damgo  (Tocris)

 
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  • 94
    Name:
    DAMGO
    Description:
    Selective μ agonist
    Catalog Number:
    1171
    Product Aliases:
    DAGO
    Price:
    None
    Category:
    μ Opioid Receptor Agonists μ Opioid Receptors Opioid Receptors 7 TM Receptors Pharmacology
    Formula:
    [D-Ala2, NMe-Phe4, Gly-ol5]-enkephalin
    Buy from Supplier


    Structured Review

    Tocris damgo
    DAMGO
    Selective μ agonist
    https://www.bioz.com/result/damgo/product/Tocris
    Average 94 stars, based on 17 article reviews
    Price from $9.99 to $1999.99
    damgo - by Bioz Stars, 2020-09
    94/100 stars

    Images

    1) Product Images from "μ and κ Opioid receptor distribution in the monogamous titi monkey (Callicebus cupreus): Implications for social behavior and endocrine functioning"

    Article Title: μ and κ Opioid receptor distribution in the monogamous titi monkey (Callicebus cupreus): Implications for social behavior and endocrine functioning

    Journal: Neuroscience

    doi: 10.1016/j.neuroscience.2015.01.023

    Selective and non-specific binding for MOR and KOR autoradiography. 1A) Selective binding of MOR using [ 3 H]DAMGO blocking DOR and KOR by co-incubating with DPDPE and U69,593, respectively 1B) Non-specific binding of MOR with [ 3 H]DAMGO and co-incubating
    Figure Legend Snippet: Selective and non-specific binding for MOR and KOR autoradiography. 1A) Selective binding of MOR using [ 3 H]DAMGO blocking DOR and KOR by co-incubating with DPDPE and U69,593, respectively 1B) Non-specific binding of MOR with [ 3 H]DAMGO and co-incubating

    Techniques Used: Binding Assay, Autoradiography, Blocking Assay

    2) Product Images from "Regional Differences in Mu and Kappa Opioid Receptor G-protein Activation in Brain in Male and Female Prairie Voles"

    Article Title: Regional Differences in Mu and Kappa Opioid Receptor G-protein Activation in Brain in Male and Female Prairie Voles

    Journal: Neuroscience

    doi: 10.1016/j.neuroscience.2015.10.047

    Representative autoradiograms of mu- and kappa-stimulated [ 35 S]GTPγS binding Coronal sections are shown of female prairie vole brains at five brain levels (top to bottom); forebrain, mid-striatum, anterior diencephalon, posterior diencephalon, and hindbrain. Sections were incubated as described in Experimental Procedures with 0.05 nM [ 35 S]GTPγS and 2 mM GDP with 3 μM DAMGO or 1 μM U50,488H.
    Figure Legend Snippet: Representative autoradiograms of mu- and kappa-stimulated [ 35 S]GTPγS binding Coronal sections are shown of female prairie vole brains at five brain levels (top to bottom); forebrain, mid-striatum, anterior diencephalon, posterior diencephalon, and hindbrain. Sections were incubated as described in Experimental Procedures with 0.05 nM [ 35 S]GTPγS and 2 mM GDP with 3 μM DAMGO or 1 μM U50,488H.

    Techniques Used: Binding Assay, Incubation

    Pharmacological specificity of DAMGO- and U50,488H-stimulated [ 35 S]GTPγS binding in vole brain Sections of male prairie vole forebrain were incubated as described in Experimental Procedures with 0.05 nM [ 35 S]GTPγS and 2 mM GDP with DAMGO (3 μM) as a mu agonist or U50,488H (1 μM) as a kappa agonist, with and without the mu antagonist naloxone (0.1 μM) or the kappa antagonist nor-BNI (0.1 μM). Similar results were obtained at 3 additional coronal levels (not shown).
    Figure Legend Snippet: Pharmacological specificity of DAMGO- and U50,488H-stimulated [ 35 S]GTPγS binding in vole brain Sections of male prairie vole forebrain were incubated as described in Experimental Procedures with 0.05 nM [ 35 S]GTPγS and 2 mM GDP with DAMGO (3 μM) as a mu agonist or U50,488H (1 μM) as a kappa agonist, with and without the mu antagonist naloxone (0.1 μM) or the kappa antagonist nor-BNI (0.1 μM). Similar results were obtained at 3 additional coronal levels (not shown).

    Techniques Used: Binding Assay, Incubation

    Concentration-effect curves of DAMGO and U50,488H in stimulating [ 35 S]GTPγS binding in striatal membranes from female prairie vole brains Membranes were prepared and assayed for agonist-stimulated [ 35 S]GTPγS binding as described in Methods, using 0.01–10 μM concentrations of DAMGO and U50,488H. Results are expressed as per cent stimulation over basal binding, and represent mean values ± SEM of three different assays each performed in triplicate.
    Figure Legend Snippet: Concentration-effect curves of DAMGO and U50,488H in stimulating [ 35 S]GTPγS binding in striatal membranes from female prairie vole brains Membranes were prepared and assayed for agonist-stimulated [ 35 S]GTPγS binding as described in Methods, using 0.01–10 μM concentrations of DAMGO and U50,488H. Results are expressed as per cent stimulation over basal binding, and represent mean values ± SEM of three different assays each performed in triplicate.

    Techniques Used: Concentration Assay, Binding Assay

    3) Product Images from "Hippocampal µ-opioid receptors on GABAergic neurons mediate stress-induced impairment of memory retrieval"

    Article Title: Hippocampal µ-opioid receptors on GABAergic neurons mediate stress-induced impairment of memory retrieval

    Journal: Molecular Psychiatry

    doi: 10.1038/s41380-019-0435-z

    EP stress impairs memory retrieval in the MWM task by activating hippocampal µRs. a The effects of systemic injection of naloxone or saline on time spent in the target/opposite quadrants and the target time ratio during probe test of the stressed and unstressed mice. *, target vs. opposite within-group, paired Student’s t -test. The target time ratio F 3,36 = 3.59, p = 0.023, one-way ANOVA; #, vs. S-Sal. b The effects of bilateral intra-hippocampal infusion of CTAP (0.5 µg/µl), naltrindole (2.26 µg/µl), nor-binaltorphimine (3.85 µg/µl), DAMGO (0.5 μg/μl), or saline on memory retrieval. The target time ratio F 8,81 = 7.00, p = 0.000, one-way ANOVA; #, vs. S-Sal. c The effects of EP stress on the levels of hippocampal µR proteins and their phosphorylation. Total µRs F 2,15 = 0.25, p = 0.784; cell surface µRs F 2,12 = 4.09, p = 0.044; phosphorylated µRs F 2,12 = 4.75, p = 0.030; one-way ANOVA; *, vs. unstressed. d Hippocampal injection of β-endorphin antiserum does not affect stress-induced memory impairment. e Hippocampal injection of enkephalin antiserum at a concentration of 1:10 before stress abolishes memory impairment. f The level of Met/Leu-enkephalin in hippocampal tissues. g The effects of systemic application of naloxone or bilateral intra-hippocampal infusion with CTAP on the level of serum corticosterone immediately after 50-min stress. F 3,12 = 4.22, p = 0.030 for naloxone groups, and F 3,12 = 8.74, p = 0.000 for CTAP groups, one-way ANOVA; *, vs. stress. One symbol, p
    Figure Legend Snippet: EP stress impairs memory retrieval in the MWM task by activating hippocampal µRs. a The effects of systemic injection of naloxone or saline on time spent in the target/opposite quadrants and the target time ratio during probe test of the stressed and unstressed mice. *, target vs. opposite within-group, paired Student’s t -test. The target time ratio F 3,36 = 3.59, p = 0.023, one-way ANOVA; #, vs. S-Sal. b The effects of bilateral intra-hippocampal infusion of CTAP (0.5 µg/µl), naltrindole (2.26 µg/µl), nor-binaltorphimine (3.85 µg/µl), DAMGO (0.5 μg/μl), or saline on memory retrieval. The target time ratio F 8,81 = 7.00, p = 0.000, one-way ANOVA; #, vs. S-Sal. c The effects of EP stress on the levels of hippocampal µR proteins and their phosphorylation. Total µRs F 2,15 = 0.25, p = 0.784; cell surface µRs F 2,12 = 4.09, p = 0.044; phosphorylated µRs F 2,12 = 4.75, p = 0.030; one-way ANOVA; *, vs. unstressed. d Hippocampal injection of β-endorphin antiserum does not affect stress-induced memory impairment. e Hippocampal injection of enkephalin antiserum at a concentration of 1:10 before stress abolishes memory impairment. f The level of Met/Leu-enkephalin in hippocampal tissues. g The effects of systemic application of naloxone or bilateral intra-hippocampal infusion with CTAP on the level of serum corticosterone immediately after 50-min stress. F 3,12 = 4.22, p = 0.030 for naloxone groups, and F 3,12 = 8.74, p = 0.000 for CTAP groups, one-way ANOVA; *, vs. stress. One symbol, p

