Opioid Receptor Trafficking and Signaling: What Happens After Opioid Receptor Activation?

Prolonged opioid treatment leads to a comprehensive cellular adaptation mediated by opioid receptors, a basis to understand the development of opioid tolerance and dependence. However, the molecular mechanisms underlying opioid-induced cellular adaptation remain obscure. Recent advances in opioid receptor trafficking and signaling in cells have extensively increased our insight into the network of intracellular signal integration. This review focuses on those important intracellular biochemical processes that play critical roles in the development of opioid tolerance and dependence after opioid receptor activation, and tries to explain what happens after opioid receptor activation, and how the cellular adaptation develops from cell membrane to nucleus. Decades of research have delineated a network on opioid receptor trafficking and signaling, but the challenge remains to explain opioid tolerance and dependence from a single cellular signal network.     

The effect of alpha-interferon, cyclosporine A, and radiation-induced immune suppression on morphine-induced hypothermia and tolerance

An interconnection between the immune and the central nervous systems has been suggested by investigators studying the actions of several types of immune modifying agents and procedures upon opiate related phenomena. These studies have included the effects of altering immune system function by administration of either alpha-interferon, cyclosporine or radiation exposure upon naloxone-precipitated opiate withdrawal and upon opioid antinociceptive effects. The present study extends these earlier investigations by examining the effect of immune modulation upon opiate induced hypothermia. The results demonstrate that interferon and cyclosporine have no effects on baseline temperature or morphine induced hypothermia, while irradiation exposure elicits hyperthermia without affecting morphine-induced hypothermia. Finally, neither cyclosporine nor irradiation affect the development of tolerance to morphine induced hypothermia, while a single injection of the immune system modifier interferon was able to prevent the development of such tolerance. These observations suggest that yet another opiate-related phenomenon may be regulated at least in part by the immune system. These results together with our previous findings are further evidence of a link between the immune system and the CNS mediated through the opioid system. In addition, these studies further support our earlier hypothesis that "Interferon" is one of the endogenous substances which serves to prevent the development of tolerance and dependence to endogenous opioids.     

Opiate receptors

The main goal of pharmacology is to establish the complete chain of causality between the combination of a drug with its specific receptor and the manifest effects of that drug in the organism. The central concern of modern molecular pharmacology is to understand the first step in that chain -- the drug-receptor interaction (1). Unusual interest attaches to opiate receptors because of the role they may play in analgesia, the euphorigenic effects of narcotics, and other aspects of narcotic addiction such as tolerance and physical dependence.     

Molecular and cellular mechanisms of the age-dependency of opioid analgesia and tolerance

ABSTRACT: The age-dependency of opioid analgesia and tolerance has been noticed in both clinical observation and laboratory studies. Evidence shows that many molecular and cellular events that play essential roles in opioid analgesia and tolerance are actually age-dependent. For example, the expression and functions of endogenous opioid peptides, multiple types of opioid receptors, G protein subunits that couple to opioid receptors, and regulators of G protein signaling (RGS proteins) change with development and age. Other signaling systems that are critical to opioid tolerance development, such as N-methyl-D-aspartic acid (NMDA) receptors, also undergo age-related changes. It is plausible that the age-dependent expression and functions of molecules within and related to the opioid signaling pathways, as well as age-dependent cellular activity such as agonist-induced opioid receptor internalization and desensitization, eventually lead to significant age-dependent changes in opioid analgesia and tolerance development.     

