Mu-opioid Receptor Internalization Research Articles

RGS9‐2 is a negative modulator of μ‐opioid receptor function

Regulators of G-protein signaling (RGS) 9-2 is a striatal enriched protein that controls G protein coupled receptor signaling duration by accelerating Galpha subunit guanosine triphosphate hydrolysis. We have previously demonstrated that mice lacking the RGS9 gene show enhanced morphine analgesia and delayed development of tolerance. Here we extend these studies to understand the mechanism via which RGS9-2 modulates opiate actions. Our data suggest that RGS9-2 prevents several events triggered by mu-opioid receptor (MOR) activation. In transiently transfected PC12 cells, RGS9-2 delays agonist induced internalization of epitope HA-tagged mu-opioid receptor. This action of RGS9-2 requires localization of the protein near the cell membrane. Co-immunoprecipitation studies reveal that RGS9-2 interacts with HA-tagged mu-opioid receptor, and that this interaction is enhanced by morphine treatment. In addition, morphine promotes the association of RGS9-2 with another essential component of MOR desensitization, beta-arrestin-2. We also show that over-expression of RGS9-2 prevents opiate-induced extracellular signal-regulated kinase phosphorylation. Our data indicate that RGS9-2 plays an essential role in opiate actions, by negatively modulating MOR downstream signaling as well as the rate of MOR endocytosis.

RGS14 prevents morphine from internalizing Mu-opioid receptors in periaqueductal gray neurons

Opioid agonists display different capacities to stimulate mu-opioid receptor (MOR) endocytosis, which is related to their ability to provoke the phosphorylation of specific cytosolic residues in the MORs. Generally, opioids that efficiently promote MOR endocytosis and recycling produce little tolerance, as is the case for [d-Ala2, N-MePhe4,Gly-ol5] encephalin (DAMGO). However, morphine produces rapid and profound antinociceptive desensitization in the adult mouse brain associated with little MOR internalization. The regulator of G-protein signaling, the RGS14 protein, associates with MORs in periaqueductal gray matter (PAG) neurons, and when RGS14 is silenced morphine increased the serine 375 phosphorylation in the C terminus of the MOR, a GRK substrate. Subsequently, these receptors were internalized and recycled back to the membrane where they accumulated on cessation of antinociception. These mice now exhibited a resensitized response to morphine and little tolerance developed. Thus, in morphine-activated MORs the RGS14 prevents GRKs from phosphorylating those residues required for β-arresting-mediated endocytosis. Moreover morphine but not DAMGO triggered a process involving calcium/calmodulin-dependent kinase II (CaMKII) in naïve mice, which contributes to MOR desensitization in the plasma membrane. In RGS14 knockdown mice morphine failed to activate this kinase. It therefore appears that phosphorylation and internalization of MORs disrupts the CaMKII-mediated negative regulation of these opioid receptors.

Distinct effects of individual opioids on the morphology of spines depend upon the internalization of mu opioid receptors

This study has examined the relationship between the effects of opioids on the internalization of mu opioid receptors (MORs) and the morphology of dendritic spines. Several opioids (morphine, etorphine, DAMGO or methadone) were applied to cultured hippocampal neurons. Live imaging and biochemical techniques were used to examine the dynamic changes in MOR internalization and spine morphology. This study reveals that MOR internalization can regulate opioid-induced morphological changes in dendritic spines: (1) Chronic treatment with morphine, which induced minimal receptor internalization, caused collapse of dendritic spines. In contrast, “internalizing” opioids such as DAMGO and etorphine induced the emergence of new spines. It reveals that opioid-induced changes in spines vary greatly depending on how the applied opioid agonist affects MOR internalization. (2) The blockade of receptor internalization by dominant negative mutant of dynamin, K44E, reversed the effects of DAMGO and etorphine. It indicates that receptor internalization is necessary for the distinct effects of DAMGO and etorphine on spines. (3) In neurons that were cultured from MOR knock-out mice and had been co-transfected with DsRed and MOR-GFP, morphine caused collapse of spines whereas DAMGO induced emergence of new spines, indicating that opioids can alter the structure of spines via postsynaptic MORs. (4) Methadone at a low concentration induced minimal internalization and had effects that were similar to morphine. At a high concentration, methadone induced robust internalization and had effects that are opposite to morphine. The concentration-dependent opioid-induced changes in dendritic spines might also contribute to the variation in the effects of individual opioids.