    Techniques Used: Injection, Mouse Assay, Concentration Assay

    4) Product Images from "S-Nitrosothiols modulate G protein-coupled receptor signaling in a reversible and highly receptor-specific manner"

    Article Title: S-Nitrosothiols modulate G protein-coupled receptor signaling in a reversible and highly receptor-specific manner

    Journal: BMC Cell Biology

    doi: 10.1186/1471-2121-6-21

    GSNO only marginally affect opiate related receptor (ORL1), μ opioid receptor (MOR) and adenosine A 1 (Ado A 1 ) receptors signaling in brain sections . [ 35 S]GTPγS autoradiography was conducted using a 3-step protocol with adenosine deaminase (ADA, 1 U/ml) present throughout steps 2 and 3, as detailed in the Methods section. Where indicated, GSNO (1 mM) was present for 60 min during the GDP loading (step 2). Protease inhibitor cocktail was included in step 2 for brain sections used for testing ORL1 and MOR responses. Receptor agonists nociceptin (ORL1), DAMGO (MOR), and 2-chloroadenosine (2ClAdo) (adenosine A 1 receptor) were present at submaximal concentrations during step 3. Note wide distribution of nociceptin-responsive brain regions, including the cerebral cortex (Cx). Note also robust response to DAMGO in the MOR-enriched striatal patches (Sp), as well as the relatively GSNO-resistant, and widely distributed adenosine A 1 receptor-dependent signal throughout the sagittal plane. Scale bar = 2 mm. For quantitative data on selected brain regions, see Supplementary Table 2 in additional file 1 .
    Figure Legend Snippet: GSNO only marginally affect opiate related receptor (ORL1), μ opioid receptor (MOR) and adenosine A 1 (Ado A 1 ) receptors signaling in brain sections . [ 35 S]GTPγS autoradiography was conducted using a 3-step protocol with adenosine deaminase (ADA, 1 U/ml) present throughout steps 2 and 3, as detailed in the Methods section. Where indicated, GSNO (1 mM) was present for 60 min during the GDP loading (step 2). Protease inhibitor cocktail was included in step 2 for brain sections used for testing ORL1 and MOR responses. Receptor agonists nociceptin (ORL1), DAMGO (MOR), and 2-chloroadenosine (2ClAdo) (adenosine A 1 receptor) were present at submaximal concentrations during step 3. Note wide distribution of nociceptin-responsive brain regions, including the cerebral cortex (Cx). Note also robust response to DAMGO in the MOR-enriched striatal patches (Sp), as well as the relatively GSNO-resistant, and widely distributed adenosine A 1 receptor-dependent signal throughout the sagittal plane. Scale bar = 2 mm. For quantitative data on selected brain regions, see Supplementary Table 2 in additional file 1 .

    Techniques Used: Autoradiography, Protease Inhibitor

    5) Product Images from "Ligand-Directed Functional Selectivity at the Mu Opioid Receptor Revealed by Label-Free Integrative Pharmacology On-Target"

    Article Title: Ligand-Directed Functional Selectivity at the Mu Opioid Receptor Revealed by Label-Free Integrative Pharmacology On-Target

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0025643

    The DMR characteristics of BNTX and the negative control (0.1% DMSO). (a) The BNTX DMR in HEK-293 cells; (b) the BNTX DMR in the buffer treated (BNTX) and CTOP pretreated (CTOP – BNTX) HEK-MOR cells; and (c) the DAMGO DMR in the BNTX pretreated cells (BNTX – DAMGO) in comparison with the BNTX DMR in the DAMGO pretreated cells (DAMGO – BNTX); (d) the DMR induced by DMSO in HEK293; (e) the DMSO DMR in HEK-MOR or the CTOP pretreated HEK-MOR cells; (f) the DAMGO DMR after pretreatment with DMSO (DMSO – DAMGO) and the DMSO DMR after pretreatment with DAMGO (DAMGO –DMSO) in HEK-MOR cells. Each curve represents the average of duplicates.
    Figure Legend Snippet: The DMR characteristics of BNTX and the negative control (0.1% DMSO). (a) The BNTX DMR in HEK-293 cells; (b) the BNTX DMR in the buffer treated (BNTX) and CTOP pretreated (CTOP – BNTX) HEK-MOR cells; and (c) the DAMGO DMR in the BNTX pretreated cells (BNTX – DAMGO) in comparison with the BNTX DMR in the DAMGO pretreated cells (DAMGO – BNTX); (d) the DMR induced by DMSO in HEK293; (e) the DMSO DMR in HEK-MOR or the CTOP pretreated HEK-MOR cells; (f) the DAMGO DMR after pretreatment with DMSO (DMSO – DAMGO) and the DMSO DMR after pretreatment with DAMGO (DAMGO –DMSO) in HEK-MOR cells. Each curve represents the average of duplicates.

    Techniques Used: Negative Control

    The DMR signals induced by levallorphan, β-funaltrexamine, and naltrindole in the DAMGO-activated HEK-MOR cells. Each curve represents the average of duplicates.
    Figure Legend Snippet: The DMR signals induced by levallorphan, β-funaltrexamine, and naltrindole in the DAMGO-activated HEK-MOR cells. Each curve represents the average of duplicates.

    Techniques Used:

    The ICI 199441-induced DMR in distinct molecule-treated cells. The cells were HEK-293 cells (HEK293), HEK-MOR (HEK-MOR), or CTOP- (CTOP), or DAMGO- (DAMGO) pretreated HEK-MOR cells. Each curve represents the average of duplicates.
    Figure Legend Snippet: The ICI 199441-induced DMR in distinct molecule-treated cells. The cells were HEK-293 cells (HEK293), HEK-MOR (HEK-MOR), or CTOP- (CTOP), or DAMGO- (DAMGO) pretreated HEK-MOR cells. Each curve represents the average of duplicates.

    Techniques Used:

    6) Product Images from "Buprenorphine-elicited alteration of adenylate cyclase activity in human embryonic kidney 293 cells coexpressing κ-, μ-opioid and nociceptin receptors"

    Article Title: Buprenorphine-elicited alteration of adenylate cyclase activity in human embryonic kidney 293 cells coexpressing κ-, μ-opioid and nociceptin receptors

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.12644

    Effects of acute exposure to U-69593, DAMGO, nociceptin or buprenorphine on forskolin-stimulated cAMP accumulation in HEK 293 cells expressing KOP, KOP+MOP, KOP+NOP and KOP+MOP+NOP receptors. HEK 293 cells expressing KOP ( open triangle ), KOP+MOP ( open square ), KOP+NOP ( open circle ) or KOP+MOP+NOP ( filled diamond ) were treated with U-69593 ( A ), DAMGO ( B ), nociceptin ( C ) or buprenorphine ( D ) for 30 min. at room temperature in the presence of 10 μM forskolin prior to HTRF cAMP assays. Each point represents the mean ± SE value of five to fourteen (numbers indicated in the parentheses) experiments performed in duplicate using different batches of cells. 100% defines forskolin-stimulated cAMP accumulation in cells not treated with aforementioned drugs.
    Figure Legend Snippet: Effects of acute exposure to U-69593, DAMGO, nociceptin or buprenorphine on forskolin-stimulated cAMP accumulation in HEK 293 cells expressing KOP, KOP+MOP, KOP+NOP and KOP+MOP+NOP receptors. HEK 293 cells expressing KOP ( open triangle ), KOP+MOP ( open square ), KOP+NOP ( open circle ) or KOP+MOP+NOP ( filled diamond ) were treated with U-69593 ( A ), DAMGO ( B ), nociceptin ( C ) or buprenorphine ( D ) for 30 min. at room temperature in the presence of 10 μM forskolin prior to HTRF cAMP assays. Each point represents the mean ± SE value of five to fourteen (numbers indicated in the parentheses) experiments performed in duplicate using different batches of cells. 100% defines forskolin-stimulated cAMP accumulation in cells not treated with aforementioned drugs.