MOR Is Not Enough: Identification of Novel mu-Opioid Receptor Interacting Proteins Using Traditional and Modified Membrane Yeast Two-Hybrid Screens

The mu-opioid receptor (MOR) is the G-protein coupled receptor primarily responsible for mediating the analgesic and rewarding properties of opioid agonist drugs such as morphine, fentanyl, and heroin. We have utilized a combination of traditional and modified membrane yeast two-hybrid screening methods to identify a cohort of novel MOR interacting proteins (MORIPs). The interaction between the MOR and a subset of MORIPs was validated in pulldown, co-immunoprecipitation, and co-localization studies using HEK293 cells stably expressing the MOR as well as rodent brain. Additionally, a subset of MORIPs was found capable of interaction with the delta and kappa opioid receptors, suggesting that they may represent general opioid receptor interacting proteins (ORIPS). Expression of several MORIPs was altered in specific mouse brain regions after chronic treatment with morphine, suggesting that these proteins may play a role in response to opioid agonist drugs. Based on the known function of these newly identified MORIPs, the interactions forming the MOR signalplex are hypothesized to be important for MOR signaling and intracellular trafficking. Understanding the molecular complexity of MOR/MORIP interactions provides a conceptual framework for defining the cellular mechanisms of MOR signaling in brain and may be critical for determining the physiological basis of opioid tolerance and addiction.     

Endocytosis of the mu opioid receptor reduces tolerance and a cellular hallmark of opiate withdrawal.

Morphine is unusual in its failure to promote robust desensitization and endocytosis of the mu opioid receptor (MOR), processes that for many receptors contribute directly to tolerance. This apparent paradox has led us to revise the idea that receptor desensitization and endocytosis are solely responsible for tolerance and withdrawal to morphine, and instead test the hypothesis that these side effects occur due to abnormally prolonged MOR signaling. We report here that MOR mutations that facilitate endocytosis reduce the development of cellular tolerance and cAMP superactivation, a cellular hallmark of withdrawal. Moreover, mutant receptors with reduced endocytosis produce exacerbated superactivation. These data demonstrate a critical role for receptor endocytosis in the development of adverse side effects associated with prolonged opiate use.     

The next frontier in the molecular biology of the opioid system

The analgesic and euphoric properties of some plant alkaloids such as morphine have been known and exploited for centuries. In contrast, only during the last twenty years have we begun to unravel the molecular basis by which opiates exert their effects, mechanisms important to our general understanding of the nervous system. The analgesic response to opiates is the result of a cascade of biochemical events that are triggered by the interaction of the opiate with specific macromolecular components found on the membranes of nervous system tissues, the opioid receptors. The endogenous ligands of these receptors are small peptides, the opioid peptides. Although much has been learned about the structures and the mode of synthesis of the opioid peptides, little is understood about the structure of their receptors. The application of molecular genetic techniques was of great importance to the studies of the opioid peptides. It is now expected that this same technology will unravel the physical mysteries of the opioid receptors.     

Multiple actions of spinophilin regulate mu opioid receptor function

Spinophilin, a dendritic spine-enriched scaffold protein, modulates synaptic transmission via multiple functions mediated by distinct domains of the protein. Here, we show that spinophilin is a key modulator of opiate action. Knockout of the spinophilin gene causes reduced sensitivity to the analgesic effects of morphine and early development of tolerance but a higher degree of physical dependence and increased sensitivity to the rewarding actions of the drug. At the cellular level, spinophilin associates with the mu opioid receptor (MOR) in striatum and modulates MOR signaling and endocytosis. Activation of MOR by opiate agonists such as fentanyl and morphine promotes these events, which feedback to suppress MOR responsiveness. Our findings support a potent physiological role of spinophilin in regulating MOR function and provide a potential new target for the treatment of opiate addiction.     

Proteomic analysis of spinal protein expression in rats exposed to repeated intrathecal morphine injection

