Neurotensin NT2 Research Articles

Radiometal-Containing Aryl Diazonium Salts for Chemoselective Bioconjugation of Tyrosine Residues.

Tyrosine is an attractive target for chemo- and site-selective protein modification. The particular chemical nature of tyrosine residues allows bioconjugation chemistry with reactive aryl diazonium salts via electrophilic aromatic substitution to produce diazo compounds. In this work, we describe the preparation of 64Cu- and 68Ga-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)-diazonium salts as building blocks for azo coupling chemistry with tyrosine and tyrosine-containing peptides and proteins under mild conditions. 2-S-(4-aminobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-NH2-Bn-NOTA) was used to form the corresponding 64Cu- and 68Ga-labeled complexes, followed by diazotization with NaNO2 in the presence of HCl. 64Cu- and 68Ga-labeled NOTA complexes were prepared in high radiochemical yields >80% starting from 20 μg of p-NH2-Bn-NOTA. Conversion of p-NH2-Bn-NOTA complexes into diazonium salts followed by azo coupling with l-tyrosine afforded 64Cu- and 68Ga-labeled tyrosine in radiochemical yields of 80 and 56%, respectively. Azo coupling with tyrosine-containing hexapeptide neurotensin NT(8–13) afforded 64Cu- and 68Ga-labeled NT(8–13) in radiochemical yields of 45 and 11%, respectively. Azo coupling of 64Cu-labeled NOTA-diazonium salt with human serum albumin (HSA) gave 64Cu-labeled HSA in radiochemical yields of 20%. The described azo coupling chemistry represents an innovative and versatile bioconjugation strategy for selective targeting of tyrosine residues in peptides and proteins.

Cardio-respiratory effects of systemic neurotensin injection are mediated through activation of neurotensin NT1 receptors

Cardio-respiratory effects of systemic neurotensin injection are mediated through activation of neurotensin NT1 receptors

Differential Virtual Screening (DVS) with Active and Inactive Molecular Models for Finding and Profiling GPCR Modulators: Case of the CCK1 Receptor.

We discovered a constitutively activating mutation (CAM) V308E for the neurotensin NT1 receptor. Molecular dynamics (MD) performed for the CAM NT1-V308E exhibiting a high spontaneous activity, and for the wild-type NT1 without basal activity, show dramatic conformational changes for the CAM. To test if the two MD models could be valuable active and inactive templates for building molecular models for other class-A GPCR, supposed active and inactive models were built by homology for the cholecystokinin CCK1 receptor. Virtual screening of a corporate library with 250 000 compounds was performed with the two CCK1 models, and a differential virtual screening analysis (DVS), led us to isolate 250 predicted agonists and 250 predicted antagonists. The two sets were merged and the compounds were tested in CCK1 agonist and antagonist cellular assays. An excellent correlation was obtained between predictions and biological results. The effective profiling provided by DVS with active and inactive molecular models, opens new perspectives for finding agonists and antagonists for other class-A GPCR, notably for orphan GPCRs for which no ligands are known.

β-Lactotensin derived from bovine β-lactoglobulin suppresses food intake via the CRF system followed by the CGRP system in mice

We found that β-lactotensin (His-Ile-Arg-Leu), which has been isolated as an ileum-contracting peptide from chymotrypsin digest of bovine β-lactoglobulin, dose-dependently suppresses food intake after intracerebroventricular (i.c.v.) or intraperitoneal administration at a dose of 40 nmol/mouse or 100 mg/kg, respectively, in fasted mice. Orally administered β-lactotensin also suppressed food intake at 500 mg/kg. We previously reported that β-lactotensin acts as an agonist for neurotensin receptors; however, the anorexigenic activity of β-lactotensin was not inhibited by i.c.v. co-administration with SR48692 or levocabastine, an antagonist for neurotensin NT 1 or NT 2 receptor, respectively. On the other hand, the anorexigenic effect of β-lactotensin was blocked by i.c.v. co-administration with astressin or calcitonin gene-related peptide (CGRP)(8–37), an antagonist for corticotropin releasing factor (CRF) or CGRP, respectively. β-Lactotensin had affinity for neither CRF nor CGRP receptor. In addition, CRF-induced anorexigenic activity after i.c.v. administration was completely blocked by CGRP(8–37), while CGRP-induced anorexigenic activity was not inhibited by astressin. These results suggest that the CGRP system is activated downstream of the CRF system in food intake regulation. Taken together, β-lactotensin may suppress food intake by activating the CRF system followed by the CGRP system, independently of the neurotensin system.

