Opioids are naturally occurring or synthetic/semisynthetic chemicals that bind to opioid receptors. There are many indications for clinical use of opioids of which the most common is pain relief (1). Opioids are also commonly abused drugs for their psychoactive effects. Opioid tolerance is a phenomenon in which an increased amount of drug is required to produce the same level of drug effect after repeated use of the drug. Although the drug effects can be any of the opioid-induced effects, such as respiratory depression and euphoric action, in this review, I will limit the discussion of opioid tolerance to its analgesic effect.
Multiple in vivo animal studies have repeatedly demonstrated the development of opioid tolerance in rodents (2,3) and have shown that opioid tolerance is pharmacologically represented by a shift to the right of the dose-response curve (a larger 50% effective dose when opioid tolerance develops) (4). Previous studies have also shown that opioids with high intrinsic efficacy (such as etorphine and fentanyl) are less likely to result in tolerance than those with low intrinsic efficacy (such as morphine) after continuous infusion (4,5). One hypothesis for this phenomenon was that opioids with low intrinsic efficacy at equieffective doses occupy and produce tolerance effects at more opioid receptors than do opioids with high intrinsic efficacy. However, intrinsic efficacy has been found to have no effect on the magnitude of tolerance after intermittent dosing (4).
Opioid tolerance also occurs in humans (6). After being exposed to opioids, patients with cancer developed a right shift of the opioid dose-response curve (7). Opioid requirements by bone marrow transplant patients increase rapidly after starting treatment (8,9). It is a common observation that patients with chronic opioid use usually require larger doses of opioids to produce postoperative pain relief than opioid naïve patients. Thus, understanding the mechanisms of opioid tolerance and developing drugs and strategies to prevent its occurrence have been a focus of opioid research so that effective analgesia with tolerable side effects can be achieved in patients.
Multiple and complex mechanisms, including opioid receptor uncoupling and post-receptor adaptations, are involved in the development of opioid tolerance. In this review, I will focus on recent, exciting findings of opioid receptor uncoupling and recycling after activation by opioids. These findings have prompted us to revise the theory about how the dynamic processes of activated opioid receptors participate in the development of opioid tolerance. Contributions of multiple post-receptor adaptations to the development of opioid tolerance have been reviewed by other authors (10,11).
Opioids, via their receptors, induce many effects, including analgesia, respiratory depression, and euphoric action. Opioid receptors are G protein-coupled (Gi and/or Go) receptors (GPCRs) (12). Molecular cloning has identified three opioid receptors: δ, κ, and μ receptors. These receptors are named OP1, OP2, and OP3, respectively, by the International Union of Pharmacology Subcommittee on Opioid Receptors (13). Because of the predominant role of μ opioid receptors in the analgesic effects and in the development of opioid tolerance (14), this receptor has often been used in studies of opioid tolerance and is the type of opioid receptor to generate the data presented in this review unless the types of opioid receptors are specified. However, other opioid receptors, especially δ receptors, also have a role in the development of opioid tolerance (15,16).
Activation of opioid receptors induces changes in two major intracellular second messenger systems via Gi/o proteins: 1) inhibition of adenylyl cyclase, and 2) activation of phospholipase C (17–22). Inhibition of adenylyl cyclase results in reduced adenosine 3′,5′-cyclic monophosphate (cAMP), and this may underlie opioid-induced modulation of the release of neurotransmitters such as substance P. Phospholipase C activation produces diacylglycerol and inositol 1,4,5-triphosphate from phosphatidylinositol-4,5-bisphosphate. Activation of opioid receptors also inhibits voltage-dependent Ca2+ channels and activates inwardly rectifying K+ channels (21). These effects are thought to be important for the action of opioids, by reducing cell excitability and inhibiting neurotransmitter release.
A Traditional Theory for Opioid Tolerance
Once opioid receptors are activated, a group of protein kinases (GPCR kinases, GRK) are recruited to the plasma membrane and specifically phosphorylate agonist-occupied receptors (10,11,23). The phosphorylation of receptors may decrease the coupling with their associated G proteins. It will also increase the binding affinity of the receptors to a group of proteins called “arrestins.” This binding ensures the uncoupling of receptors from the G proteins and receptor desensitization occurs (10,11,24). Thus, receptor desensitization is defined in this review as loss of receptor ability to connect with its effector system: a functional uncoupling of receptor and effector system. Binding of activated and phosphorylated receptors with arrestins also induces receptor internalization resulting in fewer available receptors at the cell surface for activation by opioids. Thus, receptor internalization and desensitization would reduce agonist signaling to its effector system and could explain opioid tolerance. Of note, the internalized opioid receptors can be recycled to the plasma membrane as competent receptors, a process to resensitize receptors and cells to opioids (10,25).
