FIGURE 7.

Enhanced 5-HT_2C mediated signaling when GHS-R1a ligand-independent activity is blocked. Cells stably expressing the GHS-R1a receptor were transduced with viral vectors expressing the 5-HT_2C receptor and treated with 100 nm 5-HT (light gray bars), 100 nm ghrl (dark gray bars) with or without pretreatment of GHS-R1a inverse agonist, peptide [d-Arg¹,d-Phe⁵,d-Trp^7,9,Leu¹¹]-substance P (1 μm), and intracellular calcium influx was measured. A and B, substance P (SP) has no effect on 5-HT_2C-mediated signaling (A), but a significant increase in GHS-R1a-mediated G_q activation is observed following SP pretreatment (B). C, interestingly, 5-HT_2C receptor-mediated calcium influx is enhanced after SP pretreatment when the GHS-R1a receptor is co-expressed. Intracellular calcium increase is depicted in RFU as a percentage of maximal calcium increase as elicited by control in each separate experiment (3% FBS). The data represent the means ± S.E. of two independent experiments performed in triplicate for each concentration point. ANOVA followed by a Bonferroni multiple comparison test; statistical significance is notated as follows: a, p < 0.001, 5-HT compared with ghrl; b, p < 0.001, 5-HT+SP compared with ghrl+SP; and ***, p < 0.001, treatment with SP compared with no SP.