6-O-(2-[18F]Fluoroethyl)-6-O-Desmethyl-Diprenorphine ([18F]FE-DPN) Preferentially Binds to Mu Opioid Receptors In Vivo.

PURPOSE: 6-O-(2-[18F]Fluoroethyl)-6-O-desmethyl-diprenorphine ([18F]FE-DPN) is regarded as a non-selective opioid receptor radiotracer. PROCEDURE: Here, we report the first characterization of [18F]FE-DPN synthesized from the novel precursor, 6-O-(2-tosyloxyethoxy)-6-O-desmethyl-3-O-trityl-diprenorphine (TE-TDDPN), using a one-pot, two-step nucleophilic radiosynthesis to image opioid receptors in rats and mice using positron emission tomography. RESULTS: We also show that [18F]FE-DPN and [3H]DPN exhibit negligible brain uptake in mu opioid receptor (MOR) knockout mice. CONCLUSIONS: Taken together with prior findings, our results suggest that [18F]FE-DPN and [3H]DPN preferentially bind to MOR in rodents in vivo.

Drug Binding Poses Relate Structure with Efficacy in the µ Opioid Receptor.

The µ-opioid receptor (MOPr) is a clinically important G protein-coupled receptor that couples to Gi/o proteins and arrestins. At present, the receptor conformational changes that occur following agonist binding and activation are poorly understood. This study has employed molecular dynamics simulations to investigate the binding mode and receptor conformational changes induced by structurally similar opioid ligands of widely differing intrinsic agonist efficacy, norbuprenorphine, buprenorphine, and diprenorphine. Bioluminescence resonance energy transfer assays for Gi activation and arrestin-3 recruitment in human embryonic kidney 293 cells confirmed that norbuprenorphine is a high efficacy agonist, buprenorphine a low efficacy agonist, and diprenorphine an antagonist at the MOPr. Molecular dynamics simulations revealed that these ligands adopt distinct binding poses and engage different subsets of residues, despite sharing a common morphinan scaffold. Notably, norbuprenorphine interacted with sodium ion-coordinating residues W2936.48 and N1503.35, whilst buprenorphine and diprenorphine did not. Principal component analysis of the movements of the receptor transmembrane domains showed that the buprenorphine-bound receptor occupied a distinct set of conformations to the norbuprenorphine-bound receptor. Addition of an allosteric sodium ion caused the receptor and ligand to adopt an inactive conformation. The differences in ligand-residue interactions and receptor conformations observed here may underlie the differing efficacies for cellular signalling outputs for these ligands.

Imaging endogenous opioid peptide release with [11C]carfentanil and [3H]diprenorphine: influence of agonist-induced internalization.

Understanding the cellular processes underpinning the changes in binding observed during positron emission tomography neurotransmitter release studies may aid translation of these methodologies to other neurotransmitter systems. We compared the sensitivities of opioid receptor radioligands, carfentanil, and diprenorphine, to amphetamine-induced endogenous opioid peptide (EOP) release and methadone administration in the rat. We also investigated whether agonist-induced internalization was involved in reductions in observed binding using subcellular fractionation and confocal microscopy. After radioligand administration, significant reductions in [(11)C]carfentanil, but not [(3)H]diprenorphine, uptake were observed after methadone and amphetamine pretreatment. Subcellular fractionation and in vitro radioligand binding studies showed that amphetamine pretreatment only decreased total [(11)C]carfentanil binding. In vitro saturation binding studies conducted in buffers representative of the internalization pathway suggested that µ-receptors are significantly less able to bind the radioligands in endosomal compared with extracellular compartments. Finally, a significant increase in µ-receptor-early endosome co-localization in the hypothalamus was observed after amphetamine and methadone treatment using double-labeling confocal microscopy, with no changes in δ- or κ-receptor co-localization. These data indicate carfentanil may be superior to diprenorphine when imaging EOP release in vivo, and that alterations in the ability to bind internalized receptors may be a predictor of ligand sensitivity to endogenous neurotransmitter release.