Repeated administration of morphine for treating severe chronic pain may lead to neuroadaptive changes in the spinal cord that are thought to underlie molecular mechanisms of the development of morphine tolerance and physical dependence. Here, we employed a 2-D gel-based proteomic technique to detect the global changes of the spinal cord protein expression in rats that had developed morphine tolerance. Morphine tolerance at the spinal cord level was induced by repeated intrathecal injections of morphine (20 microg/10 microL) twice daily for 5 days and evaluated by measurements of paw withdrawal latencies and maximal possible analgesic effect at day 5. After behavioral tests, the lumbar enlargement segments of spinal cord were harvested and proteins resolved by 2-DE. We found that eight proteins were significantly up-regulated or down-regulated in spinal cord after morphine tolerance development, including proteins involved in targeting and trafficking of the glutamate receptors and opioid receptors, proteins involved in oxidative stress, and cytoskeletal proteins, some of which were confirmed by Western blot analysis. Morphine-induced expressional changes of these proteins in the spinal cord might be involved in the central mechanisms that underlie the development of morphine tolerance. It is very likely that these identified proteins may serve as potential molecular targets for prevention of the development of morphine tolerance and physical dependence.     

Upregulation of the opioid receptor complex by the chronic administration of morphine: a biochemical marker related to the development of tolerance and dependence.

Studies conducted after the development of the rapid filtration assay for opiate receptors, and before the recognition of multiple opioid receptors, failed to detect changes in opioid receptors induced by chronic morphine. Recent experiments conducted in our laboratories were designed to examine the hypothesis that only one of several opioid receptor types might be altered by chronic morphine. Using binding surface analysis and irreversible ligands to increase the "resolving power" of the ligand binding assay, the results indicated that chronic morphine increased both the Bmax and Kd of the opioid receptor complex, labeled with either [3H][D-Ala2,D-Leu5]enkephalin, [3H][D-Ala2-MePhe4,Gly-ol5]enkephalin or [3H]6-desoxy-6 beta-fluoronaltreone. In the present study rats were pretreated with drugs known to attenuate the development of tolerance and dependence [the irreversible mu-receptor antagonist, beta-funaltrexamine (beta-FNA), and the inhibitor of tryptophan hydroxylase, para-chlorophenylalanine], prior to subcutaneous implantation of morphine pellets. The results demonstrated that 1) unlike chronic naltrexone, beta-FNA failed to upregulate opioid receptors and 2) both beta-funaltrexamine and PCPA pretreatment attenuated the chronic morphine-induced increase in the Bmax, but not the Kd, of the opioid receptor complex. These results provide evidence that naltrex-one-induced upregulation of the opioid receptor complex might occur indirectly as a consequence of interactions at beta-funaltrexamine-insensitive opioid receptors and that morphine-induced upregulation (increased Bmax) of the opioid receptor complex is a relevant in vitro marker related to the development of tolerance and dependence. These data collectively support the hypothesis that endogenous antiopiate peptides play an important role in the development of tolerance and dependence to morphine.     

Endogenous opiates and behavior: 2014

This paper is the thirty-seventh consecutive installment of the annual review of research concerning the endogenous opioid system. It summarizes papers published during 2014 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior (endogenous opioids and receptors), and the roles of these opioid peptides and receptors in pain and analgesia (pain and analgesia); stress and social status (human studies); tolerance and dependence (opioid mediation of other analgesic responses); learning and memory (stress and social status); eating and drinking (stress-induced analgesia); alcohol and drugs of abuse (emotional responses in opioid-mediated behaviors); sexual activity and hormones, pregnancy, development and endocrinology (opioid involvement in stress response regulation); mental illness and mood (tolerance and dependence); seizures and neurologic disorders (learning and memory); electrical-related activity and neurophysiology (opiates and conditioned place preferences (CPP)); general activity and locomotion (eating and drinking); gastrointestinal, renal and hepatic functions (alcohol and drugs of abuse); cardiovascular responses (opiates and ethanol); respiration and thermoregulation (opiates and THC); and immunological responses (opiates and stimulants).This paper is the thirty-seventh consecutive installment of the annual review of research concerning the endogenous opioid system. It summarizes papers published during 2014 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior (endogenous opioids and receptors), and the roles of these opioid peptides and receptors in pain and analgesia (pain and analgesia); stress and social status (human studies); tolerance and dependence (opioid mediation of other analgesic responses); learning and memory (stress and social status); eating and drinking (stress-induced analgesia); alcohol and drugs of abuse (emotional responses in opioid-mediated behaviors); sexual activity and hormones, pregnancy, development and endocrinology (opioid involvement in stress response regulation); mental illness and mood (tolerance and dependence); seizures and neurologic disorders (learning and memory); electrical-related activity and neurophysiology (opiates and conditioned place preferences (CPP)); general activity and locomotion (eating and drinking); gastrointestinal, renal and hepatic functions (alcohol and drugs of abuse); cardiovascular responses (opiates and ethanol); respiration and thermoregulation (opiates and THC); and immunological responses (opiates and stimulants).     

