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), [Cit 8 ]neurotensin‐(8–13), [Lys 8 ]neurotensin‐(8–13), [Cit 9 ]neurotensin‐(8–13),[Lys 9 ]neurotensin‐(8–13), [Phe 11 ]neurotensin‐(8–13), [Ala 12 ]neurotensin‐(8–13) and [Ala 13 ]‐ neurotensin‐(8–13) was verified by amino acid analyses after acid and enzymatic hydrolyses. reverse‐phase high‐ performance liquid chromatography in two systems and Edman degradation. The above analogues, those obtained after N‐acetylation of neurotensin‐(6–13), neurotensin‐(8–13), [Cit 8 ]neurotensin‐(8–13), [Cit 9 ]‐ neurotensin‐(8–13), [Lys 8 ]neurotensin‐(8–13), [Lys 9 ]neurotensin‐(8–13) and [Phe 11 ]neurotensin‐(8–13), as well as native xenopsin, were all tested for binding competition with [ 3 H]neurotensin on the specific fixation sites of rat brain synaptosomal membranes and on those of HT 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 α‐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.
The binding of [3H]neurotensin to a cell line (HT 29) derived from a human colon carcinoma was characterized and compared with [3H]neurotensin binding to rat brain synaptic membranes. Both systems were used as radioreceptor assays for neurotensin and 18 neurotensin synthetic analogs, and the binding affinities thus derived were compared to the biological potencies obtained from the peptide abilities to contract isolated longitudinal smooth muscle strips of the guinea pig ileum. Tritiated neurotensin bound specifically and reversibly to HT 29 cells. The characteristics of [3H]neurotensin binding to cells at 24°C were those of a simple, bimolecular reaction involving one class of noncooperative binding sites. A Kd value of 1.5 nM was independently obtained from association kinetic and equilibrium experiments; total binding capacity was 37 fmol/106 cells (22,000 neurotensin-binding sites/cell). Peptides structurally not related to neurotensin did not affect [3H]neurotensin binding. These binding characteristics were very similar to those observed for the binding of [3H]neurotensin to rat brain synaptic membranes. When the binding affinities of neurotensin and neurotensin analogs were compared in the extraneural (HT 29 cells) and neural (brain membranes) systems, a highly significant correlation between the two binding systems was observed. A highly significant correlation was also found when the biological potencies of neurotensin and neurotensin analogs were compared with their binding affinities in either the neural or the extraneural radioreceptor assay. The positive charge on both arginyl residues 8 and 9 and the L-configuration of Arg9 were important for binding and biological activity. An aromatic residue in the L-configuration was required in position 11 of the neurotensin molecule. The side-chain methyl groups of Ile12 and carboxy-terminal residue Leu13, as well as the presence of Leu13 in the L-configuration, were required for activity.
Iodination of [Trp11]neurotensin, a neurotensin analogue in which tyrosine 11 has been substituted by a tryptophan, led to the incorporation of one or two iodine atoms on the single tyrosine residue in position 3. Both mono- and diiodinated derivatives were purified by ion exchange chromatography and their biological activity in an in vitro bioassay involving rat ileum was found to be similar to that of native neurotensin. The 125I-labeled monoiodo derivative of [Trp11]neurotensin bound specifically and reversibly to rat brain synaptic membranes. The binding isotherm was biphasic and could be described by postulating the existence of two different classes of independent binding sites with dissociation constants of 0.1 and 4.7 nM. The specificity of a series of neurotensin analogues for both high and low affinity binding sites was the same as that previously observed in other neurotensin radioreceptor assays. The low affinity binding sites appeared to be similar to the single class of sites described in other binding studies. The high affinity binding sites which were not previously detected might represent either a new class of neurotensin receptors or a high affinity state for a fraction of a single population of neurotensin receptors.
Abstract: 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[Trp 11 ]neurotensin to homogenates of differentiated N1E115 cells is specific, saturable, and reversible. The interaction is characterized by a dissociation constant of 150 p M and a maximal binding capacity of 9 fmol/mg of protein at 0°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.
