SYNTHESIS OF A BIOLOGICALLY-ACTIVE FLUORESCENT-PROBE FOR LABELING NEUROTENSIN RECEPTORS

We synthesized a fluorescent derivative of the tridecapeptide neuroten sin (NT), with the aim of providing a new tool for the pharmacological characterization and anatomic localization of NT receptors in mammali an brain. Fluoresceinylated NT (N alpha-fluoresceinyl thiocarbamyl (FT C)-[Glu(1)]NT; fluo-NT) was synthesized using solid-phase methodology and purified to 99% homogeneity by preparative high-pressure liquid ch romatography (HPLC). Analytical HPLC, acidic and carboxypeptidase Y hy drolysis, and fast atom bombardment-mass spectroscopy confirmed that t he purified compound was selectively labeled on the [Glu(1)] terminus and that a single FTC moiety was coupled to each molecule of [Glu(1)]N T. Flow cytometric analysis of the binding of fluo-NT to SN17 septal n euroblastoma cells indicated that the fluorescent derivative bound neu ral NT receptors with an affinity comparable to that of monoiodinated NT([I-125]-NT). Competition experiments on mouse brain membrane prepar ations showed fluo-NT to inhibit specific [I-125]-NT binding with a co efficient of inhibition (K-I) virtually identical to that of the nativ e peptide (0.67 vs 0.55 nM). Conventional epifluorescence and confocal microscopic analysis of specific fluo-NT binding to sections of the r at midbrain revealed a topographic distribution of the bound fluoresce nt ligand similar to that previously observed with autoradiography usi ng [I-125]-NT. However, fluo-NT provided markedly higher cell resoluti on and enabled, in particular, the detection of hitherto unnoted intra cytoplasmic receptor dusters. Binding of fluo-NT to live SN17 hybrid c ells indicated that the fluorescent ligand had retained its ability to internalize in vivo and confirmed that this internalization process w as both time- and temperature-dependent. In sum, the present study dem onstrates that fluo-NT is applicable to both the pharmacological study of NT binding sites using now cytometry and to the regional and cellu lar localization of these sites by conventional epifluorescence and co nfocal microscopy.