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. 2014 Jul 1;172(2):364–374. doi: 10.1111/bph.12653

Opioid receptor trafficking and interaction in nociceptors

X Zhang 1, L Bao 2, S Li 3
PMCID: PMC4292953  PMID: 24611685

Abstract

Opiate analgesics such as morphine are often used for pain therapy. However, antinociceptive tolerance and dependence may develop with long-term use of these drugs. It was found that μ-opioid receptors can interact with δ-opioid receptors, and morphine antinociceptive tolerance can be reduced by blocking δ-opioid receptors. Recent studies have shown that μ- and δ-opioid receptors are co-expressed in a considerable number of small neurons in the dorsal root ganglion. The interaction of μ-opioid receptors with δ-opioid receptors in the nociceptive afferents is facilitated by the stimulus-induced cell-surface expression of δ-opioid receptors, and contributes to morphine tolerance. Further analysis of the molecular, cellular and neural circuit mechanisms that regulate the trafficking and interaction of opioid receptors and related signalling molecules in the pain pathway would help to elucidate the mechanism of opiate analgesia and improve pain therapy.

LINKED ARTICLES

This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2

Keywords: μ-opioid receptor, δ-opioid receptor, nociceptor, dorsal root ganglion, receptor trafficking, receptor interaction, morphine antinociceptive tolerance

Background

Morphine is widely used for pain therapy. However, its clinical applications are often limited by the development of antinociceptive tolerance. That is, when a dose of morphine is given repeatedly and selectively for a single condition, it gradually loses its antinociceptive potency (Fields, 2004; 2011; Manchikanti and Singh, 2008). Studies over the past few decades have demonstrated the presence of many types of opioid receptor in the nociceptive sensory neurons and their Aδ- and C-fibre terminals in the superficial dorsal horn of the spinal cord (Fields et al., 1980; Moskowitz and Goodman, 1984; Gouarderes et al., 1991; Besse et al., 1992; Mennicken et al., 2003). The μ- and δ-opioid receptors are predominantly found to be expressed in small-diameter neurons of the dorsal root ganglion (DRG). These small DRG neurons convey the signals from peripheral nociceptors, thermoreceptors and sensitive mechanoreceptors to the superficial dorsal horn of spinal cord, and cause the release of the excitatory neurotransmitter glutamate, as well as the neuropeptides substance P and calcitonin gene-related peptide (CGRP), from the afferent terminals. This excitatory neurotransmission can be presynaptically inhibited by activating μ- or δ-opioid receptors (Ueda et al., 1995; Zachariou and Goldstein, 1996; Beaudry et al., 2011). Therefore, it is of interest to explore the molecular and cellular mechanisms that regulate opioid analgesia and tolerance. The present review discusses the expression, intracellular trafficking and interaction of opioid receptors in the pain pathway with a focus on small DRG neurons and the role of opioid receptor interactions in the modulation of opiate analgesia and antinociceptive tolerance.

Co-expression of opioid receptors in nociceptive afferent neurons

Early autoradiographic studies showed that many opioid receptors and the binding sites for μ- and δ-opioid receptor agonists, are present in nociceptive afferent Aδ- and C-fibres terminating in the superficial dorsal horn of the spinal cord (Fields et al., 1980; Moskowitz and Goodman, 1984; Gouarderes et al., 1991; Besse et al., 1992; Mennicken et al., 2003). Moreover, the release of the excitatory neurotransmitter glutamate, the neuropeptides substance P and CGRP from afferent C- and Aδ-fibres could be inhibited by activating δ-opioid receptors with several δ-opioid receptor agonists (Ueda et al., 1995; Zachariou and Goldstein, 1996; Beaudry et al., 2011; Normandin et al., 2013), suggesting the presynaptic localization of δ-opioid receptors on nociceptive afferents. This notion was supported by the finding that δ-opioid receptor mRNA is present in about 70% of DRG neurons, including both peptidergic [isolectin B4 (IB4)-negative] and non-peptidergic (IB4-positive) subsets of small neurons and mechanoreceptive large neurons, while μ-opioid receptors were expressed in the subsets of small DRG neurons and some large DRG neurons (Arvidsson et al., 1995a; Minami et al., 1995a; Wang and Wessendorf, 2001; Wang et al., 2010; Gaveriaux-Ruff et al., 2011; He et al., 2011). The level of δ-opioid receptor mRNA in small DRG neurons is generally lower than that in large DRG neurons (Wang et al., 2010). About one-third of δ-opioid receptor-expressing DRG neurons contain neuropeptides, such as substance P and CGRP (Wang et al., 2010). Co-expression of μ- and δ-opioid receptors was found in a substantial population of peptidergic small DRG neurons by using single-cell PCR, in situ double hybridization and other approaches (Joseph and Levine, 2010; van Rijn et al., 2010; Wang et al., 2010; Beaudry et al., 2011; He et al., 2011). μ- and δ-opioid receptors are also co-expressed in small DRG neurons which do not contain substance P (Wang et al., 2010).

