Fig. 5
CCK-8 attenuates IA through Src-dependent JNK pathway. a CCK-8 increased the protein levels of phosphorylated JNK (p-JNK), with no significant changes in p-p38 and p-ERK. Representative western blots are shown from at least three independent experiments. b activation of JNK by CCK-8 is via CCK-BR and requires PI3K activity. Pretreatment of cells with LY225910 (1 μM) or LY294002 (20 μM) abolished CCK-8–induced JNK phosphorylation. Representative western blots are shown from at least three independent experiments. c, time course showing the effect of 100 nM CCK-8 on IA in the presence of SP600125. Insets show representative current traces. The Arabic numerals indicate the relative points utilized for exemplary current traces. d bar graph showing the effects of CCK-8 (100 nM) on IA in the presence of SP600125 (10 μM, n = 7), U0126 (1 μM, n = 9), and SB203580 (10 μM, n = 11), respectively. e Time course (left panel) and summary of results (right panel) indicating that application of anisomycin (25 ng/ml, n = 11) significantly decreased peak IA amplitude in small DRG neurons. f time course (left panel) and summary of results (right panel) indicating the effects of 100 nM CCK-8 on IA in the presence of KT-5720 (1 μM for 30 min, n = 10). Insets show exemplary current traces. The Arabic numerals indicate the relative points utilized for exemplary current traces. g bar graph showing the effects of 20 μM forskolin on PKA activity in DRG cells pretreated with KT-5720 (1 μM). The experiments were conducted in triplicate and yielded with similar results. h the level of phosphorylated Src (pTyr418, p-Src) increased following treatment with CCK-8 (100 nM). This effect was abolished by the PI3K inhibitor LY294002 (20 μM for 30 min). Representative western blots are shown from at least three independent experiments. i CCK-8-induced JNK activation is blocked by the Src inhibitor PP2 (10 μM). PP2 or its inactive structure analog PP3 (10 μM) was pre-administered for 30 min before CCK-8 addition. Representative western blots are shown from at least three independent experiments. j time course indicating the effects of CCK-8 on IA in the presence of 10 μM PP2. Insets show exemplary current traces. The Arabic numerals indicate the relative points utilized for exemplary current traces. k bar graph indicating that application of PP2 (10 μM for 30 min, n = 12), but not PP3 (10 μM for 30 min, n = 8), abolished the CCK-8-induced IA decrease. * p < 0.05 and ** p < 0.01 vs. control; # p < 0.05 vs. vehicle; & p < 0.05 vs. forskolin without KT-5720