola V, Tessarollo L, Jones KR, Nestler EJ et al.: Brain-derived neurotrophic factor is essential for opiate-induced plasticity of noradrenergic neurons. J Neurosci 2002, 22:4153-4162. 58. Hack SP, Christie MJ: Adaptations in adenosine signalling in drug dependence: therapeutic implications. Crit Rev Neurobiol 2003, 15:235-274. 59. Hack SP, Vaughan CW, Christie MJ: Modulation of GABA release during morphine withdrawal in midbrain neurons in vitro. Neuropharmacology 2003, 45:575-584. 60. Brundege JM, Williams JT: Increase in adenosine sensitivity in the nucleus accumbens following chronic morphine treatment. J Neurophysiol 2002, 87:1369-1375. 61. Ingram SL, Prasad BM, Amara SG: Dopamine transportermediated increase in excitability of midbrain dopamine neurons. Nat Neurosci 2002, 5:971-978.
45. Bohn LM, Gainetdinov RR, Caron MG: G protein-coupled receptor kinase/b-arrestin systems and drugs of abuse. Neuromolecular Med 2004, 5:41-50.
62. Ozawa T, Nakagawa T, Shige K, Minami M, Satoh M: Changes in expression of glial glutamate transporters in the rat brain accompanied with morphine dependence and naloxoneprecipitated withdrawal. Brain Res 2001, 905:254-258.
46. Gold SJ, Han MH, Herman AE, Ni YG, Pudiak CM, Aghajanian GK, Liu R-J, Potts BW, Mumby SM, Nestler EJ: Regulation of RGS proteins by chronic morphine in rat locus coeruleus. Eur J Neurosci 2003, 17:971-980.
63. Mao J, Sung B, Ji RR, Lim G: Chronic morphine induces downregulation of spinal glutamate transporters: implications in morphine tolerance and abnormal pain sensitivity. J Neurosci 2002, 22:8312-8323.
47. Lopez-Fando A, Rodriguez-Munoz M, Sanchez-Blazqeuz P, Garzon J: Expression of neural RGS-R7 and Gb5 proteins in response to acute and chronic morphine. Neuropsychopharmacology 2004, in press.
64. Xu N-J, Bao L, Fan H-P, Bao G-B, Pu L, Lu Y-J, Wu C-F, Zhang X, Pei G: Morphine withdrawal increases glutamate uptake and surface expression of glutamate transporter GLT1 at hippocampal synapses. J Neurosci 2003, 23:4775-4784.
48. Hurle´ MA: Changes in the expression of G protein-coupled receptor kinases and b-arrestin 2 in rat brain during opioid tolerance and supersensitivity. J Neurochem 2001, 77:486-492.
65. Nestler EJ: Molecular basis of long-term plasticity underlying addiction. Nat Rev Neurosci 2001, 2:119-128.
49. Patel MB, Patel CN, Rajashekara V, Yoburn BC: Opioid agonists differentially regulate m-opioid receptors and trafficking proteins in vivo. Mol Pharmacol 2002, 62:1464-1470. 50. Fan X, Zhang J, Zhang X, Yue W, Ma L: Acute and chronic morphine treatments and morphine withdrawal differentially regulate GRK2 and GRK5 gene expression in rat brain. Neuropharmacology 2002, 43:809-816. 51. Chakrabarti S, Oppermann M, Gintzler AR: Chronic morphine induces the concomitant phosphorylation and altered association of multiple signaling proteins: a novel mechanism for modulating cells signaling. Proc Natl Acad Sci USA 2001, 98:4209-4214. 52. Fan XL, Zhang JS, Zhang XQ, Yue W, Ma L: Differential gene expression of b-arrestin 1 and b-arrestin 2 gene expression in rat brain by morphine. Neuroscience 2003, 117:383-389. 53. Clark MJ, Neubig RR, Traynor JR: Endogenous regulator of G protein signaling proteins suppress Gao-dependent, m-opioid agonist-mediated adenylyl cyclase supersensitization. J Pharmacol Exp Ther 2004, 310:215-222. 54. Xu H, Wang X, Wang J, Rothman RB: Opioid peptide receptor studies. 17. Attenuation of chronic morphine effects after antisense oligodeoxynucleotides knock-down of RGS9 protein in cells expressing the cloned mu opioid receptor. Synapse 2004, 52:209-217. 55. Inoue M, Mishina M, Ueda H: Locus-specific rescue of GluRe1 NMDA receptors in mutant mice identifies the brain regions important for morphine tolerance and dependence. J Neurosci 2003, 23:6529-6536.
