The First Brazilian Purine Club Meeting

August 23–25, 2010 Águas de Lindóia, Brazil

Guest Editors and Organizing Committee:

Dr. Henning Ulrich, University of São Paulo

Dr. Robson Coutinho-Silva, Federal University of Rio de Janeiro

Dr. Ana Maria Oliveira Battastini, Federal University of Rio Grande do Sul

Sponsors:

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil

Fundação de Amparo à Pesquisa do Estado de Rio de Janeiro (FAPERJ)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

INSIGHT® Pesquisa e Ensino

Additional support:

Brazilian Societies for Experimental Biology (FESBE)

The Brazilian Purine Club and Introduction of the Event:

The Brazilian Purine Club was founded in August 2009 during the XXIV Annual Meeting of the Brazilian Societies for Experimental Biology (FESBE) in Águas de Lindoia, Brazil. This initiative rose from the desire of Brazilian scientists to bring together the scientific community working in the area of purinergic signaling and to attract further researchers to this area by periodically organizing scientific events. Dr. Robson Coutinho-Silva (Federal University of Rio de Janeiro; UFRJ) was elected the first President, Dr. Henning Ulrich (University of São Paulo; USP) Vice-President, and Dr. Ana Maria O. Battastini (Federal University of Rio Grande do Sul; UFRGS) General Secretary, and a Scientific Committee composed by Dr. Roberto Paes de Carvalho (Federal University of Fluminense, Rio de Janeiro), Maria José da Silva Fernandes (Federal University of São Paulo; UNIFESP), Dr. Wamberto Varanda (University of São Paulo in Ribeirão Preto; USP-RP), Dr. José Roberto Meyer Fernandes (Federal University of Rio de Janeiro; UFRJ) and Dr. Carla Denise Bonan (Catholic University of Rio Grande de Sul; PUCRS).

The Council of the Brazilian has organized the first Brazilian Purine Club Meeting “Purinergic Signaling: Biological and Therapeutic Implications”, which was held in Águas de Lindóia, State of São Paulo, Brazil, from August 23–25, 2010, as part of the Pre-FESBE program (Federation of Brazilian Societies of Experimental Biology). The scientific program focused on recent achievements in the field of ectonucleotidases, purines and their receptors.

Brazilian researchers and renowned scientists from abroad composed invited speakers of the First Brazilian Purine Club Meeting. Lectures focused on signaling of purinergic P1 and P2 receptors, ectonucleotidases as well as on structural aspects of purinergic receptors. In addition to invited lectures of six scientists from abroad and 14 Brazilian scientists acting in the various areas of purinergic signaling, the program also included poster and oral presentations selected from submitted abstracts by students and young scientists. A total of 138 participants were present including 18 undergraduate and 61 graduate students as well as 59 established researchers and postdoctoral fellows. The II. Meeting of the Purine Club shall be held as part of the FESBE congress to be held in Rio de Janeiro in 2011.

The list of conferences and lectures is presented below:

Opening conference:

Dr. Geoffrey Burnstock - Autonomic Neuroscience Centre, Royal Free and University College Medical School, London, UK. The struggle to establish purinergic signalling and the current explosion of interest.

Conferences:

Dr. Pablo J. Schwarzbaum - Instituto de Química y Fisicoquímica Biológicas, Universidad de Buenos Aires, Buenos Aires, Argentina. Cell volume regulation mediated by P receptors.

Dr. Gary Weisman - Department of Biochemistry, University of Missouri, Columbia, MO, USA: P2Y2 nucleotide receptors mediate neuroinflammatory and neuroprotective responses.

Dr. Peter Illes, Rudolf-Boehm-Institute for Pharmacology and Toxicology, University of Leipzig, Germany. P2X7 receptors of cortical astrocytes in rodent cell cultures and brain slice preparations; identification and functional roles.

Dr. Rodrigo Cunha - Institute of Biochemistry, Faculty of Medicine University of Coimbra, Portugal. Adenosine receptors define salience of information in neuronal circuits and control neurodegeneration and memory impairment in animal models of brain disease.

Dr. Francesco Di Virgilio – Dept. of Experimental and Diagnostic Medicine, University of Ferrara, Ferrara, Italy. Key role of purinergic signalling in cell fusion.

Lectures:

Dr. Alice Teixeira Ferreira (UNIFESP): Action of ATP and analogs on murine hematopoietic cell differentiation.

Dr. Ana Lúcia Ventura (UFF): Nucleotide signaling and the development of the avian retina.

Dr. Ana Maria Battastini (UFRGS): Knockdown of ecto-5´- nucleotidases / CD73 suppresses tumor growth in a rat glioma model.

Dr. Benedito Honório Machado (USP-RP): Purinergic modulation of synaptic transmission in the nucleus tractus solitarius of rats.

Dr. Carla Denise Bonan (PUCRS): Ectonucleotidases and adenosine deaminase in zebrafish: characterization and toxicological implications.

Dr. Guido Lenz (UFRGS): Role of the P2X7 receptor in glioblastoma cell biology and pathology.

Dr. Henning Ulrich (USP): Implications of purinergic receptors in neuronal differentiation.

Dr. José Roberto Meyer Fernandes (UFRJ): Ecto-nucleoside triphosphate diphosphohydrolases in protozoa parasites: looking for a function.

Dr. Luís Carlos Crocco Afonso (UFOP): The role of ectonucleotidases and adenosine production in the infection by Leishmania parasites.

Dr. Maria José da Silva Fernandes (UNIFESP): Purinergic P2 receptors are up regulated in the hippocampus of patients with temporal lobe epilepsy associated with hippocampal sclerosis.

Dr. Maria Rosa Schetinger (UFSM). NTPDase and 5’-nucleotidase activities in physiological and disease conditions: new perspectives for human health.

Dr. Patrícia Castelucci (USP): Effects of the intestinal inflammation, obesity and malnutrition on the P2X receptors and enteric nervous system.

Dr. Roberto Paes de Carvalho (UFF): Adenosine as a signaling molecule in the developing retina.

Dr. Robson Coutinho Silva (UFRJ): Purinergic signaling and innate immunity: implications in the defense against intracellular parasites.

Young Scientist presentations:

Camila Marques da Silva (UFRJ): Pyrimidine coupled P2Y receptors as a new target for Leishmania amazonensis amastigote elimination.

Dr. Elizandra Braganhol (UFRGS): Extracellular nucleotides control Il-8 and MCP-1 secretion in U251MG glioma cell line.

Dr. Karin Calaza (UFF): Developmental regulation of neuronal survival by adenosine in the in vitro and in vivo avian retina depends on a shift of signaling pathways leading to CREB phosphorylation or dephosphorylation.

Dr. Marilia Zaluar Passos Guimarães (UFRJ): P2X2 and P2X7 pore formation is inhibited by colchicine: a possible mechanism for its anti-inflammatory actions.

ABSTRACTS:

OPENING CONFERENCE

The struggle to establish purinergic signalling and the current explosion of interest

Geoffrey Burnstock

UCL, Autonomic Neuroscience Centre Univ. College Medical School, Royal Free Campus, Rowland Hill Street, London NW3 2PF

The talk will begin with a description of the discovery in the early 1970’s of purinergic neurotransmission (i.e. ATP acting as a neurotransmitter) and the struggle we had for the next 20 years to establish this hypothesis. The major conceptual steps leading to our current understanding of purinergic signalling will be described and reference made to some of the key scientists who have influenced its development. Finally, some of the exciting areas of current interest will be considered, including in particular the pathophysiology of purinergic signalling and its therapeutic potential.

Key words: ATP, purinergic neurotransmission, therapeutic potential.

LECTURES

Cell volume regulation mediated by P receptors

Pablo Schwarzbaum

Universidad de Buenos Aires, Pueyrredon 569, Buenos Aires, Argentina

In almost all vertebrate cells challenged by hypotonicity, swelling is followed by a regulatory volume decrease (RVD) that tends to recover the original isotonic cell volume. There is abundant information regarding the intracellular mechanisms mediating volume regulation, but relatively little on extracellular signaling events. Moreover, although several reports show that specific P receptors and ectonucleotidases are important for RVD, the issue has not been studied systematically. To understand nucleotide dependent RVD it is essential to analyse to what extent nucleotides and adenosine accumulate in the extracellular space (nucleotide transmembrane transport), how they are interconverted by ectonucleotidases (nucleotide extracellular metabolism), and activate different purine and pyrimidine receptors (P receptor signaling). Using goldfish hepatocytes challenged by hipotonicity as a cell model to study volume regulation, results will be presented showing how the interaction between extracellular ATP (and other nucleotides), P receptors and ectonucleotidases can modulate RVD. Moreover, since in goldfish hepatocytes RVD is a function of extracellular [ATP], and on the other hand hypotonic exposure leads to a non linear extracellular accumulation of the nucleotide, studying the factors governing the kinetic of extracellular [ATP] is important to characterize the nucleotide-dependent RVD response.

Key words: P receptors, Cell volume regulation, goldfish hepatocytes.

P2Y2 nucleotide receptors mediate pro-inflammatory and neuroprotective responses

Gary Weisman

University of Missouri, Columbia, Missouri 65211-7310 USA

Acute inflammation regulates tissue repair, however, chronic inflammation contributes to neurodegeneration in Alzheimer¡¦s disease (AD) and occurs when glial cells undergo prolonged activation. In the brain, stress or damage causes the release of nucleotides and subsequent activation of the Gq protein-coupled P2Y2 nucleotide receptor subtype (P2Y2R) in glial cells promotes pro-inflammatory responses including P2Y2R upregulation in neurons that mediates neuroprotective responses. P2Y2R activation also increases the proliferation of primary cortical astrocytes by inducing the phosphorylation of the epidermal growth factor receptor (EGFR), a response dependent upon the presence of a SH3 binding domain in the intracellular C-terminus of the P2Y2R that facilitates Src binding and transactivation of the EGFR. Other studies indicate that P2Y2R activation increases astrocyte migration by increasing the expression in astrocytes of alpha(V)beta(3/5) integrins that bind directly to the P2Y2R via an Arg-Gly-Asp (RGD) motif in the first extracellular loop of the P2Y2R , an interaction required for activation Go and G12 protein-dependent cytoskeletal reorganization. In primary cortical neurons (PCNs) from mouse or rat, P2Y2R expression is increased by stimulation with interleukin-1β] (IL-1 β]), a pro-inflammatory cytokine whose levels are elevated in AD, in part due to nucleotide-stimulated release from glial cells. Other results indicate that oligomeric β-amyloid peptide (Aβ 1–42), a contributor to AD, increases nucleotide release from astrocytes and microglial cells, which should serve to activate upregulated P2Y2Rs in neurons. Data with rat PCNs suggest that P2Y2R upregulation by IL-1β and subsequent activation by UTP are neuroprotective, since this increases the non-amyloidogenic cleavage of amyloid precursor protein. Furthermore, activation of IL-1β-upregulated P2Y2Rs by UTP in PCNs increases the phosphorylation of cofilin, a cytoskeletal protein that enhances neurite outgrowth. Thus, activation of pro-inflammatory P2Y2Rs in glial cells can promote neuroprotective responses, suggesting that P2Y2Rs represent a novel pharmacological target in neurodegenerative and other pro-inflammatory diseases. Supported by grants AG18357, DE07389, and DE17591 from the National Institutes of Health (USA).

Key words: P2Y2, neurodegenerative disease, neuroprotection, inflammation.

P2X7 receptors of cortical astrocytes in rodent cell cultures and brain slice preparations; identification and functional roles

Peter Illes

University of Leipzig, University of Leipzig, D-04107 Leipzig, Germany

Astrocytes, which form the most abundant cell population in the brain, were previously considered to represent only a supportive passive element mainly with trophic and homeostatic functions in the CNS. More recently, considerable evidence has been gathered to demonstrate that these cells are indispensable partners of neurons, actively involved in synaptic transmission and consequently playing an important role in astrocytic-neuronal cell communication. Cultured astrocytes from rats and mice, have been reported to possess functional P2X7 receptors (Duan et al., J. Neurosci. 23:1320–1328, 2003), although enhanced green fluorescent protein-expressing astroglia acutely dissociated from the cortex of the transgenic Tg(GFAP/EGFP) mice exhibited ATP-induced currents, which were mediated by P2X1/5 heteromeric receptors (Lalo et al., J. Neurosci., 26:2673–2683, 2008). Moreover, in brain slice preparations, where the functional integrity of the astrocytic network and the neuronal-astrocytic synaptic connections have been preserved, there were either no, or unpredictable and non-reproducible responses to high concentrations of ATP (Jabs et al., Glia 55:1648–1655, 2007). In primary cell cultures of the rat cerebral cortex, voltage-clamp protocols classified astrocytes as belonging to the outwardly rectifying or passive types. Both classes of astrocytes reacted to the application of ATP or dibenzoyl-ATP (Bz-ATP) with inward currents at a holding potential of −80 mV. These current responses considerably increased in an external medium containing no Mg²⁺ and low Ca²⁺ concentrations. The use of various receptor-type selective agonists and antagonists suggested the presence of P2X7 receptors. In brain slices of the rat prefrontal cortex, astrocytes were identified on the basis of their small cell diameters, and the absence of action potentials in response to depolarizing current injection. Immunohistochemistry experiments revealed that astrocytes labelled after recording with pipettes filled with the fluorescent dye Lucifer Yellow (LY), exhibited co-localization of LY with the astrocytic markers S100 and GFAP. In addition, P2X7 receptors were also present on the S100- and GFAP-immunoreactive (IR) astrocytes. Based on their voltage-current characteristics, astrocytes were classified into the outwardly rectifying, passive and intermediate types. Notwithstanding this classification, alls astrocytes reacted to NMDA, ATP, and BzATP at a holding potential of −80 mV. Once again, the current responses to ATP and Bz-ATP were greatly potentiated in a divalent cation-poor medium. Both the agonist and antagonist profiles of the P2 receptor-ligands investigated suggested the existence of P2X7 receptors. There was no response to ATP or BzATP in P2X7-/- mice, when compared with the respective background animals. Hence, electrophysiologically and immunohistochemically identified rodent astrocytes both in cell culture and brain slice preparations appeared to exhibit P2X7 receptors together with a range of P2Y receptor-types. These receptors may be involved in immunological defense reactions, apoptosis/necrosis as well as long-term proliferation such as gliosis.

Key words: P2X7, astrocytes, brain slices.

Adenosine receptors define salience of information in neuronal circuits and control neurodegeneration and memory impairment in animal models of brain disease

Rodrigo A Cunha

University of Coimbra, Center for Neurosciences of Coimbra, I Biochemistry, University of Coimbra, Portugal

Adenosine is a ubiquitous neuromodulator, i.e. it does not directly trigger neuronal responses but it effectively controls the efficiency of synaptic transmission. Adenosine mainly controls excitatory transmission through the activation of widespread inhibitory A1 receptors (A1Rs), which sets a global inhibitory tonus in brain circuits designed to reduce noise in a circuit. In parallel, the local activation of facilitatory A2ARs by adenosine, formed from ATP released only at high frequencies from neuronal vesicles, down-regulates A1Rs and facilitates plasticity through potentiation of NMDA receptors, selectively in the tetanised synapse. Thus, upon high-frequency firing of a given pathway, the combined exacerbation of global A1R-mediated inhibition in the circuit (through heterosynaptic depression) with the local synaptic activation of A2ARs in the activated synapse, cooperate to maximise salience between the activated and non-tetanised synapses, acting as a low-pass filter fine-tuning synaptic plasticity processes in brain circuits. This dual role of adenosine also translates into the control of neurodegeneration. Thus, bolstering A1R activation at the onset of neuronal injury attenuates brain damage, but these A1Rs undergoes rapid desensitization. In contrast, A2ARs are up-regulated in noxious brain conditions and their blockade confers robust brain neuroprotection in adult animals through a simultaneous inhibition of glutamatergic excitotoxicity and mitochondria-dependent apoptosis and controlling neuroinflammation. Thus, either caffeine (an adenosine receptor antagonist) or selective blockade of A2ARs prevents neurodegeneration and memory impairment in animal models of Alzheimer disease, epilepsy, sepsis, diabetes and chronic unpredictable stress. Accordingly, human epidemiological studies also indicate an inverse association between caffeine consumption and age-induced cognitive deficits as well as with the incidence of Alzheimer’s disease. This prompts the interest of testing and developing A2AR antagonists as novel protective agents in neurodegenerative diseases, in particular to prevent memory impairment. (Supported by FCT, Fundação Oriente, Pfizer, Sanofi-Aventis)

Key words: adenosine, caffeine, synaptic plasticity, memory, neuroprotection.

