title: Synthesis of a biologically active fluorescent probe for labeling neurotensin receptors.
abstract: We synthesized a fluorescent derivative of the tridecapeptide neurotensin (NT), with the aim of providing a new tool for the pharmacological characterization and anatomic localization of NT receptors in mammalian brain. Fluoresceinylated NT (N alpha-fluoresceinyl thiocarbamyl (FTC)-[Glu1]NT; fluo-NT) was synthesized using solid-phase methodology and purified to 99% homogeneity by preparative high-pressure liquid chromatography (HPLC). Analytical HPLC, acidic and carboxypeptidase Y hydrolysis, and fast atom bombardment-mass spectroscopy confirmed that the purified compound was selectively labeled on the [Glu1] terminus and that a single FTC moiety was coupled to each molecule of [Glu1]NT. Flow cytometric analysis of the binding of fluo-NT to SN17 septal neuroblastoma cells indicated that the fluorescent derivative bound neural NT receptors with an affinity comparable to that of monoiodinated NT([125I]-NT). Competition experiments on mouse brain membrane preparations showed fluo-NT to inhibit specific [125I]-NT binding with a coefficient of inhibition (KI) virtually identical to that of the native peptide (0.67 vs 0.55 nM). Conventional epifluorescence and confocal microscopic analysis of specific fluo-NT binding to sections of the rat midbrain revealed a topographic distribution of the bound fluorescent ligand similar to that previously observed with autoradiography using [125I]-NT. However, fluo-NT provided markedly higher cell resolution and enabled, in particular, the detection of hitherto unnoted intracytoplasmic receptor clusters. Binding of fluo-NT to live SN17 hybrid cells indicated that the fluorescent ligand had retained its ability to internalize in vivo and confirmed that this internalization process was both time- and temperature-dependent. In sum, the present study demonstrates that fluo-NT is applicable to both the pharmacological study of NT binding sites using flow cytometry and to the regional and cellular localization of these sites by conventional epifluorescence and confocal microscopy.
authors.affiliation: Neurobiology group, Montreal Neurological Institute, Quebec, Canada.
authors.forename: M P
authors.initials: MP
authors.lastname: Faure
authors.forename: P
authors.initials: P
authors.lastname: Gaudreau
authors.forename: I
authors.initials: I
authors.lastname: Shaw
authors.forename: N R
authors.initials: NR
authors.lastname: Cashman
authors.forename: A
authors.initials: A
authors.lastname: Beaudet
mesh_terms: D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000166:cytology / Q000378:metabolism*; D002460:Cell Line; D005434:Flow Cytometry; D005452:Fluoresceins / Q000138:chemical synthesis*; D005456:Fluorescent Dyes / Q000138:chemical synthesis*; D006825:Hybridomas; D007202:Indicators and Reagents; D051379:Mice; D009447:Neuroblastoma; D009496:Neurotensin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D018028:Receptors, Neurotensin / Q000032:analysis* / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D000596:Amino Acids; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D018028:Receptors, Neurotensin; C087348:neurotensin, N-alpha-(fluoresceinylthiocarbamyl)glutamyl(1)-; D009496:Neurotensin