Figure 4.

Inhibition of C-Raf partially restores opioid sensitivity after depolarization. A, Inhibition of 15 mm [K⁺]_e effects on DAMGO responses. DRG neurons were preincubated with inhibitors of Src (saracatinib, 10 μm), PKC (sotrastaurin, 1 μm), PKA (H89, 10 μm), C/B-Raf (RAF709, 10 μm), C-Raf (GW5074, 3 μm), or MEK1/2 (UO126, 10 μm) for 30 min and then stimulated with Fsk (3 μm) ± DAMGO (0.1 μm) in control media or 15 mm [K⁺]_e (K⁺15; 5 min). Effects are reported as inhibition of the control Fsk response (1.0). Drug effects were compared against the Control-DAMGO inhibition (*) or K⁺-DAMGO inhibition (#) via one-way ANOVA, followed by Dunnett's test. Significance and n values are reported on each bar. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ^##p< 0.01. B, C-Raf inhibition restores DAMGO IC₅₀ in 15 mm [K⁺]_e, but has no effect on DAMGO efficacy (E_max). GW5074 (10 μm) was applied 30 min before simultaneous addition of K⁺(15 mM), Fsk(3 mm), and DAMGO for 5 min. Per data point, Control, n = 10-21; K⁺15, n = 6-22; K⁺15+GW5074, n = 3-10; Control+GW5074, n = 3-11. Treatment effects were compared using two-way ANOVA followed by Tukey's multiple comparisons test. Individual data point comparisons using Sidak's test can be found in Table 1. C, D, Quantification of individual IC₅₀ (C) and E_max (D) values of DAMGO dose–response curves as described in B. C, Control, n = 13; K⁺15(–), n = 10; K⁺15(GW), n = 4. D, Control, n = 14; K⁺15(–), n = 10; K⁺15(GW), n = 4. Data were compared using one-way ANOVA, followed by Tukey's test. Box-and-whisker plot represents the median, mean (+), quartiles, and range of the data. Detailed statistical information is provided in Table 1.