To determine the amount of δ-opioid receptor protein, immunoblot analyses were carried out with δ-opioid receptor antibodies; these specifically detected δ-opioid receptors in the DRGs and the dorsal spinal cord of wild-type mice, but not in δ-opioid receptor-deficient mice (Wang et al., 2010; He et al., 2011; Zhao et al., 2011). It is notable that the correct dilutions of the same antibodies should be used to determine the specific immunostaining of δ-opioid receptors in the DRGs and spinal cord (Wang et al., 2010; Zhang and Bao, 2012). Although some antibodies specifically labelled δ-opioid receptors in the peptidergic small DRG neurons and large DRG neurons (Arvidsson et al., 1995a; Guan et al., 2005; Wang et al., 2010; He et al., 2011), these antibodies could not simultaneously label the δ-opioid receptors expressed in all subsets of DRG neurons and the receptors in all afferent terminals in the spinal cord. Thus, the immunostaining patterns in the spinal cord only partially represent the autoradiographic patterns of the binding sites of δ-opioid receptor agonists. For instance, the antibodies that stain δ-opioid receptors in small DRG neurons could detect the receptors accumulated in the afferent terminals in the superficial dorsal horn of spinal cord, but not the receptors in the spinal cord neurons. However, double immunostaining can be used to demonstrate the co-expression of δ- and μ-opioid receptors in small DRG neurons and their afferent terminals. Importantly, Gupta et al. have developed antibodies that recognize the δ-/μ-opioid receptor heteromer, and showed the presence of this opioid receptor heteromer in both small and large DRG neurons (Gupta et al., 2010), supporting the notion that δ- and μ-opioid receptors are co-expressed in these neurons.

In addition to the above approaches, the expression of opioid receptors fused with various fluorescent proteins or epitope tags in animals may help to study the distribution of opioid receptors. However, in the mouse the expression of δ-opioid receptors inserted with the enhanced green fluorescent protein at the C-terminus (DOPr-eGFP) was only found in ∼17% of DRG neurons by immunostaining with the antibody against GFP and most of these neurons were large neurons (Scherrer et al., 2009). Moreover, DOPr-eGFP were not detected in μ-opioid receptor-containing small DRG neurons, suggesting that the δ-opioid receptor might not coexist with the μ-opioid receptor in nociceptive DRG neurons. These data do not accord with the results obtained using other multiple approaches, and could be due to a reduced expression of DOPr-eGFP or a degradation of the fusion protein which could not correctly move into the secretory pathway and get transported to the afferent axons (Guan et al., 2005; Wang et al., 2008; 2010). A recent study was able to show the presence of DOPr-eGFP in a few substance P-containing small DRG neurons, and coexistence of DOPr-eGFP and μ-opioid receptor in a small population of large DRG neurons that contained CGRP (Bardoni et al., 2014). Therefore, it is also possible that the methods used in these studies are still not sensitive enough to detect the receptor expressed at low levels in various types of neurons. The distribution of DOPr-eGFP cannot fully represent the endogenous δ-opioid receptor in all subsets of DRG neurons (Zhang and Bao, 2012). It is still not known whether the in vivo expression of δ-opioid receptors inserted with a small tag, such as haemagglutinin (HA), Myc or Flag, might be better than GFP for showing their distribution in all subsets of DRG neurons.

The co-expression of δ- and μ-opioid receptors in peptidergic small DRG neurons is also supported by the finding that both of these receptors mediated inhibitory effects on the Ca2+ currents in the same small DRG neurons and the release of substance P from C- and Aδ-afferents (Arvidsson et al., 1995a; b; Ji et al., 1995; Zachariou and Goldstein, 1996; Zhang et al., 1998a; b; Wu et al., 2004; Guan et al., 2005; Rau et al., 2005; Walwyn et al., 2005; Beaudry et al., 2011; Kouchek et al., 2013; Normandin et al., 2013). Taken together, these results suggest that the co-expression of δ- and μ-opioid receptors in certain populations of small DRG neurons is the cellular basis for opioid receptor interactions in the pain pathway.

