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Chronic pain remains one of the most widespread and under treated medical conditions. Opioid drugs, such as morphine, are some of the most effective analgesics available. However, their utility for the treatment of chronic pain are limited by side effects including tolerance and dependence. Morphine induces its biological actions primarily through the G-protein coupled mu-opioid peptide receptor (MOP-R) , which is also the target of the endogenous opioids. However, unlike endogenous MOP-R ligands, morphine fails to promote substantial endocytosis of the MOP-R both in vitro [2–5] and in vivo [6–11]. Endocytosis of the MOP-R serves at least two important functions in regulating signal transduction. First, desensitization and endocytosis act as an “OFF” switch by uncoupling receptors from their cognate G protein. Second, endocytosis and receptor recycling serve as an “ON” switch, resensitizing receptors by recycling them to the plasma membrane where agonist can regain access to the receptors. Thus, due to poor endocytosis, both the OFF and ON function of the MOP-R are altered in response to morphine compared to endogenous ligands. To examine whether poor endocytosis contributes to morphine tolerance and dependence, we generated a knock-in mouse that expresses a mutant MOP-R that undergoes rapid endocytosis in response to morphine. Morphine remains an excellent antinociceptive agent in these mice. In addition, the mutant mice display substantially reduced antinociceptive tolerance and attenuated physical dependence. These data suggest that opioid drugs with pharmacological profile of morphine and the ability to promote endocytosis of the receptor could provide analgesia while having a reduced liability for the side effects of tolerance and dependence.
To directly examine whether endocytosis of the MOP-R in response to morphine alters the development of tolerance and dependence in vivo, we generated a knock-in mouse expressing the rMOP-R mutant receptor that internalizes in response to morphine . In this rMOP-R, a portion of the cytoplasmic tail of the MOP-R, encoded entirely within exon 3, has been replaced with sequence from the delta opioid peptide receptor (see Fig. 1a). Mice expressing the rMOP-R were identified by Southern (DNA) blot analysis (Fig. 1a and b). The specific mutation introduced to the MOP-R gene  was contained entirely within Exon 3, which is common to all splice variants that have been described. Endocytic trafficking of the wild-type MOP-R and the rMOP-R mutant receptor examined in striatal neurons cultured from wild-type and mutant mice demonstrated that morphine promote rMOP-R but not MOP-R endocytosis in response to morphine (Fig. 1c and Supplemental Fig. 1).
The mutant mice were viable, had no gross phenotypic abnormalities and showed normal baseline pain responses (hot-plate latency, 56°C: wild-type, 4.88 ± 0.33 seconds; mutant, 4.55 ± 0.32 seconds and see Fig. 4 saline treatments). Consistent with their equivalent baseline pain responses, there were no genotypic differences in MOP-R distribution in the spinal cord or multiple brain regions important for the antinociceptive and reinforcing properties of opiates (Supplemental Fig. 2 and data not shown). In addition, ligand affinity, receptor number and receptor G-protein coupling were unaltered in the rMOP-R mice (Fig. 2a-c).
Morphine-induced antinociception was evaluated by measuring response latencies in the hot-plate test. We tested a dose of morphine (10 mg/kg) known to induce robust antinociception in mice. The acute antinociceptive effect of this dose of morphine was significantly enhanced and prolonged in knock-in mice relative to their wild-type littermates (Fig. 3a). A dose of 3 mg/kg in the mutant mouse was equi-antinociceptive to 10 mg/kg in the wild-type mouse (Fig. 3b). Both genotypes reached a ceiling effect at the highest dose tested, 50 mg/kg. The opioid antagonist naloxone completely reversed the antinociceptive effects of morphine in both wild-type and mutant mice (Fig. 3b).
