ISTITUTO SUPERIORE DI SANITÀ

ISSN 0393-5620ISTISAN Congressi

04/C3

Purine ClubAnnual Meeting

Istituto Superiore di SanitàRome, 23-24 September, 2004

ABSTRACT BOOKEdited by

Patrizia Popoli (a) and Gloria Cristalli (b)(a) Dipartimento del Farmaco, Istituto Superiore di Sanità, Rome

(b) Dipartimento di Scienze Chimiche, Università di Camerino, Camerino

Presidente dell’Istituto Superiore di Sanità e Direttore responsabile: Enrico Garaci Registro della Stampa - Tribunale di Roma n. 131/88 del 1° marzo 1988 Redazione: Paola De Castro e Sandra Salinetti La responsabilità dei dati scientifici e tecnici è dei singoli autori. © 2004 Istituto Superiore di Sanità (Viale Regina Elena, 299 - 00161 Roma)

Istituto Superiore di Sanità Purine Club Annual Meeting. Istituto Superiore di Sanità. Rome, 23-24 September 2004. Abstract book. Edited by Patrizia Popoli and Gloria Cristalli 2004, vii, 48 p. ISTISAN Congressi 04/C3

The meeting is jointly organized by the Istituto Superiore di Sanità and the Purine Club. The main aim of the annual meetings of the Purine Club is to provide a forum for discussion and to allow collaborations between scientists working in the fields of chemistry, pharmacology and functions of purines. This 2004 meeting includes two authoritative lectures focused on the “fine tuning” by adenosine receptors in the brain and on the regulation of P2X receptors by hypoxia, respectively. The first session of oral communications covers the field of the Central Nervous System effects of purines, while the second one is focused on the pharmacology and functions of P2 receptors. Several other aspects (e.g., synthesis and SAR profile of purine derivatives, expression and functions of P1 and P2 receptors in central and peripheral tissues) are covered as well by the poster session.

Key words: Purines, Chemistry, Pharmacology, Functional effects

Istituto Superiore di Sanità Riunione annuale Purine Club. Istituto Superiore di Sanità. Roma, 23-24 settembre 2004. Riassunti. A cura di Patrizia Popoli e Gloria Cristalli 2004, vii, 48 p. ISTISAN Congressi 04/C3 (in English)

Il convegno è organizzato congiuntamente dall’Istituto Superiore di Sanità e dal Purine Club. L’obiettivo principale dei meeting annuali del Purine Club è quello di favorire gli scambi e di promuovere la collaborazione tra i ricercatori impegnati nello studio della chimica, della farmacologia e degli effetti funzionali delle purine. Questo meeting 2004 comprende due autorevoli letture riguardanti rispettivamente il “fine tuning” esercitato dai recettori dell’adenosina sul sistema nervoso centrale e la regolazione dei recettori P2X da parte di condizioni di ipossia/ischemia. La prima sessione di comunicazioni orali è dedicata agli effetti delle purine sul sistema nervoso centrale, mentre la seconda alla farmacologia ed alle funzioni dei recettori P2. Diversi altri aspetti (come la sintesi ed il profilo struttura-attività dei derivati purinici, o le funzioni dei recettori P1 e P2 a livello centrale e periferico) sono affrontati nell’ambito della sessione poster.

Parole chiave: Purine, Chimica, Farmacologia, Effetti funzionali

Scientific Committee Gloria Cristalli (Università degli Studi di Camerino), Patrizia Popoli (Istituto Superiore di Sanità)

Scientific Secretariat Mariangela Galluzzo, Rosa Grieco (Istituto Superiore di Sanità)

Editing: Federica Magnani

Per informazioni su questo documento scrivere a: patrizia.popoli@iss.it.

Il rapporto è disponibile online sul sito di questo Istituto: www.iss.it.

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This meeting and this book are dedicated to the memory of our friend and colleague Stefano Sagratella (1952-2004).

We are sure that Stefano, who loved pharmacology and thought us to love it as well,

will appreciate to be remembered in such a way.

Patrizia Popoli, Claudio Frank, Antonella Pèzzola, Maria Rosaria Domenici, Maria Teresa Tebano, Rosaria Reggio

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TABLE OF CONTENTS

Programme .................................................................................................................. v

LecturesHow adenosine receptors fine tune neurotransmission to protect the brain ................... 3 Modulation of P2X7 receptor-function by hypoxia/ischemia......................................... 4

First session Receptor-dependent and independent effects of nucleosides in the Central Nervous System....................................................................................... 5

Second session Pharmacology and functions of P2 receptors ................................................................. 13

Poster abstracts ........................................................................................................ 21

Authors’ index ........................................................................................................... 47

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PROGRAMME

Tuesday, September 23, 2004

11.00 Registration

13.00 Lunch

14.10 Welcome and opening remarks G. Cristalli, P. Popoli

First session RECEPTOR-DEPENDENT AND INDEPENDENT EFFECTS OF NUCLEOSIDES IN THE CENTRAL NERVOUS SYSTEM Chairpersons: M. Morelli, F. Pedata

14.30 Involvement of ERK1/2 phosphorylation in 2-chloro-2’-deoxy-adenosine-induced apoptosis and cell cycle block of human astrocytoma cells S. Ceruti, A. Mazzola, E. Beltrami, M.P. Abbracchio

14.50 Short-term regulation of A2B adenosine receptor functioning by tumor necrosis factor alpha in human astroglial cells M.L. Trincavelli, D. Tuscano, I. Tonazzini, S. Ceruti, M.P. Abbracchio, A. Lucacchini, C. Martini

15.10 Adenosine A2A antagonists and Parkinson’s disease: new findings on parkinsonian tremor N. Simola, A.R. Carta, E. Tronci, A. Pinna, M. Morelli

15.30 Adenosine A2A receptors enable metabotropic glutamate 5 receptors (mGlu5R)-mediated effects in the rodent hippocampus M.R. Domenici, M.T. Tebano, A. Martire, R. Pepponi, J.F. Chen, M.A. Schwarzschild, M.C. Grò, P. Popoli

15.50 The role of adenosine A3 receptors during mild and severe “ischemic conditions” in rat hippocampal slices A. M. Pugliese, E. Coppi, R. Corradetti, F. Pedata

16.10 Coffee break

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16.30 Lecture How adenosine receptors fine tune neurotransmission to protect the brain J.A. Ribeiro Introduced by P. Popoli

17.30 Poster viewing

18.30 Assembly of the Society members

20.30 Social dinner

Friday, September 24, 2004

Second Session PHARMACOLOGY AND FUNCTIONS OF P2 RECEPTORSChairpersons:M.P. Abbracchio, F. Caciagli

9.00 P2Y4 receptor, from differentiation to death F. Cavaliere, S. Amadio, V. Nestola, N. D’Ambrosi , G. Sancesario, G. Bernardi, C. Volonté

9.20 P2Y1 receptor activation regulates oligodendrocytes progenitor migration and proliferation C. Agresti, M.E. Meomartini, S. Amadio, E. Ambrosiani, B. Serafini, C. Volonté, F. Aloisi, S. Visentin

9.40 Upregulation of ionophoric P2X6 in chronic heart failure: interaction with TNF- and potential role in myocardial cell death

S. Ferrario, C. Banfi, O. De Vincenti, S. Ceruti, M. Fumagalli, A. Mazzola, N. D’Ambrosi, C. Volonté, P. Fratto, E. Vitali, A. Parolari, G. Polvani, P. Biglioli, E. Tremoli, M.P. Abbracchio

10.00 2-Alkynyladenosine phosphates as new ligands for the P2 platelet receptorsS. Vittori, S. Costanzi, F. R. Portino, S. Taffi, R. Volpini, M. Cattaneo,

G. Cristalli

10.20 Cloning, pharmacological characterization and distribution of the rat G-protein-coupled P2Y13 receptor M. Fumagalli, L. Trincavelli, D. Lecca, C. Martini, P. Ciana, M.P. Abbracchio

10.40 Coffee break

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11.00 Lecture Modulation of P2X7 receptor function by hypoxia P. Illes Introduced by R. Ciccarelli

11.30 Discussion of posters 1-12Chairpersons: F. Laghi-Pasini, S. Vittori, C. Volonté

12.30 Lunch

13.30 Discussion of posters 13-24 Chairpersons: R. Ciccarelli, C. Martini, M.T. Tebano

14.30 Concluding remarks G. Cristalli

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Lectures

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HOW ADENOSINE RECEPTORS FINE TUNE NEUROTRANSMISSION TO PROTECT THE BRAIN

Joaquim Alexandre Ribeiro Laboratory of Neurosciences and Institute of Pharmacology, IMM, Faculty of Medicine, University of Lisboa, Lisboa, Portugal

Adenosine is released from insulted neurones and glial cells (1), and is consensually recognised as very important in the homeostasis of the cells, being once named “a signal of life”. It modulates the activity of the nervous system by acting pre- (by inhibiting or facilitating transmitter release), post-, and/or non-synaptically, through activation of physiologically important high affinity adenosine receptors (A1, A2A) as well as of a lower affinity receptors (A2B, A3), which might be relevant in pathological conditions. Adenosine uses very subtle manners to participate in these actions, it acts as a fine tuner between its own receptors and the receptors for other synaptic mediators. From these modulatory actions it is possible to anticipate potential therapeutic applications (e.g. 2), especially when these actions are mediated by high-affinity adenosine A2A receptors in regions where clear functions for this receptor have been identified. The A2A receptor is highly expressed in the striatopallidal GABAergic neurones and expressed in lower levels in other brain regions. A2B possesses low levels of expression in the brain. A3 has apparently intermediate levels of expression in the human cerebellum and hippocampus and low levels in most of the brain. The well known striatal A2A/D2 receptor interactions may offer new therapeutic basis for basal ganglia disorders, such as Parkinson’s disease and Huntington’s chorea, as well as for schizophrenia. Inhibition of D2 receptors in the ventral striatum seems to be associated with the antipsychotic effect of neuroleptics, while inhibition of dopamine D2 receptors in the dorsal striatum is related to their extrapyramidal side effects. In the periphery, the interaction of A2A receptors with other receptors that regulate acetylcholine release from motor nerve terminals could reveal the importance of A2A receptors when deficits in acetylcholine release occur (e.g. myasthenic syndromes). The excitatory effects of A2A receptors on acetylcholine release might prove useful when developing cognitive enhancers that by increasing acetylcholine release, could be of therapeutic interest in dementia (e.g. Alzheimer’s disease). Since the adenosine A1 receptors inhibit neurotransmitter release and NMDA currents in hypoxia (3) and A2A promote the action of neurotrophic factors (4), one can also predict that adenosine-related medicines that combine adenosine A1 receptor activation with or without A2A receptor activation will be useful in situations where a harmonic neurotransmitter release is needed.

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MODULATION OF P2X7 RECEPTOR FUNCTION BY HYPOXIA/ISCHEMIA

Peter Illes (a), Heike Franke (a), Albrecht Günther (a), Kerstin Wirkner (a), Beata Sperlagh (b) (a) Rudolf-Boehm-Institute of Pharmacology and Toxicology, University of Leipzig, Leipzig,

Germany(b) Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary

High concentrations of ATP have been reported to activate a certain subtype of the ionotropic P2X receptor (P2X7). This receptor contains two transmembrane domains, a large extracellular loop containing the ATP binding site, a relatively short intracellular N-terminus, and a long C-terminal tail. P2X7 receptors allow the passage of small cations on immediate activation by agonists; long-lasting activation, however, leads to a progressive dilation of the ion channel and formation of a large pore (permeable to organic molecules), and membrane blebbing. Ion channel dilation, cell lysis and membrane blebbing, all depend on the intracellular C-terminus of the receptor. P2X7 receptors are preferentially localized on peripheral and central immunocytes (monocytes/macrophages, microglia). In addition, central immunocompetent cells such as astrocytes also possess P2X7 receptors possibly mediating inflammation and subsequent proliferation. More recently functional P2X7receptors were suggested to occur on neurons of the central and peripheral nervous system. Cerebral ischemia is known both to cause a massive release of ATP, and to activate microglial cells as well as to increase the expression of P2X7 receptors on this cell type. After middle cerebral artery occlusion of rats, a previously absent P2X7-immunoreactivity (IR) was observed in microglia, astrocytes and neurons of the cerebral cortex; in the periinfarct area an early expression of microglial P2X7-IR (~1 day after ischemia) was followed by a late expression of astrocytic and neuronal P2X7-IR (~>4 days after ischemia). ATP released [3H]GABA both from mixed astrocytic-neuronal cultures of the rat cerebral cortex and from similar cultures enriched in neurons. After in vitro ischemia, the effect of ATP was largely increased and also a previously absent stimulatory potency of the P2X7receptor agonist dibenzoyl-ATP (BzATP) became evident. Both actions could be depressed by pre-treatment with the P2X7 receptor antagonist Brilliant Blue G. Patch-clamp recordings from the cultured cortical neurons indicated a slight presynaptic (enhanced frequency of TTX-insensitive spontaneous excitatory postsynaptic currents) and a pronounced postsynaptic effect (evoked inward current) of BzATP, which could be antagonized by Brilliant Blue G. In vitro ischemia caused an up-regulation of P2X7-IR in neurons and astrocytes. In conclusion, ischemia may lead to the exocytotic release of GABA from neurons and to the non-exocytotic release of the same transmitter from astrocytes by a reversely operating uptake mechanism, connexin hemichannels or an ATP-binding cassette protein. This GABA may favourably influence the neuronal damage caused by ischemia and counterbalance the deleterious effect of glutamate also released by P2X7 receptor activation.

