BINDING AND INTERNALIZATION OF NEUROTENSIN IN HYBRID-CELLS DERIVED FROM SEPTAL CHOLINERGIC NEURONS

Autoradiographic studies from our laboratory have previously demonstra ted a selective association of high affinity neurotensin (NT) binding sites with basal forebrain cholinergic neurons. In search of an in vit ro model for further characterization of the role and regulation of th ese sites, we have examined the binding and internalization of I-125-T yr(3)-NT (I-125-NT) and fluorescein isothiocyanate (FITC)-conjugated N T (fluo-NT) on SN17 hybrid cells, produced by fusion of embryonic muri ne septal cells with neuroblastoma. I-125-NT binding to SN17 membrane preparations was specific and saturable. Scatchard analysis of the dat a 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 ob served on the parent neuroblastoma cell line, confirming that the expr ession of those sites is a neuronal trait. Incubation of whole SN17 ce lls with I-125-NT resulted in a time- and temperature-dependent intern alization of the specifically bound peptide. The ty, Of this internali zation was estimated at 13 min, a value nearly identical to that repor ted for neurons in culture. Confocal microscopic analyses using fluo-N T indicated that the internalization process was endocytic in nature i n that: 1) it was entirely blocked by the endocytosis inhibitor phenyl arsine oxide; and 2) it was mediated through small intracytoplasmic pa rticles the size and maturation of which corresponded to that of endos omes. It is proposed that the expression and internalization of NT rec eptors by SN17 hybrid cells represent a new facet of these cells' chol inergic phenotype that makes them amenable to the study of NT interact ions with cholinergic cells. (C) 1995 Wiley-Liss, Inc.