Dominique Nouel

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...

Primary structure and expression of a naturally truncated human P2X ATP receptor subunit from brain and immune system

A novel member of the ionotropic ATP receptor gene family has been identified in human brain. This 422 amino acid long P2X receptor subunit has 62% sequence identity with rat P2X5. Several characteristic motifs of ATP‐gated channels are present in its primary structure, but this P2X5‐related subunit displays a single transmembrane domain. Heterologous expression of chimeric subunits containing the C‐terminal domain of rat P2X5 leads to the formation of desensitizing functional ATP‐gated channels in Xenopus oocytes. The developmentally regulated mRNA, found in two splicing variant forms, is expressed at high levels in brain and immune system.

Receptor-induced Internalization of Selective Peptidic μ and δ Opioid Ligands

The binding and internalization of radioiodinated and fluorescent μ and δ opioid peptides in mammalian cells were quantitatively studied by biochemical techniques and directly visualized by confocal microscopy. The labeled peptides were prepared by inserting either a 125I-Bolton-Hunter group or a fluorescent probe into the C-terminal part of 5-aminopentylamide derivatives of deltorphin-I and [Lys7]dermorphin. The purified derivatives kept most of their specificity and selectivity toward δ and μ opioid receptors, respectively. Biochemical and confocal microscopy data showed that both μ and δ opioid peptides were internalized in mammalian cells transfected with the corresponding opioid receptor according to a receptor-mediated mechanism. The internalization process was time- and temperature-dependent and was completely blocked by the endocytosis inhibitor phenylarsine oxyde. Internalization of both δ and μ ligands occurred from a single large cap at one pole of the cell, indicating that polymerization of ligand-receptor complexes preceeded internalization. Finally, green and red fluorescent analogues of deltorphin-I and [Lys7]dermorphin, respectively, were found to internalize through partly distinct endocytic pathways in cells co-transfected with μ and δ receptors, suggesting that each of these receptors interacts with distinct proteins mediating intracellular sorting and trafficking.

Receptor-mediated Internalization Is Critical for the Inhibition of the Expression of Growth Hormone by Somatostatin in the Pituitary Cell Line AtT-20

The inhibitory effect of the neuropeptide somatostatin on the expression of growth hormone was measured by quantitative polymerase chain reaction in the pituitary cell line AtT-20. We demonstrate that this effect is dependent on the internalization of somatostatin-receptor complexes and that it is totally independent from the peptide-induced inhibition of adenylate cyclase. Indeed, the inhibitory effect of the peptide on growth hormone mRNA levels was totally insensitive to pertussis toxin treatment but was totally abolished under conditions which block somatostatin receptor internalization. Comparative confocal microscopic imaging of fluorescent somatostatin sequestration and fluorescence immunolabeling of sst1, sst2A, and sst5 receptors suggests that sst2A is most probably responsible of the inhibitory effect of somatostatin on growth hormone expression.

Pharmacological, molecular and functional characterization of glial neurotensin receptors

The pharmacological properties, molecular identity and physiopathological regulation of neurotensin receptors expressed by central astrocytes were investigated in primary glial cultures and sections from the adult rat brain. Binding experiments carried out on astrocytes in culture revealed the presence of a single apparent class of neurotensin binding sites. These sites bound [125]neurotensin with an affinity (6 nM) comparable to that of the recently cloned NT2 low-affinity receptor expressed in transfected cells. The glial receptor was sensitive to the antihistamine, levocabastine, but less so than the NT2 site expressed in heterologous expression systems, suggesting the presence of an additional site or a differential coupling of the NT2 receptor in glia. Reverse transcription-polymerase chain reaction experiments demonstrated that both NT2 and NT3 neurotensin receptor sub-types were in fact expressed by cortical glial cells in culture. Confocal microscopic visualization of specifically bound fluorescent neurotensin indicated that this expression concerned only a sub-population of astrocytes in culture, in conformity with earlier reports of a heterogeneous expression of neuropeptides and their receptors by glial cells. To further investigate the functionality of NT2 receptors expressed in astrocytes, dual immunohistochemical labeling of glial fibrillary acidic protein and in situ hybridization of NT2 messenger RNA was performed on sections of normal and lesioned rat brain. In sections from normal brain, only a small subset of immunolabeled astrocytes hybridized NT2 messenger RNA. By contrast, in sections of stab-wounded rat brains, there was a marked increase in the number of NT2-hybridizing astrocytes in the surround of the lesion. Furthermore, NT2 expression within immunopositive reactive astrocytes was significantly enhanced as compared to immunolabeled glial cells in the brain of control animals. These results indicate that NT2 receptor expression is up-regulated during astrocytic reaction, suggesting that NT2 receptors may play a role in regulating glial response to injury.