Opioids and breathing

This review summarizes recent developments on the effects of opiate drugs and the various endogenous opioid peptides on breathing. These developments include demonstration of receptors and site-specific effects of application of opioids in the pons and medulla, demonstration of variable tolerance of respiratory responses in addicted individuals as well as their offspring, and demonstration of an endogenous opioid influence on breathing in early neonatal life and in certain physiological settings and disease states. The validity and limitations of using naloxone as a tool to uncover postulated endogenous opioid influences are also discussed as well as the potential problems imposed by the various settings in which this opiate antagonist drug is used. It is concluded that some parallelism exists between the role of endogenous opioids in pain modulation and their role in respiration especially in adults. Although more studies are needed especially with regard to defining specific effects of the various opioid receptors and ligands, it is felt that the effects of endogenous opioids on the control of breathing will probably be one of modulating the responses to drugs or nociceptive respiratory stimuli through inhibitory pathways.     

Stress responsivity, addiction, and a functional variant of the human mu-opioid receptor gene

Discovery and characterization of the functional A118G mu-(mu)-opioid receptor variant led to hypotheses, now in part proven, about its role in alterations of endogenous human physiology and in responses to opioid antagonist administration. Differences in cellular expression levels, ligand binding, and signal transduction for variant receptors have been documented in vitro. Human genetic studies also indicate that individuals carrying one or two copies of the 118G allele may have increased risk for opiate and alcohol addictions and that this polymorphism may also explain some of the variability in success of opioid antagonist treatment for alcoholism. Future research will further define the role of the A118G variant in addictive diseases and their treatment, in pain perception and opioid analgesia, and for a myriad of other responses mediated by the mu-opioid receptor.     

Opioid receptor agonists and Ca2+ modulation in human B cell lines.

Opiates and opioid peptides have been shown to modulate lymphocyte functions; however, little attention has been given to the type of receptors or receptor signaling mechanisms that are involved. Receptor-mediated signaling via ionized free Ca2+ is an event thought to be important in the triggering of lymphocyte activities. We report use of the calcium indicator dye, indo-1, and flow cytometry to identify B lymphocyte calcium responses to physiologic concentrations of opioid peptides. The human B cell lines Nalm 6 and JY responded to the naturally occurring opioid pentapeptide methionine-enkephalin or other opiate receptor agonists with a rapid, dose-dependent rise in free cytoplasmic Ca2+. This opioid peptide effect on Ca2+ modulation was inhibited by the opiate receptor antagonist naloxone. The synthetic enkephalin analogue DAMGO with specificity for mu-type opiate receptors and the synthetic opiate receptor agonists U50,488H and U69,593 with selectivity for kappa-type sites also stimulated calcium responses when applied to the B cell lines. These studies provide evidence that human B cell lines express functional opiate receptors of the mu- and kappa-types and suggest that such receptors, coupled with Ca2+ modulation, are instrumental in the B cell response to opiates and endogenous opioid neuropeptides.     

Agonist-specific regulation of the delta-opioid receptor

Delta opioid receptor (DOR) agonists are attractive potential analgesics, since these compounds exhibit strong antinociceptive activity with relatively few side effects. In the past decade, several novel classes of delta-opioid agonists have been synthesized. Recent experimental data indicate that structurally distinct opioid agonists interact differently with the delta-opioid receptor. Consequently, individual agonist-bound DOR conformations may interact differently with intracellular proteins. In the present paper, after a brief review of the cellular processes that contribute to homologous desensitization of the DOR signaling, we shall focus on experimental data demonstrating that chemically different agonists differ in their ability to phosphorylate, internalize, and/or down-regulate the DOR. Homologous regulation of the opioid receptor signaling is thought to play an important role in the development of opioid tolerance. Therefore, agonist-specific differences in DOR regulation suggest that by further chemical modification, delta-selective opioid analgesics can be designed that exhibit a reduced propensity for analgesic tolerance.     

