Morphine inhibits Fc-mediated phagocytosis through mu and delta opioid receptors.
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Animals↗
Biomedical subjects
H H Loh
Publications and source records attributed to H H Loh.
Insights into the receptor transcription and signaling: implications in opioid tolerance and dependence.
Drug addiction has great social and economical implications. In order to resolve this problem, the molecular and cellular basis for drug addiction must be elucidated. For the past three decades, our research has focused on elucidating the molecular mechanisms behind morphine tolerance and dependence. Although there are many working hypotheses, it is our premise that cellular modulation of the receptor signaling, either via transcriptional or post-translational control of the receptor, is the basis for morphine tolerance and dependence. Thus, in the current review, we will summarize our recent work on the transcriptional and post-translational control of the opioid receptor, with special emphasis on the mu-opioid receptor, which is demonstrated to mediate the in vivo functions of morphine.
Animals↗
Lack of mu-opioid receptor leads to an increase in the NMDA receptor subunit mRNA expression and NMDA-induced convulsion.
The present study investigated in situ hybridization of N-methyl-D-aspartate (NMDA) receptor (NR) subunit mRNA and convulsion induced by intracerebroventricular injection of NMDA, in order to examine changes in NMDA receptor function in mu-opioid receptor gene knockout mice. Levels of NR1 and NR2A subunit mRNA were significantly increased in the parietal cortex (8.4 and 10.6%, respectively) and hypothalamus (8.7 and 15.2%, respectively) in mu-opioid receptor knockout mice. Levels of NR2B subunit mRNA were noted to be increased in the parietal cortex (9.1%), thalamus (7.7%), and hypothalamus (10.4%) in mu-opioid receptor knockout mice. The ED(50) for NMDA-induced convulsion in wild-type mice was 0.20 microg/10 microl/mouse. The ED(50) in mu-opioid receptor knockout mice was 0.14 microg/10 microl/mouse. There is a significant difference in the potency ratio of wild-type mice versus knockout mice (potency ratio: 1.44, P < 0.05). These results indicate that mu-opioid receptor knockout mice are more sensitive to NMDA-induced convulsion. Therefore, these results suggest that absence of mu-opioid receptor gene is accompanied by changes in the NMDA receptor system which can modulate the synaptic excitability in the process such as convulsion or epilepsy.
Animals↗
Differential effects of morphine, DPDPE, and U-50488 on apomorphine-induced climbing behavior in mu-opioid receptor knockout mice.
The present study examined the hypothesis whether the opioid receptors (mu, delta, and kappa) contribute to a behavioral dopaminergic activation produced by dopamine receptor agonist, apomorphine, by comparing responses in wild type and mu-opioid receptor knockout mice. The data suggest that expression of mu-opioid receptors plays an important role in the enhancement of climbing behavior induced by apomorphine. Compared to wild type mice, a response in the dopaminergic behavior by treatment with delta-receptor agonist, DPDPE, is more sensitive to the mice lacking mu-opioid receptor. Treatment with kappa-receptor agonist, U-50488, is potentiated the apomorphine-induced climbing behavior in wild type and mu-opioid receptor knockout mice. These responses may be independent of that through mu-opioid receptors. Therefore, the our results show that dopaminergic activation measured by climbing behavior in mu-opioid receptors knockout mice are differently regulated by mu-, delta-, and kappa-opioid receptor agonists.
3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
Transcriptional regulation of mouse delta-opioid receptor gene: role of Ets-1 in the transcriptional activation of mouse delta-opioid receptor gene.
Previously, we identified a minimum core promoter of the mouse delta-opioid receptor (DOR) gene. The DOR promoter contains an E-box that binds upstream stimulatory factor and is crucial for the DOR promoter activity in NS20Y cells, a mouse neuronal cell line that constitutively expresses DOR. In the present study, we further analyzed the DOR promoter in NS20Y cells and have demonstrated that transcription factor Ets-1 binds to an Ets-1-binding site overlapping the E-box and trans-activates the DOR promoter by synergizing with upstream stimulatory factor in specific DNA binding. In addition, the Ets-1 DNA-binding domain is sufficient to play the functional role of Ets-1 in trans-activating the DOR promoter. Furthermore, through in vivo cross-linking assays and Northern blot analyses, we have demonstrated that Ets-1 binds to the DOR promoter in the neonatal mouse brain and that overexpressed Ets-1 can significantly enhance the expression of DOR mRNA in primary neonatal mouse neuronal cells. Collectively, our data suggest that Ets-1 functions as a trans-activator of the DOR promoter in the neonatal mouse brain and thus may contribute to the development of the mouse brain DOR system.
