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Modified Mu Opioid Receptors Lead to Analgesia Without Physical Dependence

/, Biology, Health and Medicine/Modified Mu Opioid Receptors Lead to Analgesia Without Physical Dependence

Modified Mu Opioid Receptors Lead to Analgesia Without Physical Dependence

2021-05-17T18:08:05-07:00 May 14th, 2021|Biochemistry, Biology, Health and Medicine|

By Neha Madugala, Neurobiology, Physiology, and Behavior ‘21

Author’s Note: I wrote this literature review for my UWP104F class to assess new opioid-based medications for pain-relief. While opioids are the best known pain relievers we currently have, they have the severe risks of addiction and overdose. This paper analyzes literature that attempts to amplify the analgesic (pain-relief) properties of opioids, while minimizing their addictive potential. 

 

Introduction

As the opioid epidemic grows, opioids are becoming increasingly synonymous with addiction and overdose. While opioids have immense pain-relief properties, their use has been limited due to their major risks. The potential for addiction lies in the interaction between opioids and the mu-opioid receptor. Scientists are working to create a modified receptor that can have these analgesic effects but have limited risk. These findings could aid in the development of safer but effective pain-relief medications. 

Background

Drug addiction is a chronic relapsing disorder that occurs through repeated exposure to the drug, leading to physiological changes in brain chemistry [6]. While initial use is associated with improved well-being and feelings of euphoria, this repeated use in addition to environmental factors and genetics can lead to modifications in the endogenous opioid system, or the body’s natural pain killers, and alterations of stress physiology through hormonal imbalance [6, 7]. These homeostatic changes deregulate brain reward pathways, resulting in tolerance and dependence [7]. 

Opioids have a significantly higher rate of relapse compared to other addictive drugs since they induce strong physical dependence [7] and craving [7]. As a result, scientists have extensively studied the mu-opioid receptor (MOPr), which directly binds to opioids and indirectly binds to other addictive drugs, such as alcohol, cannabinoids, and nicotine [6]. Current research is focused on understanding the mechanism of the MOPr. While scientists agree that this mechanism plays a role in physical dependence, it is unclear what part of this pathway is responsible for these observed effects. 

Opioids act as agonists at the MOPr. Agonists modify a receptor through intrinsic efficacy and affinity. Affinity determines how well a ligand is able to bind to the active site of a receptor and intrinsic efficacy is a measure of how well a ligand is able to stabilize the receptor in its active conformation [1]. Intrinsic efficacy is particularly important for the MOPr because the MOPr is strongly agonist-dependent [6]. For instance, morphine acts as a full agonist at the MOPr, inducing the maximal effect. As a result, morphine is commonly used to study the function of this receptor and its role in addiction. 

At the MOPr, opioids bind to the active site inducing a conformational change and activating the receptor. The MOPr is a G protein-coupled receptor (GPCR), which is a type of receptor where activation leads to secondary pathway signaling. As a result, binding of a ligand to the MOPr causes secondary pathways to be activated, resulting in downstream signaling effects. Specifically, the MOPr is a Gi-coupled GPCR, so it inhibits the enzyme adenylyl cyclase, which converts ATP to cAMP. As a result, opioid binding inhibits the production of cAMP. 

This effect is brief. MOPr activation promotes translocation of β-arrestin from the cytosol to the plasma membrane. MOPr is rapidly phosphorylated by GPCR kinases (GRKs). This phosphorylation increases the affinity of β-arrestin to the MOPr [6]. β-arrestin binds to the MOPr. This binding uncouples the MOPr from the Gi-coupled GPCR, which halts the inhibition of cAMP production. This uncoupling further halts the signaling pathways and results in desensitization of the MOPr, since the opioid can no longer induce downstream effects.

β-arrestin also recruits components of the endocytic machinery [2] to engulf the MOPr into the cell. The desensitized receptor is internalized within the cell via endocytosis [2]. Endocytosis plays an important role in engulfing the desensitized receptor, which is no longer functional, and placing it back into the plasma membrane resensitized, or functional. This rapid resensitization process is important for having a consistent supply of available receptors for ligand binding. 

Naloxone acts to block agonists of the MOPr and is used as a treatment for drug overdose, and to induce withdrawal in experimental models. As an antagonist, naloxone has zero intrinsic efficacy, so it does not modify the constitutive activity of the receptor.  Essentially antagonists do not activate any additional pathways but have an affinity for the active site of the respective receptor [1]. As a result, naloxone competes with agonists for the binding site and prevents agonists from inducing an effect on the MOPr. Their ability to compete is dependent on their level of affinity. Opioid antagonists are used in drug experiments to quickly stop drug administration in the brain to assess signs of physical dependence. This method is efficient because it can prevent agonists from working, even when the agonist is present in the bloodstream. For this reason, antagonists are also administered following overdose to reverse opioid-induced respiratory depression, where the brain stops sending signals to the body to breathe [4]. 

