Fig. 2.

Endosomal DOPr signaling and nociceptor excitability. (A and B) Endocytosis of DOPr-eGFP in DRG neurons from DOPr-eGFP mice. Neurons were incubated with vehicle (Veh) or DADLE (1 µM, 30 min), and DOPr-eGFP was localized by immunofluorescence. Neurons were preincubated with vehicle, Dy4, or PS2. (A) Representative images from four independent experiments. Arrowheads denote plasma membrane; arrows, endosomal DOPr-eGFP. (B) Quantification of the proportion of total cellular DOPr-eGFP at the plasma membrane. Data points indicate the number of studied neurons (N). *P < 0.05, ***P < 0.001, two-way ANOVA with Tukey’s post hoc test. (C–L) Rheobase of mouse DRG neurons at 0 or 30 min after exposure to supernatant or DOPr agonists and washing. (C and D) Supernatant from cDSS, cUC, or HC biopsy specimens. (E–J) Neurons were incubated with the following agonists for 15 min and washed (W), and rheobase was measured at 0 or 30 min after washing: DOPr agonists SNC80 (E, 10 nM, internalizing), DADLE (F, 10 nM, internalizing) or ARM390 (G, 100 nM, weakly internalizing), and MOPr agonist DAMGO (H, 10 nM). In C–H, neurons were preincubated with Dy4, PS2, or vehicle. In I and J, neurons were preincubated with PKC inhibitor GF10923X or MEK1 inhibitor PD98059 before DADLE. (K and L) Neurons were incubated with the following agonists overnight and washed, and rheobase was measured at 0 or 30 min after washing: DADLE (K, 100 nM, internalizing) or ARM390 (L, 300 nM, weakly internalizing). Data points indicate the number of studied neurons from 12 to 16 mice in C, 6 mice in D, 10 to 15 mice in E–J, and 6 mice in K and L for each treatment (mean ± SEM). *P < 0.05, **P < 0.01, ***P < 0.001, one-way or two-way ANOVA with Tukey’s post hoc test.