http://http://dspace.unitus.it:80 The DSpace digital repository system captures, stores, indexes, preserves, and distributes digital research material. 2013-05-20T22:24:19Z 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/2067/1460 Title: Compensatory changes in the hippocampus of somatostatin knockout mice: upregulation of somatostatin receptor 2 and its function in the control of bursting activity and synaptic transmission Authors: Cammalleri, Maurizio; Cervia, Davide; Dal Monte, Massimo; Martini, Davide; Langenegger, Daniel; Fehlmann, Dominique; Feuerbach, Dominik; Pavan, Barbara; Hoyer, Daniel; Bagnoli, Paola Abstract: Somatostatin-14 (SRIF) colocalizes with GABA in the hippocampus and regulates neuronal excitability. A role of SRIF in the control of seizures has been proposed although its exact contribution requires some clarification. In particular, SRIF knock out (KO) mice do not exhibit spontaneous seizures, indicating that compensatory changes may occur in KO. In the KO hippocampus, we examined whether specific SRIF receptors and/or the cognate peptide cortistatin-14 (CST) compensate for SRIF’s absence. We found increased levels of both sst2 receptors (sst2) and CST and we explored the functional consequences of sst2 compensation on bursting activity and synaptic responses in hippocampal slices. Bursting was decreased by SRIF in wild type (WT) mice, but it was not affected by either CST or sst2 agonist and antagonist. sst4 agonist increased bursting frequency in either WT or KO. In WT, but not in KO, its effects were blocked by agonizing or antagonizing sst2, suggesting that sst2 and sst4 are functionally coupled in the WT hippocampus. Bursting was reduced in KO as compared to WT and was increased upon application of sst2 antagonist while SRIF, CST and sst2 agonist had no effect. At the synaptic level, we observed that in WT, SRIF decreased excitatory postsynaptic potentials which were, in contrast, increased by sst2 antagonist in KO. We conclude that sst2 compensates for SRIF absence and that its upregulation is responsible for reduced bursting and decreased excitatory transmission in KO mice. We suggest that a critical density of sst2 is needed to control hippocampal activity. Description: L'articolo è disponibile sul sito dell'editore http://onlinelibrary.wiley.com/ 2005-12-31T23:00:00Z http://hdl.handle.net/2067/1455 Title: Somatostatin receptors differentially affect spontaneous epileptiform activity in mouse hippocampal slices Authors: Cammalleri, Maurizio; Cervia, Davide; Langenegger, Daniel; Liu, Yanqiang; Dal Monte, Massimo; Hoyer, Daniel; Bagnoli, Paola Abstract: Somatostatin-14 (SRIF) reduces hippocampal epileptiform activity but the contribution of its specific receptors (sst1-5) is poorly understood. We have focused on sst1 and sst2 role in mediating SRIF modulation of epilepsy using hippocampal slices of wild type (WT) and sst1 or sst2 knock out (KO) mice. Recordings of epileptiform discharge induced by Mg2+-free medium with 4-aminopyridine were performed from the CA3 region before and after the application of SRIF compounds. In WT mice, SRIF and the sst1 agonist CH-275 reduce epilepsy whereas sst1 blockade with its antagonist SRA-880 increases bursting discharge. Activation of sst2 does not affect bursting frequency unless its agonist octreotide is applied with SRA-880, indicating that sst1 masks sst2-mediated modulation of epilepsy. In sst1 KO mice: i. bursting frequency is lower than in WT; ii. SRIF, CH-275 and SRA-880 are ineffective on epilepsy; iii. octreotide is also devoid of effects, whereas blockade of sst2 with the antagonist D-Tyr8 Cyn 154806 increases bursting frequency. In sst2 KO mice, SRIF ligand effects are similar to those in WT. In the whole hippocampus of sst1 KO mice, sst2 mRNA, protein and binding are higher than in WT and RT-PCR of the CA3 subarea confirms an increase of the sst2 messenger. We conclude that sst1 mediates inhibitory actions of SRIF and that interactions between sst1 and sst2 may prevent sst2 modulation of epilepsy. We suggest that, in sst1 KO mice, activation of over-expressed sst2 reduces bursting frequency, indicating that sst2 density represents the rate-limiting factor for sst2-mediated modulation of epilepsy. Description: L'articolo è disponibile sul sito dell'editore