http://http://dspace.unitus.it:80 The DSpace digital repository system captures, stores, indexes, preserves, and distributes digital research material. 2013-05-23T11:20:39Z http://hdl.handle.net/2067/1450 Title: Comparison of functional profiles at human recombinant somatostatin sst2 receptor: simultaneous determination of intracellular Ca2+ and luciferase expression in CHO-K1 cells Authors: Nunn, Caroline; Cervia, Davide; Langenegger, Daniel; Tenaillon, Laurent; Bouhelal, Rochdi; Hoyer, Daniel Abstract: 1. Somatostatin (somatotropin release inhibiting factor; SRIF) acts via five G protein coupled receptors (sst1-sst5) which modulate multiple cellular effectors. The aim of this study was to compare two functional effects of the human sst2 receptor stably expressed in CHO-K1 cells in a single experiment using a duplex assay for intracellular calcium and serum response element (SRE)-driven luciferase expression. 2. Intracellular calcium was measured using a FLuorometric Imaging Plate Reader II (FLIPR II). SRIF-14 rapidly and transiently increased intracellular calcium with a pEC50 of 8.74 ± 0.03 (n = 52). Five hours after FLIPR II measurements luciferase expression was determined. SRIF-14 concentration-dependently increased luciferase expression (pEC50 = 9.06 ± 0.03, n = 52). 3. Natural and synthetic agonist/antagonist ligands for SRIF receptors were tested in the duplex assay. Correlation of agonist potencies and efficacies between the two responses were significant (r2 = 0.83 and 0.90, pEC50 and Emax respectively). 4. Pertussis toxin pre-treatment reduced SRIF-14/octreotide-mediated intracellular calcium increases by 45-47% and luciferase expression by 95-98%. 5. Thapsigargin pre-treatment abolished the SRIF-14/octreotide-mediated intracellular calcium increase but had no effect on luciferase expression. 6. In conclusion, SRIF stimulates an increase in intracellular calcium and SRE-luciferase expression via human sst2 receptors in CHO-K1 cells. The increase in luciferase is mediated via Gi/Go while intracellular calcium increase is mediated by both Gi/Go proteins and pertussis toxin insensitive G proteins, and is mainly via release of calcium from intracellular stores. SRIF ligands display a similar recognition profile suggesting that the ligand/receptor/G protein/effector interaction is similar for the two parameters. Description: L'articolo è disponibile sul sito dell'editore http://onlinelibrary.wiley.com/ 2003-12-31T23:00:00Z http://hdl.handle.net/2067/1444 Title: Pharmacological characterisation of native somatostatin receptors in AtT-20 mouse tumour corticotrophs Authors: Cervia, Davide; Nunn, Caroline; Fehlmann, Dominique; Langenegger, Daniel; Schuepbach, Edi; Hoyer, Daniel Abstract: 1. The mouse corticotroph tumour cell line AtT-20, is a useful model to investigate the physiological role of native somatostatin (SRIF) receptor subtypes (sst1-sst5). The objective of this study was to characterise the pharmacological features and the functional effects of SRIF receptors expressed by AtT-20 cells using radioligand binding and cAMP accumulation. 2. [125I]LTT-SRIF-28, [125I]CGP 23996, [125I]Tyr10-cortistatin-14 and [125I]Tyr3-octreotide labelled SRIF receptor binding sites with high affinity and in a saturable manner (Bmax = 315, 274, 239 and 206 fmol mg-1, respectively). [125I]LTT-SRIF-28 labels significantly more sites than [125I]Tyr10-cortistatin-14 and [125I]Tyr3-octreotide as seen previously in cells expressing pure populations of sst2 or sst5 receptors. 3. SRIF analogues displaced the binding of the four radioligands. sst2/5 receptor-selective ligands showed much higher affinity than sst1/3/4 receptor-selective ligands. The binding profile of [125I]Tyr3-octreotide was different from that of [125I]LTT-SRIF-28, [125I]CGP 23996 and [125I]Tyr10-cortistatin-14. The sst5/1 receptor-selective ligand L-817,818 identified two binding sites, one with subnanomolar affinity (sst5 receptors) and one with micromolar affinity (sst2 receptors), however the proportions were different: 70-80% high affinity with [125I]LTT-SRIF-28, [125I]CGP 23996, [125I]Tyr10-cortistatin-14 but only 20% with [125I]Tyr3-octreotide. 4. SRIF analogues concentration-dependently inhibited the forskolin-stimulated cAMP levels. sst2/5 receptor-selective ligands were highly potent, whereas sst1/3/4 receptor-selective ligands had no significant effects. The sst2 receptor antagonist D-Tyr8-CYN 154806 competitively antagonised the effects of SRIF-14 and sst2 receptor-preferring agonists, but not those of L-817,818. The complex binding properties of SRIF receptor analogues indicates that sst2 and sst5 receptors are the predominant SRIF receptors expressed on AtT-20 cell membranes with no or only negligible presence of sst1, sst3 and sst4 receptors. In the functional studies using cAMP accumulation, only sst2 and sst5 receptors appear to play a role. However, the “predominant” receptor appears to be the sst2 receptor, although sst5 receptors can also mediate the effect, when the ligand is not able to activate sst2 receptors. This clearly adds flexibility to SRIF-mediated functional effects and suggests that the physiological role of SRIF and its analogs may be mediated preferentially via one subtype over another. Description: L'articolo è disponibile sul sito dell'editore http://onlinelibrary.wiley.com/ 2002-12-31T23:00:00Z http://hdl.handle.net/2067/1458 Title: Multiple Signalling Transduction Mechanisms Differentially Coupled to Somatostatin Receptor Subtypes: a Current View. Authors: Cervia, Davide; Nunn, Caroline; Bagnoli, Paola Abstract: Somatostatin (SRIF) is a cyclic peptide widely distributed throughout the body with important physiological effects (mostly inhibitory) on several organ systems. SRIF may act as a neurohormone, neurotransmitter, neuromodulator or as a local factor, and exhibits potent antiproliferative activity. SRIF effects have formed the basis for the clinical use of SRIF analogues in the treatment of endocrine tumours, acromegaly and gastrointestinal disorders. Several data suggest that SRIF may also be a therapeutic target in a number of different diseases. The binding of SRIF to its five G-protein coupled receptors leads to modulation of multiple transduction pathways, including adenylyl cyclase, guanylyl cyclase, phospholipase C, K+ and Ca2+ channels, phospholipase A2, nitric oxide, Na+/H+ exchanger, protein phosphatases and MAP kinases. The diversity of the transduction pathways reflects the pleiotropic actions of SRIF. However, our current understanding depicts a rather complicated picture and conflicting results have also been reported. Data are mostly based on in vitro experiments, and parallels with the real in vivo conditions are not so obvious. Due to the clinical relevance of the SRIF system, the elucidation of the intracellular role of endogenous SRIF receptors may offer new therapeutic perspectives. These will enable development of specific pharmacological signalling modulators which can be incorporated into the therapeutic arsenal. The present review represents a detailed and exhaustive summary which covers the latest advances in the transduction pathways of SRIF receptors. Description: L'articolo è disponibile sul sito dell'editore http://www.benthamscience.com/index.htm 2004-12-31T23:00:00Z