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66. Shaw-Lutchman TZ, Barrot M, Wallace T, Gilden L, Zachariou V, Impey S, Duman RS, Storm D, Nestler EJ: Regional and cellular mapping of cAMP response element-mediated transcription during naltrexone-precipitated morphine withdrawal. J Neurosci 2002, 22:3663-3672. 67. Barrot M, Olivier JDA, Perrotti LI, DiLeone RJ, Berton O, Eisch AJ, Impey S, Storm DR, Neve RL, Yin JC et al.: CREB activity in the nucleus accumbens shell controls gating of behavioral responses to emotional stimuli. Proc Natl Acad Sci USA 2002, 99:11435-11440. 68. Valverde O, Mantamadiotis T, Torrecilla M, Ugedo L, Pineda J, Bleckmann S, Gass P, Kretz O, Mitchell JM, Schutz G, Maldonado R: Modulation of anxiety-like behavior and morphine dependence in CREB-deficient mice. Neuropsychopharmacology 2004, 29:1122-1133. 69. Sweatt JD: Mitogen activated protein kinases in synaptic plasticity and memory. Curr Opin Neurobiol 2004, 14:311-317. 70. Eitan S, Bryant CD, Saliminejad N, Yang YC, Vojdani E, Keith D Jr, Polakiewicz R, Evans CJ: Brain region-specific mechanisms for acute morphine-induced mitogen-activated protein kinase modulation and distinct patterns of activation during analgesic tolerance and locomotor sensitization. J Neurosci 2003, 23:8360-8369. 71. Muller DL, Unterwald EM: In vivo regulation of extracellular signal-regulated protein kinase (ERK) and protein kinase B (Akt) phosphorylation by acute and chronic morphine. J Pharmacol Exp Ther 2004, 310:774-782. 72. Bagley EE, Gerke MB, Vaughan CW, Hack SP, Christie MJ: GABA transporter currents activated by protein kinase A excite midbrain neurons during opioid withdrawal. Neuron 2005, in press.
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Current Opinion in Pharmacology 2005, 5:60–68 This review comes from a themed issue on Neurosciences Edited by Graeme Henderson, Hilary Little and Jenny Morton Available online 19th December 2004 1471-4892/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coph.2004.08.012
Introduction Opioid drugs are used clinically as unsurpassed analgesic agents but are also illegally abused on the street to induce a sense of well-being and euphoria. Tolerance to opioids, defined as a loss of effect following repeated treatments such that a higher dose is required for equivalent effect, limits the analgesic efficacy of these drugs [1] and contributes to the social problems surrounding recreational opioid abuse. In animals, tolerance to the anti-nociceptive effects of opioids can be observed even after a single dose, and continues to develop over many weeks of drug treatment. A complex interplay of events occurring at the single cell level and also in neuronal networks are likely to contribute to whole animal opioid tolerance, with distinct mechanisms being more important at different times during chronic exposure. This review summarizes key recent advances in our understanding of the mechanisms underlying the phenomenon of cellular tolerance, which is the reduced response to opioid agonists by m-opioid receptor (MOR)expressing neurons during chronic agonist exposure. Cellular tolerance following prolonged opioid receptor activation could result from alterations in receptor coupling, receptor number, the amount of effector protein or the capacity of an effector to be regulated by opioid receptors. Recent work has focused largely on the idea that the capacity of agonists to recruit various MOR regulatory events is a major determinant of their propensity to induce both tolerance and dependence. Concurrent work is increasingly promoting the idea that tolerance and dependence/withdrawal are molecularly separable phenomena, and therefore this review also covers recent studies examining possible cellular substrates of physical dependence, including adaptations unmasked on withdrawal from chronic opioid treatment.
