A REVIEW OF THE MECHANISMS OF OPIOID

TOLERANCE
INTRODUCTION
Chronic pain affects the lives of millions of people worldwide. The opioid system provides an attractive target for mediating analgesia. Currently, -opioid receptor (MOR) agonists such as morphine are the gold standard in centrally acting strong analgesics. Opioid agonists have no intrinsic limit to their analgesic effect - a sufficient dose will relieve the most severe of pain (Glajchen, 2001). The analgesic efficacy of opioids is only limited by the development of tolerance and physical dependence. A better understanding of the neurobiological mechanisms by which the opioids produce not only their analgesic effects, but also the mechanisms which induce tolerance is necessary. With the neural basis of the opioid pathways better understood, the potent analgesia can be decoupled from tolerance. Opioid tolerance is defined as the decreased analgesic response after continuous exposure (Dang and Christie, 2012) a given dose no longer produces the same analgesic effect. Opioids can induce tolerance via multiple mechanisms (Pan, 2007; Dang and Christie, 2012) and it is difficult to distinguish the many pathways involved. There appears to be differences in the method of induction of tolerance between opioids that have different intrinsic efficacies (Hull et al., 2010; Bailey et al., 2009). However, there are many similarities in downstream paths. For example, antinociceptive action is due to the effect of opioids on individual cells as well as networks within the brain and spinal cord. The adaptive effects of opioids occur in both cells and the plasticity of these networks. Tolerance can thus be suggested to occur at multiple levels within the brain.

At the level of the opioid receptor, two distinct but related processes occur. Firstly, receptor desensitization, the decreased response of a receptor to activation, occurs with exogenous opioids (Dang and Christie, 2012; Kovoor et al., 1998). When opioid receptors are activated, another process, namely, receptor internalization, can occur. Internalization leads to a decrease in receptors in the cell membrane and can affect tolerance. The effect of receptor internalization (endocytosis) is unclear (Rodrguez-Muoz et al., 2007; Koch et al., 2005). These processes are mediated through regulatory pathways. Overall, the regulatory effects result in a decreased analgesic response. Another receptor-level process that produces tolerance occurs with the NMDA (Nmethyl-D-aspartate) and AMPA (-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) glutamate receptors. These receptors represent the classical view of opioid tolerance, i.e. learning and conditioning (Elliott et al., 1994). NMDA receptor antagonists have been shown to reduce morphine tolerance. The likely mechanism of NMDA effects is via inhibition of GABAergic interneurons (Ueda and Ueda, 2009). There is also evidence for the involvement of glial cells in NMDA-mediated tolerance (Ueda and Ueda, 2009). These receptors affect synaptic plasticity and lead to higher level network-based tolerance. Lastly, at a higher level, a confounding process, hyperalgesia the increased response to painful stimuli can occur with opioid use and is intricately related to tolerance (Mao et al., 1995). When hyperalgesia occurs, the analgesic response of opioids is diminished and increasing doses of opioids cause a painful response. Hyperalgesia is distinct from tolerance but may be induced by similar pathways. It is possible that

hyperalgesic effects also have a neurotoxic component (Mao et al., 2002a). Hyperalgesia presents a serious problem in the management of pain. Opioids affect the brain from the lowest level of the receptor in the cell membrane to the highest level networks. This review will examine the effects throughout the brain, starting at the level of the cell via receptor desensitization and internalization. The traditional view of opioid tolerance through synaptic plasticity will be considered, and the complications of hyperalgesia will be discussed. It is likely that there is no single mechanism that can both fully explain, and be targeted to reduce the development of tolerance. However, it may be possible to target the downstream effects of the many pathways. It remains to be seen whether an easy solution to the problem of opioid tolerance is possible.

