NEUROTENSIN AND NEUROMEDIN-N STIMULATE MUCIN OUTPUT FROM HUMAN GOBLET CELLS (CL.16E) VIA NEUROTENSIN RECEPTORS

The stably differentiated human intestinal goblet cell line Cl.16E was used to study the effects of two structurally related regulatory peptides, neurotensin (NT) and neuromedin N (NN), on mucus secretion. NT and NN stimulated rapid release of mucins from filter-grown Cl.16E cells, this effect being dose related with a mean effective dose of 36 nM for NT and 422 nM for NN. The order of potency of NT, three NT fragments corresponding to the NH2-terminal part [NT-(1-11)] or to the COOH-terminal part [NT-(8-13) and NT-(9-13)], and NN in promoting mucin release and in inhibiting I-125-labeled NT binding to Cl.16E cell membranes was identical with NT greater-than-or-equal-to NT-(8-13) > NN > NT-(9-13) >> NT-(1-11) supporting the hypothesis that NT and NN stimulate mucin output through interaction with a common NT-preferring receptor. Scatchard analysis of equilibrium binding data showed one population of NT binding sites in Cl.16E cell membranes with the following characteristics: binding capacity (B(max)) was 141 fmol/mg of protein and dissociation constant (K(d)) was 1.00 nM. When Scatchard analysis of I-125-NT binding to membranes was carried out in the presence of (in mM) 0.4 GTP, 2 ATP, 10 NaCl, 2 CaCl2, 30 MgCl2, and 140 KCl, i.e., the intracellular concentration of these nucleotides and ions in a typical mammalian cell, a curvilinear plot was obtained, resulting from the conversion of most receptors from a high-affinity state to a lower-affinity state (K(d):21.7 nM; B(max):129 fmol/mg of protein), which may be responsible for the NT-induced mucin output. NT-stimulated mucus secretion was found to be associated with a rise in intracellular Ca2+ concentration with no alteration of adenosine 3',5'-cyclic monophosphate level. Finally comparison and synergism of NT with other known secretagogues, i.e., carbachol and vasoactive intestinal peptide (VIP) were evaluated. VIP, a neuropeptide capable of potentiating the action of carbachol, was found to exert only an additive effect on NT-induced stimulation of mucus output. The mechanisms responsible for the distinct interactions of VIP with carbachol and NT remain to be determined.