Receptor desensitization and trafficking Abbreviations AC adenylyl cyclase CREB cAMP response element binding protein DAMGO [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin DOR d-opioid receptor GABA g-aminobutyric acid GAT-1 GABA transporter GRK G-protein-coupled receptor kinase LC locus coeruleus MAPK mitogen-activated protein kinase MOR m-opioid receptor NK1 neurokinin-1 receptor PAG periaqueductal grey PLD2 phospholipase D2 Current Opinion in Pharmacology 2005, 5:60–68
The analgesic and rewarding properties of opioid drugs occur through activation of MORs [2]. MORs are Gi/ocoupled receptors and, like many other G-proteincoupled receptors, can undergo rapid desensitization and internalization following exposure to agonist [3,4,5]. These acute receptor regulatory processes have assumed a central role in discussions into the development of cellular tolerance to morphine and other MOR agonists [4]. The generally accepted mechanism underlying MOR desensitization and internalization begins with phosphorylation of activated receptors by G-protein-coupled receptor kinases (GRKs), followed by arrestin binding. At this point, the www.sciencedirect.com
Opioids: cellular mechanisms of tolerance and physical dependence Bailey and Connor 61
receptor is in a desensitized state at the plasma membrane. Arrestin-bound receptors can then be internalized via a clathrin-dependent pathway, and either recycled to the cell surface or downregulated [3,4,6] (Figure 1). In heterologous expression systems, opioid receptor desensitization can also be modulated by second mes-
senger-linked protein kinases such as protein kinase C, protein kinase A and calcium/calmodulin-dependent kinase II [7]. The relative importance of different kinases in regulating opioid receptor activity in neurons has not yet been resolved, and the potential interactions between second-messenger-linked protein kinases, GRKs and arrestins in MOR desensitization, internalization and
Figure 1
β α P γ GDP
Newly synthesized receptors
OP Agonist Resensitization OP P
GTP α∗
β γ GRK
OP H+ OP
P
P GRK
ATP ADP
P
P
OP
Ser/Thr kinase (?)
Tyrosine kinase ? P Phosphatase
ATP ADP
Desensitization Degraded receptors
Arr3
H+ OP OP P P P
P P Arr3 P
Internalization OP P P Arr3 P
Intracellular compartment
AP-2 clathrin
Plasma membrane compartment
Current Opinion in Pharmacology
Pathways for acute m-opioid receptor regulation. The phenomena of opioid receptor activation, uncoupling and internalization are well described, but the precise mechanisms underlying the experimental observations are largely undefined. The pink shaded area represents states of the receptor that are likely to be uncoupled from signalling. Although it is assumed that once MOR is phosphorylated and bound by arrestin it is functionally desensitized, alternative mechanisms of desensitization might also occur. Opioid receptors are basally phosphorylated, but the kinases responsible for this are unknown. A role for serine/threonine protein kinases such as protein kinase A, calmodulin-dependent protein kinase and protein kinase C in receptor desensitization and/or the recruitment of GRK-dependent receptor trafficking have been suggested, but they have yet to be defined in detail. Similarly, specific tyrosine kinases that phosphorylate the MOR have not been identified. Approximately 80% of MORs that undergo endocytosis are recycled; the remaining 20% are degraded in lysosomes. AP-2, adaptor protein 2; Arr3, arrestin-3; P, phosphate. www.sciencedirect.com
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recycling are also ill-defined. Nonetheless, kinasemediated rapid receptor desensitization is likely to be an obligatory event preceding the development of cellular tolerance. Indirect evidence that arrestin-dependent MOR desensitization contributes to morphine tolerance in vivo comes from studies in arrestin-3 and GRK3 knockout mice. In arrestin-3 knockout mice, the acute antinociceptive response to morphine or heroin was enhanced and both acute and chronic tolerance to the antinociceptive effects of morphine was significantly attenuated [8,9]. However, arrestin-3 deletion had no effect on the acute antinociceptive potency of etorphine, fentanyl or methadone [8–10]. By contrast, deletion of GRK3 produced a modest inhibition of the development of tolerance to fentanyl, but not to morphine, in antinociceptive tests. Desensitization of fentanyl actions on population responses in hippocampal slices was also abolished [11], whereas desensitization of morphine actions was only slightly attenuated. Significantly, morphine withdrawal was not affected by deletion of arrestin-3, and loss of GRK3 did not affect withdrawal following chronic fentanyl treatment, suggesting that the development of tolerance and dependence are separable phenomena. In neurons, the molecular details of MOR desensitization have not been defined, although a single study in nucleus raphe magnus neurons reported that desensitization following short (<15 s) applications of DAMGO ([D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin; a MOR agonist) or morphine could be reduced by intracellular application of a GRK2 inhibitory peptide [12]. Agonist-induced rapid MOR desensitization and internalization