A third mechanism, an alternation of post-receptor signaling, may contribute to the development of opioid tolerance. It has been shown that opioids decrease cellular cAMP levels (17). However, prolonged opioid exposure induces an increase of cyclase activity (cyclase supersensitivity) in cultured cells and animals (26,27). This cyclase supersensitivity is thought to have a role in neuronal hyperactivity during opioid withdrawal (28). However, its role in the development of opioid tolerance in vivo has not been determined.
Thus, the traditional theory proposes that receptor desensitization and internalization, along with the possible contribution from alternations of post-receptor signaling such as cyclase supersensitivity, cause a reduced agonist signaling that results in the development of opioid tolerance (29).
An Alternative Theory for Opioid Tolerance
It has been realized that the traditional theory for opioid tolerance needs to be revised to explain some of the recent findings, especially those related to morphine, the prototypic opioid widely used in clinical practice. In HEK293 cells expressing μ opioid receptors, the opioids [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO), met-enkephalin, etorphine, or methadone, but not morphine, induced receptor desensitization (30,31). In hippocampal neurons that express native opioid receptors, chronic treatment with DAMGO, methadone, or morphine induced opioid tolerance (32). Receptor desensitization was not measured in the study. Two studies showed that some receptor desensitization could be induced by morphine (33,34). However, it is generally agreed that morphine is much less potent at inducing receptor desensitization than opioids with high intrinsic efficacy such as DAMGO, fentanyl, and etorphine (35). Thus, agonist efficacy seems to be an important factor in determining opioid receptor desensitization. Similarly, opioids with high intrinsic efficacy induced a rapid receptor internalization, whereas in most studies morphine did not induce receptor internalization (29,30,35,36) and in two studies induced a slower and less complete receptor internalization than opioids with high intrinsic efficacy (32,34). This inefficiency of morphine in inducing receptor desensitization and internalization may be attributable to its inability to induce receptor phosphorylation by GRK after receptor activation (36). Thus, the ligand-receptor complex, rather than the receptor itself, may determine the active conformations of opioid receptors and induction of distinct cellular responses by individual ligands (37,38).
Morphine induces tolerance in animals and humans (4,39) and the development of morphine tolerance requires opioid receptor activation (40). How does morphine induce tolerance if it is ineffective at inducing receptor desensitization and internalization? Based on their results, Whistler et al. (26,41,42) proposed that receptor internalization is an effective way to resensitize receptors and cells to opioids. According to their proposal, the internalized receptors will have two fates: 1) they can be dephosphorylated and then cycled back to the plasma membrane as competent receptors, or 2) they can be degradated by proteases in the lysosomes (Fig. 1) (43). The biological implications of the cycling of receptors may be twofold: it serves as a way to rapidly attenuate receptor-mediated signaling and it is a mechanism to resensitize the receptors and cells to agonists (41). Because morphine is an ineffective agonist to induce opioid receptor internalization, morphine stimulation does not induce the dynamic cycle and resensitization of activated receptors (30,36,42). This failure to induce receptor resensitization contributes to the development of morphine tolerance. In addition, because of the ineffective mechanism of receptor desensitization and internalization to terminate morphine signaling, compensatory, post-receptor mechanisms including cyclase supersensitivity develop to antagonize morphine signaling and to develop morphine tolerance (29).