Key role of purinergic signalling in cell fusion

Francesco DiVirgilio

Univ. Ferrara, Univ. Ferrara, Via Riviera 41, 35020 Polverara (PD) Italy

Disturbances affecting bone tissue homeostasis are a major health problem that especially affects elderly people, postmenopausal women, patients treated with corticosteroids, or bearing metastatic malignancies. Chronic inflammatory diseases (e.g. rheumatoid arthritis) are another set of disturbances that exact a heavy toll on bone as they are often accompanied by a reduction in bone mineral density. Contrary to a widely held opinion, bone far from being a rigid unchanging structure, is on the contrary a dynamic living tissue that undergoes constant reshaping in response to mechanical forces and soluble osteogenic or osteolytic factors. Osteoclasts play a central role in bone homeostasis as they are the unique cell responsible of bone resorption. An excess of osteoclast activity results in progressive bone loss and deterioration of bone architecture, leading to osteoporosis and bone fractures, while lack of osteoclast activity may cause abnormal bone deposition, thus leading to osteopetrosis. Osteoclasts are multinucleated giant cells originating from fusion of bone marrow hematopoietic precursors of the monocyte/macrophage lineage. The key factors that drive final osteoclast differentiation are RANKL and M-CSF (Lacey et al., 1998). The sole presence of these two cytokines, produced by osteoblasts and immune cells, is sufficient to drive osteoclast formation, i.e. formation of a bone-reabsorbing multinucleated giant cell. Several plasma membrane molecules likely acting via a common intracellular pathway have been implicated in macrophage or osteclast precursor fusion, but the molecular mechanism has remained elusive. In addition to surface receptors, plasma membrane phospholipids seem also to be involved as loss of the physiological phospholipid asymmetry is a required event for fusion. Over recent years increasing attention has been paid to the role played by purinergic signalling in mononuclear cell differentiation and function. Among P2 receptors, P2X7 is a hot focus of investigation for its undisputed role in inflammasome activation and cytokine release. Furthermore, there is growing awareness that this receptor plays a central role in bone homeostasis. A few years ago we observed that macrophage cultures expressing high P2X7 levels were unusually prone to form multinucleated giant cells, and hypothesized that a physiological function of P2X7 might be in supporting cell fusion. Hereby we provide evidence of an absolute requirement for the P2X7 receptor, ATP release and adenosine signaling in human osteoclast formation, as shown by the following findings: a) M-CSF/RANKL-stimulated fusion of human monocytes is fully prevented by an anti-P2X7 mAb; b) fusion-competent monocytes release ATP via the P2X7 receptor; c) accelerated degradation of released ATP by addition of either apyrase or hexokinase strongly increases fusion; d) removal of extracellular adenosine by adenosine deaminase blocks fusion; e) pharmacologic stimulation of the adenosine A2A receptor increases, while selective A2A blockade inhibits fusion. In conclusion, we show that the P2X7 receptor, ATP, adenosine and the adenosine receptors act in a concerted fashion to promote fusion of M-CSF/RANKL -stimulated monocytes into multinucleated osteoclasts.

Key words: P2X7, osteoclasts, inflammasome.

SIMPOSIUM

ECTO-NUCLEOTIDASES AND PURINERGIC SIGNALLING

Knock-down of ecto-5′-nucleotidase/CD3 suppresses tumor growth in a rat glioma model

Ana Maria Oliveira Battastini¹ , Luis Felipe Campesato¹, Elizandra Braganhol¹, Rafael Zanin¹, Andressa Bernardi¹, Patrícia Luciana Lopez¹, Emerson André Casali², Maria Isabel A. Edelweiss³, Guido Lenz⁴,

¹Departamento de Bioquímica, ICBS, UFRGS,²Centro Universitário Metodista do IPA, Porto Alegre, RS;³Hospital de Clinicas de Porto Alegre, HCPA, UFRGS,⁴Departamento de Biofísica, IB, UFRGS, Porto Alegre, RS, Brazil

Glioblastoma multiforme (GBM) is the most aggressive tumor of the central nervous system that present a high proliferative and invasive behavior. Ecto-5′-nucleotidase (e5NT/CD73) is a widely expressed enzyme that hydrolyses extracellular AMP, producing adenosine, an important molecule in several cell functions. Besides that, e5NT/CD73 has been shown to exert an important role in cell-cell and cell-matrix interactions. Several evidences suggest that e5NT/CD73 has tumor-promotion functions, being up-regulated in several tumors. In this study we aimed to better characterize the role of e5NT/CD73 by silencing its expression in C6 rat glioma cell line and analyzing the in vitro and in vivo glioblastoma growth. Knockdown (KD) of e5NT/CD73 was obtained through stable RNA interference mediated by lentivirus and KD cells were analyzed through immunofluorescence and enzyme activity methods. We also assessed parameters of malignancy in vitro, such as cell adhesion and migration. For a deeper investigation, KD C6 cells were implanted in the striatum of adult Wistar rats and haematoxylin and eosin (H&E) as well immunohistochemical analysis were performed to assess the tumor size and histological malignancy characteristics. Two stable e5NT/CD73 KD clones of C6 glioma cells were obtained, named C6-c54 and C6-c56, and these clones presented a large reduction in expression and AMPase activity of e5NT/CD73. A significant alteration in adhesion parameter of silenced cells was observed in vitro possibly due to the loss of the adherence function of e5NT/CD73 per se. In addition, e5NT/CD73 KD cells were not able to develop a tumor mass in the brain of rats, which was confirmed by immunohistochemistry analysis, whereas all rats injected with C6 wild-type (wt) cells developed tumors with all characteristics of GBMs. Tumor growth of C6 wt cells was not blocked by the co-injection with APCP, an e5NT/CD73 inhibitor. Taken together these results reinforce the crucial involvement of purinergic signaling in the GBM progression. Further studies are necessary to establish new and powerful therapies against these tumors using e5NT/CD73 as target. Financial Support: CNPq, FIPE-HCPA.

Key words: ecto-5′-nucleotidase/CD73 , glioma model, adenosine, RNAi.

Ectonucleotidases and adenosine deaminase in Zebrafish: characterization and toxicological implications

Carla Denise Bonan¹, Denis Broock Rosemberg^2,1, Eduardo Pacheco Rico^2,1, Mario Roberto Senger^2,1, Diogo Onofre Gomes de Souza², Renato Dutra Dias¹, Mauricio Reis Bogo¹

¹Pontificia Universidade Católica do Rio Grande do Sul, Avenida Ipiranga, 6681, Prédio 12 D, sala 340, 90619-900, Porto Alegre, RS;²Departamento de Bioquímica, ICBS, UFRGS, Rua Ramiro Barcelos, 2600-anexo, 90035-003, Porto Alegre, R, Brazil

Zebrafish (Danio rerio) is an ideal vertebrate model for the study of human diseases, drug screening, and toxicological assessment. Several neurotransmitter systems have been already described in zebrafish. Among them, the purinergic system, in which ATP evokes responses through the P2X and P2Y receptors. ATP can be inactivated the action of ectonucleotidases, which include NTPDase (nucleoside triphosphate diphosphohydrolase) family and ecto-5´-nucleotidase. The final product of ATP hydrolysis is adenosine, an important neuromodulator, which can be deaminated by adenosine deaminase (ADA), producing inosine. We previously identified the NTPDase and ecto-5′-nucleotidase activities in zebrafish brain membranes with kinetic characteristics similar to those found in mammals. In addition, we performed a complete analysis of NTPDase family in zebrafish and evaluated the nucleotide hydrolysis in three tissues of adult zebrafish (brain, liver, and heart), confirmed the presence of distinct NTPDase members by a phylogenetic analysis and verified their relative gene expression profiles in these tissues. A different profile of ATP and ADP hydrolysis in the brain, liver, and heart as a function of time, protein amount, and sensitivity to inhibitors was observed. Homology-based searches identified the presence of NTPDase1-6 and NTPDase8 orthologs. In addition, the phylogeny also grouped three NTPDase2 and two NTPDase5 paralogs. RT-PCR experiments revealed the existence of a distinct relative entpd1–6 and entpd8 expression profile in brain, liver, and heart. The enzymatic and kinetic properties of adenosine deaminase were characterized in both soluble and membrane preparations from zebrafish brain, which displayed a strong preference for adenine over guanine nucleosides. A phylogenetic analysis confirmed the presence of distinct ADA-related genes (ADA1, ADAL and two orthologous genes of ADA2). ADA1 had a similar expression pattern, whereas ADAL was less expressed in the heart. The highest relative amount of ADA2-1 transcripts was observed in the brain, liver, and gills and it was less expressed in the heart. RT-PCR assays revealed that the other ADA2 form (ADA2-2) was expressed ubiquitously and at comparable levels in zebrafish tissues. The enzymes involved in the control of extracellular nucleotide and nucleoside levels can be a target for the neurotoxicity induced by metals, such as mercury, lead, and copper. ATP, ADP, and AMP hydrolysis were significantly decreased after exposure to these metals. In addition, adenosine deaminase activity was decreased after both acute and subchronic exposures to mercury whereas in brain membranes the enzyme was inhibited only after subchronic exposure. However, the changes induced by mercury and lead were not promoted by transcriptional control, because there were no changes in the gene expression pattern for NTPDases, ecto-5′-nucleotidase, and adenosine deaminase. In contrast, copper induced a decrease in ATP, ADP, and AMP hydrolysis after 96-h exposure, followed by a decrease in NTPDase1, NTPDase_mv, NTPDase_mq, NTPDase_mq, and ecto-5-nucleoitdase mRNA transcript levels. The results demonstrated that changes induced by metals on extracellular nucleotide and nucleoside hydrolysis can be involved in behavioral and neurotoxic effects induced by these contaminants. Supported by: CNPq, CAPES, FAPERGS, FINEP (Rede Instituto Brazileiro de Neurociência (IBN-Net 01.06.0842-00), INCT-TM.

Key words: ecto-nucleotidases, zebrafish, NTPDases, ecto-5′-nucleotidase, adenosine deaminase.

Ecto-nucleoside triphosphate diphosphohydrolases in protozoa parasites: looking for a function

José Roberto Meyer-Fernandes

Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil

The plasma membrane of cells contains enzymes whose active sites face the external medium rather than cytoplasm. The activities of these enzymes, referred to as ecto-enzymes, can be measured using living cells. Cell membrane ecto-nucleoside triphosphate diphosphohydrolases (ecto-NTPDases) are integral membrane glycoproteins that are millimolar divalent cation-dependent, low specificity enzymes that hydrolyze extracellular nucleoside tri-and / or diphosphate. Their physiological role is still unknown. However, several hypothesis have been suggested such as (i) protection from cytolytic effects of extracellular ATP, (ii) regulation of ecto-kinase substrate concentration, (iii) termination of purinergic signaling, (iv) participation in the salvage of purines from extracellular medium, (v) involvement in signal transduction and (vi) involvement in cellular adhesion, virulence and infection. Here, using kinetic experiments, Western blotting, Flow cytometry, and immunoelectron microscopy analysis we present some evidences showing that in protozoa parasites these enzymes contribute to adenosine generation and to adhesion to host cells. Key words: ecto-ATPase, adenosine acquisition and virulence. This work was supported by MEC, CNPq, FAPERJ and CAPES.

Key words: ecto-ATPase, adenosine acquisition and virulence, protozoa parasites.

NTPDase and 5′-nucleotidase in physiological and disease conditions: new perspectives for human health

Maria Rosa Schetinger, Paula Maldonado, Vara Maria Morsch

Universidade Federal de Santa Maria, CAMPUS Camobi, Santa Maria, RS, 97105 900, Brazil

Background: We intend to report nucleotide hydrolysis in distinct human diseases. Methods: This study emphasized the involvement of NTPDase and 5′-nucleotidase activities in disease processes in several tissues and cell types. Background information is given about the general characteristics of these enzymes in diabetes, pregnancy, hypercholesterolemia and cancer. In addition, the role of these enzymes is considered under clinical conditions such as acute lymphoblastic leukemia (ALL), B-chronic lymphocytic leukemia (B-CLL) and human immunodeficiency virus (HIV). The Human Ethics Committee from the Federal University of Santa Maria approved all protocols. Results: In diabetes, the increased ATP and ADP hydrolysis by NTPDase could be compensatory, avoiding coagulation processes by increasing ADP depletion. In pregnancy, NTPDase and 5′-nucleotidase act directly in thromboregulation. In hypercholesterolemia the increased NTPDase activity is possibly related to a compensatory response to the inflammatory and pro-oxidative state associated with hypercholesterolemia. In breast cancer patients, the enhanced NTPDase activity may be related to changes in platelet activity. The altered activity in lymphocytes from patients with ALL and B-CLL shows that NTPDase may play an important role in the modulation of the disease. Increased NTPDase activity in lymphocytes of HIV patients suggests that it may play an important role in maintaining an adequate balance between the generation and consumption of ATP and preserving cellular integrity and the immune response. Conclusions: It may be suggested that despite advances made toward understanding the molecular structure of ectonucleotidases, much more investigation will be necessary to entirely grasp their role in physiological and pathological conditions.

Key words: NTPDase, 5′-nucleotidase, disease.

