Down-regulation Of Opiate Receptors Research Articles

Thr353, Located within the COOH-terminal Tail of the ´ Opiate Receptor, Is Involved in Receptor Down-regulation

Prolonged exposure to abused drugs such as opiates causes decreased response to the drug; this reduced sensitivity is thought to be due to the loss of receptors, or down-regulation. The molecular mechanism of the opiate receptor down-regulation is not known. In order to address this, we generated a number of mutants of the delta opiate receptor COOH-terminal tail. When expressed in the Chinese hamster ovary cells, both the wild type and the receptor with a deletion of 37 COOH-terminal residues bind diprenorphine with comparable affinities and show similar decreases in cAMP levels in response to D-Ala2, D-Leu5, enkephalin (DADLE). However, the truncated receptor does not show down-regulation from the cell surface upon prolonged exposure (2-48 h) to DADLE. In contrast, both the wild type receptor and the receptor with the deletion of only 15 COOH-terminal residues show substantial down-regulation upon long term DADLE treatment. These results suggest that the region located between 15 and 37 residues from the COOH terminus is involved in the receptor down-regulation. In order to identify residues that play a key role in down-regulation, point mutations of residues within this region were examined for their ability to modulate receptor down-regulation. The receptor with a mutation of Thr353 to Ala does not down-regulate, whereas the receptor with a mutation of Ser344 to Gly down-regulates with a time course similar to that of the wild type receptor. Taken together, these results suggest that the COOH-terminal tail is not essential for functional coupling but is necessary for down-regulation and that Thr353 is critical for the agonist-mediated down-regulation of the delta opiate receptor.

Quantification of opiate receptors in two patients with mesiobasal temporal lobe epilepsy, before and after selective amygdalo- hippocampectomy, using positron emission tomography

Using PET, reduced glucose metabolism (rCMRglu) and increased binding of the mu opiate receptor ligand 11C-carfentanil have been demonstrated in lateral temporal cortex overlying mesial temporal epileptic foci. Binding of the non-specific opiate receptor ligand 11C-diprenorphine (DPN) to lateral temporal cortex has not shown consistent asymmetries. We measured rCMRglu with 18F-FDG and binding of 11C-diprenorphine in two patients with temporal lobe epilepsy (TLE) before and 5 months after selective amygdalo-hippocampectomy (both patients were seizure-free post-operatively). Pre-operatively, in both patients rCMRglu was decreased in mesial temporal lobe (MTL) (asymmetry index AI = −9.1 and 9.0) ipsilateral to the EEG focus. A more marked reduction was seen in ipsilateral lateral temporal cortex (LTC) ( AI = −32.0 and 18.9). DPN binding was reduced in MTL and LTC ( AI MTL = −9.3 and 16.2; AI LTC = −8.0 and 5.5) ipsilateral to the focus, but was within 2 SD of the normal range. Post-operatively, the reduction of rCMRglu in LTC was accentuated in one patient and decreased in the other ( AI = −23.1 and 45.7) while there was a further reduction of DPN binding in LTC in both patients ( AI = −27.8 and 9.8). These preliminary results in only two patients are compatible with downregulation of opiate receptors in LTC after removal of the epileptic focus or post-operative neuronal dysfunction.

Visualization of μ opiate receptor downregulation following morphine treatment in neonatal rat brain

In order to visualize neuroanatomical alterations in specific brain regions, light microscopy autoradiography was carried out on neonatal brain sections from controls and neonates chronically treated with morphine. In the case of brains exposed to morphine from postnatal day (PD) 1–4, μ receptor density on PD 5 was non-existent in the patches of the striatum. There were also decreases in μ opiate receptor density in the surrounding matrix area, and in the nucleus accumbens and amygdala. Longer durations of morphine treatment (PD 1–8) did not show these alterations in μ opiate receptor density. These data demonstrate the unique plasticity seen in the immature opioid system. It is suggested that the differences observed in neonatal vs adult central nervous system may be due to interactions with and the differential development of the G-protein/cyclic adenosine 3′,5′-monophosphate (cAMP) system.

