http://http://dspace.unitus.it:80 The DSpace digital repository system captures, stores, indexes, preserves, and distributes digital research material. Thu, 23 May 2013 03:14:25 GMT 2013-05-23T03:14:25Z http://hdl.handle.net/2067/1479 Title: Changes in neuronal response to ischemia in retinas with genetic alterations of somatostatin receptor expression Authors: Catalani, Elisabetta; Cervia, Davide; Martini, Davide; Bagnoli, Paola; Simonetti, Elisa; Timperio, Anna Maria; Casini, Giovanni Abstract: Ischemia is a primary cause of neuronal death in retinal diseases. The repertoire of expressed transmitter receptors would determine the neurons’ responses to ischemic damage, and peptidergic receptors may be involved. With a new in vitro model of the ischemic mouse retina, we investigated whether an altered expression of somatostatin receptors could modulate retinal responses to ischemia. We used retinas of somatostatin receptor 1 (sst1) knock out (KO) mice, where sst2 are over-expressed and over-functional, and of sst2 KO mice. TUNEL analysis of ischemic retinas showed a marked reduction of cell death in sst1 KO retinas, while there were no differences between wild-type (WT) and sst2 KO retinas. In addition, caspase-3 mRNA expression was also reduced in sst1 KO as compared to WT retinas. An immunohistochemical analysis demonstrated that different cell populations responded differently to the ischemic insult, and that the persistence of some immunohistochemical markers was greater in sst1 KO than in WT or in sst2 KO retinas. In particular, rod bipolar cell survival was markedly improved in sst1 KO retinas, while it was dramatically decreased in sst2 KO retinas. Furthermore, consistent with a role of glutamate excitotoxicity in ischemia-induced neuronal death, retinal glutamate release was observed to increase under ischemic conditions, but this increase was significantly reduced in sst1 KO retinas. These observations demonstrate that an increased presence of functional sst2 protects against retinal ischemia, thus implementing the background for the use of sst2 analogs in therapies of retinal diseases such as glaucoma or diabetic retinopathy. Description: L'articolo è disponibile sul sito dell'editore http://onlinelibrary.wiley.com/ Sun, 31 Dec 2006 23:00:00 GMT http://hdl.handle.net/2067/1479 2006-12-31T23:00:00Z http://hdl.handle.net/2067/1488 Title: Modulation of the neuronal response to ischemia by somatostatin analogues in wild-type and knock-out mouse retinas Authors: Cervia, Davide; Martini, Davide; Ristori, Davide; Catalani, Elisabetta; Timperio, Anna Maria; Bagnoli, Paola; Casini, Giovanni Abstract: Somatostatin acts at five G protein-coupled receptors, sst1-sst5. In mouse ischemic retinas, the overexpression of sst2 (as in sst1 knock-out mice) results in reduction of cell death and glutamate release. Here, we reported that, in wild-type retinas, somatostatin, the multireceptor ligand pasireotide and the sst2 agonist octreotide decreased ischemia-induced cell death and that octreotide also decreased glutamate release. In contrast, cell death was increased by blocking sst2 with cyanamide. In sst2 over-expressing ischemic retinas, somatostatin analogues increased cell death, and octreotide also increased glutamate release. To explain this reversal of the anti-ischemic effect of somatostatin agonists in the presence of sst2 over-expression, we tested sst2 desensitisation due to internalisation or altered receptor function. We observed that: i) sst2 was not internalised, ii) among G protein-coupled receptor kinases (GRKs) and regulators of G protein signalling (RGSs), GRK1 and RGS1 expression increased following ischemia, iii) both GRK1 and RGS1 were downregulated by octreotide in wild-type ischemic retinas, iv) octreotide down-regulated GRK1 but not RGS1 in sst2 over-expressing ischemic retinas. These results demonstrate that sst2 activation protects against retinal ischemia. However, in the presence of sst2 over-expression sst2 is functionally desensitised by agonists, possibly due to sustained RGS1 levels. Description: L'articolo è disponibile sul sito dell'editore http://onlinelibrary.wiley.com/ Mon, 31 Dec 2007 23:00:00 GMT http://hdl.handle.net/2067/1488 2007-12-31T23:00:00Z http://hdl.handle.net/2067/1484 Title: Physiology and pathology of somatostatin in the mammalian retina: a current view Authors: Cervia, Davide; Casini, Giovanni; Bagnoli, Paola Abstract: In the retina, peptidergic signalling participates in multiple circuits of visual information processing. The neuropeptide somatostatin (SRIF) is localised to amacrine cells and, in some instances, in a subset of ganglion cells. The variegated expression patterns of SRIF receptors (sst1-sst5) and the variety of signalling mechanisms activated by retinal SRIF suggest that this peptide may exert multiple actions on retinal neurons and on retinal