Distinct subcellular distribution of opioid receptors

Newly synthesized receptors are usually processed in the Golgi complex and assemble in various microvesicles in the constitutive secretory pathway to be transported and inserted into the plasma membrane (PM) spontaneously, while secretory polypeptides and proteins are collected in large dense-core vesicles (LDCVs) in the regulated secretory pathway to be stored in the cytoplasm and released at the PM in response to stimuli that increase the intracellular levels of Ca2+. Immunostaining with antibodies against δ-opioid receptors or epitope-tag HA and Myc shows that both the endogenous δ-opioid receptor and exogenously expressed HA- and Myc-δ-opioid receptors are mainly located intracellularly and often associated with LDCVs in both peptidergic small DRG neurons and phaeochromocytoma (PC12) cells, whereas HA- or Myc-δ-opioid receptors are mostly present on the cell surface of large DRG neurons and HEK293 cells, which do not contain LDCVs and neuropeptides (Table 1; Cheng et al., 1995; Bao et al., 2003; Guan et al., 2005; Wang et al., 2010; Zhao et al., 2011; Zhang and Bao, 2012). In addition, the scattered distribution of δ-opioid receptors in the cytoplasm indicates the receptors in the constitutive pathway. In contrast to the tagged δ-opioid receptors, HA- and Myc-μ-opioid receptors often appear on the cell surface, consistent with the localization of μ-opioid receptors shown using μ-opioid receptor antibodies (Table 1; Zhang et al., 1998b; 2010; Wang et al., 2010). Thus, in the steady state, there are two pools of opioid receptors in nociceptive afferent neurons, the surface pool containing mostly μ-opioid receptors and an intracellular pool of δ-opioid receptors (Figure 1).

Table 1.

Subcellular distribution of δ-opioid receptor (DOPr) and μ-opioid receptor (MOPr) in DRG neurons, PC12 cells and HEK293 cells

Receptor subtype Subcellular localization Cell type Method Reference
DOPr PM, LDCV, MV, endosome, TGN Small DRG neuron IFM, IEM, IB Cheng et al., 1995; Zhang et al., 1998a; Bao et al., 2003; Guan et al., 2005; Wang et al., 2010; Gupta et al., 2010; Zhao et al., 2011
PM, GN Large DRG neuron IFM Wang et al., 2010; Gupta et al., 2010
PM, LDCV, MV, TGN PC12 cell IFM, IEM, IB Bao et al., 2003; Guan et al., 2005; Wang et al., 2008
PM, MV, TGN HEK293 cell (Tg) IFM, IEM Guan et al., 2005; Wang et al., 2010
DOPr-HA (or -Myc, -FLAG) PM, LDCV, TGN Small DRG neuron (Tg) IFM Wang et al., 2010
PM, GN Large DRG neuron (Tg) IFM Wang et al., 2010
PM, LDCV, MV, TGN PC12 cell (Tg) IFM, IEM Bao et al., 2003; Guan et al., 2005; Wang et al., 2008; Wang et al., 2010
PM, MV, TGN HEK293 cell (Tg) IFM, IEM Whistler et al., 2001; Whistler et al., 2002; Guan et al., 2005; Rozenfeld and Devi, 2007; Wang et al., 2010; Milan-Lobo and Whistler, 2011; He et al., 2011
DOPr-GFP (or -RFP) PM, GN Small DRG neuron (Tg) Wang et al., 2010; Pradhan et al., 2010; Pettinger et al., 2013
PM, GN Large DRG neuron (Tg) Pradhan et al., 2009; Wang et al., 2010; Pradhan et al., 2010
PM, GN PC12 cell (Tg) Wang et al., 2008
PM, GN HEK293 cell (Tg) Kabli et al., 2010
MOPr PM, MV, TGN Small DRG neuron IFM, IEM Zhang et al., 1998b
PM, GN HEK293 cell (Tg) IFM Wang et al., 2010
MOPr-HA (or -Myc, -FLAG) PM, GN PC12 cell (Tg) IFM Guan et al., 2005
PM, GN HEK293 cell (Tg) IFM Whistler et al., 1999; Whistler et al., 2002; He et al., 2002; Pfeiffer et al., 2002; Rozenfeld and Devi, 2007; Wang et al., 2010; Milan-Lobo and Whistler, 2011
MOPr-GFP PM, GN HEK293 cell (Tg) Celver et al., 2004; Kabli et al., 2010
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MV, microvesicle; TGN, trans-Golgi network; Tg, transgene; IFM, immunofluorescent microscopy; IEM, immuno-electron microscopy; IB, immunoblotting.

