Article Figures & Data
Figures
- Figure 1.
Paroxetine enhances Delta Opioid Receptor (DOR) functional competence in sensory neurons. A, Concentration response curve for paroxetine ([M], 10 min) effect on GRK2 Co-IP with DOR in crude membrane fractions from serum-starved TG cultures [IC50 = 3.840 μm (vertical black dotted line), least squares fit (best-fit) variable slope curve (black line), ANOVA summary: F(5,18) = 2.932, p = 0.415, one-way ANOVA with Bonferroni post hoc, mean ± SEM, n = 4 independent trials from 24 TG/12 total rats]. B, GRK2 Co-IP with membrane-associated DOR in serum-starved TG cultures treated for 10 min with vehicle (VEH; DMSO), paroxetine (PRX; 5 μm), CMPD101 (0.5 μm), or fluoxetine (FLX; 5 μm; *p < 0.05, ns = not significant, ANOVA summary: F(3,12) = 8.805, p = 0.0023, one-way ANOVA with Bonferroni post hoc, mean ± SEM, n = 4 independent trials from 24TG/12 total rats). C, GRK2 phosphorylation at Ser685 isolated by GRK2 IP in serum-starved TG cultures treated for 10 min with vehicle (VEH; DMSO), paroxetine (PRX; 5 μm), CMPD101 (0.5 μm), or fluoxetine (FLX; 5 μm; **p < 0.01, ns = not significant, ANOVA summary: F(3,12) = 7.328, p = 0.0047, one-way ANOVA with Bonferroni post hoc test, mean ± SEM, n = 4 independent trials from 24 TG/12 total rats). D–F, Cumulative (C) mean experimental traces, (D) average KCl response traces, and (E) quantification of DPDPE (1 μm) inhibition of KCl (50 mm)-evoked Ca2+ influx in CAP (1 μm)-sensitive serum-starved DRG pretreated for 10 min with vehicle (VEH; DMSO), paroxetine (PRX; 5 μm), CMPD101 (0.5 μm), or fluoxetine (FLX; 5 μm; ***p < 0.005, **p < 0.01, ns = not significant, ANOVA summary: F(3,92) = 18.83, p < 0.0001, one-way ANOVA with Bonferroni post hoc, mean ± SEM, n = 21–27 DRG/group collected from a minimum of 5 rats).
- Figure 2.
Paroxetine enhances Delta Opioid Receptor (DOR) functional competence via GRK2-dependent mechanism. A–C, Cumulative (A) mean experimental traces, (B) average KCl response traces, and (C) quantification of DPDPE (1 μm) inhibition of KCl (50 mm)-evoked Ca2+ influx in CAP (1 μm)-sensitive serum-starved nucleofected DRG pretreated with vehicle (VEH; DMSO) or paroxetine (PRX; 5 μm) for 10 min [***p ≤ 0.005, ns = not significant, ANOVA summary: Interaction: F(1,82) = 11.72, p = 0.0010, Overexpression (E.V. vs GRK2): F(1,82) = 9.900, p = 0.0023, Pretreatment (VEH vs PRX): F(1,82) = 19.12, p < 0.0001, two-way ANOVA with Bonferroni post hoc, mean ± SEM, n = 19–24 DRG/group collected from a minimum of 5 rats]. D–F, Cumulative (D) mean experimental traces, (E) average KCl response traces, and (F) quantification of DPDPE (1 μm) inhibition of KCl (50 mm)-evoked Ca2+ influx in CAP (1 μm)-sensitive serum-starved transfected DRG pretreated with vehicle (VEH; DMSO) or paroxetine (PRX; 5 μm) for 10 min [**p ≤ 0.01, ns = not significant, ANOVA summary: Interaction: F(1,87) = 6.377, p = 0.0134, Transfection (Mock vs FITC-GRK2 siRNA): F(1,87) = 6.141, p = 0.0151, Pretreatment (VEH vs PRX): F(1,87) = 21.27, p < 0.0001, two-way ANOVA with Bonferroni post hoc, mean ± SEM, n = 19–26 DRG/group collected from a minimum of 5 rats].
- Figure 3.