Effects of chronic infusion of neurotensin and a neurotensin NT1 selective analogue PD149163 on amphetamine-induced hyperlocomotion

Neurotensin (NT) has been proposed as an endogenous antipsychotic based in part on the similarity in behavioural effects to antipsychotic drugs, for example, attenuation of both amphetamine-induced hyperlocomotion (AH) and amphetamine disrupted pre-pulse inhibition in the rat. However, there is some evidence that repeated administration of NT or an analogue produces behavioural tolerance to such effects. The present experiments sought to confirm and extend these findings by testing the effects on AH of 7 days central administration of NT and the NT1 selective analogue PD 149163 and the effects of 21 days central administration of NT. NT and PD149163 continuously administered for 7 days produced no effect on AH (in contrast to attenuation with a single injection here and previously reported), whereas 21 days of NT administration potentiated AH. Together, these studies report that the effects of NT or a NT analogue on AH depends on the duration of administration of peptide. The results are discussed in comparison with the reported antipsychotic properties of acute administration of NT and possible mechanisms involving NT1 receptors.

β-Lactotensin, a neurotensin agonist peptide derived from bovine β-lactoglobulin, enhances memory consolidation in mice

β-Lactotensin (His-Ile-Arg-Leu) is an ileum-contracting tetrapeptide isolated from bovine β-lactoglobulin. We previously reported that a neurotensin agonist β-lactotensin shows antinociceptive effect through neurotensin NT 2 receptor. We found that centrally or orally administered β-lactotensin at a dose of 60 nmol/mouse or 300–500 mg/kg, respectively, increased memory consolidation in the step-through-type inhibitory avoidance test in mice. The memory-enhancing activity of β-lactotensin was inhibited by the dopamine D 2 receptor antagonist raclopride but not the D 1 receptor antagonist SCH23390. Taken together, β-lactotensin might improve memory consolidation through activating the dopamine D 2 receptor.

Effects of excitotoxic lesions of the medial prefrontal cortex on density of high affinity [ 125I-Tyr3]neurotensin binding sites within the ventral midbrain and striatum

The present study was aimed at determining the extent to which excitotoxic lesions of the medial prefrontal cortex reduce neurotensin receptors within the striatum, the nucleus accumbens, the ventral tegmental area and the substantia nigra. The medial prefrontal cortex was unilaterally lesioned with ibotenic acid and 10 days later brain sections were processed for neurotensin receptor autoradiographic analysis using 0.1 nM [ 125I-Tyr3]neurotensin with, or without, levocabastine. Analysis revealed at least two sites, one levocabastine-insensitive neurotensin NT 1 and one levocabastine-sensitive neurotensin NT 2-like. The proportion of the latter site was high within the caudal striatum, the nucleus accumbens and the medial prefrontal cortex. Lesions produced a 60% to 80% reduction in neurotensin NT 1 within the ipsilateral medial prefrontal cortex, but no change in the sub-cortical nuclei. An increase in neurotensin NT 2-like receptors was found in ipsilateral dorso-caudal caudate. These results show that a significant amount of neurotensin NT 1 receptors are located on neurons within the medial prefrontal cortex but not on their efferent terminals.

β-Lactotensin and neurotensin rapidly reduce serum cholesterol via NT 2 receptor

β-Lactotensin, a neurotensin NT 2 agonist derived from β-lactoglobulin, has hypocholesterolemic activity after administration for 2 days at a dose of 30 mg/kg (i.p.) or 100 mg/kg (p.o.) for 2 days in mice fed a high-cholesterol/cholic acid diet. The onset of hypocholesterolemic activity of β-lactotensin was observed 90 min after a single i.p. or p.o. administration at the same dose as described above. Neurotensin also induced hypocholesterolemic activity 90 min after single i.p. administration at a dose of 2 μg per mouse but was ineffective after oral administration. The rapid onset of hypocholesterolemic activities of β-lactotensin and neurotensin was blocked by levocabastine (50 μg/kg), an NT 2 antagonist, and raclopride (0.5 mg/kg), a dopamine D 2 antagonist.