Figure 1.

Figure 1

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Proposed models of opioid receptor trafficking and interaction in nociceptive afferent neurons in response to the acute treatment with opioid agonists. There are two pools of δ-opioid receptors (DOPr), namely the surface pool and the intracellular pool, in peptidergic small DRG neurons, whereas the μ-opioid receptor (MOPr) is mainly present on the cell surface. In the steady state, the intracellular δ-opioid receptor can be transported via the LDCVs in the regulated secretory pathway, in addition to the constitutive transport of δ-opioid receptors via microvesicles. A limited number of surface δ-opioid receptors interact with μ-opioid receptors and form μ-/δ-opioid receptor heteromers. Both δ-opioid receptors and the heteromers are internalized following treatment with a δ-opioid receptor agonist, and processed for degradation. At the same time, the δ-agonists may induce a slow but lasting cell-surface expression of δ-opioid receptors that maintains the neuronal sensitivity to δ-agonists. Both μ-opioid receptors and the heteromers are internalized by some μ-opioid receptor agonists such as DAMGO, and processed for recycling.

It is of interest to elucidate the mechanisms that regulate receptor trafficking in different cell types. The HA- or Myc-tagged δ-opioid receptors present in the LDCV of transfected small DRG neurons could be shifted to the PM in the absence of a protachykinin that interacts with δ-opioid receptors, consistent with the reduction in δ-opioid receptors located in the LDCV of small DRG neurons observed in protachykinin gene-knockout mice (Guan et al., 2005; Ma et al., 2008; Wang et al., 2010; Zhang et al., 2010). Moreover, numerous δ-opioid receptors underwent degradation in the protachykinin-deficient small DRG neurons (Guan et al., 2005). Thus, the δ-opioid receptor/protachykinin interaction is essential for the transporting of δ-opioid receptors into LDCVs in nociceptive sensory neurons. However, the mechanisms for the transfer of δ-opioid receptors into secretory vesicles could be significantly different in the various types of neurons and cells.

The distributions of exogenous HA- or Myc-δ-opioid receptors and tagged μ-opioid receptors in DRG neurons are consistent with the distribution patterns of endogenous δ- and μ-opioid receptors shown with the δ- and μ-opioid receptor-specific antibodies (Cheng et al., 1995; Zhang et al., 1998a,b; 2010; Bao et al., 2003; Guan et al., 2005; Wang et al., 2010; Zhao et al., 2011). However, in small DRG neurons and PC12 cells transfected with the plasmids expressing DOPr-eGFP, the DOPr-eGFP cannot be sorted into LDCVs, but is transported via the constitutive secretory pathway to be inserted spontaneously into the PM (Table 1; Wang et al., 2008; 2010; Zhang and Bao, 2012). Therefore, eGFP insertion may alter the intracellular trafficking of the newly synthesized receptors. It was also noticed that the δ-opioid receptor antibodies could not simultaneously label the δ-opioid receptors in LDCVs or in the PM of neurons, suggesting that the antibodies might preferentially recognize the δ-opioid receptors in different conformational or folding states. Interestingly, DOPr-eGFP synthesized in large DRG neurons could be transported to the peripheral terminals of Aβ-fibers in the skin (Bardoni et al., 2014), but not to the central terminals of Aβ-fibres in the lamina III-V of spinal cord. This polarized transport of the receptor could be true, because neither autoradiography nor immunostaining could show the enrichment of δ-opioid receptors in Aβ-fibre terminals in the deep dorsal horn (Mennicken et al., 2003; Wang et al., 2010; see Zhang and Bao, 2012). However, both methods could detect the δ-opioid receptors stored in C- and Aδ-fibre terminals in the spinal lamina I–II, although the expression level of the receptor in small DRG neurons is much lower than that in large DRG neurons. Accumulating evidence suggest that the protein levels of δ-opioid receptors in the central or peripheral terminals are not always match to the levels of receptor mRNA in the DRG neurons, due to the differential processing of the synthesized receptors for transport in different types of neurons. Therefore, the mechanism for regulating the opioid receptor trafficking is an attractive research direction for the cell biology of neurons.