Differential activation of pregnane X receptor and constitutive androstane receptor by buprenorphine in primary human hepatocytes and HepG2 cells.

Buprenorphine is a partial µ-opioid receptor agonist used for the treatment of opioid dependence that has several advantages over methadone. The principal route of buprenorphine disposition has been well established; however, little is known regarding the potential for buprenorphine to influence the metabolism and clearance of other drugs by affecting the expression of drug-metabolizing enzymes (DMEs). Here, we investigate the effects of buprenorphine on the activation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR), as well as the induction of DMEs, in both HepG2 cells and human primary hepatocytes (HPHs). In HepG2 cells, buprenorphine significantly increased human PXR-mediated CYP2B6 and CYP3A4 reporter activities. CYP2B6 reporter activity was also enhanced by buprenorphine in HepG2 cells cotransfected with a chemical-responsive human CAR variant. Real-time reverse transcription-polymerase chain reaction analysis revealed that buprenorphine strongly induced CYP3A4 expression in both PXR- and CAR-transfected HepG2 cells. However, treatment with the same concentrations of buprenorphine in HPHs resulted in literally no induction of CYP3A4 or CYP2B6 expression. Further studies indicated that buprenorphine could neither translocate human CAR to the nucleus nor activate CYP2B6/CYP3A4 reporter activities in transfected HPHs. Subsequent experiments to determine whether the differential response was due to buprenorphine's metabolic stability revealed a dramatically differential rate of elimination for buprenorphine between HPHs and HepG2 cells. Taken together, these studies indicate that metabolic stability of buprenorphine defines the differential induction of DMEs observed in HepG2 and HPHs, and the results obtained from PXR and CAR reporter assays in immortalized cell line require cautious interpretation.

Kappa2 opioid receptor subtype binding requires the presence of the DOR-1 gene.

Over the past several years substantial evidence has documented that opioid receptor homo- and heterodimers form in cell lines expressing one or more of the opioid receptors. We used opioid receptor knockout mice to determine whether in vivo pharmacological characteristics of kappa1 and kappa2 opioid receptors changed following knockout of specific opioid receptors. Using displacement of the general opioid ligand diprenorphine, we observed that occupancy or knockout of the DOR-1 gene increases the binding density of kappa1 receptors and eliminates kappa2 receptors in crude membrane preparations while the total density of kappa opioid binding sites is unchanged. Further, the analgesic potency of U69,593 in cumulative dose response curves is enhanced in mice lacking the DOR-1 gene. These results demonstrate that the DOR-1 gene is required for the expression of the kappa2 opioid receptor subtype and are consistent with the possibility that a KOR-1/DOR-1 heterodimer mediates kappa2 pharmacology.

Evidence for modulation of opioidergic activity in central vestibular processing: A [(18)F] diprenorphine PET study.

Animal and functional imaging studies had identified cortical structures such as the parieto-insular vestibular cortex, the retro-insular cortex, or the anterior cingulate cortex belonging to a vestibular cortical network. Basic animal studies revealed that endorphins might be important transmitters involved in cerebral vestibular processing. The aim of the present study was therefore to analyse whether the opioid system is involved in vestibular neurotransmission of humans or not. Changes in opioid receptor availability during caloric air stimulation of the right ear were studied with [(18)F] Fluoroethyl-diprenorphine ([(18)F]FEDPN) PET scans in 10 right-handed healthy volunteers and compared to a control condition. Decrease in receptor availability to [(18)F]FEDPN during vestibular stimulation in comparison to the control condition was significant at the right posterior insular cortex and the postcentral region indicating more endogenous opioidergic binding in these regions during stimulation. These data give evidence that the opioidergic system plays a role in the right hemispheric dominance of the vestibular cortical system in right-handers.

Effects of aquaporin4 deficiency on opioid receptors characteristics in naive and chronic morphine-treated mice.

