Mu Opioid Receptor Regulation Research Articles

Key phosphorylation sites for robust β-arrestin2 binding at the MOR revisited

Desensitisation of the mu-opioid receptor (MOR) is proposed to underlie the initiation of opioid analgesic tolerance and previous work has shown that agonist-induced phosphorylation of the MOR C-tail contributes to this desensitisation. Moreover, phosphorylation is important for β-arrestin recruitment to the receptor, and ligands of different efficacies induce distinct phosphorylation barcodes. The C-tail 370TREHPSTANT379 motif harbours Ser/Thr residues important for these regulatory functions. 375Ser is the primary phosphorylation site of a ligand-dependent, hierarchical, and sequential process, whereby flanking 370Thr, 376Thr and 379Thr get subsequently and rapidly phosphorylated. Here we used GRK KO cells, phosphosite specific antibodies and site-directed mutagenesis to evaluate the contribution of the different GRK subfamilies to ligand-induced phosphorylation barcodes and β-arrestin2 recruitment. We show that both GRK2/3 and GRK5/6 subfamilies promote phosphorylation of 370Thr and 375Ser. Importantly, only GRK2/3 induce phosphorylation of 376Thr and 379Thr, and we identify these residues as key sites to promote robust β-arrestin recruitment to the MOR. These data provide insight into the mechanisms of MOR regulation and suggest that the cellular complement of GRK subfamilies plays an important role in determining the tissue responses of opioid agonists.

Cellular Tolerance Induced by Chronic Opioids in the Central Nervous System.

Opioids are powerful analgesics that elicit acute antinociceptive effects through their action the mu opioid receptor (MOR). However opioids are ineffective for chronic pain management, in part because continuous activation of MORs induces adaptive changes at the receptor level and downstream signaling molecules. These adaptations include a decrease in receptor-effector coupling and changes to second messenger systems that can counteract the persistent activation of MORs by opioid agonists. Homeostatic regulation of MORs and downstream signaling cascades are viewed as precursors to developing tolerance. However, despite numerous studies identifying crucial mechanisms that contribute to opioid tolerance, no single regulatory mechanism that governs tolerance in at the cellular and systems level has been identified. Opioid tolerance is a multifaceted process that involves both individual neurons that contain MORs and neuronal circuits that undergo adaptations following continuous MOR activation. The most proximal event is the agonist/receptor interaction leading to acute cellular actions. This review discusses our understanding of mechanisms that mediate cellular tolerance after chronic opioid treatment that, in part, is mediated by agonist/receptor interaction acutely.

Involvement of GRK2 in modulating nalfurafine-induced reduction of excessive alcohol drinking in mice

Though it is well known that G protein-coupled receptor kinase 2 [GRK2] is involved in regulation of mu opioid receptor [MOR] desensitization and morphine-related behaviors, the potential role of GRK2 in regulation of kappa opioid receptor [KOR] functions in vivo has not been established yet. A couple of recent studies have found that GRK2 activity desensitizes KOR functions via decreasing G protein-coupled signaling with sensitizing arrestin-coupled signaling. Nalfurafine, a G protein-biased KOR full agonist, produces an inhibitory effect on alcohol intake in mice, with fewer side effects (sedation, aversion, or anxiety/depression-like behaviors). Using RNA sequencing (RNA-seq) analysis, we first identified that nuclear transcript level of grk2 [adrbk1] (but not other grks) was significantly up-regulated in mouse nucleus accumbens shell (NAcs) after chronic excessive alcohol drinking, suggesting alcohol specifically increased NAcs grk2 expression. We then tested whether selective GRK2/3 inhibitor CMPD101 could alter alcohol intake and found that CMPD101 alone had no effect on alcohol drinking. Therefore, we hypothesized that the grk2 increase in the NAcs could modulate the nalfurafine effect on alcohol intake via interacting with the G protein-mediated KOR signaling. Nalfurafine decreased alcohol drinking in a dose-related manner, and pretreatment with CMPD101 enhanced the reduction in alcohol intake induced by nalfurafine, indicating an involvement of GRK2/3 blockade in modulating G protein-biased KOR agonism of nalfurafine. Together, our study provides initial evidence relevant to the transcriptional change of grk2 gene in the NAc shell after excessive alcohol drinking. Pharmacological GRK2/3 blockade enhanced nalfurafine’s efficacy, suggesting a GRK2/3-mediated mechanism, probably through the G protein-mediated KOR signaling.

