C-terminal splice variants of the mouse mu-opioid receptor differ in morphine-induced internalization and receptor resensitization

The main analgesic effects of the opioid alkaloid morphine are mediated by the mu-opioid receptor. In contrast to endogenous opioid peptides, morphine activates the mu-opioid receptor without causing its rapid endocytosis. Recently, three novel C-terminal splice variants (MOR1C, MOR1D, and MOR1E) of the mouse mu-opioid receptor (MOR1) have been identified. In the present study, we show that these receptors differ substantially in their agonist-selective membrane trafficking. MOR1 and MOR1C stably expressed in human embryonic kidney 293 cells exhibited phosphorylation, internalization, and down-regulation in the presence of the opioid peptide [d-Ala(2),Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO) but not in response to morphine. In contrast, MOR1D and MOR1E exhibited robust phosphorylation, internalization, and down-regulation in response to both DAMGO and morphine. DAMGO elicited a similar desensitization (during an 8-h exposure) and resensitization (during a 50-min drug-free interval) of all four mu-receptor splice variants. After morphine treatment, however, MOR1 and MOR1C showed a faster desensitization and no resensitization as compared with MOR1D and MOR1E. These results strongly reinforce the hypothesis that receptor phosphorylation and internalization are required for opioid receptor reactivation thus counteracting agonist-induced desensitization. Our findings also suggest a mechanism by which cell- and tissue-specific C-terminal splicing of the mu-opioid receptor may significantly modulate the development of tolerance to the various effects of morphine.     

A single nucleotide polymorphic mutation in the human mu-opioid receptor severely impairs receptor signaling

Large scale sequencing of the human mu-opioid receptor (hMOR) gene has revealed polymorphic mutations that occur within the coding region. We have investigated whether the mutations N40D in the extracellular N-terminal region, N152D in the third transmembrane domain, and R265H and S268P in the third intracellular loop alter functional properties of the receptor expressed in mammalian cells. The N152D receptor was produced at low densities. Binding affinities of structurally diverse opioids (morphine, diprenorphine, DAMGO and CTOP) and the main endogenous opioid peptides (beta-endorphin, [Met]enkephalin, and dynorphin A) were not markedly changed in mutant receptors (<3-fold). Receptor signaling was strongly impaired in the S268P mutant, with a reduction of efficacy and potency of several agonists (DAMGO, beta-endorphin, and morphine) in two distinct functional assays. Signaling at N40D and R265H mutants was highly similar to wild type, and none of the mutations induced detectable constitutive activity. DAMGO-induced down-regulation of receptor-binding sites, following 20 h of treatment, was identical in wild-type and mutant receptors. Our data show that natural sequence variations in hMOR gene have little influence on ligand binding or receptor down-regulation but could otherwise modify receptor density and signaling. Importantly, the S268P mutation represents a loss-of-function mutation for the human mu-opioid receptor, which may have an incidence on opioid-regulated behaviors or drug addiction in vivo.     

Internalization and recycling of human mu opioid receptors expressed in Sf9 insect cells

Internalization and recycling of G protein-coupled receptors (GPCRs), such as the mu-opioid receptor, largely depend on agonist stimulation. Agonist-promoted internalization of some GPCRs has been shown to mediate receptor desensitization, resensitization, and down-regulation. In this study, we investigated whether different mu opioid agonists displayed different effects in receptor internalization and recycling, the potential mechanisms involved in ohmefentanyl-induced internalization process. In transfected Sf9 insect cells expressing 6His-tagged wild type mu opioid receptor, exposure to 100 nM ohmefentanyl caused a maximum internalization of the receptor at 30 min and receptors seemed to reappear at the cell membrane after 60 min as determined by radioligand binding assay. Ohmefentanyl-induced human mu opioid receptor internalization was concentration-dependent, with about 40% of the receptors internalized following a 30-min exposure to 1 microM ohmefentanyl. 10 microM morphine and 1 microM DAMGO could also induce about 40% internalization. The antagonist naloxone and pretreatment with pertussis toxin both blocked ohmefentanyl-induced internalization without affecting internalization themselves. Incubation with sucrose 0.45 M significantly inhibited ohmefentanyl-induced internalization of the mu receptor. The removal of agonists ohmefentanyl and morphine resulted in the receptors gradually returning to the cell surface over a 60 min period, while the removal of agonist DAMGO only partly resulted in the receptor recycling. The results of this study suggest that ohmefentanyl-induced internalization of human mu opioid receptor in Sf9 insect cells occurs via Gi/o protein-dependent process that likely involves clathrin-coated pits. In addition, the recycling process displays the differential modes of action of different agonists.     