    Techniques Used: Expressing

    Naloxone precipitation on the effects of chronic exposure to U-69593, DAMGO, nociceptin or buprenorphine on forskolin-induced cAMP accumulation in HEK 293 cells expressing KOP, KOP+MOP, KOP+NOP and KOP+MOP+NOP receptors. After exposure to U-69593 ( A ), DAMGO ( B ), nociceptin ( C ) or buprenorphine ( D ) for 4 hrs at 37°C, the incubation media were subsequently removed, and HEK 293 cells expressing KOP ( open triangle ), KOP+MOP ( open square ), KOP+NOP ( open circle ) or KOP+MOP+NOP ( filled diamond ) were treated with 1 μM naloxone accompanied by 10 μM forskolin immediately prior to HTRF cAMP assays. Each point represents the mean ± SE value of six to eleven (numbers indicated in the parentheses) experiments performed in duplicate using different batches of cells. 100% defines forskolin-stimulated cAMP accumulation in cells treated with none of the aforementioned drugs.
    Figure Legend Snippet: Naloxone precipitation on the effects of chronic exposure to U-69593, DAMGO, nociceptin or buprenorphine on forskolin-induced cAMP accumulation in HEK 293 cells expressing KOP, KOP+MOP, KOP+NOP and KOP+MOP+NOP receptors. After exposure to U-69593 ( A ), DAMGO ( B ), nociceptin ( C ) or buprenorphine ( D ) for 4 hrs at 37°C, the incubation media were subsequently removed, and HEK 293 cells expressing KOP ( open triangle ), KOP+MOP ( open square ), KOP+NOP ( open circle ) or KOP+MOP+NOP ( filled diamond ) were treated with 1 μM naloxone accompanied by 10 μM forskolin immediately prior to HTRF cAMP assays. Each point represents the mean ± SE value of six to eleven (numbers indicated in the parentheses) experiments performed in duplicate using different batches of cells. 100% defines forskolin-stimulated cAMP accumulation in cells treated with none of the aforementioned drugs.

    Techniques Used: Expressing, Incubation

    7) Product Images from "G protein βγ subunits inhibit TRPM3 ion channels in sensory neurons"

    Article Title: G protein βγ subunits inhibit TRPM3 ion channels in sensory neurons

    Journal: eLife

    doi: 10.7554/eLife.26138

    Activation of μ-opioid receptors inhibits TRPM3. ( a ) Effect of the selective μ opioid receptor agonist DAMGO, δ opioid receptor agonist SB205607 and κ opioid receptor agonist U50488 (each at 20 nM) on [Ca 2+ ] i -responses evoked by PS (20 µM, second application, see Figure 1 for protocol). *p
    Figure Legend Snippet: Activation of μ-opioid receptors inhibits TRPM3. ( a ) Effect of the selective μ opioid receptor agonist DAMGO, δ opioid receptor agonist SB205607 and κ opioid receptor agonist U50488 (each at 20 nM) on [Ca 2+ ] i -responses evoked by PS (20 µM, second application, see Figure 1 for protocol). *p

    Techniques Used: Activation Assay

    8) Product Images from "Electroacupuncture Alleviates Pain Responses and Inflammation in a Rat Model of Acute Gout Arthritis"

    Article Title: Electroacupuncture Alleviates Pain Responses and Inflammation in a Rat Model of Acute Gout Arthritis

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2018/2598975

    Effect of local administration of specific μ - and κ -opioid receptor agonists on mechanical hyperalgesia of MSU-induced acute gout arthritis rats. Opioid receptor agonists were locally administered to the ankle 7.5 h after MSU injection. PWTs were evaluated 30 min after opioid receptor application. (a-b) Effects of μ -receptor agonist DAMGO (4.9 μ g/ankle, panel (a)) and κ -receptor agonist (±) U50488 (1 μ g/ankle, panel (b)) injected (i.a.) into the ankle either ipsi (IL) or contralaterally (CL) on the mechanical hyperalgesia of MSU-induced acute gout arthritis rats. ∗∗ p
    Figure Legend Snippet: Effect of local administration of specific μ - and κ -opioid receptor agonists on mechanical hyperalgesia of MSU-induced acute gout arthritis rats. Opioid receptor agonists were locally administered to the ankle 7.5 h after MSU injection. PWTs were evaluated 30 min after opioid receptor application. (a-b) Effects of μ -receptor agonist DAMGO (4.9 μ g/ankle, panel (a)) and κ -receptor agonist (±) U50488 (1 μ g/ankle, panel (b)) injected (i.a.) into the ankle either ipsi (IL) or contralaterally (CL) on the mechanical hyperalgesia of MSU-induced acute gout arthritis rats. ∗∗ p

    Techniques Used: Injection

    9) Product Images from "Endothelin-converting enzyme 2 differentially regulates opioid receptor activity"

    Article Title: Endothelin-converting enzyme 2 differentially regulates opioid receptor activity

    Journal: British Journal of Pharmacology

    doi: 10.1111/bph.12833

    ECE2 inhibition prevents recycling of μ opioid receptors endocytosed following exposure to endogenous peptides that are the substrates of ECE2. CHO-μ-ECE2 cells were incubated with either DAMGO (1 μM), dynorphin B (Dyn B, 100 nM),
    Figure Legend Snippet: ECE2 inhibition prevents recycling of μ opioid receptors endocytosed following exposure to endogenous peptides that are the substrates of ECE2. CHO-μ-ECE2 cells were incubated with either DAMGO (1 μM), dynorphin B (Dyn B, 100 nM),

    Techniques Used: Inhibition, Incubation

    Recycling of receptors endocytosed by a peptidic agonist (and not by a non-peptidic agonist) is blocked by ECE2 inhibition. CHO-μ-ECE2 cells were incubated with DAMGO (1 μM) (A), dynorphin B (Dyn B, 100 nM) (B) or fentanyl (1 μM)
    Figure Legend Snippet: Recycling of receptors endocytosed by a peptidic agonist (and not by a non-peptidic agonist) is blocked by ECE2 inhibition. CHO-μ-ECE2 cells were incubated with DAMGO (1 μM) (A), dynorphin B (Dyn B, 100 nM) (B) or fentanyl (1 μM)

    Techniques Used: Inhibition, Incubation

    Inhibition of ECE2 activity affects μ opioid receptor resensitization. CHO-μ-ECE2 cells were incubated with DAMGO (1 μM) (A), dynorphin B (Dyn B, 100 nM) (B) or fentanyl (1 μM) (C) along with 10 μM forskolin for
    Figure Legend Snippet: Inhibition of ECE2 activity affects μ opioid receptor resensitization. CHO-μ-ECE2 cells were incubated with DAMGO (1 μM) (A), dynorphin B (Dyn B, 100 nM) (B) or fentanyl (1 μM) (C) along with 10 μM forskolin for

    Techniques Used: Inhibition, Activity Assay, Incubation

    Inhibition of endogenous ECE2 activity in F11 DRG-derived cell line impairs recycling of native μ opioid receptors. F11 cells were incubated with 1 μM DAMGO (A) or 100 nM dynorphin B (B) for 30 min ( t = 0); cells were washed and incubated
    Figure Legend Snippet: Inhibition of endogenous ECE2 activity in F11 DRG-derived cell line impairs recycling of native μ opioid receptors. F11 cells were incubated with 1 μM DAMGO (A) or 100 nM dynorphin B (B) for 30 min ( t = 0); cells were washed and incubated