Stimulus-induced cell-surface expression of δ-opioid receptors and G-protein complex

It could be expected that the LDCV-localized δ-opioid receptors would be inserted into the PM when the exocytosis of LDCVs occurs in response to various stimuli, such as membrane depolarization and treatment with capsaicin or ATP, which elevate the intracellular levels of Ca2+ (Bao et al., 2003; Wang et al., 2010; Zhao et al., 2011). The rate of δ-opioid receptor insertion is dependent on the pattern of Ca2+ elevation. The δ-opioid receptor insertion following treatment with δ-opioid receptor agonists, which induces a low level but long-lasting Ca2+ elevation, is more pronounced than that induced by K+-induced membrane depolarization which induces a fast and high Ca2+ influx (Bao et al., 2003; Figure 1). In fact, a number of chemical and behavioural stimuli, including sustained pain conditions and prolonged treatment of morphine or ethanol could induce δ-opioid receptor insertion (Cahill et al., 2001; Bao et al., 2003; Patwardhan et al., 2005; Walwyn et al., 2005; Gendron et al., 2006; Ma et al., 2006; Gupta et al., 2010; van Rijn et al., 2012; Pettinger et al., 2013) (Figure 2). It is likely that in addition to δ-opioid receptors in the regulated secretory pathway, δ-opioid receptors in the constitutive secretory pathway could be also available for the stimulus-induced membrane insertion, since morphine-induced surface expression of δ-opioid receptors was shown in both small DRG neurons and the spinal dorsal horn neurons with the antibodies that seldom detected the LDCV-localized δ-opioid receptors (Cahill et al., 2001; Gendron et al., 2006). In contrast to the δ-opioid receptor, μ-opioid receptors are expressed on the cell surface without stimulation (Zhang et al., 1998b; 2010; Wang et al., 2010). Therefore, the μ-/δ-opioid receptor interaction is enhanced by the stimulus-induced δ-opioid receptor insertion, although the opioid receptor heteromers can also be present in the cytoplasm (Gupta et al., 2010). In the stimulated state, the number of μ-/δ-opioid receptor heteromers in the surface pool is increased in nociceptive afferent neurons (Figure 2).

Figure 2.

Figure 2

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Proposed models of the stimulus-induced opioid receptor trafficking and interaction in nociceptive afferent neurons and the subsequent effects of agonist treatments. In the stimulated state induced by membrane depolarization and other stimuli, the δ-opioid receptor (DOPr) in the regulated pathway could be delivered to the surface pool and, therefore, increase the number of μ-/δ-opioid receptor heteromers as well as δ-opioid receptor monomers and homomers. When the stimulated neurons are further treated with δ-opioid receptor agonists, the heteromers, δ-opioid receptor monomers and homomers are internalized and processed for degradation. The reduction of μ-opioid receptors (MOPr) on the cell surface could be significant due to the increased number of μ-/δ-opioid receptor heteromers. In contrast, the heteromers, μ-opioid receptor monomers and homomers internalized by μ-opioid receptor agonists, such as DAMGO, could be recycled to the cell surface.

Recently, at least 298 proteins have been identified in the LDCV membrane purified from the dorsal spinal cord, including GPCRs, G-proteins and other signalling molecules, and ion channels (Zhao et al., 2011). In small DRG neurons, δ-opioid receptor/Gαi2/Gβ1γ5/PLC β2 complexes are localized in the substance P-containing LDCVs. Electrical stimulation at 10 Hz increases the cell-surface level of δ-opioid receptors and Gαi2 in small DRG neurons. In contrast to Gαi2, Go is mostly associated with the PM (Campbell et al., 1993; Menon-Johansson and Dolphin, 1993; Zhao et al., 2011). Go contributes to maximally efficient signalling and the antinociceptive effects of μ-opioid receptors (Lamberts et al., 2011). Therefore, stimuli, such as membrane depolarization and capsaicin treatment, could induce the cell-surface expression of a preassembled δ-opioid receptor/G-protein complex, which may interact with the surface μ-opioid receptor and Go (Figure 3). Furthermore, δ-opioid receptor-mediated functions may also be regulated by the δ-opioid receptor interaction with numerous membrane proteins such as Ca2+ channels and Na+, K+-ATPase that are expressed in small DRG neurons (Mata et al., 1991; Hamada et al., 2003; Deng et al., 2009; Wu et al., 2009; Heinke et al., 2011; Li et al., 2011). The δ- and μ-opioid receptors could interact with β2- and α2A-adrenoceptors in DRG neurons (Jordan et al., 2001; 2003; Overland et al., 2009; Zhao et al., 2011; Schuster et al., 2013). It is expected that the stimulus-induced insertion of δ-opioid receptors and related signalling complex would rapidly change the sensitivity of nociceptive afferent neurons to many neurotransmitters, neuromodulators and applied drugs.