Mu opioid receptor regulation of glutamate efflux in the central amygdala in response to predator odor

The amygdala plays an important role in the responses to predator threat. Glutamatergic processes in amygdala regulate the behavioral responses to predator stress, and we have found that exposure to ferret odor activates glutamatergic neurons of the basolateral amygdala [BLA] which are known to project to the central amygdala [CeA]. Therefore, we tested if predator stress would increase glutamate release in the rat CeA using in vivo microdialysis, while monitoring behavioral responses during a 1 h exposure to ferret odor. Since injections of mu opioid receptor [MOR] agonists and antagonists into the CeA modulate behavioral responses to predator odor, we locally infused the MOR agonist DAMGO or the MOR antagonist CTAP into the CeA during predator stress to examine effects on glutamate efflux and behavior. We found that ferret odor exposure increased glutamate, but not GABA, efflux in the CeA, and this effect was attenuated by tetrodotoxin. Interestingly, increases in glutamate efflux elicited by ferret odor exposure were blocked by infusion of CTAP, but CTAP did not alter the behavioral responses during predator stress. DAMGO alone enhanced glutamate efflux, but did not modulate glutamate efflux during predator stress. These studies demonstrate that ferret odor exposure, like other stressors, enhances glutamate efflux in the CeA. Further, they suggest that activation of MOR in the CeA may help shape the defensive response to predator odor and other threats.

Posttranscriptional Regulation of Mu Opioid Receptor (MOR-1): Implications of Alternative Splicing in the Neuropathogenesis of Morphine Tolerance. Literature Review and Research Approach

Posttranscriptional Regulation of Mu Opioid Receptor (MOR-1): Implications of Alternative Splicing in the Neuropathogenesis of Morphine Tolerance. Literature Review and Research Approach

Tolerance to the antinociceptive and hypothermic effects of morphine is mediated by multiple isoforms of c-Jun N-terminal kinase.

The abuse and overdose of opioid drugs are growing public health problems worldwide. Although progress has been made toward understanding the mechanisms governing tolerance to opioids, the exact cellular machinery involved remains unclear. However, there is growing evidence to suggest that c-Jun N-terminal kinases (JNKs) play a major role in mu-opioid receptor regulation and morphine tolerance. In this study, we aimed to determine the potential roles of different JNK isoforms in the development of tolerance to the antinociceptive and hypothermic effects of morphine. We used the hot-plate and tail-flick tests for thermal pain to measure tolerance to the antinociceptive effects of once-daily subcutaneous injections with 10 mg/kg morphine. Body temperature was also measured to determine tolerance to the hypothermic effects of morphine. Tolerance to morphine was assessed in wild-type mice and compared with single knockout mice each lacking the JNK isoforms (JNK1, JNK2, or JNK3). We found that loss of each individual JNK isoform causes impairment in tolerance for the antinociceptive and hypothermic effects of daily morphine. However, disruption of JNK2 seems to have the most profound effect on morphine tolerance. These results indicate a clear role for JNK signaling pathways in morphine tolerance. This complements previous studies suggesting that the JNK2 isoform is required for morphine tolerance, but additionally presents novel data suggesting that additional JNK isoforms also contribute toward this process.

