Fig. 2. 5-HT inhibits CYP1A1 activity. (A) Caco-2 cells were plated at low density and allowed to differentiate for 10-14 d in medium containing 10% serum before the assay was performed. Cells were co-incubated with 50 μM Luc-CEE and either vehicle (veh), 5-HT (5 - 500 μM), or ɑ-NF (5 μM) for 3 h in serum-free medium before measurement of luciferase activity (n = 3-4). Data represent the relative CYP1A1 activity measured as luciferase activity detected in the medium after incubation relative to the vehicle treated cells. Assays were performed using triplicate wells for each treatment and values were normalized to total protein by Bradford assay. Data analyzed by 1-way ANOVA followed by Dunett's multiple comparison's test. ****P<0.0001 (B) Caco-2 cells were split at low density into 150 cm² flasks and grown for 2 weeks before the assay was performed. Cells were loaded with 50 nM FICZ for 30 min and intracellular FICZ levels were measured by LC-MS/MS 2 h after removal of FICZ in the presence or absence of vehicle, 5-HT (5 μM), or ɑ-NF (5 μM). Intracellular FICZ levels were normalized to intracellular TRP levels also determined by LC-MS/MS and values are expressed as a percentage of initial FICZ measured at 0 h (n = 3). Data analyzed by 1-way ANOVA followed by Dunett's multiple comparison's test. **P<0.01, ****P<0.0001 vs. cells incubated with vehicle (C) NADPH-dependent metabolism of Luc-CEE (30 μM) by recombinant CYP1A1 (10 nM) was measured in presence or absence of ɑ-NF or TRP metabolites 5-HT, tryptamine (TRYPT), 5-hydroxyindoleacetic acid (5-HIAA), or indole-3-acetic acid (IAA) (1 - 1000 μM) at 37°C for 10 min. Assays were performed in triplicate and values were normalized to activity in the presence of vehicle (n = 3).