β-arrestin and Physical Dependence

Enkephalins, an endogenous opioid, at the MOPr activate the Gi-coupled GPCR and β-arrestin in equal amounts. As a result, enkephalins are “unbiased.” However, many addictive opioids, such as morphine, act as biased agonists signaling the Gi-coupled GPCR and β-arrestin asymmetrically [1]. At the MOPr, scientists hypothesize that “G-protein signaling [is] responsible for opioid-induced analgesia, while [β-arrestin is] responsible for the adverse effects of mu-receptor activation” [9]. 

In a study in the Nature Journal of β-arrestin-2 knockout mice by Bohn et al., they studied the development of antinociception tolerance following daily administration of a moderate dose of morphine (10 mg/kg) for nine days [3]. They conducted this experiment to determine whether chronic morphine use can diminish antinociception over time, a sign of addiction. They used wild-type mice as controls. To control for genetic variation, they crossed over eight generations of mice that were heterozygous for β-arrestin-2. This allowed them to develop wild-type and knockout mice (through the homozygous progeny) that were “age-matched, 3—5-month-old male siblings weighing between 20 and 30 g [3]. The wild-type mice had a significantly diminished response to morphine administration by day five. In contrast, the knockout mice had comparable antinociception throughout the entire experimental period [3]. These findings suggest that β-arrestin presence has a significant role in the development of antinociceptive tolerance. 

Moreover, β-arrestin plays multiple roles in the MOPr trafficking process. To further assess these results, researchers studied one aspect of β-arrestin, promoting endocytosis. Kim et al. hypothesized that over time morphine binding to the MOPr led to diminished antinociception, tolerance, and dependence due to morphine’s inability “to promote substantial receptor endocytosis” [10]. They generated knockout mice by genetically modifying the MOPr at exon 3 to create an experimental receptor (rMOP-R) that would be more effective at resensitizing the receptor through endocytosis [10]. They used wild-type mice as a control. 

The mice with the rMOP-R had enhanced and prolonged analgesic effects compared to the wild-type mice with the MOP-R. They suggested that these results were due to the rMOP-R being active for longer due to quicker resensitization [10]. Furthermore, they assessed tolerance and dependence over both a short-term experiment (one day) and a long-term experiment (five days). The wild-type mice displayed both acute and chronic antinociceptive tolerance to morphine, as well as withdrawal responses to any administration of naloxone following morphine administration. The knockout mice did not develop acute or chronic antinociceptive tolerance to morphine and showed much fewer withdrawal responses to administration of naloxone following morphine administration [10]. 

Kim et al. did these experiments with endogenous opioids (DAMGO), morphine, and methadone with acts similarly to endogenous opioids. They found a significant difference between the wild-type and knockout mice for morphine administration only [10]. This supports their hypothesis since DAMGO and methadone already have enhanced endocytosis. 

These results suggest that enhanced endocytosis of the MOPr can help alleviate tolerance and signs of physical dependence while maintaining the antinociceptive effects. While β-arrestin functions to recruit the endocytic machinery, it also turns off the MOPr by desensitizing the receptor. Since the MOPr is strongly agonist-dependent, morphine acting at this receptor results in an increased period of desensitization. By diminishing this off period through enhanced endocytosis, the rMOP-R receptor leads to quicker resensitization, alleviating signs of physical dependence [10]. 

To further explore this hypothesis, Berger and Whistler conducted a similar study to Kim et al. by comparing the knockout mice with rMOP-R to wild-type mice with the MOPr in a conditioned place preference (CPP) paradigm and self-administration study. A CPP paradigm is when the mice are placed in a room with two sides that are decorated in distinct manners. On one side they are administered morphine, while they are not on the other side. Preference for morphine is determined by which room they spend more time in. For the CPP paradigm, the rMOP-R mice displayed greater CPP at lower doses and the wild-type mice displayed greater CPP at higher doses [2]. This indicates that the knockout mice had a large rewarding effect at low doses, suggesting that smaller doses of morphine elicited a larger effect in the knockout mice compared to the wild-type mice. 

Furthermore, they assessed how these mice lines differed for additional determinants of addiction in a self-administration study. For each operant session, the mice were allowed to administer morphine on the first day, morphine and water for the next four days, and were given only water on the last two days of the experiment [2]. They assessed four factors: “high motivation to obtain drug,” “futile drug-seeking,” “persistent drug-seeking in the face of adverse consequences,” and “reduced preference for alternative rewards” [2]. 

Berger and Whistler found that when morphine was administered into the lever at regular intervals, the knockout mice learned these intervals while the wild-type mice attempted to administer the drug more often by pressing the lever, even though it was in between the intervals [2]. They further assessed lever presses when accompanied by an electric shock or in the presence of an alternate reward, saccharin. The wild-type mice administered the drug more often and showed a greater preference for morphine respectively for these experiments compared to the knockout mice [2]. These results indicate that the enhanced endocytosis and quicker resensitization of the MOPr helps alleviate the adverse effects of addiction beyond physical dependence. 