In isolated cells, high efficacy MOR agonists such as DAMGO have consistently been shown to cause MOR desensitization over seconds to minutes [13,14,15, 16,17,18]. However, this is not always the case for the less efficacious agonist morphine. In locus coeruleus (LC) neurons, an agonist’s ability to produce rapid desensitization of potassium channel activation was found to correlate with its effectiveness at promoting receptor internalization in HEK-293 cells [13,14]. Notably, morphine produced little desensitization in LC neurons or MOR internalization in HEK-293 cells, whereas DAMGO, methadone and met-enkephalin produced both substantial rapid desensitization in LC neurons and receptor internalization in HEK-293 cells. In contrast to effects observed in LC neurons, morphine produced a similar level of rapid desensitization in pituitary-derived AtT-20 cells as did DAMGO and methadone. However, morphine caused minimal MOR internalization in AtT20 cells, as in HEK-293 cells, in direct contrast to the robust internalization seen with DAMGO and methadone [16]. Further, a point mutation in MOR (T180A) Current Opinion in Pharmacology 2005, 5:60–68
resulted in a receptor that internalized normally following DAMGO exposure, but which did not exhibit DAMGOinduced desensitization when expressed in AtT-20 cells [17]. Thus rapid MOR desensitization does not always correlate with receptor internalization, most notably in cells challenged with morphine. The specific role of MOR internalization in MOR desensitization is not clear. Receptors are likely to be desensitized when still in the plasma membrane following receptor phosphorylation and/or arrestin binding, and internalization of the desensitized receptor might be fundamentally important for receptor resensitization, rather than being a significant component of desensitization. It is not known why morphine causes rapid MOR desensitization in some cell systems but not in others. It is possible that this is a result of the varying intracellular environment of different cells, raising the possibility that opioid receptor desensitization is mediated by different processes, and that different protein kinases are differentially activated or expressed in different cell types. For example, a recent study has shown that morphine can be converted into a rapidly desensitizing MOR agonist in LC neurons when protein kinase C is activated by phorbol esters or concomitant stimulation of Gq-coupled receptors [18]. Why does morphine cause minimal rapid MOR internalization?
The reason why morphine is less effective at producing MOR internalization remains obscure, although there is increasing evidence that morphine is not as effective as agonists such as DAMGO or etorphine at initiating specific steps in the GRK/arrestin pathway [7,10,19]. Indeed, morphine has been shown to induce substantially less phosphorylation of a putative GRK phosphorylation site (Ser375) in heterologously expressed MORs [19]. Furthermore, experiments in embryonic fibroblasts taken from arrestin-2/3 knockout mice, which completely lack native arrestin, confirmed that morphine activation of heterologously expressed MORs resulted in less translocation of fluorescent arrestin constructs to the plasma membrane than did other opioid agonists [10]. An alternative explanation for the differences between morphine and DAMGO emerged following reports that DAMGO, but not morphine, activated phospholipase D2 (PLD2) [20,21]. Activation of recombinant PLD2 in HEK-293 cells facilitated MOR internalization, whereas inhibition of this enzyme blocked it. Native MORs have not yet been shown to couple to PLD2 in neurons, but this pathway is the first reported to be activated by DAMGO but not morphine. Interestingly, a study modelling ligand/receptor/effector interactions suggested that the failure of morphine to recruit receptor regulatory mechanisms, including internalization, could be explained largely on kinetic grounds; this intriguing theory deserves more experimental attention [22]. www.sciencedirect.com
Opioids: cellular mechanisms of tolerance and physical dependence Bailey and Connor 63
Does morphine cause greater tolerance because of less MOR internalization?