A biased mu‐opioid receptor positive allosteric modulator that does not alter receptor regulation

Currently used opioid analgesics compete with endogenous opioid peptide ligands by binding to the orthosteric site of the mu opioid receptor (MOPr). Activation of this G protein‐coupled receptor (GPCR) affords analgesia but also causes respiratory depression, sedation, constipation, and euphoria leading to a high addictive liability. Our lab is currently pursuing the idea that positive allosteric modulators of MOPr (mu‐PAMs) may be a way to treat pain with a better therapeutic profile. Such compounds, including the lead ligand BMS‐986122, enhance the affinity, potency, and efficacy of opioid agonists to bind MOPr and activate G protein. On the other hand, BMS‐986122 does not stimulate G protein activation, as measured by [35S]GTPγS binding in the absence of an orthosteric ligand. Here, we sought to determine if BMS‐986122 activates non‐G protein mediated pathways downstream of MOPr and to investigate the long‐term consequences of BMS‐986122 exposure on the regulation of MOPr in vitro using C6 rat glioma cells expressing rat MOPr. The results demonstrate that BMS‐986122 initiates phosphorylation of ERK1/2 in a pertussis‐toxin insensitive manner supporting a G protein‐independent mechanism. BMS‐986122 alone did not cause any long‐term adaptations in MOPr signaling. Moreover, in contrast to its ability to enhance agonist‐mediated activation of G protein, BMS‐986122 failed to enhance agonist‐induced desensitization of MOPr as measured by receptor downregulation, receptor phosphorylation, and loss of receptor‐mediated signaling following long‐term agonist exposure. Development of biased allosteric ligands at GPCRs represents a novel avenue for analgesic drug development. Future work will determine the physiological consequences of this biased PAM in mouse models of pain.Support or Funding InformationFunded by DA039997

Simian Immunodeficiency Virus Impacts MicroRNA-16 Mediated Post-Transcriptional Regulation of mu Opioid Receptor in CEM ×174 Cells.

Although the mechanism which regulates transcription in the 5'-UTR of the mu opioid receptor gene (OPRM1) in lymphocytes has been well-studied, a question remains as to whether there is post-transcriptional regulation of OPRM1 gene in lymphocytes. In this study, the authors describe both the role played by miRNAs and the impact of SIVmac239 infection on post-transcriptional regulation of OPRM1 gene in CEM ×174 cells. Our results show that miR-16 is able to bind the target site in the range of 8699-8719 nt from the stop codon in MOR-1 mRNA 3'-UTR and suppress the expression of OPRM1 gene. Mutation of this target site reduces the effect of miR-16. Morphine (1 µM) inhibits the expression of miR-16, and this effect is reversed by the antagonist naloxone. Thus, morphine may up-regulate receptor level by both stimulating OPRM1 gene transcription and stabilizing its mRNA. SIVmac239 infection results in an apparent elevation of miR-16 and gradual reduction of OPRM1 gene expression. The inverse correlation of elevated miR-16 and reduced OPRM1 gene expression under viral loading confirmed the effect of SIVmac239 on post-transcriptional regulation of OPRM1 gene in lymphocytes. The authors conclude that miR-16 is a primary factor in post-transcriptional regulation of OPRM1 gene. SIVmac239 upregulates miR-16 levels and consequently suppresses OPRM1 gene expression. This finding will be helpful for full understanding of the regulatory mechanism of OPRM1 gene in lymphocytes, as well as the synergistic mechanism of HIV infection and morphine addiction in the pathogenesis of AIDS.

Methadone Reverses Analgesic Tolerance Induced by Morphine Pretreatment.

Background:Opiates such as morphine are the most powerful analgesics, but their protracted use is restrained by the development of tolerance to analgesic effects. Recent works suggest that tolerance to morphine might be due to its inability to promote mu opioid receptor endocytosis, and the co-injection of morphine with a mu opioid receptor internalizing agonist like [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin reduces tolerance to morphine. So far, no studies have been conducted to evaluate the ability of methadone to reduce morphine tolerance in morphine-pretreated animals, a treatment sequence that could be encountered in opiate rotation protocol. We investigated the ability of methadone (a mu opioid receptor internalizing agonist used in therapy) to reverse morphine tolerance and the associated cellular mechanisms in the periaqueductal gray matter, a region involved in pain control.Methods:We measured analgesic response following a challenge dose of morphine in the hot plate test and investigated regulation of mu opioid receptor (coupling and endocytosis) and some cellular mechanisms involved in tolerance such as adenylate cyclase superactivation and changes in N-methyl-d-aspartate receptor subunits expression and phosphorylation state.Results:A chronic treatment with morphine promoted tolerance to its analgesic effects and was associated with a lack of mu opioid receptor endocytosis, adenylate cyclase overshoot, NR2A and NR2B downregulation, and phosphorylation of NR1. We reported that a methadone treatment in morphine-treated mice reversed morphine tolerance to analgesia by promoting mu opioid receptor endocytosis and blocking cellular mechanisms of tolerance.Conclusions:Our data might lead to rational strategies to tackle opiate tolerance in the frame of opiate rotation.