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Session IReceptor-dependent and independent effects

of nucleosides in the Central Nervous System Chairpersons

M. Morelli, F. Pedata

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INVOLVEMENT OF ERK1/2 PHOSPHORYLATION IN 2-CHLORO-2’-DEOXY-ADENOSINE-INDUCED APOPTOSIS AND CELL CYCLE BLOCK OF HUMAN ASTROCYTOMA CELLS

Stefania Ceruti, Alessia Mazzola, Elena Beltrami, Maria P. Abbracchio Laboratory of Molecular and Cellular Pharmacology of Purinergic Transmission, Department of Pharmacological Sciences, University of Milan, Milan, Italy

We have previously demonstrated that 2-chloro-2’-deoxyadenosine (Cladribine, 2CdA), the drug of choice in lymphoproliferative disorders, acts as a pro-apoptotic stimulus also in human ADF astrocytoma cells. 2CdA does not recruit the classical caspase-8- or caspase-9-dependent apoptotic pathways, but time-dependently activates caspase-2 as “initiator” caspase, followed by “effector” caspase-3 responsible for the appearance of the nuclear signs of apoptosis. This represents the first demonstration of the possible involvement of caspase-2 as upstream caspase in drug-induced apoptosis of cancer cells without any involvement of the caspase-9/mitocondrial pathway. Aim of the present study was the in-depth identification of the mechanisms at the basis of 2CdA-induced ADF cell death.

Induction of apoptosis by 2CdA was crucially dependent upon the activation of the ERK1/2 members of the MAP kinase family, which are usually involved in the stimulation of cell proliferation. This atypical pro-apoptotic role for ERK1/2 was demonstrated by the ability of a selective inhibitor (PD098059) to prevent both 2CdA-induced ERK1/2 phosphorylation and appearance of nuclear signs of apoptosis. Other pro-apoptotic members of the MAP kinase family (e.g, p38) and the PI3K/Akt pathway of death were only partially involved in 2CdA-induced effects.

Besides acting as a pro-apoptotic stimulus, 2CdA was also able to induce a G0/G1 cell cycle block of ADF cells after a short incubation period (7-9 hours). Also this effect was crucially dependent upon the intracellular phosphorylation of the drug and activation of ERK1/2 and was accompanied by inhibition of the retinoblastoma protein (pRb) phosphorylation. Microarray analysis confirm these results, by showing a significant induction of immediate early genes (i.e., Fos and JunB), of genes involved in cell cycle block (i.e., GADD45A and GADD34) and in MAP kinase activation (i.e., GADD45B, DUSP1, 2 and 5) in 2CdA-treated cultures with respect to Control cells. The link between cell cycle block and induction of cell death is currently under evaluation in our laboratory. Taken together our results support the possibility to activate atypical cell death pathways involving caspase-2 and ERK1/2 by using 2CdA which may represent a useful alternative to kill tumoral cells resistant to “classical” pro-apoptotic stimuli.

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SHORT-TERM REGULATION OF A2B ADENOSINE RECEPTOR FUNCTIONING BY TUMOR NECROSIS FACTOR ALPHA IN HUMAN ASTROGLIAL CELLS

Maria Letizia Trincavelli (a), Daniela Tuscano (a), Ilaria Tonazzini(a), Stefania Ceruti (b), Maria Pia Abbracchio (b), Antonio Lucacchini (a), Claudia Martini (a) (a) Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, University

of Pisa, Pisa, Italy (b) Department of Pharmacological Science, University of Milan, Milan, Italy

Astrocytes are the most important source of extracellular adenine-based purines in the brain and also express specific adenosine receptors (ARs) including A1, A2A, A2B and A3subtypes. Among all ARs, A2BAR is quite unique, since it exhibits relatively low affinity for adenosine and seem to be activated only under hypoxic or ischemic conditions. Under these pathological conditions other endogenous molecules are released in a large amount, including cytokines. The aim of this work has been to investigate the effect of short-term exposure of human astrocytoma cells (ADF) to the proinflammatory cytokine tumor necrosis factor alpha (TNFα) on A2BAR signalling and regulation processes.

ADF cells were treated with medium alone or TNFα (10-1000U/ml) for different time (5’-12 hours) and then harvested for cAMP and [35S]GTPγS binding assay. Receptor expression and phosphorylation levels were evaluated by immunoblotting analysis. To investigate the intracellular kinases involved in TNFα-mediated A2BAR regulation, we used GF109203X, H89 and Worthmannin as inhibitors of PKC, PKA and PI3K activity respectively.

In a previous work the presence of A2BAR in ADF cells was demonstrated by immunoblotting assay. Moreover we demonstrated that A2BAR activated adenylyl cyclase through Gs protein. The effect of short-time cell exposure to the cytokine TNF alpha on A2BAR responsiveness was tested pre-treating ADF cells with TNFα at different concentrations (10-1000U/ml) for different time (5 min-12 hours). Results showed that TNFα induced an impairment in A2BAR-G protein coupling in a time and concentration-dependent manner, with a maximal effect after 3 hours (1000U/ml). These data suggested that TNFα is able to induce A2BAR desensitization. Cell pre-treatment with PKA and PI3Kinhibitors counteracted this event, for 76 and 66.6% respectively, suggesting that both PKA and PI3K kinases are involved in TNFα heterologous regulation. On the contrary no significant effects were obtained following cell pre-incubation with PKC inhibitors. Moreover the A2BAR agonist induced ERK1/2 activation in a concentration dependent manner with a maximum (3 folds vs control) after 15’. TNFα per se is also able to induce ERK1/2 phosphorylation, with a maximum value of activation (5.5 folds vs control) after 5’. Nevertheless, ADF cells pre-treatment with TNFα (1000U/ml, 3hours) counteracts A2BARagonist-mediated ERK1/2 activation, suggesting a TNFα control on A2BAR-mediated intracellular phosphorylative pathway. Functional studies are in progress to evaluate the effect of TNFα on A2BAR-mediated cAMP pathway.

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ADENOSINE A2A ANTAGONISTS AND PARKINSON’S DISEASE: NEW FINDINGS ON PARKINSONIAN TREMOR

Nicola Simola (a), Anna R. Carta (a,b), Elisabetta Tronci (a), Annalisa Pinna (a,c), Micaela Morelli (a,b) (a) Department of Toxicology, University of Cagliari, Cagliari, Italy (b) Center of Excellence for Neurobiology of Dependence, Cagliari, Italy (c) CNR, Institute of Neurogenetic and Neuropharmacology, section of Cagliari, Italy

Studies in animal models of Parkinson’s disease (PD) and preliminary clinical trials have shown that adenosine A2A antagonists might be useful in the treatment of the disease. Using the 6-hydroxydopamine (6-OHDA) model of PD, previous studies of our group have shown that the A2A receptor antagonist SCH 58261 administered in combination with L-DOPA: I) potentiated L-DOPA-induced contralateral turning II) maintained the efficacy after subchronic administration and produced less dyskinetic effects than a full dose of L-DOPA III) induced minimal or not chances in GAD 67 mRNA in striatum, globus pallidus or substantia nigra and in striatal mRNA levels of dynorphin and enkephalin. In the present study we have evaluated the effect of the A2A antagonist SCH 58261 on tremulous jaw movements induced by tacrine, a valuable rat model of parkinsonian tremor. Parenteral administration of SCH 58261 (5 mg/kg) counteracted the number of bursts (- 35%) and the number of jaw movements per burst (-50%) induced by tacrine (2.5 mg/kg). Local infusion into the striatum of the water-soluble analog SCH BT2 did not antagonize the number of bursts and partially counteracted the number of jaw movements (- 50%) when infused in the dorsa-medial striatum. In contrast, local infusion in the ventro-lateral striatum almost completely counteracted the number of bursts (- 68%) and the number of jaw movements (- 76%) induced by tacrine. A2A receptor antagonists administered with low doses of L-DOPA effectively counteract motor impairments in experimental models of PD and produce less dyskinetic effects than a full dose of L-DOPA. With several A2A receptor antagonists emerging as therapeutic candidates for managing the movement disorders associated with PD, the additional antitremor benefit reported in the present study enhances their therapeutic potential.

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ADENOSINE A2A RECEPTORS ENABLE METABOTROPIC GLUTAMATE 5 RECEPTORS (mGLU5R)-MEDIATED EFFECTS IN THE RODENT HIPPOCAMPUS

Maria Rosaria Domenici (a), Maria Teresa Tebano (a), Alberto Martire (a), Rita Pepponi (a), Jang-Fan Chen (b), Michael A. Schwarzschild (c), Maria Cristina Grò (a), Patrizia Popoli (a) (a) Dipartimento del Farmaco, Istituto Superiore di Sanità, Rome, Italy (b) Boston University, School of Medicine, Boston, MA, USA (c) Massachusetts General Hospital, Boston, MA, USA

The metabotropic glutamate receptors 5 (mGlu5Rs) and the adenosine A2A receptors (A2ARs) show a functional interaction in the rat striatum, where they are colocalized on striopallidal neurons. The aim of the present study was to investigate whether the Glu5R/A2A interaction occurs also in other areas, such as the hippocampus. In electrophysiological experiments in rat hippocampal slices (450 um thick) the selective mGlu5R agonist CHPG (1 mM) induced a reduction of the slope of the excitatory post-synaptic field potentials (fEPSPs) recorded in the CA1 region (-15.56%±3.16). CHPG 500 uM did not modify the slope of fEPSP on its own, but together with the selective A2ARagonist CGS 21680 (50 nM), ineffective on itself, induced a reduction of fEPSPs slope (-20.13±5.2% P<0.05 vs basal). Slice perfusion with 8 uM NMDA induced a slope reduction of fEPSP (-32.4%±3.1) that was potentiated by co-application of CHPG 500 uM (-76.91%±4.14; P<0.05 vs NMDA alone). The potentiating effect of CHPG on NMDA was counteracted by the A2A antagonist ZM 241385 (30 nM) (-50.10±8.29; P<0.05 vsNMDA+CHPG). The key role of A2ARs in modulating mGlu5 effects was confirmed by the observation that in hippocampal slices from A2AR knock out (KO) mice, CHPG lost its ability to potentiate NMDA-induced effects, in contrast with wild type littermates (WT). In addition, western blot experiments showed a reduced expression of mGlu5R immunoreactivity in the hippocampus of KO mice as compared with WT littermates. To evaluate the effect of this interaction on excitotoxicity, we performed experiments on primary cultures of rat hippocampal neurons (ED 17). The application of NMDA (300 uM) induced a significant increase in LDH release (216±34%) that was potentiated by 1 mM CHPG (340±56%; P<0.05 vs NMDA) and prevented by the application of ZM 241385 (25-100 nM; 170±18%; P<0.01 vs CHPG+NMDA). These results suggest that A2A and mGlu5 receptors functionally interact in the hippocampus and that the activation of A2ARs results in a potentiation of mGlu5R-induced effects (facilitatory role) while the blockade or the genetic inactivation of A2ARs prevents the NMDA potentiating effects of mGlu5Rs (permissive role).

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THE ROLE OF ADENOSINE A3 RECEPTORS DURING MILD AND SEVERE “ISCHEMIC CONDITIONS” IN RAT HIPPOCAMPAL SLICES

Anna Maria Pugliese, Elisabetta Coppi, Renato Corradetti and Felicita Pedata Department of Preclinical and Clinical Pharmacology, University of Florence, Florence, Italy

Adenosine activates receptors of the four subtypes: A1, A2A, A2B and A3. Stimulation of adenosine A1 receptors, by reducing excitatory glutamatergic neurotransmission, is involved in protection from hypoxic/ischemic insults, while the role of A2A and/or A3receptors in these phenomena is still controversial.

In this study we investigated the role of adenosine A3 receptors during ischemia, induced by oxygen and glucose deprivation (OGD), in the rat hippocampus in vitro.Excitatory post-synaptic potentials (fEPSP) were extracellularly recorded in the CA1 region of rat hippocampal slices. Two min OGD induced a reversible and reproducible (40 min interval) depression of the fEPSP amplitude (-79%±4, n=10). Conversely, seven min OGD produced an irreversible depression in synaptic transmission (-99±3.5%, n=25).

MRS 1523 (0.1-100 nM), a selective adenosine A3 antagonist, applied 10 min before and during 7 min OGD, did not modify fEPSP in normoxic conditions, but, starting from a concentration of 1 nM, it significantly protected hippocampal slices from the irreversible depression of synaptic transmission induced by 7 min OGD. The recovery of fEPSP amplitude after 7 min OGD carried out in the presence of 100 nM MRS 1523 was 83±12% (P<0.05, n=9). Similar results were obtained using the selective adenosine A3 receptor antagonist MRS 1220 (100 nM, n=6). In addition, MRS 1523 (100 nM, n=6) significantly decreased the fEPSP amplitude depression evoked by 2 min OGD (from –75±8% to –44±11%; P<0.05).

To better clarify the role of A3 adenosine receptors during OGD, we investigated the effects of Cl-IB-MECA, a selective A3 adenosine receptor agonist, on fEPSP applied 5 min before and during 2 min OGD. In the presence of the A3 agonist, applied at a concentration of 10 nM, a significant reduction in the fEPSP depression peak (-41±10, n=6, P<0.05) was found. Furthermore, Cl-IB-MECA (10 nM, n=6) fully induced recovery of synaptic potential amplitude after 7 min OGD (105±4%, P<0.05).