To quantitatively illustrate this alternative theory, Whistler et al. (41) and Alvarez et al. (44) proposed to use a ratio of relative activity of an opioid to induce effector responses (potassium currents in their studies) versus its ability to cause receptor endocytosis/internalization, termed RA/VE, to predicate tolerance liability of an opioid and mechanisms for the tolerance development after the use of the opioid. Agonists with a high RA/VE value (for example, morphine that has a good ability to induce effector responses and a poor ability to internalize receptors) would have a high potential to induce tolerance after continuous application. These agonists would have compensatory post-receptor mechanisms and inability to induce receptor recycling to mediate the tolerance (Fig. 1). However, agonists with a low RA/VE value (for example, etorphine, and fentanyl that have high signaling efficacy and high receptor internalization potency) would have a low potential to induce tolerance after continuous application. Receptor desensitization and internalization would be the major mechanisms for the development of tolerance to these agonists (Fig. 1).
The evidence to support this alternative theory for opioid tolerance comes mainly from three published articles from Whistler’s group. In the first study (41), the authors measured potassium currents and μ opioid receptor internalization induced by a saturated concentration of morphine, methadone, or DAMGO to calculate RA/VE value for each opioid. Their data support an inverse relationship between RA/VE value and receptor uncoupling measured by nucleotide exchange activity. In the second study (26), a wild type μ receptor and two mutants of μ receptors were used. Consistent with their previous study, morphine did not induce internalization of the wild type μ receptor but induced internalization of both mutants. Whereas morphine induced cyclase supersensitivity in cells expressing the wild type μ receptor, this morphine-induced cyclase supersensitivity was reduced or abolished in the cells expressing mutants. These results suggest that receptor internalization reduces post-receptor adaptational changes after opioid stimulation. In their latest study (42), the authors performed both in vitro and in vivo studies to implicate a protective role of receptor internalization against the development of opioid tolerance. In the in vitro study, they carefully chose DAMGO concentrations which were below the threshold for inducing internalization by itself, but which would induce receptor internalization in the presence of morphine. It is proposed that this dragging of receptors to intracellular compartments is the result of receptor oligomerization (42,45). Binding of DAMGO to an oligomerized receptor complex occupied by morphine may initiate the complex internalization. Under these experimental conditions, the authors showed that DAMGO inhibited the cyclase supersensitivity induced by morphine. These results were reproduced in rat spinal cord in their in vivo experiments. A very exciting finding in their in vivo study is that analgesic effects of morphine after continuous use were enhanced by a subanalgesic concentration of DAMGO.
The findings of Whistler’s group are supported by a study by Walker and Young (46). They investigated the development of tolerance to morphine, etonitazene, and buprenorphine in rats and found that the ability to induce tolerance is inversely related to efficacy of the opioids. However, one study failed to confirm a facilitating effect of DAMGO on receptor internalization in the presence of morphine in rat locus ceruleus neurons in brainstem slice preparations (47). Obviously, more studies are needed to support or to disagree with the findings of Whistler’s group and to refine the alternative theory for opioid tolerance.
Interestingly, Koch et al. (48) and Schulz et al. (49) have proposed that morphine tolerance develops because morphine-activated μ receptors do desensitize but cannot be resensitized because of their inability to internalize. Similar to Whistler et al.’s RA/VE theory, this proposal also emphasizes the role of receptor internalization in reducing morphine tolerance. However, this proposal is different from Whistler et al.’s RA/VE theory in that it includes receptor desensitization after morphine activation as an important component for the development of morphine tolerance. It is important to point out that Koch et al. and Schulz et al. used decrease in inhibition of cAMP production to measure receptor desensitization, which should reflect changes in at least two components of opioid signaling: receptor coupling and cyclase activity. This method is different from assaying nucleotide exchange activity that mainly measures receptor uncoupling to reflect receptor desensitization as used by other investigators (33,41). Two lines of evidence provided by Koch et al. and Schulz et al. support their proposal: 1) C-terminal splice variants of mouse μ opioid receptors that were not phosphorylated and internalized after morphine application showed a faster desensitization and no resensitization compared with the splice variants with robust morphine-induced phosphorylation and internalization (48); 2) whereas DAMGO induced phosphorylation in multiple Ser/Thr residues of the μ receptors and these DAMGO-desensitized receptors rapidly internalized, dephosphorylated, and resensitized; morphine induced phosphorylation of Ser (375) only and these morphine-desensitized receptors remain at the plasma membrane in a Ser (375)-phosphorylated state for prolonged periods (49).
As discussed in the section “A Traditional Theory for Opioid Tolerance,” arrestins have an important role in receptor desensitization and internalization. I will expand the discussion on this role in the context of opioid tolerance in the next three sections after briefly discussing the molecular biology of arrestins in this section.