Our previous study indicated that aquaporin4 (AQP4) deficiency potentiated morphine analgesia, but attenuated tolerance and physical dependence induced by chronic exposure to morphine. However, the mechanisms remained to be explored. In the present study, effects of AQP4 deficiency on opioid receptor characteristics were investigated by [(3)H]-diprenorphine binding assays. In basal condition, the K(d) values of opioid receptors increased from 0.27+/-0.03 nM in wild-type mice to 0.44+/-0.04 nM in AQP4 deficient mice. Meanwhile, the density (B(max) values) of opioid receptors increased from 0.40+/-0.04 pmol/mg protein in wild-type mice to 0.66+/-0.04 pmol/mg protein in AQP4 deficient mice. After chronic morphine treatment, the affinity of opioid receptors decreased in wild-type mice, in which the K(d) value increased from 0.27+/-0.03 nM to 0.40+/-0.04 nM, while no change in the density of opioid receptors was observed. In AQP4 knockout mice, the effects of chronic morphine treatment on opioid receptors were similar to that in wild-type mice, in which the K(d) values increased from 0.44+/-0.04 nM to 0.64+/-0.08 nM, whereas the density had no significant change. Taken together, at the first time, we found that AQP4 deficiency decreased the affinity and increased the density of opioid receptors. Additionally, AQP4 deficiency did not affect chronic morphine-induced alterations of opioid receptor characteristics.

Increased opioid release in specific brain areas in animals exposed to prenatal morphine and emotional stress later in life.

Prenatal morphine treatment and emotional stress both have been shown to increase sensitivity to reward-related behaviors. It has been postulated that this increased sensitivity to rewarding stimuli may be the result of an enhanced release of endogenous opioids. In the present study, in vivo autoradiography was employed to investigate the endogenous opioid release in specific brain areas in rats. Pregnant animals were exposed to morphine or saline from day 8 of gestation till birth. Development of pups was monitored and play behavior was tested on postnatal day 21. Adult rats were exposed to repeated emotional stress or control treatment for five consecutive days and tested in a small open field 5 days later. [(3)H]-Diprenorphine was injected following this test to investigate endogenous opioid release. Prenatal morphine treatment increased play behavior and endogenous opioid release in a number of cortical and subcortical brain areas after being subjected to an open field challenge later in life. Emotional stress exposure increased locomotor activity in the open field irrespective of the type of prenatal treatment and increased endogenous opioid release in some specific brain areas. It is suggested that the increased release of endogenous opioids in the substantia nigra, the piriform cortex and the septum observed after both types of treatments is related to the increased sensitivity to reward.

Activation of the mu opioid receptor involves conformational rearrangements of multiple transmembrane domains.

We previously demonstrated that D3.49(164)Y or T6.34(279)K mutation in the rat mu opioid receptor (MOPR) resulted in agonist-independent activation. Here, we identified the cysteine(s) within the transmembrane domains (TMs) of the D3.49(164)Y mutant that became accessible in the binding-site crevice by use of methanethiosulfonate ethylammonium (MTSEA) and inferred conformational changes associated with receptor activation. While the C7.38(321)S mutant was insensitive to MTSEA, the D3.49(164)Y/C7.38(321)S mutant showed similar sensitivity as the D3.49(164)Y, suggesting that, in the D3.49(164)Y mutant, C7.38(321) becomes inaccessible while other cysteines are accessible in the binding-site crevice. Each of the other seven cysteines in the TMs was mutated to serine on the background of D3.49(164)Y/C7.38(321)S, and the resulting triple mutants were evaluated for [3H]diprenorphine and [d-Ala2,NMe-Phe4,Gly5-ol]-enkephalin (DAMGO) binding and effect of MTSEA on [3H]diprenorphine binding. The D3.49(164)Y/C7.38(321)S mutant and the triple mutants, except the C6.47(292)S triple mutant, retained similar affinities for [3H]diprenorphine and DAMGO as the D3.49(164)Y mutant. The second-order rate constants for MTSEA reactions showed that C3.44(159)S, C4.48(190)S, C5.41(235)S, and C7.47(330)S significantly reduced sensitivity to MTSEA, compared with the D3.49(164)Y/C7.38(321)S. These results suggest that the four cysteines may be rotated and/or tilted to become accessible. While the D3.49(164)Y/C7.38(321)S was similarly sensitive to MTSEA as the D3.49(164)Y mutant, the T6.34(279)K/C7.38(321)S was much less sensitive to MTSEA than the T6.34(279)K mutant, suggesting that the two constitutively active mutants assume different conformations and/or possess different dynamic properties. Molecular models of the MOPR monomer and homodimer, using the crystal structures of rhodopsin, the beta2-adrenergic receptor, and the ligand-free opsin, which contains several features characteristic of the active state, were employed to analyze these experimental results in a structural context.