Our results indicate that the selective block of adenosine A3 receptors during severe OGD (7 min) is neuroprotective, suggesting a deleterious role of these receptors during lethal ischemia. On the contrary, the block of adenosine A3 receptors during mild OGD (2 min) have a detrimental effect, indicating an inhibitory and protective role of A3 adenosine receptors during mild ischemia.

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Session IIPharmacology and functions of P2 receptors

Chairpersons M.P. Abbracchio, F. Caciagli

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P2Y4 RECEPTOR, FROM DIFFERENTIATION TO DEATH

Fabio Cavaliere (a,b), Susanna Amadio (a), Valeria Nestola (a), Nadia D’Ambrosi (a,c), Giuseppe Sancesario (c), Giorgio Bernardi (c) and Cinzia Volonté (a,d) (a) Fondazione Santa Lucia, Neurobiology Unit, Rome, Italy (b) FAN-GmbH, Magdeburg, Germany (c) Department of Neuroscience, University of Rome Tor Vergata, Rome, Italy (d) CNR Institute of Neurobiology and Molecular Medicine, Rome, Italy

It is well established that extracellular nucleotides are involved in the regulation of many basic functions, including cell proliferation, differentiation, survival or even death, both in physiological and pathological conditions. In this work we studied the actions of the endogenous or transiently transfected metabotropic P2Y4 receptor in primary cultures and in diverse cell lines. We found that neuronal differentiation of the neuroblastoma SH-SY5Y increased the expression of the P2Y4 protein and that the activation of both endogenous and exogenous P2Y4 receptor by its physiological pyrimidine agonist UTP facilitated neuritogenesis, as detected by morphological phase contrast analysis and confocal examination of neurofilament proteins. This was concurrent with increased transcription of immediate-early genes linked to differentiation such as cdk-5 and NeuroD6, and activity of AP-1 transcription family members such as c-fos, fos-B and jun-D. Nevertheless, a prolonged activation of the P2Y4 receptor by UTP was able to induce cell death in naive, differentiated and transfected SH-SY5Y cells. Among the impairing actions mediated by P2 receptors, emerging studies indicate that several subunits are directly involved in metabolic stress and identify a strong relationship between purinergic and glutamatergic signals. Therefore, we have then studied the molecular interplay between P2Y4 and the glutamatergic ionotropic receptor NMDAR1 during hypoglycemic cell death. We found that, in cultures of cerebellar granule neurons, these proteins were oppositely modulated during glucose starvation (P2Y4 was induced, whereas NMDAR1 was inhibited) and that both P2 and NMDA antagonists could restore basal protein expression levels. Moreover, double immunofluorescence and co-immunoprecipitation experiments revealed membrane co-localization between P2Y4 and NMDAR1 receptors, both in cerebellar granule and in HEK-293 cells transiently transfected with these receptors. Finally, the heterologous over-expression of P2Y4 in SH-SY5Y cells during hypoglycemia was sufficient to cause severe cell death and simultaneous down-regulation of the NMDAR1 protein.Taken together, our data indicate that the activation and high expression of the P2Y4 receptor are responsible for different final effects ranging from differentiation to cell death in both physiological and stress-mediated conditions.

The research presented was supported by grants from FIRB and MURST 2001-2002.

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P2Y1 RECEPTOR ACTIVATION REGULATES OLIGODENDROCYTE PROGENITOR MIGRATIONAND PROLIFERATION

C. Agresti (a), M.E. Meomartini (b), S. Amadio (b), E. Ambrosiani (a), B. Serafini (a), C. Volonté (b), F. Aloisi (a) and S. Visentin (a) (a) Dipartimento di Biologia Cellulare e Neuroscienze, Istituto Superiore di Sanità, Rome, Italy (b) CNR-Fondazione Santa Lucia, Rome, Italy

ATP released in a controlled manner by specialized mechanisms or following cell lysis is known to act as potent signalling molecule via the activation of ionotropic (P2X) and metabotropic (P2Y) receptors.

In the central nervous system, oligodendrocytes are responsible for myelin production and are generated by the proliferation and differentiation of migratory bipolar oligodendrocyte progenitors (OPs). With the aim to better comprehend the role played by purinergic receptor stimulation in oligodendrocyte development, we have investigated the expression of P2X and P2Y receptor subtypes and their functional activity in oligodendrocyte progenitors purified from rat brain cultures. Western blot analysis demonstrates that OPs express several P2X (P2X1,2,3,4,7) and P2Y (P2Y1,2,4) receptor subtypes. Intracellular Ca2+ recording by Fura-2 video imaging allowed to determine the rank potency order of the P2 agonists tested: ADP beta S = ADP = Benzoyl ATP > ATP >ATP gamma S > UTP, alpha,beta-meATP ineffective. The sensitivity to P2 receptor agonists and the inhibition by the specific antagonists oxATP and MRS2179 reveal thatCa2+ signalling in OPs is mediated mainly by P2X7 and P2Y1 receptor activation. Whole–cell recording confirmed that the P2X7 subtype was the only P2 ionotropic receptor active in OPs, since only BzATP was able to induce an inward current. Beside P2Y1, a second ADP sensitive metabotropic receptor was hypothesized on the basis of the incomplete inhibition by MRS2179 and on the lack of reciprocal cross-desensitization between ATP and ADP. Searching for the roles played by P2 receptors, we found that ATP, ADP and the more selective P2Y1 receptor agonist ADP beta S, but not UTP, induce OP migration, mainly acting as chemokinetic agents. Moreover, ATP and ADP were able to inhibit OP proliferation induced by platelet-derived growth factor and to promote oligodendrocyte differentiation, both in purified cultures and in cerebellar tissue slices. The effect of ATP and ADP on migration and proliferation was prevented by the P2Y1 antagonist MRS2179. By confocal laser scanning microscopy, P2Y1 receptor expression was detected in NG2+

OPs in the developing rat brain. Our results suggest that P2Y1 receptors may be involved in regulating OP functions during development and remyelination processes.

This research was supported by Projects 1AC/F and 1AC/F3 of the Italian Ministry of Health (to F.A. and C.V., respectively).

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UPREGULATION OF IONOPHORIC P2X6 IN CHRONIC HEART FAILURE: INTERACTION WITH TNF-AND POTENTIAL ROLE IN MYOCARDIAL CELL DEATH

Silvia Ferrario (a)§, Cristina Banfi (a)§, Ombretta De Vincenti (a), Stefania Ceruti (a), Marta Fumagalli (a), Alessia Mazzola (a), Nadia D’Ambrosi (b), Cinzia Volonté (b), Pasquale Fratto (c), Ettore Vitali (c), Alessandro Parolari (d), GianLuca Polvani (d), Paolo Biglioli (d), Elena Tremoli (a)(d)§, Maria Pia Abbracchio (a)§ (a)Dipartimento di Scienze Farmacologiche and (d) Centro Cardiologico Monzino IRCCS-Università degli Studi di Milano, Milan; (b)Fondazione Santa Lucia, Rome; (c) Ospedale Niguarda Cà Granda , Milan, Italy

Both neurohumoral factors (e.g., cathecholamines) and pro-inflammatory cytokines (e.g., TNFα) have been implicated in myocardial remodelling and progression of left ventricle dysfunction in chronic heart failure (CHF). These responses may act initially as compensatory mechanisms and eventually become deleterious. Despite available drug therapies, including those targeting enzymes, channels and G-protein-coupled receptors (GPCRs), CHF remains the major cause of ill health in industrial societies, suggesting that other transmitter systems are involved and further therapeutic targets remain to be discovered. So far ATP has been merely regarded as a major fuel utilized during myocardial physiology and its involvement in CHF has never received adequate attention, despite evidence that ATP can be released from heart sympathetic terminals and hypoxic cardiomyocytes. ATP may mediate important effects via G-protein-coupled-P2Y and ionophoric P2X receptors, but a full map of myocardial P2 receptors is missing, and their roles in non-diseased and CHF hearts have never been investigated. On this basis, we aimed at determining the expression and presence of all cloned P2 receptors (and of ten selected “orphan” GPCRs structurally related to the P2Y family) in the explanted hearts of five CHF patients undergoing cardiac transplantation in comparison with five control subjects. RT-PCR and Western Blot analysis revealed comparable expression, in all subjects, of seven out of the eight P2Y receptors cloned so far (only P2Y2 was not found) and of two “P2Y-like”orphan GPCRs. All the seven known P2X1-7 receptors (which are channels for Na+ and Ca++) were also found; of these, only P2X6 was upregulated in CHF patients. The potential significance of this change was verified in cultured cardiac fibroblasts freshly isolated from healthy heart. We found that prolonged exposure to ATP or the relatively selective P2X-agonist benzoylATP (BzATP) induced apoptosis, which was exacerbated by TNFα.Moreover, exposure of cardiac fibroblasts to ATP or BzATP resulted in a marked down-regulation of P2X6 mRNA which was prevented by co-exposure to TNFα. This effect could result in overactivation of the P2X6 receptor, enhanced Ca2+entry into cells and, ultimately, in increased cell death. These data provide a potential significance to the detected P2X6upregulation, thus suggesting a novel pathogenic mechanism in heart disease. We suggest that, in human failing hearts, this upregulation may reflect an initial attempt to increase cardiac function but eventually contribute to the progression of the disease, hence representing a novel biological target for the pharmacological modulation of CHF. § These authors equally contributed to this work

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2-ALKYNYLADENOSINE PHOSPHATES AS NEW LIGANDS FOR THE P2 PLATELET RECEPTORS

Sauro Vittori (a), Stefano Costanzi (a), Floriana R. Portino (a), Sara Taffi (a), Rosaria Volpini (a), Marco Cattaneo (b), Gloria Cristalli (b) (a) Dipartimento di Scienze Chimiche, Università di Camerino, Camerino (MC), Italy (b) Dipartimento di Medicina, Chirurgia e Odontoiatria, Università di Milano, Milan, Italy

To date seven P2X (P2X1-P2X7) and nine P2Y (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11-P2Y15)subtypes have been cloned from human cells. The existence of multiple P2 receptor subtypes and their widespread expression in the human body suggest that extracellular nucleotides play important pathophysiological roles and that alterations of these systems may be at the basis of several human diseases. Hence, elucidation of the role of these receptors is expected to advance our knowledge on important physiological functions and provide the basis for identifying novel therapeutic approaches to several incurable diseases. However, the characterization of P2 receptors is currently hampered by the lack of subtype-selective ligands. In fact, most of the currently available ligands are highly limited in terms of kinetics of action, receptor affinity, subtype selectivity, and P2X/P2Y receptor specificity. Moreover, their use is restricted by their suspected ability to be substrates or inhibitors of ecto-nucleotidases, the enzymes responsible for nucleotide degradation. Recently, a series of mono-, di-, and triphosphate of 2-alkynyl adenosines have been synthesized and well characterized in our laboratory. In this work the ability of these derivatives to inhibit or stimulate the human platelet aggregation and platelet adenylyl cyclase activity via P2 receptors have been thoroughly investigated. These studies showed that the 2-hexynyladenosine di- and triphosphate produced shape change and aggregation with an EC50 ranging from 10 to 100 microM. The extent of platelet aggregation induced by 100 microM of these compounds was slightly lower than that elicited by 10 microM ADP, indicating that these compounds activate both P2Y1 and P2Y12 receptors. On the contrary, the 2-phenylethynyladenosine mono-, di- and triphosphates and the 2-hexynyl monophosphate derivative, at concentrations between 10 and 130 microM, did not induce platelet shape change or aggregation, but concentration-dependently inhibited platelet aggregation induced by 10 microM ADP. Hence, they displayed primarily an antagonistic effect on the platelet P2Y1 receptor, while a partial inhibitory effect on the platelet P2Y12receptor was observed only at the highest nucleotide concentration.

In conclusion, the 2-hexynyladenosine di- and triphosphate behave as agonists at P2Y1and P2Y12, while the 2-hexynyladenosine monophosphate and the mono-, di-, and tri-phosphate of the 2-pheynylethynyladenosine behave as antagonists.