    Techniques Used: Inhibition, Activity Assay, Derivative Assay, Incubation

    ECE2 expression leads to enhanced recycling of μ opioid receptors. CHO-μ or CHO-μ-ECE2 cells were incubated with 1 μM DAMGO or 100 nM dynorphin B (Dyn B) for 30 min ( t = 0); cells were washed and incubated for either 120
    Figure Legend Snippet: ECE2 expression leads to enhanced recycling of μ opioid receptors. CHO-μ or CHO-μ-ECE2 cells were incubated with 1 μM DAMGO or 100 nM dynorphin B (Dyn B) for 30 min ( t = 0); cells were washed and incubated for either 120

    Techniques Used: Expressing, Incubation

    Inhibition of endogenous ECE2 activity affects native μ opioid receptor resensitization in F11 cells. F11 cells were incubated with DAMGO (1 μM) (A), dynorphin B (Dyn B, 100 nM) (B) or fentanyl (1 μM) (C), along with 10 μM
    Figure Legend Snippet: Inhibition of endogenous ECE2 activity affects native μ opioid receptor resensitization in F11 cells. F11 cells were incubated with DAMGO (1 μM) (A), dynorphin B (Dyn B, 100 nM) (B) or fentanyl (1 μM) (C), along with 10 μM

    Techniques Used: Inhibition, Activity Assay, Incubation

    ECE2 and an acidic pH are required for modulation of μ opioid receptor recycling following endocytosis by agonists that are ECE2 substrates. CHO-μ-ECE2 cells were incubated with DAMGO (1 μM) (A, B) or dynorphin B (Dyn B, 100 nM)
    Figure Legend Snippet: ECE2 and an acidic pH are required for modulation of μ opioid receptor recycling following endocytosis by agonists that are ECE2 substrates. CHO-μ-ECE2 cells were incubated with DAMGO (1 μM) (A, B) or dynorphin B (Dyn B, 100 nM)

    Techniques Used: Incubation

    10) Product Images from "Role of RGS12 in the differential regulation of kappa opioid receptor-dependent signaling and behavior"

    Article Title: Role of RGS12 in the differential regulation of kappa opioid receptor-dependent signaling and behavior

    Journal: Neuropsychopharmacology

    doi: 10.1038/s41386-019-0423-7

    RGS12 expression reduces potency of the KOR agonist U50,488, yet augments agonist-stimulated recruitment of β-arrestin to KOR via independent mechanisms. a - c RGS12 expression reduces potency of the KOR agonist U50,488 to a greater extent than for the MOR agonist DAMGO or the DOR agonist DADLE. GloSensor-22F (Promega) luciferase-based measurements of cAMP levels within HEK293T cells stimulated with 100 nM isoproterenol and simultaneously treated with opioid receptor-selective agonists following transient co-expression of ( a ) KOR cDNA plus indicated RGS12 expression plasmids, b MOR cDNA with or without wildtype (WT) RGS12, or c DOR cDNA with or without WT RGS12. ( a , inset ) PTX (200 ng/mL) pretreatment abolished KOR agonist-driven cAMP inhibition, confirming that the assay is dependent on G i/o -mediated signaling. Wildtype RGS12 expression reduced U50,488 potency by 20-fold, i.e., 6 to 8-fold more than its effects on DAMGO (3-fold) and DADLE (2.5-fold) potency. Loss-of-function point mutants of RGS12 curtailed U50,488 potency reductions from 20-fold down to 3-to-4-fold. Data were normalized to vehicle control conditions and are expressed as the mean ± SEM from multiple experiments ( N = 3). Concentration-response curves were fit by four-parameter non-linear regression (Prism 7). d - f The PDZ domain docking site of KOR (-NKPV-c) is necessary, but not sufficient, to completely account for the specificity of RGS12 for KOR over MOR in assays of GPCR-mediated G protein signaling. d Schematic representing creation of KOR expression cDNA containing mutation to the C-terminus (-NKPV-c to -AAAA-c) and MOR expression cDNA containing mutation of its wildtype C-terminus (MOR: -APLP-c) to KOR’s wildtype C-terminus (-NKPV-c). e Mutation to the C-terminus of KOR curtailed RGS12-mediated U50,488 potency reductions from 20-fold (e.g., panel a ) to 4-fold. f RGS12 reduced MOR agonist potency by 6-fold following substitution of the MOR C-terminus with the wildtype KOR C-terminus, relative to a 3-fold potency reduction observed with wildtype MOR (e.g., panel b ). Data are normalized to vehicle control and expressed as mean ± SEM from N = 3 experiments. Dose-response curves fit by four parameter non-linear regression (Prism). g - j ] stimulated with indicated agonists following transient co-expression of ( g ) KOR cDNA plus indicated RGS12 expression plasmids, ( h ) KOR cDNA with or without WT RGS12 following pretreatment with PTX or vehicle, ( i ) MOR cDNA with or without WT RGS12, or ( j ) DOR cDNA with or without WT RGS12. Agonist potency (pEC 50 values) did not differ between conditions. U50,488-induced β-arrestin recruitment efficacy was ~3-fold greater when either WT RGS12, or loss-of-function point mutants of RGS12, was co-expressed with KOR, whereas WT RGS12 only increased β-arrestin recruitment to MOR by 1.2-fold and DOR by 1.1-fold. PTX pretreatment did not affect U50,488-induced β-arrestin recruitment to KOR (panel H) in the presence or absence of WT RGS12. Data were normalized to vehicle control conditions (fold change) and are expressed as the mean ± SEM from multiple experiments ( N = 3). Curves were analyzed by three-parameter non-linear regression and panel H was analyzed by two-way ANOVA with Sidak’s post hoc test. k [ 3 H]U69,593 saturation binding analysis of vSTR membranes from β-arrestin-2 knockout (βarr2 KO ) mice and wildtype (WT) controls. l As derived from data in panel K and parallel binding data from dSTR membrane samples, B max was increased in vSTR (but not dSTR) of β-arrestin-2 knockout (βarr2 KO vSTR B max = 150.1 ± 17.1 fmol/mg protein vs wildtype vSTR B max = 93.1 ± 14.6 fmol/mg protein; βarr2 KO dSTR B max = 74.5 ± 9.8 fmol/mg protein vs wildtype dSTR B max = 69.3 ± 7.7 fmol/mg protein). The K D for [ 3 H]U69,593 did not differ across genotypes (βarr2 KO vSTR: 7.1 ± 1.8 nM; WT vSTR: 8.4 ± 2.9 nM; βarr2 KO dSTR: 7.8 ± 2.3 nM; WT dSTR: 6.6 ± 1.7 nM;). Data are the mean ± SEM and panel L was analyzed by two-way ANOVA with Sidak’s post hoc test ( n = 12 mice per group) (* p
    Figure Legend Snippet: RGS12 expression reduces potency of the KOR agonist U50,488, yet augments agonist-stimulated recruitment of β-arrestin to KOR via independent mechanisms. a - c RGS12 expression reduces potency of the KOR agonist U50,488 to a greater extent than for the MOR agonist DAMGO or the DOR agonist DADLE. GloSensor-22F (Promega) luciferase-based measurements of cAMP levels within HEK293T cells stimulated with 100 nM isoproterenol and simultaneously treated with opioid receptor-selective agonists following transient co-expression of ( a ) KOR cDNA plus indicated RGS12 expression plasmids, b MOR cDNA with or without wildtype (WT) RGS12, or c DOR cDNA with or without WT RGS12. ( a , inset ) PTX (200 ng/mL) pretreatment abolished KOR agonist-driven cAMP inhibition, confirming that the assay is dependent on G i/o -mediated signaling. Wildtype RGS12 expression reduced U50,488 potency by 20-fold, i.e., 6 to 8-fold more than its effects on DAMGO (3-fold) and DADLE (2.5-fold) potency. Loss-of-function point mutants of RGS12 curtailed U50,488 potency reductions from 20-fold down to 3-to-4-fold. Data were normalized to vehicle control conditions and are expressed as the mean ± SEM from multiple experiments ( N = 3). Concentration-response curves were fit by four-parameter non-linear regression (Prism 7). d - f The PDZ domain docking site of KOR (-NKPV-c) is necessary, but not sufficient, to completely account for the specificity of RGS12 for KOR over MOR in assays of GPCR-mediated G protein signaling. d Schematic representing creation of KOR expression cDNA containing mutation to the C-terminus (-NKPV-c to -AAAA-c) and MOR expression cDNA containing mutation of its wildtype C-terminus (MOR: -APLP-c) to KOR’s wildtype C-terminus (-NKPV-c). e Mutation to the C-terminus of KOR curtailed RGS12-mediated U50,488 potency reductions from 20-fold (e.g., panel a ) to 4-fold. f RGS12 reduced MOR agonist potency by 6-fold following substitution of the MOR C-terminus with the wildtype KOR C-terminus, relative to a 3-fold potency reduction observed with wildtype MOR (e.g., panel b ). Data are normalized to vehicle control and expressed as mean ± SEM from N = 3 experiments. Dose-response curves fit by four parameter non-linear regression (Prism). g - j ] stimulated with indicated agonists following transient co-expression of ( g ) KOR cDNA plus indicated RGS12 expression plasmids, ( h ) KOR cDNA with or without WT RGS12 following pretreatment with PTX or vehicle, ( i ) MOR cDNA with or without WT RGS12, or ( j ) DOR cDNA with or without WT RGS12. Agonist potency (pEC 50 values) did not differ between conditions. U50,488-induced β-arrestin recruitment efficacy was ~3-fold greater when either WT RGS12, or loss-of-function point mutants of RGS12, was co-expressed with KOR, whereas WT RGS12 only increased β-arrestin recruitment to MOR by 1.2-fold and DOR by 1.1-fold. PTX pretreatment did not affect U50,488-induced β-arrestin recruitment to KOR (panel H) in the presence or absence of WT RGS12. Data were normalized to vehicle control conditions (fold change) and are expressed as the mean ± SEM from multiple experiments ( N = 3). Curves were analyzed by three-parameter non-linear regression and panel H was analyzed by two-way ANOVA with Sidak’s post hoc test. k [ 3 H]U69,593 saturation binding analysis of vSTR membranes from β-arrestin-2 knockout (βarr2 KO ) mice and wildtype (WT) controls. l As derived from data in panel K and parallel binding data from dSTR membrane samples, B max was increased in vSTR (but not dSTR) of β-arrestin-2 knockout (βarr2 KO vSTR B max = 150.1 ± 17.1 fmol/mg protein vs wildtype vSTR B max = 93.1 ± 14.6 fmol/mg protein; βarr2 KO dSTR B max = 74.5 ± 9.8 fmol/mg protein vs wildtype dSTR B max = 69.3 ± 7.7 fmol/mg protein). The K D for [ 3 H]U69,593 did not differ across genotypes (βarr2 KO vSTR: 7.1 ± 1.8 nM; WT vSTR: 8.4 ± 2.9 nM; βarr2 KO dSTR: 7.8 ± 2.3 nM; WT dSTR: 6.6 ± 1.7 nM;). Data are the mean ± SEM and panel L was analyzed by two-way ANOVA with Sidak’s post hoc test ( n = 12 mice per group) (* p