Equianalgesic doses of opioid drugs do not produce equivalent amounts of tolerance in the whole animal. In particular, tolerance to the antinociceptive effects of morphine is greater than that seen with higher efficacy agonists such as etorphine or fentanyl [23]. Although this could be attributable to the fact that morphine would occupy more receptors than etorphine or fentanyl at equianalgesic doses, this finding, coupled with the repeated observation that morphine produces little MOR internalization, has generated two contrasting ideas concerning the role of MOR internalization in the development of cellular tolerance and dependence in vivo. If MOR resensitization is dependent upon internalization and dephosphorylation in endosomes (see Figure 1), then morphine might produce greater cellular tolerance because desensitized morphine-bound MORs cannot be internalized and resensitized; the other hypothesis suggests that, because morphine does not promote receptor internalization, receptors remain at the plasma membrane and continue to signal, thus disproportionately recruiting adaptations that induce cellular tolerance [24]. The idea that MOR resensitization following morphine treatment is comparatively less than with other opioid drugs is most strongly supported by evidence from cells heterologously expressing high densities of MORs. In these cells, resensitization of the MOR response following morphine-induced desensitization was significantly slower than resensitization following DAMGO treatment [19,25,26]. This has been attributed to relatively slower dephosphorylation of morphine-activated receptors compared with DAMGO-activated receptors [19], although direct evidence for MOR dephosphorylation in endosomes is still lacking. Unfortunately, the interpretation of these studies is significantly hampered by a lack of correlation between the time-course of MOR desensitization (maximal in approximately 4 h) and receptor internalization (maximal in less than 30 min), as well as by the failure to demonstrate any correlation between receptor re-insertion and recovery of agonist responses [3]. The notion that morphine produces more profound cellular adaptations than do other agonists because of its reduced capacity to initiate receptor internalization remains controversial [14,15,24,27]. One cellular consequence of chronic morphine is ‘superactivation’ of adenylyl cyclase (AC); it is claimed that this occurs as an adaptive response to prolonged MOR activation by morphine [24]. However, acute ‘superactivation’ of AC is an adaptation produced by all MOR agonists, including those that cause rapid MOR desensitization and internalization. Superactivation might be a correlate of the upregulated AC activity reported in chronically opioidtreated animals, but this in vivo phenomenon is also likely to involve increased AC protein [28]. Morphine produced www.sciencedirect.com
greater AC superactivation in HEK-293 cells transfected with MORs than it did in cells expressing mutant MORs that internalized in response to morphine; however, the differences in AC superactivation produced by methadone acting at these internalizing and non-internalizing MOR variants was modest [24]. Furthermore, the observation that a low concentration of DAMGO reduced morphine-induced AC superactivation by ‘dragging’ MORs into the cell via activation of one half of a putative MOR dimer [27] could not be reproduced using other measurements of receptor desensitization in neurons or receptor internalization in HEK-293 cells [14,15]. The relationship between processes involved in MOR desensitization, internalization and trafficking and the development of cellular tolerance in neurons remains to be established. Studies in neurons have not yet directly correlated MOR desensitization with receptor internalization, and as the vast majority of desensitization and internalization studies have focused on the first hour of agonist treatment we do not know how the processes underlying rapid desensitization relate to loss of function following longer-term opioid agonist treatment. There are, however, hints that the mechanisms of acute receptor desensitization and resensitization are altered by chronic morphine treatment of animals, because the recovery of MOR responses in LC neurons following an acute desensitizing stimulus is slower and less complete in neurons from morphine-treated animals than in controls [29]. Further studies in neurons are essential, and the observation that morphine produces redistribution and aggregation of MORs in the dendrites but not soma of cultured nucleus accumbens neurons [30] reinforces the idea that commonly utilized non-neuronal cell models might not be appropriate for studying opioid receptor regulation owing to their lack of crucial components of regulatory pathways that exist in neurons.
