Characterization and Autoradiographic Localization of Neurotensin Binding Sites in Human Sigmoid Colon

• Published in: The Journal of pharmacology and experimental therapeutics 2001-06, Vol.297 (3), p.1074-1081
• Main Authors: Azriel, Yael, Burcher, Elizabeth
• Format: Article
• Language: English
• Subjects: Adult; Aged; Autoradiography; Binding, Competitive - drug effects; Colon, Sigmoid - drug effects; Colon, Sigmoid - innervation; Colon, Sigmoid - metabolism; Dose-Response Relationship, Drug; Enteric Nervous System - metabolism; Female; Histamine H1 Antagonists - pharmacology; Humans; In Vitro Techniques; Iodine Radioisotopes; Male; Middle Aged; Muscle Contraction - drug effects; Muscle, Smooth - drug effects; Muscle, Smooth - innervation; Muscle, Smooth - metabolism; Neurotensin - metabolism; Neurotensin - pharmacology; Peptide Fragments - metabolism; Peptide Fragments - pharmacology; Phenanthrolines - pharmacology; Piperidines - pharmacology; Protease Inhibitors - pharmacology; Receptors, Neurotensin - metabolism; Tetrodotoxin - pharmacology
• ISSN: 0022-3565; 1521-0103
• DOI: 10.1016/S0022-3565(24)29635-X

Radioiodinated neurotensin (125I-NT) was used to characterize and localize NT binding sites in normal human sigmoid colon. Specimens were obtained from patients (30–77 years old) undergoing resection for colon carcinoma. Specific binding of125I-NT to sigmoid circular muscle membranes was enhanced by o-phenanthroline (1 mM) but other peptidase inhibitors were ineffective. 125I-NT bound to a high-affinity site of Kd = 0.88 ± 0.09 nM and Bmax = 4.03 ± 0.66 fmol/mg of wet weight tissue (n = 14), although in the majority of patients another site, of low but variable affinity, could also be detected. Specific binding of 50 pM 125I-NT was inhibited by NT(8-13) > NT > SR142948A ≥ neuromedin N ≥ SR48692, consistent with binding to the NT1 receptor. In autoradiographic studies, dense specific binding of125I-NT was seen over myenteric and submucosal ganglia, moderate binding over circular muscle, and sparse binding over longitudinal muscle and taenia coli. Levocabastine, which has affinity for the NT2 receptor, did not inhibit specific binding of125I-NT in membrane competition or autoradiographic studies. NT contracted sigmoid colon circular muscle strips with a pD2 value of 6.8 ± 0.2 nM (n = 25). The contractile responses to NT were significantly potentiated in the presence of tetrodotoxin (1 μM), indicating a neural component. Results from functional studies support actions for NT on both muscle and enteric neurons, consistent 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.