    Techniques Used: Expressing, Luciferase, Inhibition, Concentration Assay, Mutagenesis, Binding Assay, Knock-Out, Mouse Assay, Derivative Assay

    11) Product Images from "Presynaptic Mechanisms and KCNQ Potassium Channels Modulate Opioid Depression of Respiratory Drive"

    Article Title: Presynaptic Mechanisms and KCNQ Potassium Channels Modulate Opioid Depression of Respiratory Drive

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2019.01407

    Pharmacological interrogations of in vitro preBötC slices suggests a modulatory role for KCNQ potassium channels in opioid-induced respiratory depression (OIRD), and a minor role for GIRK potassium channels. ( A , left) Dose-response curves of inspiratory burst frequencies in response to increasing titers of DAMGO, and two activators of KCNQ potassium channels (ICA 69673, Retigabine) which act on different structural domains of the channel subunit. Both activators inhibit burst frequencies with an IC 50 = ∼0.7–1.0 μM, comparable to their EC 50 for activation of KCNQ channels. ( A , right) Dose-response curves of inspiratory burst frequencies in response to increasing titers of ML297, a GIRK1 subunit specific activator. Modest depression of burst frequencies, at concentrations 10–100-fold higher than the EC 50 s (0.16–0.9 μM) for GIRK1 containing heteromeric channels. ( B , left) Dose-response curves of inspiratory burst frequency in response to increasing titers of the KCNQ blockers XE991 and Chromanol 293B (293B), applied in the presence of DAMGO (100 nM) to suppress respiratory rhythms. ( B , right) Dose-response curves of inspiratory burst frequency in response to increasing titers of TertiapinQ (TPQ), a GIRK-specific blocker, applied in the presence of DAMGO (100 nM). No recovery of respiratory burst frequency was observed at the highest concentration of TPQ (100 nM). By contrast, both KCNQ blockers partially rescued respiratory rhythms suppressed by DAMGO, at relatively high concentrations (20–100 μM). Shown above plots are the EC 50 s and IC 50 s of each compound for specific molecular species of homo- and hetero-tetrameric GIRK and KCNQ channels reported in the literature (see text and Table 2 for references). Mean and SE plotted for DAMGO and TertiapinQ; median and IQR plotted for all other datasets, along with individual replicant values: DAMGO ( N = 35), ICA 69673 ( N = 14), Retigabine ( N = 6), ML297 ( N = 6), XE991 ( N = 7), Chromanol 293B ( N = 5), TertiapinQ ( N = 9).
    Figure Legend Snippet: Pharmacological interrogations of in vitro preBötC slices suggests a modulatory role for KCNQ potassium channels in opioid-induced respiratory depression (OIRD), and a minor role for GIRK potassium channels. ( A , left) Dose-response curves of inspiratory burst frequencies in response to increasing titers of DAMGO, and two activators of KCNQ potassium channels (ICA 69673, Retigabine) which act on different structural domains of the channel subunit. Both activators inhibit burst frequencies with an IC 50 = ∼0.7–1.0 μM, comparable to their EC 50 for activation of KCNQ channels. ( A , right) Dose-response curves of inspiratory burst frequencies in response to increasing titers of ML297, a GIRK1 subunit specific activator. Modest depression of burst frequencies, at concentrations 10–100-fold higher than the EC 50 s (0.16–0.9 μM) for GIRK1 containing heteromeric channels. ( B , left) Dose-response curves of inspiratory burst frequency in response to increasing titers of the KCNQ blockers XE991 and Chromanol 293B (293B), applied in the presence of DAMGO (100 nM) to suppress respiratory rhythms. ( B , right) Dose-response curves of inspiratory burst frequency in response to increasing titers of TertiapinQ (TPQ), a GIRK-specific blocker, applied in the presence of DAMGO (100 nM). No recovery of respiratory burst frequency was observed at the highest concentration of TPQ (100 nM). By contrast, both KCNQ blockers partially rescued respiratory rhythms suppressed by DAMGO, at relatively high concentrations (20–100 μM). Shown above plots are the EC 50 s and IC 50 s of each compound for specific molecular species of homo- and hetero-tetrameric GIRK and KCNQ channels reported in the literature (see text and Table 2 for references). Mean and SE plotted for DAMGO and TertiapinQ; median and IQR plotted for all other datasets, along with individual replicant values: DAMGO ( N = 35), ICA 69673 ( N = 14), Retigabine ( N = 6), ML297 ( N = 6), XE991 ( N = 7), Chromanol 293B ( N = 5), TertiapinQ ( N = 9).

