Neurotensin and several sequence analogues have been synthesized using solid-phase technology. The purity of the following derivatives: neurotensin, neurotensin-(10 -13), neurotensin-(9 -13). neurotensin-(8 -13), neurotensin-(6 -13), neurotensin-(4-13), [Cit8]neurotensin-(8 -13), [Lys8]neurotensin-( 8 -13), [Cit'lneurotensin-(S -13), [Lys9]neurotensin-(8 -13), [Phe"]neurotensin-(8 -13), [Ala12]neurotensin-( X -13) and [AlaI3]-neurotensin-(8 -13) was verified by amino acid analyses after acid and enzymatic hydrolyses. reverse-phase highperformance liquid chromatography in two systems and Edman degradation. The above analogues, those obtained after N-acetylation of neurotensin-(6 -13), neurotensin-( , and [Phe"]neurotensin-(8 -13), as well as native xenopsin, were all tested for binding competition with [3H]neurotensin on the specific fixation sites of rat brain synaptosomal membranes and on those of H T 29 cells. In addition to these radioreceptor assays on neural and extraneural targets, a pharmacological test (contraction of guinea pig ileum in the presence of neostigmine) was used to compare the behavior of the synthetic analogues. The use of these three biological systems enabled us to obtain consistent results. A good parallel was observed between the degree of fixation and pharmacological effects for entire neurotensin and for C-terminal region analogues up to the size of neurotensin-(8-13). The two peptides neurotensin-(6-13) and neurotensin-(4-13) had an abnormally high affinity for rat brain synaptic membrane binding sites compared to a relatively low contracting activity. The C-terminal peptide -Arg-Arg-Pro-Tyr-Ile-Leu fulfills all the structural requirements for mimicking the entire sequence, provided its a-amino end is protected by acetylation. The guanidinium structure of residues 8 and 9 are not of vital importance, since they could be efficiently replaced by amino groups of lysyl side chains. Xenopsin, which can be considered as a natural analogue of neurotensin-(8-13), acts similarly to acetyl-neurotensin-(8 -13). Removal of the phenolic function of residue 11 induces a decrease in neurotensin effects. The C-terminal isoleucyl and leucyl residues could not be replaced by alanine without complete loss of the three activities tested.Neurotensin is a 13-residue peptide (< Glu-Leu-Tyr-GluAsn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu), originally isolated by Carraway and Leeman from calf hypothalamus [I] and subsequently purified from bovine [2] and human [3] small intestine. It has stimulated considerable research, since it displays a wide spectrum of pharmacological activities when injected peripherally (hypotension, hyperglycemia, gut contraction, increased vascular permeability, increased secretion of growth hormone) or centrally (hypothermic effect on coldexposed rats, analgesic activity). There are both central and peripheral neurotensin fixation sites (for the above mentioned effects, see review of Fernstrom et al. role and the significance of its ubiquitous distribution, however, remain unknown.In the l...

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This paper describes the interaction of neurotensin with mouse neuroblastoma N1E115 cells. Neurotensin binding sites are undetectable in nondifferentiated neuroblastoma cells. They appear during cell differentiation in the presence of a low serum concentration and dimethyl sulfoxide, and reach a maximal level after 50-60 h of incubation under these conditions. The binding of monoiodo[Trp11]neurotensin to homogenates of differentiated N1E115 cells is specific, saturable, and reversible. The interaction is characterized by a dissociation constant of 150 pM and a maximal binding capacity of 9 fmol/mg of protein at 0 degrees C, pH 7.5. These binding parameters, as well as the specificity toward a series of neurotensin analogues, are similar for neurotensin receptors in N1E115 cells and for the high-affinity binding sites that had been previously characterized in rat brain synaptic membranes by means of the same radiolabeled ligand. The presence of high-affinity binding sites for neurotensin in the neuroblastoma N1E115 provides a useful model to study the cellular responses that are generated by the association of neurotensin to its receptor in electrically excitable cells.

This work deals with the preparation and binding properties to rat brain synaptic membranes of a biologically active derivative of neurotensin that can be labeled at a specific radioactivity of 2,000 Cilmmol. [zH]neurotensin is the only radioactive ligand of the neurotensin receptor that has been prepared in a pure state. However the specific radioactivity of this tritiated derivative is only 40 to 60 Cil mmol. Because the number of neurotensin binding sites is small in brain and peripheral tissues, there is a need for a biologically active derivative of neurotensin with a much higher specific radioactivity. Iodination of neurotensin gives a complex mixture of peptides in which both Tyr, and Tyr,, have incorporated 1 or 2 iodine atoms per residue. Moreover, we have found that iodination of Tyr,, in acetyl n e u r o t e n~i n~-'~ , a fully active analogue of neurotensin, decreased the biological activity of the hexapeptide by a factor of 20.Trpll-neurotensin, a neurotensin analogue in which Tyr" is substituted by a Trp, was synthesized by the Menifield solid-phase procedure. The biological activity of this analogue was found to be almost identical to that of native neurotensin in an in vitro bioassay involving rat ileum. Iodination of Tyr? in Trp"neurotensin by the lactoperoxidase method was carried out in the presence of nonradioactive [ Y ] N a , containing tracer amounts of radioactive [ 12511]Na. The resulting mixture of diiodo, monoiodo, and unmodified Trp"-neurotensin was purified in one step by ion-exchange chromatography at pH 8.6. The mono and diiodo derivatives of Trp"-neurotensin were pure as judged by UV spectrophotometry, calculation of the number of iodine atoms per mole of peptide, HPLC, and amino-acid analysis. The activity of rnonoiodo Trp'l-neurotensin in the rat ileum bioassay was very similar to that of the unmodified peptide. The use of [ 12"I]Na as the sole source of iodine in the lactoperoxidase-catalyzed iodination reaction allowed to prepare [ 12~I]m~n~iodo-Trp11-ne~r~ten~in with a specific radioactivity of 2,000 Cil mmol. Binding of this analogue to synaptic membranes from rat brain was specific and reversible. The binding isotherm was biphasic and