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Functional characterization of enterochromaffin cells in mice and humans
Biochemistry and Molecular Biology
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Serotonin (5-HT) is a key regulator of gastrointestinal (GI) motility. Abnormal concentrations of 5-HT are found in GI disorders. About 90-95% of bodily 5-HT is produced by enterochromaffin (EC) cells in the GI. The synthesis of 5-HT is catalyzed by the enzyme tryptophan hydroxylase (Tph1). Given the dubious data collected from the previously generated animal models, to elucidate the role of EC cells in the GI tract, we have generated an EC cell-specific tamoxifen inducible Cre line (Tph1CreERT2/+), a reporter mouse (Tph1CreERT2/+;Rosa26tdTom/+), and an EC cell depleted mouse (Tph1CreERT2/+;Rosa26DTA/+). We examined changes of the EC cells and 5-HT in the GI tract of the transgenic mice using various in vivo and in vitro experimental techniques. We also measured levels of 5-HT, SERT, and TPH1 in stomach biopsy and/or blood samples from idiopathic gastroparesis patients by ELISA and western blot. We confirmed EC cell-restricted Cre activity in the reporter line (Tph1CreERT2/+;Rosa26tdTom/+). The reporter mice were then used to map EC cell distribution in the stomach (fundus, corpus, and antrum), small intestine (duodenum, jejunum, and ileum), and colon (proximal, mid, and distal colon). Proximal colon and antrum have the most EC cells while the fundus contains no observable EC cells. The distribution pattern is consistent with levels of 5-HT in the tissue of the GI tract. Furthermore, we examined EC cell turnover time using the tdTom reporter mice. We found two different types of EC cells, fast-turnover EC cells, and slow-turnover EC cells. Most fast-turnover EC cells are located in the epithelial layer and lost within ten days. Slow-turnover EC cells are located in the crypt and express stem cell markers (LGR5 and REG4) and survive over one year. Next, to access the functional role of EC cells in GI motility, we generated Tph1CreERT2/+;Rosa26DTA/+ mice. These EC cells were conditionally removed upon tamoxifen injection. 5-HT in blood and GI tissue were significantly reduced in tamoxifen injected mice. The reduction of 5-HT resulted in slower gastric emptying, slow colonic transit time, slow colonic migrating motor complex, slow total GI transit time, and decreased pellet output. We next examined human stomach biopsy samples (fundus, gastric body, and antrum) with idiopathic gastroparesis along with healthy controls. Consistent with the mouse stomach, we found the highest 5-HT concentration detected in human antrum and a lower concentration was detected in the gastric body, and levels were negligible in healthy fundus. However, in idiopathic gastroparesis samples, we found 5-HT was significantly reduced in the antrum and body from all patients. 5-HT was also reduced in the patient’s plasma and platelets. Finally, we assessed the gain-of-function of 5-HT in regulating GI motility in the mouse model via oral administration of 5-HT. The oral gavage of 5-HT to the EC cell-depleted mice rescue the gastroparesis and constipation. In summary, our new Tph1CreERT2/+ mice provide a novel and powerful tool to study the functional roles of EC cells in the GI tract which lead to the discovery of serotonin deficiency in idiopathic gastroparesis.