PURINERGIC RECEPTORS AND IMMUNE SYSTEM

Purinergic signaling and innate immunity: implications in the defense against intracellular parasites

Robson Coutinho-Silva^1,2

¹IBCCF-UFRJ, Immunobiology Program, Inst. Biophysics Carlos Chagas Filho, Av. Carlos Chagas Filho, 373, CCS, Ilha do Fundão, Rio de Janeiro, RJ, Brazil;²(INPeTAm/UFRJ), Inst Nacional de Pesq. Translacional em Saúde da Amazônia, Av. Carlos Chagas Filho, 373, CCS, Ilha do Fundão, Rio de Janeiro, RJ, Brazil

The interest of immunologists in the effects of nucleotides during the immune responses is growing very fast. During inflammation and cell death a variety of danger signals is released, including nucleotides. The outcome of immune response might depends on the amount of ATP released, varying from silent repair to strong inflammatory response. In addition, the release of extracellular ATP may alert the immune system to the presence of infected cells with parasites, and arm them to respond against the invader. At same time, different intracellular parasites have evolved enzymes or other mechanisms to block or reduce nucleotide-mediated signaling. We found that extracellular nucleotides stimulate the expression of cell adhesion molecules involved in recognition and phagocytosis of apoptotic and necrotic dead cells, while having no effect on the adhesion and internalization of viable cells. We will thus discuss how this mechanism can participate in Leishmania amazonensis uptake by macrophages and how infections with intracellular parasites such as Leishmania amazonensis and Toxoplasma gondii can be differently controlled by P2 receptors members’ activation. Supported by: CNPq, FAPERJ-PRONEX, INCT-INPeTAm

Key words: Intracellular parasites, P2 receptors, phagocytosis of apoptotic bodies, Leishmania, Toxoplasma

The role of ectonucleotidases and andenosine production in the infection by Leishmania parasites

Luiz Carlos Crocco Afonso^1,2

¹DECBI/NUPEB , Laboratório de Imunoparasitologia, Universidade de Ouro Pret, Campus Universitário, Morro do Cruzeiro, 35400-000 Ouro Preto – MG;²UFV, Departamento de Biologia Geral e Departamento de Bioquímica , Viçosa. Brazil

Leishmania are obligatory parasites of macrophages in the mammalian host. To avoid activation of these cells by the immune system, the parasite interferes with a series of macrophage function such as antigen presentation and cytokine and nitric oxide production. In addition, proper activation of T cells is frequently found in more severe forms of the disease such as diffuse leishmaniasis. In face of the expression of ectonucleotidases in the parasite’s membrane and the known effects of ATP and adenosine on the regulation of the immune response, our laboratory has been interested in investigating the role of adenosine and its receptors on the regulation of the immune response against Leishmania parasites. Initially, we observed a significant correlation between ectonucleotidasic activity of different Leishmania species and their ability to cause infection in the murine model of cutaneous leishmaniasis. Furthermore, we observed that alterations in the parasite’s culture conditions that led to altered expression of these enzymatic activities directly correlated with the parasites infectivity, both in vitro and in vivo. The role of adenosine in the regulation of the infection was first demonstrated in Leishmania braziliensis infection, where addition of a A2b receptor antagonist led to faster lesion resolution while administration of adenosine together with the parasite caused increased parasitism and lesion development. More recently, we demonstrated that infection of dendritic cells (DC) by Leishmania amazonensis causes a significant reduction in DC activation characterized by decreased expression of MHCII and CD86. Interestingly, we demonstrated that infection caused a significant increase in ecto-5′-nucleotidase (CD73) expression by infected DC without affecting bystanders uninfected cells. Finally we demonstrate that inhibition of CD39 activity by suramin or blockage of A2a or A2b receptors were able to partially restore DC activation as measured by MHCII and CD86 expression as well as by their ability to induce T cell proliferation.; In summary, our results show that adenosine production by dendritic cells infected by Leishmania parasites represents another layer of regulation of the immune response during the infection by these protozoans.

Key words: Leishmania, Immunoregulation, adenosine, adenosine receptors.

Action of ATP and analogs on murine hematopoietic cell differentiation

Alice Teixeira Ferreira

UNIFESP, Universidade Federal de São Paulo, Rua Botucatú, 862 São Paulo, SP 04023-062, Brazil

The role of intracellular Ca2+ (Ca2 + i) on hematopoiesis was investigated in long term bone marrow cells (LTBMC) cultures using agonists of P2 receptors (ATP,ADP and UTP). With the images and registers obtained in the Confocal Microscopy with fluo-4 as Ca2+ fluophore, it was observed that ATP, ADP, and UTP promoted a large increase in [Ca2+]i, moderate proliferation (6 times), a reduction in the primitive Gr-1-Mac-1-c-Kit + population(selected through Flow Cytometry), and differentiation into macrophages. Increase in Ca2 + i concentration ([Ca2+]i) consequent the activation of phospholipase C(PLC) beta and gamma by stimulation of 2P receptors triggered the cascate of MEK1/2, and Ca2+/calmodulin kinase II(CaMKII),observed through immunotargeting in Confocal Scanning Micoscopy. Flow cytometry analysis showed, too,the expression of Ca2 + -dependent PKC alpha, beta and gamma in these cells. It is likely that Ca2 + i participates as a regulator of hematopoietic signaling: high increases in [Ca2+]i would be related to macrophage differentiation without maintenance of the primitive population. Participation of different subtypes of Ras, MAPK, PLC-gamma, PLC-beta, PKC,and CaMK proteins was investigated. These results suggest that activation of P2Y subtype receptors triggers two different [Ca2+]iincrase pathways,one inositol-tris-phosphate(IP3)dependent and other kinase-dependet. This cross-talk between Ca2i signaling and Ras-Raf-MEK-MAPK pathways are under investigation in murine and human HSCs. We can state that the activation of P2 receptors increasing [Ca2+]i has an important role in hematopoiesis. Financiamento: FAPESP.

Key words: calcium, cellular differentiation, cellular signaling, hematopoesis, 2P receptors.

PURINERGIC SIGNALLING IN THE NERVOUS SYSTEM

Implications of purinergic receptors in neuronal differentiation

Henning Ulrich

Depto de Bioquímica, Instituto de Química, Universidade de São Paulo, Av. Professor Lineu Prestes, Butantan, São Paulo, S.P. 05508-900, Brazil.

Elevations in cytosolic calcium concentration [Ca²⁺]_i controlled by the activity of metabotropic and ionotropic receptors are crucial for differentiation of stem or progenitor cells into neurons. The outcome of a defined neuronal phenotype depends on the participation of a network of receptors which induce [Ca²⁺]_i transients at defined time points, thereby triggering the transition to the next differentiation stage. We have used the murine embryonal carcimoma P19 cell line as an in vitro model for studying functions of ATP-activated purinergic receptors during early neurogenesis. Following induction of P19 cells to differentiation, the expression of marker proteins specific for embryonic cells decreased, whereas at the same time expression of proteins characteristic for neural progenitor cells and neurons augmented. Purinergic P2Y and P2X receptor expression was modulated during differentiation of P19 cells. In embryonic cells mainly P2Y1 and P2Y2 receptors were responsible for ATP-induced [Ca²⁺]_i transients. In neuronal-differentiated cells, however, P2Y2, P2Y26 and P2X2 receptors were the major mediators of the [Ca²⁺]_i response. In addition to demonstrating participation of P2Y1 and P2Y2 receptors in triggering proliferation of P19 embryonic and progenitor cells, we have collected evidence for the involvement of purinergic receptors in the progress of differentiation, shown by increased expression of neuronal marker proteins of differentiating cells in the presence of ATP. Purinergic P2Y1, P2Y2 and P2X2 receptor activities were also implicated to participate in neuronal maturation, as cholinergic and glutamate-NMDA receptor mediated calcium responses were affected when cells had been differentiated in the presence of purinergic receptor antagonists PPADS, suramin or reactive-blue-2. As overall result, purinergic receptors were involved in all stages of differentiation of embryonic cells into functional neurons. Expression and activities of purinergic receptors were further characterized during in vitro differentiation of neural stem and progenitor cells isolated from fetal rat telencephalon (E 14). These cells proliferated as neurospheres, and their differentiation into neural and glial phenotypes closely resembled events occurring during in vivo development. In conditions of increased neurogenesis, P2X2 and P2X6 receptor subunit expression was significantly enhanced being in agreement with our previous studies in P19 cells. P2X3 and P2X4 receptors had their expression reduced when neurospheres differentiated into neurons, while P2X1 receptors were not expressed. P2X3, P2X4 and P2X5 receptor expression was not differentially regulated during differentiation. Metabotropic P2Y1 and P2Y2 receptors were already present in undifferentiated cells, while P2Y6 receptor expression increased along the differentiation process. Further studies will reveal whether P2 receptor activities are essential for phenotype specification during neurosphere differentiation.

Key words: purinergic receptors, P19 embryonal carcinoma cells, neurogenesis, neural stem and progenitor cells, calcium signaling.

Purinergic modulation of synaptic transmission in the nucleus tractus solitarius of rats

Benedito Machado, Daniela Accorsi-Mendonça

FMRP-USP, Universidade de São Paulo, Av. Bandeirantes, 3900, São Paulo, SP, Brazil

The first synapse of the peripheral chemoreflex afferents is located in the nucleus tractus solitarius (NTS). However, the mechanisms involved in the modulation of the synaptic transmission of this reflex in the NTS are not yet completely understood. Using whole cell patch clamp combined with fluorescence, we analyzed the interaction of ATP and L-glutamate in NTS neurons projecting to rostral ventro lateral medulla (RVLM), since these cells are part in the sympathoexcitatory pathways of chemoreflex. A fluorescent membrane tracer (DiI) was previously microinjected in the RVLM of adult rats in order to identify the neuronal projection from NTS and RVLM. One or two days later transversal brainstem slices were obtained and tractus solitarii evoked excitatory pos-synaptic currents (TS-eEPSCs) were recorded. DNQX, an antagonist of ionotropic non-NMDA glutamatergic receptors, abolished completely the TS-eEPSCs, showing that they are mediated by L-glutamate. To indentify the purinergic receptors activated by endogenous ATP during glutamate release we used different purinergic antagonists. PPADS (100 μM, P2X1/2/3/5/7 receptors antagonist), TNP-ATP (1 μM, P2X1/2/3/4/7 receptors antagonist) and A317491 (100 μM, P2X3 receptor antagonist) decreased the amplitude of TS-eEPSC, indicating a major role of pre-synaptic P2X3 receptor in the synaptic modulation. In order to evaluate the possible role of glia as source of ATP release, we used FAC, an inhibitor of glia metabolism. FAC also decreased the peak amplitude of excitatory events, indicating that glia is involved in this response. To confirm that ATP is released by glia we applied FAC after bath perfusion with PPADS. FAC produced no additional changes in the TS-eEPSCs after PPADS, demonstrating that after the purinergic receptors antagonism the inhibition of glia did not affect the pre-synaptic release of glutamate. These results suggest that during TS stimulation ATP is released by glia and modulates glutamatergic transmission in NTS-RVLM neurons. We also verified that PPADS did not produce any additional effect in the TS-eEPSC after FAC perfusion, indicating that glia is the unique source of endogenous ATP in the NTS. These data show that after TS stimulation glutamatergic vesicles are released onto NTS neurons projecting to RVLM and endogenous ATP, released by glia, play a facilitatory effect via activation of pre-synaptic P2X3 receptor. These findings support the concept that L-glutamate is the excitatory neurotransmitter during evoked neurotransmission in NTS neurons involved with the sympathoexcitatory component of chemoreflex and ATP, released by glia, facilitates this synaptic transmission acting on P2X3 receptor.

Key words: NTS, chemoreflex, RVLM.

Nucleotide signaling and the development of the avian retina.

Ana Lucia Marques Ventura, Isis Moraes Ornelas, Roxana Mamani Anccasi, Erick Correia Loiola, Thayane Martins Silva, Guilherme Rapozeiro França, Mariana Siqueira Oliveira, Karina Lima Tosto

Departamento de Neurobiologia- Instituto de Biologia- Universidade Federal Fluminense, UFF, Niterói- RJ, Brazil.

In the present work we characterized the involvement of nucleotides in the development of the chick retina. ATP is released in a calcium dependent way when glial retinal cells in culture are stimulated by glutamate. Release of ATP induced by glutamate is mediated by both NMDA and non-NMDA ionotropic receptors and seems to occur by exocytosis. Activation of P2Y1 receptors by exogenous ATP or ADP promotes the proliferation of glial/bipolar progenitors through activation of phospholipase C, PKC and ERKs. The proliferative activity of nucleotides also involves activation of PI3K/AKT pathway, trans-activation of growth factor receptors and an increase in the expression of cyclin D1. In contrast to the effect of P2Y1 receptor stimulation, activation of P2X7 receptors by high concentrations of ATP or by the selective agonist Bz-ATP induces cell permeabilization and apoptosis of developing postmitotic neuroblasts in culture. Moreover,besides inducing cell death, activation of P2X7 receptors inhibits the proliferation of late developing progenitors, thus antagonizing the proliferative activity induced by activation of P2Y1 receptors. If retinal cells are incubated with the P2X7 antagonists BBG or oxidized-ATP, P2Y1-induced proliferation can be observed in more differentiated cultures that normally do not proliferate under control conditions. Our results suggest that ATP, by interacting with different receptors, acts as a morphogen during the development of the avian retina. Supported by: CNPq, FAPERJ, CAPES, Proppi-UFF.

Key words: cell proliferation, cell death, developing retina, P2 receptors.

Expression of P2 receptors in the SNC of patients and rats with temporal lobe epilepsy

Maria Jose da Silva Fernandes¹, Ricardo Silva Centeno¹, Carolina Batista Ariza¹, Marimélia Porcionatto¹, Henrique Carrete Jr.¹, Elza Marcia Yacubian¹, Henning Ulrich², Mauro Canzian³, Esper Abrão Cavalheiro¹

1 Unifesp, Universidade Federal de São Paulo, Rua Pedro de Toledo, 781 - 6 andar CEP 04039-032; 2. USP, Universidade de São Paulo, Instituto de Química, Bioquímica, Cidade Universitária; SP; 3. Incor-FCMUSP, Instituto do Coração, Faculdade de Ciencias Médicas USP, Av. Eneas de Carvalho, Incor, USP, São Paulo, SP, Brazil.

Purpose: Although the involvement of P2 receptors remains speculative in neurodegenerative diseases, our previous experimental data suggests participation of P2X receptors in the pathophysiology of pilocarpine-induced temporal lobe epilepsy (Vianna et al, Epilepsia, 43, 227, 2002; Dona et al., Epilepsy Res.,83, 157–167, 2009). In the present work, we have employed immunohistochemistry, Western blot and RT-PCR, to study changes in P2X4, P2X7 and P2Y1 in the hippocampus of rats subjected to pilocarpine model and of patients with temporal lobe epilepsy (TLE) associated with hippocampal sclerosis subjected to surgical treatment for seizures control. Methods: Rats subjected to pilocarpine-induced status epilepticus (SE) were studied during at 60 days after SE. Selected patients with TLE (N = 5, age 42 ± 9) had detailed anamnesis, video EEG and MRI, all procedures approved by the Institutional Ethics Committee of the University (Unifesp). Non-epileptic autopsy tissue served as controls (N = 5). Results: Immunoblots analysis showed up-regulation of all studied purinoceptors in the hippocampus of rats and patients with TLE compared to control. The amount of P2X4, P2X7 and P2Y1 receptor were respectively 10, 3 and 7-fold greater than in control hippocampus (p < 0,0001, Student “t” test). The pattern of immunostaining of purinoceptors in the hippocampus was similar in rats and patients. Intense immunostaining for P2X7 and P2Y1 receptor was detected in neurons and glial cells in the dentate gyrus, hilus, CA1, and CA3. Strong punctate staining for P2X7 was detected in the stratum lucidum of CA3. P2X4-immunoreactive cells were detected in the dentate hilus and CA1. Conclusions: Our data indicate that P2X4, P2X7 and P2Y1 receptors are up-regulated in the hippocampus of rats and patients with TLE and these receptors may play a significant role in neuronal firing as regulators of calcium influx and glutamate release. The role of these purinoceptors in hyperexcitability and neuronal death are in investigation. Financial support: Fapesp-Cinapce, CNPq, CAPES, FAP-Unifesp and INNT-MCT-CNPq.

Key words: epilepsia, hipocampo, purinoceptores, P2X, P2Y.