Phospholipase D2 modulates agonist-induced mu-opioid receptor desensitization and resensitization

Receptor phosphorylation, arrestin binding, uncoupling from G protein and subsequent endocytosis have been implicated in G protein-coupled receptor desensitization after chronic agonist exposure. In search of proteins regulating the mu-opioid receptor endocytosis, we have recently established that activation of phospholipase D (PLD)2 is required for agonist-induced mu-opioid receptor endocytosis. In this study, we determined the effect of PLD2 activity on the desensitization and resensitization rate of the mu-opioid receptor. We clearly demonstrated that inhibition of PLD2-mediated phosphatidic acid formation by alcohol (1-butanol or ethanol) or overexpression of a dominant negative mutant of PLD2 prevented agonist-mediated endocytosis and resulted in a faster desensitization rate of the mu-opioid receptor after chronic (D-Ala2, Me Phe4, Glyol5)enkephalin treatment in human embryonic kidney 293 cells. Moreover, inhibition of PLD2 activity led to an impairment of the resensitization rate of the mu-opioid receptor. In summary, our data strongly suggest that PLD2 is a modulator of agonist-induced endocytosis, desensitization and resensitization of the mu-opioid receptor.     

Mu-opioid receptor desensitization: role of receptor phosphorylation,internalization, and representation

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.     

Identification of morphine-6-glucuronide in chromaffin cell secretory granules

We report for the first time that morphine-6-glucuronide, a highly analgesic morphine-derived molecule, is present in adrenal chromaffin granules and secreted from chromaffin cells upon stimulation. We also demonstrate that phosphatidylethanolamine-binding protein (alternatively named Raf-1 kinase inhibitor protein or RKIP) acts as an endogenous morphine-6-glucuronide-binding protein. An UDP-glucuronosyltransferase 2B-like enzyme, described to transform morphine into morphine-6-glucuronide, has been immunodetected in the chromaffin granule matrix, and morphine-6-glucuronide de novo synthesis has been characterized, demonstrating the possible involvement of intragranular UDP-glucuronosyltransferase 2B-like enzyme in morphine-6-glucuronide metabolism. Once secreted into the circulation, morphine-6-glucuronide may mediate several systemic actions (e.g. on immune cells) based on its affinity for mu-opioid receptors. These activities could be facilitated by phosphatidylethanolamine-binding protein (PEBP), acting as a molecular shield and preventing morphine-6-glucuronide from rapid clearance. Taken together, our data represent an important observation on the role of morphine-6-glucuronide as a new endocrine factor.     

Isolation and characterization of new exon 11-associated N-terminal splice variants of the human mu opioid receptor gene

Alternative splicing of the mu opioid receptor genes to create multiple mu receptor subtypes has been demonstrated in animals and humans. Previously, we identified a number of C-terminal variants in mice, rats and human, followed by several N-terminal variants associated with a new upstream exon in mice (exon 11). Behavioral studies in exon 11 knockout mice suggest an important role for the exon 11 variants in the analgesic actions of heroin and morphine-6β-glucuronide, but not morphine or methadone. We now have identified a homologous human exon 11 and three similar human exon 11-associated variants, suggesting conservation of exon 11 and its associated variants across species. hMOR-1i has an additional 93 amino acids at the tip of the N-terminus but is otherwise identical to hMOR-1. When expressed in Chinese hamster ovary cells, the additional 93 amino acids in hMOR-1i had little effect on opioid binding, but significantly altered agonist-induced G-protein activation. hMOR-1G1 and hMOR-1G2 predicted six transmembrane domain variants, similar to those seen in mice. The regional expression of these exon 11-associated variants, as determined by RT-PCR, varied markedly, implying region-specific alternative splicing. The presence of exon 11-associated variants in humans raises questions regarding their potential role in heroin and morphine-6β-glucuronide actions in people as they do in mice.     

Mu receptor binding of some commonly used opioids and their metabolites.

The binding affinity to the mu receptor of some opioids chemically related to morphine and some of their metabolites was examined in rat brain homogenates with 3H-DAMGO. The chemical group at position 6 of the molecule had little effect on binding (e.g. morphine-6-glucuronide Ki = 0.6 nM; morphine = 1.2 nM). Decreasing the length of the alkyl group at position 3 decreased the Ki values (morphine less than codeine less than ethylmorphine less than pholcodine). Analgesics with high clinical potency containing a methoxyl group at position 3 (e.g. hydrocodone, Ki = 19.8 nM) had relatively weak receptor binding, whilst their O-demethylated metabolites (e.g. hydromorphone, Ki = 0.6 nM) had much stronger binding. Many opioids may exert their pharmacological actions predominantly through metabolites.     

