Contribution à l'étude du rôle du PACAP et du VIP dans le cerveau ainsi que dans des tumeurs d'origine neuroectodermique et hypophysaire

Doctorat en sciences médicalesinfo:eu-repo/semantics/nonPublishe

Contribution à l'étude du rôle du PACAP et du VIP dans le cerveau ainsi que dans des tumeurs d'origine neuroectodermique et hypophysaire

Doctorat en sciences médicalesinfo:eu-repo/semantics/nonPublishe

Identification of key residues for the binding of glucagon to the N-terminal domain of its receptor: an alanine scan and modeling study.

Glucagon plays an essential role in the glycemia maintenance during fasting, but also aggravates hyperglycemia in diabetic patients. A series of analogues of glucagon were synthesized replacing each amino acid of the C-terminal region (residues 15-29) with alanine. The residues affecting the binding to the glucagon receptor are found to be located on one face of the glucagon helix. Several 3-dimensional models of the N-terminal domain of the glucagon receptor in complex with its ligand peptide were built and used to analyze the peptide-receptor interface in terms of the nature of the peptide residues and the interactions they form with the receptor. The models suggest that glucagon keeps its native helical structure upon binding, and that a large part of the interface formed with the receptor is hydrophobic. We find that in the C-terminal region, F22, V23, M27, and D15 are the most important residues for peptide binding. They bury a large portion of their solvent accessible surface area and make numerous interactions with the receptor mainly of the hydrophobic type.Journal ArticleSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Pituitary Adenylate Cyclase-Activating Polypeptide: Development of Selective Ligands for Pac1, Vpac1 and Vpac2 Receptors

At the time of its isolation, purification and sequencing in 1970-1972 by Said and Mutt (Said and Mutt, 1972;Mutt and Said, 1974), VIP was the third identified member of a peptide group that included secretin (Mutt et al, 1970) and glucagon (Bromer et al, 1957). The family signature was a N-terminal histidylseryl sequence separated by three amino acid residues from a phenylalanylthreonyl sequence. Synthesis of secretin, VIP, glucagon and analogs as well as a careful study of their biological activities revealed that there was no, or only negligible, interference of glucagon with secretin and VIP mediated responses, nor of secretin and VIP with the glucagon response. It was also rapidly recognized that VIP and secretin shared common in vitro properties and that each peptide interacted with its cognate receptor and with the other receptors (Bataille et al, 1974; Robberecht et al, 1976). It was also established for the three peptides that the N-terminal sequence was necessary for the high affinity recognition of the receptor and for induction of the biological activity, and that the C-terminus was necessary for a high affinity recognition. It was thus not possible to shorten significantly the molecule without a loss of the biological properties or peptide potency. This was a major point for the rational design of agonists and antagonists as well as for the interest of industry. Binding studies of125I-labeled peptides characterized the receptors and for each peptide high and low affinity sites were described (Christophe et al, 1976; Laburthe et al, 1978; Prieto et al, 1979).info:eu-repo/semantics/publishe

Role of scaffolds, subchondral, intra-articular injections of fresh autologous bone marrow concentrate regenerative cells in treating human knee cartilage lesions: Different approaches and different results

The value of bone marrow aspirate concentrates for treatment of human knee cartilage lesions is unclear. Most of the studies were performed with intra-articular injections. However, subchondral bone plays an important role in the progression of osteoarthritis. We investigated by a literature review whether joint, subchondral bone, or/and scaffolds implantation of fresh autologous bone marrow aspirate concentrated (BMAC) containing mesenchymal stem cells (MSCs) would improve osteoarthritis (OA). There is in vivo evidence that suggests that all these different approaches (intra-articular injections, subchondral implantation, scaffolds loaded with BMAC) can improve the patient. This review analyzes the evidence for each different approach to treat OA. We found that the use of intra-articular injections resulted in a significant relief of pain symptoms in the short term and was maintained in 12 months. However, the clinical trials indicate that the application of autologous bone marrow concentrates in combination with scaffolds or in injection in the subchondral bone was superior to intra-articular injection for long-term results. The tendency of MSCs to differentiate into fibrocartilage affecting the outcome was a common issue faced by all the studies when biopsies were performed, except for scaffolds implantation in which some hyaline cartilage was found. The review suggests also that both implantation of subchondral BMAC and scaffolds loaded with BMAC could reduce the need for further surgery.SCOPUS: re.jinfo:eu-repo/semantics/publishe

Differential alternative splicing of PACAP receptor in pituitary cell subpopulations

The capability of rat pituitary cells to express receptors for pituitary adenylate cyclase activating polypeptide (PACAP) and VIP was evaluated by binding studies and measurement of adenylate cyclase activity on whole gland preparations and by reverse transcriptase-polymerase chain reaction (RT-PCR) using specific primers on preparations from isolated cell populations enriched in PRL- and GH-producing cells. Data obtained on whole gland preparations indicated that selective PACAP receptors (PACAP Type I) predominated. The mRNA coding for PACAP Type I and for the non-selective PACAP receptor Type II VIP2 (but not VIP1) were identified. The mRNA coding for four different spliced variants of the PACAP Type I receptor were detected. In PRL producing cells, three variants and the VIP, mRNA were detected, whereas in GH-producing cells the mRNA coding for the variant having a 28-amino acid insert (termed HOP) in the third intracellular loop was the only present.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Relative quantitative analysis of glucagon receptor mRNA in rat tissues

