Characterization and autoradiographic localization of neurotensin binding sites in human sigmoid colon

Radioiodinated neurotensin (I-125-NT) was used to characterize and localize NT binding sites in normal human sigmoid colon. Specimens were obtained fr om patients (30-77 years old) undergoing resection for colon carcinoma. Spe cific binding of I-125-NT to sigmoid circular muscle membranes was enhanced by o-phenanthroline (1 mM) but other peptidase inhibitors were ineffective . I-125-NT bound to a high-affinity site of K-d = 0.88 +/- 0.09 nM and B-ma x = 4.03 +/- 0.66 fmol/mg of wet weight tissue (n = 14), although in the ma jority of patients another site, of low but variable affinity, could also b e detected. Specific binding of 50 pM I-125-NT was inhibited by NT(8-13) > NT > SR142948A greater than or equal to neuromedin N greater than or equal to SR48692, consistent with binding to the NT1 receptor. In autoradiographi c studies, dense specific binding of I-125-NT was seen over myenteric and s ubmucosal ganglia, moderate binding over circular muscle, and sparse bindin g over longitudinal muscle and taenia coli. Levocabastine, which has affini ty for the NT2 receptor, did not inhibit specific binding of I-125-NT in me mbrane competition or autoradiographic studies. NT contracted sigmoid colon circular muscle strips with a pD(2) value of 6.8 +/- 0.2 nM (n = 25). The contractile responses to NT were significantly potentiated in the presence of tetrodotoxin (1 muM), indicating a neural component. Results from functi onal studies support actions for NT on both muscle and enteric neurons, con sistent with the presence of NT receptors on circular muscle and ganglia of human sigmoid colon. The lack of inhibition by levocabastine suggests that the second binding site detected does not correspond to the NT2 receptor.