physiology, although our current understanding reflects a rather complicated picture. SRIF, mostly through sst2, may act as a positive factor in the retina by regulating retinal homeostasis and protecting neurons against damage. In this respect, SRIF analogues seem to constitute a promising therapeutic arsenal to cure different retinal diseases, as for instance ischemic and diabetic retinopathies. However, further investigations are needed not only to fully understand the functional role of the SRIF system in the retina but also to exploit new chemical space for drug-like molecules. Description: L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com/ Mon, 31 Dec 2007 23:00:00 GMT http://hdl.handle.net/2067/1484 2007-12-31T23:00:00Z http://hdl.handle.net/2067/1486 Title: The cyclooxygenase-2/prostaglandin E2 pathway is involved in the somatostatin-induced decrease of epileptiform bursting in the mouse hippocampus Authors: Ristori, Chiara; Cammalleri, Maurizio; Martini, Davide; Pavan, Barbara; Casini, Giovanni; Cervia, Davide; Bagnoli, Paola Abstract: The neuromodulatory peptide somatostatin-14 (SRIF) plays an important inhibitory role in epilepsy, but little is known on the signalling mechanisms coupled to this effect of SRIF. We have previously demonstrated that SRIF induces reduction of epileptiform bursting in a model of interictal-like activity in mouse hippocampal slices. In this same model, we investigated whether the cyclooxygenase 2 (COX-2)/prostaglandin E2 (PGE2) pathway is part of those signalling mechanisms mediating SRIF anti-epileptic actions. Both the expression of COX-2 (mRNA and protein) and the endogenous release of PGE2 increased in concomitance with epileptiform bursting. In particular, COX-2 protein increased in CA1/CA3 pyramidal layer and in the granular layer of the dentate gyrus. In addition, the selective inhibition of COX-2 by NS-398 markedly decreased endogenous PGE2 release induced by epileptiform bursting and the epileptiform bursting itself. Similar effects on epileptiform bursting were obtained with another COX-2 inhibitor, i.e., meloxicam. SRIF application counteracted the increase of both COX-2 expression and PGE2 release which occurred in concomitance with epileptiform bursting. Interestingly, SRIF and NS-398 comparably reduced epileptiform bursting in a non-additive manner and PGE2 abolished the inhibitory effect of SRIF on epileptiform bursting. These results demonstrate that: i) the COX-2/PGE2 pathway facilitates epileptiform bursting; and ii) SRIF exerts an anti-epileptic role by coupling to the COX-2/PGE2 pathway. In conclusion, we have identified a key set of signalling events that underlie anti-convulsant effects of SRIF in a mouse model of hippocampal bursting, thus providing useful data not only to identify alternative intervention points for the modulation of SRIF function, but also to exploit new chemical space for drug-like molecules. Description: L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com/ Mon, 31 Dec 2007 23:00:00 GMT http://hdl.handle.net/2067/1486 2007-12-31T23:00:00Z http://hdl.handle.net/2067/1464 Title: An update on somatostatin receptor signaling in native systems and new insights on their pathophysiology Authors: Cervia, Davide; Bagnoli, Paola Abstract: The peptide somatostatin (SRIF) has important physiological effects (mostly inhibitory) which have formed the basis for the clinical use of SRIF compounds. SRIF binding to its five G-protein coupled receptors leads to the modulation of multiple transduction pathways. However, our current understanding of signalling exerted by receptors endogenously expressed in different cells/tissues reflects a rather complicated picture. On the other hand, the complexity of SRIF receptor signalling in pathologies, including pituitary and nervous system diseases, may be studied not only as alternative intervention points for the modulation of SRIF function but also to exploit new chemical space for drug-like molecules. Description: L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com/ Sun, 31 Dec 2006 23:00:00 GMT http://hdl.handle.net/2067/1464 2006-12-31T23:00:00Z 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/ Fri, 31 Dec 2004 23:00:00 GMT http://hdl.handle.net/2067/1456 2004-12-31T23:00:00Z http://hdl.handle.net/2067/1430 Title: Somatostatin-induced control of cytosolic free calcium in pituitary tumour cells Authors: Petrucci, Cristina; Cervia, Davide; Buzzi, Marco; Biondi, Carla; Bagnoli, Paola Abstract: 1 In rat pituitary tumor cells (GC cells), spontaneous oscillations of the intracellular concentration of Ca2+ ([Ca2+]i) induce growth hormone (GH) secretion that is inhibited by octreotide, a somatostatin (SRIF) agonist which binds to SRIF subtype (sst) receptor 2. The effects of its functional activation on the control of [Ca2+]i were investigated using fluorimetric measurements of [Ca2+]i. 2 SRIF decreases the basal [Ca2+]i and the [Ca2+]i rise in response to forskolin (FSK) through the inhibition of L-type voltage-dependent Ca2+ channels. 