Molecular mechanism of drug tolerance and dependence

Morphine and ethanol drugs known to develop tolerance and dependence, induce changes in the adenylate cyclase system. Morphine inhibits the adenylate cyclase activity in NG108-15 cells and causes increases in adenylate cyclase synthesis and the down-regulation of opiate receptors in cells treated for several days. Chronic exposure of NG108-15 cells to ethanol also causes a decrease in the mRNA of the GTP-binding protein (Gs). These observations suggest the possibility that a group of genes is expressed in response to morphine or ethanol during the acquisition of tolerance and dependence. Recently, it has been reported that cAMP regulates a number of genes through a cAMP response element (CRE) in their promotor regions and that nuclear CRE-binding proteins bind specifically to the CRE to stimulate the transcription of cAMP-responsive genes. The gel shift assay with a single stranded oligo-DNA of CRE in a somatostatin promotor region was employed to examine the possibility of transcriptional regulation of cAMP-inducible genes by chronic morphine or ethanol treatment of NG108-15 cells. When the nuclear proteins from the cells treated with morphine or ethanol for several days were provided for the assay, the amounts of DNA-protein complex were decreased. The decreased complexes were recovered by 1-2 days after morphine withdrawal. The nuclear proteins were purified partially by a combination of chromatography on Q-Sepharose, Sephacryl S-300 and DNA affinity-Sepharose. Changes in CRE-binding proteins from the cells treated chronically with morphine or ethanol suggest that these drugs can modulate the expression of cAMP-inducible genes through which tolerance and dependence may develop.

Enhancement of dynorphin gene expression in spinal cord following experimental inflammation: stimulus specificity, behavioral parameters and opioid receptor binding

The stimulus specificity for enhancement of dynorphin gene expression in rat spinal cord was studied by combined measurements of the peptide dynorphin A 1–8 and preprodynorphin mRNA levels during peripheral inflammation induced by several agents. The density of kappa receptors, the putative receptor for dynorphin peptides, was examined using receptor binding with autoradiographic visualization. Mu and delta receptor classes were also studied. All inflammatory agents tested (carrageenan, phorbol ester, yeast and Freund's adjuvant) rapidly induced edema and thermal hyperalgesia. All agents also induced a rapid (within 8 h) elevation in dynorphin mRNA and, in comparison, a delayed (within 2 days) elevation of dynorphin A 1–8 peptide; peak peptide levels were reached at 4 days. No alteration of kappa, mu or delta receptor binding was observed at 4 h or 4 days post inflammation. The rapid development of thermal hyperalgesia and elevation of dynorphin mRNA and peptide content indicates that the involvement of dynorphin-containing neurons in nociceptive processing does not require a chronic abnormality and a dynamic picture of opioid modulation of sensory processing emerges. These data also demonstrate that activation of dynorphin biosynthesis in spinal cord is a feature common to hyperalgesia and peripheral inflammation and is not restricted to any one type of inflammatory agent. The lack of alteration in receptors suggests that the physiological effects of an increased biosynthesis are not accompanied by a concurrent down-regulation of opiate receptors.

Neurochemical correlates of opiate receptor regulation

The actions of opiates and opioid peptides upon nervous tissue are mediated by p, 6, K, and oreceptors [for reviews see Refs. l-31. The p receptor is the high affinity site at which morphine-like opiates produce analgesia and a variety of other classical opiate effects. The 6 receptor exhibits a higher affinity for the naturally occurring enkephalins (a class of shorter opioid peptides) than for morphine. The K receptor is that site at which ketocyclazocine-like opiates produce analgesia, as well as their unique ataxic and sedative effects. More recently, it has been defined as a receptor highly selective for dynorphin (a 17 amino acid opioid peptide). The areceptor mediates the psychotomimetic and stimulant effects of SKF-10,047 (N-allylnorcyclazocine) and other o opiates. These receptors exhibit rather different ligand selectivity patterns, are widely distributed in the central nervous system, and exhibit diverse neuroanatomical distributions [4-81. Findings from studies in neuronal cell lines demonstrating opiateinduced inhibition of adenylate cyclase [9, 101 and in brain and cell lines showing guanyl nucleotide modulation of agonist binding [ll-141 make it likely that the p and 6 receptors are coupled to cyclase through an inhibitory guanyl nucleotide binding protein (Ni). It is now well-established that the p, 6, and K receptors are subserved by three classes of opioid peptides, pendorphin, the enkephalins, and dynorphin-related peptides. It is, however, unknown whether these receptors represent different gene products or a single gene product which has been post-translationally modified. Moreover, the question arises as to whether they are regulated in a coordinated or an independent manner. Opiate analgesics are well-known to produce tolerance and dependence in vivo and desensitization in vitro. The observation of these phenomena has raised the question as to whether opiate receptors undergo upor downregulation in vivo, in response to long-term administration of opiate drugs. Opiate receptor downregulation has been difficult to document. Several groups have reported that chronic administration of narcotic agonists in vivo does not produce any change in either receptor number or affinity [15-211. On the other hand, receptor downregulation has been observed in neurotumor cell

Effect of pertussis toxin treatment on the down-regulation of opiate receptors in neuroblastoma X glioma NG108-15 hybrid cells.