Figure 3.

Figure 3

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Subcellular translocation of δ-opioid receptors (DOPr) and related signalling molecules in nociceptive afferent neurons. Preassembled DOPr/Gαi2/Gβ1γ5/PLCβ2 complexes associated with the LDCV membrane are delivered to the cell surface in response to the Ca2+ influx. The receptor/G-protein complex might be also associated with the microvesicles in the constitutive secretory pathway to be delivered spontaneously. In the PM, the DOPr/Gαi2/Gβ1/PLCβ2 complexes interact with μ-opioid receptors (MOPr) which may interact with Go during MOPr agonist treatment. Following agonist treatment, the receptor heteromers are internalized, and processed for either recycling to the cell surface or degradation in the lysosomes.

Post-endocytic pathways for the opioid receptor complex

The GPCRs activated by selective agonists are often internalized and processed in either the recycling pathway for re-sensitization or the degradation pathway that leads to receptor down-regulation (Trapaidze et al., 2000; Tsao and von Zastrow, 2000). Accumulated evidence has shown that internalized μ- and δ-opioid receptors are differentially processed in post-endocytotic pathways. Internalized μ-opioid receptors can be recycled to the PM and re-sensitized after treatment with a specific agonist, [D-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin (DAMGO; Arden et al., 1995; Law et al., 2000; Finn and Whistler, 2001). In contrast, internalized δ-opioid receptors can be processed in the lysosomal compartments for degradation after treatment with their agonists (Trapaidze et al., 1996; Tsao and von Zastrow, 2000; Hislop et al., 2009). Agonist-induced receptor phosphorylation and ubiquitination are involved in the endocytosis and down-regulation of opioid receptors (Finn and Whistler, 2001; Hislop et al., 2009).

The δ-/μ-opioid receptor interaction plays an important role in regulating the opioid receptor trafficking, signalling and metabolism (Zhang et al., 2006; Berger and Whistler, 2010; Chao and Xia, 2010; van Rijn et al., 2010; Pradhan et al., 2011; Stockton and Devi, 2011). Given that the δ-opioid receptor interacts with the μ-opioid receptor and forms a heteromer (Gomes et al., 2004; Law et al., 2005), it would be interesting to know whether or how the receptor complexes are internalized and processed following agonist stimulation. In transfected cells, treatment with either δ-opioid receptor agonists or the μ-opioid receptor agonist DAMGO and methadone results in endocytosis of the μ-/δ-opioid receptor heteromers (He et al., 2011; Milan-Lobo and Whistler, 2011). The δ-opioid receptor agonist-induced slow but constant membrane insertion of δ-opioid receptors could be an intrinsic mechanism for replenishing the loss of surface δ-opioid receptors and maintaining the neuronal sensitivity to the agonists (Bao et al., 2003). β-Arrestin mediates the internalization of many GPCRs. The μ-/δ-opioid receptor heteromers could constitutively recruit β-arrestin, while the δ-opioid receptor but not the μ-opioid receptor is normally coupled with β-arrestin (Cheng et al., 1998; Rozenfeld and Devi, 2007). Moreover, the heteromers internalized by δ-opioid receptor agonists are often processed for lysosomal degradation, resulting in a reduction in both surface δ- and μ-opioid receptors (He et al., 2011; Figures 2 and 3). The basal level of co-degradation of μ-/δ-opioid receptor heteromers in the dorsal spinal cord may be caused by the opiate peptide enkephalin released from the local neurons (Cesselin et al., 1989; He et al., 2011). The receptor co-degradation was enhanced by exogenously applied δ-opioid receptor agonists (He et al., 2011) or persistent release of endogenous opioid peptides.