3 Pretreatment with octreotide or with L-Tyr8Cyanamid 154806, a sst2 receptor antagonist, abolishes the SRIF-induced inhibition of [Ca2+]i. Octreotide is known to operate through agonist-induced desensitization, while the antagonist operates through receptor blockade. 4 sst1 and sst2 receptor-immunoreactivities (-IRs) are localized to cell membranes. sst2, but not sst1 receptor-IR, internalizes after cell exposure to octreotide. 5 SRIF-induced inhibition of basal [Ca2+]i or FSK-induced Ca2+ entry is blocked by pertussis toxin (PTX). 6 FSK-induced cAMP accumulation is only partially decreased by SRIF or octreotide, indicating that sst2 receptors are coupled to intracellular pathways other than adenylyl cyclase (AC) inhibition. 7 In the presence of H-89, an inhibitor of cAMP-dependent protein kinase (PKA), SRIF-induced inhibition of basal [Ca2+]i is still present, although reduced in amplitude. 8 SRIF inhibits [Ca2+]i by activating sst2 receptors. Inhibition of AC activity is only partly responsible for this effect, and other transduction pathways may be involved. Description: L'articolo è disponibile sul sito del nuovo editore http://onlinelibrary.wiley.com/ Fri, 31 Dec 1999 23:00:00 GMT http://hdl.handle.net/2067/1430 1999-12-31T23:00:00Z http://hdl.handle.net/2067/1459 Title: Cytotoxic effects and apoptotic signalling mechanisms of the sesquiterpenoid euplotin C, a secondary metabolite of the marine ciliate Euplotes crassus, in tumour cells Authors: Cervia, Davide; Martini, Davide; Garcia-Gil, Mercedes; Di Giuseppe, Graziano; Guella, Graziano; Dini, Fernando; Bagnoli, Paola Abstract: Most antitumour agents with cytotoxic properties induce apoptosis. The lipophilic compound euplotin C, isolated from the ciliate Euplotes crassus, is toxic to a number of different opportunistic or pathogenic microorganisms, although its mechanism of action is currently unknown. We report here that euplotin C is a powerful cytotoxic and pro-apoptotic agent in mouse AtT-20 and rat PC12 tumour-derived cell lines. In addition, we provide evidence that euplotin C treatment results in rapid activation of ryanodine receptors, depletion of Ca2+ stores in the endoplasmic reticulum (ER), the release of cytochrome c from the mitochondria, activation of caspase-12, and activation of caspase-3, leading to apoptosis. Intracellular Ca2+ overload is an early event which induces apoptosis and is parallelled by ER stress and the release of cytochrome c, whereas caspase-12 may be activated by euplotin C at a later stage in the apoptosis pathway. These events, either independently or concomitantly, lead to the activation of the caspase-3 and its downstream effectors, triggering the cell to undergo apoptosis. These results demonstrate that euplotin C may be considered for the design of cytotoxic and pro-apoptotic new drugs. Description: L'articolo è disponibile sul sito dell'editore http://www.springerlink.com/ Sat, 31 Dec 2005 23:00:00 GMT http://hdl.handle.net/2067/1459 2005-12-31T23:00:00Z http://hdl.handle.net/2067/1441 Title: Inhibitory control of growth hormone secretion by somatostatin in rat pituitary GC cells: sst2 but not sst1 receptors are coupled to an inhibition of single-cell intracellular free calcium concentrations Authors: Cervia, Davide; Petrucci, Cristina; Bluet-Pajot, Marie Thérèse; Epelbaum, Jacques; Bagnoli, Paola Abstract: Rat pituitary tumor cells (GC cells) exhibit spontaneous oscillations of intracellular free calcium concentration ([Ca2+]i) that allow a continuous release of growth hormone (GH). Of the SRIH receptor subtypes (sst receptors) mediating SRIH action, sst1 and sst2 receptors are highly expressed by GC cell membranes. In the present study, the effects of sst1 or sst2 receptor activation on single-cell [Ca2+]i were investigated in GC cells by confocal fluorescence microscopy. In addition, the effects of sst1 or sst2 receptor activation on GH secretion were also studied. Our results demonstrate that SRIH decreases [Ca2+]i baseline and almost completely blocks Ca2+ transients through the activation of sst2 but not of sst1 receptors. In contrast, SRIH effectively inhibits GH secretion through the activation of both sst1 and sst2 receptors. Blocking Ca2+ transients is less efficient than SRIH to inhibit GH release. The cyclic octapeptide, CYN-154806, antagonizes sst2 receptors at [Ca2+]i since it abolishes the sst2 receptor-mediated inhibition of [Ca2+]i without affecting single-cell Ca2+ signals. Unexpectedly, CYN-154806 alone potently inhibits GH secretion through the involvement of pertussis toxin-sensitive G proteins. In conclusion, the present results demonstrate that SRIH inhibition of GH release in GC cells involves mechanisms either dependent or independent on