AB9370 Sigma-AldrichAnti-Corticotropin-Releasing Factor Receptor 1 Antibody

CRH and 5-hydroxytryptamine (5-HT) are expressed in human colonic enterochromaffin (EC) cells, but their interactions at the cellular level remain largely unknown. The mechanistic and functional relationship between CRH and 5-HT systems in EC cells was investigated in a human carcinoid cloned BON cell line (BON-1N), widely used as an in vitro model of EC cell function. First, we identified multiple CRH(1) splice variants, including CRH(1a), CRH(1c), CRH(1f), and a novel form lacking exon 4, designated here as CRH(1i), in the BON-1N cells. The expression of CRH(1i) was also confirmed in human brain cortex, pituitary gland, and ileum. Immunocytochemistry and immunoblot analysis confirmed that BON-1N cells were CRH(1) and 5-HT positive. CRH, urocortin (Ucn)-1, and cortagine, a selective CRH(1) agonist, all increased intracellular cAMP, and this concentration-dependent response was inhibited by CRH(1)-selective antagonist NBI-35965. CRH and Ucn-1, but not Ucn-2, stimulated significant ERK1/2 phosphorylation. In transfected human embryonic kidney-293 cells, CRH(1i) isoforms produced a significant increase in pERK1/2 in response to CRH(1) agonists that was sensitive to NBI-35965. CRH and Ucn-1 stimulated 5-HT release that reached a maximal increase of 3.3- and 4-fold at 10(-8) m over the basal level, respectively. In addition, exposure to CRH for 24-h up-regulated tryptophan hydroxylase-1 mRNA levels in the BON-1N cells. These findings define the expression of EC cell-specific CRH(1) isoforms and activation of CRH(1)-dependent pathways leading to 5-HT release and synthesis; thus, providing functional evidence of a link exists between CRH and 5-HT systems, which have implications in stress-induced CRH(1) and 5-HT-mediated stimulation of lower intestinal function.