NEUROTRANSMISSION AND DEVELOPMENT

Adenosine as a signaling molecule in the developing retina

Roberto Paes de Carvalho, M. R. Pereira, A. Santos-Rodrigues , C.C. Portugal , R. Socodato , E. Vardiero , S.A. Rodrigues

Laboratory of Cellular Neurobiology, Program of Neurosciences, UFF, Niterói, RJ, Brazil

Since the original discovery that adenosine promotes cyclic AMP accumulation in the developing retina, a great progress was achieved in the knowledge of functions of this nucleoside in the retina. Here we will review some of these findings, specially those related to the regulation of cell survival, receptor expression and interaction with other neurotransmitter systems. A1 and A2a receptors are expressed in the developing chick retina and both receptors are coupled to cyclic AMP production. Interestingly, recent data show that cell aggregation or activation of A2a receptors regulate the expression of A1 receptors both in vitro and in vivo. While A2a receptors are expressed in neurons as well as glial cells, A1 receptors appear to be expressed only in neurons. However, A1 receptors are present in purified glial cultures and promote the activation of the MAP kinase cascade. On the other hand, stimulation of A2a receptors promotes inhibition of ERK phosphorylation. Adenosine is also a potent neuroprotective molecule in the retina, as cell death induced by glutamate or culture re-feeding is blocked when cultures are pre-incubated for long periods with A2a receptor agonists or cyclic AMP analogs, but not A1 receptor agonists. These events are related to PKA activation and CREB phosphorylation. Recent evidence indicates that adenosine also induces cell death through the activation of A2a receptors in earlier developmental stages but this effect is related to the stimulation of the PLC/PKC pathway and CREB dephosphorylation. These results suggest that adenosine receptors can be coupled to different signaling pathways and playing different functions during development. Glutamate promotes adenosine release from cultured retinal cells by activating NMDA and AMPA/kainate receptors. Ascorbate release is also observed after glutamate receptor stimulation and this effect is partly mediated by adenosine release and A2a receptor stimulation. The ascorbate release is mediated by different signaling pathways and through the specific ascorbate transporter SVCT2. The results indicate that adenosine participates in several events during retina development and can be considered as an important neuromodulator and signaling molecule in the developing retina. Supported by Faperj, Capes, CNPq.

Key words: Adenosine receptors, retina, signaling pathways, cyclic AMP, CREB.

Role of the P2X7 receptor in glioblastoma cell biology and pathology

ASK Tamajusuku¹, ES Villodre¹, R Paulus¹, R Coutinho-Silva⁴, AMO Battastini², MR Wink³, Guido Lenz¹

1.UFRGS, Departamento de Biofísica - UFRGS, Porto Alegre, RS, Brazil 2. UFRGS, Departamento de Bioquímica - UFRGS, Porto Alegre, RS, Brazil; 3.UFCSPA, Departamento de Bioquímica, UFCSPA, Porto Alegre, RS, Brazil; 4. UFRJ, Instituto de Biofísica Carlos Chagas Filho, Rio de Janeiro, RJ, Brazil.

Gliomas have one of the worst prognosis among cancers. Their resistance to cell death induced by endogenous neurotoxic agents, such as extracellular ATP, seems to play an important role in their pathobiology since alterations in the degradation rate of extracellular ATP drastically affects glioma growth in rats. In the present work we characterized the mechanisms of cell death induced by extracellular ATP in a murine glioma cell line, GL261. ATP and BzATP induced cell death at concentrations that are described to activate the P2X7 receptor in mouse while oATP blocked the ATP-induced cell death. A sub-population of cells more sensitive to ATP expressed more P2X7 when compared to a less sensitive subpopulation. Accordingly, RNA interference of the P2X7 receptor drastically reduced ATP-induced cell death, suggesting that this receptor is necessary for this effect. The mechanism of ATP-induced cell death is predominantly necrotic, since cells presented shrinkage accompanied by membrane permeabilization, but not apoptotic, since no phosphatidylserine externalization or caspase activity was observed. Median survival time of glioma patients with high expression of P2X7 is significantly lower when compared to patients with low P2X7 expression in two different sets of patients analyzed at mRNA and protein levels, pointing to the importance of P2X7 in ATP-induced cell death and glioma tumor progression.

Key words: glioblastoma multiforme, cell death, necrosis, glioma, P2X7.

Effects of the intestinal inflammation, obesity and malnutrition on the P2X receptors and enteric nervous system

Patricia Castelucci

Departamento de Anatomia/ICB, USP, São Paulo, SP, Brazil.

Two classes of receptors mediate extracellular effects of adenosine triphosphate (ATP): P2X (ionotropic) and P2Y (metabotropic) receptors. There are seven known P2X receptor subunits (P2X1-7), which can form both homomeric and heteromeric channels, with different pharmacological properties, including different responses to agonists and antagonists, and differences in desensitization properties (Ralevic and Burnstock, 1998). The enteric nervous system contains intrinsic sensory neuron, interneurons and motor neurons, in the guinea-pig small intestine where these have been most studied; there are 14 functionally defined neurons types that form the intrinsic circuits that control motility, transmucosal fluid transport and local blood flow (Furness, 2000). The P2X2 subtype of purine receptor was localized by immunohistochemistry to nerve cells of the myenteric ganglia and submucosal neurons of the stomach, small and large intestine of the guinea-pig. In the all regions of the myenteric neurons, 90% of NOS (nitric oxide sinthase)-immunoreactivity (NOS-IR) neurons were strongly P2X2 receptor immunoreactivity (P2X2-IR), in the ileum 90% were calbindinin-IR were P2X2-IR. In the submucosal ganglia, all vasointestinal peptide-IR were P2X2-IR. These concluded that P2X2 receptor was expressed by specific subtypes of enteric neurons, including inhibitory motor neurons, non-cholinergic secretomotor neurons and intrinsic primary afferent neurons (Castelucci et al., 2002). The distribution of P2X3 receptor showed in myenteric neurons was in calretinin, enkephalin and NOS. In submucosal ganglia, all calretinin-IR nerve cells were P2X3, the P2X3 subunit is not expressed in intrinsic primary neurons, in type the major types of neurons are excitatory and inhibitory muscle motor neurons, ascendening interneurons and cholinergic secretomotor neurons (Poole et al., 2002). Changes in neuronal P2X1-7 expression are not only seen as a result of maturation and neuronal differentiation, but also after various acute insults to the central nervous system, such as ischemia, hypoxia, mechanical stress, axotomy, and inflammation. Purinergic mechanisms are involved in the etiology of many neurodegenerative conditions, due to the large extracellular release of ATP, adenosine, and other neurotransmitters with neural damage. Prolonged stimulation of ATP receptors results in changes in the location and density of P2 receptors at the cell membrane (Franke et al., 2006). Previous studies have reported changes in the expression of P2X2 and 7 purinergic receptors of the myenteric plexus in dietary conditions suggesting that dietary protein low levels affect protein expression and could be a modulator of purinoceptor function (Girotti et al., 2008; Misawa et al., 2010). The ischemia/reperfusion intestinal (I/R-i) affects the expression of the P2X2 and 7 receptors, neuronal density, and size of neurons in the myenteric and submucosal plexus (Palombit et al., 2009; Paulino et al., 2010). In obese female mice group (OFM) Mizuno et al. (2009) have shown that neuronal density values (neuron/cm2) of P2X2 receptor exhibited increase about 62%, while NOS and ChAT neurons had a reduction in 49% and 57%, respectively, in OBF. Also, morphometric studies have demonstrated in OFM group that NOS-ir, ChAT-ir and CalR-ir neurons have been an increase of 34%, 20% and 55% respectively in the cell body profile area, and P2X2-ir neurons have not showed morphological alterations. The distinct expression pattern of the P2X in enteric nervous system has possible therapeutic implications, as activity of specific neurons could be altered pharmacologically to treat disorders of the gastrointestinal tract.

Key words: intestinal inflammation, ischemia/reperfusion, malnutrition, obesity, P2X receptors

YOUNG SCIENTISTS

Pyrimidine coupled P2Y receptors as a new target on Leishmania amazonensis amastigotes elimination

Camila Marques da Silva, Mariana Martins Chaves, Vanessa Figliuolo, Bartira Rossi-Bergman, Robson Coutinho-Silva

Universidade Federal do Rio de Janeiro, Ilha do Fundão, RJ, Brazil.

Recently, the existence of a pyrimidinergic transmission has arisen from the discovery that several G protein-coupled P2Y receptors can be activated also or exclusively by uracil nucleotides and sugar conjugates. Therefore, new unforeseen effects mediated by uracil derivatives have emerged, in particular in the nervous system, and previously unexplored avenues for the pharmacological manipulation of this system are currently under investigation. Such receptors are involved in physiologically relevant actions such as reactive astrogliosis, cell proliferation and differentiation, modulation of microglia activation and of pain signaling. Infections with intracellular parasites elicit diverse responses in the host that include activation of the innate immune system, inflammation and cell death. Data from our group have shown that the P2X7 receptors are up regulated in macrophages infected with protozoan Leishmania amazonensis, if compared with non infected ones. Also, when coupled to ATP, P2X7 receptors promote elimination of intracellular amastigotes, and consequent apoptosis in infected macrophages (Microbes Infect. 11: 842, 2009). In this work we analyzed the role of UTP in control of Leishmania amazonensis infection in vitro (infected macrophages), and in vivo (BALB/c mice paw). Methods: Macrophages were obtained by intraperitoneal lavage of BALB/c mice and platted with 2 × 105 per well. Cells were infected or not with 10:1 (parasites: macrophages) with L.amazonensis promastigotes for 4 h. Non-internalized parasites were washed away with PBS and the cells were cultured for a further 24 h at 37•C in 5% CO2 to allow parasite replication. Cells were treated or not with nucleotides 0,1 mM UTP,0,1 mM UDP or 3 mM ATP for 30 min at 37°C plus 5% CO2. For in vivo experiments, BALB/c mice were infected in the footpad with 2 × 106 L. amazonensis, promastigotes in 20ìl of PBS. After lesion appearance, mice were treated in the infect paws twice a week with 10 mM UTP. After 4 weeks of treatment, the feet were aseptically excised, skinned, and minced. During the treatment, mice were weighted and the lesion measured twice a week. Results: Infected macrophages treated with UTP presented a reduction of 73 ± 1% of parasitic load over non-treated cells. The UTP action started with a reduction of 22 ± 1% within 1 h of treatment reaching a maximum reduction with 6 h after treatment. After 5 h treatment, we observed that UTP induced apoptosis in 20 ± 1% in infected cells, reverted by suramin in 71 ± 14%. UDP was also, capable of inducing apoptosis only in infected cells in 13 ± 1%, partially reverted by suramin in 62 ± 9%. Results confirmed by tunnel and annexin-v staining. Thus, our data suggests that the L.amazonensis, elimination starts to occur independently of hosts apoptosis. One of macrophage arsenals is to kill internalized pathogens is the production of toxic reactive oxygen species (ROS). Nucleotides are linked to ROS production. We observed that UTP induced ROS production only in infected cells. In the in vivo approach, animals treated with UTP, presented a reduction in 44 ± 1% in lesion size and 55 ± 6% in parasitic load in infected mice. We did not observe differences in cellularity and weight of the infected animals treated in comparison to untreated ones. Our results suggest that UTP signaling is an important part on the Leishmania amazonensis control host arsenal. This may indicate that host co-evolved with parasite in a way to survive parasitic infections, and these receptors are possible targets for resolving infections. Grants: CNPq, CAPES, PRONEX, FAPERJ.

Key words: UTP, Infection, apoptosis.

Extracellular nucleotides control IL-8 and MCP-1 secretion in U251MG glioma cell line

Elizandra Braganhol¹, Filip Kukulski², Sébastien A. Lévesque², Michel Fausther², Elise G. Lavoie², Fariborz Bahrami², Fethia Ben Yebdri², Jean Sévigny², Ana Maria Oliveira Battastini¹

¹Departamento de Bioquímica, ICBS, UFRGS, Porto Alegre, RS, Brazil;²Université Laval, Centre de Recherche en Rhumatologie et Immunologie, 2705, Boulevard Laurier, local T1-49, Québec, QC, G1V4G2, Canada.

Chronic inflammation is implicated in the pathogenesis of several types of cancer. For example, cancer cells can recruit inflammatory cells by the release of IL-8 and MCP-1 that stimulate angiogenesis and tumour formation. In addition, ATP accumulates in the tumor interstitium and regulates chemokine release in monocytes. Extracellular nucleotides may therefore participate in gliomagenesis via autocrine induction of chemokine release. Here, we investigated the role of extracellular nucleotides in the release of chemokines from U251MG human glioma cell line that expresses multiple nucleotide P2 receptors and exhibits negligible ecto-ATP/ADPase activities. We found that human U251MG glioma cells have a basal ATP release which could be markedly increased upon stimulation with LPS. We further showed that extracellular nucleotides trigger basal and TLR-induced IL-8 and MCP-1 secretion. Indeed, the administration of apyrase, suramin, the general P2 receptor antagonist, and MRS2578, a specific P2Y6 antagonist, significantly reduced the cytokine secretion either by unstimulated or LPS-treated U251MG cells. The involvement of P2Y6 in the release of IL-8 (basal and LPS-induced) and MCP-1 (basal only) was further confirmed with specific shRNAs, as the cells with knocked down P2Y6 expression produced markedly less IL-8 compared to the control cells. P2Y6 knockdown had no a significant effect on LPS-induced MCP-1 secretion but there was a clear tendency indicating a decrease in this chemokine release in cells treated with specific P2Y6 siRNA. We also found that high milimolar concentrations of exogenous ATP can markedly increase IL-8 and MCP-1 release by glioma cells stimulated with LPS. The effect of ATP was probably mediated by P2X7 receptor as the antagonists of this receptor significantly inhibited this response. Finally, in line with the role of TLRs in tumor progression, we showed that LPS treatment induced glioma cell proliferation which correlated with P2 receptor-dependent cytokine secretion. Altogether, these data suggest that extracellular nucleotides control glioma IL-8 and MCP-1 secretion via distinct P2 receptors and support the hypothesis that nucleotides may modulate angiogenesis and therefore glioma’s growth.

Key words: Gliomas, ATP, P2 receptors, IL-8, MCP-1.

Developmental regulation of neuronal survival by adenosine in thein vitroandin vivoavian retina depends on a shift of signaling pathways leading to CREB phosphorylation or dephosphorylation

Renato Socodato, Rafael Brito, Karin Calaza, Roberto Paes de Carvalho

UFF, Universidade Federal Fluminense, Outeiro São João Batista, s/n, Niterói, RJ, Brazil.

Previous studies have shown a cAMP/PKA-dependent neuroprotective effect of adenosine on glutamate or refeeding-induced apoptosis in chick retina neuronal cultures. In the present work, we have studied the effect of adenosine on the survival of retinal progenitor cells. Cultures obtained from six day-old (E6) or from eight day-old (E8) chick embryos were challenged 2 h (C0) or 1 day (C1) after seeding and analyzed at the fourth day in vitro (C4). Surprisingly, treatment with DPMA (100 nM), a selective A2a adenosine receptor agonist, promoted cell death when added at E6C0 but not at E6C1 or E8C0. DPMA-induced cell death involved activation of A2a receptors and the phospholipase C/PKC but not the cAMP/PKA pathway, and was not correlated with early modulation of precursor cells proliferation. Regarding CREB phosphorylation, cultures from E6 embryos behave in an opposite manner from that from E8 embryos, both in vitro and in vivo. While the phospho-CREB level was high at E6C0 cultures and could be diminished by DPMA, it was lower at E8C0 and could be increased by DPMA. Similar to what was observed in cell survival studies, CREB dephosphorylation induced by DPMA in E6C0 cultures was dependent on the PLC/PKC pathway. Accordingly, cell death induced by DPMA was inhibited by okadaic acid, a PP1 phosphatase blocker. Moreover, DPMA modulates cell survival and CREB phosphorylation in a population of cells in the ganglion cell layer (GCL) in vivo. These data suggest that A2a adenosine receptors and CREB may display a novel and important function by controlling the repertoire of developing retinal neurons.

Key words: retina, A2a receptors, PKC, CREB, development.