Effects of chronic opioid exposure on guinea pig mu opioid receptor in Chinese hamster ovary cells: Comparison with human and rat receptor

Chronic opioid treatment leads to agonist-specific effects at the mu opioid receptor. The molecular mechanisms resulting from chronic opioid exposure include desensitization, internalization and down-regulation of membrane-bound mu opioid receptors (MOP). The purpose of this study was to compare the cellular regulation of guinea pig, human and rat MOP expressed in Chinese hamster ovary (CHO) cells, following exposure to two clinically important opioids, morphine and methadone. MOP expressing CHO cells were treated in culture with methadone or morphine for up to 48 h. Radioligand diprenorphine and [ d-AIa 2, N-Me-Phe 4,Gly 5-ol]-enkephalin (DAMGO)-stimulated GTPγS binding assays were carried out using paired control and opioid-exposed CHO cells. Methadone induced downregulation of the mu opioid receptor, while morphine induced desensitization of the receptor for all three species. Furthermore, morphine predominantly decreased the potency of DAMGO to stimulate GTPγS binding, whereas methadone primarily reduced its efficacy. Changes in DAMGO potency and efficacy differed among species and depended on the opioid used to treat the cells. Our results showed similarities between guinea pig and human MOP for morphine-induced desensitization, but identified differences between the two for methadone-induced desensitization. In contrast, human and rat MOP differed in response to morphine treatment, but were not distinct in their response to methadone treatment. The guinea pig is an excellent and established animal model to study opioid effects, but its molecular opioid pharmacology has not been investigated thus far. These results can assist in understanding species differences in the effects of opioid ligands activating the mu opioid receptor.

Enhancement of Morphine Analgesic Effect with Induction of μ-Opioid Receptor Endocytosis in Rats

Morphine can desensitize mu-opioid receptor (MOR), but it does not cause internalization of the receptor after binding. Acute desensitization of MOR impairs the efficiency of signaling, whereas the receptor internalization restores the cell responsiveness to the agonists. Thereby, the property of morphine may limit the analgesic effects of this opiate drug. It has been shown that [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAMGO), a potent MOR agonist inducing the internalization, facilitates morphine to internalize MOR, suggesting that MOR agonists with low relative activity versus endocytosis (RAVE) values such as DAMGO can potentiate analgesic effects of morphine through stimulating MOR internalization. The authors examined whether the acute analgesic effect of morphine can be potentiated by low relative activity versus endocytosis agonists DAMGO and fentanyl. Rats injected intrathecally with opioids were subjected to a hot plate test for antinociceptive effect. Immunostained spinal dorsal horn was analyzed by confocal microscopy. Fentanyl induced MOR internalization to a lesser extent than DAMGO at equianalgesic doses. Coadministration of fentanyl promoted morphine-induced MOR internalization. The analgesic effect of morphine was greatly potentiated together with decrease in the relative activity versus endocytosis value when MOR internalization was induced by coadministration of a subanalgesic dose of DAMGO or fentanyl. In contrast, the combination of DAMGO and fentanyl increased neither the analgesic effect nor the internalization of MOR. The results suggest that the coadministration of morphine with MOR-internalizing agonist is clinically applicable to develop successful pain-management regimens to achieve satisfactory analgesia using less morphine.

Agonist-Selective Mechanisms of μ-Opioid Receptor Desensitization in Human Embryonic Kidney 293 Cells

The ability of two opioid agonists, [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) and morphine, to induce mu-opioid receptor (MOR) phosphorylation, desensitization, and internalization was examined in human embryonic kidney (HEK) 293 cells expressing rat MOR1 as well G protein-coupled inwardly rectifying potassium channel (GIRK) channel subunits. Both DAMGO and morphine activated GIRK currents, but the maximum response to DAMGO was greater than that of morphine, indicating that morphine is a partial agonist. The responses to DAMGO and morphine desensitized rapidly in the presence of either drug. Expression of a dominant negative mutant G protein-coupled receptor kinase 2 (GRK2), GRK2-K220R, markedly attenuated the DAMGO-induced desensitization of MOR1, but it had no effect on morphine-induced MOR1 desensitization. In contrast, inhibition of protein kinase C (PKC) either by the PKC inhibitory peptide PKC (19-31) or staurosporine reduced MOR1 desensitization by morphine but not that induced by DAMGO. Morphine and DAMGO enhanced MOR1 phosphorylation over basal. The PKC inhibitor bisindolylmaleimide 1 (GF109203X) inhibited MOR1 phosphorylation under basal conditions and in the presence of morphine, but it did not inhibit DAMGO-induced phosphorylation. DAMGO induced arrestin-2 translocation to the plasma membrane and considerable MOR1 internalization, whereas morphine did not induce arrestin-2 translocation and induced very little MOR1 internalization. Thus, DAMGO and morphine each induce desensitization of MOR1 signaling in HEK293 cells but by different molecular mechanisms; DAMGO-induced desensitization is GRK2-dependent, whereas morphine-induced desensitization is in part PKC-dependent. MORs desensitized by DAMGO activation are then readily internalized by an arrestin-dependent mechanism, whereas those desensitized by morphine are not. These data suggest that opioid agonists induce different conformations of the MOR that are susceptible to different desensitizing and internalization processes.

