Neurotensin (NT), a neuropeptide released in the gastrointestinal tract in response to several stimuli, is involved in the pathophysiology of colonic inflammation. However, the molecular mechanism(s) mediating this proinflammatory response remains unclear. We found that NCM460, non-transformed human colonocytes, express a functional high affinity NT receptor that mediates NT-induced Erk activation. By using NCM460 cells stably transfected with NTR1, we show that NTR1 activation leads to interleukin (IL)-8 secretion that is mediated via both NF-B-and Erk-dependent pathways. In addition, NT-stimulated NF-B activation is dependent on intracellular calcium release. NT-stimulated Erk activity requires Ras activation because overexpression of the dominant negative Ras mutant Ras-17N almost completely inhibits the Erk activation. Furthermore, NT directly stimulates Ras-GTP formation as shown by a Ras-GTP pull-down assay. By using reporter gene constructs containing targeted substitutions in the IL-8 promoter, we show that the NF-B, AP-1, and to a lesser degree the C/EBP sites in the IL-8 promoter region are required for IL-8 gene expression induced by NT. In summary, our results demonstrate that NT stimulates calcium-dependent NF-B and Ras-dependent Erk pathways that mediate the release of IL-8 from non-transformed human colonocytes. We speculate that these NT-related proinflammatory pathways are important in the pathophysiology of colonic inflammation.Neurotensin (NT), 1 a 13-amino acid neuropeptide originally isolated by Carraway and Leeman (1), is highly expressed in the gastrointestinal tract (2). In the ileal mucosa NT is synthesized and secreted by specific endocrine cells (3), in response to diverse stimuli (4). NT increases small bowel, colonic, and gastric motility and stimulates ileal, pancreatic, and biliary secretion (4, 5) as well as Cl Ϫ secretion from human colonic mucosa (6), indicating that this peptide may contribute to the pathophysiology of human diarrhea. NT also stimulates growth of the intestinal mucosa under physiological and pathological conditions and causes proliferation of intestinal epithelial cells in vivo and in vitro (7-11). Two G-protein-coupled receptors (GPCRs) have been described for NT, a high affinity (NTR1) and a low affinity (NTR2) receptor (12). Administration of the specific NTR1 antagonist SR 48692 to rats inhibits colonic mucin and prostaglandin E 2 secretion in response to immobilization stress (13), suggesting the importance of NTR1 in stress-mediated colonic responses. Our recent studies (14) demonstrate that NT is a proinflammatory peptide in the colon because blockage of the NT-NTR1 interaction with SR 48692 inhibited colonic secretion and inflammation mediated by Clostridium difficile toxin A. We also showed that, compared with normal colonic epithelial cells, there was a dramatic up-regulation of NTR1 during human colonic inflammation (15) as well as in Clostridium difficile toxin A-mediated colitis (14). NT exerts its proinflammatory effects by interacting with severa...