Sex differences in peripheral mu-opioid receptor mediated analgesia in rat orofacial persistent pain model.

Unilateral ligation of the tendon of anterior superficial part of rat masseter muscle (TASM) leads to long-lasting allodynia. Sex differences in peripheral mu-opioid receptor (MOR)-mediated analgesia under persistent myogenic pain are not well understood. In this study, we examined (1) whether locally applied MOR agonists attenuate persistent pain following TASM ligation in a sex dependent manner, (2) whether there are sex differences of MOR expression changes in rat trigeminal ganglia (TG). The effects of MOR agonist, D-Ala2, N–Me-Phe4, Gly5-ol]-Enkephalin acetate salt (DAMGO), were assessed 14 days after TASM ligation in male, female and orchidectomized (GDX) male rats. MOR mRNA and protein levels in TG 14 days following tendon ligation were also determined. The mechanical thresholds of the injured side were significantly decreased in both male and female rats, from 3 days to 28 days after TASM ligation. A10 μg DAMGO significantly attenuated allodynia in male rats. A 10-fold higher dose of DAMGO was required in female and GDX male rats to produce the level of anti- allodynia achieved in male rats. The level of MOR mRNA in TG from male rats was significantly greater 14 days after TASM ligation compared with the sham-operated male rats, but not from female and GDX male rats. After TASM ligation, males had significantly more MOR immunoreactivity in TG compared to sham-operated males. The MOR levels increased to 181.8% of the sham level in male rats receiving tendon injury. But there was no significant change in female rats receiving tendon injury compared to the sham female rats. Taken together, our data suggest that there were sex differences in the effects of peripheral MOR agonists between male and female rats under TASM ligation developing long-lasting pain condition, which is partly mediated by sex differences in the changes of MOR expressions and testosterone is an important factor in the regulation of MOR.

Knockdown of ventral tegmental area mu-opioid receptors in rats prevents effects of social defeat stress: Implications for amphetamine cross-sensitization, social avoidance, weight regulation and expression of brain-derived neurotrophic factor

Social defeat stress causes social avoidance and long-lasting cross-sensitization to psychostimulants, both of which are associated with increased brain-derived neurotrophic factor (BDNF) expression in the ventral tegmental area (VTA). Moreover, social stress upregulates VTA mu-opioid receptor (MOR) mRNA. In the VTA, MOR activation inhibits GABA neurons to disinhibit VTA dopamine neurons, thus providing a role for VTA MORs in the regulation of psychostimulant sensitization. The present study determined the effect of lentivirus-mediated MOR knockdown in the VTA on the consequences of intermittent social defeat stress, a salient and profound stressor in humans and rodents. Social stress exposure induced social avoidance and attenuated weight gain in animals with non-manipulated VTA MORs, but both these effects were prevented by VTA MOR knockdown. Rats with non-manipulated VTA MOR expression exhibited cross-sensitization to amphetamine challenge (1.0 mg/kg, i.p.), evidenced by a significant augmentation of locomotion. By contrast, knockdown of VTA MORs prevented stress-induced cross-sensitization without blunting the locomotor-activating effects of amphetamine. At the time point corresponding to amphetamine challenge, immunohistochemical analysis was performed to examine the effect of stress on VTA BDNF expression. Prior stress exposure increased VTA BDNF expression in rats with non-manipulated VTA MOR expression, while VTA MOR knockdown prevented stress-induced expression of VTA BDNF. Taken together, these results suggest that upregulation of VTA MOR is necessary for the behavioral and biochemical changes induced by social defeat stress. Elucidating VTA MOR regulation of stress effects on the mesolimbic system may provide new therapeutic targets for treating stress-induced vulnerability to substance abuse.