Differential sorting of human delta-opioid receptors after internalization by peptide and alkaloid agonists

Desensitization and internalization of G protein-coupled receptors observed after agonist activation are considered two important regulatory processes of receptor transduction. Endogenous human delta-opioid receptors (hDOR) are differentially regulated in terms of desensitization by peptide ([d-Pen2,5]enkephalin (DPDPE) and Deltorphin I) and alkaloid (etorphine) agonists in the neuroblastoma cell line SK-N-BE (Allouche, S., Roussel, M., Marie, N., and Jauzac, P. (1999) Eur. J. Pharmacol. 371, 235-240). In the present study, we examined the role of hDOR internalization and down-regulation in this differential desensitization. Sustained activation by peptides for 30 min caused a marked decrease of both [3H]diprenorphine binding sites and hDOR immunoreactivity, observed in a Western blot, whereas a moderate reduction by 30% was observed after a 30- and 60-min etorphine exposure in binding experiments without opioid receptor degradation. Using fluorescence microscopy, we visualized hDOR internalization promoted by different agonists in SK-N-BE cells expressing FLAG-tagged hDOR. Agonist withdrawal results in a greater recycling process correlated with a stronger hDOR resensitization after etorphine treatment compared with DPDPE or Deltorphin I, as shown in binding, immunocytochemical, and functional experiments. This suggests a distinct sorting of opioid receptors after their internalization. We demonstrated a lysosomal hDOR targeting upon peptides by using chloroquine in binding, Western blot, and immunocytochemical experiments and by colocalization of this receptor with a late endosome marker. In contrast, when the recycling endosome blocker monensin was used, acceleration of desensitization associated with a strong intracellular immunostaining was observed upon etorphine treatment. The possibility of separate endocytic pathways responsible for the differential sorting of hDOR upon peptide and alkaloid ligand exposure was ruled out by binding and immunocytochemical experiments using sucrose hypertonic solution. First, these results showed complex relationships between hDOR internalization/down-regulation and desensitization. Second, we demonstrated for the first time that the same receptor could undergo a distinct sorting after internalization by peptide and alkaloid agonists.     

Agonist-induced internalization and recycling of the human A(3) adenosine receptors: role in receptor desensitization and resensitization

A(3) adenosine receptors have been proposed to play an important role in the pathophysiology of cerebral ischemia with a regimen-dependent nature of the therapeutic effects probably related to receptor desensitization and down-regulation. Here we studied the agonist-induced internalization of human A(3) adenosine receptors in transfected Chinese hamster ovary cells, and then we evaluated the relationship between internalization and signal desensitization and resensitization. Binding of N(6)-(4-amino-3-[(125)I]iodobenzyl)adenosine-5'-N-methyluronamide to membranes from Chinese hamster ovary cells stably transfected with the human A(3) adenosine receptor showed a profile typical of these receptors in other cell lines (K:(D) = 1.3+/-0.08 nM; B(max) = 400+/-28 fmol/mg of proteins). The iodinated agonist, bound at 4 degrees C to whole transfected cells, was internalized by increasing the temperature to 37 degrees C with a rate constant of 0.04+/-0.034 min(-1). Agonist-induced internalization of A(3) adenosine receptors was directly demonstrated by immunogold electron microscopy, which revealed the localization of these receptors in plasma membranes and intracellular vesicles. Moreover, short-term exposure of these cells to the agonist caused rapid desensitization as tested in adenylyl cyclase assays. Subsequent removal of the agonist led to restoration of the receptor function and recycling of the receptors to the cell surface. The rate constant of receptor recycling was 0.02+/-0.0017 min(-1). Blockade of internalization and recycling demonstrated that internalization did not affect signal desensitization, whereas recycling of internalized receptors was implicated in the signal resensitization.     

