Binding and internalization of neurotensin in hybrid cells derived from septa1 cholinergic neurons

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 125I-Tyr3-NT (125I-NT) and fluorescein isothiocyanate (FITC)-conjugated NT (fluo-NT) on SN17 hybrid cells, produced by fusion of embryonic murine septal cells with neuroblastoma. 125I-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 (Kd = 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 125I-NT resulted in a time- and temperature-dependent internalization of the specifically bound peptide. The t1/2 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.

Alzheimer's beta-amyloid precursor protein is expressed on the surface of immediately ex vivo brain cells: A flow cytometric study

“…Inhibition of this pathway may result in the accumulation of PAPP molecules on the surface of brain cells. It was observed that when immediately ex vivo dissociated brain cells were placed in culture with the general endocytosis inhibitor PheAsO (10 pM; Wallace and Ho, 1972;Low et al, 1981;Chabry et al, 1993;Faure et al, 1995) for 30 min and subsequently stained for flow cy- 1 *0° 1 C tometric analysis, the amount of PAPP detectable on the surface of these brain cells was increased compared to untreated cells (Fig. 5).…”

“…These data are consistent tion, cells were cultured in MEM + 15% fetal bovine serum (FBS) + 0.1% dextrose + 2 mM L-glutamine + 50 pg/ml DNase I, pH 7.2, for 30-60 min at 37.5"C/5% CO, to allow for PAPP expression. The general endocytosis inhibitor phenylarsine oxide (PheAsO, 10 pM; Wallace and Ho, 1972;Low et al, 1981;Chabry et al, 1993;Faure et al, 1995) was added to the medium during the incubation period in another set of experiments. The cells were washed thoroughly with PBS then stained for flow cytometric analysis.…”