Animals↗
Role of hypothalamic-pituitary axis in morphine-induced alteration in thymic cell distribution using mu-opioid receptor knockout mice.
Mu-opioid receptor knockout mice (MORKO), were used to address two questions: (1) if morphine induced decrease in thymic weight and cell distribution is mediated by the mu-opioid receptor and (2) the role of corticosteroids in morphine mediated alteration in thymic cell distribution. Our result show that morphine mediated increase in plasma corticosterone is mediated by the mu-opioid receptor since morphine at doses as high as 25 mg/kg-body weight does not increase plasma corticosterone levels in the MORKO. In addition, we have also shown that morphine treatment results in the differentiation of CD4+CD8+ (double positive cells) to single positive CD4+ cells while dexamethasone treatment results in the deletion of CD4+CD8+ (double positive) cells.
Adrenocorticotropic Hormone↗
Covalently induced activation of the delta opioid receptor by a fluorogenic affinity label, 7'-(phthalaldehydecarboxamido)naltrindole (PNTI).
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Affinity Labels↗
Region specific increase of dopamine receptor D1/D2 mRNA expression in the brain of mu-opioid receptor knockout mice.
Previous pharmacological studies have indicated the possible existence of functional interactions between opioidergic and dopaminergic neurons in the CNS. In this study, the expression of mRNAs encoding dopamine receptor D1/D2 was examined to investigate whether there is a change in the dopamine pathway of mice lacking the mu-opioid receptor by in situ hybridization technique. In the mu-opioid receptor knockout mice, the expression of dopamine receptor D1 mRNA was increased in the olfactory tubercle, nucleus accumbens, caudate putamen, and the layer VI of the neocortex compared with that of wild-type mice. The expression of dopamine receptor D2 mRNA was also increased in the olfactory tubercle, caudate putamen, and the nucleus accumbens of mu-opioid receptor knockout mice. These results indicate that there are compensational changes in the dopaminergic systems of mu-opioid receptor knockout mice.
Animals↗
Effects of morphine on pentobarbital-induced responses in mu-opioid receptor knockout mice.
Effects of morphine on the potentiation of pentobarbital-induced responses were investigated using mu-opioid receptor knockout mice. The duration of loss of righting reflex, hypothermia, and loss of motor coordination induced by pentobarbital were measured after pretreatment with either morphine or saline. Morphine pretreatment failed to show potentiation of both pentobarbital-induced loss of righting reflex and hypothermia in mu-opioid receptor knockout mice, while it significantly potentiated these responses in the wild-type controls. For motor incoordination test, morphine potentiated pentobarbital-induced motor incoordination in the wild-type mice. However, morphine may have opposite effects in the mu-opioid receptor knockout mice. These results demonstrate that synergism between morphine and pentobarbital is not detected in mu-opioid receptor knockout mice and that potentiation of pentobarbital-induced loss of righting reflex and hypothermia by morphine is mediated through mu-opioid receptor. It was interesting to note that pentobarbital-induced decrease in body temperature was less severe in mu-opioid receptor knockout mice than in wild-type mice.
Animals↗
Regulation of mouse kappa opioid receptor gene expression by retinoids.
The effect of retinoids on the expression of kappa opioid receptor (KOR) gene was examined in normal and transgenic animals. KOR-lacZ transgene expression was specifically elevated in KOR-positive areas of the developing CNS by depleting vitamin A from animal diets. The endogenous KOR mRNA species, including all three isoforms, were also upregulated by depleting vitamin A in developing animals. Change in the expression of isoforms a and b is similar in prenatal stages but differs during postnatal development. Interestingly, upregulation of isoform c is most significant postnatally. The regulation of KOR gene by vitamin A was substantiated in a mouse embryonal carcinoma P19 culture system in which retinoic acid (RA), the most potent ingredient of vitamin A, was able to suppress the expression of all the three KOR isoforms and KOR protein. The RA-mediated suppression was blocked by an RA receptor antagonist and a histone deacetylase (HDAC) inhibitor. By using a reporter transfection assay in P19 cells, the potential genetic element responsible for RA-mediated suppression of KOR gene expression was located to intron 1 of the mouse KOR gene, which could also be blocked by HDAC inhibitor. Furthermore, suppression of KOR gene expression by RA in P19 cells appeared to be an indirect event and required protein synthesis. A role of RA in KOR gene regulation during developmental stages was discussed.