Opioid receptor PET reveals the psychobiologic correlates of reward processing.

UNLABELLED: Little is known about the neurobiologic correlates of human personality. On the basis of the key role of the central opioidergic system in addiction and substance abuse, we investigated the relationship between certain personality traits that are supposed to be relevant in addiction and the opioid receptor status in healthy subjects. METHODS: We investigated 23 healthy male volunteers who were extensively clinically tested to exclude substance abuse. All of the subjects underwent 1 PET scan with the subtype-nonselective opioidergic radioligand 18F-fluoroethyl-diprenorphine under resting conditions without sensory or cognitive stimulation. Subsequently, the subjects were psychologically tested for the personality traits novelty seeking, harm avoidance, reward dependence, and persistence, according to Cloninger's biosocial model of personality. The binding potential (BP) as a parameter of regional cerebral opioid receptor availability was computed by means of the modified Logan plot using the occipital cortex as a reference region. Further imaging data analysis was performed using statistical parametric mapping; after stereotactic normalization, the correlations were calculated between the regional BP and the psychologic scores on a voxel-by-voxel basis. RESULTS: The correlation analysis between personality dimensions and opioid receptor availability showed a significant (P < 0.001) positive correlation between the scores of reward dependence and the BP of the bilateral ventral striatum with nucleus accumbens (z scores, 4.52 and 4.33, respectively). The additionally performed region-of-interest-based correlation analysis yielded correlation coefficients of r = 0.84 and r = 0.81 for the left and right ventral striata, respectively. No further significant correlations were detectable between the other personality dimensions and cerebral opioid receptor binding. CONCLUSION: In healthy subjects, personality traits, which might be predisposing for addictive behavior, are correlated to the opioidergic neurotransmission in core structures of the human reward system.

CXCR2 chemokine receptor antagonism enhances DOP opioid receptor function via allosteric regulation of the CXCR2-DOP receptor heterodimer.

Opioid agonists have a broad range of effects on cells of the immune system, including modulation of the inflammatory response, and opioid and chemokine receptors are co-expressed by many white cells. Hetero-oligomerization of the human DOP opioid and chemokine CXCR2 receptors could be detected following their co-expression by each of co-immunoprecipitation, three different resonance energy transfer techniques and the construction of pairs of individually inactive but potentially complementary receptor G-protein alpha subunit fusion proteins. Although DOP receptor agonists and a CXCR2 antagonist had no inherent affinity for the alternative receptor when either receptor was expressed individually, use of cells that expressed a DOP opioid receptor construct constitutively, and in which expression of a CXCR2 receptor construct could be regulated, demonstrated that the CXCR2 antagonist enhanced the function of DOP receptor agonists only in the presence of CXCR2. This effect was observed for both enkephalin- and alkaloid-based opioid agonists, and the effective concentrations of the CXCR2 antagonist reflected CXCR2 receptor occupancy. Entirely equivalent results were obtained in cells in which the native DOP opioid receptor was expressed constitutively and in which expression of the isolated CXCR2 receptor could be induced. These results indicate that a CXCR2 receptor antagonist can enhance the function of agonists at a receptor for which it has no inherent direct affinity by acting as an allosteric regulator of a receptor that is a heterodimer partner for the CXCR2 receptor. These results have novel and important implications for the development and use of small-molecule therapeutics.

Novel trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidines as mu opioid receptor antagonists with improved opioid receptor selectivity profiles.

A series of N-substituted trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidines, mu opioid receptor antagonists, analogs of alvimopan, were prepared using solid phase methodology. This study led to the identification of a highly selective mu opioid receptor antagonist, which interacts selectively with mu peripheral receptors.