    Techniques Used: In Vitro, Activated Clotting Time Assay, Activation Assay, Concentration Assay

    mEPSCs recorded from Dbx1 -derived ( Dbx1 + ) preBötC neurons support a presynaptic site of action for DAMGO and KCNQ potassium channels. (A) Dbx1 + preBötC neuron labeled with tdTomato, without tamoxifen, imaged with Dodt-IR (left) and RFP fluorescence (right). Slice homozygous for Dbx1 Cre–ERT2 and Ai14 ( Dbx1 Cre–ERT2/Cre–ERT2 ; Rosa26 Ai14/Ai14 ). (B) Representative mEPSCs recorded from a identified Dbx1 + inspiratory neuron in response to sequential bath application of TTX (1 μM), DAMGO (100 nM), and XE991 (20 μM). Currents measured at a holding potential of -60 mV. (C) Representative cumulative fractional distribution plot of mEPSC inter-event intervals (IEIs) recorded from an inspiratory Dbx1 + neuron, in response to TTX (black), DAMGO,TTX (red), and XE991,DAMGO,TTX (blue). DAMGO,TTX (red) distribution is significantly shifted toward longer IEIs relative to either TTX (black) or XE991,DAMGO,TTX (blue) distributions; p = 0.05 (Paired Wilcox–Signed Rank test; modified Kolmogorov-Smirnov), whereas TTX (black) and XE991,DAMGO,TTX (blue) distributions are not significantly different (Paired Wilcox–Signed Rank test; modified Kolmogorov–Smirnov). (D) Summary of pairwise comparisons of mEPSC cumulative fractional inter-event interval distributions (IEIs) from individual neurons in response to TTX ( N = 7), DAMGO,TTX ( N = 7) and XE991,DAMGO,TTX ( N = 7), by paired Wilcox–Signed Rank tests (modified Kolmogorov-Smirnov). Significant ranked differences in mEPSC IEI distributions denoted by colored boxes [ p = 0.05; orange ( > ), blue (
    Figure Legend Snippet: mEPSCs recorded from Dbx1 -derived ( Dbx1 + ) preBötC neurons support a presynaptic site of action for DAMGO and KCNQ potassium channels. (A) Dbx1 + preBötC neuron labeled with tdTomato, without tamoxifen, imaged with Dodt-IR (left) and RFP fluorescence (right). Slice homozygous for Dbx1 Cre–ERT2 and Ai14 ( Dbx1 Cre–ERT2/Cre–ERT2 ; Rosa26 Ai14/Ai14 ). (B) Representative mEPSCs recorded from a identified Dbx1 + inspiratory neuron in response to sequential bath application of TTX (1 μM), DAMGO (100 nM), and XE991 (20 μM). Currents measured at a holding potential of -60 mV. (C) Representative cumulative fractional distribution plot of mEPSC inter-event intervals (IEIs) recorded from an inspiratory Dbx1 + neuron, in response to TTX (black), DAMGO,TTX (red), and XE991,DAMGO,TTX (blue). DAMGO,TTX (red) distribution is significantly shifted toward longer IEIs relative to either TTX (black) or XE991,DAMGO,TTX (blue) distributions; p = 0.05 (Paired Wilcox–Signed Rank test; modified Kolmogorov-Smirnov), whereas TTX (black) and XE991,DAMGO,TTX (blue) distributions are not significantly different (Paired Wilcox–Signed Rank test; modified Kolmogorov–Smirnov). (D) Summary of pairwise comparisons of mEPSC cumulative fractional inter-event interval distributions (IEIs) from individual neurons in response to TTX ( N = 7), DAMGO,TTX ( N = 7) and XE991,DAMGO,TTX ( N = 7), by paired Wilcox–Signed Rank tests (modified Kolmogorov-Smirnov). Significant ranked differences in mEPSC IEI distributions denoted by colored boxes [ p = 0.05; orange ( > ), blue (

    Techniques Used: Derivative Assay, Labeling, Fluorescence, Modification

    Exemplary records of fictive inspiratory bursts recorded from in vitro preBötC slices in response to increasing titers of activators or blockers of KCNQ and GIRK potassium channels, mimicking or reversing opioid-induced respiratory depression (OIRD). Integrated recording (top), instantaneous burst frequency (below). (A) Inspiratory bursts as a function of increasing titers of ICA 69673, a KCNQ activator (0.1, 0.5, 2.0, in mM), mimicking OIRD. (B) Inspiratory bursts as a function of increasing titers of retigabine (RTG), an FDA-approved KCNQ activator (0.1, 0.5, 1.0, 3.0, in mM), mimicking OIRD. (C) Rescue of DAMGO-induced OIRD (100 nM) with increasing titers of Chromanol 293B (293B), a KCNQ blocker (10, 50, 100, in mM). (D) Rescue of DAMGO-induced OIRD (100 nM) with increasing titers of XE991, a KCNQ blocker (1, 3, 30, 60, in mM). (E) Failure to rescue DAMGO-induced OIRD (100 nM) with increasing titers of TertiapinQ (TPQ), a GIRK blocker (5, 15, 60, 100, in nM). (F) Failure to mimic OIRD by increasing titers of ML297, a GIRK activator (3, 10, 30, in mM). Transient apneas observed at the beginning of washes (A–D) are an artifact of temperature drop or oxygen desaturation from perfusion exchanges.
    Figure Legend Snippet: Exemplary records of fictive inspiratory bursts recorded from in vitro preBötC slices in response to increasing titers of activators or blockers of KCNQ and GIRK potassium channels, mimicking or reversing opioid-induced respiratory depression (OIRD). Integrated recording (top), instantaneous burst frequency (below). (A) Inspiratory bursts as a function of increasing titers of ICA 69673, a KCNQ activator (0.1, 0.5, 2.0, in mM), mimicking OIRD. (B) Inspiratory bursts as a function of increasing titers of retigabine (RTG), an FDA-approved KCNQ activator (0.1, 0.5, 1.0, 3.0, in mM), mimicking OIRD. (C) Rescue of DAMGO-induced OIRD (100 nM) with increasing titers of Chromanol 293B (293B), a KCNQ blocker (10, 50, 100, in mM). (D) Rescue of DAMGO-induced OIRD (100 nM) with increasing titers of XE991, a KCNQ blocker (1, 3, 30, 60, in mM). (E) Failure to rescue DAMGO-induced OIRD (100 nM) with increasing titers of TertiapinQ (TPQ), a GIRK blocker (5, 15, 60, 100, in nM). (F) Failure to mimic OIRD by increasing titers of ML297, a GIRK activator (3, 10, 30, in mM). Transient apneas observed at the beginning of washes (A–D) are an artifact of temperature drop or oxygen desaturation from perfusion exchanges.

    Techniques Used: In Vitro

    12) Product Images from "HINT1 protein cooperates with cannabinoid 1 receptor to negatively regulate glutamate NMDA receptor activity"

    Article Title: HINT1 protein cooperates with cannabinoid 1 receptor to negatively regulate glutamate NMDA receptor activity

    Journal: Molecular Brain

    doi: 10.1186/1756-6606-6-42

    MOR and CNR1 associate with the NR1 subunits via HINT1: Implications for NMDAR activity. (A) The association of GPCRs, MOR and CNR1 with the NMDAR NR1 subunits is dependent on HINT1. The GPCRs were immunoprecipitated from solubilized mouse brain cortical synaptosomes from WT and HINT1 -/- mice, and the proteins that co-precipitated with the NR1 subunits were analyzed by western blot. (B) The association of CNR1 with NR1 in WT and HINT1 -/- cortical cell cultures uninfected or infected with 10 μL/well of HINT1 -/- lentiviral particles. The presence/absence of HINT1 was determined, and the co-precipitates of the NR1 subunits and CNR1 were then assessed by immunoprecipitation and western blot. For each determination, cells from 10 wells were pooled, and the assay was repeated twice with identical results. (C) Opioid agonists promoted a higher level of responsiveness of the NMDAR by inhibiting the association between MOR and NR1. Morphine or DAMGO was icv-injected into the mice 24 h prior to the analysis of MOR/NMDA NR1 association and CaMKII activating autophosphorylation on Thr286. At 7 days post-surgery, the mice from the CCI neuropathic pain group displayed increased pThr286 CaMKII and reduced MOR-NR1 association compared to the sham-operated controls. Changes of Mechanical Withdrawal Threshold . Following ligation, animals developed significant mechanical allodynia by day 3 that remained until day 21. Naïve control and sham – operated mice failed to exhibit mechanical allodynia. Data are mean ± SEM. *P
    Figure Legend Snippet: MOR and CNR1 associate with the NR1 subunits via HINT1: Implications for NMDAR activity. (A) The association of GPCRs, MOR and CNR1 with the NMDAR NR1 subunits is dependent on HINT1. The GPCRs were immunoprecipitated from solubilized mouse brain cortical synaptosomes from WT and HINT1 -/- mice, and the proteins that co-precipitated with the NR1 subunits were analyzed by western blot. (B) The association of CNR1 with NR1 in WT and HINT1 -/- cortical cell cultures uninfected or infected with 10 μL/well of HINT1 -/- lentiviral particles. The presence/absence of HINT1 was determined, and the co-precipitates of the NR1 subunits and CNR1 were then assessed by immunoprecipitation and western blot. For each determination, cells from 10 wells were pooled, and the assay was repeated twice with identical results. (C) Opioid agonists promoted a higher level of responsiveness of the NMDAR by inhibiting the association between MOR and NR1. Morphine or DAMGO was icv-injected into the mice 24 h prior to the analysis of MOR/NMDA NR1 association and CaMKII activating autophosphorylation on Thr286. At 7 days post-surgery, the mice from the CCI neuropathic pain group displayed increased pThr286 CaMKII and reduced MOR-NR1 association compared to the sham-operated controls. Changes of Mechanical Withdrawal Threshold . Following ligation, animals developed significant mechanical allodynia by day 3 that remained until day 21. Naïve control and sham – operated mice failed to exhibit mechanical allodynia. Data are mean ± SEM. *P