P2X2 and P2X7 pore formation is inhibited by colchicine: a possible mechanism for its anti-inflammatory actions

Camila Marques da Silva, Mariana M. Chaves, Robson Coutinho Silva, Marilia Zaluar Passos Guimarães

UFRJ, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil

Introduction. The two longest C-termini of the purinergic P2X receptors belong to P2X2 and P2X7. This region of the channel is thought to Interact with multiple cytoplasmatic proteins, which are important for the function of these channels. Among the proteins are members of the cytoskeleton, including microtubules. In this work we asked whether disrupting the microtubule cytoskeleton might affect these channels functions. Methods. P2X2 and P2X7 were expressed in Xenopus oocytes and HEK293 cells and evaluated with electrophysiology and dye uptake following ATP, after treatment with colchicine. Peritoneal mouse macrophages were plated and assayed for permeabilization and reactive oxygen species (ROS) formation induced by ATP with or without colchicine treatment. Finally, mice were inoculated with LPS and ATP, with or without colchicine, for subsequent ROS and cytokine production measurements. Results. Disrupting the microtubule network did not affect currents generated by ATP in P2X2 and P2X7-expressing oocytes. However, it affected YO-PRO-1 uptake, by inhibiting in approximately 72 and 89% in oocytes expressing P2X2 and P2X7, respectively. In HEK293 cells the same was observed and the kinetics of YO-PRO-1 uptake was reduced by colchicine pre-treatment. Macrophages freshly plated or cultured also showed less ATP-induced permeabilization to ethidium bromide in the presence of colchicine (58% and 39% reduction, respectively). In addition, plated macrophages produced less ATP-evoked ROS when treated with colchicine (55%), but not phorbol ester-induced ROS formation. Finally, in vivo treatment with colchicine diminished ROS, IL1&beta, IL12, INF&gamma and nitric oxide. Discussion. In search for the function of cytoskeletal interactions with P2X receptors, we found out a new tool to study the biophysics of these channels, since colchicine specifically blocked pore formation and not other functions of these channels. Colchicine has known anti-inflammatory actions and is currently used to treat several conditions known to involve P2X receptors, such as gout and familial Mediterranean fever. Here we show that perhaps some of the anti-inflammatory actions of colchicine, microtubule dependent or not, might be due to inhibition of pore formation by P2X receptors.

Key words: permeabilization, electrophysiology, oocytes, P2X, microtubule

POSTER SESSION

01 - ECTO-NUCLEOTIDASES

01-001

Ectonucleotidases activities in serum and cardiac synaptosomes of diabetic rats treated with insulin or subjected to forced swimming

César Eduardo Jacintho Moritz¹, Gustavo de Abreu Vieira¹, Camila Piroli¹, Priscyla Nunes Senna¹, Angela DÁvilla Hartmann¹, Valesca Veiga Cardoso¹, Bárbara Rücker², Cristina Ribas Fürstenau^2,3, Marcia Wink⁴, Emerson André Casali^1,2

¹IPA, Centro Universitário Metodista do IPA, Laboratório de Bioquímica, Centro de Pesquisa e Pós-Graduação;²UFRGS, Departamento de Bioquímica, ICBS;³USP, Laboratório de Biologia Celular e Anatomia Funcional, Instituto de Ciências Biomédicas;⁴UFCSPA, Departamento de Bioquímica, UFCSPA, Rua Sarmento Leite, Brazil.

Introduction: Diabetes is a pathological change characterized by increased serum glucose due to decreased levels and/or effects of insulin. Among its complications are cardiovascular disorders and altered blood homeostasis and purinergic signaling. The adenine nucleotide can to control functions such as heart rate, inflammation, tissue regeneration and platelet aggregation; and are regulated mainly by ectonucleotidases. Objective: The objective of this study was to determine the ectonucleotidases activities in serum and cardiac synaptosomes of diabetic rats treated with insulin or physical training. Methods: Male rats received streptozotocin (60 mg/kg in citrate 0.1 M, ip.) or only vehicle. After 1 week, blood glucose was checked and was considered diabetic who had blood glucose above 250 mg/dL. Insulin treatment was started 30 days after induction of diabetes the animals received insulin (NPH) 2U and 4U in the morning and late afternoon for 6 days while control animals received saline. To evaluate the influence of physical training, the animals were divided into trained diabetic (DT), diabetic sedentary (DS), control trained (CT) and sedentary controls (CS). The training was forced swimming with overload of 5% of body weight for 4 weeks (5 days a week for 60 min). On the last day of training or insulin treatment was measured blood glucose levels and the animals were sacrificed. In both experiments, blood was collected and the hydrolysis of ATP, ADP, AMP and P-TMP determined in serum as previously described (Oses, 2004). The cardiac synaptosomes were obtained and incubated as described by Rücker et al. (2007). Results and Conclusions: We demonstrated a significant increase in the degradation of ATP, ADP and AMP in the serum of diabetic sedentary compared to other groups, the changes were reversed with insulin treatment and training. These results demonstrate the important role of physical training as a factor in restoring blood homeostasis in diabetics. In cardiac synaptosomes was decreased in the hydrolysis of ATP and ADP in diabetic animals and a reversal in the group treated with insulin, leading to changes in the degradation of AMP. Training caused an increase in AMP hydrolysis was not observed in sedentary diabetic. The significance of this enzyme modulation requires more data to be evaluated, but indicate a possible modulatory role of adenosine produced on the diabetic heart.

Key words: ectonucleotidases, diabetes, cardiovascular system, physical training, insulin.

01-002

Study of the ectonucleotidases in medulloblastoma cell lines

Angélica Regina Cappellari¹, Fabrícia Dietrich¹, Elizandra Braganhol¹, Caroline Brunetto De Farias², Ana Lúcia Abujamra², Rafael Roesler², Ana Maria Oliveira Battastini¹.

^1.Departamento de Bioquímica, ICBS, UFRGS;²HCPA, Hospital de Clínicas de Porto Alegre, UFRGS, Porto Alegre, RS, Brazil.

The medulloblastomas are the embryonic neuroepithelial tumors that affecting mainly children with 9 years old. This tumor affects the cerebellum and the median survival of patients is 5 years. ATP is proposed as extracellular signaling molecule and shows many functions in the central nervous system. ENTPDases and ecto-5´-NT are enzymes that catalyze the extracellular nucleotide metabolism and present different expression in brain tumors. Some evidences suggest that these enzymes are involved in the malignance process of these tumors. Considering these data, the objective of the present study was to investigate the involvement of the ENTPDases in medulloblastoma progression. The Daoy, ONS76 and D283 medulloblastoma cell lines were used. All cell lines did not show significant ATPase⁄ADPase activities and only Daoy presented the highest AMPase activity as well as for other monophosphate nucleotides. All lineages showed ATP as main secreted nucleotide. Inorganic pyrophosphate is the main substrate hydrolyzed by inorganic pyrophophatase. Taken together, these results showed that Daoy cell line presented a preference to monophospho nucleotides and ATP is the main nucleotide secreted, suggesting that it is important molecule for medulloblatoma progression, as previously showed by our group for gliomas. As perspectives for this work and confirm the presence of these enzymes in this cells, we will study the expression of ENTPDases and ecto-5´-NT in Daoy, ONS76 and D283 medulloblastoma cell lines and evaluate the phosphodiesterase activity. Financial support: CNPq; FIPE-HCPA.

Key words: medulloblastoma, ectonucleotidases, ENTPDases, ecto-5´-nucleotidase, CD73.

01-003

Ecto-5´-nucleotidase/CD73 inhibition by quercetin in the T24 human bladder cancer cell line

Liliana Rockenbach¹, Luci Bavaresco¹, Patrícia Fernandes Farias¹, Angélica Regina Cappellari¹, Carlos Henrique Barrios², Fernanda Bueno Morrone², Ana Maria Oliveira Battastini¹

¹Departamento de Bioquímica, ICBS, UFRGS;²Faculdade de Farmácia, PUCRS, Porto Alegre-RS, Brazil.

The bladder cancer is the most prevalent tumor in the genitourinary tract and the current treatments are not efficient to prevent recurrence and progression tumor cases. Nucleotides are important molecules that regulate many pathophysiological functions in the extra cellular space. Nucleotide-mediate signaling is controlled by a high efficient action of ectonucleotidases. The ENTPDases hydrolyses the extracellular ATP to ADP, this to AMP and the ecto-5′-nucleotidase hydrolyses the AMP to adenosine. Quercetin is the most flavonoid widespread in the plant kingdom and occurs in apple, onion, garlic, in red wine, grape juice and black tea. Recent studies have demonstrated the antineoplasic potential, elicited by antiproliferative effects, enzymatic inhibition, inhibition of mutant genes and others. Therefore, the aim of the present study was to investigate the effect of quercetin in the ectonucleotidases. The quercetin had antiproliferative effect with 47,65 μM IC50. It was able to cause an increase of ADP hydrolyses and inhibit the ecto-5′-nucleotidase activity both in 24, 48, 72 h and with a quickly treatment of 10 min. The APCP treatment, a classic inhibitor of ecto-5′-nucleotidase, had antiproliferative effect too. Moreover Quercetin caused inhibitory effect in the migration cells. With the flow cytometry analyses was observed that there was no alteration in protein expression on the cell surface. Taken together, these results suggest the participation of purinergic system in the antiproliferative and antimigratory effects of quercetin through the ecto-5′-nucleotidase inhibition. Financial support: CNPq.

Key words: bladder cancer, ectonucleotidases, ecto-5′-nucleotidase, quercetin.

01-004

Effect of inhalatory corticosteroids administration upon nucleotidases activities in blood serum of rats with induced periodontal disease

Vanessa Leal Scarabelot¹, Joanna Ripoll Rozisky¹, Bernardo Detanico¹, Liciane Medeiros¹, Juliano Cavagni^1,3, Isabel Cristina Macedo¹, Andressa de Souza¹, Ana Maria Oliveira Battastini², Cassiano K. Rosing¹, Iraci Lucena da Silva Torres^1,3.

¹Departamento de Farmacologia, ICBS, UFRGS;²Departamento de Bioquímica, ICBS, UFRGS;³Faculdade de Odontologia , UFRGS, Porto Alegre, RS, Brazil.

Corticosteroids are widely used in the treatment of chronic obstructive pulmonary diseases. However, the use of inhaled corticosteroids (IC) aims to direct effects on the lungs, and the association with systemic absorption in the long run may cause decrease of bone density. Alveolar bone loss was identified only after the use of corticosteroids. This relationship with the use of IC has not been sufficiently proven. Previous studies of our group have demonstrated that steroids treatment can produce changes in the extracellular nucleotides hydrolysis (ATP, ADP and AMP). The adenine nucleotides are involved in a number of physiological and pathological processes. The NTPDases are enzymes presents in the interstitial and biological fluids, and hydrolyzes ATP and ADP, while 5′nucleotidase hydrolyses AMP to adenosine. Objectives: the aim of this study was evaluate the effect of IC administration upon nucleotidases activities in blood serum of rats with induced periodontal disease. Materials and Methods: were utilized 30 male Wistar rats (250 ∼ 300 g) treated with IC for 15 days, randomly and divided into four groups: control (C), ligature (L - 0.9% NaCl), Ligature-Budesonide 1 (LB1-30 μg of budesonide) Ligature-Budesonide-2 (LB2 - 100 μg of budesonide). The induction of periodontitis was utilized by the method previously described by Galvão et al. (2003). The rats were killed 24 h after the last administration, and the serum were obtained by centrifuged (5 min, 5000xg). The enzymatic assays were performed by the method described by Oses et al. (2004), and it was utilized Coomassie Blue method for protein measures (Bradford, 1976). The results were expressed as mean ± standard error of mean of nmol of Pi/min/mg of protein, and analyzed by one-way ANOVA followed by multiple comparison test (Bonferroni). Differences were considered statistically significant when P <0.05. Results: The L group showed an increase in ATP hydrolysis (C = 1.045 ± 0.10, L = 2.259 ± 0.48, LB1 = 1.648 ± 0.48, LB2 = 1.385 ± 0.10, ANOVA, P < 0.05) and LB2 showed an increase in the ADP hydrolysis when compared to other groups (C = 1.827 ± 0.15, L = 2.087 ± 0.25, LB1 = 1.542 ± 0.14, LB2 = 4.0 ± 0.44, ANOVA, P < 0.05). Discussion: it is known that tissue injury results in release of pro-nociceptive mediators, including ATP. This may suggest that the increase of ATP hydrolysis observed in this study can be a compensatory effect due to increase of ATP release caused by tissue injury of ligature placement. This differs from the others groups that received corticosteroids and had return of ATP hydrolysis to basal levels. Furthermore, we observed that the treated animals showed increase of ADP hydrolysis. This nucleotide has procoagulant effect, so we can suggest that greater dose of corticosteroids had a protective effect with regard to thromboembolism. Considering that NTPDase1 hydrolyses ATP and ADP equally (1:1), we suggest that this enzyme is involved with different ATP and ADP hydrolysis observed in this study. In conclusion, the chronic treatment with IC at high doses may promote physiological changes significant as evidenced by the alteration of nucleotides hydrolysis in blood serum of rats.

Key words: budesonidae, periodontal disease, nucleotidases.

01-005

Modulation of ecto-ATPase activity by purinergic receptor activation

Marcelle de Carvalho Ribeiro¹, Dilza Balteiro Pereira de Campos¹, Robson Coutinho Silva¹, Celso Caruso Neves^1,2, Ana Acacia de Sá Pinheiro^1,3

¹IBCCF/UFRJ, Instituto de Biofísica Carlos Chagas Filho;²INBEB/INCT, Institutos Nacionais de Ciência e Tecnologia;³INPTAm/INCT, Institutos Nacionais de Ciência e Tecnologia, Rio de Janeiro, Brazil.

OBJETIVE: Nucleotides are released along the nephron to the extracellular milieu and act as an autocrine/paracrine factor through their binding to purinergic/pirimidinergic receptors. Depending on the receptor type, mechanisms such as solute reabsorption, cell proliferation or cell death can be modulated. The extracellular nucleotide concentration is tightly regulated by ecto-enzymes able to hydrolyze these nucleotides and their activity ultimately controls the activation of purinergic receptors. It has been shown that ecto-ATPase activity can be modulated during pathophysiological conditions, but little is known whether these activities could be under purinergic/piriminergic modulation. The aim of this work is to verify if purinergic receptor activation can modulate ecto-ATPase activity in renal proximal tubule, which has been characterized as the nephron segment that presents the most elevated ATP concentrations. METHODS AND RESULTS: LLC-PK1 cells, a well characterized porcine proximal tubule cell line, were grown in DMEM supplemented with 10% FBS, 1% antibiotic/antimycotic at 5% CO₂/37 ⁰ C. After confluence, these cells were starved and treated overnight with different concentration of ATP and ADP. When indicated, agonists and antagonists of P2 receptors were incubated before adding the nucleotides. Ecto-ATPase activity was assayed using a reaction medium containing 20 mM Hepes-tris buffer pH 7,5, 4 mM KCl, 5 mM glucose, 116 mM NaCl, 5 mM MgCl₂ and (&Gamma-32Pi)ATP/ 0,7 mM ATP-Na⁺. The reaction was stopped after 10 min by the addition of charcoal activated in 0.1 N HCl. The amount of Pi in the supernatant was determined by liquid cintilography. It is noteworthy that cells were washed with PBS before ecto-ATPase activity assay; therefore variations can not be due to high substrate availability, since nucleotides are the physiological substrates of these enzymes. Apoptotic cells were detected through Annexin-FITC/propidium iodide assay kit (BD Biosciences, San Diego, CA). We observed that 3 mM ATP is able to induce apoptosis in LLC-PK1 cells (3.82 ± 1.00% of Annexin-FITC positive cells in control group versus 9,99 ± 2,53% in 3 mM ATP-treated cells, p < 0,05). When cells were preincubated with different concentrations of ATP, ecto-ATPase activity was only stimulated when cells were treated with elevated ATP dose (3 mM). Ecto-ATPase activity increased from 17.72 ± 3 to 33.25 ± 7 nmol Pixmg⁻¹xmin-1. (68% of control). The agonist of P2X7 receptor, BzATP enhanced the ecto-ATPase activity likewise. Furthermore, the antagonist oATP per se decreased the ecto ATPase activity in 71% (34.39 ± 4.5 vs 9.81 ± 1.3 nmol Pixmg⁻¹x min-1. Incubation with ADP did not alter ecto-ATPase activity. CONCLUSION: Our results indicated that ecto-ATPase could be modulated by a purinergic receptor, possibly by P2X₇ receptor. Further experiments should be performed to confirm that this receptor is indeed responsible for our observations. We propose a possible molecular mechanism induced by P2 receptors that can decrease extracellular ATP levels in LLC-PK1 cells to rescue them from ATP-induced apoptosis.