Chronic treatment of neuroblastoma X glioma NG108-15 hybrid cells with 10 nM [D-Ala2,D-Leu5] enkephalin (DADLE) results in a reduction of cell-surface opiate delta receptors. Whether opiate receptor internalization requires the activation of the guanine nucleotide-binding protein (Ni) is unclear. Hence, activation of Ni was attenuated by treating hybrid cells with 100 ng/ml pertussis toxin (PT) for 3 h, which resulted in a decrease in DADLE's ability to inhibit adenylate cyclase activity. Despite this prior treatment with PT, chronic exposure of these cells to 10 nM DADLE resulted in a time-dependent decrease in both [3H]diprenorphine and [3H]DADLE binding. This reduction in 3H-ligand binding in cells previously treated with PT represented internalization of the receptors because translocation was observed of bound [3H]DADLE from plasma membrane fractions to the lysosomal fractions in the Percoll gradients. Thus, opiate receptors internalize without activation of Ni. The internalization of opiate receptors was not accompanied by Ni. By measuring the amount of the 41-kDa alpha subunit being labeled by PT with [32P]NAD+, it was determined that plasma membrane preparations, of both the control cells and cells treated with 10 nM of DADLE for 4 h, contained equal concentrations of Ni, 2 pmol of Ni/mg of protein. Additionally, there was no measurable alteration in the amount of PT substrate in the lysosomal fractions of the DADLE-treated cells as compared to that of control cells. Chronic DADLE treatment resulted in a decrease in Km value of NAD+ in the ADP-ribosylation of 41-kDa subunit of Ni. In summary, opiate receptors internalize as agonist-receptor complexes without the guanine nucleotide-binding component.

Effects of cycloheximide and tunicamycin on opiate receptor activities in neuroblastoma X glioma NG108-15 hybrid cells

The molecular mechanism of opiate receptor down-regulation and desensitization was investigated by studying the effects of cycloheximide and tunicamycin on opiate receptor activities in neuroblastoma X glioma NG108-15 hybrid cells. Cycloheximide inhibited [ 35S]methionine and [ 3H]-glucosamine incorporation by hybrid cells, while tunicamycin inhibited [ 3H]glucosamine incorporation only. Exposing hybrid cells to these two agents did not alter the viability of the cell. Treatment of NG108-15 cells with cycloheximide or tunicamycin produced a decrease in [ 3H]diprenorphine binding dependent on both time and concentrations of inhibitors, with no measurable modification in the ability of etorphine to regulate intracellular cyclic AMP production. Cycloheximide attenuated [ 3H]-diprenorphine binding by decreasing both the number of sites, B max, and the affinity of the receptor, K d . Tunicamycin treatment produced a decrease in B max with no apparent alteration in K d values. Cycloheximide and tunicamycin did not potentiate the rate or magnitude of etorphine-induced down-regulation or desensitization of opiate receptor in NG108-15 cells. Furthermore, there was an apparent antagonism in cycloheximide action on receptor down-regulation. The reappearance of opiate binding sites after agonist removal was affected by these two inhibitors. Cycloheximide and tunicamycin eliminated the increase in [ 3H]diprenorphine binding in the chronic etorphine-treated cells after agonist removal. These two inhibitors did not alter the resensitization of hybrid cells to etorphine. Thus, the site of opiate agonist action to induce receptor down-regulation and desensitization is not at the site of protein synthesis or protein glycosylation. These data substantiate previously reported observations that receptor down-regulation and receptor desensitization are two different cellular adaptation processes.

Selectivity in the control of opiate receptor density in the animal and in cultured fetal brain cells

Two aspects of the mechanisms controlling down-regulation of opiate receptors were studied: 1 . The possibility that morphine does not induce down-regulation of δ receptors is an observation confined to in vitro conditions was investigated by studying the regulation of receptors in neuroblastomaglioma cells in diffusion chambers implanted in ICR mice peritonea. Injection of morphine for 9 days at a dose inducing opiate tolerance did not change the number of receptors in the chamber-implanted cells, whereas etorphine at a 1 100 dose had a profound effect. 2 . Embryonic cells from rat forebrain or hindbrain were cultured with μ type opiate alkaloid (morphine) or peptide (morphiceptin) to further establish the selectivity of opiate action. A partial effect of morphiceptin but not morphine on the number of receptors in hindbrain aggregates was observed. Thus, conclusions derived from experiments with morphine may not be applicable to μ type peptides. The results suggest that the mammalian brain may contain sub-types of μ receptors. Alternatively, although interacting with a common μ receptor, morphine and μ opioid peptides may induce different regulatory mechanisms.