From inhibition to excitation: functional effects of interaction between opioid receptors

Opioids have excitatory effects in multiple regions of the nervous system. Excitation by opioids is generally attributed to inhibition of inhibitory pathways (disinhibition). However, recent studies indicate that opioids can directly excite individual cells. These effects may occur when opioid receptors interact with other G protein coupled receptors, when different subtypes of opioid receptors interact, or when opioids transactivate other receptors such as receptor tyrosine kinases. Changes in the relative level of expression of different receptors in an individual cell may therefore determine its functional response to a given ligand. This phenomenon could represent an adaptive mechanism involved in tolerance, dependence and subsequent withdrawal.     

NMDA受体拮抗剂对阿片类药物耐受和依赖的阻断作用

本文综述阻断ＮＭＤＡ受体离子通道复合药物对阿惩耐受和成瘾发生的影响。行为药理学研究显示,非竞争性ＮＭＤＡ受体拮抗剂、竞争性ＮＭＤＡ受体拮抗剂和甘氨酸受占拮抗剂能抑制阿片耐受和戒断反应,其药理学特性明显不同于其他类型抗阿片耐受和成瘾的药物,阐述了ＮＭＤＡ受体拮抗剂治疗阿片类芗耐受和领事的系列化机制。并指出ＮＭＤＡ受体拮抗剂具有神经毒性。     

Effect of naltrindole on the development of physical dependence on morphine in mice: A behavioral and biochemical study

The effect of pretreatment with a δ opioid receptor antagonist, naltrindole (NTI), on the development of physical dependence on morphine was investigated in mice. Several withdrawal signs, an increase in cortical noradrenaline (NA) turnover and a decrease in dopamine (DA) turnover in the limbic forebrain were observed following naloxone challenge in morphine-dependent mice. Pretreatment with NTI (0.3–5 mg/Kg, S.c.) during chronic morphine treatment dose-dependently suppressed the behavioral and biochemical changes after withdrawal. The blocking effects of NTI suggest that δ opioid receptors may play a significant role in modulating the development of physical dependence on morphine.     

Functional dissociation of mu opioid receptor signaling and endocytosis: implications for the biology of opiate tolerance and addiction.

Opiate analgesia, tolerance, and addiction are mediated by drug-induced activation of the mu opioid receptor. A fundamental question in addiction biology is why exogenous opiate drugs have a high liability for inducing tolerance and addiction while native ligands do not. Studies indicate that highly addictive opiate drugs such as morphine are deficient in their ability to induce the desensitization and endocytosis of receptors. Here, we demonstrate that this regulatory mechanism reveals an independent functional property of opiate drugs that can be distinguished from previously established agonist properties. Moreover, this property correlates with agonist propensity to promote physiological tolerance, suggesting a fundamental revision of our understanding of the role of receptor endocytosis in the biology of opiate drug action and addiction.     

Endogenous opiates and behavior: 2007

This paper is the thirtieth consecutive installment of the annual review of research concerning the endogenous opioid system. It summarizes papers published during 2007 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior, and the roles of these opioid peptides and receptors in pain and analgesia; stress and social status; tolerance and dependence; learning and memory; eating and drinking; alcohol and drugs of abuse; sexual activity and hormones, pregnancy, development and endocrinology; mental illness and mood; seizures and neurologic disorders; electrical-related activity and neurophysiology; general activity and locomotion; gastrointestinal, renal and hepatic functions; cardiovascular responses; respiration and thermoregulation; and immunological responses.