Endogenous heptapeptide Met-enkephalin-Gly-Tyr binds differentially to duplicate delta opioid receptors from zebrafish.

Met-enkephalin-Gly-Tyr (MEGY) is an endogenous peptide that binds to opioid sites in zebrafish and in rat brain homogenates. The aim of this work is to characterize the binding profile of this opioid ligand on two duplicate delta receptors from zebrafish, ZFOR1 and ZFOR4. Our results show that, while ZFOR1 presents one single binding site for [(3)H]-MEGY (K(D)=4.0+/-0.4 nM), the experimental data from ZFOR4 fit better to the two-site binding model (K(D1)=0.8+/-0.2 nM and K(D2)=30.2+/-10.2 nM). Two other MEGY synthetic analogues, (D-Ala(2))-MEGY and (D-Ala(2), Val(5))-MEGY were also prepared and tested, together with the original peptide MEGY and other opioid ligands, in competition binding assays. While these peptides presented K(i) values on the nanomolar range when using [(3)H]-MEGY as radioligand, these parameters were two orders higher in competition binding assays with the antagonist [(3)H]-diprenorphine. Functional [(35)S]GTPgammaS stimulation analysis has revealed that these two receptors can be activated by several opioid agonists. Our results prove that although the MEGY peptide acts as an agonist on ZFOR1 and ZFOR4, there are subtle pharmacological differences between these two delta opioid receptors from zebrafish.

Agonist treatment did not affect association of mu opioid receptors with lipid rafts and cholesterol reduction had opposite effects on the receptor-mediated signaling in rat brain and CHO cells.

Lipid rafts are small cholesterol- and glycosphingolipid-enriched membrane subdomains. Here we compared the mu opioid receptor (MOR)-lipid rafts relationship in the rat brain, where neurons have non-caveolae rafts, and in CHO cells stably transfected with HA-rat MOR (CHO-HA-rMOR), which are enriched in caveolae. Membranes of rat caudate putamen (CPu) and thalamus or CHO-HA-rMOR cells were homogenized, sonicated in a detergent-free 0.5 M Na(2)CO(3) buffer and fractionated through sucrose density gradients. Western blot and [(3)H]diprenorphine binding showed that approximately 70% of MOR in CHO-HA-rMOR was present in low-density (5-20% sucrose) fractions enriched in cholesterol and/or ganglioside M1 (GM1) (lipid rafts) in plasma membranes, whereas about 70% and 45% of MOR in CPu and thalamus, respectively, were associated with lipid rafts. Incubation with a saturating concentration of etorphine or morphine at 37 degrees C for 30 min failed to change the MOR location in rafts in CHO-HA-rMOR, indicating that the internalized MOR does not move out of rafts, in contrast to the delta opioid receptor. In vivo, rafts association of MOR in CPu and thalamus was not affected significantly in rats implanted with two 75-mg morphine pellets for 72 h. In addition, cholesterol reduction by methyl-beta-cyclodextrin (MCD) disrupted rafts and shifted MOR to higher density fractions in both CHO-HA-rMOR and CPu membranes. However, MCD treatment had opposite impacts on MOR signaling in the two tissues: it attenuated MOR-mediated [(35)S]GTPgammaS binding in CPu but enhanced it in CHO-HA-rMOR.

Brain opioid receptor binding in early abstinence from opioid dependence: positron emission tomography study.

BACKGROUND: Although opioid receptor function in humans is clearly reduced during opioid dependence, what happens to the receptor in early abstinence is not understood. AIMS: This study sought to examine changes in opioid receptor availability in early abstinence from opioid dependence. METHOD: Ten people with opioid dependence who had completed in-patient detoxification and 20 healthy controls underwent [11C]-diprenorphine positron emission tomography. Clinical variables were assessed with structured questionnaires. Opioid receptor binding was characterised as the volume of distribution of [11C]-diprenorphine using a template of predefined brain volumes and an exploratory voxel-by-voxel analysis. RESULTS: Compared with controls, participants with opioid dependence had increased [11C]-diprenorphine binding in the whole brain and in 15 of the 21 a priori regions studied. CONCLUSIONS: This study suggests that opioid receptor binding is increased throughout the brain in early abstinence from dependent opioid use. These data complement the findings in cocaine and alcohol dependence.