Other proteins that may affect cellular tolerance The rewarding, dependence-producing and analgesic effects of opioids result from activation of MORs in several brain regions simultaneously. Functional and gene expression studies have suggested an ever-growing number of target proteins that are likely to be involved in in vivo adaptations to chronic opioid treatment. These proteins and their impact on opioid tolerance have been studied by diverse techniques, including inhibition with pharmacological antagonists, knockdown of expression with antisense oligonucleotides and genomic deletion in mice. d-opioid receptors
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responses to acute and chronic morphine. Deletion of the genes for DORs and preproenkephalin, the precursor of both m and d endogenous peptide agonists, inhibits the development of morphine tolerance in antinociceptive tests without affecting the development of adaptations expressed during morphine withdrawal [31,32]. Although the potency of morphine is unchanged in DOR knockout mice [31], morphine analgesia in wildtype animals is made more effective by co-administration of either DOR agonists or antagonists [33]. MORs and DORs exist on overlapping populations of neurons in pain-modulating regions of the CNS, and an intriguing explanation for MOR/DOR synergy is that MOR and DOR can exist as heterodimers [33,34]. More evidence for a close association between MORs and DORs comes from studies showing that treatment of rats or mice with MOR agonists for several days leads to translocation of DORs to neuronal plasma membranes [35,36]. In sensory neurons, activation of DORs leads to translocation of intracellular DORs to the cell surface via membrane fusion of vesicles that also contain the pro-nociceptive peptide calcitonin gene-related peptide, the release of which might be expected to oppose the antinociceptive effect of morphine and thus could contribute to morphine tolerance [37]. Neurokinins and other neurotransmitters
Genetic deletion of an ever-growing number of neurotransmitters and/or their receptors also modifies the development of tolerance to the antinociceptive effects of morphine, the rewarding effects of morphine and the expression of opioid withdrawal [38], although in most cases the anatomical substrates and molecular mechanisms of these effects are not established. The most intriguing studies implicate recently identified neurotransmitters such as orexin, as well as well-known transmitters such as neurokinins [39,40]. Neurokinin-1 receptor (NK1) knockout mice do not self-administer morphine or exhibit conditioned place preference to morphine administration, although their withdrawal signs are largely preserved [40,41]. Possible target sites for NK1-mediated effects include the nucleus accumbens and amygdala [42]. Putative heterodimerization between NK1 receptors and MORs affects receptor trafficking in HEK-293 cells, but the role of heterodimerization with NK1 receptors in normal MOR function remains to be established [43]. G protein regulator and receptor trafficking proteins
Deletion of genes encoding proteins involved in regulating G protein function (e.g. RGS-9) [44] or receptor desensitization and trafficking (e.g. arrestin-3) [8–10,45] have profound effects on the potency of morphine to produce reward or analgesia, as well as the development of tolerance. There is considerable evidence that chronic opioid treatment can affect neuronal mRNA and protein levels of RGS [44,46,47] and GRKs [48–50], as well as Current Opinion in Pharmacology 2005, 5:60–68
the protein level and phosphorylation state of arrestin-3 [45,48,51,52]. Evidence from heterologous systems indicates that RGS proteins are also involved in opioidinduced AC supersensitization [53,54]. However, although it is tempting to speculate that disruption of the direct interaction between MORs and these proteins abrogates adaptations to chronic opioid treatment, deletion of these proteins will affect the signalling of multiple G-protein-coupled receptors both on morphinesensitive neurons and on other neurons in MOR-related circuits; currently, there are no data from RGS-9, GRK3 or arrestin-3 knockout animals to demonstrate that cellular or synaptic adaptations to chronic morphine treatment are affected. Direct evidence that an RGS protein in MOR-containing neurons contributes to morphine tolerance comes from a study that demonstrated increased RGS4 proteins levels following chronic morphine treatment; intracellular application of this protein attenuated the acute response to morphine in these neurons [46]. Synaptic transmission and plasticity
It is perhaps not surprising that deletion of genes encoding proteins involved in synaptic transmission and synaptic plasticity, such as the NR2A subunit of the N-methyl-D-aspartate receptor [55,56], also affects the development and expression of morphine tolerance and dependence. Clever experiments have extended the utility of gene knockout mice by re-introducing the deleted gene into specific brain regions using either virally mediated transfer (e.g. for RGS-9) or electroporation (e.g. for NR2A). NR2A re-introduction in the ventral tegmental area and periaqueductal grey (PAG) regions partially restored the expression of tolerance to the antinociceptive effects of morphine, whereas reintroduction into the nucleus accumbens partially restored morphine withdrawal signs [55]. In addition to the potential role of RGS proteins in tolerance, selective expression in the nucleus accumbens of an RGS-9 construct also abolished the exaggerated sensitivity to the rewarding properties of morphine present in RGS-9 knockout mice [44].
