Jean Mazella

Differential Internalization of Somatostatin in COS-7 Cells Transfected with SST1 and SST2 Receptor Subtypes: A Confocal Microscopic Study Using Novel Fluorescent Somatostatin Derivatives

A growing body of evidence suggests that neuropeptide binding to G protein-linked receptors may result in internalization of receptor-ligand complexes, followed by intracellular mobilization and degradation of the ligand into its target cells. Because of discrepant results in the literature concerning the occurrence of such a mechanism for the tetradecapeptide somatostatin (SRIF), we have reinvestigated this question by comparing the binding and internalization of iodinated and fluorescent derivatives of the metabolically stable analog of SRIF,[ d-Trp8]SRIF, in COS-7 cells transfected with complementary DNA encoding the sst1 or sst2A receptor subtype. A series of fluoresceinyl and Bodipy fluorescent derivatives of[ d-Trp8]SRIF-14 was purified by HPLC, analyzed for purity by mass spectrometry, and tested for biological activity in a membrane binding assay. Of the six compounds tested, fluoresceinyl and Bodipy derivatives labeled in position α (fluo-SRIF) retained high affinity for SRIF receptors. COS-7 cel...

Mutagenesis and modeling of the neurotensin receptor NTR1. Identification of residues that are critical for binding SR 48692, a nonpeptide neurotensin antagonist.

The two neurotensin receptor subtypes known to date, NTR1 and NTR2, belong to the family of G-protein-coupled receptors with seven putative transmembrane domains (TM). SR 48692, a nonpeptide neurotensin antagonist, is selective for the NTR1. In the present study we attempted, through mutagenesis and computer-assisted modeling, to identify residues in the rat NTR1 that are involved in antagonist binding and to provide a tentative molecular model of the SR 48692 binding site. The seven putative TMs of the NTR1 were defined by sequence comparison and alignment of bovine rhodopsin and G-protein-coupled receptors. Thirty-five amino acid residues within or flanking the TMs were mutated to alanine. Additional mutations were performed for basic residues. The wild type and mutant receptors were expressed in COS M6 cells and tested for their ability to bind125I-NT and [3H]SR 48692. A tridimensional model of the SR 48692 binding site was constructed using frog rhodopsin as a template. SR 48692 was docked into the receptor, taking into account the mutagenesis data for orienting the antagonist. The model shows that the antagonist binding pocket lies near the extracellular side of the transmembrane helices within the first two helical turns. The data identify one residue in TM 4, three in TM 6, and four in TM 7 that are involved in SR 48692 binding. Two of these residues, Arg327 in TM 6 and Tyr351 in TM 7, play a key role in antagonist/receptor interactions. The former appears to form an ionic link with the carboxylic group of SR 48692, as further supported by structure-activity studies using SR 48692 analogs. The data also show that the agonist and antagonist binding sites in the rNTR1 are different and help formulate hypotheses as to the structural basis for the selectivity of SR 48692 toward the NTR1 and NTR2.

Regional and cellular distribution of low affinity neurotensin receptor mRNA in adult and developing mouse brain

Levocabastine‐sensitive neurotensin receptor (NTRL) mRNAs were localized by in situ hybridization in adult and developing mouse brain. NTRL hybridization signal was widely distributed throughout the neuraxis. The highest concentrations of NTRL mRNA were detected in the olfactory system, olfactory tubercle, cerebral and cerebellar cortices, hippocampal formation, and selective hypothalamic nuclei. Moderate to dense hybridization signal was also observed in association with a variety of auditory, visual, and somatosensory relay nuclei, suggesting that the NTRL might be involved in a widespread modulation of primary afferent pathways. Finally a high expression of NTRL was evident in brainstem structures implicated in descending antinociceptive influences (e.g., the periaqueductal gray, nucleus raphe magnus, gigantocellular reticular nucleus, pars alpha, and lateral paragigantocellular nucleus) consistent with the proposed mediation of NT‐induced analgesia by the NTRL. Although most of the regions found here to express NTRL mRNA were previously reported to be devoid of mRNA encoding the high affinity NT receptor (NTRH), a few areas (e.g., the anterior olfactory nucleus, medial septum, diagonal band of Broca, reticular thalamic nucleus, suprachiasmatic hypothalamic nucleus, and pontine nucleus) were enriched in both receptor subtypes, suggesting a possible coexpression of these receptors by the same cells. Ontogenic studies revealed that in the mouse brain, NTRL mRNA was detected only from postnatal day 14 and did not reach adulthood concentrations before day 30. In cerebral cortex, the developmental increase in NTRL expression was correlated over time with the decrease in NTRH expression previously documented in the rat, suggesting a progressive takeover of the latter by the former for transduction of the effects of NT in this structure. J. Comp. Neurol. 394:344–356, 1998.