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CLONING, PHARMACOLOGICAL CHARACTERIZATION AND DISTRIBUTION OF THE RAT G-PROTEIN-COUPLED P2Y13 RECEPTOR

Marta Fumagalli (a), Letizia Trincavelli (a), Davide Lecca (a), Claudia Martini (b), Paolo Ciana (a,c) and Maria P. Abbracchio (a) (a) Department of Pharmacological Sciences, University of Milan, Milan, Italy (b) Dip. Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, University of Pisa, Italy (c) Center of Excellence on Neurodegenerative Diseases, University of Milan, Italy

The human P2Y13 receptor is a new receptor characterized by coupling to Gi, responsiveness to adenine di-phospho-nucleotides and blockade by the P2Y antagonist AR-C69931MX. The mouse P2Y13 ortholog has also been reported. Here we report, for the first time, the cloning of rat P2Y13 receptor, its pharmacological characterization and tissue distribution. Rat P2Y13 is 79% and 87% identical to human and mouse P2Y13 respectively and clusters in the same region of the rat chromosome 2q31 where some of the cloned P2Y receptors (P2Y1, P2Y12, P2Y14) and several orphan GPCRs are also present (GPR87, H963), suggesting that these receptors (like their human orthologs) may have arisen from a common ancestral receptor by gene duplication. Expression of rP2Y13 receptor in 1321N1 cells induced the appearance of responses to the typical P2Y13 receptor agonists ADP and 2MeSADP, as detected by stimulation of [35S]GTPγS binding. Similarly to the human receptor, agonist activities were higher in cells transfected with rP2Y13 receptor in the presence of hGα16 subunit; in all cases agonist effects were abolished by pertussis toxin pre-treatment. At variance from both human and mouse receptors, ADP was more potent than 2MeSADP. Other nucleotides and sugar-nucleotides were ineffective. Both in the absence and presence of Gα16, activation of rP2Y13 receptor by ADP and 2MeSADP was completely inhibited by nM concentrations of AR-C69931MX. In contrast, no inhibition of rP2Y13 receptor was induced by the selective P2Y1 receptor antagonist MRS2179. rP2Y13showed highest expression levels in spleen, followed by liver and brain (with particulary high levels in cortex and striatum as reported in man), suggesting roles in the nervous and immune systems. Expression levels comparable to those of the other cloned P2Y receptors were found in primary rat astrocytes, indicating a possible role in reactive astrogliosis. Hence, rat P2Y13 receptor displays several similarities but also interesting differences with its human and mouse orthologs, that will have to be taken into account when dissecting the effects mediated by this receptor in the rat animal models.

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Poster abstracts

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1. SYNTHESIS AND BINDING ACTIVITY OF 2-(ARYL)ALKYLTHIOADENOSINE DERIVATIVES

Rosaria Volpini, Stefano Costanzi, Catia Lambertucci, Floriana R. Portino, Sara Taffi, Sauro Vittori, and Gloria Cristalli Dipartimento di Scienze Chimiche, Università di Camerino, Camerino (MC), Italy

It is now well known that the coronary vasodilation induced by adenosine (Ado) in different species is mediated by activation of A2AAR and a compound capable of producing coronary vasodilation through activation of A2AAR, but devoided of A1- and A3-agonist activity, would have advantage over Ado for use in myocardial perfusion imaging studies. Other potential therapeutic applications of selective A2AAR agonists are as anti-aggregatory, anti-inflammatory, anti-psychotic, and anti-Huntington’s disease agents. On this basis, the synthesis of a new series of 2-(aryl)alkylthio derivatives of Ado and N-ethylcarboxamidoadenosine (NECA) was designed to improve the A2A affinity of 2-(aryl)alkylthioadenosines, which were reported to possess coronary vasodilating activity and platelet aggregation inhibitory activity.

The synthesis of the new compounds was previously accomplished by the opening-closure method, firstly reported by Kikugawa and Suehido in 1975 for the 2-(aryl)alkylthio derivatives of adenosine. Unfortunately, this procedure was unsuccessful in the case of 2-(aryl)alkylthio derivatives of NECA, owing to the basic condition and the high temperature used in the opening step. Alternatively, a new synthesis has been performed by reacting 2-iodoadenosine and 2-iodoNECA with the appropriate mercaptans in dry DMF at 120 °C using potassium carbonate as a catalyst

Binding studies (A1, A2A, and A3) and adenylyl cyclase activity (A2B) at human adenosine receptor subtypes stably transfected in Chinese hamster ovary (CHO) cells demonstrated that it is possible to modulate the activity at the A1, A2A, and A3 adenosine receptors by introducing different (aryl)alkylthio substituents in the 2-position of adenosine and NECA.

Hence, two adenosine derivatives were the most active compounds at both A1 and A3receptors (2-(1-pentyl)thioadenosine, Ki A1 = 91 nM, and 2-phenylmethylthioadenosine, KiA3 = 68 nM), while a NECA derivative was the best compound at A2A receptor. Thus, the 2-phenylethylthioNECA, with a Ki A2A = 24 nM, resulted one of the most potent and selective human A2A agonist reported so far.

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2. A NOVEL APPROACH TO GENERATE LIGAND-BASED 3D-QSARS: AUTOCORRELATION OF MOLECULAR ELECTROSTATIC POTENTIAL SURFACE PROPERTIES IN TANDEM WITH PARTIAL LEAST SQUARES ANALYSIS

Magdalena Bacilieri (a), Cristina Ferrari (a), Christian Montopoli (a), Andrea Tralli (a), Giampiero Spalluto (b), Stefano Moro (a) (a) Molecular Modeling Section, Dipartimento di Scienze Farmaceutiche, Università di

Padova, Padova, Italy (b) Dipartimento di Scienze Farmaceutiche, Università degli Studi di Trieste, Trieste, Italy

Ligand based 3D-QSAR is an ensemble of strategies to study new compounds starting from molecule structures and their corresponding chemical properties. Throughout the different approaches, CoMFA (Comparative Molecular Field Analysis) is a widely used methodology to develop molecular models which enable the better understanding of molecular activity. CoMFA computes steric and electrostatic field variables, calculated at grid points surrounding the whole molecule. A subsequent PLS (Partial Least Squares) on these 3D structural descriptors and on the biological activity is derived. An essential and critical step of the CoMFA strategy is the molecular alignment of the molecule structures.

The peculiarity of this work is the introduction of autocorrelation vectors as molecular descriptors for the PLS analysis. The autocorrelation allows in fact to compare molecules (and their properties) with different structures and with different spatial orientation without any previous alignment. In particular, Molecular Electrostatic Potential (MEP) was the property computed and its information encoded in autocorrelation vectors. The 3D spatial distribution and the values of the electrostatic potential is in fact largely responsible for the binding of a substrate to its receptor binding site.

A database of 106 human adenosine A3 antagonists was used to derive two alternative PLS models: one starting from CoMFA descriptors and the other starting from the autocorrelation descriptors. Validation was done with an external test set and the results of the two models were compared. Interestingly, our preliminary results seem to indicate that this new alternative approach could robustly compete with the already well consolidate CoMFA approach. In particular, we have demonstrated that it could be a very interesting tool to filter large structural database in several virtual screening applications.

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3. A REVISITED PHARMACOPHORE MODEL OF HUMAN A3 ADENOSINE RECEPTOR ANTAGONISTS BASED ON THE COMBINATION OF TARGET-BASED AND LIGAND-BASED DRUG DESIGN APPROACH

Stefano Moro (a), Paolo Braiuca (b), Francesca Deflorian (a), Giorgia Pastorin (b), Cristina Ferrari (a), Barbara Cacciari (c), Pier Giovanni Baraldi (c), Katia Varani (d), Pier Andrea Borea (d) and Giampiero Spalluto (b) (a) Molecular Modeling Section, Dipartimento di Scienze Farmaceutiche, Università di

Padova, Italy (b) Dipartimento di Scienze Farmaceutiche, Università degli Studi di Trieste, Italy (c) Dipartimento di Scienze Farmaceutiche, Università degli Studi di Ferrara, Italy (d) Dipartimento di Medicina Clinica e Sperimentale - Sezione di Farmacologia, Università degli

Studi di Ferrara, Italy

In the recent past, an attractive series of pyrazolo-triazolo-pyrimidines bearing different substitutions at the N5 and N8 positions has been described as highly potent and selective human A3 adenosine receptor antagonists. All the synthesized compounds showed A3adenosine receptor affinity in the nanomolar or sub-nanomolar ranges and high levels of selectivity versus the human A3 adenosine receptor.

A combined target-based and ligand-base drug design approach has been carried out to define a novel pharmacophore model of the human A3 receptor antagonists. High throughput molecular docking and CoMFA has been used in tandem to build a new target based pharmacophore model. Indeed, to provide more accurate information about the putative binding site of these A3 inhibitors, a rhodopsin-based model of the human A3receptor was built, and more than hundred pyrazolotriazolopyrimidine ligands were docked in the putative ligand binding site. A novel Y-shape binding motif has been proposed for this class of A3 inhibitors. Docking-based structure superimposition has been used to perform a quantitative study of the structure activity relationships for binding of these pyrazolotriazolopyrimidines to adenosine A3 receptor using comparative molecular field analysis (CoMFA). A remarkable correlation coefficient (cross-validated r2

cv) of 0.840 was obtained. Both steric and the electrostatic contour plots obtained from the CoMFA analysis nicely fit on hypothetical binding site obtained by molecular docking. Based on the combined hypothesis, we have designed, synthesized and tested 17 new derivatives. Impressively, the predicted pKi were very closed to the experimental values.

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4. SAR PROFILE OF A NEW SERIES OF 5-HETEROARYLCARBAMOYLAMINO-PYRAZOLO-TRIAZOLO-PYRIMIDINES AS WATER SOLUBLE hA3ADENOSINE RECEPTOR ANTAGONISTS

Stefano Moro (a), Christian Montopoli (a), Claudia Cusan (b), Giorgia Pastorin (b), Barbara Cacciari (c), Pier Giovanni Baraldi (c), Katia Varani (d), Pier Andrea Borea (d) and Giampiero Spalluto (b) (a) Molecular Modeling Section, Dipartimento di Scienze Farmaceutiche, Università di

Padova, Padova, Italy (b) Dipartimento di Scienze Farmaceutiche, Università degli Studi di Trieste, Trieste, Italy (c) Dipartimento di Scienze Farmaceutiche, Università degli Studi di Ferrara, Ferrara, Italy (d) Dipartimento di Medicina Clinica e Sperimentale - Sezione di Farmacologia, Università degli

Studi di Ferrara, Ferrara, Italy

Water-soluble derivatives represents ideal instruments for biological studies. In the field of hA3 adenosine receptor antagonists we have recently reported a completely water soluble derivative (15 mM) bearing at the 5-position of the pyrazolo-triazolo-pyrimidine core a 4-pyridyl moiety which showed an incredible increased potency (10 pM) in binding studies with respect to the no-water soluble derivative substituted with a phenyl ring (0.16 nM).

With the help of molecular modelling studies we demonstrated that this increased potency could be attributed to strong electrostatic interactions between the pyridinium moiety of and the side chain carbonyl oxygen atoms of Asn274 and Asn278, both located on TM7.

To better examine this effect we synthesized an enlarged series of heteroaryl derivatives in which both the position of the nitrogen and the kind of heteroatom have been modified.

In particular we observed that nitrogen at the four position resulted to be the best pattern of substituion, in fact isomers with nitrogen at 2 or 3 –positions proved to be less potent with respect reference compound (0.2-0.3 nM). Also methylation or oxidation of pyridine nitrogen produce a significant loss of activity (range 5-30 nM). Also the bioisosteric substitutions of nitrogen with oxygen of sulfur led to a decrease of affinity at human A3

adenosine receptors.

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5. SYNTHESIS AND BIOLOGICAL EVALUATION OF 5’-CARBAMOYL- DERIVATIVES OF A1 ADENOSINE RECEPTOR AGONISTS MODIFIED IN THE RIBOSE MOIETY

Loredana Cappellacci (a), Palmarisa Franchetti (a), Patrizia Vita (a), Riccardo Petrelli (a), Michela Pasqualini (a), Barbara Costa (b), Francesca Spinetti (b), Claudia Martini (b), Mario Grifantini (b)(a) Dipartimento di Scienze Chimiche, Università di Camerino, Camerino, Italy (b) Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Università

di Pisa, Pisa, Italy

Selective agonists have been developed for the A1 adenosine receptor (AR) subtype for their potential use as antiarrhythmic, antinociceptive, antilipolytic, cerebro- and cardio-protective, antidiabetic and antiinflammatory agents. However, attempts to apply adenosine A1 receptor agonists as drugs have been hampered by serious side effects, owing to the ubiquitous distribution and wide range of physiological actions mediated by A1 receptors, and also because of their considerable affinity at A3 receptor. One way to circumvent these side effects is the use of A1 AR partial agonists, which may exploit the differerences in receptor-effector coupling in various tissues and can achieve selective activity in vivo.Thus, for example, partial agonists selective for the A1 AR inhibit lypolysis in vivo while they are devoid of cardiovascular effects.

It is known that the substitution at the 5’-position of adenosine with different groups, such as the ethoxy, carbamate or thiocarbamate ones, induces partial agonism at A1 AR. Recently, we synthesized a series of 2’-C-methyl derivatives of potent and selective A1agonists. From structure-activity relationship studies 2-chloro-2’-C-methyl-N6-cyclopentyl-adenosine (2’-Me-CCPA) emerged as a potent and the most selective full agonist so far known for human A1 AR (2,903-fold A1 -selective vs A2A and 341-fold vs A3). The A1 high affinity and selectivity of 2’-Me-CCPA prompted us to synthesize a series of 5’-carbamate or thiocarbamate derivatives of A1 AR agonists in which the hydrogen atom in 2’-position was substituted by a methyl group. The new compounds were tested in radioligand binding assays for affinity, and their intrinsic activities were determined in guanosine 5’-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding assays.