Paroxetine targets GRK2 to modulate of DOR-mediated antinociception. A, B, DPDPE (20 μg) inhibition of PGE2 (0.3 μg)-induced mechanical allodynia in (A) ipsilateral and (B) contralateral hindpaws following systemic treatment. Readings were collected at 5-min intervals for 20 min following initial (intraperitoneal) injection [black arrow, vehicle (VEH; 10%DMSO/90%DPBS), paroxetine (PRX; 5.0 mg/kg), CMPD101 (0.5 mg/kg), or fluoxetine (FLX; 5.0 mg/kg)] and a second (i.pl.) injection (gray arrow, co-injection DPDPE (20 μg)/PGE2 (0.3 μg); DPDPE inhibition of PGE2-induced allodynia: A, VEH versus PRX (***p < 0.005 at 30, 35 min, **p < 0.01 at 40, 45 min) or CMPD1010 (**p < 0.01 at 30 min, ***p < 0.005 at 35, 40, 45 min), FLX versus PRX (**p < 0.01 at 30 and ***p < 0.005 at 35) and CMPD101 (***p < 0.005 at 35); ipsilateral ANOVA summary: Interaction: F(24,180) = 4.358, p = 0.0001, Treatment: F(3,180) =19.16, p = 0.0001, Time: F(8,180) = 14.72, p = 0.0001; repeated measures two-way ANOVA Bonferroni post hoc; mean ± SEM, n = 6 rats/group. C, D, DPDPE (20 μg) inhibition of PGE2 (0.3 μg)-induced mechanical allodynia in (C) ipsilateral and (D) contralateral hindpaws following the timeline described above, but initial injection was peripherally-administered [i.pl., VEH or PRX (1.50 μg or 150 μg); C, PRX-induced allodynia: VEH vs 1.50 μg (**p < 0.01 at 5 min) or 150 μg (***p < 0.005 at 5 min; *p < 0.05 at 10–15 min); DPDPE inhibition of PGE2-induced allodynia: VEH vs 1.50 μg (*p < 0.05 at 30 min, **p < 0.01 at 35 min); ipsilateral ANOVA summary: Interaction: F(16,135) = 2.915, p = 0.0004, Treatment: F(2,135) = 11.21, p = 0.0001, Time: F(8,135) = 5.410, p = 0.0001; repeated measures two-way ANOVA Bonferroni post hoc; mean ± SEM, n = 6 rats/group]. E–G, Molecular changes 50 min following systemic (intraperitoneal) injection [vehicle (VEH; 10%DMSO/90%DPBS), paroxetine (PRX; 5.0 mg/kg), CMPD101 (0.5 mg/kg), or fluoxetine (FLX; 5.0 mg/kg)] in spinal cord (SC), dorsal root ganglia (DRG), trigeminal ganglia (TG), and midbrain (MB)]. PKA-dependent phosphorylation of GRK2 at Ser685 (E), GRK2 translocation (F), and GRK2 association with membrane-bound DOR [G; *p < 0.05, **p < 0.01, ANOVA summary (phosphoGRK2-Ser685): Interaction: F(9,27) = 1.830, p = 0.1085, Tissue: F(3,9) = 25.17, p = 0.0001, Treatment: F(3,27) = 8.393, p = 0.0004, Matching: F(9,27) = 2.272, p = 0.0480, ANOVA summary (GRK2 translocation): Interaction: F(9,27) = 1.441, p = 0.2202, Tissue: F(3,9) = 4.558, p = 0.0332, Treatment: F(3,27) = 7.930, p = 0.0006, Matching: F(9,27) = 3.103, p = 0.0108, ANOVA summary (DOR:GRK2 association): Interaction: F(9,27) = 2.025, p = 0.0758, Tissue: F(3,9) = 5.469, p = 0.0204, Treatment: F(3,27) = 3.468, p = 0.0299, Matching: F(9,27) = 4.691, p = 0.0008, n = 3–4 independent trials of tissue collected from 16 total rats, matched two-way ANOVA with Bonferroni correction]. See Extended Data Figure 3-1 for representative WB images.
Extended Data
Extended Data Figure 3-1
A, Representative WB images used to calculate values in Figure 3E. B, Representative WB images used to calculate values in Figure 3F. C, Representative WB images used to calculate values in Figure 3G. Download Figure 3-1, TIF file.
In this issue