High-affinity receptor sites and rapid proteolytic inactivation of neurotensin in primary cultured neurons.

Abstract: The present article describes the interaction of neurotensin with specific receptors in pure primary cultured neurons and the mechanisms by which this peptide is inactivated by these cells. Neurotensin binding sites are not detectable in nondifferentiated neurons and appear during maturation. The binding at 37°C of [monoiodo‐Tyr3 neurotensin to monolayers of neurons 96 h after plating is saturable and characterized by a dissociation constant of 300 pM and a maximal binding capacity of 178 fmol/mg of protein. The binding parameters as well as the specificity of these receptors toward neurotensin analogues reveal close similarities between the binding sites present in primary cultured neurons and those described in other membrane preprations or cells. Neurotensin is rapidly degraded by primary cultured neurons. The sites of primary inactivating cleavages are the Pro7‐Arg8, Arg8‐Arg9, and Pro10‐Tyr11 bonds. Proline endopeptidase is totally responsible for the cleavage at the Pro7‐Arg8 bond and contributes to the hydrolysis mainly at the Pro10‐Tyr11 site. However, the latter breakdown is also generated by a neurotensin‐degrading neutral metallopeptidase. The cleavage at the Arg8‐Arg9 bond is due to a peptidase that can be specifically inhibited by N‐[1 (R, S)‐carboxy‐2‐phenylethyl]‐alanyl‐alanyl‐phenylalanyl‐p‐aminobenzoate. The secondary processing occurring on neurotensin degradation products are: (1) a bestatin‐sensitive aminopeptidasic conversion of neurotensin, 11‐13 to free Tyr11, and (2) a rapid cleavage of neurotensin8‐13 by proline endopeptidase. A model for the inactivation of neurotensin in primary cultured neurons is proposed and compared to that previously described for purified rat brain synaptic membranes.

Neurotensin protects pancreatic beta cells from apoptosis

The survival of pancreatic beta cells depends on the balance between external cytotoxic and protective molecular systems. The neuropeptide neurotensin (NT) has been shown to regulate certain functions of the endocrine pancreas including insulin and glucagon release. However, the mechanism of action of NT as well as the identification of receptors involved in the pancreatic functions of the peptide remained to be studied. We demonstrate here that NT is an efficient protective agent of pancreatic beta cells against cytotoxic agents. Both beta-TC3 and INS-1E cell lines and the mouse pancreatic islet cells express the three known NT receptors. The incubation of beta cells with NT protects cells from apoptosis induced either by staurosporine or by IL-1beta. In beta-TC3 cells, NT activates both MAP and PI-3 kinases pathways and strongly reduces the staurosporine or the Il-1beta-induced caspase-3 activity by a mechanism involving Akt activation. The NTSR2 agonist levocabastine displays the same protective effect than NT whereas the NTSR1 antagonist is unable to block the effect of NT suggesting the predominant involvement of the NTSR2 in the action of NT on beta cells. These results clearly indicate for the first time that NT is able to protect endocrine beta cells from external cytotoxic agents, a role well correlated with its release in the circulation after a meal.

In vitro and in vivo regulation of synaptogenesis by the novel antidepressant spadin

We have described a novel antidepressant peptide, spadin, that acts by blocking the TWIK‐related‐potassium channel, type 1 (TREK‐1). Here, we examined possible mechanisms of action of spadin at both molecular and cellular levels.

Focal adhesion kinase dependent activation of the PI3 kinase pathway by the functional soluble form of neurotensin receptor-3 in HT29 cells

The neurotensin (NT) receptor-3 (NTSR3), also called sortilin, is thought to display several functions including a role as a receptor or a co-receptor, in the sorting to plasma membrane and to lysosomes, and in the regulated secretion. The aim of this study was to investigate the function of the soluble form of NTSR3 (sNTSR3) released from several cell lines including colonic cancer cells. The human adenocarcinoma epithelial cell line HT29 has been used to monitor the release, the binding and internalization of sNTSR3 by radioreceptor assays and confocal microscopy. The modulation of the intracellular signaling pathways by the protein has been investigated by using Fura-2 fluorescence calcium imaging microscopy and Western blots analysis. We demonstrated that sNTSR3 specifically binds and internalizes into HT29 cells. This binding, independent from the transactivation of the epidermal growth factor receptor, leads to the increase of intracellular calcium concentration and to the activation of a FAK/Src-dependent activation of the PI3 kinase pathway. In conclusion, sNTSR3 released from the membrane bound NTSR3 is a functional protein able to activate intracellular pathways involved in cell survival but probably not in cell growth.