Development of Functionally Selective, Small Molecule Agonists at Kappa Opioid Receptors

The kappa opioid receptor (KOR) is widely expressed in the CNS and can serve as a means to modulate pain perception, stress responses, and affective reward states. Therefore, the KOR has become a prominent drug discovery target toward treating pain, depression, and drug addiction. Agonists at KOR can promote G protein coupling and βarrestin2 recruitment as well as multiple downstream signaling pathways, including ERK1/2 MAPK activation. It has been suggested that the physiological effects of KOR activation result from different signaling cascades, with analgesia being G protein-mediated and dysphoria being mediated through βarrestin2 recruitment. Dysphoria associated with KOR activation limits the therapeutic potential in the use of KOR agonists as analgesics; therefore, it may be beneficial to develop KOR agonists that are biased toward G protein coupling and away from βarrestin2 recruitment. Here, we describe two classes of biased KOR agonists that potently activate G protein coupling but weakly recruit βarrestin2. These potent and functionally selective small molecule compounds may prove to be useful tools for refining the therapeutic potential of KOR-directed signaling in vivo.

Inhibition of c-Jun NH2-terminal kinase stimulates mu opioid receptor expression via p38 MAPK-mediated nuclear NF-κB activation in neuronal and non-neuronal cells

Despite its potential side effects of addiction, tolerance and withdrawal symptoms, morphine is widely used for reducing moderate and severe pain. Previous studies have shown that the analgesic effect of morphine depends on mu opioid receptor (MOR) expression levels, but the regulatory mechanism of MOR is not yet fully understood. Several in vivo and in vitro studies have shown that the c-Jun NH2-terminal kinase (JNK) pathway is closely associated with neuropathic hyperalgesia, which closely resembles the neuroplastic changes observed with morphine antinociceptive tolerance. In this study, we show that inhibition of JNK by SP600125, its inhibitory peptide, or JNK-1 siRNA induced MOR at both mRNA and protein levels in neuronal cells. This increase in MOR expression was reversed by inhibition of the p38 mitogen-activated protein kinase (MAPK) pathway, but not by inhibition of the mitogen-activated protein/extracellular signal-regulated kinase (MEK) pathway. Further experiments using cell signaling inhibitors showed that MOR upregulation by JNK inhibition involved nuclear factor-kappa B (NF-κB). The p38 MAPK dependent phosphorylation of p65 NF-κB subunit in the nucleus was increased by SP600125 treatment. We also observed by chromatin immunoprecipitation (ChIP) analysis that JNK inhibition led to increased bindings of CBP and histone-3 dimethyl K4, and decreased bindings of HDAC-2, MeCP2, and histone-3 trimethyl K9 to the MOR promoter indicating a transcriptional regulation of MOR by JNK inhibition. All these results suggest a regulatory role of the p38 MAPK and NF-κB pathways in MOR gene expression and aid to our better understanding of the MOR gene regulation.

Neuroplasticity, axonal guidance and micro‐RNA genes are associated with morphine self‐administration behavior