We propose that the enhanced antinociception in the mutant mice reflects the restoration of the ON function provided by receptor endocytosis and recycling. Specifically, we propose that morphine-occupied MOP receptors become partially desensitized in wild-type mice and fail to resensitize due to poor endocytosis; whereas in the rMOP-R knock-in mice, receptors are also desensitized but are rapidly resensitized by endocytosis and recycling. Consistent with this hypothesis, MOP receptors in wild-type mice given a single 10 mg/kg dose of morphine showed significant receptor-G protein uncoupling (Fig. 3c, left panel). Clearly not all MOP receptors in these mice were desensitized, since morphine is still an excellent acute antinociceptive agent in wild-type mice. Nevertheless, MOP-Rs in the brainstem of wild-type mice treated with morphine showed a 200-fold shift in the EC50 of DAMGO (Fig. 3c, left panel) compared to wild-type mice treated with vehicle. In contrast, receptors in rMOP-R mice given the same dose of morphine, showed no desensitization (Fig. 3c, right panel). These data suggest that the reduced morphine antinociception in the wild-type compared to the rMOP-R mice reflects partial desensitization of MOP-Rs that is not reversed by endocytosis and recycling.
If this were the case, we would expect mice of both genotypes to show equivalent antinociception to an agonist that promotes endocytosis of the receptor in both genotypes. Indeed, there were no significant genotypic differences in antinociception induced by methadone (1–10 mg/kg; Fig. 3d), a MOP-R agonist that promotes rapid internalization of both the wild-type MOP-R and mutant rMOP-R. Thus, the enhanced opioid antinociception observed in the rMOP-R knock-in mice is specific to morphine. Together with our immunohistochemical and pharmacological data (Supplemental Fig. 2 and Fig. 2), these data suggest that the enhanced morphine antinociception in the rMOP-R knock-in mice cannot be accounted for by differences in MOP-R distribution, ligand affinity, receptor number or receptor G-protein coupling. Rather, these data suggest that facilitating MOP-R endocytosis enhances morphine antinociception by reversing rapid desensitization.
It has been hypothesized that MOP-R desensitization contributes to acute morphine tolerance. If this were the case, rMOP-R mice would be expected to develop reduced acute tolerance compared to wild-type mice. To examine this, we evaluated the acute antinociceptive effect of equi-antinociceptive doses of morphine (3 mg/kg in rMOP-R and 10 mg/kg in MOP-R, see Fig 3b) 24 hours following pretreatment with a high dose of morphine (100 mg/kg) or saline. The day following pretreatment, baseline response latencies between genotypes were similar (rMOP-R, 5.76 ± 0.47 secs; MOP-R, 5.86, ± 0.51 secs). Indicative of the acute tolerance that is typically observed in this paradigm , wild-type MOP-R mice that had been pretreated with 100 mg/kg of morphine showed a 43% reduction in antinociception compared to mice that had received saline the day before (Fig. 4a). In contrast, the rMOP-R knock-in mice maintained similar levels of morphine antinociception regardless of whether they had received morphine or saline pretreatment the day before (Fig. 4a). Thus, the rMOP-R knock-in mice did not develop acute antinociceptive tolerance to morphine.
While acute tolerance to high doses of opioids is most relevant to acute pain, during the treatment of chronic pain, analgesic tolerance typically develops over the course of repeated administrations of moderate levels of drug. Thus, we evaluated the development of tolerance following twice daily administrations of morphine (10 mg/kg) over 5 days. Wild-type mice in this paradigm developed antinociceptive tolerance (Fig. 4b, squares). In contrast, their rMOP-R littermates, treated with the same dose of morphine (10 mg/kg) at the same intervals, showed no evidence of tolerance, exhibiting as much antinociception on the last day of drug treatment as they did on the first day (Fig. 4b, circles).
To rule out the possibility that the lack of tolerance in the mutant mice was an artifact of enhanced morphine antinociception (Fig. 3a,b), a separate group of knock-in mice were treated chronically with an equi-antinociceptive dose of morphine (3 mg/kg, see Fig. 3b) given at the same intervals. These rMOP-R knock-in mice still showed reduced tolerance, maintaining similar levels of antinociception over the course of treatment (Fig 4b, triangles). Thus, reduced morphine tolerance in the knock-in relative to wild-type mice cannot be attributed to enhanced morphine antinociception.