Adenylyl Cyclase Superactivation Induced by Long-Term Treatment with Opioid Agonist Is Dependent on Receptor Localized within Lipid Rafts and Is Independent of Receptor Internalization

Long-term opioid agonist treatment results in adenylyl cyclase superactivation. A recent "RAVE" theory implicates a direct correlation between the ability of agonist to induce receptor internalization and the magnitude of adenylyl cyclase superactivation. We decided to test such a theory by examining the adenylyl cyclase superactivation after long-term activation of mu-opioid receptor (MOR) in an EcR293 cell model. We examined the magnitudes of adenylyl cyclase superactivation in the presence of naloxone after long-term treatment with morphine, etorphine, and methadone, three agonists reported to have differential activities in promoting MOR internalization. It can be shown that the magnitudes of adenylyl cyclase superactivation after treating with these three agonists, although different, were dependent on MOR density. Blunting MOR internalization with the dominant-negative mutant of dynamin, K44E, did not alter the magnitude of either morphine- or etorphine-induced adenylyl cyclase superactivation. In the presence of diprenorphine, the magnitude of adenylyl cyclase superactivation after etorphine treatment was identical to that observed with morphine. It could be demonstrated further that adenylyl cyclase superactivation is dependent on the cell surface-located MOR. Sucrose gradient fractionation demonstrated the colocalization of MOR and adenylyl cyclase V/VI with caveolin-1, a marker for lipid rafts. After long-term agonist treatment, the majority of MOR remained at the lipid rafts. Methyl-beta-cyclodextrin (MbetaCD) completely blunted the adenylyl cyclase superactivation and agonist-induced receptor internalization. These MbetaCD actions were reversed by incubating the cells with cholesterol. Thus, the adenylyl cyclase superactivation is not dependent on agonist-induced receptor internalization. Rather, the location of MOR at lipid rafts is an absolute requirement for the observed adenylyl cyclase superactivation.

An Opiate Cocktail that Reduces Morphine Tolerance and Dependence

Morphine is an exceptionally effective analgesic whose utility is compromised by the development of tolerance and dependence to the drug. Morphine analgesia and dependence are mediated by its activity at the mu opioid peptide (MOP) receptor [1]. The MOP receptor is activated not only by morphine, but also by other opiate drugs such as methadone and endogenous opioids such as endorphins. Morphine, however, is a unique opioid agonist ligand because it fails to induce endocytic trafficking of the MOP receptor [2], whereas the endogenous ligands and methadone do facilitate endocytosis [3]. Using the unique pharmacology of the MOP receptor and its proposed existence as an oligomeric structure [4], we designed a pharmacological cocktail that facilitates endocytosis of the MOP receptor in response to morphine. This cocktail consists of morphine and a small dose of methadone. Importantly, this cocktail, while retaining full analgesic potency, does not promote morphine dependence. We further demonstrate that dependence is reduced, at least in part, because endocytosis of the MOP receptor in response to morphine prevents the upregulation of N-methyl-D-aspartate (NMDA) receptors.