SRIH modulation of [Ca2+]i. The implications of CYN-154806 inhibition of GH secretion are discussed. Description: L'articolo è disponibile sul sito dell'editore http://www.karger.com Mon, 31 Dec 2001 23:00:00 GMT http://hdl.handle.net/2067/1441 2001-12-31T23:00:00Z 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/ Wed, 31 Dec 2003 23:00:00 GMT http://hdl.handle.net/2067/1450 2003-12-31T23:00:00Z http://hdl.handle.net/2067/1469 Title: Action Mechanisms of the Secondary Metabolite Euplotin C: Signaling and Functional Role in Euplotes Authors: Trielli, Francesca; Cervia, Davide; Di Giuseppe, Graziano; Ristori, Chiara; Kruppel, Thomas; Burlando, Bruno; Guella, Graziano; Viarengo, Aldo; Bagnoli, Paola; Delmonte Corrado, Maria Umberta; Dini, Fernando Abstract: Among secondary metabolites, the acetylated hemiacetal sesquiterpene euplotin C has been isolated from the marine, ciliated protist Euplotes crassus, and provides an effective mechanism for reducing populations of potential competitors through its cytotoxic properties. However, intracellular signaling mechanisms and their functional correlates mediating the ecological role of euplotin C are largely unknown. We report here that, in E. vannus (an Euplotes morphospecies which does not produce euplotin C and shares with E. crasssus the same interstitial habitat), euplotin C rapidly increases the intracellular concentration of both Ca2+ and Na+, suggesting a generalized effect of this metabolite on cation transport systems. In addition, euplotin C does not induce oxidative stress, but modulates the electrical properties of E. vannus through an increase of the amplitude of graded action potentials. These events parallel the disassembling of the ciliary structures, the inhibition of cell motility, the occurrence of aberrant cytoplasmic vacuoles, and the rapid inhibition of phagocytic activity. Euplotin C also increases lysosomal pH and decreases lysosomal membrane stability of E. vannus. These results suggest that euplotin C exerts a marked disruption of those homeostatic mechanisms whose efficiency represents the essential prerequisite to face the challenges of the interstitial environmental. Description: L'articolo è disponibile sul sito dell'editore http://onlinelibrary.wiley.com/ Mon, 31 Dec 2007 23:00:00 GMT http://hdl.handle.net/2067/1469 2007-12-31T23:00:00Z http://hdl.handle.net/2067/1457 Title: Distinct functional properties of native somatostatin receptor subtype 5 compared with subtype 2 in the regulation of ACTH release by corticotroph tumor cells Authors: Van der Hoek, Joost; Waaijers, Marlijn; Van Koetsveld, Peter M.; Sprij-Mooij, Diana; Feelders, Richard A.; Schmid, Herbert A.; Schoeffter, Philippe; Hoyer, Daniel; Cervia, Davide; Taylor, John E.; Culler, Michael D.; Lamberts, Steven W.J.; Hofland, Leo J. Abstract: In a series of human corticotroph adenomas, we recently found predominant mRNA expression of somatostatin (SS) receptor subtype 5 (sst5). After 72 h, the multiligand SS analog SOM230, which has a very high sst5 binding affinity, but not Octreotide (OCT), significantly inhibited basal ACTH release. To further explore the role of sst5 in the regulation of ACTH release, we conducted additional studies with mouse AtT-20 cells. SOM230 showed a 7-fold higher ligand binding affinity and a 19-fold higher potency in stimulating guanosine 5 -O- (3-thiotriphosphate) binding in AtT-20 cell membranes compared with OCT. SOM230 potently suppressed CRH-induced ACTH release, which was not affected by 48-h dexamethasone (DEX) pretreatment. However, DEX attenuated the inhibitory effects of OCT on ACTH release, whereas it increased the inhibitory potency of BIM-23268, an sst5-specific analog, on ACTH release. Quantitative PCR analysis showed that DEX lowered sst2A 2B mRNA expression significantly after 24 and 48 h, whereas sst5 mRNA levels were not significantly affected by DEX treatment. Moreover, Scatchard analyses showed that DEX suppressed maximum binding capacity (Bmax) by 72% when 125I-Tyr3-labeled OCT was used as radioligand, whereas Bmax declined only by 17% when AtT-20 cells were treated with [125I-Tyr11]SS-14. These data suggest that the sst5 protein, compared with sst2, is more resistant to glucocorticoids. Finally, after SS analog preincubation, compared with OCT both SOM230 and BIM-23268 showed a significantly higher inhibitory effect on CRHinduced ACTH release. In conclusion, our data support the concept that the sst5 receptor might be a target for new therapeutic agents to treat Cushing’s disease. Description: L'articolo è disponibile sul sito dell'editore http://www.the-aps.org/index.htm Fri, 31 Dec 2004 23:00:00 GMT http://hdl.handle.net/2067/1457 2004-12-31T23:00:00Z 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 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/2067/1472 Title: Identification and functional characterization of