    Techniques Used: Activity Assay, Immunoprecipitation, Mouse Assay, Western Blot, Infection, Injection, Ligation

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    Article Snippet: .. U-69593, DAMGO and nociceptin (Tocris Bioscience) — which are a KOP, a MOP and a NOP receptor agonist, respectively — were included as the positive control. .. To evaluate AC superactivation, desired concentrations of drugs were added to the compound buffer and incubated at 37°C for 4 hrs; the compound buffer was then replaced by 10 μM forskolin with 1 μM naloxone.

    Blocking Assay:

    Article Title: μ and κ Opioid receptor distribution in the monogamous titi monkey (Callicebus cupreus): Implications for social behavior and endocrine functioning
    Article Snippet: .. Sections were dipped in Tris buffer (50mM Tris base; pH 7.4) for 60 minutes at room temperature then incubated for 90 minutes in 3.0 nM [3 H]U69,593 (PerkinElmer, Inc., Boston, MA) and co-incubated with 400nM of DPDPE and 400nM DAMGO (Tocris Bioscience, Minneapolis, MN) at room temperature to block DOR and MOR, respectively. ..

    Concentration Assay:

    Article Title: Regional Differences in Mu and Kappa Opioid Receptor G-protein Activation in Brain in Male and Female Prairie Voles
    Article Snippet: .. Concentration-effect curves of agonist-stimulated [35 S]GTPγS binding included 0.01–10 μM DAMGO (D-Ala2 ,NMe-Phe4 ,Gly-ol5 ]-enkephalin, Tocris Bioscience, Ellisville, MO) or U-50,488H ( trans -(-)-3,4-dichloro- N -methyl- N -[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide HCl, Tocris Bioscience), 30 μM GDP, 0.05 nM [35 S]GTPγS (1200 Ci/mmol, Perkin Elmer, Waltham, MA), 100 nM DPCPX (8-cyclopentyl-1,3-dipropylxanthine, an adenosine A1 receptor antagonist) , 5 μg membrane protein and TME assay buffer in a final volume of 1 ml. .. Basal binding was determined in the presence of GDP and absence of drug, and nonspecific binding was assessed in the presence of 10 μM GTPγS.

    Incubation:

    Article Title: μ and κ Opioid receptor distribution in the monogamous titi monkey (Callicebus cupreus): Implications for social behavior and endocrine functioning
    Article Snippet: .. Sections were dipped in Tris buffer (50mM Tris base; pH 7.4) for 60 minutes at room temperature then incubated for 90 minutes in 3.0 nM [3 H]U69,593 (PerkinElmer, Inc., Boston, MA) and co-incubated with 400nM of DPDPE and 400nM DAMGO (Tocris Bioscience, Minneapolis, MN) at room temperature to block DOR and MOR, respectively. ..

    other:

    Article Title: The orphan receptor GPR88 blunts the signaling of opioid receptors and multiple striatal GPCRs
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    Article Title: Ligand-Directed Functional Selectivity at the Mu Opioid Receptor Revealed by Label-Free Integrative Pharmacology On-Target
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    Binding Assay:

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    Tocris drugs damgo
    Spinal μ-opioid receptor <t>(MOR)</t> activation reduces nociceptive responses in all ages. (A) Typical electromyographic (EMG) traces in postnatal day (P)10, P21, and adult rats during baseline (without any stimulation), predrug (a typical threshold response before application of drug) and postdrug (A: <t>DAMGO</t> 30 ng; B: CTOP 120 ng) periods. The application of MOR opioid agonist DAMGO onto the spinal cord produced antinociceptive responses in all ages tested. Spinal reflex excitability (C) was decreased when compared to age-matched saline and predrug responses in all ages, both saline and CTOP had no significant effect. Mechanical threshold (D) was increased when DAMGO was administered in P10 and P21 rats. ++ , ++++ P
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    Spinal μ-opioid receptor (MOR) activation reduces nociceptive responses in all ages. (A) Typical electromyographic (EMG) traces in postnatal day (P)10, P21, and adult rats during baseline (without any stimulation), predrug (a typical threshold response before application of drug) and postdrug (A: DAMGO 30 ng; B: CTOP 120 ng) periods. The application of MOR opioid agonist DAMGO onto the spinal cord produced antinociceptive responses in all ages tested. Spinal reflex excitability (C) was decreased when compared to age-matched saline and predrug responses in all ages, both saline and CTOP had no significant effect. Mechanical threshold (D) was increased when DAMGO was administered in P10 and P21 rats. ++ , ++++ P

    Journal: Pain

    Article Title: Postnatal maturation of endogenous opioid systems within the periaqueductal grey and spinal dorsal horn of the rat

    doi: 10.1016/j.pain.2013.09.022

    Figure Lengend Snippet: Spinal μ-opioid receptor (MOR) activation reduces nociceptive responses in all ages. (A) Typical electromyographic (EMG) traces in postnatal day (P)10, P21, and adult rats during baseline (without any stimulation), predrug (a typical threshold response before application of drug) and postdrug (A: DAMGO 30 ng; B: CTOP 120 ng) periods. The application of MOR opioid agonist DAMGO onto the spinal cord produced antinociceptive responses in all ages tested. Spinal reflex excitability (C) was decreased when compared to age-matched saline and predrug responses in all ages, both saline and CTOP had no significant effect. Mechanical threshold (D) was increased when DAMGO was administered in P10 and P21 rats. ++ , ++++ P

    Article Snippet: Drugs DAMGO (MOR-agonist, 30 ng; Tocris, Abingdon, Oxon, UK) and CTOP (MOR-antagonist, 100 ng; Tocris) were administered at doses determined from previously published studies in adult brainstem .

    Techniques: Activation Assay, Produced

    Recruitment of β-arrestin2 by activation of μ-opioid receptors. Stably transfected HEK293 cells coexpressing μ-opioid receptors and β-arrestin2 were pretreated for 60 min with DAMGO or PnPP-19 at the indicated concentrations. The group “DAMGO + PnPP-19” represents prior incubation of the cells with 10 μM of PnPP-19 for 30 min. β-arrestin2 recruitment was quantified by high content imaging complementation assay as described in Materials and Methods ( n = 5).

    Journal: Toxins

    Article Title: The Peptide PnPP-19, a Spider Toxin Derivative, Activates μ-Opioid Receptors and Modulates Calcium Channels

    doi: 10.3390/toxins10010043

    Figure Lengend Snippet: Recruitment of β-arrestin2 by activation of μ-opioid receptors. Stably transfected HEK293 cells coexpressing μ-opioid receptors and β-arrestin2 were pretreated for 60 min with DAMGO or PnPP-19 at the indicated concentrations. The group “DAMGO + PnPP-19” represents prior incubation of the cells with 10 μM of PnPP-19 for 30 min. β-arrestin2 recruitment was quantified by high content imaging complementation assay as described in Materials and Methods ( n = 5).