N-Methyl- d-Aspartate Receptors Mediate Endogenous Opioid Release in Enteric Neurons After Abdominal Surgery

We tested the hypothesis that N-methyl-D-aspartate (NMDA) receptors mediate surgery-induced opioid release in enteric neurons. We used mu opioid receptor (muOR) internalization as a measure of opioid release with immunohistochemistry and confocal microscopy. MuOR internalization was quantified in enteric neurons from nondenervated and denervated ileal segments of guinea pig after abdominal laparotomy with and without pretreatment with NMDA-receptor antagonists acting at different recognition sites (+)-5-methyl-10,11-dihydro-5H-dibenzo [a,b] cyclohepten-5,10-imine (MK-801) or (D) 2-amino-5-phosphopenoic acid (AP-5) at .5, 1 mg/kg; 8-chloro-4-hydroxy-1-oxo-1,2-dihydropyridazinol [4,5-]quinoline-5-oxide choline (MRZ 2/576) or 8-chloro-1,4-dioxo-1,2,3,4-tetrahydropyridazinol [4,5-]quinoline choline salt (MRZ 2/596) at .3, 1 mg/kg, or with an antagonist for the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (1, 3 mg/kg). To determine whether NMDA stimulation induces opioid release, (1) ilea were exposed to NMDA (100 micromol/L) and D-serine (10 micromol/L) with or without the antagonist MK-801 or AP-5 (50 micromol/L); and (2) neuromuscular preparations of the ileum were stimulated electrically (20 Hz, 20 min) with or without MK-801 or AP-5 (50 micromol/L). MuOR endocytosis induced by abdominal laparotomy was inhibited significantly by NMDA-receptor antagonists in nondenervated and denervated ileal segments, but not by the AMPA-receptor antagonist. MuOR endocytosis in neurons exposed to NMDA or electrical stimulation was prevented by NMDA-R antagonists. Abdominal laparotomy evokes local release of glutamate that results in endogenous opioid release through the activation of peripheral NMDA receptors. This suggests an interaction between the glutamatergic and opioid systems in response to the noxious and perhaps mechanosensory stimulation of surgery.

Internalization and down-regulation of mu opioid receptors by endomorphins and morphine in SH-SY5Y human neuroblastoma cells

The human neuroblastoma cell line, SH-SY5Y, was used to examine the effects of morphine and the endogenous opioid peptides, endomorphin-1 (EM-1) and endomorphin-2 (EM-2), on mu opioid receptor (MOR) internalization and down-regulation. Treatment for 24 h with EM-1, EM-2 or morphine at 100 nM, 1 μM and 10 μM resulted in a dose-dependent down-regulation of mu receptors. Exposure of cells to 10 μM EM-1 for 2.5, 5 and 24 h resulted in a time-dependent down-regulation of mu receptors. Down-regulation of mu receptors by morphine and EM-1 was blocked by treatment with hypertonic sucrose, consistent with an endocytosis-dependent mechanism. Sensitive cell-surface binding studies with a radiolabeled mu antagonist revealed that morphine was able to induce internalization of mu receptors naturally expressed in SH-SY5Y cells. EM-1 produced a more rapid internalization of mu receptors than morphine, but hypertonic sucrose blocked the internalization induced by each of these agonists. This study demonstrates that, like morphine, the endomorphins down-regulate mu opioid receptors in a dose- and time-dependent manner. This study also demonstrates that morphine, as well as EM-1, can induce rapid, endocytosis-dependent internalization of mu opioid receptors in SH-SY5Y cells. These results may help elucidate the ability of mu agonists to regulate the number and responsiveness of their receptors.

P.5.042 Gene transcription alterations associated to naltrexone treatment induces a decrease in alcohol intake in alcohol-preferring Wistar rats

The heptapeptide Met-enkephalin-Arg6-Phe7 (MERF) with the sequence of YGGFMRF is a potent endogenous opioid located at the C-terminus of proenkephalin-A (PENK), the common polypeptide precursor of Met- and Leu-enkephalin. Our systematic bioinformatic survey revealed considerable sequence polymorphism at the heptapeptide region of different PENK prepropeptides among 56 vertebrate animals. Four orthologous heptapeptides with single or double amino acid replacements were identified among 15 animals, such as YGGFMGY (zebrafish), YGGFMRY (newt), YGGFMKF (hedgehog tenrek) and YGGFMRI (mudpuppy). Each novel heptapeptide, together with the mammalian consensus MERF and Met-enkephalin, were chemically synthesized and subjected to functionality studies, using radioligand binding competition and G-protein activation assays in rat brain membranes. Equilibrium binding affinities changed from good to modest as measured by receptor type selective [3H]opioid radioligands. The relative affinities of the heptapeptides reveal slight mu-receptor (MOP) preference over the delta-receptors (DOP). [35S]GTPγS assay, which measures the agonist-mediated G-protein activation, has demonstrated that all the novel heptapeptides were also potent in stimulating the regulatory G-proteins. All peptides were effective in promoting the agonist induced internalization of the green fluorescence protein-tagged human mu-opioid receptor (hMOP-EGFP) stably expressed in HEK293 cells. Thus, the C-terminally processed PENK heptapeptide orthologs exhibited satisfactory bioactivities, moreover they represent further members of the so-called “natural combinatorial neuropeptide library” emerged by evolution.