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6. ON THE IMPORTANCE OF SPECIFIC STRUCTURAL MOTIFS OF P2Y RECEPTORS IN LIGAND BINDING:A STUDY WITH THE “ORPHAN” RECEPTOR GPR34

Davide Lecca (a), Marta Fumagalli (a), Letizia Trincavelli (b), Claudia Martini (b), Claudia Verderio (c), Stefano Costanzi (d), Kenneth A. Jacobson (d), Paolo Ciana (a) and Maria P. Abbracchio (a) (a) Department of Pharmacological Sciences, University of Milan, Italy (b) Dip. Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, University of Pisa, Italy (c) CNR Institute of Neuroscience, Cellular and Molecular Pharmacology and Department

of Medical Pharmacology, Milan, Italy (d) Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute

of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, USA

Most G-protein-coupled 7 transmembrane domain receptors (GPCRs) on human genoma still lack a defined physiologically-relevant ligand (“orphan” receptors, oGPCRs). Their “deorphanization” will advance our knowledge of human pathophysiology and unveil new pharmacological targets for the therapy of human diseases. The eight cloned hP2Y receptors (hP2Y1-2-4-6-11-12-13-14) are GPCRs responding to extracellular nucleotides. Mutagenesis studies have suggested that, in these receptors, four aminoacid residues in transmembrane (TM) 6 and 7 may be essential for ligand binding. A first aim of the present study was to identify by bioinformatic analysis novel putative P2Y receptors present on the human genoma. We identified six oGPCRs sharing significant structural and phylogenetic similarity with known P2Y receptors, and found that two of them are present in astrocytoma cells reported to express a yet-uncharacterized “P2Y-like” receptor. One of these two receptors (i.e. GPR34) fully maintains some typical TM6 and TM7 aminoacid motifs believed to be essential for ligand binding. On this basis, we cloned GPR34 cDNA from human astrocytoma cells and expressed it in 1321N1 cells for pharmacological characterization. Single cell calcium imaging and [35S]GTPγS binding have been utilised, to detect, respectively, receptor-mediated increases of intracellular calcium and receptor coupling to G-proteins by exogenously-added nucleotides. A variety of adenine and uracyl nucleotides, including sugar nucleotides were tested. No responses were found. On this basis we conclude that the structural motifs in TM6 and TM7 proposed to be necessary for nucleotide binding may be essential but are not sufficient to determine functional responses to extracellular nucleotides. Thus, additional yet-unidentified structural motifs may also contribute to addressing binding to endogenous ligands. In this respect, molecular modeling on GPR34 revealed that mutations of specific aminoacidic residues (i.e. substitution of Met203 with Lys, and Asn309 with Lys) could result in a gain of function and allow nucleotide binding. With site directed mutagenesis of GPR34 we have generated engineered receptors (“neoceptors”) carrying either one or both mutations and are currently characterising these neoceptors with [35S]GTPγS binding assays, in order to assess if they have acquired the capability to respond to nucleotides.

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7. P2X3 RECEPTOR: N-GLYCOSYLATION AND FUNCTIONAL EXPRESSION

Fabrizio Vacca (a), Valeria Nestola (a), Giuseppe Sancesario (b), Giorgio Bernardi (a,b), Cinzia Volonté (a) (a) Fondazione Santa Lucia, Rome, Italy (b) Department of Neuroscience, University of Rome Tor Vergata, Rome

P2X receptors are integral membrane proteins containing two transmembrane domains. The extracellular portion of these receptors contains several consensus sequences for N-linked glycosylation that may contribute to the functional expression of the channel and to its specific localization. Indeed it has been previously demonstrated the role of N-glycosylation in the functional expression of P2X1 and P2X2 receptors at the plasma membrane. We recently investigated the subcellular localization of the P2X3 receptor, reporting the insertion of this receptor subtype in particular domains of the plasma membrane called lipid rafts. In the present study, we analyze the role of N-glycosylation in P2X3 receptor subcellular localization. We used site-directed mutagenesis to eliminate hypothetical glycosylation sites on rat P2X3 receptor (Asn139; Asn170; Asn194 ;Asn290), chosen by homology with the known P2X1 glycosylated residues. Double, triple and quadruple mutants were also produced. The effect of removing individual or multiple N-glycans was analyzed on cell surface expression (by extracellular labeling with biotin) as well as on lipid raft localisation of P2X3 receptor. As a model system for this experiments we used transiently transfected SH-SY5Y neuroblastoma cells, not expressing apreciable amounts of endogenous P2X3 receptor. Stably P2X3-transfected SH-SY5Y cells were also produced in order to investigate the specific localisation of this receptor during in vitroinduced differentiation and the involvement of lipid raft localization and N-linked gycosylation in this process.

The research presented was supported by Cofinanziamento MURST 2002.

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8. GENOTYPE AND ALLELE DISTRIBUTION OF AN EXONIC POLYMORPHISM IN THE PURINE NUCLEOSIDE PHOSPHORYLASE (PNP) GENE IN HEALTHY CONTROLS AND PATIENTS WITH ACUTE MYOCARDIAL INFARCTION

F. Licastro (a), E. Tumini (a), E. Porcellini (a), A. Branzi (b), A. Beraudi (a), F. Caciagli (c), C.M. Caldarera (d), A. Poli (a) (a) Department of Experimental Pathology and Biology, University of Bologna, Bologna, Italy (b) Institute of Cardiology, University of Bologna, Bologna, Italy (c) Department of Biomedical Sciences, School of Medicine, University of Chieti, Chieti, Italy (d) Department of Biochemistry, University of Bologna, Bologna, Italy

Purine Nucleoside Phosphorylase (PNP) is a ubiquitously expressed enzyme involved in the catabolism and recycling of nucleotides. In particular, PNP catalyses the reversible phosphorolysis of inosine, guanosine and adenosine and their respective deoxynucleosides, using inorganic phosphate as co-substrate to generate free base and (deoxy) ribose-1-phosphate. The human PNP can be considered a “housekeeping gene”, one which is constitutively expressed within a relative narrow range in all tissues and whose product is an enzyme involved in basic cellular metabolism. The absence of PNP activity is associated with T-cells mediated immune defects in humans and these rare recessive diseases became clinically evident during the early years of life. Recently, apart from being associated with traditional risk factors (smoking, diabetes, hypercholesterolemia, etc.), atherosclerosis might also be considered as an immune-mediated condition. Infact, there is a growing increase of investigations regarding the relationship of immune system with atherosclerosis. In the present communication is showed the distribution of an exonic polymorphism in PNP gene: it consists in a transition G/A at +3052 position, which results in the conservative amino acid substitution serine to glycine. Healthy controls of different age ranges and a group of patients with acute myocardial infarction (AMI) has been investigated. Genotype and allele frequencies was similar in two groups of healthy controls of different age ranges (<=50 and 65-69 years old). On the other hand a pilot study of 59 patients with AMI showed that the genotype GG is over represented in patients in comparison to age matched controls, in patients the frequency of the GG is 72.9% (n=43) versus 62.9% (n=66) in controls. Moreover in patients with AMI, was also present a decrement of the A allele, whose frequency is 27.1% (n=16) versus 37.1% (n=39) in controls. These differences are on the limit of statistical significant, probably due to the small number of subjects analysed. In conclusion our data suggest that PNP gene variations may be a genetic risk factor for complication associated with atherosclerosis and reinforce the notion that impaired immune responses are linked with the pathogenesis of atherosclerosis.

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9. ELEVATED EXPRESSION OF A3 ADENOSINE RECEPTORS IN HUMAN COLORECTAL CANCER IS REFLECTED IN PERIPHERAL BLOOD CELLS

Stefania Gessi (a), Elena Cattabriga (a), Arianna Avitabile (a), Roberta Gafa’(b), Giovanni Lanza (b), Luigi Cavazzini (b), Nicoletta Bianchi (c), Roberto Gambari (c), Carlo Feo (d), Alberto Liboni (d), Sergio Gullini (e) and Pier Andrea Borea (a) (a) Department of Clinical and Experimental Medicine, Pharmacology Unit and

“Interdisciplinary Center for the Study of Inflammation (ICSI)” (b) Department of Experimental and Diagnostic Medicine, St. Anna Hospital, University of

Ferrara, Ferrara, Italy (c) Department of Biochemistry and Molecular Biology, St. Anna Hospital, University of

Ferrara, Ferrara, Italy (d) Department of Surgery, Anesthesiology and Radiology, St. Anna Hospital, University of

Ferrara, Ferrara, Italy (e) Department of Gastroenterology, St. Anna Hospital, University of Ferrara, Ferrara,

Italy

Adenosine is a ubiquitous nucleoside that accumulates at high levels in hypoxic regions of solid tumors and A3 adenosine receptors have been recently demonstrated to play a pivotal role in the adenosine-mediated inhibition of tumor cell proliferation. In the present work we addressed the question of the putative relevance of A3 subtypes in colorectal adenocarcinomas. 73 paired samples of tumour and surrounding peritumoral normal mucosa at a distance of 2 cm and 10 cm from the tumor, and blood samples obtained from a cohort of 30 patients with colorectal cancer were investigated to determine the presence of A3 receptors by means of binding, immunocytochemistry and real-time RT-PCR studies. As measured by receptor-binding assays, the density of A3 receptor was higher in colon carcinomas as compared with normal mucosa originating from the same individuals (P<0.05). Overexpression of A3 receptors at the protein level was confirmed by immunohistochemical studies, whilst no changes in A3 mRNA accumulation in tumors as compared to the corresponding normal tissue were revealed. The overexpression of A3receptors in tumors was reflected in peripheral blood cells where the density was approximately three-fold higher compared with healthy subjects (P<0.01). In a cohort of 10 patients, studied longitudinally, A3 receptors in circulating blood cells returned to normal after surgical resection for colorectal cancer. This study provides the first evidence that the A3 receptor plays a role in colon tumorigenesis, and more importantly, it can potentially be used as a diagnostic marker or a therapeutic target for colon cancer.

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10. EXPRESSION OF A3 ADENOSINE RECEPTORS IN HUMAN LYMPHOCYTES: UP-REGULATION IN T CELL ACTIVATION

Stefania Gessi (a), Katia Varani (a), Stefania Merighi (a), Elena Cattabriga (a), Arianna Avitabile (a), Riccardo Gavioli (b), Cinzia Fortini (b), Pier Andrea Borea (a) (a) Department of Clinical and Experimental Medicine, University of Ferrara, Italy and

“Centro Nazionale di Eccellenza per lo Sviluppo di Metodologie Innovative per lo Studio ed il Trattamento delle Patologie Infiammatorie” Ferrara, Italy

(b) Department of Biochemistry and Molecular Biology University of Ferrara, Italy

The present study describes mRNA and protein levels of A3 adenosine receptors in resting (R) and activated (A) human lymphocytes. The receptors were investigated by means of the antagonist radioligand [3H]5-N-(4-methoxyphenyl-carbamoyl)amino-8-propyl-2(2furyl)-pyrazolo-[4,3e]-1,2,4-triazolo-[1,5-c]-pyrimidine ([3H]MRE 3008F20), which revealed density values of 125±15 and 225±23 fmol/mg of protein and affinity values of 1.79±0.30 and 1.85±0.25 nM in R and A cells, respectively. The protein appears to be induced with remarkable rapidity starting at 15 min and reaching a plateau at 30 min. Western blot assays showed that the up-regulation of the A3 subtype after lymphocytes activation was due to an increase in an enriched CD4+ cells fraction. Real-time RT-PCR experiments confirmed the rapid increase of A3 mRNA after T cell activation. Competition of radioligand binding by adenosine ligands displayed a rank order of potency typical of the A3 subtype. Thermodynamic data indicated that the binding is enthalpy and entropy-driven in both R and A cells suggesting that the activation process does not involve, at a molecular level, receptor alterations leading to modifications in the A3-related binding mechanisms. Functionally, the up-regulation of A3 adenosine receptors in A versus R cells corresponded to a potency increase of the A3 agonist N6-(3-iodo-benzyl)-2-chloro-adenosine-5’-N-methyluronamide (Cl-IB-MECA) in inhibiting cAMP accumulation (IC50=1.5±0.4 and 2.7±0.3 nM, respectively); this effect was antagonized by MRE 3008F20 (IC50=5.0±0.3 nM). In conclusion our results provide, for the first time, an in-depth investigation of A3receptors in human lymphocytes and demonstrate that, under activating conditions, they are upregulated and may contribute to the effects triggered by adenosine.

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11. PATTERNS OF EXPRESSION OF P2 PURINOCEPTORS AND PROTEOMIC ANALYSIS OF PERIPHERAL BLOOD MONONUCLEAR CELLS FROM HEALTHY AND CHRONIC HEART FAILURE SUBJECTS

Alessia Mazzola (a,c), Luca Bini (b), Gerarda Pompella (c), Francesca Caporali (c), Riccardo Cianti (b), Ivano Eberini (a), Maria P. Abbracchio (a)* and Franco Laghi-Pasini (c)* (a) Department of Pharmacological Sciences, University of Milan; (b)Department of Molecular Biology, University of Siena, (c) Department of Clinical Medicine and Immunological Sciences, Division of Clinical Immunology, Univerity of Siena

Chronic heart failure (CHF) still remains a leading cause of morbidity and mortality in industrialized countries, despite significant advancement in the understanding of its aetiopathology and despite the currently available drug therapies. This suggests that other yet-to-be discovered causative agents may be involved and purine and pyrimidine nucleotides acting through G-protein-coupled P2Y and ionophoric P2X receptors are likely candidates. Eight members of the P2Y and seven members of the P2X subfamilies have been cloned in humans but scarce data exist regarding their expression and possible changes related to CHF in the heart and circulating peripheral blood cells (e.g., PBMC).

In recent years, genomics and proteomics approaches have opened the possibility of viewing the disease in a broader context. In particular, the proteomic approach might provide important indications towards the identification of the molecular pathways involved in the development and progression of this disease. Moreover, the identification of proteins in PBMC undergoing specific changes in CHF may allow the development of new diagnostic/prognostic biomarkers of the disease that may also be useful to assess the efficacy of pharmacological and non-pharmacological treatments.