The μ-/δ-opioid receptor heteromers internalized by DAMGO are not subjected to the lysosomal degradation pathway, but might be recycled (He et al., 2011). Early studies showed that δ-opioid receptor agonists phosphorylated these receptors, leading to their degradation (Trapaidze et al., 1996; Gaudriault et al., 1997; Tsao and von Zastrow, 2000; Bao et al., 2003; Hislop et al., 2009). In contrast, the μ-opioid receptor, which is phosphorylated and internalized by DAMGO, is recycled to the cell surface and re-sensitized (Arden et al., 1995; Law et al., 2000; Finn and Whistler, 2001; Qiu et al., 2003). In the μ-/δ-opioid receptor heteromers, the μ- and δ-opioid receptors could be phosphorylated by their respective agonists without cross-phosphorylation between the two receptors (He et al., 2011). Such a segregated biochemical process in the receptor heteromers may lead to different fates for the internalized μ-/δ-opioid receptor heteromers following treatment with opiate ligands. We propose that the surface pool of μ-/δ-opioid receptor heteromers could be reduced by δ-opioid receptor agonists, but recycled by some μ-opioid receptor agonists such as DAMGO (Figure 2). However, it is not known whether the G-proteins might be involved in the internalization and post-endocytic trafficking of receptor heteromers. Moreover, it would be interesting to explore the differential regulation of μ-/δ-opioid receptor trafficking in nociceptive neurons following the application of various opioid receptor agonists.

Pharmacological significance of opioid receptor redistribution

Opioid receptor ligands are known to bind to opioid receptor subtypes with various affinities (Janecka et al., 2004; Trescot et al., 2008). The opioid agonists preferentially targeting one type of opioid receptor often also bind to two other opioid receptors with low affinities (Janecka et al., 2004). For instance, endogenous Leu-enkephalin has only modest affinity and selectivity for δ-opioid receptors. Deltorphin II binds to δ-opioid receptors with ∼3000-fold higher affinity than μ-opioid receptors. DAMGO has ∼1000-fold higher affinity for μ- compared to δ-opioid receptors. HEK293 cells and other cell lines expressing one type of opioid receptor are often used to analyse the pharmacological properties and selectivity of opioid ligands. However, when such analyses are applied for in vivo analyses of pain modulation, the data interpretation sometimes appears to be complicated.

In the resting state, only a limited number of δ-opioid receptors is present on the cell surface of nociceptive afferent neurons while μ-opioid receptors are present in abundance (Figure 2). A high dose of a δ-opioid receptor agonist might activate surface μ-opioid receptors, whereas a low dose of this agonist could only be enough to induce a δ-opioid receptor-specific effect when the number of δ-opioid receptors on the cell surface is markedly increased after various stimulations (Figure 2). This hypothesis may explain some seemingly conflicting observations suggesting that δ-opioid receptor agonist-induced antinociception is mediated by μ-opioid receptors under basal conditions, but mainly by δ-opioid receptors following physiological or pathological stimuli (Matthes et al., 1998; Zhu et al., 1999; Scherrer et al., 2004; 2009; van Rijn and Whistler, 2009; Dubois and Gendron, 2010). This could also explain why the presynaptic inhibition of sensory afferents in the spinal cord by a high dose of a δ-opioid receptor agonist could be mediated by μ-opioid receptors in basal conditions, but by δ-opioid receptors after treatment with a TRPV1 agonist (Wrigley et al., 2010), which may increase the cell-surface expression of δ-opioid receptors (Bao et al., 2003; Zhang et al., 2006). Therefore, changes in the number of opioid receptors and the ratio of δ- to μ-opioid receptors may contribute to the pharmacological properties of opioid ligands in vivo. The role the surface delivery of the δ-opioid receptor/Gαi2/Gβ1/PLCβ2 complex and its interaction with μ-opioid receptors and Go plays in the pharmacological effects of opiate analgesics remains to be investigated.

Role of opioid receptor interaction in morphine antinociceptive tolerance

Recent studies have shown that both thermal and mechanical hyperalgesia are inhibited by either δ- or μ-opioid receptor agonists through the activation of δ- or μ-opioid receptors, respectively, in nociceptive afferents (Joseph and Levine, 2010; Gaveriaux-Ruff et al., 2011; He et al., 2011; Kim et al., 2011; Normandin et al., 2013). These results are consistent with the coexistence of δ- and μ-opioid receptors in peptidergic small DRG neurons, and support the notion that δ- and μ-opioid receptors interact in the nociceptive sensory circuit. Such an interaction would affect many therapeutic aspects of opiate drugs.

