Quantitative changes in intracellular calcium and extracellular-regulated kinase activation measured in parallel in CHO cells stably expressing serotonin (5-HT) 5-HT2A or 5-HT2C receptors

We optimized the Cai++ assay for stable cell lines expressing either 5-HT2AR or 5-HT2CR (including dye use and measurement parameters; cell density and serum requirements). We adapted a quantitative 96-well plate immunoassay for pERK in the same cell lines. Similar cell density optima and time courses were observed for 5-HT2AR- and 5-HT2CR-expressing cells in generating both types of signaling. Both cell lines also require serum-free preincubation for maximal agonist responses in the pERK assay. However, 5-HT2AR-expressing cells showed significant release of Cai++ in response to 5-HT stimulation even when preincubated in serum-replete medium, while the response was completely eliminated by serum in 5-HT2CR-expressing cells. Response to another serotonergic ligand (DOI) was eliminated by serum-replete preincubation in both cells lines.These data expand our knowledge of differences in ligand-stimulated signaling cascades between 5-HT2AR and 5-HT2CR. Our parallel assays can be applied to other cell and receptor systems for monitoring and dissecting concurrent signaling responses.The serotonin (5-HT) 2A and 2C receptors (5-HT2AR and 5-HT2cR) are seven-transmembrane, G protein-coupled receptors (GPCRs) that are expressed in numerous brain regions. The 5-HT2CR protein is expressed predominantly in the central nervous system while the 5-HT2AR is also prominent in peripheral tissues, such as platelets and smooth muscle cells of the gut and vasculature [1]. Both receptors are involved in a wide range of physiological (e.g., temperature regulation, feeding) and psychological processes in mammals [2] and are implicated in psychological disorders (e.g., addiction, anxiety, depression, and learning and memory) [3-5]. These receptors share a high degree of homology, have overlapping pharmacological profiles, and utilize many of the same and richly diverse second messenger signaling systems. The most commonly studied downstream signaling pathway of the 5-HT2AR and 5-HT2CR is the a