OPIOID RECEPTOR REGULATION

Tolerance at the level of the opioid receptor is mediated by receptor regulatory mechanisms. A function MOR is coupled on the intracellular domain to a G-protein effector (Davis et al., 2009). It is this effector that produces the downstream effects in the cell. When the ability of the receptor to couple to its G-protein effector is compromised the receptor becomes desensitized. The specific pathway is dependent on the efficacy and affinity of the agonist at the MOR. Phosphorylation of the receptor produces desensitization by interrupting this effector coupling (Davis et al., 2009). Opioids of different efficacies affect this phosphorylation through different kinase and second messenger pathways (Hull et al., 2010). It is thought that the binding of an agonist changes the conformation of the receptor and makes it a better substrate for a

particular kinase (Davis et al., 2009). This explains why efficacy of the agonist effects the kinase which acts on the receptor. It was found that inhibition of G-protein coupled receptor kinase (GRK) reversed tolerance to high efficacy agonists, and inhibition of protein kinase C (PKC) reversed tolerance to low efficacy agonists (Bailey et al., 2009; Hull et al., 2010). High efficacy agonists affect GRK2- (Li and Wang, 2001), GRK3- and potentially GRK5- mediated phosphorylation (Kovoor et al., 1998). There is also evidence for the involvement of Ca2+/calmodulin-dependent kinase II (Koch et al., 2005). Morphine, but not the high efficacy agonist [D-Ala2, N-MePhe4, Gly-ol5] enkephalin (DAMGO) triggers CaMKII phosphorylation (Rodrguez-Muoz et al., 2007). The amino acid target of phosphorylation depends on the efficacy of agonist (Grecksch et al., 2011) in the case of high efficacy agonists, this is threonine-370 (T370) and serine-375 (S375); the low efficacy agonists do not effect S375. Phosphorylation of the receptor is responsible for desensitization, but that is not by itself the cause of reduction of opioid effect. Phosphorylation of the receptor can lead to internalization and subsequent endocytosis. This may proceed via arrestin-2 (arr-2) binding (Dang et al., 2011). GRK phosphorylation mediates arr-2 binding with high efficacy agonists (Kovoor et al., 1998). The effect of arr-2 is unclear some authors have found tolerance attenuation in arr-2 knockouts (Dang et al., 2011; Dang et al., 2009) , whereas the classical view is that it leads to re-sensitized receptors through endocytosis (Koch et al., 2005). The effect that arr-2 has on endocytosis is likely dependent on the agonist. At least for high efficacy agonists, arr-2 serves as a linkage to the intracellular machinery responsible for endocytosis. In the classical view, the desensitized receptor is internalized and recycled or degraded. If the receptor is degraded, this would lead to a reduction in opioid effect. There seems to be

some mechanism that preferentially recycles the receptor into an active state when it has been activated by a high efficacy agonist (Koch et al., 2005). The recycled receptor can be trafficked back to the cell membrane and participate in MOR signalling. Low efficacy agonists (such as morphine) do not efficiently promote receptor resensitization and this leads to an accumulation of non-functional opioid receptors in the cell membrane. It is also possible that high efficacy, highly internalizing agonists do not induce as much tolerance because they activate less of the opioid receptors on the cell membrane for a given analgesic effect. This causes fewer receptors to be desensitized. In any case, it is generally accepted that in order for receptor resensitization, dephosphorylation must occur (Dang et al., 2009; Garzon et al., 2005). The receptor-level effects lead to changes in neural networks via synaptic plasticity.

