Synthesis of a biologically active fluorescent probe for labeling neurotensin receptors.

Faure, Marie‐Pierre; Gaudreau, Pierrette; Shaw, Ivan; Cashman, Neil R.; Beaudet, Alain

doi:10.1177/42.6.8189037

Cited by 32 publications

(25 citation statements)

References 12 publications

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“…Previous experiments have shown that in rat basal forebrain slices and in septal neuroblastoma cells (SN17), NT-NTR internalized complex is transported from the cell periphery to the perinuclear region by endosomes (34,35). This result suggested that NTR internalization may play a role in NT signaling.…”

Section: Ntr Gene Activation Requires Protein Synthesis-mentioning

confidence: 52%

Neurotensin Agonist Induces Differential Regulation of Neurotensin Receptor mRNA

Souazé

Rostène

Forgez

1997

Journal of Biological Chemistry

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The binding of neurotensin (NT) to specific receptors triggers the multiple functions that NT exerts in both periphery and brain. By studying the effect of the concentration and time of NT agonist exposure, two separate regulatory mechanisms were detected for the neurotensin receptor (NTR) gene in human colonic adenocarcinoma cells (HT-29).The incubation of cells for 6 h with the NT agonist, JMV 449, resulted in an increase of 270% in NTR mRNA levels. These changes were the direct result of new NTR gene transcription, as indicated by run-on and half-life experiments. In addition, the transcriptional activation of the NTR gene was dependent on NT-receptor complex internalization and de novo protein synthesis.A second response was detected with prolonged exposure to JMV 449. In this case, a decrease of 70% was detected in NTR mRNA levels. Unlike the initial phase, this change was mediated by a post-transcriptional event as the half-life of NTR mRNA from treated cells decreased by 50% as compared with control cells.NT agonist appears to regulate the synthesis of NTR mRNA. In HT-29 cells, this feedback is exerted by a biphasic response. These phases are apparently independent and mediated by two separate mechanisms. Neurotensin (NT)1 is a tridecapeptide, widely distributed in the central nervous system and peripheral tissues, exerting multiple functions (1). In the central nervous system, NT is a neurotransmitter as well as a neuromodulator of other neurotransmitters such as dopamine, acetylcholine, serotonin, and noradrenaline (2, 3). NT also possesses neuroendocrine actions inducing the release of several pituitary hormones (4). In the periphery, NT is secreted from mucosal endocrine cells of the small intestine into the circulation (5). In the gastrointestinal tract, NT causes many physiological effects including the stimulation of pancreatic secretion, the facilitation of colonic motility and fatty acid translocation, and tissue growth (6).In rat, NT actions are mediated by the stimulation of several specific receptors exhibiting high or low affinity for NT (7,8).The high affinity neurotensin receptor (NTR) is composed of 424 amino acids and belongs to the seven-transmembrane domain receptor family coupled to the G-proteins (7). The human NTR counterpart has also been cloned from human colonic adenocarcinoma cells (HT-29) (9). When HT-29 cells are challenged with a NT agonist, phosphatidylinositols are hydrolyzed leading to Ca 2ϩ mobilization (10). In contrast to N1E-115 cells, stimulation by NT in HT-29 cells is not associated with protein kinase C activation (10, 11).In addition to triggering cellular responses by specific ligands, receptors are often themselves regulated by their own agonists. In the case of NT, several studies have shown that variations in NTR expression were caused by changes in NT levels. For example, acute agonist stimulation of NTR induces desensitization and down-regulation of receptor in primary cultures of rat forebrain and HT-29 cells (12,13). Prolonged exposure of N1E-115 cells to N...

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Section: Ntr Gene Activation Requires Protein Synthesis-mentioning

confidence: 52%

Neurotensin Agonist Induces Differential Regulation of Neurotensin Receptor mRNA

Souazé

Rostène

Forgez

1997

Journal of Biological Chemistry

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“…In all cases, it is imperative to test the pharmacological properties of any new fluorescent ligand by determining its radioligand binding displacement properties on membranes from cells expressing the native re-ceptor or transfected with the recombinant receptor of interest (4)(5)(6). These binding experiments should be complemented by imaging studies on whole cells that either naturally express the receptor under study or are transfected with the appropriate recombinant DNA to measure concentration-dependent binding in the presence and absence of nonlabeled competitors.…”