Antinociception induced by β-lactotensin, a neurotensin agonist peptide derived from β-lactoglobulin, is mediated by NT 2 and D 1 receptors

In this study, we examined the antinociceptive effect of β-lactotensin, a neurotensin agonist that has been isolated from the chymotrypsin digest of β-lactoglobulin as an ileum-contracting peptide. β-Lactotensin showed naloxone-insensitive antinociceptive activity by the tail-pinch test after i.c.v. (200 nmol/mouse) or s.c. (300 mg/kg) administration in ddY mice. Tolerance was not developed to antinociception induced by β-lactotensin after repeated s.c. administration for 5 days. The antinociceptive activity of β-lactotensin was blocked by treatment with the neurotensin NT 2 receptor antisense ODN, while treatment with the NT 1 receptor antisense ODN had no effect. The antinociceptive activity was also blocked by a dopamine D 1 receptor antagonist, SCH23390 (1 μg/mouse, i.c.v.), while a D 2 receptor antagonist, raclopride (0.5 μg/mouse, i.c.v.), did not block the activity. These results indicate that the antinociceptive activity of β-lactotensin is mediated by NT 2 and D 1 receptors.

Neurotensin effect on Na+, K+-ATPase is CNS area- and membrane-dependent and involves high affinity NT1 receptor.

We have previously shown that peptide neurotensin inhibits cerebral cortex synaptosomal membrane Na+, K+-ATPase, an effect fully prevented by blockade of neurotensin NT1 receptor by antagonist SR 48692. The work was extended to analyze neurotensin effect on Na+, K+-ATPase activity present in other synaptosomal membranes and in CNS myelin and mitochondrial fractions. Results indicated that, besides inhibiting cerebral cortex synaptosomal membrane Na+, K+-ATPase, neurotensin likewise decreased enzyme activity in homologous striatal membranes as well as in a commercial preparation obtained from porcine cerebral cortex. However, the peptide failed to alter either Na+, K+-ATPase activity in cerebellar synaptosomal and myelin membranes or ATPase activity in mitochondrial preparations. Whenever an effect was recorded with the peptide, it was blocked by antagonist SR 48692, indicating the involvement of the high affinity neurotensin receptor (NT1), as well as supporting the contention that, through inhibition of ion transport at synaptic membrane level, neurotensin plays a regulatory role in neurotransmission.

Regulation of the neurotensin NT(1) receptor in the developing rat brain following chronic treatment with the antagonist SR 48692.

The aim of the present study was to investigate the role of neurotensin in the regulation of NT(1) receptors during postnatal development in the rat brain. Characterization of the ontogeny of neurotensin concentration and [(125)I]neurotensin binding to NT(1) receptors in the brain at different embryonic and postnatal stages showed that neurotensin was highly expressed at birth, reaching peak levels at postnatal day 5 (P5) and decreasing thereafter. The transient rise in neurotensin levels preceded the maximal expression of NT(1) receptors, observed at P10, suggesting that neurotensin may influence the developmental profile of NT(1) receptors. Using primary cultures of cerebral cortex neurons from fetal rats, we showed that exposure to the neurotensin agonist JMV 449 (1 nM) decreased (-43%) the amount of NT(1) receptor mRNA measured by reverse transcription-PCR, an effect that was abolished by the nonpeptide NT(1) receptor antagonist SR 48692 (1 microM). However, daily injection of SR 48692 to rat pups from birth for 5, 9, or 15 days did not modify [(125)I]neurotensin binding in brain membrane homogenates. Moreover, postnatal blockade of neurotensin transmission did not alter the density and distribution of NT(1) receptors assessed by quantitative autoradiography nor NT(1) receptor mRNA expression measured by in situ hybridization in the cerebral cortex, caudate-putamen, and midbrain. These results suggest that although NT(1) receptor expression can be regulated in vitro by the agonist at an early developmental stage, neurotensin is not a major factor in the establishment of the ontogenetic pattern of NT receptors in the rat brain.

Differential ontogenetic patterns of levocabastine-sensitive neurotensin NT 2 receptors and of NT 1 receptors in the rat brain revealed by in situ hybridization

The postnatal ontogeny of the levocabastine-sensitive neurotensin receptor (NT 2) mRNA was studied by in situ hybridization in the rat brain and compared with the distribution of the levocabastine-insensitive NT 1 receptor. NT 2 receptor mRNA was absent at birth from all brain structures except the ependymal cell layer lining the ventricles. The development of NT 2 receptor mRNA followed three ontogenetic patterns. The first pattern, involving the majority of the cerebral gray matter, was characterized by a continuous increase from postnatal day 5 (P5) to P30. The second one, involving regions rich in myelinated fibers such as the corpus callosum and lacunosum moleculare layer of the hippocampus, exhibited a pronounced increase between P5 and P10, peaked at P15 and was followed by a plateau or a slight decrease. The third pattern was observed in the ependymal cell layer lining the olfactory and lateral ventricles, where the high labeling already present at birth continued to increase during development. These different developmental patterns could reflect the variety of cells expressing NT 2 receptor mRNA, including neurons, protoplasmic astrocytes in gray matter, fibrous astrocytes present in myelinated fibers tracts, and ependymal cells. In contrast, NT 1 receptor mRNA, which seems to be associated only with neurons, was highly and transiently expressed during the perinatal period in the cerebral cortex, hippocampus and striatal neuroepithelium. Other regions, notably the ventral tegmental area and substantia nigra compacta, exhibited a gradual increase in NT 1 receptor signal, reaching adult levels by P21. Both the differential localization and ontogenetic profiles of NT 1 and NT 2 receptor mRNAs suggest different involvement of these two receptors in brain functions and development.