These results suggest that endocytosis of the receptor reduces the development of antinociceptive tolerance. If this were the case, one would expect that opiate agonists, such as methadone, that promote endocytosis of the MOP-R would have reduced liability for promoting tolerance in wild-type mice. In addition, wild-type and rMOP-R mice should show equivalent responsiveness to chronic methadone. To examine this hypothesis, we evaluated the development of tolerance to methadone in MOP-R and rMOP-R mice. In order to directly compare tolerance to morphine versus methadone, a dose of methadone was chosen (4 mg/kg, see Fig. 3d) that was equi-antinociceptive to the morphine dose administered in Fig. 4b. At this dose, neither genotype showed evidence of tolerance across treatment days (Fig. 4c). In addition, responsiveness to methadone during all treatment days was equivalent in MOP-R (Fig. 4c, squares) and rMOP-R mice (Fig. 4c circles). Thus, reduced chronic opioid tolerance in rMOP-R mice relative to MOP-R mice is specific to morphine.
As was the case for reduced acute tolerance, reduced chronic tolerance to morphine in the mutant mice may reflect, at least in part, that MOP-Rs in the wild-type mice are desensitized (Fig. 3c) but are unable to resensitize due to poor endocytosis of the receptor. Facilitating receptor internalization and recycling (i.e., restoring the ON function of endocytosis) may protect against the development of both acute (Fig. 4a) and chronic tolerance (Fig. 4b).
However, receptor desensitization alone cannot explain antinociceptive tolerance to morphine. Specifically, if all MOP-Rs were desensitized in morphine tolerant mice, then displacement of morphine from these non-signaling receptors with antagonist should have no behavioral effect. However, morphine tolerant animals show substantial naloxone-precipitated withdrawal signs (see for example ), indicating that receptors continue to signal actively in morphine tolerant animals despite the lack of antinociception. Hence, mechanisms other than receptor desensitization are likely contributing to tolerance.
We next examined whether facilitating endocytosis in the rMOP-R mice affected the development of morphine dependence. Following chronic treatment with morphine, mice were challenged with the opioid antagonist, naloxone (2 mg/kg), 30 min following the final morphine injection. Global withdrawal responses were scored by an observer who was blind to genotype (Fig 4d). Wild-type mice expressed robust withdrawal responses compared to mutant mice, which were chronically treated with the same amount of morphine (10 mg/kg) but at a functionally higher dose (see Fig. 3a, b). Consistent with the hypothesis that enhanced receptor endocytosis decreases withdrawal, chronic methadone treatment (4 mg/kg given at the same intervals as morphine), promoted substantially less withdrawal than did morphine in wild-type mice (Fig. 4d). In fact, the moderate level of methadone withdrawal in wild-type mice was equivalent to that produced by either morphine or methadone in the rMOP-R mice (Fig. 4d). Hence, we have generated a mouse line that retains morphine antinociceptive potency with markedly reduced morphine tolerance and dependence.
In summary, here we report that mice expressing a mutant rMOP-R with altered receptor trafficking properties in response to morphine show enhanced morphine-induced antinociception, reduced morphine tolerance, and reduced naloxone-precipitated withdrawal compared to their wild-type littermates. These knock-in mice otherwise show normal ligand affinity, receptor number, receptor G-protein coupling and receptor distribution, consistent with the fact that both their basal pain responses as well as methadone antinociception are equivalent to that of their wild-type littermates.