On this basis, aims of our work were: (i) to determine the expression of all cloned P2 receptors in the PBMC from healthy and CHF subjects and (ii) to characterize the proteome maps of the same cells in order to compare and eventually identify by mass spectrometry the proteins that change in the disease with particular focus on P2 receptors.

Regarding (i) RT-PCR analysis revealed the expression of seven out of the eight cloned P2Y receptors (P2Y11 was not found) and only four out of the seven P2X receptors (P2X6were absent). We are currently evaluating changes in the expression of these receptors between PBMC from healthy and CHF subjects to evaluate their potential as peripheral diagnostic/prognostic biomarkers of the disease.

Regarding (ii) we have also constructed 2D-electrophoretic maps of PBMC isolated from healthy (n=5) and CHF (n=7) subjects. Maps were analyzed by using the PDQuest software, showing at least 30 proteins undergoing significant changes between the two groups. We are currently identifying these proteins by mass spectrometry. Moreover, some proteins were only found in CHF and not in control subjects suggesting that they may be specifically induced in the disease. The characterization of these proteins could provide new peripheral markers to monitor the progression of the disease and the efficacy of new therapies.

* These authors equally contributed to this work

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12. INVOLVEMENT OF A1 ADENOSINE RECEPTORS IN THE ACQUISITION OF FERTILIZING CAPACITY

Alba Minelli (a) , Lavinia Liguori (a), Ilaria Bellezza (a), Björn Johansson (b), Bertil B. Fredholm (b) (a) Dipartimento di Scienze Biochimiche e Biotecnologie Molecolari, Sezione Biochimica

Cellulare, Perugia, Italy (b) Department of Physiology and Pharmacology, Section of Molecular

Neuropharmacology, Karolinska Institutet, Stockholm, Sweden

Ejaculated mammalian spermatozoa acquire competence to fertilize oocytes by a two-step process: capacitation followed by acrosome reaction.

The biochemical and biophysical modifications occurring in vivo in the female reproductive tract can be reproduced in vitro, and previous studies have suggested a capacitative role for adenosine A1 receptor (A1R). Mice with a targeted disruption of the Adora 1 gene (A1R-/- mice) provide a useful model for better understanding the role of the A1R in fertility. Murine spermatozoa express A1R in the head, neck, mid-piece region, and in the tail. The number of capacitated spermatozoa incubated in human tubal fluid was significantly reduced in A1R-/- compared to A1R+/+ and A1R+/- spermatozoa. The difference between A1 R+/+ and A1R-/- mouse spermatozoa was mainly in the time necessary to reach the maximum percentage of capacitation. A1R+/+ murine sperm obtained the full state of capacitation within 90 minutes whereas A1R-/- sperm required 240 minutes. Caffeine, a known antagonist of A1 and A 2A adenosine receptors, lowered the number of capacitated sperm and affected the time of capacitation in a dose-dependent manner mimicking the effects of the lack of A1 receptors.

Although number, motility, and viability of A1R /- murine sperm was not significantly different from A1 R+/+ mouse spermatozoa, a significant reduction of the number of pups produced by A1R-/- male mice suggests that A1 receptors must be fully operative to accomplish the optimal degree of capacitation and thereby fertilization.

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13. 8-SUBSTITUTED DERIVATIVESOF THE 9-ETHYLADENINE AS ADENOSINE RECEPTOR ANTAGONISTS

Catia Lambertucci (a), Rosaria Volpini (a), Sara Taffi (a), Sauro Vittori (a), Maria Letizia Trincavelli (b), Claudia Martini (b), Annalisa Pinna (c), Micaela Morelli (c), Gloria Cristalli (a) (a) Dipartimento di Scienze Chimiche, Università di Camerino, Camerino (MC), Italy (b) Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Università

di Pisa, Pisa, Italy (c) Dipartimento di Tossicologia, Università di Cagliari, Cagliari, Italy

Adenosine is a nucleoside involved in the regulation of a number of cellular function through the activation of at least four receptor subtypes named: A1, A2A, A2B e A3. Most of the adenosine receptor agonists possess a structure derived from that of the natural ligand adenosine, while the adenosine antagonists belong to different chemical classes and can be in general defined as substituted heterocycles bearing nitrogen atoms.

In this work our attention has been directed toward the synthesis of adenosine antagonists, which are adenine derivatives, since the presence of a bromine atom in the 8-position of the 9-ethyladenine led to a compound endowed with increased affinity at all human adenosine receptor subtypes in comparison to the unsubstituted derivative 9-ethyladenine (8-bromo-9-ethyladenine, Ki A1 = 280 nM, Ki A2A = 52 nM, Ki A2B = 840 nM, Ki A3 = 28,000 nM, vs 9-ethyladenine, Ki A1 = 7,400 nM, Ki A2A = 2,200 nM, Ki A2B >30,000 nM, Ki A3 > 100,000 nM).

Starting from these observation and in the search for potent and selective adenosine receptor antagonists, the synthesis of a new series of 8-substituted 9-ethyladenines was undertaken.

In particular, 9-ethyladenine substituted in 8-position with halogens (chloro and iodo), alkyl and alkoxy groups (methyl and methoxy), aromatic (phenyl) and heteroaromatic rings (thiophene and furane), have been synthesized.

Binding studies performed at the rat adenosine receptor subtypes demonstrated that the 8-substituted 9-ethyladenine derivatives possess good affinity for the adenosine receptors and that it is possible to modulate such affinity by introducing different groups in the 8-position. In fact, the presence in the 8-position of halogens like bromo, chloro and iodo led to derivatives which are selective for the A2A adenosine receptor subtype, while the presence in the same position of aromatic substituents like phenyl, furane and thiophene led to derivatives endowed with high affinity and selectivity for the A1 receptor subtype.

Some of the A2A selective derivatives, tested in in vivo experiments, showed promising activity for their potential application on the therapy of Parkinson’s disease.

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14. STRIATAL CHANGES OF ZIF-268 mRNA LEVELS AFTER AMPHETAMINE ADMINISTRATION IN RATS SUBCHRONICALLY TREATED WITH CAFFEINE

Elisabetta Tronci (a), Anna Rosa Carta (a,b), Nicola Simola (a) and Micaela Morelli (a,b) (a) Dept. Toxicology, University of Cagliari, Cagliari, Italy (b) Center of Excellence Neurobiol. of Drug Abuse, Cagliari, Italy

Antagonists of A2A adenosine receptors, like caffeine, modulate motor response of dopamine agonists through an opposite interaction between adenosine A2A and dopamine D2 receptors in the striatum.

In this study, one group of rats (1st experiment) was subchronically treated with caffeine (15 mg/kg, i.p., for two weeks, on alternated days) or with vehicle and three days after the last injection received a challenge with caffeine (15 mg/kg, i.p.) or vehicle. Another group of rats (2nd experiment) received the same subchronic treatment with caffeine or vehicle and three days after the last injection received a challenge with amphetamine (0.5 mg/kg s.c.) or vehicle. Subchronic treatment with caffeine did not produce any modifications in the locomotor effects of caffeine itself. By contrast, a challenge with amphetamine induced a sensitized locomotor behavioural response in rats pretreated with caffeine as compared to rats pretreated with vehicle.

Changes of mRNA levels for the immediate early gene zif-268 and for A2A receptors we evaluated in the striatum by in situ hybridization.

1st experiment: acute caffeine decreased zif-268 mRNA but not A2A mRNA expression as compared to vehicle in the striatum. Subchronic treatment with caffeine decreased zif-268 mRNA and A2A mRNA level in the striatum which was not modify further modified by a subsequent caffeine challenge

2nd experiment: acute amphetamine did not modify zif-268 mRNA levels with respect to vehicle in the striatum. In contrast, a challenge with amphetamine increased zif-268 mRNA level in the medial striatum of caffeine pretreated as compared to vehicle pretreated rats.

Results suggest that caffeine pretreatment induced a sensitization of the motor response to amphetamine. Reduction of zif-268 mRNA and A2A mRNA receptor levels induced by subchronic treatment with caffeine might be related to the sensitized behavioural response to amphetamine. Indeed, downregulation of striatopallidal neurons, resulting in an inhibition of basal ganglia output neurons, is know to exert a positive effect on motor responses.

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15. THE PROTECTIVE EFFECT OF SUBCHRONIC ADMINISTRATION OF THE A2A RECEPTOR ANTAGONIST SCH 58261 ON NEUROLOGICAL DEFICIT AND BRAIN DAMAGE IN A RAT MODEL OF FOCAL CEREBRAL ISCHEMIA

A. Melani, M. Gianfriddo, D. Turchi, M.G. Vannucchi, M.G. Giovannini, F. Pedata Department of Preclinical and Clinical Pharmacology, University of Florence, Italy

A protective effect of SCH 58261 (0.01 mg/kg, i.p.), administered 5 min after ischemia, on cortical ischemic damage and on contralateral turning behavior, but not on neurological deficit, was recently observed after permanent middle cerebral artery occlusion (MCAo). The protective effects induced by the A2A antagonist were ascribed to reduced glutamate outflow in the first hours after ischemia. Due to the short half-life of SCH 58261, we decided to study the effect of subchronic treatment of the A2A receptor antagonist SCH 58261 in a model of focal cerebral ischemia induced by permanent MCAo in the rat. SCH 58261 (0.01 mg/kg i.p.) was administered 5 min, 6 h and 15 h after MCAo. Contralateral turning behavior was evaluated as the number of rotations per hour between 3 and 4 h after MCAo. 24 h after ischemia, neurological deficit and ischemic brain damage were evaluated. Furthermore, immunohistochemistry studies for phospho-p38 MAPK evaluation were performed.

SCH 58261-treated rats (n=8) showed a significant improvement both in turning behavior (mean±S.E: 222.5±173.8 vs 932.5±227.4 for vehicle-treated rats, n=8, p<0.01) and in neurological deficit (mean±S.E: 11.0±0.5 vs 8.8±0.6, p<0.01). 24 h after ischemia, brain damage evaluated, as the pallid area, was (% of the volume of the ischemic hemisphere) 17.6±2.3% in the striatum and 39.2±2.3% in the cortex. SCH 58261 significantly reduced the extent of the damage by 43% (p<0.02) in the striatum and 18% (p<0.03) in the cortex. Phospho-p38 immunoreactivity was located on cellular processes and bodies of glial cells in the peri-ischemic area.

The results demonstrate that, considering both functional and histological parameters, subchronic administration of an A2A antagonist is protective after ischemia. Our data indicate that A2A antagonism may be a useful tool soon after and up to several hours after ischemia.

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16. GLUTAMATE, ADENOSINE AND GABA EXTRACELLULAR LEVELS, AND P38 MAPK ACTIVATION IN THE STRIATUM OF HUNTINGTON’S DISEASE TRANSGENIC MICE. A2A ANTAGONISM REDUCES GLUTAMATE EXTRACELLULAR CONCENTRATIONS

Marco Gianfriddo, Alessia Melani, Daria Turchi, Maria Grazia Giovannini, Felicita Pedata Department of Preclinical and Clinical Pharmacology, University of Florence, Italy.

Adenosine is the principal neuromodulator in the central nervous system acting on four different receptors: A1, A2A, A2B and A3. A2A receptors are highly expressed in the striatum both pre-synaptically, on glutamatergic terminals, and post-synaptically, on GABAergic neurons. Huntington’s disease (HD) is an inherited, neurodegenerative disorder that induces degeneration of the striatum and the deep layer of the cerebral cortex. Many studies suggest a pathogenetic role of excitotoxicity. The objective of this study was to characterize striatal glutamate, GABA and adenosine levels and the effect of the selective adenosine A2Areceptor antagonist SCH 58261 on striatal glutamate and adenosine outflow using in vivomicrodialysis in the transgenic mice model (R6/2) of HD. Furthermore, we investigated p38 and ERK MAPKinase (MAPK) activation as functional and vitality cellular parameters using immunohistochemistry. Adenosine extracellular concentration increases in transgenic mice compared to wild-type mice (0.032 ± 0.003 µM vs 0.023 ± 0.002 µM; n=5; p<0.05 using the Student’s t test), while glutamate and GABA levels are not modified. The increased adenosine levels may represent an index of mitochondrial dysfunction described in the post-mortem HD brain and in the transgenic mice models. SCH 58260, 50 nM, administered through microdialysis fiber into the striatum, significantly decreases the outflow of glutamate (-46.6%: 53.45 ± 5.00% vs pre-drug value: 100.0 ± 6.25%; n=5; p<10-

4 by Student’s t test) The result could be related to the inhibition of a stimulatory effect on glutamate outflow by A2A receptors localized on pre-synaptic terminals Furthermore, the drug reduces adenosine outflow (-39%: 60.13 ± 2.09% vs pre-drug value: 98.59 ± 9.11%; n=5; p<10-4 using the Student’s t test) in R6/2 mice. Since glutamate exerts a stimulatory effect on all striatal circuits, the decreased glutamate levels may represent the cause for the decrease in adenosine levels. Immunohistochemistry studies revealed increased p38 MAPK activation in the R6/2 mice striatum (number of positive cells ± S.E.: 611.8 ± 27.1 vs 402.9 ± 20.9; +52%; n= 5; p<10-5 using the Student’s t test), but not in the cortex, whereas ERK activation was unchanged. P38 MAPK is a protein that is deep implicated in the processes of the cell suffering and death, while ERK MAPK, is involved in survival and differentiation cell pathways. In addiction we performed, a confocal laser microscopy immunohistochemistry study to determinated which type of cells express the phospho-p38 increase. The results shows that all the phospho p-38 positive cells are neurons.

