Radioligand Binding Assay

The affinity of bilirubin and the 5-HT_3A receptor was measured by radioligand binding assay. 5-HT (non-specific agonist of 5-HT_3A receptor) was used as a positive control and 8-OH-DPAT (5-HT_1A receptor agonist) as a negative control. Glycodesoxycholic acid (GDCA, one kind of bile acid), which increased in cholestasis, was also tested. HEK293-5-HT3A cells were cultured, trypsinized, and collected. After centrifugation, supernatants were collected and protein concentrations of membrane samples were determined by bicinchoninic acid protein assay kit. Membrane samples were diluted to 10 doses at five-fold serial dilution in dimethylsulfoxide (DMSO). Then, [3H]-Granisetron was transferred into wells. Meanwhile, the filters within Unifilter-96 GF/C filter plates (PerkinElmer, Waltham, MA, United States) were soaked in 0.5% polyethyleneimine for 30 min. When binding assays were completed, the contents of the binding assay were vacuumed through the filters. After being washed and dried, the bottom of the filter-plate wells was sealed using Unifilter-96 Backing Seal Tape (PerkinElmer), followed by the addition of 50 μL of Microscint^TM 20 Cocktail (PerkinElmer) and sealing of the top of the filter plates with TopSeal-A Sealing Film (PerkinElmer). Finally, [3H] trapped on filters was counted using a TopCount NXT HTS Reader (PerkinElmer). Data were analyzed using GraphPad Prism 5 (San Diego, CA, United States). Inhibition (%) was calculated using the following equation:

The half-maximal inhibitory concentration (IC50) was determined using four-parameter logistic non-linear regression analysis employing the following equation:

where Y is the inhibition rate and X is the log concentration of the sample.