Methadone: does it really have low efficacy at μ-opioid receptors?

Rodríguez-Martín, Iván; Braksator, Ellen; Bailey, Christopher; Goodchild, Sam; Marrion, Neil V.; Kelly, Eamonn; Henderson, Graeme

doi:10.1097/wnr.0b013e3282f97b64

Cited by 18 publications

(17 citation statements)

References 13 publications

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“…Many studies have reported the endomorphins as being partial agonists, 18,21 whereas methadone has been reported to be an agonist with a similar efficacy to DAMGO. 11,22,23 The most likely explanation for the discrepancy in our study is that methadone appeared to have a reduced maximal effect because of its propensity to block K channels, including the GIRK channels likely to contribute to the hyperpolarization measured in the present study. 22,24 Conversely, the observation that the maximum effect of the endomorphins was similar to that of well-recognized high-efficacy agonists suggests the presence of some spare receptors in our system, as noted previously with similar cell lines.…”

Section: Discussioncontrasting

confidence: 66%

“…11,22,23 The most likely explanation for the discrepancy in our study is that methadone appeared to have a reduced maximal effect because of its propensity to block K channels, including the GIRK channels likely to contribute to the hyperpolarization measured in the present study. 22,24 Conversely, the observation that the maximum effect of the endomorphins was similar to that of well-recognized high-efficacy agonists suggests the presence of some spare receptors in our system, as noted previously with similar cell lines. 11 It is also possible that the efficacy discrepancies reflect subtle bias in ligand signaling to one pathway over another in different tissues, and it is worth noting that endomorphins have recently been reported to show such bias in assays of β-arrestin recruitment.…”

Section: Discussioncontrasting

confidence: 66%

“…The dye was reconstituted with assay buffer supplied with the kit or with a low-K modification. The standard assay buffer contained (mM) NaCl 145, HEPES 22 4, osmolarity 315 ± 5). The modified buffer was formulated without the addition of 5.33 mM KCl.…”

Section: Membrane Potential Assaymentioning

confidence: 99%

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A Continuous, Fluorescence-based Assay of µ-Opioid Receptor Activation in AtT-20 Cells

Knapman

Santiago

et al. 2013

SLAS Discovery

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Opioids are widely prescribed analgesics, but their use is limited due to development of tolerance and addiction, as well as high variability in individual response. The development of improved opioid analgesics requires high-throughput functional assays to assess large numbers of potential opioid ligands. In this study, we assessed the ability of a proprietary "no-wash" fluorescent membrane potential dye to act as a reporter of µ-opioid receptor (MOR) activation and desensitization via activation of G-protein-coupled inwardly rectifying potassium channels. AtT-20 cells stably expressing mouse MOR were assayed in 96-well plates using the Molecular Devices FLIPR membrane potential dye. Dye emission intensity decreased upon membrane hyperpolarization. Fluorescence decreased in a concentration-dependent manner upon application of a range of opioid ligands to the cells, with high-efficacy agonists producing a decrease of 35% to 40% in total fluorescence. The maximum effect of morphine faded in the continued presence of agonist, reflecting receptor desensitization. The effects of opioids were prevented by prior treatment with pertussis toxin and blocked by naloxone. We have demonstrated this assay to be an effective method for assessing ligand signaling at MOR, which may potentially be scaled up as an additional high-throughput screening technique for characterizing novel opioid ligands.

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Section: Discussioncontrasting

confidence: 66%

Section: Discussioncontrasting

confidence: 66%

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A Continuous, Fluorescence-based Assay of µ-Opioid Receptor Activation in AtT-20 Cells

Knapman

Santiago

et al. 2013

SLAS Discovery

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“…HEK293 cells were maintained at 37°C in 95% O 2 , 5% CO 2 , in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, 10 U/ml penicillin, and 10 mg/ml streptomycin. In addition, the culture medium for the HEK293 cells stably expressing T7-tagged MOPr (Bailey et al, 2003;Rodriguez-Martin et al, 2008) contained 250 g/ml G-418 (Geneticin), a selective antibiotic (PAA, Pasching, Austria).…”

Section: Methodsmentioning

confidence: 99%

μ-Opioid Receptors: Correlation of Agonist Efficacy for Signalling with Ability to Activate Internalization

McPherson

Rivero²,

Baptist³

et al. 2010

Mol Pharmacol

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322

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We have compared the ability of a number of -opioid receptor (MOPr) ligands to activate G proteins with their abilities to induce MOPr phosphorylation, to promote association of arrestin-3 and to cause MOPr internalization. For a model of G protein-coupled receptor (GPCR) activation where all agonists stabilize a single active conformation of the receptor, a close correlation between signaling outputs might be expected. Our results show that overall there is a very good correlation between efficacy for G protein activation and arrestin-3 recruitment, whereas a few agonists, in particular endomorphins 1 and 2, display apparent bias toward arrestin recruitment. The agonist-induced phosphorylation of MOPr at Ser 375 , considered a key step in MOPr regulation, and agonist-induced internalization of MOPr were each found to correlate well with arrestin-3 recruitment. These data indicate that for the majority of MOPr agonists the ability to induce receptor phosphorylation, arrestin-3 recruitment, and internalization can be predicted from their ability as agonists to activate G proteins. For the prototypic MOPr agonist morphine, its relatively weak ability to induce MOPr internalization can be explained by its low agonist efficacy.