THE ROLE OF SYNAPTIC PLASTICITY

In addition to cellular actions, at higher levels within the brain synaptic changes lead to tolerance. The classical view of opioid tolerance is through long term changes resulting from NMDA activation. NMDA antagonists reduce the development of tolerance to morphine independently of receptor desensitization (Elliott et al., 1994). Morphine may cause inhibition of GABAergic interneurons that synapse on glutamate-releasing neurons. The release of glutamine may lead to NMDA activity in the cell expressing MOR and a countering of opioid action (Ueda and Ueda, 2009). The chronic activation of NMDA receptors leads to the receptor being up-regulated in the MOR-expressing cell and a reduction in the intracellular effect when an agonist binds to the MOR. This explains why NMDA antagonists reduce morphine tolerance: the GABAergic interneuron is still inhibited,

but the released glutamate does not lead to NMDA activation. Both competitive and noncompetitive antagonists of the receptor appear to have similar effects (Elliott et al., 1994). Antagonism of AMPA receptors has also been shown to play a role, likely through NMDA and AMPA interaction (Pan, 2007). It appears that NMDA receptor activation may lead to tolerance through nitric oxide (NO) second messenger pathways (Elliott et al., 1994). Inhibitors of nitric oxide synthase (NOS) reduced morphine tolerance to the same degree and over the same time to action as NMDA antagonism (Elliott et al., 1994), suggesting the interaction of these pathways. The ultimate effect of the second messenger pathways is the phosphorylation of NMDA-regulated ion channels (Mao et al., 2002b). In the case of morphine, it has been shown that the PKC activity induced also acts of NMDA gated Ca2+ channels by alleviating the Mg2+ blockade (Elliott et al., 1994). The NMDA activation ultimately leads to long term potentiation (LTP) of the excitatory responses which act to oppose opioid effect. Specific NMDA receptor subunits that have been implicated include NR2A and NR2B (Ueda and Ueda, 2009). The NMDA effect occurs via neuronal receptor interactions but also via glial mediated interactions. The pathways by which glial cells interact with NMDA signalling are complex. Astrocytes responsible for the uptake of glutamate from the synaptic cleft experience a down-regulation of glutamate transporter (GLAST) (Ueda and Ueda, 2009; Mao et al., 2002b) following chronic morphine exposure. GLAST in the spinal cord was shown to be down-regulated in a dose-dependent manner after chronic morphine administration (Mao et al., 2002b). This leads to excitatory action which opposes the inhibitory action of the opioids. Interactions mediated through brain-derived neurotrophic factor (BDNF) action on microglia may also up-regulate the NMDA receptors in the MOR cell. The injection of an

anti-BDNF antibody into the central nervous system (CNS) has been shown to attenuate morphine tolerance. It appears morphine acts to up-regulate BDNF only in microglia (Ueda and Ueda, 2009). Overall, it appears that NMDA-mediated excitatory responses are a part of the process responsible for tolerance. The NMDA response leads to anti-opioid effects and there appears to be a role for NMDA antagonism in the reduction of tolerance. These processes occur at the level of the cell but do not necessarily interact with the MOR receptor-level mechanisms. Antagonism of NMDA receptors would likely prevent at least some synaptic plasticity adaptive effects. It is this plasticity that is responsible for sensitization to opioid effect.

HYPERALGESIA AND NEUROTOXICITY

Finally, chronic opioid administration has been shown to induce a sensitization response (partly through NMDA-mediated synaptic potentiation). This sensitization response ultimately leads to hyperalgesia. The intended effect of opioids is the reduction of pain (analgesia). With chronic usage, there is a danger that opioids will induce a nociceptive response to non-noxious stimuli (Pan, 2007). When opioids have induced a hyperalgesic response the normal course of dosage increase in response to increased pain serves a counter-productive purpose and will make the experience of pain worse. This is a serious problem because opioids become absolutely useless for analgesia. There are no other analgesics in current usage which relieve pain as effectively as opioids (Davis et al., 2009) so hyperalgesia presents an unacceptable side effect in chronic usage. There appears