Characterization and down-regulation of opiate receptors in aggregating fetal rat brain cells

Aggregating brain cells prepared from embryonic rats bind radioactive opiates in a stereospecific manner. The drug selectivity, receptor content during culturing and down-regulation of these apparent opiate receptors were studied in aggregates prepared from the embryonic hindbrain or forebrain. The receptor content in hindbrain but not forebrain aggregates was increased up to 3-fold after 21 days in culture. Differences between the receptors of the two types of aggregates were also observed in the affinity of opiate alkaloids and d-Ala 2, d-Leu 5-enkephalin (DADL). The potent opiate alkaloid etorphine induced down-regulation of opiate receptors in aggregates prepared from either brain region whereas DADL was a potent down-regulator in the forebrain but not in the hindbrain aggregates and morphine had no effect in both tissues. The implications of these results concerning the control of various types of opiate receptors in the whole animal are discussed.

Down-regulation of opiate receptor in neuroblastoma x glioma NG108-15 hybrid cells. Chloroquine promotes accumulation of tritiated enkephalin in the lysosomes.

Opiate receptor down-regulation in neuroblastoma X glioma NG108-15 hybrid cells possibly involved the internalization of ligand-receptor complexes during chronic treatment. However, receptor internalization was not supported by the observed decrease in [3H] enkephalin(D-Ala2,D-Leu5) ( [3H]DADLE) associated with the hybrid cells during prolonged incubation with 10 nM [3H]DADLE at 37 degrees C. This decrease in [3H]DADLE bound was determined to be due to degradation of the ligand-receptor complexes, for a time-dependent increase in [3H]DADLE bound was observed when the incubations were carried out in the presence of 0.1 mM chloroquine. The increase did not exceed the amount of down-regulated receptor, could be blocked by naloxone, and was not observed at 24 degrees C. The [3H]DADLE bound in the presence of chloroquine was not sensitive to trypsin or to 20 microM diprenorphine. The accumulated [3H]DADLE was demonstrated to be intracellularly located by the fractionation of the homogenates in self-generating Percoll gradients. In the presence of chloroquine, a time-dependent translocation of [3H]DADLE from the plasma membrane-enriched fractions to the lysosome-enriched fractions was observed. The translocation was not observed at 24 degrees C in the presence of chloroquine or at 37 degrees C in the absence of chloroquine. The [3H]DADLE in the lysosome-enriched fractions was not sensitive to trypsin and remained bound in the presence of chloroquine. With the removal of chloroquine, an increase in the release of [3H]DADLE into the medium was observed. Sephadex G-50 column chromatography of the sodium deoxycholate extracts of the lysosome-enriched fractions suggested that the [3H]DADLE was bound to macromolecules intracellularly. Thus, chronic [3H]DADLE treatment of the hybrid cells resulted in an internalization of ligand-receptor complexes which were degraded in the lysosomes. Subsequently, the [3H]DADLE was regurgitated by the hybrid cells.

Down regulation of opiate receptors on 108CC15 hybrid cells is inhibited by receptor occupancy

This study reports on the molecular mechanisms of δ-opiate receptor down regulation on 108CC15 neuroblastoma × glioma hybrid cells. The down regulation induced by culture in the presence of 10 −5 M 2- d-Ala, 5- d-Leu-enkephalin (DADLE) can be prevented by continued exposure to ligand concentrations greater than 4 nM, the K d of the binding site. Below this concentration, down regulation is a rapid and irreversible process. It is deduced that the internalization process in this cell line is initiated when unoccupied receptor dimers are present. These results have important implications for down regulation studies using cultured cell lines and studies of receptor regulation in vivo after chronic treatment with neuroactive drugs.

Ligand induced down regulation of opiate receptors on 108CC15 cells is inhibited by receptor occupation

Ligand induced down regulation of opiate receptors on 108CC15 cells is inhibited by receptor occupation

Down-regulation of opiate receptors in serum-free cultures of aggregating fetal brain cells

Brain cells from rat embryos that were cultured in serum-free medium form aggregates which possess stereospecific binding of radioactive opiates. The specificity of these opiate receptors and their regulation by opiate alkaloids and enkephalin was studied. D-ala 2, D-leu 5-enkephalin (DADL) induced down-regulation of opiate receptors in aggregates prepared from the forebrain but had no effect on hindbrain aggregates, whereas the potent alkaloid etorphine induced down-regulation of the receptors in both tissues. Interestingly, morphine did not induce down-regulation in both tissues. The significance of these findings and their relationship to the control of different types of opiate receptors are discussed.