http://onlinelibrary.wiley.com/ 2003-12-31T23:00:00Z http://hdl.handle.net/2067/1456 Title: Binding and functional properties of the novel somatostatin analogue KE 108 at native mouse somatostatin receptors Authors: Cervia, Davide; Langenegger, Daniel; Schuepbach, Edi; Cammalleri, Maurizio; Schoeffter, Philippe; Schmid, Herbert A.; Bagnoli, Paola; Hoyer, Daniel Abstract: Clinically used somatostatin (SRIF) analogs octreotide and lanreotide act primarily by binding to SRIF receptor subtype 2 (sst2). In contrast, the recently described multiligand SOM230 binds with high affinity to sst1-3 and sst5 and KE 108 is characterised as a high affinity ligand for all five SRIF receptors. In tumoural mouse corticotrophs (AtT-20 cells) and in mouse hippocampus, binding and functional features of KE 108 were examined and compared to SRIF-14, octreotide and SOM230. In AtT-20 cells, KE 108 bound with high affinity at [125I]LTT-SRIF-28-labelled sites similarly to SRIF, octreotide and SOM230. At the functional level, all four ligands increased guanosine-5’--(3-35Sthio)-triphosphate binding and decreased cAMP accumulation or intracellular Ca2+ concentration through Gi/o proteins. In hippocampal slices, KE 108, octreotide and SOM230 also bound with high affinity at [125I]LTT-SRIF-28-labelled sites similarly to SRIF, but KE108, octreotide or SOM230 did not influence spontaneous epileptiform activity which was, in contrast, inhibited by SRIF. In conclusion, this study demonstrates that KE 108 has high affinity for native mouse SRIF receptors. Functionally, KE 108 mediates SRIF action at sst2/5 in corticotrophs whereas it does not mimic the SRIF-induced inhibition of hippocampal excitation suggesting that the high potency and efficacy of a synthetic ligand to all known SRIF receptors may not reproduce entirely the effects of the natural SRIF. Description: L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com/ 2004-12-31T23:00:00Z http://hdl.handle.net/2067/1443 Title: Biological activity of somatostatin receptors in GC rat tumour somatotrophs: evidence with sst1-sst5 receptor-selective nonpeptidyl agonists Authors: Cervia, Davide; Zizzari, P.; Pavan, Barbara; Schuepbach, Edi; Langenegger, Daniel; Hoyer, Daniel; Biondi, Carla; Epelbaum, Jacques; Bagnoli, Paola Abstract: The physiological actions of somatostatin-14 (SRIF) receptor subtypes (sst1-sst5), which are endogenously expressed in GC cells, have not yet been elucidated, although there is evidence that sst2 receptors are negatively coupled to cytosolic free Ca2+ concentration ([Ca2+]i) and cAMP accumulation. In addition, both sst1 and sst2 receptors are negatively coupled to growth hormone (GH) secretion in GC cells. Here we report on studies concerning the expression, the pharmacology and the functional role of native SRIF receptors in GC cells with the use of five nonpeptidyl agonists, highly selective for each of the SRIF receptors. Radioligand binding studies show that sst2 and sst5 receptors are present at different relative densities, while the presence of sst3 and sst4 receptors appears to be negligible. The absence of sst1 receptor binding was unexpected in view of sst1 receptor functional effects on GH secretion. This suggests very efficient receptor-effector coupling of a low density population of sst1 receptors. Functionally, only sst2 receptors are coupled to the inhibition of [Ca2+]i and cAMP accumulation and the selective activation of sst5 receptors facilitates the stimulation of adenylyl cyclase activity through Gi/o proteins. This effect was not observed when sst2 and sst5 receptors were simultaneously activated, suggesting that there is a functional interaction between sst2 and sst5 receptors. In addition, sst1, sst2 and sst5 receptor activation inhibits GH release, further indicating that SRIF can modulate GH secretion in GC cells through mechanisms both dependent and independent on [Ca2+]i and cAMP-dependent pathways. The present data suggest SRIF-mediated functional effects in GC cells to be very diverse and provide compelling arguments to propose that multiple native SRIF receptors expressed in the same cells are not simply redundant, but contribute to marked signalling diversity. Description: L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com/ 2002-12-31T23:00:00Z