to be a link between hyperalgesic response and synaptic plasticity induced by NMDA interactions (Pan, 2007). There is ample evidence for the implication of NMDA-glutamate in opioid-induced hyperalgesia. Chronic administration of opioids appears to induce the hyperalgesic response in the spinal cord (Pan, 2007). This may proceed via GLAST downregulation as discussed. Exogenous glutamate applied to the spinal cord produced increased hyperalgesic response leading to implication of spinal glutamate transporters (Mao et al., 2002b). It is also known that chronic activation of the NMDA receptor sites in the spinal cord produces spinal sensitization. This spinal sensitization results in increased nociceptive transmission within the spinal dorsal horn neurons (Ossipov et al., 2005). Descending projections from the rostral ventromedial medulla (RVM) are effected by chronic morphine and enhance spinal pain transmission (Pan, 2007). It is difficult to separate the spinal sensitization from true tolerance. Chronic NMDA receptor activation has been associated with excitotoxicity and neuronal apoptosis. Hyperalgesia may also be mediated through neurotoxic effects of opioids on neurons in the spinal cord (Mao et al., 2002a). Opioid tolerance was observed when neuronal apoptosis was induced in spinal cord dorsal horn neurons. Hyperalgesic responses are likely induced because GABAergic neurons within the spinal cord experience apoptosis and their inhibitory effect on nociception is lost (Mao et al., 2002b). Spinal GLAST down-regulation is implicated in morphine-induced apoptosis via increased synaptic glutamate by the same mechanisms that lead to increased NMDA excitability. Neuronal

apoptosis presents a unique problem because this suggests that opioid tolerance is unlikely to fully abate in patients who have already become tolerant. It appears that hyperalgesia and tolerance occur via similar mechanisms. It is currently unclear whether hyperalgesia and tolerance are truly distinct, or if one inevitable leads to the other. The evidence seems to suggest that they are intimately linked and as a consequence, finding a solution to one would provide a good basis for combatting the other. In order to achieve the maximal benefit of opioids in pain relief, more study of the pathways involved must be undertaken.

CONCLUSION
In conclusion, the opioid system is clearly a powerful mediator of analgesia. With no viable alternatives to opioids for the treatment of chronic pain, solving the problem of opioid tolerance is of paramount importance. As more about the neuronal mechanisms of tolerance becomes known novel solutions can be developed. Like the analgesic response to opioids, the problem of tolerance is multilayered and occurs at many levels within the CNS. It is unclear if addressing any target individually would significantly reduce long-term tolerance as studies of the duration needed to determine this have yet to be conducted. Future research should include a focus on long-term reduction of tolerance. In any case, there is sufficient evidence for the involvement of multiple, sometimes interacting pathways in the induction of opioid tolerance. This review addressed the mechanisms of opioid tolerance starting from the cellular level, going to higher levels within the CNS. At the lowest level of -opioid receptor, regulatory processes involving agonist-specific kinases promote desensitization and

endocytosis. In a more general and agonist independent manner, opioid induced inhibition of GABAergic interneurons lead to NMDA signalling and synaptic plasticity. Within the spinal cord this NMDA activity leads to sensitization, hyperalgesia and excitotoxicity. It is clear that much is currently known about how opioids induce tolerance; however this knowledge is also incomplete. In order to address opioid tolerance more basic research must be conducted. It may be a difficult problem but as this review has shown the problem is not intractable. There are multiple viable approaches of study, and it is likely that at least one will prove successful. There is still hope for those living in chronic pain that a solution will be found within this century.

