Fig. 2.

Orexin A-mediated regulation of GnRH gene expression in GT1-7 cells involves the PKC, PKA and MAPK pathways. a GT1-7 cells were treated for 4 h with orexin A (0.001–10 nm) or vehicle alone (CONT). Total RNA was purified and reverse transcribed, and the level of GnRH expression was assayed by real-time PCR. In each sample, the amount of GnRH mRNA, calculated from the standard curve, was compared with the amount of GAPDH mRNA. Data represent the mean ± SEM of at least three experiments performed in triplicate. ^#Significant difference from CONT as determined by one-way ANOVA and Tukey’s post-hoc test, p < 0.05. b GT1-7 cells were treated for the different time points shown with orexin A (0.1 nm) or vehicle alone (CONT). Total RNA was purified and reverse transcribed, and the level of GnRH expression was assayed by real-time PCR. In each sample, the amount of GnRH, calculated from the standard curve, was compared with the amount of GAPDH. Data represent the mean ± SEM of at least three experiments performed in triplicate. ^#Significant difference from CONT as determined by one-way ANOVA and Tukey’s post-hoc test, p < 0.05. c GT1-7 cells were pretreated (30 min) and co-treated with a specific antagonist to OX1-R (1 µm of SB-334867), PKA inhibitor (10 µm H-89), PKC inhibitor (100 nm calphostin C), MEK inhibitor (100 nm UO-126), or with vehicle alone (CONT) prior to and during treatment with orexin A (Ox; 0.1 nm) or vehicle alone for 4 h. ^#Significant difference from CONT as determined by one-way ANOVA and Tukey’s post-hoc test, p < 0.05.