    Article Snippet: Plates were kept in a humidified incubator at 37 °C filled with 5% CO2 for 24 h. HEK293T were stimulated with the selective opioid agonist DAMGO (Tocris, Minneapolis, MN, USA) or the synthetic peptide PnPP-19 in HEPES-buffered saline solution (HBSS) including 0.1% v / v BSA (10−10 M–10−4 M) for 60 min at 37 °C.

    Techniques: Activation Assay, Stable Transfection, Transfection, Incubation, Imaging

    GPR88 inhibits opioid receptor signaling and trafficking in vitro. ( A ) We assessed whether the polyclonal anti-HA HRP-conjugated antibody allowed to satisfyingly detect modifications in the cell surface expression of HA-tagged opioid receptors. ( a ) The antibody displayed a linear response to increasing amounts of HA-tagged GPCR-expressing cells in the 50,000 to 200,000 range (n = 2–4 per receptor, in triplicates). Further tests used 50,000 cells per assay (black arrow). ( b ) When the cell number was set at 50,000, the anti-HA HRP-conjugated antibody allowed reliable detection of increasing amounts of transfected HA-tagged µOR (n = 4, in triplicates). Further used 30 ng of HA-tagged GPCR cDNA for transfection (black arrow). ( c ) At the 1:10,000 concentration (black arrow), the antibody allows optimal detection of HA-µOR (n = 1, in triplicates). This concentration was used for further testing. ( B ) Increasing amounts of GPR88 cDNA did not affect HA-δOR cell surface expression (left panel) but blunted SNC80 (10 µM)-induced Ypet-β-arrestin1 (β-arr1) recruitment and δOR-Rluc8 trafficking (early endosome sensor: Rab5-Venus; late endosome sensor: Rab7-Venus) in HEK293FT cells. ( C ) Confocal microscopy images show ( a ) HA-GPR88 expression at cell surface, ( b ) HA-δOR localized at cell surface, ( c ) internalized under SNC80-stimulation, ( d ) HA-δOR (red) and GPR88-Venus (green) co-localized at cell surface, ( e ) GPR88 inhibition of δOR internalization with cell nuclei stained using DAPI coloration. ( D ) Co-expressing GPR88 with HA-κOR did not affect κOR cell surface expression but blunted U50488H (10 µM)-induced Ypet-β-arr1 recruitment and κOR trafficking (early endosome sensor: Rab5-Venus) in HEK293FT cells. ( E ) Co-expressing GPR88 with HA-µOR did not affect µOR cell surface expression but blunted DAMGO (10 µM)-induced Ypet−β-arr1 recruitment and µOR-Rluc8 trafficking (early endosome sensor: Rab5-Venus; late endosome sensor: Rab7-Venus) in HEK293FT cells. Data ( B–E ) are presented as mean ± SEM of n = 3–5 independent experiments (performed in triplicates). BRET1 values are presented as induced BRET (normalized as the percentage of maximal BRET values in absence of GPR88) by Venus/Rluc8 BRET ratio. Asterisks: Kruskal-Wallis ANOVA, multiple comparison of mean ranks *p

    Journal: eLife

    Article Title: The orphan receptor GPR88 blunts the signaling of opioid receptors and multiple striatal GPCRs

    doi: 10.7554/eLife.50519

    Figure Lengend Snippet: GPR88 inhibits opioid receptor signaling and trafficking in vitro. ( A ) We assessed whether the polyclonal anti-HA HRP-conjugated antibody allowed to satisfyingly detect modifications in the cell surface expression of HA-tagged opioid receptors. ( a ) The antibody displayed a linear response to increasing amounts of HA-tagged GPCR-expressing cells in the 50,000 to 200,000 range (n = 2–4 per receptor, in triplicates). Further tests used 50,000 cells per assay (black arrow). ( b ) When the cell number was set at 50,000, the anti-HA HRP-conjugated antibody allowed reliable detection of increasing amounts of transfected HA-tagged µOR (n = 4, in triplicates). Further used 30 ng of HA-tagged GPCR cDNA for transfection (black arrow). ( c ) At the 1:10,000 concentration (black arrow), the antibody allows optimal detection of HA-µOR (n = 1, in triplicates). This concentration was used for further testing. ( B ) Increasing amounts of GPR88 cDNA did not affect HA-δOR cell surface expression (left panel) but blunted SNC80 (10 µM)-induced Ypet-β-arrestin1 (β-arr1) recruitment and δOR-Rluc8 trafficking (early endosome sensor: Rab5-Venus; late endosome sensor: Rab7-Venus) in HEK293FT cells. ( C ) Confocal microscopy images show ( a ) HA-GPR88 expression at cell surface, ( b ) HA-δOR localized at cell surface, ( c ) internalized under SNC80-stimulation, ( d ) HA-δOR (red) and GPR88-Venus (green) co-localized at cell surface, ( e ) GPR88 inhibition of δOR internalization with cell nuclei stained using DAPI coloration. ( D ) Co-expressing GPR88 with HA-κOR did not affect κOR cell surface expression but blunted U50488H (10 µM)-induced Ypet-β-arr1 recruitment and κOR trafficking (early endosome sensor: Rab5-Venus) in HEK293FT cells. ( E ) Co-expressing GPR88 with HA-µOR did not affect µOR cell surface expression but blunted DAMGO (10 µM)-induced Ypet−β-arr1 recruitment and µOR-Rluc8 trafficking (early endosome sensor: Rab5-Venus; late endosome sensor: Rab7-Venus) in HEK293FT cells. Data ( B–E ) are presented as mean ± SEM of n = 3–5 independent experiments (performed in triplicates). BRET1 values are presented as induced BRET (normalized as the percentage of maximal BRET values in absence of GPR88) by Venus/Rluc8 BRET ratio. Asterisks: Kruskal-Wallis ANOVA, multiple comparison of mean ranks *p

    Article Snippet: Chemical and drugs DAMGO, U50488H, SNC80, CGS21680, AVP (arginine vasopressin), IBMX, Forskolin, stromal cell-derived factor 1 (SDF-1 or CXCL12), carbamoylcholine chloride (carbachol), quinpirole hydrochloride, SKF 81297, ionomycin calcium salt, were purchased from Tocris Bioscience (Bristol, UK) and diluted in DMSO (diméthylsulfoxyde) at 10−2 M (except SDF1 at 125 µM and IBMX at 200 mM) for frozen stock aliquots and coelenterazine H substrate from Interchim (Montluçon, France) was diluted in 100% ethanol and kept at −20°C.

    Techniques: In Vitro, Expressing, Transfection, Concentration Assay, Confocal Microscopy, Inhibition, Staining, Bioluminescence Resonance Energy Transfer

    Selective and non-specific binding for MOR and KOR autoradiography. 1A) Selective binding of MOR using [ 3 H]DAMGO blocking DOR and KOR by co-incubating with DPDPE and U69,593, respectively 1B) Non-specific binding of MOR with [ 3 H]DAMGO and co-incubating

    Journal: Neuroscience

    Article Title: μ and κ Opioid receptor distribution in the monogamous titi monkey (Callicebus cupreus): Implications for social behavior and endocrine functioning

    doi: 10.1016/j.neuroscience.2015.01.023

    Figure Lengend Snippet: Selective and non-specific binding for MOR and KOR autoradiography. 1A) Selective binding of MOR using [ 3 H]DAMGO blocking DOR and KOR by co-incubating with DPDPE and U69,593, respectively 1B) Non-specific binding of MOR with [ 3 H]DAMGO and co-incubating

    Article Snippet: Sections were dipped in Tris buffer (50mM Tris base; pH 7.4) for 60 minutes at room temperature then incubated for 90 minutes in 3.0 nM [3 H]U69,593 (PerkinElmer, Inc., Boston, MA) and co-incubated with 400nM of DPDPE and 400nM DAMGO (Tocris Bioscience, Minneapolis, MN) at room temperature to block DOR and MOR, respectively.

    Techniques: Binding Assay, Autoradiography, Blocking Assay