Differential agonist efficacy for G-protein signalling and endocytosis
The discovery that different opioid agonists have different efficacies for G-protein signalling and mediation of receptor endocytosis has provided impetus to determine whether MOR regulatory mechanisms contribute to tolerance, which could explain why MOR function is lost in the absence of reduced MOR expression. It has been hypothesized that the poor ability of morphine to initiate efficient MOR endocytosis gives morphine high liability for causing tolerance. Many studies have established that morphine activates MOR but poorly induces endocytosis (Arden et al., 1995; Keith et al., 1996; Sternini et al., 1996; Whistler and von Zastrow, 1998; Borgland et al., 2003), as widely reviewed (see von Zastrow et al., 2003; Connor et al., 2004; Martini and Whistler, 2007; Koch and Hollt, 2008; Berger and Whistler, 2010; von Zastrow, 2010). Most quantitative studies of signalling efficacy have concluded that intrinsic efficacy to activate G-proteins versus endocytosis or βarr-2 association with MOR are not linearly related (Borgland et al., 2003; Molinari et al., 2010; but also see McPherson et al., 2010). Molinari et al. (2010) using RET methods, reported a hyperbolic relationship between intrinsic activity for G-protein and βarr-2, consistent with earlier studies. By contrast, McPherson et al. (2010) found a more linear relationship for both βarr-2 recruitment and endocytosis, with some outliers (but not morphine). This discrepancy could be due to the methods used to determine G-protein activation (GTPγS binding for 2 h by McPherson et al., 2010 and RET methods by Molinari et al., 2010), the expression of different densities of RET donors and acceptors in the two studies or the use an operational model by McPherson et al. (2010) but not Molinari et al. (2010). It is well established that overexpression of GRKs or arrestins can profoundly enhance induction of endocytosis by morphine (e.g. Whistler and von Zastrow, 1998; Bohn et al., 2004). Morphine also fails to induce MOR endocytosis in spinal cord in vivo (Trafton et al., 2000), but it efficiently induces endocytosis in medium spiny striatal neurons (Haberstock-Debic et al., 2003; Yu et al., 2009).
Do strongly internalizing opioid agonists produce less tolerance than weakly internalizing agonists?
Morphine produces more behavioural tolerance than strongly internalizing agonists. This finding has been widely cited to support the notion that MOR recycling influences tolerance. Morphine, for instance, produced greater opioid tolerance when compared with agonists like DAMGO, sufentanyl or etorphine, when equivalent induction doses and continuous infusions were used to control for pharmacokinetic differences (Stevens and Yaksh, 1989; Duttaroy and Yoburn, 1995; Madia et al., 2009). Whilst this seems to support the notion that strongly internalizing agonists produce less tolerance than weakly internalizing agonists, the interpretation is seriously confounded by large differences in intrinsic efficacy for G-protein activation among these agonists. Etorphine, sufentanyl and DAMGO all exhibit much higher intrinsic efficacy for G-protein activation than morphine (Traynor and Nahorski, 1995; Emmerson et al., 1996; Selley et al., 1998; McPherson et al., 2010; Molinari et al., 2010), although some studies using GTPγS binding have reported that the intrinsic efficacy of sufentanyl is comparable with morphine (Emmerson et al., 1996; Selley et al., 1998). Low intrinsic efficacy agonists usually produce larger rightward shifts in concentration-response curves than high efficacy agonists. This occurs when MOR–effector coupling is impaired either by irreversible antagonists or chronic drug treatment presumably because low intrinsic efficacy agonists such as morphine must occupy a greater fraction of the total receptor population to produce a given level of effect, due to lesser receptor reserve (e.g. Christie et al., 1987; Stevens and Yaksh, 1989; Mjanger and Yaksh, 1991; Connor et al., 1999).
To properly test the notion that strongly versus weakly internalizing opioids produce differential tolerance would therefore require direct comparison of the extent of tolerance produced by morphine with opioids that exhibit comparable intrinsic efficacy for G-protein activation but much higher efficacy for endocytosis than morphine, while ensuring equivalent receptor stimulation and duration of action. Methadone and endomorphins have been considered good candidates because their intrinsic efficacies for G-protein activation appear similar to morphine and both efficiently induce MOR endocytosis. However, the intrinsic efficacy of methadone is more similar to DAMGO than morphine in GTPγS assays (Selley et al., 1998; McPherson et al., 2010) and in vivo (Adams et al., 1990). The apparently low efficacy of methadone in electrophysiological studies is caused by non-MOR actions on ion channels (Rodriguez-Martin et al., 2008). Methadone and endomorphins also have very different pharmacokinetic properties and toxicity compared with morphine that can further complicate interpretations. Although He and Whistler (2005) did examine this issue using methadone and morphine, the results are very difficult to interpret because i.c.v. dose equivalence was not established. By contrast, Soignier et al. (2004) reported comparable rates of tolerance development and completely symmetrical cross-tolerance during continuous i.c.v. infusion of morphine, endomorphin-1 and endomorphin-2, suggesting tolerance may not be different between strongly and weakly internalizing agonists when intrinsic efficacy is matched. Furthermore, there is no clear evidence that strongly internalizing agonists produce differential tolerance compared with weakly internalizing opioids in humans (Morgan and Christie, 2011). For example, comparison of tolerance development in pain patients during continuous administration of transdermal fentanyl (high efficacy, moderate endocytosis) versus buprenorphine (low efficacy, non-internalizing) found fentanyl produced greater tolerance (Sittl et al., 2006). Therefore, it remains uncertain whether or not strongly internalizing agonists produce less tolerance than weakly internalizing agonists.