Morphine-related metabolites differentially activate adenylyl cyclase isozymes after acute and chronic administration

Morphine-3- and morphine-6-glucuronide are morphine’s major metabolites. As morphine-6-glucuronide produces stronger analgesia than morphine, we investigated the effects of acute and chronic morphine glucuronides on adenylyl cyclase (AC) activity. Using COS-7 cells cotransfected with representatives of the nine cloned AC isozymes, we show that AC-I and V are inhibited by acute morphine and morphine-6-glucuronide, and undergo superactivation upon chronic exposure, while AC-II is stimulated by acute and inhibited by chronic treatment. Morphine-3-glucuronide had no effect. The weak opiate agonists codeine and dihydrocodeine are also addictive. These opiates, in contrast to their 3-O-demethylated metabolites morphine and dihydromorphine (formed by cytochrome P450 2D6), demonstrated neither acute inhibition nor chronic-induced superactivation. These results suggest that metabolites of morphine (morphine-6-glucuronide) and codeine/dihydrocodeine (morphine/dihydromorphine) may contribute to the development of opiate addiction.     

Roles of phosphorylation-dependent and -independent mechanisms in the regulation of histamine H2 receptor by G protein-coupled receptor kinase 2

It is widely assumed that G protein-coupled receptor kinase 2 (GRK2)-mediated specific inhibition of G protein-coupled receptors (GPCRs) response involves GRK-mediated receptor phosphorylation followed by β-arrestin binding and subsequent uncoupling from the heterotrimeric G protein. It has recently become evident that GRK2-mediated GPCRs regulation also involves phosphorylation-independent mechanisms. In the present study we investigated whether the histamine H2 receptor (H2R), a Gα(s)-coupled GPCR known to be desensitized by GRK2, needs to be phosphorylated for its desensitization and/or internalization and resensitization. For this purpose we evaluated the effect of the phosphorylating-deficient GRK2K220R mutant on H2R signaling in U937, COS7, and HEK293T cells. We found that although this mutant functioned as dominant negative concerning receptor internalization and resensitization, it desensitized H2R signaling in the same degree as the GRK2 wild type. To identify the domains responsible for the kinase-independent receptor desensitization, we co-transfected the receptor with constructions encoding the GRK2 RGS-homology domain (RH) and the RH or the kinase domain fused to the pleckstrin-homology domain. Results demonstrated that the RH domain of GRK2 was sufficient to desensitize the H2R. Moreover, disruption of RGS functions by the use of GRK2D110A/K220R double mutant, although coimmunoprecipitating with the H2R, reversed GRK2K220R-mediated H2R desensitization. Overall, these results indicate that GRK2 induces desensitization of H2R through a phosphorylation-independent and RGS-dependent mechanism and extends the GRK2 RH domain-mediated regulation of GPCRs beyond Gα(q)-coupled receptors. On the other hand, GRK2 kinase activity proved to be necessary for receptor internalization and the resulting resensitization.     

Immunoaffinity extraction of morphine, morphine-3-glucuronide and morphine-6-glucuronide from blood of heroin victims for simultaneous high-performance liquid chromatographic determination

The development of an immunoaffinity-based extraction method for the determination of morphine and its glucuronides in human blood is described. For the preparation of an immunoadsorber, specific antisera (polyclonal, host: rabbit) against morphine, morphine-3-glucuronide and morphine-6-glucuronide were coupled to 1,1′-carbonyldiimidazole-activated trisacrylgel and used for immunoaffinity extraction of morphine and its glucuronides from coronary blood. The resulting extracts were analysed by HPLC with native fluorescence detection. The mean recoveries from spiked blood samples were 71%, 76% and 88% for morphine, morphine-3-glucuronide and morphine-6-glucuronide, respectively. The limit of detection was 3 ng/g blood and the limit of quantitation was 10 ng/g blood for all three analytes. The results of the analysis of coronary blood samples from 23 fatalities due to heroin are presented.     

Antinociception, tolerance and withdrawal symptoms induced by 7-hydroxymitragynine, an alkaloid from the Thai medicinal herb Mitragyna speciosa

7-Hydroxymitragynine is a potent opioid analgesic alkaloid isolated from the Thai medicinal herb Mitragyna speciosa. In the present study, we investigated the opioid receptor subtype responsible for the analgesic effect of this compound. In addition, we tested whether development of tolerance, cross-tolerance to morphine and naloxone-induced withdrawal signs were observed in chronically 7-hydroxymitragynine-treated mice. Subcutaneous (s.c.) administration of 7-hydroxymitragynine produced a potent antinociceptive effect mainly through activation of mu-opioid receptors. Tolerance to the antinociceptive effect of 7-hydroxymitragynine developed as occurs to morphine. Cross-tolerance to morphine was evident in mice rendered tolerant to 7-hydroxymitragynine and vice versa. Naloxone-induced withdrawal signs were elicited equally in mice chronically treated with 7-hydroxymitragynine or morphine. 7-Hydroxymitragynine exhibited a potent antinociceptive effect based on activation of mu-opioid receptors and its morphine-like pharmacological character, but 7-hydroxymitragynine is structurally different from morphine. These interesting characters of 7-hydroxymitragynine promote further investigation of it as a novel lead compound for opioid studies.     