Neuroadaptations in the ventral striatum (VS) and ventral midbrain (VMB) following chronic opioid administration are thought to contribute to the pathogenesis and persistence of opiate addiction. In order to identify candidate genes involved in these neuroadaptations, we utilized a behavior-genetics strategy designed to associate contingent intravenous drug self-administration with specific patterns of gene expression in inbred mice differentially predisposed to the rewarding effects of morphine. In a Yoked-control paradigm, C57BL/6J mice showed clear morphine-reinforced behavior, whereas DBA/2J mice did not. Moreover, the Yoked-control paradigm revealed the powerful consequences of self-administration versus passive administration at the level of gene expression. Morphine self-administration in the C57BL/6J mice uniquely up- or down-regulated 237 genes in the VS and 131 genes in the VMB. Interestingly, only a handful of the C57BL/6J self-administration genes (<3%) exhibited a similar expression pattern in the DBA/2J mice. Hence, specific sets of genes could be confidently assigned to regional effects of morphine in a contingent- and genotype-dependent manner. Bioinformatics analysis revealed that neuroplasticity, axonal guidance and micro-RNAs (miRNAs) were among the key themes associated with drug self-administration. Noteworthy were the primary miRNA genes H19 and micro-RNA containing gene (Mirg), processed, respectively, to mature miRNAs miR-675 and miR-154, because they are prime candidates to mediate network-like changes in responses to chronic drug administration. These miRNAs have postulated roles in dopaminergic neuron differentiation and mu-opioid receptor regulation. The strategic approach designed to focus on reinforcement-associated genes provides new insight into the role of neuroplasticity pathways and miRNAs in drug addiction.

Post-transcriptional regulation of mu-opioid receptor: role of the RNA-binding proteins heterogeneous nuclear ribonucleoprotein H1 and F

Classical opioids have been historically used for the treatment of pain and are among the most widely used drugs for both acute severe pain and long-term pain. Morphine and endogenous mu-opioid peptides exert their pharmacological actions mainly through the mu-opioid receptor (MOR). However, the expression of opioid receptor (OR) proteins is controlled by extensive transcriptional and post-transcriptional processing. Previously, the 5'-untranslated region (UTR) of the mouse MOR was found to be important for post-transcriptional regulation of the MOR gene in neuronal cells. To identify proteins binding to the 5'-UTR as potential regulators of the mouse MOR gene, affinity column chromatography using 5'-UTR-specific RNA oligonucleotides was performed using neuroblastoma NS20Y cells. Chromatography was followed by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. We identified two heterogeneous ribonucleoproteins (hnRNPs) that bound to RNA sequences of interest: hnRNP H1 and hnRNP F. Binding of these proteins to the RNA region was M4-region sequence-specific as confirmed by Western-blot analysis and RNA supershift assay. Furthermore, a cotransfection study showed that the presence of hnRNP H1 and F resulted in repressed expression of the mouse MOR. Our data suggest that hnRNP H1 and F can function as repressors of MOR translation dependent on the M4 (-75 to -71bp upstream of ATG) sequences. We demonstrate for the first time a role of hnRNPs as post-transcriptional repressors in MOR gene regulation.

Membrane-delimited proteolytic regulation of opioid receptors

Prolonged exposure of opioid receptors to agonists leads to their regulation by the classical process of clathrin-dependent internalization, followed by their intracellular degradation (down regulation). We have previously shown that the opioid agonist etorphine induced an additional process of down regulation of mu-opioid receptors (MOR) that occurred in intact MOR-transfected HEK-293 cells, as well as in isolated membranes. In the present study we show that etorphine similarly down regulated rat kappa-opioid receptors (KORs), which do not undergo the classical process of internalization and down regulation. This process was resistant to inhibitors of clathrin-coated pit formation (hypertonic sucrose, mono-dansyl-cadaverine) and was mainly mediated by membranous serine- and amino-peptidases. We further show that various opioid ligands, besides etorphine, induced down regulation of either KOR or MOR in isolated membranes. The ability of the various opioid ligands to induce membrane-delimited KOR or MOR down regulation did not correlate to their classical pharmacological profile, suggesting functional selectivity of the effect. Levorphanol, but not its stereoisomer dextrophan, induced membrane-delimited down regulation of both KOR and MOR, indicating that stereoselective binding to the receptor was necessary to initiate the process. Our findings that this proteolytic regulation of opioid receptors occurs not only in isolated membranes but also in intact cells and that it occurs even when the receptors are resistant to the conventional process of down regulation indicate its possible physiological role in the regulation of opioid activity.