There are at least 4 arrestins: visual arrestin, cone arrestin, β-arrestin 1, and β-arrestin 2 (43). All arrestins are proteins approximately 420 amino acids long (50). In the rat, the overall amino acid homology between β-arrestin 1 and β-arrestin 2 is approximately 78% (50). Mature arrestin proteins have mobility consistent with a molecular weight of approximately 50 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both visual and cone arrestins are restrictively expressed in the eyes where they quench the signaling of rhodopsin and cone opsins, respectively (43). β-Arrestin 1 and β-arrestin 2 are predominantly expressed in neuronal tissues (low expression can be detected in most of tissues) (50) and regulate GPCR coupling and signaling (51). In the central nervous system, β-arrestin 1 and β-arrestin 2 are concentrated at synapses, especially at the postsynaptic density regions (50). Overall, β-arrestin 1 is more abundant than β-arrestin 2 in the adult rat central nervous system (52). Although a differential pattern of high expression regions and differential regulation of expression in these regions after opioid administration and withdrawal have been shown for β-arrestin 1 and β-arrestin 2 (50,52,53), the expression of these two β-arrestins overlaps significantly in many brain regions, including the periaqueductal gray, cerebral cortex and hippocampus, structures that are involved in pain pathways (50,52,53).
In contrast to the one-to-one receptor specificity of visual and cone arrestins, β-arrestin 1 and β-arrestin 2 regulate >1000 different GPCRs (54). It has been proposed that the GPCRs can be divided into two classes based on their binding affinities for β-arrestins. One class of receptors, such as the β2-adrenergic and the μ opioid receptors, has a higher affinity for β-arrestin 2 than for β-arrestin 1. The second class of receptors that include the angiotensin type 1 and the vasopressin type 2 receptors does not discriminate between β-arrestin 1 and β-arrestin 2 (55). Of note, the μ receptor agonist used in the study to generate the data for this classification was not identified in the article. Interestingly, it has been realized that β-arrestin 2 may be more potent in mediating internalization of activated GPCRs and induces faster recycling of GPCRs than β-arrestin 1 (56).
In addition to the well defined role of arrestins in receptor uncoupling and internalization, β-arrestins have been shown to work as adaptor molecules triggering additional signaling events such as activation of extracellular signal regulated kinase pathway and C-jun N-terminal kinase 3 pathway (57).
Evidence for the Involvement of Arrestins in Opioid Tolerance
Evidence for the role of β-arrestins in opioid analgesic effects and tolerance is provided by studies using β-arrestin 2 knockout mice. The analgesic effects of morphine were enhanced and prolonged in the β-arrestin 2 knockout mice (58). Whereas morphine induced both acute and chronic tolerance in wild type animals, morphine did not induce tolerance in the β-arrestin 2 knockout mice when the hotplate test was used (33) or induced a delayed onset and a small degree of tolerance in these mutated mice when the warm water tail-immersion test was used (59). These in vivo data strongly suggest a role of β-arrestin 2 in the development of morphine tolerance.
Could Different Arrestins Mediate Opioid Receptor Desensitization and Opioid Tolerance Induced by Different Opioids?
The in vivo data suggesting a role for β-arrestin 2 in the development of morphine tolerance seem difficult to understand. Morphine is a poor inducer of opioid receptor desensitization and internalization, suggesting that β-arrestins do not have a major role in opioid receptor responses to morphine. In addition, if receptor internalization and recycling were critical processes to maintain sufficient numbers of competent receptors at the cell surface after opioid stimulation, β-arrestin depletion would be expected to induce opioid tolerance and to reduce analgesic effect by progressive depletion of the competent receptors in the plasma membrane. A recent study provides data to at least partially explain the seemingly paradoxical evidence obtained by studies using β-arrestin 2 knockout mice (60). In this study, the analgesic effects of morphine were enhanced in the β-arrestin 2 knockout mice, confirming the previous in vivo results. However, the analgesic effects of etorphine, fentanyl, and methadone, drugs that induced a robust β-arrestin 2 recruitment to cell membranes in the study, are not affected in these β-arrestin 2 knockout mice compared with wild type mice. The authors also found that morphine induced membrane recruitment of β-arrestin 2 but not β-arrestin 1 in their in vitro experiment and that the analgesic effects of morphine were not affected in the β-arrestin 1 knockout mice compared with wild type mice. These results suggest that different arrestins mediate opioid receptor desensitization and opioid tolerance induced by different opioids. These data also support the idea that the ligand-receptor complex, rather than the receptor itself, determines which cellular responses are induced. Although this recent study did not provide a direct explanation of enhanced analgesic effects and reduced tolerance after morphine administration in the β-arrestin 2 knockout mice, the known signaling role of β-arrestins such as activation of protein kinases (57) implies changes of cell signaling induced by morphine-receptor complex in β-arrestin 2 knockout animals compared with wild type animals.