Adaptive changes following chronic opioid treatment that might underlie physical dependence Adaptive changes in neurons and neuronal communication are a hallmark of chronic morphine treatment; such adaptations must underlie altered behaviour associated with morphine dependence and withdrawal syndrome, as well as drug-induced craving and relapse to drug use (see also review by Weiss in this issue). Adaptations affecting neuronal excitability and synaptic transmission
The most well-described consequence of morphine withdrawal is a rebound increase in cAMP levels, probably www.sciencedirect.com
Opioids: cellular mechanisms of tolerance and physical dependence Bailey and Connor 65
produced by ‘superactivation’ of AC and upregulation of the amount of enzyme. There is also evidence that activation of TrkB receptors by brain-derived neurotrophic factor can contribute to cAMP superactivation in LC neurons [57]. Earlier studies are reviewed by Williams, Christie and Manzoni [28], and the strong evidence for enhanced g-aminobutyric acid (GABA)mediated synaptic transmission during morphine withdrawal in brain regions such as the PAG, ventral tegmental area, nucleus accumbens and dorsal raphe is discussed in detail. AC-mediated effects on GABA release are largely mediated by protein kinase A, but some effects are attributable to the conversion of cAMP to adenosine (Figure 2), [58]. For example, in mouse (but not rat) PAG, there is a protein kinase A-dependent increase in GABA release and a concomitant elevation of extracellular adenosine, which acts on inhibitory A1 receptors to reduce excess GABA release [59]. Inhibitors of cAMP metabolism and nucleoside transport block adenosine-dependent inhibition of GABA release, implying that the withdrawal-induced adenosine tone results
from excessive AC activation. Another study has demonstrated enhanced adenosine inhibition of synaptic inputs to nucleus accumbens neurons [60], an effect attributed to a reduction in adenosine transport. Although there is considerable evidence for adaptations to chronic morphine treatment at the level of nerve terminals, little progress has been made in elucidating the mechanisms that underlie changes in neuronal cell body firing rates that occur during opioid withdrawal [28]. The identity of ‘cation channels’ proposed to underlie withdrawal excitation of opioid-sensitive PAG or LC neurons has not been established; however, the recent observation that currents mediated by the dopamine transporter can profoundly affect the firing rate of dopaminergic neurons [61] indicates that the activity of similar transporters (e.g. those for noradrenaline or GABA) during opioid withdrawal is worth examining, particularly in light of the dramatic changes in neurotransmitter release during this process. There is evidence for alterations of glutamate transporter expression following
Figure 2
?↑ cAMP
SOMA
Rebound AC superactivation / AC upregulation +
PKA
Cations
Nerve terminal K+ +
Rebound AC superactivation / AC upregulation
cAMP
MOR
Altered sensitivity of signal transduction pathway – – ?↑ cAMP
–
+
–
PKA +
Ca2+
Transmitter release
Adenosine A1 receptor Current Opinion in Pharmacology
Hyperexcitability and enhanced transmitter release during opioid withdrawal. Under normal conditions, MOR activation can result in inhibition of AC, inhibition of Ca2+ channels and opening of voltage-gated K+ channels. Following withdrawal from chronic opioid treatment, many neurons exhibit hyperexcitability at the soma, a process thought to be mediated, at least in part, by induction of a cation current through protein kinase A (PKA) activation following rebound cAMP superactivation or by increased levels of AC. Additionally, transmitter release at the nerve terminal can be enhanced during opioid withdrawal. The mechanisms underlying this process include rebound elevated cAMP levels or increased amounts of AC protein, causing enhanced transmitter release through PKA activation. Upregulated AC is, in turn, more sensitive to inhibition by opioids. In addition, enhanced cAMP production can also enhance extracellular levels of adenosine, causing inhibition of transmitter release through adenosine A1 receptors. Figure adapted from [28]. www.sciencedirect.com