Opiates, overeating and obesity: a psychogenetic analysis

This study provides an original perspective on the associations among endogenous opiates, overeating and obesity. The aim was to assess whether variability in the OPRM1 gene, as assessed by seven single-nucleotide polymorphisms, relates to individual differences in the preference for sweet and fatty foods. We also anticipated that these food preferences would be positively associated with binge eating, hedonic eating and emotionally driven eating-patterns of overeating that would, in turn, predict higher body mass index (BMI). Analysis of variance procedures examined genotype differences in food preferences; bivariate correlation coefficients examined the relationships among food preferences and the overeating variables; and a regression analysis tested the combined influences of the overeating variables on BMI. DNA was extracted from whole blood for the genotyping, and measures of food preferences and eating behaviours were obtained from well-validated self-report questionnaires. Participants were 300 healthy adult men and women recruited from the community. All the predicted associations were supported by statistically significant results. In particular, the G/G genotype group of the functional A118G marker of the OPRM1 gene reported higher preferences for sweet and fatty foods compared with the other two groups. Food preferences were also related to all overeating measures, which in turn accounted for a substantial proportion of the variance in BMI. Our findings suggest that some of the diversity in the preference for highly palatable foods can be explained by genotypic differences in the regulation of mu opioid receptors. The associations reported in this paper are important from a public-health perspective because of the abuse potential of sweet-fat foods and their strong relationship with obesity.

An Opioid Deficit in Borderline Personality Disorder: Self-Cutting, Substance Abuse, and Social Dysfunction

An Opioid Deficit in Borderline Personality Disorder: Self-Cutting, Substance Abuse, and Social Dysfunction

Dual regulation of mu opioid receptors in SK-N-SH neuroblastoma cells by morphine and interleukin-1β: Evidence for opioid-immune crosstalk

Treatment of SK-N-SH cells with morphine and interleukin-1beta (IL-1β) produced dual regulation of the mRNA for the human mu opioid receptor (MOR) protein. Morphine produced a decrease in the MOR mRNA while IL-1β increased it, as assessed by real-time quantitative PCR. These data were consistent with immunocytochemical studies of treated and untreated cells. Morphine-mediated down-regulation of MOR was blocked by naltrexone and IL-1β-induced up-regulation of MOR was blocked by interleukin-1 receptor type 1 antagonist. Immune-opioid crosstalk was examined by IL-1β and morphine co-treatment. These data are the first to show dual regulation of MOR in neuroblastoma cells.

μ‐Opioid Receptor Redistribution in the Locus Coeruleus Upon Precipitation of Withdrawal in Opiate‐Dependent Rats

Administration of mu-opioid receptor (MOR) agonists is known to produce adaptive changes within noradrenergic neurons of the rat locus coeruleus (LC). Alterations in the subcellular distribution of MOR have been shown to occur in the LC in response to full agonists and endogenous peptides; however, there is considerable debate in the literature whether trafficking of MOR occurs after chronic exposure to the partial-agonist morphine. In the present study, we examined adaptations in MOR after chronic opioid exposure using immunofluorescence and electron microscopy (EM), using receptor internalization as a functional endpoint. MOR trafficking in LC neurons was characterized in morphine-dependent rats that were given naltrexone at a dose known to precipitate withdrawal. After chronic morphine exposure, a subtle redistribution of MOR immunoreactivity from the membrane to the cytosol was detected within dendrites of LC neurons. Interestingly, an acute injection of naltrexone in rats exposed to chronic morphine produced a robust internalization of MOR, whereas administration of naltrexone failed to do so in naïve animals. These findings provide anatomical evidence for modified regulation of MOR trafficking after chronic morphine treatment in brain noradrenergic neurons. Adaptations in the MOR signaling pathways that regulate internalization may occur as a consequence of chronic treatment and precipitation of withdrawal. Mechanisms underlying this effect might include differential MOR regulation in the LC, or downstream effects of withdrawal-induced enkephalin (ENK) release from afferents to the LC.