The role of opioid receptor phosphorylation and trafficking in adaptations to persistent opioid treatment

Mu-opioid receptor activation underpins clinical analgesia and is the central event in the abuse of narcotics. Continued opioid use produces tolerance to the acute effects of the drug and adaptations that lead to physical and psychological dependence. Continued mu-receptor signaling provides the engine for these adaptations, with most evidence suggesting that chronic agonist treatment produces only limited alterations in primary mu-opioid receptor signaling. Here we examine agonist regulation of mu-opioid receptor function, and whether this is altered by chronic treatment. Receptor phosphorylation is thought to be the key initial event in agonist regulation of the mu-opioid receptor, providing a signal for acute receptor desensitization and also subsequent receptor resensitization. Morphine appears to produce qualitatively and quantitatively different mu-receptor phosphorylation than other agonists, but the consequences of this remain obscure, at least in neurons. There is no evidence that agonist-induced mu-opioid receptor phosphorylation changes in chronically morphine-treated animals, although receptor regulation appears to be altered. Thus, as receptor phosphorylation and resensitization appear to maintain continued signaling through the mu-opioid receptor, these two events are crucial in facilitating adaptations to chronic opioid treatment, and the possibility that agonist-specific phosphorylation can contribute to the development of different adaptations remains open.     

Membrane glycoprotein M6a interacts with the micro-opioid receptor and facilitates receptor endocytosis and recycling

Using a yeast two-hybrid screen, the neuronal membrane glycoprotein M6a, a member of the proteolipid protein family, was identified to be associated with the mu-opioid receptor (MOPr). Bioluminescence resonance energy transfer and co-immunoprecipitation experiments confirmed that M6a interacts agonist-independently with MOPr in human embryonic kidney 293 cells co-expressing MOPr and M6a. Co-expression of MOPr with M6a, but not with M6b or DM20, exists in many brain regions, further supporting a specific interaction between MOPr and M6a. After opioid treatment M6a co-internalizes and then co-recycles with MOPr to cell surface in transfected human embryonic kidney 293 cells. Moreover, the interaction of M6a and MOPr augments constitutive and agonist-dependent internalization as well as the recycling rate of mu-opioid receptors. On the other hand, overexpression of a M6a-negative mutant prevents mu-opioid receptor endocytosis, demonstrating an essential role of M6a in receptor internalization. In addition, we demonstrated the interaction of M6a with a number of other G protein-coupled receptors (GPCRs) such as the delta-opioid receptor, cannabinoid receptor CB1, and somatostatin receptor sst2A, suggesting that M6a might play a general role in the regulation of certain GPCRs. Taken together, these data provide evidence that M6a may act as a scaffolding molecule in the regulation of GPCR endocytosis and intracellular trafficking.     

Involvement of mitogen-activated protein kinase in agonist-induced phosphorylation of the mu-opioid receptor in HEK 293 cells

Agonist exposure of many G protein-coupled receptors stimulates an activation of extracellular signal-regulated protein kinases (ERKs) 1 and 2, members of the mitogen-activated protein kinase (MAPK) family. Here, we show that treatment of human embryonic kidney (HEK) 293 cells stably transfected to express the rat micro-opioid receptor (MOR1) with [D-Ala2,MePhe4,Gly5-ol]enkephalin (DAMGO) stimulated a rapid and transient (3-5-min) activation and nuclear translocation of MAPK. Exposure of these cells to the MAPK kinase 1 inhibitor PD98059 not only prevented MAPK activation but also inhibited homologous desensitization of the mu-opioid receptor. We have therefore determined the effect of PD98059 on agonist-induced mu-receptor phosphorylation. DAMGO stimulated a threefold increase in MOR1 phosphorylation within 20 min that could be reversed by the antagonist naloxone. PD98059 produced a dose-dependent inhibition of agonist-promoted mu-receptor phosphorylation with an IC50 of 20 microM. DAMGO also induced MOR1 internalization that peaked at 30 min. Confocal microscopy revealed that DAMGO-induced MOR1 internalization was also largely inhibited in the presence of PD98059. U0126, another chemically unrelated inhibitor of the MAPK cascade, mimicked the effect of PD98059 on mu-receptor phosphorylation and desensitization. MOR1 itself, however, appears to be a poor substrate for MAPK because mu-receptors immunoprecipitated from stably transfected HEK 293 cells were not phosphorylated by exogenous ERK 2 in vitro. The fact that morphine also triggered MAPK activation but did not induce MOR1 internalization indicates that receptor internalization was not required for MOR1-mediated mitogenic signaling. We conclude that MOR1 stimulates a rapid and intemalization-independent MAPK activation. Activation of the MAPK cascade in turn may not only relay mitogenic signals to the nucleus but also trigger initial events leading to phosphorylation and desensitization of the mu-opioid receptor.     