Key words: Ecto-nucleotidase, ATP, kidney.

01-007

Caffeic acid alters in vitro the NTPDase and 5´-nucleotidase activities from human platelets.

Javed Anwar, Roselia Maria Spanevello, Vera Maria Morsch, Maria Rosa Chitolina Schetinger

Universidade Federal De Santa Maria, UFSM Av. Roraima Sn Predio 18 SL 2208, Brazil.

The enzymes NTPDase and 5′-nucleotidase are important enzymes involved in the processes of platelets activation and aggregation by controlling the extracellular levels de adenine nucleotides such as ATP, ADP and AMP. Caffeic acid is a phenolic acid with important biological proprieties such as antioxidant, anti-inflammatory and antiagregant. However, the mechanisms involved in beneficial effect of caffeic acid has not yet been fully understood. In this context, the objective of this work was to evaluate the in vitro effects of caffeic acid on NTPDase and 5´-nucleotidase activities in human platelets. Samples of human blood were collected with sodium citrate and platelets were separated. The NTPDase and 5´-nucleotidase activities in platelets were determined according to methods previously described and the solutions of caffeic acid was introduced in the assays in the concentrations of 0; 0.25; 0.5; 0.75 and 1 mM. Data were analysed by one-way ANOVA following by Duncan´s multiple range tests. All data were expressed as mean ± SEM and the enzyme specific activities are reported as nmol Pi released/min/mg of protein. The results showed a significant increase in the NTPDase activity when ATP was used as substrate in the concentrations of 0.75 mM (32.33 ± 5.96, p < 0.05) and 1 mM (33.79 ± 5.96, p < 0.05) of caffeic acid when compared to control group (15.49 ± 0.92). When ADP was used with substrate the NTPDase activity was increased in the concentration of the 0.5 mM (19.44 ± 0.82; p < 0.05); 0.75 mM (19.36 ± 0.82, p < 0.05) and 1 mM (18.08 ± 0.70, p < 0.05) of caffeic acid when compared to the control group (19.44 ± 0.82). In relation the 5´-nucleotidase, the results showed that caffeic acid inhibited the activity of this enzyme only in the concentration of 1 mM (1.68 ± 0.53 p < 0.05) when compared to the control group (2.76 ± 0.12). The alterations in the NTPDase and 5´-nucleotidase activities in platelets by caffeic acid can be important in controlling the platelet coagulant status by removing the extracellular ADP, the main agonist of platelet aggregation.

Key words: NTPDase, 5′-nucleotidase, platelets.

01-008

Effects of red wine on NTPDase and 5′-nucleotidase activities of diabetic rats

Roberta Schmatz, Thaís Rapachi Mann, Jessié Martins Gutierres, Jeandre Augusto Jaques, Victor Camera Pimentel, Daniela Zanini, João Felipe Rezer, Jader Ruchel, Maria Rosa Schetinger, Vera Maria Morsch.

Programa de Pós Graduação em Bioquímica Toxicológica, Departamento de Química, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil.

Introduction: Diabetes mellitus is associated with platelet alterations that may contribute to the development of cardiovascular complications. NTPDase e 5′-nucleotidase enzymes are present in platelet membrane and play an important role in the thromboregulation mechanisms through of extracellular nucleotide adenine hydrolysis. Several studies have shown that moderate consume of red wine is associated with the reduction of cardiovascular diseases. This beneficial effect has been attributed mainly the high concentration of polyphenols present in the red wine, which have antiplatelet and antioxidant properties. Objective: In this context, the aim this study was to evaluate the effects of treatment with red wine on NTPDase e 5′-nucleotidase activities of platelets from diabetic rats. Methods: Adult male Wistar rats (75 days; 200–250 g) were divided into four groups (n = 10): Control/saline; Control/wine; Diabetic/saline; Diabetic/wine. Diabetes was induced by a single intraperitoneal injection of 55 mg/kg streptozotocin (STZ) and after 72 h the animals with fasting glycemia over 250 mg/dL were considered diabetic and used for the present study. The red wine was administered daily, by gavage, during 30 days, at a volume equal 5.71 mL/kg of body weight. After the treatment period, the animals were submitted to euthanasia, and the platelets were prepared and used to determine enzymatic activities (nmol Pi released/min/mg of protein) as described by Lunkes et al. (2004). The statistical analysis used was one-way ANOVA, followed by Duncan’s multiple range tests (p < 0.05). All data were expressed as mean ± S.E.M. Results and Conclusions: The results demonstrated that NTPDase (ATP: 21.84 ± 3.81; ADP: 15.12 ± 2.47) and 5′-nucleotidase (AMP: 7.56 ± 0.98) activities were significantly increased in the diabetic/saline group when compared to control/saline group (ATP: 17.36 ± 1.53; ADP: 9.64 ± 1.28; AMP: 4.57 ± 1.21). The treatment with red wine significantly increased NTPDase (ATP: 29.17 ± 6.24; ADP: 18.49 ± 2.61) and 5′-nucleotidase (AMP: 10.84 ± 2.85) activities in the diabetic/wine group when compared to diabetic/saline group. When resveratrol was administered per se, there was also an increase in NTPDase (ATP: 28.16 ± 4.78; ADP: 16.29 ± 2.82) and 5′-nucleotidase (AMP: 10.49 ± 1.78) activities in the control/wine group when compared to control/saline group. The treatment with red wine increased the activity of NTPDase and 5′-nucleotidase enzymes in both diabetic and control rats. These results suggest that the modulation of NTPDase and 5′nucleotidase activities caused by red wine, may contributes to decrease the levels of extracellular ADP, the main promote of platelet aggregation, and to increase the adenosine production, an important molecule inhibitory of platelet aggregation and vasodilators, and this mechanism can have beneficial effects in the prevention and treatment of cardiovascular complications associated with the diabetic state.

Key words: diabetes, NTPDase, 5′-nucleotidase, red wine, platelets.

01-009

Enzymes that hydrolyze adenine nucleotides in patients with ischemic heart disease

Margarete Dulce Bagatini, Caroline Curry Martins, Andréia Machado Cardoso, Maria Rosa Chitolina Schetinger, Vera Maria Morsch

Programa de Pós Graduação em Bioquímica Toxicológica, Departamento de Química, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, UFSM, RS, Brazil.

Objectives: The aim of the present study was to evaluate activities of enzymes that hydrolyze adenine nucleotides in platelets from patients with ischemic heart disease (IHD). Methods and results: Sixty IHD patients from the Federal University of Santa Maria Hospital were selected for the study. The IHD group was divided in two groups: Acute Coronary Syndrome (ACS - 30 patients) and Stable Angina Pectoris (SAP- 30 patients), characterized by clinical criteria, such as chest pain, associated to clinical evidence of ischemia through a history of coronary arterial disease (CAD), the electrocardiographic alterations and changes in serum concentrations of creatine kinase (CK), creatine kinase MB isoform (CK-MB) and troponins. All subjects gave written informed consent to participate in the study. The Human Ethics Committee of the Health Science Center from the Federal University of Santa Maria, protocol number 0132.0.243.000-08, Brazil, approved the protocol. Ten milliliters of blood was obtained from each patient and used for platelet-rich plasma preparation. The activities of ectonucleoside triphosphate diphosphohydrolase (NTPDase) and ecto-5′-nucleotidase were studied in isolated platelets of these patients, as well as the NTPDase expression. The results shows that ATP and ADP hydrolysis were significantly increased in ACS patients when compared with the control group and SAP patients, P < 0.0001. However, no differences for ATP and ADP hydrolysis were observed when comparing SAP patients and the control group (P = 0.19). AMP hydrolysis was modified by IHD demonstrating a significant increase in AMP hydrolysis in ACS patients when compared with the control group and SAP patients (P < 0.00001). Differences in AMP hydrolysis were also observed in SAP patients when compared with the control group (P < 0.0001). Statistical analysis of the content of CD39 - positive cells by flow cytometry using labeled antibodies against NTPDase revealed that ACS patients had a significant increase in NTPDase expression when compared with SAP patients and the control group, P < 0.0001. No differences were observed between SAP patients and the control group (P = 0.25). Conclusions: Our results suggest that the pathological condition in IHD generates alterations in ectonucleotidase activities as a compensatory organic response to thrombotic events that occur in IHD. Financial support: CNPq and CAPES.

Key words: Ischemic Heart Diseases, platelets, enzymes, nucleotides, ischemia.

01-010

Effects of resveratrol on NTPDase and 5′-nucleotidase activities in synaptosomes from diabetic rats

Jader Betsch Ruchel, Roberta Schmatz, Roselia Spanevello, Thaís Rapachi Mann, Naiara Stefanello , Daniela Zanini, João Felipe Rezer, Maria Rosa Schetinger ,Vera Maria Morsch

Programa de Pós Graduação em Bioquímica Toxicológica, Departamento de Química, Centro de Ciências Naturais e Exatas, UFSM, Santa Maria, RS, Brazil.

Objective: Diabetes mellitus is associated with neurochemical, neurophysiological, and vascular modifications and in the brain impairing its functional and structural integrity. NTPDase and 5′-nucleotidase participate in the control of extracellular ATP and adenosine levels in the synaptic cleft and in the control of purinergic neuromodulation and purinergic neurotransmission, play an important role in the functioning of the central nervous system. In addition, the compound resveratrol (RSV), a polyphenol found in grapes and red wine has been proposed to have neuroprotective effects. Objective: In this context, the aim this of study was to investigate the effects of treatment with RSV on NTPDase e 5′-nucleotidase activities in cerebral cortex synaptosomes from diabetic rats. Methods: Adult male Wistar rats (75 days; 200–250 g) were divided into four groups (n = 10): Control/saline; Control/RSV 10 mg/Kg; Diabetic/saline; Diabetic/ RSV 10 mg/Kg. Diabetes was induced by a single intraperitoneal injection of 55 mg/kg streptozotocin (STZ) and after 72 h the animals with fasting glycemia over 250 mg/dL were considered diabetic and used for the present study. The RSV was administered intraperitoneally, during 30 days. After the treatment period, the animals were submitted to euthanasia and the cerebral cortex was removed for synaptosomes preparation and enzymatic assays of NTPDase and 5′-nucleotidase (nmol Pi released/min/mg of protein) as described by Schetinger et al. (2000). The statistical analysis used was one-way ANOVA, followed by Duncan’s multiple range tests (p < 0.05). All data were expressed as mean ± S.E.M. Results and Conclusions: The results demonstrated that NTPDase and 5′-nucleotidase activities were significantly increased in the diabetic/saline group (ATP: 316.68 ± 6.37; ADP: 182.51 ± 4.38; AMP: 83.51 ± 4.38) compared to control/saline group (ATP: 269.66 ± 3.98; ADP: 165.55 ± 1.72; AMP: 70. 93 ± 1.90). Treatment with RSV significantly increased NTPDase and 5′-nucleotidase activities in the diabetic/RSV10 group (ATP: 378.42 ± 6.46; ADP: 241 ± 3.16; AMP: 139.27 ± 3.16) compared to diabetic/saline group (ATP: 316.68 ± 6.37; ADP: 182.51 ± 4.38; AMP: 83.51 ± 4.38). When resveratrol was administered per se there was also an increase in the activities of these enzymes in the control/RSV10 group (ATP: 357.58 ± 5.14; ADP: 213.61 ± 3.4; AMP: 100.35 ± 1.68) compared to control/saline group (ATP: 269.66 ± 3.98; ADP: 165.55 ± 1.72; AMP: 70. 93 ± 1.90). The treatment with RSV increased NTPDase and 5′-nucleotidase activities in the synaptic cleft of diabetic rats. These results suggest that the modulation of NTPDase and 5′nucleotidase activities caused by resveratrol may be very important because the increase in ATP, ADP and AMP hydrolysis may contributes to enhance in adenosine production, an important neuroprotective molecule that may protects the CNS from neurological complications caused by the diabetic state.

Key words: diabetes mellitus, NTPDase, 5′-nucleotidase, resveratrol, sinaptosomes

01-011

Activity of enzymes that hydrolyze adenine nucleotides in cerebral cortex synaptosomes of rats exposed to cadmium and treated with curcumin

Jamile Fabbrin Gonçalves¹, Amanda Maino Fiorenza², Fátima Husein Abdalla², Roberta Schmatz², Margarete Dulce Bagatini², Victor Camera Pimentel², Jessié Martins Gutierres², Juliano Marchi Vieira², Vera Maria Morsch², Maria Rosa Chitolina Schetinger^2,1.

¹UFRGS, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS²UFSM, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil.

Cadmium (Cd) is a ubiquitous environmental toxicant, which adversely affects biological systems. Recent reports have shown the impairment of Cd on synaptic neurotransmission and neurotransmitter and antioxidant levels in animal brain. Antioxidants and plant phenolics are becoming increasingly popular in oxidative stress-related disorders and hold promise as therapeutic agents. Curcumin is a hydrophobic polyphenol which exhibit wide variety of biological and pharmacological activities namely antioxidant, anti-inflammatory, antimicrobial, anticarcinogenic and neuroprotective activities. Therefore, the Cd toxicity is associated with neurobehavioral disturbances, the aim of this study was to investigate the effect of curcumin on ectonucleotidases NTPDase and 5′-nucleotidase activities in cerebral cortex synaptosomes (CCS) of Cd-exposed rats. Adult male Wistar rats (80 days; 328 ± 12 g) from the Central Animal House of the Federal University of Santa Maria (UFSM) were used in this experiment. The animals were maintained at a constant temperature (23 ± 1°C) on a 12 h light/dark cycle with free access to food and water. In the present study the rats received cadmium as CdCl2.H2O (Cd; 1 mg/kg) and curcumin (30 mg/kg) by gavage 5 days per week during 3 months. The animals were randomly divided into four groups (n = 5–7 per group): Control (saline/oil), Curcumin, Cd and Cd/Curcumin. The last group received Curc 30 min after Cd. The solutions were freshly prepared in saline or corn oil and were administered (1 mL/kg) between 9 and 11 a.m. At the end of the treatment, the animals were anesthetized and submitted to euthanasia. The cranium was opened and the cerebral cortex was gently removed for synaptosomes preparation and enzymatic assays. The synaptosomes were isolated as described by Nagy and Delgado-Escueta (J. Neurochem. 43:1114, 1984). The activity of NTPDase and 5′-nucleotidase was performed as determined by Schetinger et al. (Neurochem. Res. 25:949, 2000) and Heymann et al. (J. Neurochem. 43:971, 1984), respectively. The data evaluated were analyzed by two-way ANOVA, followed by Duncan’s multiple range tests. A significant Cd × Curc interaction for NTPDase activity of CCS was observed (F1,19 = 5.989; p < 0.05) when ATP was used as substrate. Post hoc comparisons demonstrate that rats exposed to Cd presented an increase (22.7%) in NTPDase activity (substrate ATP) of CCS. A significant Cd × Curc interaction for NTPDase activity of CCS was observed (F1,20 = 11.609; p < 0.01) when ADP was used as substrate. Post hoc comparisons demonstrate that rats exposed to Cd presented an increase (117.2%) in NTPDase activity (substrate ADP) of CCS. The increase in NTPDase activity (ATP or ADP as substrate) induced by Cd was abolished by curcumin administration. A significant main effect of Cd (p < 0.05) and Curc (p < 0.05) in CCS 5′-nucleotidase activity was observed when AMP was used as substrate. Results demostrate that Cd exposure caused a decrease (38.9%) in 5´-nucleotidase activity of CCS. Curcumin treatment showed a tendency to restore the decreased 5′-nucleotidase activity induced by Cd. The exposure to Cd resulted in enhancement of ATP and ADP hydrolysis associated with a decreased hydrolysis of AMP suggesting that the production of adenosine could be reduced. The adenosine is an important molecule in neuromodulation, homeostatic regulation and neuroprotection of the central nervous system. In conclusion, the curcumin interferes with the purinergic neurotransmission by altering NTPDase and 5′-nucleotidase activities in CCS of Cd-intoxicated rats. In this context, we can suggest that curcumin is a promising drug, which should be investigated in future, studies in order to improve therapeutic alternatives for brain injury associated with Cd-induced neurotoxicity.