Protein kinase C activation enhances morphine-induced rapid desensitization of mu-opioid receptors in mature rat locus ceruleus neurons.

Recent studies have shown that morphine, in contrast to other agonists at the mu-opioid receptor, causes very little rapid mu-opioid receptor desensitization or internalization in adult rat mammalian neurons, raising important questions about how morphine tolerance is induced. Here we show that morphine can indeed cause marked rapid desensitization of mu-opioid receptors in mature rat locus ceruleus neurons when protein kinase C is also activated. Thus, activation of Gq-coupled M3 muscarinic receptors or application of a phorbol ester enhanced the desensitization of the mu-opioid receptor-evoked potassium current in rat locus ceruleus neurons. The enhancement of desensitization was reversible by the protein kinase C inhibitors chelerythrine and 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF109203X) and resulted from an effect at the level of the mu-opioid receptor rather than the potassium channel. This is the first finding that morphine can induce rapid mu-opioid receptor desensitization in adult rat neurons, and because reduced protein kinase C activity in vivo attenuates morphine tolerance, we propose that G-protein coupled receptor cross-talk and the level of protein kinase C activity may play critical roles in the desensitization of the mu-opioid receptor and could underlie the development of morphine tolerance.

Intra-amygdalar injection of DAMGO: effects on c-Fos levels in brain sites associated with feeding behavior

It is well known that the mu opioid agonist, Tyr- d-Ala-Gly-(me) Phe-Gly-ol (DAMGO), increases food intake in rats when injected into a variety of brain sites including the central nucleus of the amygdala (CeA). Immunohistochemical studies measuring c-Fos immunoreactivity (IR) suggest that the CeA contributes to opioid-related feeding. In the current study, we injected 2 nmol of DAMGO and measured food intake, c-Fos IR levels in various brain sites involved in feeding behavior, and mu opioid receptor internalization. We also studied the effect of CeA-injected DAMGO on LiCl-induced increases in c-Fos IR in the amygdala. As was expected, intra-CeA injection of DAMGO increased food intake of rats over a 4-h period. DAMGO injection into the CeA also resulted in mu opioid receptor internalization in the CeA, indicating activation of mu opioid receptor expressing neurons in this site. Administration of DAMGO into the CeA increased c-Fos IR levels in the shell of the nucleus accumbens (NAcc), but not in 17 other brain sites that were studied. We also found that intra-CeA injection of DAMGO, prior to LiCl injection, decreased c-Fos IR levels in the CeA compared to vehicle-injected rats. Thus, intra-CeA administration of DAMGO may increase feeding, in part, by activating neurons in the shell of the nucleus accumbens and by inhibiting activity of selected neurons in the CeA.

Distinct domains of the mu-opioid receptor control uncoupling and internalization.

Homologous desensitization of the micro opioid receptor (muOR) can be resolved into distinct processes that include the uncoupling of the muOR from its G-protein effectors and internalization of cell surface receptors. Using electrophysiological recordings of muOR activation of G-protein-coupled K+ channels (Kir3) in Xenopus laevis oocytes and AtT20 cells, confocal microscopy of receptor localization, and radioligand binding of cell surface receptors, we resolved these desensitization mechanisms to determine the domain of muOR important for receptor uncoupling. Activation of muOR by saturating concentrations of [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO), methadone, or fentanyl, but not morphine, produced robust internalization of a green fluorescent protein-tagged muOR. A subsaturating concentration of DAMGO (100 nM) did not cause receptor internalization but markedly reduced the subsequent responsiveness of Kir3 by uncoupling muOR. muOR desensitization in AtT20 cells was confirmed to be homologous, because desensitization by 100 nM DAMGO was blocked by dominant-negative forms of either G protein-coupled receptor kinase (GRK) or arrestin, and pretreatment with DAMGO did not affect the Kir3 response to somatostatin receptor activation. Alanine substitution of a single threonine in the second cytoplasmic loop of the muOR (Threonine 180) blocked agonist-dependent receptor uncoupling without affecting receptor internalization. These results suggest that GRK-dependent phosphorylation of muOR required threonine 180 for uncoupling but that a different GRK and arrestin-dependent mechanism controlled muOR internalization in AtT20 cells.