loss-of-function mutations of the calcium-sensing receptor in four italian kindreds with familial hypocalciuric hypercalcemia Authors: Cetani, Filomena; Lemmi, Monica; Cervia, Davide; Borsari, Simona; Cianferotti, Luisella; Pardi, Elena; Ambrogini, Elena; Banti, Chiara; Brown, Edward M.; Bagnoli, Paola; Pinchera, Aldo; Marcocci, Claudio Abstract: Objective: Identification and characterization of calcium-sensing receptor (CASR) mutations in four unrelated Italian kindreds with familial hypocalciuric hypercalcemia. Design: Clinical evaluation and genetic analysis of CASR gene. Functional characterization of mutated CASRs. Methods: Direct sequencing of CASR gene in genomic DNA. Studies of CASR-mediated increases in cytosolic calcium concentration [Ca2C]i in CASR-transfected COS-7 cells in vitro. Results: Four unreported heterozygous CASR mutations were identified, including three missense (H595Y, P748H, and C765W) and one splice site (IVS2C1GOC) mutation. The H595Y, P748H, and C765W mutant receptors, although expressed at normal levels on the cell surface, showed a reduced response in [Ca2C]i relative to the wildtype (WT) CASR to increasing extracellular calcium concentrations. Cotransfection experiments showed that the H595Y and P748H mutants did not affect the apparent affinity of the WT CASR for calcium, suggesting that they do not exert a dominant-negative effect. On the other hand, the co-transfected C765W mutant decreased the maximum response of the WT CASR to calcium, suggesting that it may reduce the effective concentration of the normal CASR on the cell surface or impair its maximal signaling capacity. Conclusions: Four CASR mutations were identified. The reduced functional responses to extracellular calcium and normal expression of the mutant receptors suggest that conformational changes account for altered CASR activity. Moreover, a reduced complement of normal CASRs in these heterozygous patients, perhaps combined with a mutant receptor-induced decrease in maximal activity of the WT receptor, may contribute to defective calcium-sensing in vivo. Description: L'articolo è disponibile sul sito dell'editore http://www.euro-endo.org/default.aspx Wed, 31 Dec 2008 23:00:00 GMT http://hdl.handle.net/2067/1472 2008-12-31T23:00:00Z http://hdl.handle.net/2067/1449 Title: Genetic deletion of somatostatin receptor 1 alters somatostatinergic transmission in the mouse retina Authors: Dal Monte, Massimo; Petrucci, Cristina; Vasilaki, Anna; Cervia, Davide; Grouselle, Dominique; Epelbaum, Jacques; Kreienkamp, Hans-Jurgen; Richter, Dietmar; Hoyer, Daniel; Bagnoli, Paola Abstract: In the mammalian retina, sparse amacrine cells contain somatostatin-14 (SRIF) which acts at multiple levels of neuronal circuitry through distinct SRIF receptors (sst1-5). Among them, the sst1 receptor has been localized to SRIF-containing amacrine cells in the rat and rabbit retina. Little is known about sst1 receptor localization and function in the mouse retina. We have addressed this question in the retina of mice with deletion of sst1 receptors (sst1 KO mice). In the retina of wild type (WT) mice, sst1 receptors are localized to SRIF-containing amacrine cells whereas in the retina of sst1 KO mice, sst1 receptors are absent. sst1 receptor loss causes a significant increase in retinal levels of SRIF whereas it does not affect SRIF messenger RNA indicating that sst1 receptors play a role in limiting retinal SRIF at the post-transcriptional level. As another consequence of sst1 receptor loss, levels of expression of sst2 receptors are significantly higher than in control retinas. Together, these findings provide the first demonstration of prominent compensatory regulation in the mouse retina as a consequence of a distinct SRIF receptor deletion. The fact that in the absence of the sst1 receptor, retinal SRIF increases in concomitance with an increase in sst2 receptors suggests that SRIF may regulate sst2 receptor expression and that this regulatory process is controlled upstream by the sst1 receptor. This finding can be important in the design of drugs affecting SRIF function, not only in the retina, but also elsewhere in the brain. Description: L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com/ Tue, 31 Dec 2002 23:00:00 GMT http://hdl.handle.net/2067/1449 2002-12-31T23:00:00Z 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/ Tue, 31 Dec 2002 23:00:00 GMT http://hdl.handle.net/2067/1443 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/2067/1461 Title: Expression, pharmacology and functional role of somatostatin receptor subtypes 1 and 2 in human macrophages Authors: Armani, Chiara; Catalani, Elisabetta; Balbarini, Alberto; Bagnoli, Paola; Cervia, Davide Abstract: Somatostatin (SRIF)-14 is recognized as an important mediator between the nervous and the immune system, although the functional role of its receptors (sst1-sst5) is poorly understood in humans. In our study, we demonstrate that human macrophages differentiated from peripheral blood mononuclear cell-derived monocytes express both sst1 and sst2 mRNAs. Both sst1 and sst2 are mostly localized at the cell surface and display active binding sites. In particular, sst1/sst2 activation results in a weak internalization of sst1 while the sst2 internalization appears more efficient. At the functional level, the activation of SRIF receptors by the multiligand analogues SOM230 and KE108, but not by SRIF-14 or cortistatin-14, reduces macrophage viability. Their effects are mimicked by the selective activation of sst1 and sst2 using CH-275 and SMS 201-995/L-779,976, respectively. Further, both sst1- and sst2-mediated effects are reversed by the sst1 antagonist SRA-880 or the sst2 antagonist CYN, respectively. CH-275, SMS 201-995 and L-779,976, but not SRIF-14, decrease both mRNA expression and secretion of the monocyte chemotactic protein-1. In addition, SRIF-14, CH-275, SMS 201-995 and L-779,976 decrease interleukine-8 secretion while they do not affect interleukine-8 mRNA expression. In contrast, SRIF-14 and sst1/sst2 agonists do not affect the secretion of matrix metalloproteinase-9. Collectively, our results suggest that the SRIF system, through sst1 and sst2, exerts mainly an immunosuppressive effect in human macrophages and may, therefore, represent a therapeutic window that can be exploited for the development of new strategies in pharmacological therapy of inflammation. Description: L'articolo è disponibile sul sito dell'editore http://leukocytebiology.org/default.aspx Sun, 31 Dec 2006 23:00:00 GMT http://hdl.handle.net/2067/1461 2006-12-31T23:00:00Z http://hdl.handle.net/2067/1476 Title: Interleukin 18 in the CNS Authors: Alboni, Silvia; Cervia, Davide; Sugama, Shuei; Conti, Bruno Abstract: Interleukin (IL)-18 is cytokine isolated as an important modulator of immune responses and subsequently shown to be pleiotropic. IL-18 and its receptors are expressed in the central nervous system (CNS) where they participate in neuroinflammatory/neurodegenerative processes but also influence homeostasis and behaviour. Work on IL-18 null mice, the localization of the IL-18 receptor complex in neurons and the neuronal expression of decoy isoforms of the receptor subunits are beginning to reveal the complexity and the significance of the IL-18 system in the CNS. This review summarizes current knowledge on the central role of IL-18 in health and disease. Description: L'articolo è disponibile sul sito dell'editore http://www.biomedcentral.com/ Thu, 31 Dec 2009 23:00:00 GMT http://hdl.handle.net/2067/1476 2009-12-31T23:00:00Z http://hdl.handle.net/2067/1475 Title: Mapping of the full length and the truncated interleukin-18 receptor alpha transcripts in the mouse brain Authors: Alboni, Silvia; Cervia, Davide; Ross, Brendon; Montanari, Claudia; Sanchez-Gonzales, Alejandro; Sanchez-Alavez, Manuel; Garibaldi-Marcondes, Maria Cecilia; De Vries, David; Sugama, Shuei; Brunello, Nicoletta; Blom, Joan; Tascedda, Fabio; Conti, Bruno Abstract: The cytokine IL-18 acts on the CNS both in physiological and pathological conditions. Its action occurs through the heterodimeric receptor IL-18Rα\β. To better understand IL-18 central effects, we investigated in the mouse brain the distribution of two IL-18Rα transcripts, a full length and an isoform lacking the intracellular domain hypothesized to be a decoy receptor. Both isoforms were expressed in neurons throughout the brain primarily with overlapping distribution but also with some unique pattern. These data suggest that IL-18 may modulate neuronal functions and that its action may be regulated through expression of a decoy receptor. Description: L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com/ Wed, 31 Dec 2008 23:00:00 GMT http://hdl.handle.net/2067/1475 2008-12-31T23:00:00Z http://hdl.handle.net/2067/1470 Title: Modulation of the neuronal response to ischemia by somatostatin analogues in wild-type and knock-out mouse retinas Authors: Cervia, Davide; Martini, Davide; Ristori, Chiara; Catalani, Elisabetta; Timperio, Anna Maria; Bagnoli, Paola; Casini, Giovanni Abstract: Somatostatin acts at five G protein-coupled receptors, sst1-sst5. In mouse ischemic retinas, the over-expression of sst2 (as in sst1 knock-out mice) results in reduction of cell death and glutamate release. Here, we reported that, in wild-type retinas, somatostatin, the multireceptor ligand pasireotide and the sst2 agonist octreotide decreased ischemia-induced cell death and that octreotide also decreased glutamate release. In contrast, cell death was increased by blocking sst2 with cyanamide. In sst2 over-expressing ischemic retinas, somatostatin analogues increased cell death, and octreotide also increased glutamate release. To explain this reversal of the anti-ischemic effect of somatostatin agonists in the presence of sst2 over-expression, we tested sst2 desensitisation due to internalisation or altered receptor function. We observed that: i) sst2 was not internalised, ii) among G protein-coupled receptor kinases (GRKs) and regulators of G protein signalling (RGSs), GRK1 and RGS1 expression increased following ischemia, iii) both GRK1 and RGS1 were down-regulated by octreotide in wild-type ischemic retinas, iv) octreotide down-regulated GRK1 but not RGS1 in sst2 over-expressing ischemic retinas. These results demonstrate that sst2 activation protects against retinal ischemia. However, in the presence of sst2 over-expression sst2 is functionally desensitised by agonists, possibly due to sustained RGS1 levels. Description: L'articolo è disponibile sul sito dell'editore http://onlinelibrary.wiley.com/ Mon, 31 Dec 2007 23:00:00 GMT http://hdl.handle.net/2067/1470 2007-12-31T23:00:00Z http://hdl.handle.net/2067/1463 Title: Molecular mechanisms of euplotin C-induced apoptosis: involvement of mitochondrial dysfunction, oxidative stress and proteases Authors: Cervia, Davide; Garcia-Gil, Mercedes; Simonetti, Elisa; Di Giuseppe, Graziano; Guella, Graziano; Bagnoli, Paola; Dini, Fernando Abstract: The metabolite euplotin C (EC), isolated from the marine ciliate Euplotes crassus, is a powerful cytotoxic and pro-apoptotic agent in tumour cell lines. For instance, EC induces the rapid depletion of ryanodine Ca2+ stores, the release of cytochrome c from the mitochondria, and the activation of caspase-3, leading to apoptosis. The purpose of this study was to gain further insight into the mechanisms of EC-induced apoptosis in rat pheochromocytoma PC12 cells. We found that EC increases Bax/Bcl-2 ratio and that Bax is responsible of the EC-induced dissipation of the mitochondrial membrane potential (Δψm). In addition, EC induces the generation of reactive oxygene species (ROS) without involvement of p53. The inhibition of ROS generation prevents, at least in part, the pro-apoptotic effects of EC as well as the effects of EC on Bax, Δψm and intracellular free Ca2+, indicating a cross-talk between different pathways. However, definition of the effector cascade turns out to be more complex than expected and caspase-independent mechanisms, acting in parallel with caspases, should also be considered. Among them, EC increases the expression/activity of calpains downstream of ROS generation, although calpains seem to exert protective effects. Description: L'articolo è disponibile sul sito dell'editore http://www.springerlink.com Sun, 31 Dec 2006 23:00:00 GMT http://hdl.handle.net/2067/1463 2006-12-31T23:00:00Z 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 Fri, 31 Dec 2004 23:00:00 GMT http://hdl.handle.net/2067/1458 2004-12-31T23:00:00Z http://hdl.handle.net/2067/1477 Title: Peptidergic receptors Authors: Casini, Giovanni; Cervia, Davide Abstract: The term neuropeptides refers to a relatively large number of biologically active molecules localized to discrete cell populations of nervous system, including autonomic ganglion neurons and their effector cells. Different combinations of transmitters and peptides are found in ganglionic cells of the autonomic system which comprise a functional “chemical code” for neurons subserving specific actions. Generally, peptidergic receptors are G-protein coupled receptors acting through multiple transduction pathways which reflect the pleiotropic actions of peptides. This section represents a current view of the most important actions of peptides and their receptors in the autonomic nervous system and in peripheral organs. Description: L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com/ Wed, 31 Dec 2008 23:00:00 GMT http://hdl.handle.net/2067/1477 2008-12-31T23:00:00Z http://hdl.handle.net/2067/1432 Title: Somatostatin (SRIF) modulates distinct signaling pathways in rat pituitary tumor cells. Negative coupling of SRIF receptor subtypes 1 and 2 to arachidonic acid release Authors: Cervia, Davide; Fiorini, Sara; Pavan, Barbara; Biondi, Carla; Bagnoli, Paola Abstract: The somatotropin release inhibiting factor somatostatin-14 (SRIF) is known to activate distinct receptor subtypes (sst1-5). In rat pituitary tumor cells (GC cells), sst2 but not sst1 receptors mediate the SRIF-induced inhibition of intracellular concentration of Ca2+ ([Ca2+]i) and are negatively coupled to cAMP-dependent pathways. In the present study, transduction mechanisms coupling distinct SRIF receptors to their specific functional role were investigated with the use of both SRIF agonists with well known affinity at individual SRIF receptors and the sst2 receptor antagonist L-Tyr8 isomer of Cyanamid 154806 (CYN-154806). Our results demonstrate that sst1 and sst2 receptors are coupled to distinct signaling pathways in GC cells. In particular, sst2 receptors are negatively coupled to the cAMP-dependent pathway and this pathway is partially responsible for the sst2 receptor-mediated inhibition of [Ca2+]i. In addition, sst1 and sst2 receptors are both coupled to a decrease of arachidonic acid (AA) release with an efficacy similar to that of SRIF, suggesting that SRIF reduces AA release through either a partial activation of both receptors or the activation of one at a time.This finding is important given the well accepted role for phospholipase A2 (PLA2) as a positive signaling component in transduction pathways of SRIF receptors. sst1 and sst2 receptor negative coupling to PLA2/AA-pathways does not seem to be implicated in the SRIF-induced inhibition of [Ca2+]i. The possible role for the SRIF-mediated inhibition of AA release in GC cell function remains to be elucidated. Description: L'articolo è disponibile sul sito dell'editore http://www.springerlink.com/ Mon, 31 Dec 2001 23:00:00 GMT http://hdl.handle.net/2067/1432 2001-12-31T23:00:00Z http://hdl.handle.net/2067/1453 Title: Somatostatin coupling to adenylyl cyclase activity in the mouse retina Authors: Pavan, Barbara; Fiorini, Sara; Dal Monte, Massimo; Lunghi, Laura; Biondi, Carla; Bagnoli, Paola; Cervia, Davide Abstract: The peptide somatostatin-14 (SRIF) acts in the mammalian retina through its distinct receptors (sst1-5). Scarce information is available on SRIF function in the retina, including the elucidation of transduction pathways mediating SRIF action. We have investigated SRIF and SRIF receptor modulation of adenylyl cyclase (AC) activity in both wild type (WT) retinas and sst1 or sst2 knock-out (KO) retinas which are known to over-express sst2 or sst1 receptors, respectively. In WT retinas, application of SRIF compounds does not affect forskolin-stimulated AC activity. In contrast, activation of sst1 or sst2 receptors inhibits AC in the presence of sst2 or sst1 receptor antagonists, respectively. Results from sst1 KO retinas demonstrate that either SRIF or octreotide, pertussis toxin-dependently inhibit AC activity. In contrast, in sst2 KO retinas, neither SRIF nor CH-275, an sst1 receptor agonist, are found to influence AC activity. As revealed by immunoblotting experiments, in sst1 KO retinas, levels of Goα proteins are 60% higher than in WT retinas and this increase in Goα protein levels is concomitant with an increase in sst2A receptor expression. We conclude that interactions between sst1 and sst2 receptors may prevent SRIF effects on AC activity. In addition, we suggest that the density of sst2 receptors and/or Goα proteins may represent the rate-limiting factor for the sst2 receptor-mediated inhibition of AC. Description: L'articolo è disponibile sul sito dell'editore http://www.springerlink.com/ Wed, 31 Dec 2003 23:00:00 GMT http://hdl.handle.net/2067/1453 2003-12-31T23:00:00Z 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/ Wed, 31 Dec 2003 23:00:00 GMT http://hdl.handle.net/2067/1455 2003-12-31T23:00:00Z http://hdl.handle.net/2067/1474 Title: The secondary metabolite euplotin c induces apoptosis-like death in the marine ciliated protist euplotes vannus Authors: Cervia, Davide; Di Giuseppe, Graziano; Ristori, Chiara; Martini, Davide; Gambellini, Gabriella; Bagnoli, Paola; Dini, Fernando Abstract: The sesquiterpenoid euplotin C is a secondary metabolite produced by the ciliated protist Euplotes crassus and provides a mechanism for damping populations of potential competitors. Indeed, E. crassus is virtually resistant to its own product while different non-producer species representing an unbiased sample of the marine, interstitial, ciliate diversity are sensitive. For instance, euplotin C exerts a marked disruption of different homeostatic mechanisms in Euplotes vannus. We demonstrate by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay that euplotin C quickly decreases viability and mitochondrial function of E. vannus with a very high efficacy and at micromolar potency. In addition, euplotin C induces apoptosis in E. vannus as 4,6-diamino-2-phenylindole and Terminal Transferase dUTP Nick End Labeling staining show the rapid condensation and fragmentation of nuclear material in cells treated with euplotin C. These effects occur without detectable permeabilisation or rupture of cell membranes and with no major changes in the overall morphology, although some traits, such as vacuolisation and disorganised microtubules, can be observed by transmission electron microscopy. In particular, E. vannus show profound changes of the mitochondrial ultrastructure. Finally, we also show that caspase activity in E. vannus is increased by euplotin C. These data elucidate the pro-apoptotic role of euplotin C and suggest a mechanism for its impact on natural selection. Description: L'articolo è disponibile sul sito dell'editore http://onlinelibrary.wiley.com/ Wed, 31 Dec 2008 23:00:00 GMT http://hdl.handle.net/2067/1474 2008-12-31T23:00:00Z