Journal: Pharmacognosy Research

Article Title: Effects of mitragynine and 7-hydroxymitragynine (the alkaloids of Mitragyna speciosa Korth) on 4-methylumbelliferone glucuronidation in rat and human liver microsomes and recombinant human uridine 5’-diphospho-glucuronosyltransferase isoforms

doi: 10.4103/0974-8490.159580

Figure Lengend Snippet: Inhibitory effects of mitragynine, 7-hydroxymitragynine, ketamine, buprenorphine, and diclofenac on 4-methylumbelliferone glucuronide formation by rat liver microsomes. Panels to the right show inhibitor concentrations. Each bar represents the mean percentage activity relative to control ± standard error of mean for three independent measurements ( n = 3). Statistical analysis was conducted using one-way ANOVA and Dunnett test. * P < 0.05 versus control (no inhibitor)

Article Snippet: Ketamine hydrochloride and buprenorphine hydrochloride were purchased from Toronto Research Chemicals Inc. (North York, ON, Canada).

Techniques: Activity Assay, Control

Journal: Pharmacognosy Research

Article Title: Effects of mitragynine and 7-hydroxymitragynine (the alkaloids of Mitragyna speciosa Korth) on 4-methylumbelliferone glucuronidation in rat and human liver microsomes and recombinant human uridine 5’-diphospho-glucuronosyltransferase isoforms

doi: 10.4103/0974-8490.159580

Figure Lengend Snippet: Inhibitory effects of mitragynine, 7-hydroxymitragynine, ketamine, buprenorphine, and diclofenac on 4-methylumbelliferone glucuronide formation by recombinant human UGT2B7 isoform. Panels to the right show inhibitor concentrations. Each bar represents the mean percentage activity relative to control ± standard error of mean for three independent measurements ( n = 3). Statistical analysis was conducted using one-way ANOVA and Dunnett test. * P < 0.05 versus control (no inhibitor)

Article Snippet: Ketamine hydrochloride and buprenorphine hydrochloride were purchased from Toronto Research Chemicals Inc. (North York, ON, Canada).

Techniques: Recombinant, Activity Assay, Control

Journal: Pharmacognosy Research

Article Title: Effects of mitragynine and 7-hydroxymitragynine (the alkaloids of Mitragyna speciosa Korth) on 4-methylumbelliferone glucuronidation in rat and human liver microsomes and recombinant human uridine 5’-diphospho-glucuronosyltransferase isoforms

doi: 10.4103/0974-8490.159580

Figure Lengend Snippet: Inhibitory effects of mitragynine, 7-hydroxymitragynine, ketamine, buprenorphine, and diclofenac on 4-methylumbelliferone glucuronide formation by human liver microsomes. Panels to the right show inhibitor concentrations. Each bar represents the mean percentage activity relative to control ± standard error of mean for three independent measurements ( n = 3). Statistical analysis was conducted using one-way ANOVA and Dunnett test. * P < 0.05 versus control (no inhibitor)

Article Snippet: Ketamine hydrochloride and buprenorphine hydrochloride were purchased from Toronto Research Chemicals Inc. (North York, ON, Canada).

Techniques: Activity Assay, Control

Journal: Pharmacognosy Research

Article Title: Effects of mitragynine and 7-hydroxymitragynine (the alkaloids of Mitragyna speciosa Korth) on 4-methylumbelliferone glucuronidation in rat and human liver microsomes and recombinant human uridine 5’-diphospho-glucuronosyltransferase isoforms

doi: 10.4103/0974-8490.159580

Figure Lengend Snippet: Inhibitory effects of mitragynine, 7-hydroxymitragynine, ketamine, buprenorphine, and diclofenac on 4-methylumbelliferone glucuronide formation by recombinant human UGT1A1 isoform. Panels to the right show inhibitor concentrations. Each bar represents the mean percentage activity relative to control ± standard error of mean for three independent measurements ( n = 3). Statistical analysis was conducted using one-way ANOVA and Dunnett test. * P < 0.05 versus control (no inhibitor)

Article Snippet: Ketamine hydrochloride and buprenorphine hydrochloride were purchased from Toronto Research Chemicals Inc. (North York, ON, Canada).

Techniques: Recombinant, Activity Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Opioid-Modulated Receptor Localization and Erk1/2 Phosphorylation in Cells Coexpressing μ-Opioid and Nociceptin Receptors

doi: 10.3390/ijms24021048

Figure Lengend Snippet: HEK 293 cells transiently transfected with MOP-CFP and NOP-YFP were serum-starved overnight; vehicle (ddH 2 O), morphine (1 μM), or buprenorphine (1 μM) was added to cells immediately before FRET/SPT measurements. ( A – C ) present the results obtained under the conditions of pre-treatment, post-bup, and post-mor, respectively. The images of MOP-CPF (shown in cyan), NOP-YFP (shown in yellow), FRET ratio (%), and a magnified view from a white box in the respective FRET ratio image are shown for each condition from left to right. ( D ) is the control image taken from the MOP-CFP-channel at the pretreatment condition, which is used to confirm whether the receptors are properly expressed on the cell surface. ( E ) The histograms of the FRET ratio obtained from each condition were fitted to Gaussian distributions, indicating the peak value with a standard deviation. Scale bar = 20 μm. The bin size of the histogram = 0.01. Twelve cells were used for statistical analysis in each condition.