G-Protein Biased Agonists 

Based on these findings, researchers hypothesize that creating drugs that are G-protein biased agonists could help improve the antinociceptive effects of opioids while diminishing the adverse effects of delayed desensitization due to β-arrestin [9]. There is ongoing research to develop G-protein-biased agonists of the MOPr. For instance, oliceridine, a G-protein-biased agonist, is currently in Phase 3 of clinical trials [8]. However, current research is bringing into question whether only β-arrestin is responsible for the adverse effects of addiction [8]. 

There is now evidence suggesting that “MOPr activation in [the preBotzinger (preBotC) and Kolliker-Fuse (KF) neurons] … inhibits neuronal activity via G protein signaling” [8]. The preBotC and KF neurons are located in regions of the brain associated with respiratory control [8]. They also found that the activation of GRKs, which promotes arrestin binding to Gi-coupled GPCRs, is mediated by G protein signaling [8].

In a study in the British Journal of Pharmacology of morphine-induced respiratory depression independent of β-arrestin-2 by Kliewer et al., they used β-arrestin knockout mice to assess respiratory depression when administered an opioid. This experiment was conducted in three laboratories, located in Jena, Germany; Sydney, Australia; and Bristol, United Kingdom [11]. Each laboratory used a similar experimental set-up with slight variations. 

They all found a “dose-dependent depression of respiratory rate by morphine” [11]. The laboratory in Jena used a nose-out plethysmography system; the laboratories in Sydney and Bristol used whole-body plethysmography [11]. (Plethysmography methods assess the difference in the volume of air present in the lungs prior to and post-exhaling.) 

While biased agonism has been suggested as a possible mechanism to isolate the analgesic properties of opioids from the adverse effects, these studies point out weaknesses in this model. First, the initial study assessing β-arrestin-2 knockout mice has not been extensively repeated to verify accuracy and the pathway for respiratory depression cannot be isolated to only β-arrestin [11]. This new evidence indicates that both G-protein signaling and β-arrestin play a role in the physical dependence on opioids. However, more research is needed to determine the extent of how much each protein influences these physiological symptoms. 

Discussion

While the original findings indicated that only β-arrestin is responsible for the adverse effects of opioid use, new research suggests that these effects cannot be easily isolated to just β-arrestin. The results indicate that β-arrestin plays a role in tolerance and dependence, while G-protein plays a role in respiratory depression during an overdose. More research on G-protein signaling and its connection to the dopamine reward pathway is necessary to understand the extent of its involvement in the MOPr trafficking system. Furthermore, more research is needed to replicate the studies done on β-arrestin-2 knockout mice because these studies have not been extensively replicated, bringing into question the accuracy of these past findings. Also, our understanding of the role of β-arrestin-2 in the desensitization of the MOPr is based on research done in HEK 293 cells, found in the human embryonic kidney; these results have not been replicated in neurons [5], yet the MOPr trafficking system occurs within neurons. Overall, further research is needed to establish the reliability of past findings on β-arrestin and to understand the adverse effects of G-protein signaling. The latter is especially important because current research is focused on developing G-protein-biased agonists to act as analgesics; however, these new analgesics may still pose the risk of respiratory depression.  

Conclusion

Current research of the mu-opioid receptor trafficking system indicates that both β-arrestin and Gi-coupled GPCR are responsible for the adverse effects of opioids, including tolerance, dependence, and respiratory depression. More extensive research is required to determine the exact roles of the β-arrestin and Gi-coupled GPCR, while also verifying the results of past studies. These findings can help determine the risk of respiratory depression in the development of new analgesics and extend our understanding of the development of tolerance and withdrawal in addiction. Moreover, these results can help diminish the risk of more potent opioids, while acting as effective pain medications. 

 

References

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  2. Berger, Amy C, and Jennifer L. Whistler. “Morphine-Induced Mu Opioid Receptor Trafficking Enhances Reward yet Prevents Compulsive Drug Use.” EMBO Molecular Medicine, vol. 3, no. 7, 2011, pp. 385-97. NCBI, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3394511/.
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  7. Gerrits, Mirjam, et al. “Drug Dependence and the Endogenous Opioid System.” European NeuroPsychoPharmacology, vol. 13, 2003, pp. 424-434. Elsevier Ltd., doi:10.1016/j.euroneuro.2003.08.003.
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  10. Kim, Joseph A., et al. “Morphine-Induced Receptor Endocytosis in a Novel Knockin Mouse Reduces Tolerance and Dependence.” Cell Press Journal, vol. 18, no. 2, 2008, pp. 129-35. NIH, https://pubmed.ncbi.nlm.nih.gov/18207746/. Kliewer, Andrea, et al. “Morphine-Induced Respiratory Depression is Independent of β-arrestin-2 Signalling.” British Journal of Pharmacology, vol. 177, 2020, pp. 2923-2931. Wiley Online Library, https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.15004.