Journal: Genes to Cells
Article Title: Novel Ligands for the Orphan Receptor GPR151 Modulate Morphine Action
doi: 10.1111/gtc.70119
Figure Lengend Snippet: In vitro and in vivo characterization of GUM3. (a) Stimulation of [ 35 S]‐GTPγS binding in the presence of GUM3 in membrane fractions expressing GPR151‐Giα. Datapoints represent the mean ± standard error of the mean across three measurements and are representative of at least three independent experiments. The chemical structure of GUM3 is displayed in the box. (b) Reporter gene assay using transiently transfected CHO cells with an HA‐tagged GPR151 expression vector. Each datapoint represents mean ± standard error of the mean across three measurements, representative of at least three independent experiments. Representative images of GPR151 localization by fluorescence microscopy in transiently transfected CHO cells with HA‐tagged GPR151 expression vector treated for 6 h with (c) vehicle and (d) 100 μM GUM3, showing GPR151 internalization after GUM3 stimulation. The scale bars correspond to 10 μm. (e) Effects of GUM3 on thermal pain in native rats. Rats were administered morphine (closed squares, N = 8), GUM3 (open circles, N = 8), or both morphine and GUM3 (closed circles, N = 7), and PWL to heat stimulation was measured. Data are expressed as mean ± standard error of the mean. Individual means for morphine alone versus morphine and GUM3 pairs were compared two‐way repeated‐measures ANOVA, followed by multiple comparisons. The p ‐value was corrected versus morphine alone using Bonferroni's method and was 0.0454, 0.0091, 0.0132, 0.0064 at 30, 60, 90, and 120 min, respectively. * p < 0.05, ** p < 0.01. (f) Time course of serum GUM3 concentrations. White circles and black circles represent individual rats, respectively. GUM3 was persistently present in serum during the antinociception test. Drug concentrations during antinociception test correlated closely with the active GUM3 concentrations observed in the CHO cell assay.
Article Snippet: In an earlier study, we successfully identified novel ligand compounds for GPCRs by expressing GPCR‐Gα fusion proteins in insect cells and using them in [ 35 S]‐GTPγS binding assays, which served as the screening system in the present study (Takeda et al. ).
Techniques: In Vitro, In Vivo, Binding Assay, Membrane, Expressing, Reporter Gene Assay, Transfection, Plasmid Preparation, Fluorescence, Microscopy
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![In vitro and in vivo characterization of GUM3. (a) Stimulation of [ <t>35</t> <t>S]‐GTPγS</t> binding in the presence of GUM3 in membrane fractions expressing GPR151‐Giα. Datapoints represent the mean ± standard error of the mean across three measurements and are representative of at least three independent experiments. The chemical structure of GUM3 is displayed in the box. (b) Reporter gene assay using transiently transfected CHO cells with an HA‐tagged GPR151 expression vector. Each datapoint represents mean ± standard error of the mean across three measurements, representative of at least three independent experiments. Representative images of GPR151 localization by fluorescence microscopy in transiently transfected CHO cells with HA‐tagged GPR151 expression vector treated for 6 h with (c) vehicle and (d) 100 μM GUM3, showing GPR151 internalization after GUM3 stimulation. The scale bars correspond to 10 μm. (e) Effects of GUM3 on thermal pain in native rats. Rats were administered morphine (closed squares, N = 8), GUM3 (open circles, N = 8), or both morphine and GUM3 (closed circles, N = 7), and PWL to heat stimulation was measured. Data are expressed as mean ± standard error of the mean. Individual means for morphine alone versus morphine and GUM3 pairs were compared two‐way repeated‐measures ANOVA, followed by multiple comparisons. The p ‐value was corrected versus morphine alone using Bonferroni's method and was 0.0454, 0.0091, 0.0132, 0.0064 at 30, 60, 90, and 120 min, respectively. * p < 0.05, ** p < 0.01. (f) Time course of serum GUM3 concentrations. White circles and black circles represent individual rats, respectively. GUM3 was persistently present in serum during the antinociception test. Drug concentrations during antinociception test correlated closely with the active GUM3 concentrations observed in the CHO cell assay.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6533/pmc13156533/pmc13156533__GTC-31-0-g014.jpg)
![Evaluation of GUM4, as described in Figure . (a) [ 35 S]‐GTPγS binding assay. The chemical structure of GUM4 is displayed in the box. (b) Reporter gene assay. (c) GUM4‐stimulated intracellular translocation. The scale bar corresponds to 10 μm. (d) Effects on thermal pain in native rats. Rats were administered morphine (closed squares, N = 8), GUM4 (open circles, N = 6), or a combination of morphine and GUM4 (closed circles, N = 9), and PWL to heat stimulation was measured. Data are expressed as mean ± standard error of the mean. Individual means for morphine alone versus morphine and GUM4 pairs were compared two‐way repeated‐measures ANOVA, followed by multiple comparisons. The p ‐value was corrected versus morphine alone using Bonferroni's method and was less than 0.0001 at 30 min. ** p < 0.01. (e) Time course of serum GUM4 concentrations. White circles and black circles represent individual rats, respectively. GUM4 was persistently present in serum during the antinociception test. Drug concentrations during antinociception test correlated closely with the active GUM4 concentrations observed in the CHO cell assay.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6533/pmc13156533/pmc13156533__GTC-31-0-g002.jpg)