Complex actions of neurotensin in human colon: A binding and functional study

Abstract

Neurotensin (NT) is a brain-gut peptide with endocrine and neuronal actions. It has complex actions on gastrointestinal muscle, depending on the location and the species studied. In rodents, the actions have been attributed to a direct action on NT receptors on smooth muscle as well as indirect mechanisms involving release of acetylcholine, tachykinins, histamine, nitric oxide and/or prostaglandins. In this study, we have used [lz5I][Tyr]NT to characterize and localize NT binding sites and have investigated the actions of NT in human ascending and sigmoid colon. Normal colon was obtained from patients (age range 60-76 years) undergoing resection for colon carcinoma. Radioligand binding studies were carried out in membranes prepared from sigmoid circular muscle (CM) (which also contained longitudinal muscle and myenteric ganglia), in 50 mM Tris-HCI (pH 7.4) containing I roM MgClz and I mM O-phenanthroline. Nonspecific binding was defined using I pM NT. One site analysis of saturation data gave K, 2.2 :t 0.4 nM and Bma• 3.5 :t 0.4 fmol/mg wet weight tissue (n=13), but 2 distinct sites were found in some specimens. Binding was inhibited by NT(8-13) > NT 2= SR142948A 2= neuromedin N 2= SR48692; levocabastine £artly inhibited binding. In autoradiographic studies, specific binding of [' 5I][Tyr]NT was seen over CM and myenteric ganglia, but not on blood vessels or the mucosa. Strips of CM and taenia coli (TC) were suspended under isometric tension in Krebs-Henseleit solution at 37°C. The magnitude of contractile responses was variable (19-29% in CM; 38-41% in TC, of maximum response to acetylcholine) and on several occasions, biphasic responses were observed. The potency in CM (pDz 6.8-7.1) and TC (pDz 6.4-6.5) was no different in ascending or sigmoid colon. In sigmoid colon CM, contractile responses to NT were potentiated by 1 pM tetrodotoxin (P < 0.05) and inhibited (P < 0.05) by 1 pM MEN10627, the tachykinin NK2 receptor antagonist. In ascending colon CM, a potentiation (P < 0.01) of the contractile response was observed in the presence of indomethacin (l /-LM) as well as IT (l /-LM). Atropine (1 /LM) had no effect in any region of the colon. These inhibitors had little effect on NT responses in TC. Thus, NT appears to have both direct actions on smooth muscle and neuronal actions (supported by the presence of binding on myenteric ganglia). NT may also cause the release of relaxant prostanoids in ascending colon. Many effects of NT appear to be regionally specific. Supported by the National Health and Medical Research Council of Australia