Serotonin antagonist profiling on 5HT2A and 5HT2C receptors by nonequilibrium intracellular calcium response using an...

Characterization of the potencies of agonists and antagonists in cell-based assays can be complicated by nonequilibrium conditions of functional response. We assessed the potencies of a series of serotonin (5HT) antagonists by inhibition of intracellular calcium response in HEK 293 cells expressing 5HT(2A) or 5HT(2C) receptors. An automated system, HT-PS 100, was used to profile the antagonists in two experimental setups: coadministration of agonist and antagonist to cells and preincubation of the cells with antagonist prior to agonist administration. We showed that the antagonist potencies (pIC(50) values) determined in the preincubation configuration were close to or exceeded those measured in the coadministration configuration. Closeness of the potencies determined in the two configurations supposedly reflected a rapid antagonist-receptor equilibration, whereas a significantly higher preincubation potency implied slow antagonist dissociation from the receptor. Schild analysis of the inhibition of serotonin-induced cell response by a competitive 5HT(2A) antagonist, spiperone, showed a typical competitive inhibition pattern when both the agonist and antagonist were applied simultaneously. Contrary to this, an insurmountable diminishing of the maximal cell response to serotonin was observed when the cells were preincubated with spiperone. We conclude that a combination of the coadministration and preincubation experimental setups is necessary for appropriate mechanistic interpretation and quantitative assessment of the antagonist activity when using transient functional readouts.