There is additional evidence in the literature to suggest that β-arrestin 1 does not modulate morphine signaling and that both β-arrestin 1 and β-arrestin 2 are involved in signaling by opioids with high intrinsic efficacy (Fig. 1). An in vitro study has shown that morphine failed to induce recruitment of β-arrestin 1 to the plasma membrane in μ or δ opioid receptor expressing cells (61). It has been shown that etorphine induced μ receptor binding to β-arrestin 2, and that the protein complex was then internalized (36). In vivo studies have shown that acute and chronic application of sufentanil, an opioid with high intrinsic efficacy, induced tolerance associated with μ opioid receptor desensitization and down-regulation and an increased β-arrestin 2 expression in rat cerebral cortex (62–64). Etorphine and [D-Ala2, D-Leu5]enkephalin (DADLE) induced opioid receptor desensitization and internalization and the recruitment of β-arrestin 1 to cell membrane (61). Although morphine did not increase μ receptor internalization in cells overexpressing β-arrestin 1, etorphine induced an increased μ receptor internalization in these cells compared with cells without overexpression of β-arrestin 1 (36). The analgesic effects of etorphine, fentanyl, and methadone in the β-arrestin 2 knockout mice were similar to those in control mice and etorphine induced β-arrestin 1 recruitment to the plasma membrane in cells expressing β-arrestin 1 only (60), supporting a role of β-arrestin 1 in regulating the signaling induced by these opioids. However, there is no direct evidence that β-arrestin 1 is indeed involved in the analgesic effects and tolerance development of opioids with higher efficacy.
Although the clinical implications of the new concepts and findings discussed in this review may be numerous, two new concepts deserve particular attention. The first is the determination of cellular responses by the agonist-receptor complex but not by activated receptor alone. When this concept is applied to opioid tolerance, it implies that different opioids that work on the same receptor may induce different cell responses to cause tolerance. This theory may explain the well known asymmetric cross-tolerance between drugs that activate the same receptor but have different intrinsic activity (for example, a patient who develops morphine tolerance may have good analgesic effects from sufentanil) (39) and a commonly used technique of opioid rotation for patients requiring chronic opioid use (65). Along this line, different β-arrestins have been found to mediate cell responses after different opioids that are all μ receptor agonists. Thus, β-arrestins may be intracellular targets for modifying cell responses such as tolerance induced by individual opioid.
The second new concept is that receptor internalization and recycling reduce opioid tolerance. If this is proved true in humans, one simple way to reduce opioid tolerance in clinical practice would be to combine morphine with a subanalgesic dose of an opioid with high intrinsic efficacy. (These opioids usually induce receptor internalization.) Development of drugs that induce receptor internalization but do not activate opioid receptor may be another useful approach to reduce morphine tolerance.
Coadministration of two or more opioids is not commonly used in clinical practice. Patients must be carefully monitored when morphine and an opioid with high efficacy are coadministered. Although the opioid with high efficacy is at small dose, the coadministration may significantly increase the side effects such as respiratory inhibition. In addition, the mechanisms for opioid tolerance are complex. For example, receptor internalization can be a mechanism for opioid tolerance and can be a process for receptor resensitization and reduction of opioid tolerance. Thus, fine adjustment in each case would be needed to achieve the desirable outcome, reducing opioid tolerance, during the coadministration of opioids.
Because of the obvious implication of opioid tolerance in clinical practice, many basic scientific studies have been performed and the theory for opioid tolerance has been revised. The correctness of the theory and the associated new concepts will need to be verified by more studies of basic science and clinical research.
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