Section: Selection Of a Fluorescent Ligandmentioning

confidence: 99%

Fluorescent ligands for studying neuropeptide receptors by confocal microscopy

Beaudet

Nouel

Stroh

et al. 1998

Braz J Med Biol Res

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This paper reviews the use of confocal microscopy as it pertains to the identification of G-protein coupled receptors and the study of their dynamic properties in cell cultures and in mammalian brain following their tagging with specific fluorescent ligands. Principles that should guide the choice of suitable ligands and fluorophores are discussed. Examples are provided from the work carried out in the authors laboratory using custom synthetized fluoresceinylated or BODIPYtagged bioactive peptides. The results show that confocal microscopic detection of specifically bound fluorescent ligands permits high resolution appraisal of neuropeptide receptor distribution both in cell culture and in brain sections. Within the framework of time course experiments, it also allows for a dynamic assessment of the internalization and subsequent intracellular trafficking of bound fluorescent molecules. Thus, it was found that neurotensin, somatostatin and muand delta-selective opioid peptides are internalized in a receptordependent fashion and according to receptor-specific patterns into their target cells. In the case of neurotensin, this internalization process was found to be clathrin-mediated, to proceed through classical endosomal pathways and, in neurons, to result in a mobilization of newly formed endosomes from neural processes to nerve cell bodies and from the periphery of cell bodies towards the perinuclear zone. These mechanisms are likely to play an important role for ligand inactivation, receptor regulation and perhaps also transmembrane signaling. Correspondence

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“…That bound NT molecules were indeed internalized in SN17 cells was further confirmed here by confocal laser microscopy using fluo-NT, a fluoresceinylated NT conjugate, as NT receptor marker. This compound was previously shown by us to display both the pharmacological and the electrophysiological properties of native NT and to provide for selective in vitro labelling of high affinity NT receptors (Alonso et al, 1994;Faure et al, 1994).…”

Section: Discussionmentioning

confidence: 99%

Binding and internalization of neurotensin in hybrid cells derived from septa1 cholinergic neurons

Faure

Labbé-Jullié

Cashman

et al. 1995

Synapse

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Autoradiographic studies from our laboratory have previously demonstrated a selective association of high affinity neurotensin (NT) binding sites with basal forebrain cholinergic neurons. In search of an in vitro model for further characterization of the role and regulation of these sites, we have examined the binding and internalization of 125I-Tyr3-NT (125I-NT) and fluorescein isothiocyanate (FITC)-conjugated NT (fluo-NT) on SN17 hybrid cells, produced by fusion of embryonic murine septal cells with neuroblastoma. 125I-NT binding to SN17 membrane preparations was specific and saturable. Scatchard analysis of the data was suggestive of an interaction with a single population of sites, the affinity (Kd = 1.7 nM) and pharmacological profile of which were comparable to those of neural NT receptors. No specific binding was observed on the parent neuroblastoma cell line, confirming that the expression of those sites is a neuronal trait. Incubation of whole SN17 cells with 125I-NT resulted in a time- and temperature-dependent internalization of the specifically bound peptide. The t1/2 of this internalization was estimated at 13 min, a value nearly identical to that reported for neurons in culture. Confocal microscopic analyses using fluo-NT indicated that the internalization process was endocytic in nature in that: 1) it was entirely blocked by the endocytosis inhibitor phenylarsine oxide; and 2) it was mediated through small intracytoplasmic particles the size and maturation of which corresponded to that of endosomes. It is proposed that the expression and internalization of NT receptors by SN17 hybrid cells represent a new facet of these cells' cholinergic phenotype that makes them amenable to the study of NT interactions with cholinergic cells.

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Synthesis of a biologically active fluorescent probe for labeling neurotensin receptors.

Abstract: We synthesized a fluorescent derivative of the tridecapeptide neurotensin (NT), with the aim ofproviding a new tool for the pharmacological characterization and anatomic localization of NT receptors in " a l i an brain. Fluoresceinylated NT (Na-fluorrsceinyl thiocarbamyl (FTc)-

Cited by 32 publications

References 12 publications

Neurotensin Agonist Induces Differential Regulation of Neurotensin Receptor mRNA

Neurotensin Agonist Induces Differential Regulation of Neurotensin Receptor mRNA

Fluorescent ligands for studying neuropeptide receptors by confocal microscopy

Binding and internalization of neurotensin in hybrid cells derived from septa1 cholinergic neurons

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