Low sensitivity of the positron emission tomography ligand [11C]diprenorphine to agonist opiates.

Previously, we reported minimal opioid receptor occupancy following a clinical dose of the micro-opioid agonist, methadone, measured in vivo using positron emission tomography (PET) with [(11)C]diprenorphine and subsequently used rats to obtain experimental data in support of a high receptor reserve hypothesis (Melichar et al., 2005). Here, we report on further preclinical studies investigating opioid receptor occupancy with oxycodone (micro- and kappa-receptor agonist), morphine (micro-receptor agonist), and buprenorphine (partial agonist at the micro-receptor and antagonist at the delta- and kappa-receptors), each given at antinociceptive doses. In vivo binding of [(11)C]diprenorphine was not significantly reduced after treatment with the full agonists but was reduced by approximately 90% by buprenorphine. In addition, given that [(11)C]diprenorphine is a non-subtype-specific PET tracer, there was no regional variation that might feasibly be interpreted as due to differences in opioid subtype distribution. The data support minimal competition between the high-efficacy agonists and the non-subtype-selective antagonist radioligand and highlight the limitations of [(11)C]diprenorphine PET to monitor in vivo occupancy. Alternative means may be needed to address clinical issues regarding opioid receptor occupancy that are required to optimize treatment strategies.

Salvinorin A: allosteric interactions at the mu-opioid receptor.

Salvinorin A [(2S,4aR,6aR,7R,9S,10aS,10bR)-9-(acetyloxy)-2-(3-furanyl)-dodecahydro-6a,10b-dimethyl-4,10-dioxo-2h-naphtho[2,1-c]pyran-7-carboxylic acid methyl ester] is a hallucinogenic kappa-opioid receptor agonist that lacks the usual basic nitrogen atom present in other known opioid ligands. Our first published studies indicated that Salvinorin A weakly inhibited mu-receptor binding, and subsequent experiments revealed that Salvinorin A partially inhibited mu-receptor binding. Therefore, we hypothesized that Salvinorin A allosterically modulates mu-receptor binding. To test this hypothesis, we used Chinese hamster ovary cells expressing the cloned human opioid receptor. Salvinorin A partially inhibited [(3)H]Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol (DAMGO) (0.5, 2.0, and 8.0 nM) binding with E(MAX) values of 78.6, 72.1, and 45.7%, respectively, and EC(50) values of 955, 1124, and 4527 nM, respectively. Salvinorin A also partially inhibited [(3)H]diprenorphine (0.02, 0.1, and 0.5 nM) binding with E(MAX) values of 86.2, 64, and 33.6%, respectively, and EC(50) values of 1231, 866, and 3078 nM, respectively. Saturation binding studies with [(3)H]DAMGO showed that Salvinorin A (10 and 30 microM) decreased the mu-receptor B(max) and increased the K(d) in a dose-dependent nonlinear manner. Saturation binding studies with [(3)H]diprenorphine showed that Salvinorin A (10 and 40 microM) decreased the mu-receptor B(max) and increased the K(d) in a dose-dependent nonlinear manner. Similar findings were observed in rat brain with [(3)H]DAMGO. Kinetic experiments demonstrated that Salvinorin A altered the dissociation kinetics of both [(3)H]DAMGO and [(3)H]diprenorphine binding to mu receptors. Furthermore, Salvinorin A acted as an uncompetitive inhibitor of DAMGO-stimulated guanosine 5'-O-(3-[(35)S]thio)-triphosphate binding. Viewed collectively, these data support the hypothesis that Salvinorin A allosterically modulates the mu-opioid receptor.

The opioid ligand binding of human mu-opioid receptor is modulated by novel splice variants of the receptor.