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chronic morphine treatment in glia [62], spinal cord neurons [63] and hippocampal neurons [64], although it is not known whether these changes in glutamate transporters occur in opioid-sensitive neurons. Transcription factors underlying adaptations to opioids
Synaptic plasticity is thought to be important in mediating certain behavioural effects of chronic opioid treatment (see review by Jones and Bonci in this issue). Activation of the cAMP-activated transcription factor — cAMP response element binding protein (CREB) — is heavily implicated in various forms of synaptic plasticity and, because elevations in cAMP have long been considered central to the expression of opioid withdrawal, CREB is thought to be important for withdrawal-dependent synaptic plasticity [65]. Region-specific increases in phosphorylated CREB have been demonstrated in the rat brain following morphine withdrawal, notably in the nucleus accumbens, amygdala and hypothalamus [66]; manipulating CREB activity in the nucleus accumbens affects behavioural responses to rewarding stimuli, including morphine [67]. Few reports have linked changes in CREB activity to any specific cellular adaptation to acute or chronic opioid treatment, with the exception of one study that demonstrated attenuated hyperexcitability in LC neurons following morphine withdrawal in CREB1 knockout mice, or in mice with a CREB1 mutant that showed decreased transcriptional activity [68].
phine, and which of these adaptations give rise to the persistently altered behaviours found in animals and humans exposed to chronic opioids. The next few years should yield further progress towards defining the cellular molecular processes underlying the behavioural adaptations to opioid drugs.
Update The mechanism linking upregulated AC activity and morphine-withdrawal-induced hyperexcitability of PAG neurons has been established in a recent paper [72], which demonstrated that most PAG neurons in brain slices from morphine-dependent mice exhibit a novel opioid-sensitive cation conductance following morphine withdrawal. This conductance was produced by a PKAdependent increase in the activity of the GABA transporter (GAT-1), and inhibitors of GAT-1 blocked the withdrawal-induced hyperexcitability of PAG neurons. GAT-1 inhibitors are currently used to treat epilepsy; this study suggests they may also have efficacy in treating opioid withdrawal symptoms.
Acknowledgements This work was funded in part by the Wellcome Trust (CPB) and the National Health and Medical Research Council of Australia Project Grant #302002 (MC).
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
Mitogen-activated protein kinases
Mitogen-activated protein kinases (MAPKs) are activated by many G-protein-coupled receptors, including opioid receptors, and their activation has been implicated in synaptic plasticity [69]. Acute morphine treatment increases levels of phosphorylated (activated) MAPK in some brain regions including the anterior cingulate, somatosensory and association cortices and LC, but decreases activated MAPK levels in the nucleus accumbens and amygdala [70,71]. Interestingly, with the exception of the LC, the increases in activated MAPKs were seen in neurons adjacent to those expressing MORs, suggesting that opioid modulation of intercellular communication is important in modulating MAPK activity.
Conclusions The cellular consequences of MOR activation are well understood, and significant progress has been made in understanding the acute regulation of MORs heterologously expressed in cell lines. Furthermore, the involvement of diverse neurotransmitter systems and intracellular signalling proteins in acute and chronic opioid actions has been revealed by the use of knockout mice. However, the challenge for biologists interested in understanding the consequences of chronic morphine exposure is to establish which of the defined actions of morphine at the molecular level are important for producing cellular tolerance and synaptic adaptation to morCurrent Opinion in Pharmacology 2005, 5:60–68
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