Development and validation of a liquid chromatography-mass spectrometry assay for the determination of opiates and cocaine in meconium

A procedure based on liquid chromatography-mass spectrometry (LC-MS) is described for determination of 6-monoacetylmorphine, morphine, morphine-3-glucuronide, morphine-6-glucuronide, codeine, cocaine, benzoylecgonine and cocaethylene in meconium using nalorfine as the internal standard. The analytes are initially extracted from the matrix by methanol (6-monoacetylmorphine, morphine, codeine, cocaine, benzoylecgonine and cocaethylene) or 0.01 M ammonium hydrogen carbonate buffer (morphine-3-glucuronide, morphine-6-glucuronide). Subsequently a solid-phase extraction with Bondelut Certify columns (6-monoacetylmorphine, morphine, codeine, cocaine, benzoylecgonine and cocaethylene) or ethyl solid-phase extraction columns (morphine-3-glucuronide, morphine-6-glucuronide) was applied. Chromatography was performed on a C(8) reversed-phase column using a gradient of acetic acid 1%-acetonitrile as a mobile phase. Analytes were determined in LC-MS single ion monitoring mode with atmospheric pressure ionisation-electrospray (ESI) interface. The method was validated in the range 0.005-1.00 microg/g using 1 g of meconium per assay and applied to analysis of meconium in newborns to assess fetal exposure to opiates and cocaine.     

Synthesis and in vitro biological evaluation of a carbon glycoside analogue of morphine-6-glucuronide

Attachment of a glucose moiety to 6-beta-aminomorphine afforded compound 3, where the glucose moiety was linked to the C-6 nitrogen atom by a two-carbon bridge. The synthesis of 3 was accomplished in eight steps from 3-triisopropylsilyl-6-beta-aminomorphine and 2,3,4,6-tetra-O-benzyl-D-glucose. The C-glycoside 3 was prepared with the objective of examining a metabolically stable analogue of morphine-6-glucuronide and determining the potency and selectivity of opioid receptor binding. Competition binding assays showed that 3 bound to the mu opioid receptor with a Ki value of 3.5 nM. The C-glycoside 3 exhibited delta/mu and kappa/mu selectivity ratios of 76 and 165, respectively. The synthetic intermediate (i.e., benzyl precursor, compound 11) bound to the mu opioid receptor with a Ki value of 0.5 nM, was less selective for the mu opioid receptor. The [35S]GTPgammaS assay was used to evaluate the functional properties of compounds 3 and 11. Compound 3 was determined to be a full agonist at the mu opioid receptor, whereas compound 11 was found to be a partial agonist. Compound 3 was determined to be very stable in the presence of human liver S9, and rat and monkey liver microsomes: no detectable loss of 3 was observed up to 90 min. Compound 3 was also very stable at pH 2 and pH 7.4, suggesting that 3 possessed properties for sustained duration of action.     

Differential effects of chronic agonist administration on mu-opioid receptor- and muscarinic receptor-regulated adenylate cyclase in rat striatal neurons.

In cultured rat striatal neurons exposed to 10 microM morphine or oxotremorine for 24 hours, we observed an increased (about 30%) dopamine D1 receptor-stimulated cyclic AMP production, whereas no desensitization of mu-opioid receptor or muscarinic cholinergic receptor was found. However, whereas upregulation of dopamine D1 receptor-stimulated adenylate cyclase activity upon 7 days morphine exposure was at least as pronounced as observed after 24 hours of exposure to the opioid, this adaptive phenomenon was virtually absent following one week of oxotremorine treatment. This reduced adenylate cyclase sensitization upon 7 days oxotremorine exposure appeared to coincide with desensitization of muscarinic cholinergic receptors. A possible role of the resistance of mu receptors to desensitization and the (resulting) upregulation of the neuronal adenylate cyclase system upon chronic receptor activation in the development of opiate tolerance and dependence is suggested.