The pharmacological actions of morphine and morphine-like drugs, such as heroin, mediate primarily through the mu-opioid receptor (MOR). It has been proposed that the functional diversity of MOR may be related to alternative splicing of the MOR gene. Although a number of MOR mRNA splice variants have been reported, their biological function has been controversial. In this study, two novel splice variants of the human MOR gene were discovered. Splice variants 1 and 2 (here called the SV1 and SV2) retain different portions of intron I. In vitro translation of SV1 and SV2 produced proteins with the predicted molecular weights. The splice variant proteins were identical to the wild-type MOR-1 up to the first transmembrane domains, but were different after the first intracellular loop domains. SV1 and SV2 of hMOR were present in human neuroblastoma NMB cells and human whole brain confirmed by RT-PCR. In a receptor binding assay, cells expressing the SV1 and SV2 do not exhibit binding to [(3)H]diprenorphine. The formations of MOR.SV1 and MOR.SV2 heterodimers were demonstrated by co-immunoprecipitation and bioluminescence resonance energy transfer between MOR and splice variants. Co-transfection of MOR-GFP and SV-DsRed gene showed that MOR and SV protein co-localized at the cytoplasmic membrane. In NMB cells expressing human MOR gene, transfection of SV1 or SV2 reduced binding activity of the endogenous MOR. These data support a potential role of SV1 and SV2 proteins as possible biological modulator of human mu-opioid receptor.

Comparison of the antinociceptive response to morphine and morphine-like compounds in male and female Sprague-Dawley rats.

Male rats are more sensitive to the antinociceptive effects of morphine than female rats. This difference is seen across several rat strains using a variety of nociceptive stimuli. However, the literature in regard to sex differences in antinociceptive responses to mu-opioids other than morphine is less consistent. The present study was designed to examine whether there is a structure-activity rationale that determines which mu-opioids will show a differential antinociceptive response between male and female rats. A series of morphinans closely related in structure to morphine, namely, codeine, heroin, hydrocodone, hydromorphone, oxymorphone, and oxycodone, were examined for their antinociceptive activity in male and female Sprague-Dawley rats and compared with the structurally unrelated mu-opioid agonists methadone and fentanyl. Antinociception was measured by the warm-water tail-withdrawal assay. The results show that morphine is more potent in males compared with females > hydromorphone = hydrocodone = oxymorphone, but there was no observable sex difference in the antinociceptive potency of codeine, heroin, oxycodone, methadone, or fentanyl. The potency to stimulate guanosine 5'-O-(3-[35 S]thio)triphosphate ([35S]GTPgammaS) binding and binding affinity of the various morphinans was compared in rat glioma C6 cells expressing the rat mu-opioid receptor; relative efficacy was also compared by stimulation of [35S]GTPgammaS binding in slices of rat brain thalamus. The presence of a sex difference in antinociceptive responsiveness was not related to drug potency, efficacy, or affinity. Consequently, it is likely that differential metabolism of the opioid, possibly by glucuronidation, determines the presence or absence of a sex difference.

Inhibition of ligand binding to G protein-coupled receptors by arachidonic acid.

Arachidonic acid (AA), released in response to muscarinic acetylcholine receptor (mAChR) stimulation, previously has been reported to function as a reversible feedback inhibitor of the mAChR. To determine if the effects of AA on binding to the mAChR are subtype specific and whether AA inhibits ligand binding to other G protein-coupled receptors (GPCRs), the effects of AA on ligand binding to the mAChR subtypes (M1, M2, M3, M4, and M5) and to the micro-opioid receptor, beta2-adrenergic receptor (beta2-AR), 5-hydroxytryptamine receptor (5-HTR), and nicotinic receptors were examined. AA was found to inhibit ligand binding to all mAChR subtypes, to the beta2-AR, the 5-HTR, and to the micro-opioid receptor. However, AA does not inhibit ligand binding to the nicotinic receptor, even at high concentrations of AA. Thus, AA inhibits several types of GPCRs, with 50% inhibition occurring at 3-25 MuM, whereas the nicotinic receptor, a non-GPCR, remains unaffected. Further research is needed to determine the mechanism by which AA inhibits GPCR function.