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17. NEUROPROTECTIVE ACTIVITY OF A2A RECEPTOR ANTAGONIST SCH 58261 IN THE QUINOLINIC RAT MODEL OF HUNTINGTON’S DISEASE: CHARACTERISATION OF EXPLORATORY AND EMOTIONAL BEHAVIOUR

Maria Luisa Scattoni (a), Angela Valanzano (a), Patrizia Popoli (b), Antonella Pèzzola (b), Rosaria Reggio (b), Gemma Calamandrei (a) (a) Department of Cell Biology and Neurosciences Istituto Superiore di Sanità, Rome, Italy (b) Department of Drug Research and Evaluation, Istituto Superiore di Sanità, Rome, Italy

Intrastriatal injection of the excitotoxin quinolinic acid (QA), an N-methyl-D-aspartate receptor agonist, has been found to reproduce in rats some of the clinical features of human Huntington’s disease (HD), included the motor and cognitive disturbances. We found that QA lesion induced in rats a progressive deterioration in motor and exploratory behaviours from two weeks to six months post-lesion (Scattoni et al., Behav Brain Res 2004). Furthermore, six months after surgery QA-lesioned animals showed a marked atrophy of the frontal cortex, which was not evident at earlier time points. The main implication of these findings is that, besides genetic mice models of HD, QA-lesioned rats may represent a suitable mean to test the ability of new drugs to slow down disease progression.

In the present study we assessed whether the adenosine A2A receptor antagonist SCH 58261 (which was recently reported to prevent the QA-induced EEG, neurochemical and neuropathological alterations) prevented the behavioural changes induced by QA lesion. Different groups of male Wistar rats (SCH + QA, SCH + vehQA, vehSCH + QA, vehSCH + vehQA) were administered i.p. with SCH 58261 (0.01 mg/kg) or its vehicle and after 20 min, bilaterally injected with QA (300 nmol/1 µl) or its vehicle in the dorsal striatum (coordinates: A=+1.7, L±2.7, V=-4.8). Two weeks after the lesion, rats were subjected to an open field test to assess exploratory behaviour and to a plus maze test to evaluate anxiety levels. Rats pre-treated with SCH58261 appeared to be protected from some of the exploratory alterations (i.e. wall rearing and object exploration increase) induced by the lesion. However, the emotional alterations induced by the QA lesion (reduced anxiety levels and perseverative behaviour) were not affected by SCH 58261 pretreatment. These findings suggest a new approach for further research concerning the role of A2A receptor antagonists in the prevention of striatal neurodegenerative disorders.

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18. REGULATION OF GLUTAMATE REUPTAKEIN THE STRIATUM AS A POSSIBLE NEUROPROTECTIVE MECHANISM OF ADENOSINE A2ARECEPTOR ANTAGONISTS

Annita Pintor, Rita Pepponi, Mariangela Galluzzo, Rosa Grieco, Antonella Pèzzola, Rosaria Reggio, Patrizia Popoli Dipartimento del Farmaco, Istituto Superiore di Sanità, Rome, Italy

Reuptake mechanisms are essential to maintain glutamate at physiological extracellular concentrations in the Central Nervous System, and thus to protect neurons from excitotoxic injury. An impairment in glutamate reuptake has been hypothesized to be involved in neurodegenerative diseases such as Huntington’s Disease (HD). The selective A2ARantagonist SCH 58261 has been reported to prevent the damage induced by intrastriatal injection of quinolinic acid in the rat, a model of HD (Popoli et al., 2002 J Neurosci 2002), mainly by reducing quinolinic acid-induced raise in extracellular glutamate.

The aim of the present work was to investigate the regulation of glutamate uptake in the rat striatum as a possible mechanism of the neuroprotective effects of A2AR antagonists. To this end, we have investigated by in vivo and in vitro experiments the influence of the A2Areceptor antagonist ZM 241385 on the effects elicited by the glutamate uptake inhibitors L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC, a non-selective blocker) and dihydrokainic acid (DHK, which is selective for the glial GLT-1 transporter).

Microdialysis experiments: male Wistar rats were stereotaxically implanted under Equithesin anaesthesia with a concentric dialysis probe into the striatum (A= +2.0, L= +2.5; V= -6.8 mm). Twenty-four hours later, the probe was perfused at a rate of 2 µl/min with a Ringer’s solution and basal samples (= 4-5 samples/animal) were collected. PDC (4mM) or DHK (10 mM) were then infused over 40 min alone or in presence of ZM 241385 (5nM via probe). As expected, PDC and DHK induced an increase in extracellular glutamate of about 4-fold respect to basal levels (P<0.05 versus basal, N=4). ZM 241385 significantly reduced the effects of both PDC and DHK (P<0.05, N=4/group). In experiments on primary cultures of rat striatal neurons (ED 17, DIV 13-15), DHK didn’t exert any significant effects on the release of lactic dehydrogenase (LDH, an index of cell death) up to the concentration of 1000 µM, probably because of the low percentage of astrocytes in our culture conditions. PDC (12.5, 25, 50 and 100 µM) significantly and dose dependently increased LDH release. The toxicity induced by PDC 50 µM (LDH release 179±18% of basal) was significantly prevented by the application of 30nM ZM 241385 (137±9%; N=7/group, P< 0.01 vs PDC).

These results indicate that A2A receptor blockade could represent a suitable approach to the treatment of neurodegenerative diseases and in particular to those diseases in which striatal excitotoxicity associated with compromised glutamate transporter function plays a pivotal pathogenetic role.

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19. CHANGES IN A2AR-MEDIATED EFFECTS IN THE STRIATUM OF MICE TRANSGENIC FOR HUNTINGTON’S DISEASE

Alberto Martire, Maria Teresa Tebano, Maria Rosaria Domenici, Patrizia Popoli Department of Drug Research and Evaluation, Istituto Superiore di Sanità, Rome, Italy

Huntington’s disease (HD) is an inherited neurodegenerative disease characterized by marked striatal neurodegeneration and for which only symptomatic treatments are available to date. Adenosine A2ARs are involved in excitotoxic/neurodegenerative processes and increasing evidence suggests that A2AR antagonists may be neuroprotective. The neuroprotective potential of A2AR antagonists mainly depend on their ability to reduce the striatal levels of glutamate. On the other hand, adenosine A2AR blockade could also elicit detrimental effects according to the mechanisms involved in toxicity and to the stages of striatal neurodegeneration. Whether, for instance, the HD mutation influences A2A R-mediated effects on basal synaptic transmission and on NMDA-dependent toxicity is not known.

The aim of this study was to verify whether the effects elicited by A2AR activation or blockade are altered in the striatum of symptomatic HD versus wild-type (WT) mice. Transgenic R6/2 mice in a frankly symptomatic phase (12-13 weeks) and age-matched WT mice were used. The animals were decapitated under ether anaesthesia, the brain quickly removed and coronal slices (300 µm thick), including the neostriatum and the neocortex, cut with a vibratome. Extracellular field potentials (FPs) were recorded in the striatum by stimulation of the white matter between the cortex and the striatum. The mean basal FP amplitude was calculated, and the effects of the drugs expressed as percentage variation with respect to basal values. No differences were observed in the mean FP amplitude of R6/2 vs WT mice. In WT mice the application of 75 µM NMDA induced a FP disappearance followed by an almost complete recovery at the washout (FP amplitude after 30 and 50 min of washout: 67.89 ±8.2 and 89.29±9% of baseline). In R6/2 mice, NMDA was significantly more toxic (FP amplitude after 30 and 50 min of washout: 40.3±4 and 49.8±6.4% of baseline, P<0.05 vs WT). Neither the A2AR antagonist ZM 241385 or the A2AR agonist CGS 21680 influenced basal FP amplitude on their own. The effects of NMDA were not influenced by ZM 241385 (30-200 nM) in either WT or R6/2 mice. Conversely, CGS 21680 (100 nM) significantly potentiated NMDA toxicity in WT mice (30 min recovery 35.2 ±10 vs 67.89 ±8.2 P<0.05), while it tended to reduce it in R6/2 mice (58.65 ±9.7 vs 40.3±4, P=0.07).

These results show that A2ARs oppositely modulate NMDA-induced toxicity in the striatum of HD vs WT mice. These findings may have important implications for the neuroprotective potential of A2A R antagonists in HD.

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20. STRIATAL ATP OUTFLOW IN A MODEL OF FOCAL CEREBRAL ISCHEMIA IN VIVOAND THE NEUROPROTECTIVE EFFECT OF A P2 PURINOCEPTOR ANTAGONIST

Daria Turchi, Alessia Melani, Marco Gianfriddo, Maria Giuliana Vannucchi and Felicita Pedata Department of Preclinical and Clinical Pharmacology, University of Florence

Adenosine triphosphate (ATP) is now generally considered a neurotransmitter both in the central and peripheral nervous systems. ATP acts through P2 receptors, identified in P2X, ionotropic receptors and P2Y, metabotropic receptors. Recent evidence suggest that in hypoxic/ischemic conditions, ATP exerts an excitotoxic role in the brain through P2receptors, even if there is no direct evidence of an increase in extracellular ATP levels during cerebral ischemia in vivo. The aim of the present study was to evaluate ATP outflow from the rat striatum using a microdialysis technique associated to a model of focal cerebral ischemia in vivo by intraluminal occlusion of the middle cerebral artery (MCA). Since extracellular ATP is rapidly metabolized to adenosine, we also assessed ATP outflow in presence of the ecto-5’-nucleotidase inhibitor, α,β-methylene adenosine diphosphate (AOPCP, 1 mM). ATP in our samples was analyzed by the luciferine-luciferase reaction. The basal ATP concentration found in this study was (mean±S.E.M.): 3.10±0.34 nM in vehicle-treated rats (n=8) and 9.57±0.26 nM in AOPCP-treated rats (n=8). The drug induced a 3-fold increase in the basal ATP level. During cerebral ischemia, the extracellular ATP levels significantly increased by 2-fold in vehicle-treated rats (5.90±0.61 nM, p<0.01 vs basal level, mean±S.E.M) and by 1.3-fold in AOPCP-treated rats (12.62±0.65 nM, p<0.01 vs the basal level, mean±S.E.M). After 24 h from MCA occlusion, neurological deficit and brain damage were evaluated. The vehicle- and AOPCP-treated rats showed clear neurological impairment (neurological score: 9.9±0.5 and 9.4±0.6 respectively, -43% vs sham-operated rats) and definite striatal (19.0±1.4 mm3 and 23.4±2.6 mm3 respectively) and cortical (52.0±6.5 mm3 and 59.9±4.3 mm3 respectively) damage. In a second group of animals, we evaluated the effect of the P2 receptor antagonist Basilen Blue (10-100 mg/kg i.p.). Basilen blue administered at the dose of 10 mg/kg, significantly reduced the ischemic damage in the striatum (-29% vs vehicle-treated rats). The drug, at the higher dose of 100 mg/kg, significantly reduced both striatal (-49% vs vehicle-treated rats) and cortical (-43% vs vehicle-treated rats) damage. A significant improvement in the neurological deficit (+45 and +35% vs vehicle-treated rats) was induced by both doses of Basilen Blue. The present study confirms that ATP is continuously released in the brain and demonstrates for the first time that ATP outflow increases during ischemia in vivo. Moreover, ATP can be included among the potential causes of neuronal death after ischemia in vivo and P2 antagonists may be regarded as potential neuroprotective drugs in this disease.

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21. P2 RECEPTORS AND CORTICAL-STRIATAL DEGENERATION

Susanna Amadio (a,b), Giuseppe Sancesario (b), Giorgio Bernardi (a,b) and Cinzia Volonté (a,c) (a) Neurobiology Unit, Fondazione Santa Lucia, Rome, Italy (b) Department of Neuroscience, University of Rome Tor Vergata, Rome, Italy (c) CNR Institute of Neurobiology and Molecular Medicine, Rome, Italy

ATP is known to be released in the extracellular space where it acts as an important intercellular signalling molecule. Therefore it can mediate both short term effects, such as neurotransmission, and long-term effects such as trophic and cytotoxic actions. Moreover, ATP can interact with other neurotransmitters or growth factors and act at both receptor- and signal-transduction levels, modulating their effects. Direct and indirect cross talk between P2 receptors and other classes of receptors is well documented. For example, ATP is often co-released with acetylcholine, noradrenaline and GABA. Synapses expressing P2X receptors are also glutamatergic in the cerebellum and hippocampus. In previous works, in cerebellar granule cells, we proposed a cross-talk between glutamate and P2 receptors or NGF and ATP. Mutual interactions between ATP and dopamine have also been observed in the striatum, although the effects of ATP on dopamine release are still not clear.

In this work, we use primary cortical-striatal cultures, as a model of dopaminergic deafferentation in vitro. These are treated with the neurotoxins 6-OHDA and MPP+, normally utilized to induce the loss of dopaminergic neurons, a prominent feature of cerebral degeneration in Parkinson’s disease. Particularly, we investigate the involvement of purinergic neurotransmission, analyzing the presence and modulation of P2 receptor subtypes after neurotoxins’ treatment.

Under these conditions we observe strong and specific modulation of selected P2 receptors. Our results indicate the potential involvement of purinergic mechanisms in cortical-striatal cell loss, mimicking, in vitro, the Parkinson’s neurodegeneration.