Total RNA prepared from nine rat tissues were analyzed for their content in glucagon receptor mRNA by two independent hybridization approaches: (1) simple dot blot analysis using labelled oligodeoxynucleotide; (2) highly specific RNase protection assay using labelled antisense RNA. Hybridization signal was quantified by laser densitometric scanning of autoradiographies. Results were expressed for each method relative to the liver content (100%) for either a constant amount of total RNA or for a constant amount of β-actin mRNA. We obtained similar relative values of glucagon receptor mRNA per constant amount of total RNA by the two hybridization methods: in liver (100 and 100), in kidney (38 and 34), and in heart (12 and 11). The glucagon receptor mRNA was overestimated by the less specific dot assays, in adrenal glands (21 versus 10) and in adipose tissues (24 versus 5). In the stomach, brain, duodenum and lung, the signal was equal to or below the reliable quantification limit. Reverse transcription and polymerase chain reaction (RT-PCR) of glucagon receptor mRNA with limited cycle number were performed using two sets of primers: the first set amplified a single band at the 3' coding end, and the second, 3-6 bands at the 5' coding end, revealing tissue-specific polymorphism. RT-PCR data confirmed the presence of glucagon receptor mRNA in liver, kidney, heart, adrenal glands and adipose tissue, and allowed the detection of a very low amount of glucagon receptor mRNA in the stomach, the duodenum and brain but not in the lung. Although a simple dot hybridization assay could be used for analysis of glucagon receptor mRNA level in liver, heart and kidney, the more sophisticated RNase protection assay is necessary to obtain reliable results in tissues with a lower expression of messenger. The glucagon receptor presented high, tissue-specific mRNA polymorphism. This may contribute to the difference of the expression of the glucagon receptor in different tissues.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Autoradiographic visualization of the receptor subclasses for vasoactive intestinal polypeptide (VIP) in rat brain. Ann N.Y. Acad Sci, 865: 412-415

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Autoradiographic visualization of the receptor subclasses for vasoactive intestinal polypeptide (VIP) in rat brain.

Vasoactive Intestinal Polypeptide (VIP) exerts its biological effects through interaction with two high affinity receptors named the VIP1- and the VIP2 receptors. Their messenger RNAs have been mapped in rat brain by in situ hybridization. A cyclic peptide (RO 25-1553) and a secretion analogue ([R16]chicken secretin) were identified as selective agonist peptides for the VIP2- and VIP1 receptors, respectively. The iodinated peptides retained the high affinity and selectivity of the unlabelled peptides and were used for the mapping of each receptor subclass in rat brain. VIP1 receptors were present in the cerebral cortex, the piriform cortex, the claustrum, the caudate-putamen, the dentate gyrus, the lateral amygdaloïd nucleus, the anteroventral thalamic nucleus, the rhomboïd nucleus, the supraoptic nucleus and the choroïd plexus. VIP2 receptors were present in the cerebral cortex, the claustrum, the caudate-putamen, the nucleus accumbens, the lateral septal nuclei, the bed nucleus of the stria terminalis, the basolateral amygdaloïd nucleus, the Ammon's horn, the thalamic nuclei except some centromedial nuclei, the medial habenula, the suprachiasmatic nucleus, the periventricular nucleus, the mammilary nucleus, the superior colliculus and the choroïd plexus.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Ac His1 [D-Phe2, K15, R16, L27] VIP (3-7)/GRF (8-27)--a VPAC1 receptor antagonist--is an inverse agonist on two constitutively active truncated VPAC1 receptors.

C-terminally truncated human VPAC(1) receptors were constructed and stably transfected in Chinese hamster ovary (CHO) cells. Selected clones expressing comparable receptor densities were studied for ligand's binding properties, basal and stimulated adenylate cyclase activity. The wild-type (1-457) receptor served as reference. The binding properties of all the constructions were preserved. As judged by the intrinsic activity of the partial agonist Q(3)-VIP, the shortest receptors have a moderate impairment of the coupling efficacy to G(alpha s) protein. Cells expressing the VPAC(1) (1-436) and (1-441) truncated receptors had a two- to three-fold higher basal adenylate cyclase activity than those expressing the wild-type or the VPAC(1) (1-444), (1-433), (1-429), (1-421) and (1-398) receptor. The stimulatory effect of VIP and other agonist was preserved. This suggested that VPAC(1) (1-436) and (1-441) receptors had a constitutive activity. The selective VPAC(1) receptor antagonist Ac His(1) [D-Phe(2), K(15), R(16), L(27)] VIP (3-7)/GRF (8-27) reduced by 60% the basal activity with an EC(50) value of 3 nM comparable to its IC(50) value for binding. This agonist behaved thus like an inverse agonist on the constitutively active VPAC(1) receptors generated by C-terminal truncation and expressed in CHO cells.Comparative StudyJournal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe