http://http://dspace.unitus.it:80 The DSpace digital repository system captures, stores, indexes, preserves, and distributes digital research material. Sat, 18 May 2013 08:09:26 GMT 2013-05-18T08:09:26Z 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/ Tue, 31 Dec 2002 23:00:00 GMT http://hdl.handle.net/2067/1444 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/ Sat, 31 Dec 2005 23:00:00 GMT http://hdl.handle.net/2067/1460 2005-12-31T23:00:00Z http://hdl.handle.net/2067/1446 Title: Native somatostatin sst2 and sst5 receptors functionally coupled to Gi/o-protein, but not to the serum response element in AtT-20 mouse tumour corticotrophs Authors: Cervia, Davide; Fehlmann, Dominique; Hoyer, Daniel Abstract: Of the five cloned somatostatin (SRIF: somatotropin release inhibitory factor) receptors (sst1-5), only sst2 and sst5 receptors appear to be endogenously expressed and functionally active in AtT-20 mouse anterior pituitary tumour cells. In this study, the presence and the functional coupling of SRIF receptors to G-protein in AtT-20 cells was evaluated by receptor autoradiography and guanosine-5′-O-(3-[35S]thio)-triphosphate ([35S]GTPγS) binding, respectively. In addition, transcriptional effects via the serum response element (SRE) were assessed in AtT-20-SRE-luci cells, engineered to express constitutively SRE upstream of the luciferase reporter gene. [125I]LTT-SRIF-28, [125I]CGP 23996 and [125I]Tyr3-octreotide binding illustrates the high level of sst2/5 receptor in AtT-20 cell membranes. SRIF-14 and SRIF-28 produced a concentration-dependent increase in [35S]GTPγS binding (pEC50 = 6.72 and 7.45; Emax = 79 and 74.9, respectively) which was completely abolished by pertussis toxin, sst2/5 receptor-selective ligands caused a concentration-dependent increase in [35S]GTPγS binding (pEC50 = 7.74-5.84; Emax = 76.6-20.2) while SSt1/3/4 receptor-selective ligands were devoid of activity. The binding profiles of [125I]LTT-SRIF-28 and the inhibition of cAMP accumulation correlated highly significantly with their corresponding [35S]GTPγS binding profiles (r=0.862 and 0.874, respectively). The effects of the sst2 receptor-preferring agonists Tyr3-octreotide and BIM 23027 on [35S]GTPγS binding, but not those of SRIF-14 and the sst5/1 receptor selective-agonist L-817,818, were competitively antagonised by the sst2 receptor antagonist D-Tyr8-CYN 154806 (pKB = 7.36 and 7.72, respectively; slope factors not significantly different from unity). In AtT-20-SRE-luci cells, which carry a SRE-luciferase construct functioning in a very efficient manner, SRIF and its analogues did not affect luciferase activity. Taken together, these results demonstrate that in AtT-20 cells the expression of sst2 and sst5 receptors fit with their functional coupling to Gi/o-proteins. The pharmacological implications of the existence of different ligand/receptor complexes are discussed. However, the intracellular pathways coupled to the activation of sst2 and sst5 receptors appear not to modulate the SRE-mediated transcriptional activity, suggesting that SRIF effects on gene expression coupled to mechanisms that have promoters other than SRE. Description: L'articolo è disponibile sul sito dell'editore http://www.springerlink.com/ Tue, 31 Dec 2002 23:00:00 GMT http://hdl.handle.net/2067/1446 2002-12-31T23:00:00Z