Article Snippet: Morphine (1 μM, Toronto Research Chemicals, North York, ON, Canada) or buprenorphine (1 μM, Toronto Research Chemicals) was added to cells shortly before starting FRET/SPT measurements and present in the serum-free medium throughout the whole experiment.

Techniques: Transfection, Control, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Opioid-Modulated Receptor Localization and Erk1/2 Phosphorylation in Cells Coexpressing μ-Opioid and Nociceptin Receptors

doi: 10.3390/ijms24021048

Figure Lengend Snippet: Representative confocal images ( A ) and colocalization rates of MOP and NOP on lipid rafts (red) ( B ) from HEK 293 cells stably expressing HA-tagged MOP (green) and/or myc-tagged NOP (magenta) after exposure to morphine (1 μM) or buprenorphine (1 μM) for 20 min at 4 °C. The inset panels display higher magnification images highlighting the colocalization. Scale bars are equal to 10 μm. Each bar represents the mean ± SE value of the percentage of ROI colocalization derived from five independent experiments with 4–6 individual cell images in each group.

Article Snippet: Morphine (1 μM, Toronto Research Chemicals, North York, ON, Canada) or buprenorphine (1 μM, Toronto Research Chemicals) was added to cells shortly before starting FRET/SPT measurements and present in the serum-free medium throughout the whole experiment.

Techniques: Stable Transfection, Expressing, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Opioid-Modulated Receptor Localization and Erk1/2 Phosphorylation in Cells Coexpressing μ-Opioid and Nociceptin Receptors

doi: 10.3390/ijms24021048

Figure Lengend Snippet: Representative immunoblots of Erk1/2 (p44/p42 MAPK) phosphorylation after stimulation with ( A ) morphine and ( B ) buprenorphine in cells stably expressing MOP, NOP, and MOP+NOP receptors. Immunoblotting using anti-phospho-p44/p42 MAPK (Erk1/2) rabbit polyclonal and anti-p44/42 MAPK (Erk1/2) rabbit monoclonal antibodies was performed to visualize opioid-induced phosphorylation of Erk1/2 (p44 MAPK-P and p42 MAPK-P) and total Erk1/2 (p44 MAPK and p42 MAPK), respectively. ( C ) Dose–response curves of the immunoblots. The density of phospho-p44/p42 MAPK versus the density of p44/42 MAPK in control cells (with vehicle treatment) was considered to be 1. All the experiments were repeated no less than four times, and the values represent the average ± SE.

Article Snippet: Morphine (1 μM, Toronto Research Chemicals, North York, ON, Canada) or buprenorphine (1 μM, Toronto Research Chemicals) was added to cells shortly before starting FRET/SPT measurements and present in the serum-free medium throughout the whole experiment.

Techniques: Western Blot, Phospho-proteomics, Stable Transfection, Expressing, Bioprocessing, Control

Journal: Science signaling

Article Title: Agonist-selective NOP receptor phosphorylation correlates in vitro and in vivo and reveals differential post-activation signaling by chemically diverse agonists

doi: 10.1126/scisignal.aau8072

Figure Lengend Snippet: (A) HEK293 cells stably expressing HA-tagged hNOP receptors were preincubated with HA antibody and then stimulated with 10 μM N/OFQ, Ro64–6198, MCOPPB, SCH221510, NNC 63–0532, AT-202, buprenorphine (BUP), norbuprenorphine (norBUP), cebranopadol or vehicle (according to the solvent) for 60 min at 22 °C. Cells were fixed, permeabilized, immunofluorescently stained, and subsequently examined using confocal microscopy. Images are representative, n=3 independent experiments. Scale bar, 20 μm. (B) NOP receptor-expressing HEK293 cells were treated with the compounds listed in (A) (–, vehicle solvent) at concentrations ranging from 10−9 to 10−5 M for 10 min at 37 °C, and lysates were immunoblotted with antibodies to pSer346, pSer351, or pThr362/Ser363. Blots were stripped and reprobed for NOPR. Blots are representative of n=3–5 experiments. (C) HEK293 cells stably expressing hNOP receptors were preincubated with antibody to HA-tag and treated with vehicle (solvent) or 10 μM N/OFQ, Ro64–6198, MCOPPB, SCH221510, NNC 63–0532, AT-202, BUP, norBUP or cebranopadol for 60 min at 37 °C. Cells were fixed and labeled with a peroxidase-conjugated secondary antibody. Receptor internalization was measured by ELISA and quantified as the percentage of internalized receptors in agonist-treated cells. Data are means ± SEM from twelve independent experiments performed in quadruplicate. (D) Maximum NOP receptor ligand-induced phosphorylation at Thr362/Ser363 from data in (B). Data are mean ± SEM from three independent experiments. *P<0.05 vs. N/OFQ by two-way ANOVA with Bonferroni post-test. (E) Correlation between NOP receptor phosphorylation and internalization induced by different ligands in HEK293 cells, from data in (A to D). Abscissae: ligand-induced internalization in percentage (normalized to N/OFQ). Ordinates: ligand-induced phosphorylation in percentage (normalized to N/OFQ). Solid line, linear regression of the data points; correlation coefficient r = 0.8581.

Article Snippet: Norbuprenorphine (BUP-982-FB) and buprenorphine (BUP-399-HC) were purchased from Lipomed. γ-Endorphin (60893–02-9) was obtained from Bachem and tramadol from Grunenthal.

Techniques: Stable Transfection, Expressing, Staining, Confocal Microscopy, Labeling, Enzyme-linked Immunosorbent Assay