Animals↗
Phosphorylation of Ser363, Thr370, and Ser375 residues within the carboxyl tail differentially regulates mu-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.
Alanine↗
Single-stranded DNA-binding complex involved in transcriptional regulation of mouse mu-opioid receptor gene.
Previously, we reported the presence of dual (distal and proximal) promoters in mouse mu-opioid receptor (mor) gene, with mor transcription in mouse brain predominantly initiated by the proximal promoter. Sp factors, bound to double-stranded (ds) cis-regulatory elements, are critical for proximal promoter activity. Here, we further report that a single-stranded (ss) cis-regulatory element and trans-acting protein factor are also important for proximal promoter activity. A 26-bp mor polypyrimidine/polypurine region (PPy/u) can adopt ss DNA conformation, as demonstrated by S1 nuclease sensitivity. Using electrophoretic mobility shift analysis with nuclear extracts from mor-expressing SH-SY5Y cells, we demonstrate that the sense strand of PPy/u interacts with a major nuclear protein, termed mor polypyrimidine-binding protein (mPy), which is not related to Sp factors. Southwestern blot analysis indicated that mPy protein is approximately 25 kDa in size. Functional analysis suggests that mPy protein can trans-activate mor promoter as well as a heterologous promoter. Moreover, combinatorial activation of ss (mPy) and ds (Sps) DNA binding factors, interacting with an overlapping DNA (PPy/u) region, is necessary for proximal promoter activation. Thus our results suggest that transcription of mouse mor gene is regulated by an interplay of ss and ds DNA binding factors.
Animals↗
Role of mu-opioid receptor in immune function.
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Animals↗
Affinity labels as tools for the identification of opioid receptor recognition sites.
Affinity labels have proven to be useful tools in opioid research. We review experiments carried out with the mu opioid receptor affinity label, beta-funaltrexamine (2), that support the concept of different recognition sites for mu opioid agonists and antagonists. The data are interpreted in the context of a dimeric receptor that contains two allosterically coupled binding sites: one that binds endogenous agonist, and the second that functions as an inhibitory modulator of agonism. It is proposed that exogenous antagonists bind selectively to the second site. The first of a new class of affinity labels, PGNA (5), that contains the phthaldehyde moiety attached to an opioid antagonist pharmacophore, is described. This class of ligands has been named 'reporter affinity labels' because covalent association leads to the formation of a fluorescent isoindole that is diagnostic for cross-linking of lysine and cysteine residues. PGNA binds opioid receptors covalently, as suggested by (a) irreversible binding to cloned opioid receptors, (b) irreversible opioid antagonism in the guinea pig ileum preparation, and (c) ultra-long opioid antagonism in mice. Since flow cytometry experiments revealed specific enhancement of fluorescence in cloned mu receptors after a 1 min exposure to 5, it is concluded that covalent binding has occurred via the formation of an isoindole, presumably by cross-linking neighboring lysine and cysteine residues in the vicinity of the receptor recognition site.
Affinity Labels↗
mu-Opioid receptor gene expression: the role of NCAM.
We have investigated the mechanisms regulating the expression of the mu-opioid receptor, using P19 mouse embryonal carcinoma cells, which normally lack this receptor, but which can be induced to express it in aggregated cells by retinoic acid treatment. The expression level of mu-opioid receptor mRNA was found to be closely correlated with aggregation status, and more specifically by cell to cell interaction requiring neural cell adhesion molecules (NCAM). We showed that NCAM activates the mu-opioid receptor gene through a pathway involving phospholipase C-arachidonic acid-calcium channel-calcium/calmodulin kinase II. A similar pathway was previously shown to promote neurite outgrowth, however, with distinct specificity, including the role of calcium channels. Activation of L-type calcium channels elevated mu-opioid receptor expression, while N-type-channel activity had the opposite effect. The effect of anti-NCAM-antibody treatment was not due to retardation of general neural development and was specific to the mu-opioid receptor gene. Our results indicate that the P19 system is an useful model to study the expression of the mu-opioid receptor gene.