Decreased MOR–effector coupling contributes to opioid tolerance
Chronic exposure to opioids can cause profound tolerance in both animals and humans (Christie, 2008; Morgan and Christie, 2011).Tolerance measured in whole animals is mediated by multiple adaptive mechanisms ranging from molecular mechanisms of MOR–effector coupling in neurons, second messenger systems in opioid sensitive cells, non-neuronal cells (including glia) and neural networks interacting with opioid sensitive neurons, to learned behaviour in animals (see Christie, 2008). Nonetheless, there is very solid evidence that impaired MOR–effector coupling contributes to tolerance in vivo.
Opioid tolerance has been extensively quantified in isolated tissues, neurons and membrane preparations from morphine tolerant animals, as well as in cell culture models. Functional measurements of impaired MOR–effector coupling in isolated tissues and cells after chronic morphine treatment consistently show a loss of functional receptors without consistent changes in MOR binding density (down-regulation, reviewed by Christie, 2008; Koch and Hollt, 2008). Agonists that strongly promote MOR endocytosis, such as etorphine, are an exception because they do induce receptor down-regulation (Stafford et al., 2001), presumably because a small proportion of endocytosed MOR is degraded during each internalization cycle (Whistler et al., 2002).
Operational models (or Fuchgott analysis) used to quantify the loss of functional MOR–effector coupling in isolated systems (e.g. Chavkin and Goldstein, 1984; Christie et al., 1987; Bailey et al., 2009a) after chronic morphine have calculated a loss of approximately 80% of functional surface MOR is required to account for the observed shift in agonist concentration–response curves. Studies using physiological end-points (direct Gγβ interactions with ion channels) in single opioid sensitive neurons have reported impaired MOR–effector coupling in a range of neuronal cell types from animals that have been chronically treated with morphine in vivo (except Ingram et al., 2008), including rat and mouse periaqueductal grey (PAG; Bagley et al., 2005), rat and mouse locus coeruleus (LC; Christie et al., 1987; Connor et al., 1999; Dang and Williams, 2004; Bailey et al., 2009a; Dang et al., 2011; Quillinan et al., 2011) and mouse trigeminal ganglion neurons (Johnson et al., 2006b). Similar results were also reported for inhibition of GABAergic synaptic transmission in nerve terminals in PAG (Hack et al., 2003; Fyfe et al., 2010). These findings are consistent with those examining MOR-activated GTPγS binding in brainstem in parallel with MOR binding density (Bohn et al., 2000) and GTPγS binding in some brain regions but not others (Sim et al., 1996; Kim et al., 2008). Taken together, these results are consistent with earlier reports in cultured cells showing that chronic morphine exposure impaired MOR–effector coupling (GTPγS binding) without greatly affecting MOR binding density (Puttfarcken et al., 1988; Puttfarcken and Cox, 1989).
β-Arrestin-2 and endocytic mechanisms are involved in opioid tolerance
Although the phenomenon that chronic morphine impairs MOR–effector coupling without much effect on MOR binding density has been known for more than 20 years, the mechanisms responsible are still uncertain and controversial. There is, however, accumulating evidence that the MOR regulatory mechanisms involved in acute desensitization, including association with βarr-2 and endocytosis, are intimately involved in the development of opioid tolerance.
Bohn et al. (2000; 2002; 2004) established that development of morphine anti-nociceptive tolerance (but not withdrawal) is blunted in βarr-2 k.o. mice. Concurrently, GTPγS assays in brainstem membranes from the k.o. mice also showed a blunted shift in the concentration–response curve (tolerance). Acute anti-nociceptive responses to morphine (but not etorphine, fentanyl or methadone; Bohn et al., 2004; or other opioid actions of morphine, Raehal et al., 2005) were also enhanced in the βarr-2 k.o. It was suggested that MOR is resistant to desensitization in the absence of βarr-2 (see contrary evidence; Bradaia et al., 2005; Walwyn et al., 2007; Dang et al., 2009; 2011). Additional support for the involvement of MOR regulatory processes in the development of opioid tolerance comes from study using GRK3 k.o. mice. This study showed MOR tolerance was reduced in hippocampal neurons from GRK3 k.o. mice (Terman et al., 2004). Development of behavioural tolerance to fentanyl was attenuated; however, there was no effect on morphine tolerance. Together, these studies suggest that arrestin-dependent MOR regulation is linked to morphine tolerance.