Neurotensin is an antagonist of the human neurotensin NT 2 receptor expressed in Chinese hamster ovary cells

The human levocabastine-sensitive neurotensin NT 2 receptor was cloned from a cortex cDNA library and stably expressed in Chinese hamster ovary (CHO) cells in order to study its binding and signalling characteristics. The receptor binds neurotensin as well as several other ligands already described for neurotensin NT 1 receptor. It also binds levocabastine, a histamine H 1 receptor antagonist that is not recognised by neurotensin NT 1 receptor. Neurotensin binding to recombinant neurotensin NT 2 receptor expressed in CHO cells does not elicit a biological response as determined by second messenger measurements. Levocabastine, and the peptides neuromedin N and xenin were also ineffective on neurotensin NT 2 receptor activation. Experiments with the neurotensin NT 1 receptor antagonists SR48692 and SR142948A, resulted in the unanticipated discovery that both molecules are potent agonists on neurotensin NT 2 receptor. Both compounds, following binding to neurotensin NT 2 receptor, enhance inositol phosphates (IP) formation with a subsequent [Ca 2+] i mobilisation; induce arachidonic acid release; and stimulate mitogen-activated protein kinase (MAPK) activity. Interestingly, these activities are antagonised by neurotensin and levocabastine in a concentration-dependent manner. These activities suggest that the human neurotensin NT 2 receptor may be of physiological importance and that a natural agonist for the receptor may exist.

Distinct functional characteristics of levocabastine sensitive rat neurotensin NT 2 receptor expressed in Chinese hamster ovary cells

Neurotensin has been shown to produce pharmacological effects both in brain and periphery. Several of these effects are mediated by a high-affinity neurotensin NT 1 receptor. On the other hand, a low-affinity levocabastine-sensitive neurotensin NT 2 receptor was molecularly cloned from rodent brain recently. In this study, in contrast to NT 1 receptor, levocabastine (a histamine H 1 receptor antagonist) and SR48692 (an antagonist for NT 1 receptor) strongly stimulated intracellular Ca 2+ mobilization in transfected Chinese hamster ovary cells expressing rat NT 2 receptor, thus acting as potent NT 2 receptor. Furthermore, despite of their affinities for NT 2 receptor, the Ca 2+ responses to potent NT 1 agonists, neurotensin or JMV449 ([Lys 8-( ch 2 nh)-Lys 9]Pro-Tyr-Ile-Leu, a peptidase resistant analogue of neurotensin) were much smaller than that observed with SR48692. These findings suggest that NT 1 and NT 2 receptors present distinct functional characteristics and that SR48692 may act as a potent agonist for NT 2 receptor.

Pro-neurotensin/Neuromedin N Expression and Processing in Human Colon Cancer Cell Lines

The regulatory peptide neurotensin NT has been proposed to exert an autocrine trophic effect on human colon cancers. In the present study, pro-neurotensin/neuromedin N (proNT/NN) expression and processing were investigated in 13 human colon cancer cell lines using a combination of radioimmunoassay and HPLC techniques. All 13 cell lines displayed low to moderate levels of proNT/NN ranging from 10 to 250 fmol/mg protein. However, only 6 (HCT8, LoVo, HT29, C119A, LS174T, and coloDM320) processed the precursor. Three of the latter (HCT8, LS174T, and coloDM320) were analysed in detail with regard to proNT/NN processing pattern and were found to produce NT and large precursor fragments ending with the NT or NN sequence. They had no detectable level of NN. Such a processing pattern resembles that generated by the prohormone convertase PC5. Northern and Western blot analysis of prohormone convertase expression in the 3 cell lines revealed that they were devoid of PC1 and PC2, whereas they all expressed PC5. These data indicate that proNT/NN is a good marker of human colon cancer cell lines while NT is found in only about half of the cell lines. They also suggest that, in addition to NT, several proNT/NN-derived products, possibly generated by PC5, might exert an autocrine positive effect on human colon cancer growth.