Key words: adenine, synaptosomes, cadmium, curcumin

01-012

Aqueous stem bark extract ofScutia buxifolia reissekinhibits NTPDase and 5′-nucleotidase activity in platelets from rats.

Victor Camera Pimentel, Roberta Schmatz, Margarete Bagatini, Daniela Zanini, Jessié Martins Gutierres, Jeandre Augusto Jaques, Aline Boligon, Margareth Linde Athayde, Vera Maria Morsch, Maria Rosa Chitolina Schetinger.

UFSM, Universidade Federal de Santa Maria, Av. Roraima N:1000, Santa Maria, RS, Brazil.

Introduction: The World Health Organization (WHO) estimates that 65–80% of the population of the developing countries depends on medicinal plants for basic pharmaceutical care. Scutia buxifolia Reissek belongs to the Rhamnaceae family and is popularly know as “coronilha”. It is a native plant from South America, with a dispersion area that includes the Rio Grande do Sul state, Brazil. Infusions of the root bark are popularly used as a cardiotonic, antihypertensive and diuretic. The nucleotides are messengers that modulate the exocrine and endocrine systems, the vasculature and hemostatic mechanisms and musculoskeletal, immune and inflammatory cells. Their levels are controlled by a complex cell surface-located group of enzymes called ectonucleotidases, anchored in the plasmatic membrane of platelets. The cascade of ecto-enzymes, with this function, is formed by an NTPDase that hydrolyzes ATP and ADP to AMP and by a 5′-nucleotidase that hydrolyzes AMP to the end product, adenosine. Therefore, the aim of this study was to investigate the effect of aqueous stem bark extract of S. buxifolia on enzymes that hydrolyze adenine nucleotides in platelets from rats. Methods: Adult male Wistar rats (200–250 g) obtained from the Central Animal Facility from the Federal University of Santa Maria, RS, Brazil, were utilized from experiments. Rats were euthanized and the platelets were prepared for the NTPDase and 5´-nucleotidase activities assays (nmol Pi/min/mg of protein) according to (Diabetes Res. Clin. Pract. 65:1, 2004). Twenty microliters of enzyme preparation (8–12 μg of protein) were added to the reaction mixture and pre-incubated at 370 C for 10 min with extract of S. buxifolia at concentrations of 0.001, 0.01, 0.05, 0.1 and 0.2 μg/mL. The reaction was initiated by the addition of ATP, ADP or AMP and incubation proceeded for 60 min. The results were analyzed by one-way ANOVA, followed by Duncan’s multiple range test. P < 0.05 was considered to represent a significant difference in the analyses used. Results: The results demonstrated that ATP hydrolysis was inhibited in vitro by extract of stem bark, at final concentrations of 10, 50, 100 and 200 μg/mL (P < 0.001), when compared to control enzyme activity. Hydrolysis of ADP, in the same type of experiment, presented a similar profile when compared to ATP, although when ADP was utilized as substrate the inhibitory effect was observed at final concentrations of 1, 10, 50, 100 and 200 μg/mL. The inhibition of 5′-nucleotidase was showed only with stem bark extract in concentration of 100 and 200 μg/mL (P < 0.05). Conclusion: We observed a significant in vitro inhibition in the NTPDase and 5´-nucleotidase activities from platelets when incubated with the stem bark extract of S. buxifolia. This study suggests that alterations in the ectonucleotidases activities by aqueous stem bark extract of S. buxifolia may contribute to the understanding of the beneficial effect of this plant described in the literature. Acknowledgments: CAPES, FAPERGS.

Key words: NTPDase, Platelets, Scutia buxifolia, 5′-nucleotidase.

01-013

Nicotine changes NTPDase and 5′-nucleotidase in rat hippocampus synaptossomes

Gustavo Roberto Thomé¹, João Felipe Peres Rezer¹, Jonas Serres¹, Nicéia Calgaroto¹, Jucimara Baldissarelli¹, Victor Pimentel¹, Cinthia Melazzo Mazzanti², Vera Maria Morsch¹, Maria Rosa Schetinger¹.

¹Program of Postgraduate in Biochemistry and Toxicology, CCNE, UFSM, Santa Maria, RS, Brazil;²Department of Small Animal Clinic, CCR, UFSM, RS, Brazil.

Introduction: Nicotine is the primary alkaloid found in tobacco and promotes numerous effects on the human body as a reduction of cell growth in cell culture and stimulation of the central nervous system. Objectives: The study was conducted to evaluate the enzyme activity of purinergic system (NTPDase and 5′-nucleotidase) in synaptosomes from hippocampus of rats subjected to treatment with nicotine. Methods: The synaptosomes were prepared from hippocampus of rats (n = 6) according to the method of Nagy et al (1984), using discontinuous percoll gradient, and NTPDase activity was determined according to Schetinger et al (2000) and 5′-nucleotidase according to Heymann et al (1984). The enzyme activities are expressed in nmol Pi/min/mg protein. The nicotine concentrations used in the treatment (sc) were: control (saline) 0.0, 0.25, 0.5, 1.0 and 2.0 mg/kg/day for a period of 10 days. Results: The activity of NTPDase (ATP as substrate) was changed (Figure 1A) in synaptosomes from rat hippocampus and post hoc comparisons by Duncan’s test revealed that was significantly decreased in all nicotine concentrations studied when compared with control group (p < 0.05). For NTPDase activity (ADP as substrate) (Figure 1B) significantly was no change when compared with the control group (p < 0.05). However, 5’-nucleotidase activity (Figure 1 C), AMP as substrate, the post hoc comparisons by Duncan’s test revealed that was significantly increased at concentrations of nicotine 1.0 and 2.0 mg/kg/day when compared with the control group (p <0.05). Conclusion: We can suggest that nicotine alters the activity of the enzymes NTPDase and 5′-nucleotidase, which affects the functionality of purinergic system in the brain of rats as a result of exposure. Moreover, the purinergic system may be promoting an increase of neuroprotective adenosine nucleoside to combat the neurotoxic effects caused by nicotine. Financial Support: UFSM, CNPq e CAPES.

Key words: ectonucleotidases, NTPDase, 5′-nucleotidase, nicotine, synaptosomes.

01-014

L-NAME-treatment alters ectonucleotidase activities in kidney membranes of rats

Cristina Ribas Fürstenau¹, Denise Barbosa Ramos¹, Fernanda Cenci Vuaden¹, Emerson André Casali², Priscilla de Souza Monteiro³, Danielle da Silva Trentin¹, Agnes Nogueira Gossenheimer¹, Maurício Reis Bogo⁴, Carla Denise Bonan³, Maria Luiza Morais Barreto-Chaves⁵, João José Freitas Sarkis¹, Susana Tchernin Wofchuk¹.

¹Departamento de Bioquímica, ICBS, UFRGS;²IPA, Centro Universitário Metodista do Sul – IPA, Porto Alegre, RS, Brazil;³PUCRS, Pontífica Universidade Católica do Rio Grande do Sul, Lab Neuroq e Psicofarmacol;⁴PUCRS, Pontifícia Universidade Católica do Rio Grande do Sul, Lab Biol e Genôm Mol, Porto Alegre, RS, Brazil;⁵USP, Universidade de São Paulo, Depto de Anatomia São Paulo, SP, Brazil.

Background: The kidneys serve to maintain fluid and electrolyte homeostasis by adapting renal excretion to bodily needs. Extracellular nucleotides (ATP, ADP, UTP and UDP) as well as the nucleoside adenosine are widely accepted as autocrine or paracrine signaling agents in different tissues, including the kidneys. The breakdown of these molecules involves several ectoenzymes called ectonucleotidases, which influence the extent of purinergic receptors activation. In the kidney, ATP and adenosine are known to participate in the regulation of renal microcirculation, renin secretion, and tubular function. Aims: To investigate the effect of N&omega-Nitro-L-arginine methyl ester (L-NAME) treatment, known to induce a sustained elevation of blood pressure, on ectonucleotidase activities in kidney membranes of rats. Main methods: L-NAME (30 mg/kg/day) was administered to Wistar rats for 14 days in the drinking water. Enzyme activities were determined colorimetrically and their gene expression patterns were analyzed by semi-quantitative RT-PCR. The metabolism of ATP and the accumulation of adenosine were evaluated by HPLC in kidney membranes from control and hypertensive rats. PKC phosphorylation state was investigated by Western blot. Key findings: We observed an increase in systolic blood pressure from 115 ± 12 mmHg (control group) to 152 ± 18 mmHg (L-NAME-treated group). Furthermore, the hydrolysis of ATP, ADP, AMP, and p-Nph-5′TMP was also increased (17%, 35%, 27%, 20%, respectively) as was the gene expression of NTPDase2, NTPDase 3 and NPP3 in kidneys of hypertensive animals. Phospho-PKC was increased in hypertensive rats. Significance: The general increase in ATP hydrolysis and in ecto-5′-nucleotidase activity suggests a rise in renal adenosine levels and in renal autoregulatory responses in order to protect the kidney against the threat presented by hypertension. Supported by CNPq-Brazil, FINEP research grant (IBN-Net) # 01.06.0842-00, INCTen (CNPq-FINEP).

Key words: ecto-nucleotidase, hypertension, ATP, adenosine, kidney.

01-016

Effect of morphine exposure upon nucleotide hydrolysis in blood serum of rats.

Isabel Cristina de Macedo¹, Joanna Ripoll Rozisky^1,2, Bernardo Carraro Detanico¹, Liciane Fernandes Medeiros¹, Vinicius Souza dos Santos¹, Ana Maria Oliveira Battastini³, Iraci Lucena da Silva Torres^1,2,4

¹Departamento de Farmacologia, ICBS, UFRGS;²PPGCM-UFRGS, Programa de Pós-Graduação em Medicina, UFRGS;³Departamento de Bioquímica, ICBS, UFRGS;⁴HCPA-POA, Unidade de Experimentação Animal do HCPA,Porto Alegre, RS, Brazil.

Objectives: Studies have shown that exposure to drugs in early life can have implications for the developing of physiologic systems in adult life. It has been proposed that adenosine is involved in opioid antinociception, and ATP is involved in nociception. The NTPDase family that hydrolyzes ATP and ADP to AMP and the 5′-nucleotidase that breakdown AMP to adenosine are co-expressed on endothelial and hematopoietic cells and are the major regulators of purinergic signaling in the blood. The aim of this study was to evaluate the long-term effect of morphine exposure upon nucleotides hydrolysis in blood serum of rats. Methods and Results: Were utilized 8-day-old male Wistar rats at the beginning of the experiment, which were divided into two groups: control (C) and morphine (M), which received saline (0.9% NaCl) or morphine (5 μg s.c. in the mid-scapular area) at postnatal day 8 (P8), once a day for 7 days. At P80 the groups were divided into 4 groups which received saline (0.9% NaCl) [Control-saline (CS, n = 6), Morphine-saline (MS, n = 6)] or morphine (5 mg/kg i.p.) [Control-morphine (CM, n = 6), Morphine-morphine (MM, n = 6)] at P80 until P86, once a day. At P88 the rats were killed and the serum samples were obtained by centrifuged (5 min at 5000 x g). The enzymatic assays were performed by the method described by Oses and colleagues (Life Sci. 74:3275, 2004). The protein concentration was measured by the Coomassie Blue method using bovine albumin as standard (Anal. Biochem. 72: 248, 1976). Enzyme activities were expressed as nmolPi/min/mg protein. Statistical analyses were performed by one-way ANOVA followed by a multiple comparisons test (Bonferroni) when indicated and data was expressed as mean + standard error of the mean. Differences were considered statistically significant if P < 0.05. The groups did not show difference amongst themselves in ATP and ADP hydrolysis, but morphine-morphine group presented a decrease of AMP hydrolysis in comparison to other groups (ATP: CS = 0.91 + 0.14, CM = 0.78 + 0.12, MS = 0.9 + 0.06, MM = 0.73 + 0.09; ADP: CS = 0.89 + 0.16, CM = 0.92 + 0.12, MS = 0.73 + 0.13, MM = 0.81 + 0.11, one-way ANOVA, P > 0.05; AMP: CS = 1.04 + 0.11, CM = 0.9 + 0.12, MS = 0.67 + 0.07, MM = 0.49 + 0.14, one-way ANOVA, P < 0.05). Conclusions: In this study we observed that sustained morphine exposure in early life and adult life can result in decrease of AMP hydrolysis in blood serum, and probable decrease of adenosine. Previous study has demonstrated that adenosine attenuates the morphine withdrawal symptoms, and leads adaptative changes in adenosinergic system. Moreover, our group has demonstrated that these exposures are capable to alter the opioid response in adult life. Therefore, our findings indicate that two treatments promote adaptations in opioid and purinergic system. Thus, further studies are required to elucidate the mechanism of these changes involving the adenosinergic system after repeated opioid exposure. Financial support: CNPq, FIPE-HCPA.

Key words: morphine, nucleotide hydrolysis, opioid antinoception, NTPDase.

01-017

HPLC method for the quantification of adenine and guanine based purines.

Ana Elisa Böhmer, Gisele Hansel, Denise Barbosa Ramos, Luis Valmor Portela, Diogo Onofre Souza.

Departamento de Bioquímica ICBS, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.

Purines act as important signaling molecules that induce a multiplicity of effects in various biological systems and play important roles in physiological and pathological conditions. The purpose of this study was to improve and validate a simple, rapid and sensitive high performance liquid chromatography (HPLC) method to analyze all adenine- and guanine-based purines at CSF and plasma of human, mice and rat samples. HPLC was performed to measure ATP, ADP, AMP, adenosine, GTP, GDP, GMP, guanosine, IMP, inosine, hypoxanthine, xanthine and uric acid. Two phosphate buffers pH 6.0 were used, one being with acetonitrila 15%. The mobile phase flowed at a rate of 1.2 mL/min during 17 min and the C18 column temperature was 22 °C. An UV detector (254 nm) was used. The analytical method was validated through the analysis of accuracy, precision, limit quantification, linearity, and stability. Standard and samples remained stable for up to 6 months at -20 °C. Linearity range was observed: from 0.5 to 2000 nmol for AMP, GTP, GDP, GMP and IMP; from 0.5 to 100 nmol for UA; from 1 to 2000 nmol for ATP, GUO, INO, ADO, HIPOX, XANT; and from 5 to 2000 nmol for ADP. The precision (0.05% to 3.9% for intra-day and 1.39% to 4.92% for inter-day) and accuracy test (89.3% to 105.2% for intra-day and 86.1% to 111.8% for inter-day) suggest that the applied method have high precision and accuracy for all analyzed purines. This method proved to be simple, rapid, stable, sensitive, specific, and accurate, and allows measuring all these purines in a single running. The usefulness of this method is demonstrated by successful application for human, mouse and rat CSF and plasma samples.