The research presented was supported by Cofinanziamento MURST 2002.

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22. THE STATE OF AGGREGATION OF PURINE NUCLEOSIDE PHOSPHORYLASE (PNP) IN RAT ASTROCYTES IS DEPENDENTON THE CELL COMPARTMENT

A. Frassanito (a), A. Beraudi (a), G. Tasco (b), R. Casadio (b), F. Caciagli (c) and A. Poli (a) (a) Dept. of Biology, University of Bologna, Bologna, Italy (b) Biocomputing Unit, Dept. of Biology/CIRB, University of Bologna, Bologna, Italy (c) Dept. of Biomedical Sciences, School of Medicine, University of Chieti, CHieti, Italy

The purine nucleoside phosphorylase (PNP) is a cytosolic enzyme involved in the purine salvage pathway that catalyzes the reversible hydrolysis of nucleosides adenosine, guanosine and inosine, in the corresponding nucleobases adenine, guanine, ipoxantine and ribose-1-posphate. Other crucial enzymes involved in the purine metabolism are adenosine deaminase (ADA) and 5’-nucleotidase, that are distributed in the cytosol as well on the external surface of neuronal and astrocytic membranes where they are anchored by a complexing protein (CD26) and phospholipid anchor (GPI), respectively. Besides the key role in the regulation of extracellular purines released from brain cells, ecto-ADA and ecto-5’-nucleotidase seem to be involved in the interaction with the cell surface proteins and in the receptor signal transduction. For PNP the question is still under debate. Recently, by means of a western blotting analysis, a PNP has been localized on the external membrane of rat astrocytes with a specific activity lower than that of the cytosolic form.

This work focuses on the characterization of the membrane bound PNP as compared to the cytosolic protein.Using a partial-denaturing preparation of cytosol and membrane astrocyte samples in SDS-PAGE, we detected the presence of the enzyme by means of a western blotting analysis. The cytosolic component is endowed with two bands, corresponding to the putative forms of the PNP monomer (about 33 kDa) and trimer (about 96 kDa), respectively. Alternatively, membrane preparations are only characterised by the presence of the monomeric form of the enzyme.

The monomeric form of PNP is solved with atomic resolution (1ULB, in the PDB data base). It is known however that the functional cytosolic PNP is a homo-trimeric enzyme; therefore we modelled the trimer on the basis of its homology with the PNP counterpart from Bos taurus (1LVU) and highlighted with computational tools the properties of the protein surface both for the monomeric and the trimeric form of the enzyme. We find that each monomer interacts with the other partners in the trimer mainly by means of hydrofobic patches that are buried. In the monomeric form, the hydrophobic patch is solvent-accessible and likely to be stabilized by the interaction with the membrane surface.

Based on both experimental and computational results, we suggest that in the membrane the protein is present as a monomer and that its interaction with the membrane may be mediated by hydrophobic interactions and/or by a specific anchor.

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23. GUANOSINE INHIBITS CD40 RECEPTOR EXPRESSION INDUCED BY CYTOKINES AND β AMYLOID IN MOUSE MICROGLIA CELLS

I. D’Alimonte (a), M. D’Auro (a), E. Toniato (b), S. Martinotti (b), V. Flati (c), M.P. Rathbone (d), F. Caciagli (a), R. Ciccarelli (a) (a) Departments of Biomedical Sciences, Section of Pharmacology, and of (b) Oncology

and Neuroscience, University of Chieti, Italy (c) Department of Experimental Medicine, University of L’Aquila, Italy (d) Department of Medicine, Division of Neurology, McMaster University Hamilton, ON,

Canada

CD40, a member of TNF receptor superfamily, is expressed on the surface of various cells including microglia. Aberrant expression of CD40, that promotes brain inflammatory responses and ultimately neuronal injury and death, has been implicated in Alzheimer’s disease (AD) pathogenesis. Thus, strategies to suppress CD40 expression may be of benefit in such a disorder. Here, we investigated whether guanosine, a purine nucleoside that exerts a number of neurotrophic and neuroprotective effects, modulated the expression of CD40 receptors induced by exposure of mouse microglia cultures to different pro-inflammatory agents and the signal transduction pathways involved in this effect. Interferon gamma (IFNγ) is a potent microglia-activating factor. We confirmed that microglia exposure to IFNγ (10 ng/ml; 1-48 h) increased in a time-dependent manner the expression of CD40 receptor (evaluated by western blot analysis). The effect was evident at 6 h, peaked at 24 h and remained sustained up to 48 h. CD40 expression was potentiated by cell exposure to IFNγ plus tumour necrosis factor alpha (TNFα; 50 ng/ml) or to IFNγ plus beta amyloid (βA) peptides (βA1-42; 500 nM). In both cases, CD40-induced expression was linked to the activation of IκBα/NF-κB pathway, that was maximal after 15 and 30 min of IFNγ/TNFαor IFNγ/βA treatment, respectively. Culture pre-treatment with 300 µM guanosine inhibited either cytokine- and βA-induced NF-κB translocation to the nucleus (evaluated by electrophoretic mobility shift assay) or IκBα degradation (evaluated by western blot analysis as an increase in its phosphorylation), reducing also the levels of NF-κB p65 (RelA) free subunit in the cytosol. We looked at the molecular pathways activated by guanosine to induce these inhibitory effects. Microglia exposure to guanosine induced an increased phosphorylation of ERK1/2 and p38 mitogen-activated protein kinases (MAPKs). Culture pre-treatment with selective antagonists of ERK1/2 and p38 MAPKs (PD98059 or SB202190, respectively) counteracted the inhibition caused by guanosine on IFNγ/TNFα-or IFNγ/βA-induced CD40 expression and NF-κB activation, indicating an involvement of these pathways in the regulation of CD40 expression by guanosine. These findings, together with the evidence that guanosine protects SH-SY5Y human neuroblastoma cells against Aβ-induced apoptosis, suggest a role for guanosine as a potential drug in the experimental therapy of AD.

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24. UTP-INDUCED Ca2+ TRANSIENTS IN MICROGLIA ARE INHIBITED BY THE COOPERATIVE ACTION OF ADENOSINE AND ATP

Sergio Visentin, Teresa Radica Dipartimento di Biologia Cellulare e Neuroscienze, Istituto Superiore di Sanità, Rome, Italy

Several CNS cell types have been found to release ATP in vitro: astrocytes during Ca2+

waves propagation, neurons as result of electrical activity, microglia challenged by infecting agents. Interestingly, microglial cells have been described to respond to cell derived ATP, and ATP has been proposed as one of the signals warning microglia of an occurring pathology. On the other hand, extracellular nucleotides are metabolized down to nucleosides such as adenosine, by an efficient ensemble of ecto-enzymes (ATPase and nucleotidases). In an in vivo tissue, following the release of nucleotides, it can be envisaged their transient presence together with that of their degradation products, among which adenosine.

In order to investigate on the possible relationship between the responses induced by nucleotides and those of their degradation products, we focused on the effect of adenosine. Among other effects, microglial cells responded to nucleotide stimulation with a rapid build up of intracellular Ca2+ due to the stimulation of ionotropic (mainly P2X7) and metabotropic (P2Y) receptors sensitive to adenine-based (i.e. ATP, ADP) and uracil-based (i.e. UTP, UDP) nucleotides. The result of the presence of these different subpopulation of receptors is that ATP and UTP induce Ca2+ transients clearly distinguishable, with the latter characterized by a more sustained plateau likely due to capacitative Ca2+ entry.

Adenosine, known to act on a different subset of receptors (P1 receptors), was found to induce a Ca2+ transient only in a subpopulation of cells. However, when it was applied right before the challenge with a nucleotide, the Ca2+ transient induced by this latter was almost completely inhibited. The effect was evident at low doses of adenosine (10 µM), it was reversible, it was mimicked by guanosine and it discriminated between nucleotides, being more evident on Ca2+ transients induced by UTP than to ATP. Interestingly, the inhibitory effect of adenosine increased significantly when cells were pre-treated with ATP. We propose a adenosine/ATP cooperative action aimed to the inhibition of the Ca2+

transient induced by UTP, and that such action could be part of a regulatory mechanism between purinergic and pirimidinergic signaling systems in microglia.

This research is supported by grant no. 2110/RI of the ISS and by the Research Project “Alzheimer” no. ALZ 3 and ALZ 6 of the Italian Ministry of Health.

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AUTHORS’ INDEX

Abbracchio, M.P.; 7; 8; 17; 19; 28; 33 Agresti, C.; 16 Aloisi, F.; 16 Amadio, S.; 15; 16; 43 Ambrosiani, E.; 16 Avitabile, A.; 31; 32 Bacilieri, M.; 24 Banfi, C.; 17 Baraldi, P.G.; 25; 26 Bellezza, I.; 34 Beltrami, E.; 7 Beraudi, A.; 30; 44 Bernardi, G.; 15; 29; 43 Bianchi, N.; 31 Biglioli, P.; 17 Bini, L.; 33 Borea, P.A.; 25; 26; 31; 32 Braiuca, P.; 25 Branzi, A.; 30 Cacciari, B.; 25; 26 Caciagli, F.; 30; 44; 45 Calamandrei, G.; 39 Caldarera, C.M.; 30 Caporali, F.; 33 Cappellacci, L.; 27 Carta, A.R.; 9; 36 Casadio, R.; 44 Cattabriga, E.; 31; 32 Cattaneo, M.; 18 Cavaliere, F.; 15 Cavazzini, L.; 31 Ceruti, S.; 7; 8; 17 Chen, J-F; 10 Ciana, P.; 19; 28 Cianti, R.; 33 Ciccarelli, R.; 45 Coppi, E.; 11 Corradetti, R.; 11 Costa, B.; 27 Costanzi, S.; 18; 23; 28 Cristalli, G.; 18; 23; 35 Cusan, C.; 26 D’Alimonte, I.; 45 D’Ambrosi, N.; 15; 17 D’Auro, M.; 45 De Vincenti, O.; 17 Deflorian, F.; 25 Domenici, M.R.; 10; 41 Eberini, I.; 33

Feo, C.; 31 Ferrari, C.; 24; 25 Ferrario, S.; 17 Flati, V.; 45 Fortini, C.; 32 Franchetti, P.; 27 Franke, H.; 4 Frassanito, A.; 44 Fratto, P.; 17 Fredholm, B.B.; 34 Fumagalli, M.; 17; 19; 28 Gafa’, R.; 31 Galluzzo, M.; 40 Gambari, R.; 31 Gavioli, R.; 32 Gessi, S.; 31; 32 Gianfriddo, M.; 37; 38; 42 Giovannini, M.G.; 37; 38 Giuseppe Sancesario, G.; 29 Grieco, R.; 40 Grifantini, M.; 27 Grò, M.C.; 10 Gullini, S.; 31 Günther, A.; 4 Illes, P.; 4 Jacobson, K.A.; 28 Johansson, B.; 34 Laghi-Pasini, F.; 33 Lambertucci, C.; 23; 35 Lanza, G.; 31 Lecca, D.; 19; 28 Liboni, A.; 31 Licastro, F.; 30 Liguori, L.; 34 Lucacchini, A.; 8 Martini, C.; 8; 19; 27; 28; 35 Martinotti, S.; 45 Martire, A.; 10; 41 Mazzola, A.; 7; 17; 33 Melani, A.; 37; 38; 42 Meomartini, M.E.; 16 Merighi, S.; 32 Minelli, A.; 34 Montopoli, C.; 24; 26 Morelli, M.; 9; 35; 36 Moro, S.; 24; 25; 26 Nestola, V.; 15; 29 Parolari, A.; 17 Pasqualini, M.; 27

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Pastorin, G.; 25; 26 Pedata, F.; 11; 37; 38; 42 Pepponi, R.; 10; 40 Petrelli, R.; 27 Pèzzola, A.; 39; 40 Pinna, A.; 9; 35 Pintor, A.; 40 Poli, A.; 30; 44 Polvani, G.; 17 Pompella, G.; 33 Popoli, P.; 10; 39; 40; 41 Porcellini, E.; 30 Portino, F.R.; 18; 23 Pugliese, A.M.; 11 Radica, T.; 46 Rathbone, M.P.; 45 Reggio, R.; 39; 40 Ribeiro, J.A.; 3 Sancesario, G.; 15; 43 Scattoni, M.L.; 39 Schwarzschild, M.A.; 10 Serafini, B.; 16 Simola, N.; 9; 36 Spalluto, G.; 24; 25; 26 Sperlagh, B.; 4 Spinetti, F.; 27

Taffi, S.; 18; 23; 35 Tasco, G.; 44 Tebano, M.T.; 10; 41 Tonazzini, I.; 8 Toniato, E.; 45 Tralli, A.; 24 Tremoli, E.; 17 Trincavelli, L.; 19; 28 Trincavelli, M.L.; 8; 35 Tronci, E.; 9; 36 Tumini, E.; 30 Turchi, D.; 37; 38; 42 Tuscano, D.; 8 Vacca, F.; 29 Valanzano, A.; 39 Vannucchi, M.G.; 37; 42 Varani, K.; 25; 26; 32 Verderio, C.; 28 Visentin, S.; 16; 46 Vita, P.; 27 Vitali, E.; 17 Vittori, S.; 18; 23; 35 Volonté, C.; 15; 16; 17; 29; 43 Volpini, R.; 18; 23; 35 Wirkner, K.; 4

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