Animals↗
Transcriptional modulation of mouse mu-opioid receptor distal promoter activity by Sox18.
Previously, we reported the presence of dual promoters, referred to as distal (DP) and proximal, with a negative regulatory element between them in the mouse mu-opioid receptor (mor) gene. Here we have identified a positive regulatory element influencing mor DP transcription, which contains multiple consensus binding motifs for Sox factors (sex-determining Sry-like high mobility group box-containing genes). In gel supershift assays, the Sox family member Sox18 bound directly to the multiple Sox consensus binding motifs of the mor DP enhancer. Overexpression of Sox18 cDNA increased luciferase activity regulated by the mor DP, and did so in a Sox18 concentration-dependent manner. In contrast, overexpression of another Sox member, Sox5, triggered no such trans-activation of mor DP-driven luciferase activity or DNA-protein binding activity. These results suggest that Sox18 directly and specifically stimulates mor gene expression, by trans-activating the mor DP enhancer.
5' Untranslated Regions↗
delta-Opioid receptor gene: effect of Sp1 factor on transcriptional regulation in vivo.
delta-Opioid receptor (DOR) promoter exhibited a cell-type-specific expression pattern. Protein-DNA interactions in this promoter were identified by dimethyl sulfate in vivo footprinting analysis of NG108-15 cells, expressing endogenous DOR. Complete protection of the putative Sp1 cis-element and partial protection of the sequence defined as X-NotI in the basal promoter were observed only in the G0/G1 phase of the cell cycle. No protection was detected in Neuro2A cells that do not express DOR. In vivo formaldehyde cross-linking confirmed Sp1 factor binding to its cis-acting element during the G0/G1 phase. The functional significance of these Sp1 and X-NotI sites was evaluated by transient transfection analysis. Northern blot analysis and nuclear run-off assays revealed maximum DOR mRNA level and transcription rate, respectively, during the G0/G1 phase of NG108-15 cells. In summary, the protein-DNA interactions at the Sp1 and X-NotI sites are necessary for cell cycle-dependent and cell-type-specific up-regulated DOR gene expression.
Alternative Splicing↗
Opioid and cannabinoid modulation of precipitated withdrawal in delta(9)-tetrahydrocannabinol and morphine-dependent mice.
The goal of the present study was to elucidate the relationship between cannabinoid and opioid systems in drug dependence. The CB(1) cannabinoid receptor antagonist SR 141716A precipitated both paw tremors and head shakes in four different mouse strains that were treated repeatedly with Delta(9)-tetrahydrocannabinol (Delta(9)-THC). SR 141716A-precipitated Delta(9)-THC withdrawal was ameliorated in mu-opioid receptor knockout mice compared with the wild-type control animals and failed to occur in mice devoid of CB(1) cannabinoid receptors. An acute injection of morphine in Delta(9)-THC-dependent mice undergoing SR 1417161A-precipitated withdrawal dose dependently decreased both paw tremors, antagonist dose 50 (AD(50)) (95% CL) = 0.035 (0.03--0.04), and head shakes, AD(50) (95% CL) = 0.07 (0.04--0.12). In morphine-dependent mice, the opioid antagonist naloxone precipitated head shakes, paw tremors, diarrhea, and jumping. As previously reported, naloxone-precipitated morphine withdrawal failed to occur in mu-opioid knockout mice and was significantly decreased in CB(1) cannabinoid receptor knockout mice. Acute treatment of Delta(9)-THC in morphine-dependent mice undergoing naloxone-precipitated withdrawal blocked paw tremors, AD(50) (95% CL) = 0.5 (0.3--1.0), and head shakes AD(50) (95% CL) = 0.6 (0.57--0.74) in dose-dependent manners, but failed to diminish the occurrence of diarrhea or jumping. Finally, naloxone and SR 141716A failed to elicit any overt effects in Delta(9)-THC-dependent and morphine-dependent mice, respectively. These findings taken together indicate that the mu-opioid receptor plays a modulatory role in cannabinoid dependence, thus implicating a reciprocal relationship between the cannabinoid and opioid systems in dependence.
Animals↗