Opioid analgesics (e.g. morphine) with high affinity for μ-opioid receptors are still the most powerful analgesics available for pain relief. However, their long-term use may lead to the development of antinociceptive tolerance and dependence (Fields, 2004; 2011; Manchikanti and Singh, 2008). Early studies showed that blockage of δ-opioid receptors enhanced morphine analgesia, and reduced analgesic tolerance (Abdelhamid et al., 1991; Schiller et al., 1999a; b; Schiller, 2010). Further studies revealed that morphine tolerance can be reduced by intrathecal application of the antisense oligodeoxynucleotide of the δ-opioid receptor gene (Oprd1), deleting either Oprd1 or the preproenkephalin gene (Penk1), preventing δ-opioid receptor phosphorylation or deleting Tac1, which reduces the transport of δ-opioid receptors to the spinal dorsal horn via LDCVs (Standifer et al., 1994; Zhu et al., 1999; Nitsche et al., 2002; Guan et al., 2005; Xie et al., 2009; Chen et al., 2012).

Daniels et al. (2005) reported that the bivalent ligands targeting the δ-/μ-opioid receptor heterodimer with the spacer length between the two pharmacophores longer than 22 Å did not induce morphine tolerance and dependence, suggesting that this heterodimer could be a signalling unit mediating tolerance and dependence through specific signal transducers that recognize and coupled the heterodimer but not μ-opioid receptor monomers/homomers. Recently, δ-opioid receptor agonist-induced co-degradation of μ-opioid receptors was found to be one of the mechanisms for morphine antinociceptive tolerance, and the tolerance could be reduced by disrupting the δ-/μ-opioid receptor interaction in the PM of nociceptive afferents with a TAT- and glutathione S-transferase-fused first transmembrane domain of the μ-opioid receptor that mediates the interaction with δ-opioid receptors (Filizola et al., 2002; He et al., 2011). This is direct evidence that the physical dissociation of μ- from δ-opioid receptors in nociceptive afferents in vivo improves opioid analgesia. Although some mechanisms of receptor internalization have been studied (He et al., 2002; 2011; Puthenveedu et al., 2010; Yu et al., 2010; Milan-Lobo and Whistler, 2011; Patierno et al., 2011; Anselmi et al., 2013), it would be interesting to further study the regulatory mechanisms for post-endocytic trafficking of the μ-/δ-opioid receptor heteromers following the application of different μ-opioid receptor agonists such as DAMGO, methadone and other opioid analgesics.

Conclusions

There is accumulating evidence that δ- and μ-opioid receptors are co-expressed in the nociceptive afferent neurons. The δ-opioid receptors can be distributed into both the constitutive and regulated secretory pathways. In contrast, μ-opioid receptors are mainly transported via the constitutive pathway. Thus, the δ-opioid receptor is often transferred into the PM in a stimulus-dependent manner, while the μ-opioid receptor moves there spontaneously. In the PM, these two types of opioid receptor interact and form heteromers to modulate the neuronal sensitivity to the opiate analgesics. The δ-opioid receptor agonist-induced co-degradation of μ-opioid receptors could be one of the mechanisms of morphine antinociceptive tolerance. It would be interesting to further study the translocation and interaction of opioid receptors and related signalling molecules in the nociceptive afferents, and their contribution to the pharmacological mechanisms of opiate analgesia.

Acknowledgments

This work was supported by National Natural Science Foundation of China (31130066 and 30930044), National Basic Research Program of China (2010CB912001) and the Strategic Priority Research Program (B) of Chinese Academy of Sciences (XDB01020300).

Glossary

Abbreviations

CGRP

calcitonin gene-related peptide

DOPr-eGFP

δ-opioid receptors inserted with the enhanced green fluorescent protein at the C-terminus

DAMGO

Tyr-D-Ala-Gly-MePhe-Gly-ol

DRG

dorsal root ganglion

HA

haemagglutinin

IB4

isolectin B4

LDCV

large dense-core vesicle

PC12 cell

phaeochromocytoma cell

PM

plasma membrane

Conflict of interest

The authors have no conflicts of interest.

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