Colocalization of corticotrophin releasing factor and 5-hydroxytryptamine and their cellular and mechanistic interactions in the gut

A series of experiments were conducted to assess the presence of CRF and 5-HT in the sheep and rat duodenum, and the function of their respective receptors in mediating physiological changes. The first study began with characterizing CRF and 5-HT in the sheep and rat duodenum using immunohistochemistry. Characterization of CRF, CRF receptors, 5-HT, and 5-HT receptors was also carried out on BON cells, an enterochromaffin cell-like cell line. In vitro experiments on BON cells were performed to assess cellular activation measured as changes in the phosphorylation of ERK1/2 and intracellular calcium ([Ca++]i) induced by CRF and 5-HT and their ligands. The second study measured functional changes caused by CRF receptor and 5-HT 3R activation in net ileal fluid flux and fecal pellet output. Assays measuring ileal fluid flux was conducted as a measure of fluid secretion and fecal pellet output as a measure of colonic motility. CRF and 5-HT colocalize within enterochromaffin cells in the crypt region of sheep and rat duodenal tissue. BON cells were immunopositive for CRF, CRF-R1/2, 5-HT, and 5-HT 3R. Data show that CRF and 5-HT induced activation of a second messenger system associated with ERK1/2 phosphorylation and to a lesser extent by urocortin1 and 3 (CRF related ligands), but not 2-Me-5-HT, a 5-HT3R agonist. CRF and both CRF-R1 and R2 agonists led to significant increases in [Ca ++]i. 5-HT3R, but not 5-HT4R, activation caused increases in [Ca++]i. Extra and intracellular calcium play essential roles in the ability of the ligands to induce changes in [Ca++]i. CRF-R1, CRF-R2 and 5-HT3R activation led to net ileal fluid secretion. Coadministration of CRF and 2-Me-5-HT was no different from CRF alone, but did reduce CRF-mediated mucous production. Fecal pellet output was increased significantly by CRF and 2-Me-5-HT. CRF treatment induced rapid diarrhea expulsion whereas 2-Me-5-HT caused a gradual increase in fecal pellets. When coadministered, rats did not develop diarrhea and this effect was seen even when CRF was administered 60 minutes after 2-Me-5-HT treatment. CRF and 5-HT are present within the same enteric endocrine cells and may functionally interact during the stress response in the gut.