REFERENCES
Bailey CP, Oldfield S, Llorente J, Caunt CJ, Teschemacher AG, Roberts L, McArdle CA, Smith FL, Dewey WL, Kelly E, Henderson G (2009) Involvement of PKC alpha and G-proteincoupled receptor kinase 2 in agonist-selective desensitization of mu-opioid receptors in mature brain neurons. Br J Pharmacol (England) 158:157-164. Dang VC, Christie MDJ (2012) Mechanisms of rapid opioid receptor desensitization, resensitization and tolerance in brain neurons. Br J Pharmacol 165:1704-1716. Dang VC, Napier IA, Christie MDJ (2009) Two distinct mechanisms mediate acute -opioid receptor desensitization in native neurons. The Journal of Neuroscience 29:3322-3327. Dang VC, Chieng B, Azriel Y, Christie MDJ (2011) Cellular morphine tolerance produced by arrestin-2-dependent impairment of -opioid receptor resensitization. The Journal of Neuroscience 31:7122-7130. Davis MP, Glare PA, Hardy J, Quigley C (2009) Opioids in cancer pain. OUP Oxford. Elliott K, Minami N, Kolesnikov YA, Pasternak GW, Inturrisi CE (1994) The NMDA receptor antagonists, LY274614 and MK-801, and the nitric oxide synthase inhibitor, NG-nitro-Larginine, attenuate analgesic tolerance to the mu-opioid morphine but not to kappa opioids. Pain 56:69-75. Garzon J, Rodriguez-Munoz M, de la Torre-Madrid E, Snchez-Blzquez P (2005) Effector antagonism by the regulators of G protein signalling (RGS) proteins causes desensitization of mu-opioid receptors in the CNS. Psychopharmacology (Berl ) 180:1-11. Glajchen M (2001) Chronic pain: Treatment barriers and strategies for clinical practice. The Journal of the American Board of Family Practice 14:211-218. Grecksch G, Just S, Pierstorff C, Imhof AK, Glck L, Doll C, Lupp A, Becker A, Koch T, Stumm R (2011) Analgesic tolerance to high-efficacy agonists but not to morphine is diminished in phosphorylation-deficient S375A -opioid receptor knock-in mice. The Journal of Neuroscience 31:13890-13896. Hull LC, Llorente J, Gabra BH, Smith FL, Kelly E, Bailey C, Henderson G, Dewey WL (2010) The effect of protein kinase C and G protein-coupled receptor kinase inhibition on tolerance induced by mu-opioid agonists of different efficacy. J Pharmacol Exp Ther (United States) 332:1127-1135. Koch T, Widera A, Bartzsch K, Schulz S, Brandenburg LO, Wundrack N, Beyer A, Grecksch G, Hllt V (2005) Receptor endocytosis counteracts the development of opioid tolerance. Mol Pharmacol 67:280-287. Kovoor A, Celver JP, Wu A, Chavkin C (1998) Agonist induced homologous desensitization of -opioid receptors mediated by G protein-coupled receptor kinases is dependent on agonist efficacy. Mol Pharmacol 54:704-711.

Li AH, Wang HL (2001) G proteincoupled receptor kinase 2 mediates opioid receptor desensitization in GABAergic neurons of the nucleus raphe magnus. J Neurochem 77:435444. Mao J, Price DD, Mayer DJ (1995) Mechanisms of hyperalgesian and morphine tolerance: A current view of their possible interactions. Pain 62:259-274. Mao J, Sung B, Ji RR, Lim G (2002a) Neuronal apoptosis associated with morphine tolerance: Evidence for an opioid-induced neurotoxic mechanism. The Journal of Neuroscience 22:7650-7661. Mao J, Sung B, Ji RR, Lim G (2002b) Chronic morphine induces downregulation of spinal glutamate transporters: Implications in morphine tolerance and abnormal pain sensitivity. The Journal of Neuroscience 22:8312-8323. Ossipov MH, Lai J, King T, Vanderah TW, Porreca F (2005) Underlying mechanisms of pronociceptive consequences of prolonged morphine exposure. Peptide Science 80:319324. Pan ZZ (2007) Mechanisms of opioid tolerance. Molecular Pain 413-422. Rodrguez-Muoz M, de la Torre-Madrid E, Gaitn G, Snchez-Blzquez P, Garzn J (2007) RGS14 prevents morphine from internalizing mu-opioid receptors in periaqueductal gray neurons. Cell Signal 19:2558-2571. Ueda H, Ueda M (2009) Mechanisms underlying morphine analgesic tolerance and dependence. Front Biosci (United States) 14:5260-5272.