μ-Opioid Receptor Desensitization: ROLE OF RECEPTOR PHOSPHORYLATION, INTERNALIZATION, AND RESENSITIZATION

It is generally accepted that the internalization and desensitization of mu-opioid receptor (MOR) involves receptor phosphorylation and beta-arrestin recruitment. However, a mutant MOR, which is truncated after the amino acid residue Ser363 (MOR363D), was found to undergo phosphorylation-independent internalization and desensitization. As expected, MOR363D, missing the putative agonist-induced phosphorylation sites, did not exhibit detectable agonist-induced phosphorylation. MOR363D underwent slower internalization as reflected in the attenuation of membrane translocation of beta-arrestin 2 when compared with wild type MOR, but the level of receptor being internalized was similar to that of wild type MOR after 4 h of etorphine treatment. Furthermore, MOR363D was observed to desensitize faster than that of wild type MOR upon agonist activation. Surface biotinylation assay demonstrated that the wild type receptors recycled back to membrane after agonist-induced internalization, which contributed to the receptor resensitization and thus partially reversed the receptor desensitization. On the contrary, MOR363D did not recycle after internalization. Hence, MOR desensitization is controlled by the receptor internalization and the recycling of internalized receptor to cell surface in an active state. Taken together, our data indicated that receptor phosphorylation is not absolutely required in the internalization, but receptor phosphorylation and subsequent beta-arrestin recruitment play important roles in the resensitization of internalized receptors.

Estrogen receptor‐α is required for estrogen‐induced μ‐opioid receptor internalization

Endogenous opioid circuits are pivotal for the regulation of sexual receptivity. Treatment of mice with morphine, a preferential mu-opioid receptor (MOR) agonist, severely attenuates lordosis. Estrogen induces internalization of MOR in cell groups of the limbic-hypothalamic lordosis-regulating circuit. Because rapid MOR internalization is mediated by estrogen release of endogenous opioid peptides, internalization has been used as a neurochemical signature of estrogen action in the central nervous system. Together these results indicate that estrogen induces a MOR mediated inhibition of sexual receptivity. To determine which estrogen receptor, estrogen receptor-alpha (ERalpha) or estrogen receptor-beta (ERbeta), mediates MOR internalization, ERalpha knockout (ERalphaKO), ERbeta knockout (ERbetaKO) and wild-type (WT) mice were used in the present study. WT, ERalphaKO and ERbetaKO mice had similar MOR distributions in the limbic-hypothalamic lordosis-regulating circuit. Estrogen treatment internalized MOR in the medial preoptic nucleus of ovariectomized WT and ERbetaKO, but not ERalphaKO mice. Treatment of ERalphaKO mice with the selective endogenous MOR ligand, endomorphin-1, induced levels of MOR internalization similar to WT mice suggesting that MOR in ERalphaKO mice could be activated and were probably functional. The results of the present experiments indicate that ERalpha is required for estrogen-induced MOR internalization and suggest that ERalpha can mediate rapid actions of estrogen.

Internalization of mu-opioid receptors produced by etorphine in the rat locus coeruleus

Chronic administration of mu-opioid receptor agonists is known to produce adaptive changes within noradrenergic neurons of the locus coeruleus. Although mu-opioid receptors are densely expressed by locus coeruleus neurons, the effects of acute and chronic administration of agonists on the subcellular distribution of mu-opioid receptors remain poorly understood. Therefore, we examined the ultrastructural distribution of mu-opioid receptor immunoreactivity in the locus coeruleus of rats subjected to either acute morphine, or etorphine, or chronic morphine treatment. In the locus coeruleus of control rats receiving acute saline injections or placebo pellet implants, immunogold–silver labeling for mu-opioid receptors was localized to parasynaptic and extrasynaptic portions of the plasma membranes of perikarya and dendrites. Only 8% of the gold–silver particles analyzed were distributed within the cytoplasm of dendrites and perikarya in vehicle-treated rats. Immunolabeling for mu-opioid receptors was distributed along portions of the plasma membrane that were often apposed by astroglial sheaths. After acute injections of etorphine, there was a dramatic internalization of mu-opioid receptors to intracellular compartments. Quantitative analysis of gold–silver particles indicative of mu-opioid receptors showed that a substantial number of gold particles shifted from the plasma membrane to early endosomes in dendrites from etorphine-treated rats. In dendrites sampled from etorphine-treated rats, 85% of the gold–silver grains indicative of mu-opioid receptor labeling were located in intracellular compartments as compared to 15% that were distributed along the plasma membrane. In animals that received either acute morphine injections or chronic morphine via pellet implantation, no change in the subcellular distribution of immunogold particles indicative of mu-opioid receptors was detected when compared to matched control animals.These results provide the first ultrastructural evidence that mu-opioid receptors are internalized by agonists such as etorphine, but not the partial agonist morphine, in the locus coeruleus.

