Synthesis of a biologically active fluorescent probe for labeling neurotensin receptors

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.