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“…In addition to 5-HT 3 receptors, methadone directly interacts with several other ion channels, including hERG K ϩ channels (Katchman et al, 2002;Eap et al, 2007), inwardly rectifying K ϩ channels (Rodriguez-Martin et al, 2008), the NMDA subtype of the glutamate receptor (Ebert et al, 1995;Callahan et al, 2004), and the ␣3␤4 and ␣7 nicotinic receptors (Xiao et al, 2001;Pakkanen et al, 2005). (R)-Methadone, the isomer that preferentially binds to -opioid receptors is also more potent than (S)-methadone as an inhibitor of NMDA receptors (Kristensen et al, Callahan et al, 2004), whereas (S)-methadone has a higher potency than (R)-methadone as an inhibitor of hERG channels (Eap et al, 2007).…”

Section: Discussionmentioning

confidence: 99%

Direct Subunit-Dependent Multimodal 5-Hydroxytryptamine₃Receptor Antagonism by Methadone

Deeb

Sharp²,

Hales

2009

Mol Pharmacol

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Homomeric 5-hydroxytryptamine (5-HT) 3A and heteromeric 5-HT 3AB receptors mediate rapid excitatory responses to serotonin in the central and peripheral nervous systems. The alkaloid morphine, in addition to being a -opioid receptor agonist, is a potent competitive inhibitor of 5-HT 3 receptors. We examined whether methadone, an opioid often used to treat morphine dependence, also exhibited 5-HT 3 receptor antagonist properties. Racemic (R/S)-methadone inhibited currents mediated by human homomeric 5-HT 3A receptors (IC 50 ϭ 14.1 Ϯ 2.5 M). Incorporation of the 5-HT 3B subunit into heteromeric 5-HT 3AB receptors reduced the potency of inhibition by (R/S)-methadone (IC 50 ϭ 41.1 Ϯ 0.9 M). (R/S)-Methadone also increased apparent desensitization of both 5-HT 3 receptor subtypes. The inhibition of the 5-HT 3A receptor was competitive; however, incorporation of the 5-HT 3B subunit caused the appearance of inhibition that was insurmountable by 5-HT. In the absence of rapid desensitization, when dopamine was used as an agonist of 5-HT 3AB receptors, the inhibition by (R/S)-methadone was voltage-dependent. The antagonist and desensitization-enhancing effects of (R/S)-methadone were shared by pure (R)-and (S)-methadone enantiomers, which had similar actions on 5-HT-evoked currents mediated by 5-HT 3 receptors. However, (R)-methadone exhibited a larger voltage-dependent inhibition of dopamine-evoked currents mediated by 5-HT 3AB receptors than did (S)-methadone. Inhibition of 5-HT 3A receptors by (R/S)-methadone was not influenced by voltage. Thus, methadone displays multimodal subunit-dependent antagonism of 5-HT 3 receptors.The 5-hydroxytryptamine (5-HT) type 3 receptor is a ligand-gated cation channel that mediates rapid serotonergic excitatory synaptic transmission (Sugita et al., 1992). It contains binding sites for 5-HT and several allosteric modulators. The 5-HT 3 receptor is a member of the Cys-loop superfamily of pentameric receptors, which also includes the nicotinic acetylcholine, ␥-aminobutyric acid, and glycine receptors, and the Zn 2ϩ -activated ion channel (Barnes et al., 2009). The 5-HT 3A subunit forms homomeric receptors and can also combine into heteromeric receptors with the 5-HT 3B

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Methadone: does it really have low efficacy at μ-opioid receptors?

Cited by 18 publications

References 13 publications

A Continuous, Fluorescence-based Assay of µ-Opioid Receptor Activation in AtT-20 Cells

A Continuous, Fluorescence-based Assay of µ-Opioid Receptor Activation in AtT-20 Cells

μ-Opioid Receptors: Correlation of Agonist Efficacy for Signalling with Ability to Activate Internalization

Direct Subunit-Dependent Multimodal 5-Hydroxytryptamine₃Receptor Antagonism by Methadone

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Methadone: does it really have low efficacy at μ-opioid receptors?

Cited by 18 publications

References 13 publications

A Continuous, Fluorescence-based Assay of µ-Opioid Receptor Activation in AtT-20 Cells

A Continuous, Fluorescence-based Assay of µ-Opioid Receptor Activation in AtT-20 Cells

μ-Opioid Receptors: Correlation of Agonist Efficacy for Signalling with Ability to Activate Internalization

Direct Subunit-Dependent Multimodal 5-Hydroxytryptamine3Receptor Antagonism by Methadone

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Direct Subunit-Dependent Multimodal 5-Hydroxytryptamine₃Receptor Antagonism by Methadone