Axonal and dendritic transport of internalized neurotensin in rat mesostriatal dopaminergic neurons

Previous studies have demonstrated that neurotensin is internalized and retrogradely transported in neurons of the substantia nigra following its intracerebral injection in the neostriatum. The aim of the present study was to compare the intracellular distribution of retrogradely transported material with that observed following internalization of the peptide at the somatodendritic level and to confirm that the internalization was confined to dopamine neurons. To document somatodendritic internalization, slices (350 microns) from the rat ventral midbrain were incubated in vitro with 20 mM fluoresceinylated neurotensin, a fluorescent derivative of neurotensin, and immunostained 5-60 min later for tyrosine hydroxylase. To document retrograde transport, rats were injected with the same compound into the neostriatum and the brains processed for tyrosine hydroxylase immunohistochemistry 4.5 and 8 h later. Confocal laser microscopic examination of superfused slices revealed that fluoresceinylated neurotensin was internalized at the level of the perikarya and processes of neurons in the substantia nigra, ventral tegmental area and interfascicular nucleus. At short time intervals, the label was detected in the form of small, intensely fluorescent particles distributed within the cytoplasm of both perikarya and dendrites. At longer time intervals, these fluorescent particles were larger, less numerous and confined to the perikarya where they eventually clustered against the nucleus. Following intrastriatal injection of fluoresceinylated neurotensin, retrogradely labeled cells were apparent throughout the substantia nigra, pars compacta, as well as in the lateral part of the ventral tegmental area. Here again, the label took the form of small fluorescent particles, comparable in size, shape and distribution to those detected following superfusion of midbrain slices. In both labeling conditions, fluoresceinylated neurotensin was almost exclusively confined to tyrosine hydroxylase-immunoreactive cells. These results indicate that neurotensin is internalized throughout the terminal and dendritic arborization of mesostriatal dopamine cells and that the internalized peptide is transported centripetally from both locations to the soma of the cells. The clustering of fluorescent particles in the perinuclear region of the cells further suggests that the internalized process may play a role in the long term transcellular signalling.

Preferential expression of the neuropeptide Y Y1 over the Y2 receptor subtype in cultured hippocampal neurones and cloning of the rat Y2 receptor

Neuropeptide Y (NPY) and NPY receptors are most abundant in the hippocampal formation where they modulate cognitive functions. Expression of NPY receptors in rat cultured primary hippocampal cells was investigated in the present study by use of combined molecular, pharmacological and immunohistochemical approaches, including the cloning of the rat Y2 receptor described here for the first time. More than 70% of the hippocampal neurones were endowed with [125I]‐[Leu31,Pro34]PYY Y1‐like receptor silver grain accumulations and Y1 receptor immunostaining. These radio‐ and immuno‐labelling signals were distributed over cell bodies and processes of bipolar, stellate and pyramidal‐like neuronal cells, as confirmed by neurone‐specific enolase and MAP‐2 staining. Competition binding profiles revealed that specific [125I]‐[Leu31,Pro34]PYY binding was competitively displaced according to a ligand selectivity pattern prototypical of the Y1 receptor sub‐type with [Leu31,Pro34]substituted NPY/PYY analogues>>C‐terminal fragments=pancreatic polypeptides, with the non‐peptide antagonist BIBP3226 being most potent. This profile excludes the possible labelling by [125I]‐[Leu31,Pro34]PYY of the newly cloned Y4, Y5 and Y6 receptors. The expression of the genuine Y1 receptor was confirmed by RT–PCR in hippocampal cultures. In contrast, negligible levels of Y2‐like/[125I]‐PYY3–36 binding were detected in these cultures in spite of the presence of its mRNA, as characterized by RT–PCR. The expression of both the Y1 and the Y2 receptor mRNAs was also noted in normal embryonic hippocampal tissues showing that signals expressed in cultured neurones were also present in utero. Taken together, these results suggest that the Y1 receptor subtype may be of critical importance in the normal functioning of the rat hippocampus, especially during brain development and maturation.