These studies suggest that blocking MOR endocytosis, which is presumably impaired in the βarr-2 k.o. (but see Arttamangkul et al., 2008; Quillinan et al., 2011), attenuates tolerance but others have provided seemingly contradictory evidence that induction of MOR endocytosis and recycling limits morphine tolerance, and suppression of endocytosis or recycling enhances it. He et al. (2002) reported that inclusion of an extremely low dose of a strongly-internalizing agonist, DAMGO (that had no anti-nociceptive effect on its own), with constantly infused i.t. morphine limited the development of tolerance and also stimulated MOR endocytosis in spinal cord and cultured cells (but see contrary evidence; Bailey et al., 2003; Koch et al., 2005). This was not observed with either drug alone at the doses used. The authors hypothesized that a very low concentration of DAMGO, which does not induce detectable endocytosis by itself, can stimulate endocytosis of morphine-occupied MOR and thereby reduces tolerance, perhaps via interaction with homomultimers of MOR. Similarly, Kim et al. (2008) studied a transgenic MOR mouse, in which part of the C-terminal region of the DOR is substituted into MOR (rMOR). This conferred the ability of morphine to efficiently mediate MOR endocytosis and recycling. The rMOR mice showed similar anti-nociceptive sensitivity to morphine as wild-types but developed less morphine anti-nociceptive tolerance, as well as less reduction in MOR-activated GTPγS binding in brainstem membranes. Consistent with these studies, the converse has also been reported in spinophilin k.o. mice (Charlton et al., 2008); development of morphine tolerance was enhanced in spinophilin k.o. mice. Spinophilin is a neuronal scaffolding protein that facilitates MOR endocytosis, so endocytosis should be impaired in the k.o., although other regulatory actions of spinophilin cannot be ruled out. Taken together, these studies suggest that MOR endocytosis limits tolerance and, therefore, opioids that do not promote receptor endocytosis should produce greater tolerance than agonists that do promote MOR endocytosis (but see above for lack of direct behavioural evidence that this is the case).
The findings described above appear contradictory in terms of the relationship between endocytosis and tolerance. On the one hand, blocking βarr-2 association with MOR (which should impair endocytosis) inhibits morphine tolerance and, on the other hand, manipulations that enhance MOR endocytosis (and vice versa) impair development of morphine tolerance. Various explanations have been proposed to account for these disparate findings. In the case of manipulations that prevent βarr-2 binding, it was proposed that βarr-2 association is necessary for, or facilitates MOR desensitization (Bohn et al., 2002; 2004, but see below). However, desensitization (as defined above) was not directly examined in those studies. But examination of MOR desensitization in both sensory and LC neurons show that it is unaffected by βarr2- deletion. It is therefore unclear how βarr-2 deletion can account for blunted tolerance in the k.o. mice. Two general interpretations (not mutually exclusive) for the inhibition of tolerance were developed by Whistler and co-workers that are in line with findings from other groups (e.g. Berger and Whistler, 2010). One interpretation is that strongly internalizing agonists produce less tolerance because the cycles of endocytosis promote dephosphorylation of MOR in endosomes and resensitized receptors are then recycled to the cell surface. Because morphine poorly stimulates endocytosis of phosphorylated and desensitized MOR (whether or not MOR associated with arrestins) the desensitized receptors accumulate at the cell surface causing tolerance. The other interpretation is that morphine causes persistent signalling that contributes to secondary adaptations involved in tolerance in vivo, whereas endocytosis terminates persistent signalling, limiting downstream adaptations and tolerance. These concepts are summarized in Figure 1. These authors have also provided extensive evidence that such secondary adaptations are more pronounced following chronic morphine stimulation of wild-type MOR compared with chimeric MOR that can undergo endocytosis and recycling when stimulated by morphine (ibid.).
Figure 1. Previous models to explain how strongly internalizing opioid agonists can proiduc less tolerance than weakly internalizing agonists. (A) Strongly internalizing agonists induce rapid desenisitization of MOR coupling. GRK2-mediated phosphorylation is pivotal for βarr-2 binding and endocytosis, both process that were considered ireversible at the cell surface, so MOR slowly resensitizes over the time course of endocytosis and recycling. (B) With weakly internalizing agonists, MOR desensitizes slowly (accelerated by PKC activity) but accumulates in a phosphorylated desensitized state at the cell surface because it stimulates GRK2 and βarr-2 binding very weakly, so cannot resensitize causing tolerance. As discussed in the text, the crucial assumption that endocytosis (and recycling) is necessary for resensitization is incorrect.
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In essence, both types of study described above include the notion that arrestin-dependent endocytosis and recycling is necessary for MOR resensitization to occur. This notion is based largely on the model established for β2-adrenoceptor recycling (Gainetdinov et al., 2004). Although some studies appear to support this for MOR (Koch et al., 1998; 2001; Law et al., 2000; Qiu et al., 2003), more recent findings discussed below clearly establish that MOR dephosphorylation proceeds efficiently at the cell surface, as does resensitization in the βarr-2 k.o. or when endocytosis is blocked.