These data are consistent with the hypothesis that enhanced endocytosis of the MOP-R in response to morphine can reduce antinociceptive tolerance and dependence while retaining the antinociceptive efficacy of morphine. It is important to note that endocytosis is only one step in a cascade of highly conserved events that occurs following G-protein coupled receptor activation. When receptors are activated by endogenous ligand, they are rapidly desensitized by phosphorylation and interaction with arrestin and then endocytosed. Following endocytosis, MOP-Rs are functionally resensitized by recycling to the plasma membrane. Many groups have demonstrated in vitro that morphine-activated receptors elude this natural cycle of receptor desensitization, endocytosis and resensitization that is induced by endogenous MOP-R ligands [15-18]. Similarly, morphine has been found to be a poor inducer of receptor endocytosis in vivo [6–10]. However, in vivo, subtleties also emerge, because in some cases, desensitization has not been detected , whereas in other cases, desensitization of the morphine-activated receptor by GRK/arrestin and/or PKC does appear to occur [20–27]. Thus, receptor desensitization may be either brain region specific, incomplete, or both.
In the context of regionally-specific or incomplete receptor desensitization, the failure to endocytose the morphine-bound receptor has the potential to affect signal transduction in at least two ways. First, in cells or brain regions where morphine does not cause substantial receptor desensitization, prolonged receptor activation may trigger downstream adaptive responses that contribute to morphine tolerance and dependence. In rMOP-R mice, this prolonged receptor activation is replaced by pulsatile receptor activation due to restoration of the OFF/ON switch of endocytosis. Second, in cells or brain regions where receptors do become desensitized after morphine activation, failure to endocytose would prevent functional resensitization of the receptor. In rMOP-R mice, resensitization would be restored. Notably, even in regions where desensitization appears to occur (Fig. 3c), a significant number of receptors remain coupled. These remaining receptors would exhibit prolonged activation in wild-type mice and pulsatile activation in knock-in mice.
Disruption of arrestin appears to enhance morphine antinociception  and delay tolerance , presumably by decreasing the degree of morphine-induced desensitization. However, arrestin knock-out mice show levels of withdrawal equivalent to their wild-type littermates , indicating that there are still a substantial number of functionally coupled MOP-Rs even in animals with intact arrestin. Promoting morphine-induced endocytosis would be expected to both facilitate receptor resensitization and alleviate the compensatory adaptive changes associated with dependence. Thus, while both preventing receptor desensitization and facilitating receptor endocytosis/resensitization are effective strategies to enhance morphine antinociception and prevent tolerance, the latter has the added benefits of 1) reducing morphine dependence and 2) specificity to the MOP-R.
All opioids, when given at high enough concentration for a long enough period of time, including methadone, can induce tolerance and dependence. However, when given at equi-antinociceptive doses, opioids induce different degrees of tolerance and dependence [29–31] and see Fig. 4c, and some ligands even appear to cause these effects by different mechanisms [32, 33]. Hence, given the complex pharmacology of the various opioid ligands, it has been difficult to isolate the effect of endocytosis on tolerance and dependence.
The present results provide a genetic “proof-of-concept” that endocytosis, is an important mechanism that can delay tolerance and dependence. Notably, the use of the rMOP-R knock-in mice allowed the same opioid drug to be compared in mice that appear to differ only in their MOP-R trafficking properties. Importantly, even if mechanisms other than endocytosis are contributing to the behavioral differences in the MOP-R and rMOP-R mice, these mice will provide a powerful tool for delineating which of the adaptive changes that have been observed in wild-type animals following chronic morphine treatment are relevant to behavioral tolerance and dependence.
The authors thank Mark von Zastrow for support in the development of this project and Brigitte Kieffer for supplying the pBK2 plasmid containing the genomic fragment used to generate the targeting vector. We thank Randall Armstrong for assistance with the DNA blot, Yuichiro Inoue, Ling Wang, and Marian Logrip for assistance with primary culture and Brigitte Kieffer, Mark Von Zastrow, Howard L. Fields, Dorit Ron and Randy Hampton for critical reading of the manuscript. This work was supported by the National Institute on Drug Abuse (NIDA) grant DA015232 and funds provided by the state of California for medical research on alcohol and substance abuse through the University of California San Francisco (UCSF), both to J.L.W.
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