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Binding and internalization of neurotensin in hybrid cells derived from septa1 cholinergic neurons
Author(s) -
Faure MariePierre,
LabbéJullié Catherine,
Cashman Neil,
Kitabgi Patrick,
Beaudet Alain
Publication year - 1995
Publication title -
synapse
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.809
H-Index - 106
eISSN - 1098-2396
pISSN - 0887-4476
DOI - 10.1002/syn.890200203
Subject(s) - internalization , microbiology and biotechnology , population , biology , phenylarsine oxide , endocytosis , cholinergic , clathrin , receptor , neurotensin , chemistry , biochemistry , neuropeptide , endocrinology , demography , sociology
Abstract Autoradiographic studies from our laboratory have previously demonstrated a selective association of high affinity neurotensin (NT) binding sites with basal forebrain cholinergic neurons. In search of an in vitro model for further characterization of the role and regulation of these sites, we have examined the binding and internalization of 125 I‐Tyr 3 I‐NT( 125 I‐NT) and fluorescein isothiocyanate (FITC)‐conjugated NT (fluo‐ NT) on SN17 hybrid cells, produced by fusion of embryonic murine septa1 cells with neuroblastoma. 125 I‐NT binding to SN17 membrane preparations was specific and saturable. Scatchard analysis of the data was suggestive of an interaction with a single population of sites, the affinity (K d = 1.7 nM) and pharmacological profile of which were comparable to those of neural NT receptors. No specific binding was observed on the parent neuroblastoma cell line, confirming that the expression of those sites is a neuronal trait. Incubation of whole SN17 cells with 125 I‐NT resulted in a time‐ and temperature‐dependent internalization of the specifically bound peptide. The t ½ of this internalization was estimated at 13 min, a value nearly identical to that reported for neurons in culture. Confocal microscopic analyses using fluo‐NT indicated that the internalization process was endocytic in nature in that: (1)it was entirely blocked by the endocytosis inhibitor phenylarsine oxide; and (2) it was mediated through small intracytoplasmic particles the size and maturation of which corresponded to that of endosomes. It is proposed that the expression and internalization of NT receptors by SN17 hybrid cells represent a new facet of these cells' cholinergic phenotype that makes them amenable to the study of NT interactions with cholinergic cells. © 1995 Wiley‐Liss, Inc.