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.