Functional and Molecular Evidence for the Expression of Autocrine Serotonin Receptors on an Enterochromaffin Cell Line
Jarrod D. Day, Roberto C. Iglesias, Jessie G. Howell, Dorne R. Yager, John M. Kellum; General Surgery, Medical College of Virginia, Richmond, VA
Introduction: Recent studies suggest that the exclusively neuronal 5-HT3 receptor, important in the serotonin secretory pathway, is expressed at the mucosal level in the enterochromaffin (EC) cell. There is also evidence that the 5-HT4 receptor may be an autocrine inhibitory receptor for 5-HT release from these cells. These studies have all been done in vascularly-perfused organ or intact tissue models. The BON cell is a unique human pancreatic tumor cell line with many EC cell characteristics. The aim of this study is to demonstrate the existence of autocrine receptors on BON cells. Methods: BON cells were subcloned by serial dilution, and a clone was chosen based on having the largest amount of 5-HT released by a calcium ionophore. Cells were propagated and maintained as a monolayer culture using standard tissue culture techniques. Cells were treated with a 5-HT3 receptor agonist, 2-methyl-5-HT (2Me5HT), in the absence and presence of either a 5-HT4 receptor agonist (GR113808) or antagonist (HTF919). 5-HT was measured by HPLC an hour after the addition of ligands. Secondly, BON cell protein extracts were prepared and used for immunoprecipitation for 5-HT3 and 5-HT4 receptors using rabbit polyclonal antibodies. Finally, RNA was isolated from BON cells and served as a template for real-time RT-PCR to generate and clone a partial cDNA which corresponds to the 5-HT3 receptor transcript. Results: 2Me5HT was associated with a 200% increase (p<0.05, one-way RM ANOVA, paired data, n=10, table) in 5-HT release from baseline. Neither GR113808 nor HTF919 caused a significant change in 5-HT release compared to control. The combination of 2Me5HT with GR113808 did not increase release compared to 2Me5HT alone. Immunoprecipitation of the BON cell lysate revealed an immunogenic band at the expected 55 kilodaltons for the 5-HT3 receptor but no identifiable band for the 5-HT4 receptor. The RT-PCR product for the 5-HT3 receptor was of the expected size of 250 base pairs and verified by DNA sequencing. Real-time RT-PCR revealed detection thresholds of 25 and 35 cycles for 5-HT3 and 5-HT4 transcripts, respectively. Conclusions: There exists autocrine 5-HT3 receptors on BON cells which mediate release of 5-HT. However, even though 5-HT4 receptors are expressed, they appear to have no functional role in the regulation of serotonin release.
| LIGAND | Serotonin Release (Mean ± SEM) | P Value (difference from Control) |
| Control (No Drug) | 5.7 ± 1.7 ng/ml | --- |
| 2Me5HT (50 μM) | 15.2 ± 3.7 ng/ml | p<0.05 |
| 2Me5HT (50 μM) + GR113808 (10 nM) | 15.9 ± 3.5 ng/ml | p<0.05 |
| GR113808 (10 nM) | 5.9 ± 2.1 ng/ml | Not significant |
| HTF919 (10 nM) | 7.3 ± 2.2 ng/ml | Not significant |
Back to 2006 Program and Abstracts