Progesterone Blockade of Estrogen Activation of μ-Opioid Receptors Regulates Reproductive Behavior

The mu-opioid receptor (MOR), a G-protein-coupled receptor, is internalized after endogenous agonist binding. Although receptor activation and internalization are separate events, internalization is a good assay for activation because endogenous opioid peptides all induce internalization. Estrogen treatment of ovariectomized rats induces MOR internalization, providing a neurochemical signature of estrogen activation of the medial preoptic nucleus. MOR activation appears to be the mechanism via which estrogen acts in the medial preoptic area to prevent the display of female reproductive behavior during the first 20-24 hr after estrogen treatment. Naltrexone, an alkaloid universal opioid receptor antagonist, prevented MOR internalization, suggesting that estrogen induces the release of endogenous opioid peptides that in turn activate the MOR. Enkephalins and beta-endorphin are nonselective endogenous MOR ligands. The most selective endogenous MOR ligands are the endomorphins. Infusions of selective MOR agonists, H-Tyr-d-Ala-Gly-N-Met-Phe-glycinol-enkephalin (DAMGO) or endomorphin-1, into the medial preoptic nucleus attenuated lordosis, and their effects were blocked with the MOR antagonist H-d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH(2) (CTOP). Infusion of endomorphin-1 internalized MOR. To determine whether progestin also acts via the MOR system to facilitate reproductive behavior, ovariectomized rats were primed with 17beta-estradiol and progesterone. Progestin facilitation of lordosis was correlated with a reduction of estrogen-induced MOR internalization. Progestin reversed estrogen-induced MOR internalization, suggesting that progesterone blocked estrogen-induced endogenous opioid release, relieving estrogen inhibition and facilitating lordosis. These results indicate a central role of MOR in the mediation of sex steroid activation of the CNS to regulate female reproductive behavior.

Phosphorylation of Ser363, Thr370, and Ser375 Residues within the Carboxyl Tail Differentially Regulates μ-Opioid Receptor Internalization

Prolonged activation of opioid receptors leads to their phosphorylation, desensitization, internalization, and down-regulation. To elucidate the relationship between mu-opioid receptor (MOR) phosphorylation and the regulation of receptor activity, a series of receptor mutants was constructed in which the 12 Ser/Thr residues of the COOH-terminal portion of the receptor were substituted to Ala, either individually or in combination. All these mutant constructs were stably expressed in human embryonic kidney 293 cells and exhibited similar expression levels and ligand binding properties. Among those 12 Ser/Thr residues, Ser(363), Thr(370), and Ser(375) have been identified as phosphorylation sites. In the absence of the agonist, a basal phosphorylation of Ser(363) and Thr(370) was observed, whereas [d-Ala(2),Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO)-induced receptor phosphorylation occurs at Thr(370) and Ser(375) residues. Furthermore, the role of these phosphorylation sites in regulating the internalization of MOR was investigated. The mutation of Ser(375) to Ala reduced the rate and extent of receptor internalization, whereas mutation of Ser(363) and Thr(370) to Ala accelerated MOR internalization kinetics. The present data show that the basal phosphorylation of MOR could play a role in modulating agonist-induced receptor internalization kinetics. Furthermore, even though mu-receptors and delta-opioid receptors have the same motif encompassing agonist-induced phosphorylation sites, the different agonist-induced internalization properties controlled by these sites suggest differential cellular regulation of these two receptor subtypes.

Abdominal surgery induces Mu opioid receptor internalization

Abdominal surgery induces Mu opioid receptor internalization