Abstract
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.
Footnotes
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Send reprint requests to: Professor E. Burcher, School of Physiology and Pharmacology University of New South Wales, NSW 2052, Australia. E-mail: e.burcher{at}unsw.edu.au
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This study was supported by the National Health and Medical Research Council of Australia. Y.A. is the recipient of an Australian Postgraduate Award. This work was previously published as an abstract (no. 2091, P-322) at the 2000 meeting of Digestive Diseases Week, San Diego, CA (Azriel Y, Lubowski DZ and Burcher E Complex actions of neurotensin in human colon: a binding and functional study. American Gastroenterological Society, San Diego, CA, May, 2000).
- Abbreviations:
- NT
- neurotensin
- cFP
- N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate
- BSA
- bovine serum albumin
- Ach
- acetylcholine
- TTX
- tetrodotoxin
- GTPγS
- guanosine-5′-O-(3-thio)triphosphate
- Received November 13, 2000.
- Accepted January 30, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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