Retroinverso analogs of spadin display increased antidepressant effects

RationaleAlthough depression is the most common mood disorder, only one third of patients are treated with success. Finding new targets, new drugs, and also new drug intake way are the main challenges in the depression field. Several years ago, we identified a new target with the TWIK-related potassium channel-1 (TREK-1) potassium channel, and more recently, we have discovered a peptide of 17 amino acids with antidepressant properties. This peptide, that we called spadin, can be considered as a new concept in antidepressant drug design. Spadin derives from a larger peptide resulting to a posttranslational maturation of sortilin; consequently, spadin can be considered as a natural molecule. Moreover, spadin acts more rapidly than classical antidepressants and does not induce side effects.ObjectivesIn this work, we sought analogs of spadin displaying a better affinity on TREK-1 channels and an increased action duration.MethodsAnalogs were characterized by electrophysiology measurements, by behavioral tests, and by their ability to induce neurogenesis.ResultsWe identified two retro-inverso peptides that have kept the antidepressant properties of spadin; particularly, they increased the hippocampal neurogenesis after a 4-day treatment. As spadin, these analogs did not induce side effects on either pain, epilepsy processes, or at the cardiac level.ConclusionsTogether, our results indicated that spadin retro-inverso peptides could represent new potent antidepressant drugs. As exemplified by spadin in the field of depression, retro-inverso strategies could represent a useful technique for developing new classes of drugs in a number of pathologies.

Neurotensin-induced Erk1/2 phosphorylation and growth of human colonic cancer cells are independent from growth factors receptors activation.

Neurotensin (NT) promotes the proliferation of human colonic cancer cells by undefined mechanisms. We already demonstrated that, in the human colon adenocarcinoma cell line HT29, the effects of NT were mediated by a complex formed between the NT receptor-1 (NTSR1) and-3 (NTSR3). Here we examined cellular mechanisms that led to NT-induced MAP kinase phosphorylation and growth factors receptors transactivation in colonic cancer cells and proliferation in HT29 cells. With the aim to identify upstream signaling involved in NT-elicited MAP kinase activation, we found that the stimulatory effects of the peptide were totally independent from the activation of the epidermal growth factor receptor (EGFR) both in the HT29 and the HCT116 cells. NT was unable to promote phosphorylation of EGFR and to compete with EGF for its binding to the receptor. Pharmacological approaches allowed us to differentiate EGF and NT signaling in HT29 cells since only NT activation of Erk1/2 was shown to be sensitive to PKC inhibitors and since only NT increased the intracellular level of calcium. We also observed that NT was not able to transactivate Insulin-like growth factor receptor. Our findings indicate that, in the HT29 and HCT116 cell lines, NT stimulates MAP kinase phosphorylation and cell growth by a pathway which does not involve EGF system but rather NT receptors which transduce their own intracellular effectors. These results indicate that depending on the cell line used, blocking EGFR is not the general rule to inhibit NT-induced cancer cell proliferation.

Increased Brain Neurotensin and NTSR2 Lead to Weak Nociception in NTSR3/Sortilin Knockout Mice

The neuropeptide neurotensin (NT) elicits numerous pharmacological effects through three different receptors (NTSR1, NTSR2, and NTSR3 also called sortilin). Pharmacological approaches and generation of NTSR1 and NTSR2-deficient mice allowed to determine the NT-induced antipsychotic like behavior, the inhibitory of weak fear memory and the nociceptive signaling in a rat formalin tonic pain model to NTSR1. Conversely, the effects of NT on thermal and tonic nociceptions were mediated by NTSR2. However, the role of NTSR3/sortilin on the neurotensinergic system was not investigated. Here, by using C57Bl/6J mouse model in which the gene coding for NTSR3/sortilin has been inactivated, we observed a modification of the expression of both NTSR2 and NT itself. Quantitative PCR and protein expression using Western blot analyses and AlphaLisa™ technology resulted in the observation that brain NTSR2 as well as brain and blood NT were 2-fold increased in KO mice leading to a resistance of these mice to thermal and chemical pain. These data confirm that NTSR3/sortilin interacts with other NT receptors (i.e., NTSR2) and that its deletion modifies also the affinity of this receptor to NT.