Key words: purines, HPLC, adenine, guanine, quantification.

01-018

Overexpression of CD39L1/NTPDase2 in gliomas promotes platelet activation and metastasis

Elizandra Braganhol¹, Rafael Fernandes Zanin¹, Andressa Bernardi¹, Leticia Scussel Bergamin¹, Angelica Regina Capellari¹, Luis Felipe Campesato¹, Fernanda Morrone², Maria M Campos², João Calixto⁶, Maria Isabel Edelweiss⁵, Marcia Wink³, Simon Robson⁴, Ana Maria Oliveira Battastini¹

¹Departamento de Bioquímica, ICBS, UFRGS, Porto Alegre, RS, Brazil;²PUCRS, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, RS, Brazil;³UFCSPA, Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre, RS, Brazil;⁴Harvard University, Harvard University – USA;⁵HCPA, Hospital de Clinicas de Porto Alegre, Porto Alegre, RS, Brazil;⁶UFSC, Universidade Federal de Santa Catarina, Campus Universitário Reitor João David Ferreira Lima - Trindade – Florianópolis, SC, Brazil

Gliomas are the most common and devastating type of primary brain tumor. Many non-neoplastic cells, such as immune cells comprise the tumor microenvironment and create an inflammatory milieu fundamental for cancer development. Disruption of purinergic signalling has been implicated in cancer. ATP and its breakdown products ADP and adenosine display cytokine-like properties and may participate in the interactions among cancer and immune cells. The nucleotide receptor-mediated cell communication is controlled by ectonucleotidases, such as ectonucleoside triphosphate diphosphohydrolases (E-NTPDases), which hydrolyze extracellular nucleotides. We have shown that, in opposite to astrocytes, gliomas, exhibit a low E-NTPDase activity; ATP induces glioma cell proliferation and the co-injection of apyrase/NTPDase1 with gliomas decreases glioma progression in vivo. However, recently we have shown that the NTPDase2 restoration to gliomas dramatically increased tumor growth in vivo. The contradictory results obtained using different ATP scavengers suggest those interactions between tumor, immune cells and purines as key elements in the anti- or pro-tumor responses. Here we evaluated whether the NTPDase2 restoration to gliomas could modulate the systemic inflammatory response and the distant metastasis. NTPDase2 overexpression increased the in vitro C6 glioma cell adhesion properties, modulated the pro-inflammatory cytokine production and the platelet reactivity in vivo. Alterations on lung tissue suggestive of glioma metastasis were also observed. These results suggest that disruption of purinergic signaling creates an inflammatory microenvironment that modulates tumor cell migration and malignity in vivo. Financial support: CNPQ; FIPE-HCPA.

Key words: ATP, E-NTPDases, Gliomas, Inflammation, Lung.

01-019

Assessing levels of purinergic compounds in serum of patients with multiple sclerosis

C.E.J. Moritz¹, R. Spanevello², M.R.C Schetinger³, C. Mazzanti³, R. Schmatz³, M. Bagatini³, N. Stefanello³, V. Morsch³, E.A Casali^1,2

¹Laboratório de Bioquímica, Centro Universitário Metodista do IPA, Porto Alegr,RS²Departamento de Bioquímica da UFRGS, Porto Alegre/RS,³Departamento de Química, UFSM, Santa Maria, RS, Brazil.

Objective: The aim of this study was determinate the levels of purines in the serum of patients with multiple sclerosis. Methods and Results: Were selected 10 patients diagnosed with multiple sclerosis and 10 healthy controls. All procedures were approved by Ethics in Research of University of Santa Maria (23081.007854/2007-44) and the free consent was obtained from all individuals. Whole blood samples were collected by venipuncture and later centrifuged to obtain serum. The samples of plasma protein were precipitated with 0,6 N perchloric acid followed by centrifugation at 16.000gX10min., the supernatant was neutralized with KOH followed by new centrifugation at 16.000gX10min. The samples levels of ATP, ADP, AMP, adenosine and inosine were determined by HPLC. Results were expressed as nmol compound/ ml of serum. The results showed a reduction in the levels of nucleotides ATP (4,24 ± 0,62), ADP (4,4 ± 0,88) and AMP (3,26 ± 0,67), in patients with multiple sclerosis, when compared to control individuals, ATP (6,66 ± 1,12), ADP (2,74 ± 0,75) and AMP (2,32 ± 0,47). Despite reduction in levels nucleotides, patients with multiple sclerosis showed increase in concentration of nucleoside adenosine 12,34 ± 1,63) and inosine (3,52 ± 0,88), when compared to control group (9,89 ± 0,94) e (2,62 ± 0,41), respectively. Conclusion: These findings indicate that patients with multiple sclerosis has a decrease in the levels of ATP, a molecule pro-inflammatory action and increased adenosine and inosine levels, anti-inflammatory and immunosuppressive compounds. These changes in the levels of nucleotides and nucleosides in serum may represent a compensatory response to minimize the immune and inflammatory process characteristic of disease.

Key words: Ectonucleotidases, Multiple Sclerosis, Extracellular Purines, HPLC analysis.

01-020

Effects of restraint stress upon temporal patterns of adenine nucleotides hydrolysis in rat blood serum.

Andressa de Souza², Bernardo Carraro Detânico², Liciane Fernandes Medeiros², Joanna Ripoll Rozisky², Wolnei Caumo², Ana Maria Oliveira Batastini¹, Iraci Lucena da Silva Torres²

¹Departamento de Bioquímica, ICBS, UFRGS;²HCPA, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil.

Objectives: Adenosine 5′-triphosphate (ATP) and its breakdown products, ADP, AMP and adenosine can act as extracellular messenger in a range of biological processes through binding to the purinergic receptors, and are involved in a variety of pathological conditions including cerebral ischemia, neuroinflammatory, neuropsychiatric, neurodegenerative and cardiovascular diseases. Extracellular adenine nucleotides are metabolized to adenosine by a number of enzymes including NTPDases and ecto-5´-nucleotidase that are considered to be the major regulators of purinergic signaling in the blood. These enzymes may also have a protective function by keeping extracellular ATP/ADP and adenosine levels within physiological conditions. Previous works of our group demonstrate that ATPase and ADPase activities exhibit a 24-hour temporal pattern in blood serum rat. Circadian rhythms are present in a large number of organisms, representing an important mechanism for preparing the organism to environmental changes. Alterations of enzyme activities involved in nucleotide hydrolysis have also been reported after repeated and acute restraint stress. Moreover, it was demonstrated that stress could cause disruptions in biological circadian rhythms in humans and in rodents. Therefore, the aim of the present study was to examine the influence of acute restraint stress exposure upon temporal patterns in NTPDase and 5′-nucleotidase enzymes activities in rat blood serum. Methods: Adult male Wistar rats the animals were divided into 4 groups according to daytime (ZT 0, ZT 6, ZT 12 and ZT 18) and each of these was subdivided in 4 groups according to time of death (control group, 0 h, 6 h and 24 h after acute stress exposure). It was observed significant differences on effect of stress on 24-h profile on ATPase, ADPase and AMPase activities (n = 7–11, One way ANOVA, P < 0.001 for all activities). The control groups showed significant higher ATPase (82.1%) and ADPase (64.0%) activities at ZT 12 and 18 when compared with control group. All stressed groups showed significant decrease in all enzymatic activities at ZT 12 and ZT 18 when compared with control group. Conclusion: In conclusion, the activities of nucleotidase enzymes suffer a higher influence during night hours than at daylight hours by acute stress and this influence seems to persist at least 24 h. The findings of this work suggest that stress can deregulate the circadian timing presents in nucleotidase enzymes. It may be proposed that altered levels of nucleotides in serum can be involved in cardiovascular events more frequently during the day in humans, and with its etiology induced by stress. Financial Support: FIPE-HCPA; CNPq.

Key words: stress, cronobiology, nucleotidases.

01-021

Enzymes that hydrolyze adenine nucleotides in patients with ischemic heart disease

Vera Maria Morsch, Margarete Bagatini, Caroline Martins, Maria Rosa Schetinger

UFSM, UNIVERSIDADE FEDERAL DE SANTA MARIA, Campus Universitário, Camobi, Depto. Química, Santa Maria, RS, Brazil.

Ischemic heart disease (IHD) is an increasingly common cause of death in the world. This disease affects millions of people annually and results in morbidity and mortality. In most cases, ischemia occurs when an atherosclerotic plaque fissures, ruptures or ulcerates and when conditions favor thrombogenesis, so that a mural thrombus forms at the site of rupture and leads to coronary artery occlusion.Platelets are one the most important blood components that participate in and regulate thrombus formation by releasing active substances such as ADP. It is known that micromolar concentrations of ADP are sufficient to induce human platelet aggregation, whereas adenosine (the final product of ATP hydrolysis) can inhibit platelet aggregation. On the other hand, ATP, in low concentrations, enhances collagen, thromboxane A2 and thrombin, induced platelet aggregation. At high concentrations, it inhibits platelet aggregation induced by ADP, probably because ATP is rapidly hydrolyzed to adenosine, which demonstrates antiplatelet action. Adenosine displays functions that will depend on the type of receptor in each tissue and on the origin of the damage, one important action being the reduction of vascular injury by platelet aggregation inhibition.The extracellular nucleotides, ATP and ADP, as well as the nucleoside adenosine have been implicated in a great number of pathologic and physiological functions. Extracellular adenine nucleotide levels are controlled by a complex cell surface-located group of enzymes called ectonucleotidases. Taking into account the importance of IHD and adenine nucleotides in the regulation of platelet aggregation, as well as their importance in adenosine formation, the aim of the present study was to evaluate the activity of enzymes that hydrolyze adenine nucleotides and nucleosides in platelets from patients with IHD. Sixty IHD patients were selected for the study. The activities of ectonucleoside triphosphate diphosphohydrolase (NTPDase, CD39), ectonucleotide pyrophosphatase/phosphodiesterase (E-NPP), ecto-5′-nucleotidase and adenosine deaminase (ADA) were studied in isolated platelets of these patients, as well as the platelet aggregation and NTPDase expression. The results show that NTPDase, ecto-5′-nucleotidase, E-NPP activities and NTPDase expression were increased in platelets of IHD patients when compared with the control group (p < 0.05). On the other hand, ADA activity and platelet aggregation were decreased in IHD patients, when compared with the control group (p < 0,05). Our results suggest that the pathological condition in IHD generates alterations in ectonucleotidase activities as a compensatory organic response to thrombotic events that occur in IHD.

Key words: ectonucleotideases, ischemic heat disease, platelets, nucleotides.

01-022

Adenosine deaminase activity in intact trophozoites ofTrichomonas vaginalis

Patrícia de Brum Vieira¹, Marina Weizenmann¹, Geraldo Attilio De Carli², Maurício Reis Bogo², Carla Denise Bonan², Tiana Tasca¹

¹UFRGS, Universidade Federal do Rio Grande do Sul, Avenida Ipiranga, 2752;²PUCRS, Pontifícia Universidade Católica do Rio Grande do Sul, Avenida Ipiranga, 6681, Porto Alegre, RS, Brazil.

Aim: To characterize the adenosine deaminase (ADA) activity from intact trophozoites of Trichomonas vaginalis, the etiologic agent of trichomonosis. Methods: ADA activity was characterized by a colorimetric assay carried out at 635 nm for measuring the ammonia released. The ADA protein sequences obtained from BLASTP function via the GenBank database were aligned with ClustalX program and a phylogenetic tree was built with MEGA 4.0 program using statistical Neighbor-Joining method with proportional (p) distance. Results: Considering adenosine as substrate, the protein curve was linear between 50 and 150 mg protein/mL, and the time course was linear up to 40 min. The optimal pH for deamination was 7.5. Adenosine and 2-deoxyadenosine were substrates for ADA, while guanosine and 2-deoxyguanosine were not deaminated. The apparent values for KM and Vmax were, respectively, 1.13 ± 0.07 mM and 2.61 ± 0.054 nmol NH3/min/mg of protein. Adenosine deamination was strongly inhibited in the presence of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA). Calcium and magnesium also inhibited the activity; effect prevented by EDTA. Furthermore, when ecto-5′-nucleotidase was inhibited, there also was no ADA activity, strongly suggesting the cascade association between these enzymes. The phylogenetic tree revelead four well-resolved terminal clades supported by high bootstrap values, confirming the presence of two ADA orthologues for T .vaginalis, which composed the second clade. Conclusion: Our data suggest the presence of an ecto-ADA in the parasite surface. The occurrence of an ADA activity in T. vaginalis may represent important implications for the purinergic system in the immune response during trichomonosis.

Key words: adenosine deaminase, enzymatic characterization, phylogeny, Trichomonas vaginalis.

01-023

Trichomonas vaginalisnucleoside triphosphate diphosphohydrolase and ecto-5´-nucleotidase activities are inhibited by lycorine and candimine

Raquel Brandt Giordani¹, Marina Weizenmann¹, Denis Broock Rosemberg¹, Geraldo Attilio De Carli², Maurício Reis Bogo², José Angelo Zuanazzi¹, Tiana Tasca¹

¹UFRGS, Universidade Federal do Rio Grande do Sul, Avenida Ipiranga, 2752;²PUCRS, Pontifícia Universidade Católica do Rio Grande do Sul, Avenida Ipiranga, 6681, Porto Alegre, RS, Brazil.

Drug discovery from plants plays an important role in the pharmaceutical therapy field and the alkaloids lycorine and candimine are candidates for this purpose. Trichomonas vaginalis is a parasite that infects the human urogenital tract and causes trichomonosis, the most prevalent non-viral sexually transmitted disease. Ecto-nucleotidases including nucleoside triphosphate diphosphohydrolase (NTPDase) members, which hydrolyses extracellular ATP (adenosine triphosphate) and ADP (adenosine diphosphate), and ecto-5′-nucleotidase, which hydrolyses AMP (adenosine monophosphate), have been characterized in T. vaginalis. Because purine nucleotides are released from cells under physiological and stress conditions, the aim of this study was to evaluate the effect of lycorine and candimine on T. vaginalis NTPDase and ecto-5′-nculeotidase activities. The alkaloids (50 to 250 μM) were tested against both long-term-grown and clinical isolates. Specific enzymatic activities were expressed as nmolPi realeased/min/mg protein. The effect of both alkaloids at NTPDase A and B expression level was investigated. When the alkaloids were added directly to the reaction mixture, no effect on ATP, ADP or AMP hydrolysis was observed. In contrast, NTPDase and ecto-5′-nucleotidase activities were strongly inhibited by candimine and lycorine on 24 h-treated parasites. This effect was abolished when 24-treated parasites were innoculated in a culture medium without alkaloid. Transcript levels of NTPDase A or B were not altered by the alkaloids. Considering the cytotoxic and proinflammatory role of ATP besides the anti-inflammatory effects of adenosine, the regulation of extracellular nucleotide levels could be relevant in increasing susceptibility of T. vaginalis to host immune response in presence of lycorine and candimine.

Key words: Candimine, ecto-nucleotidases, Lycorine, NTPDase, Trichomonas vaginalis.

01-025

Bovine serum limitation increases the activity and expression of enzymes involved in atp, adp and amp extracellular hydrolysis inTrichomonas vaginalis

Amanda Piccoli Frasson¹, Denis Broock Rosemberg², Mariele Feiffer Charão¹, Geraldo Attilio De Carli³, Solange Cristina Garcia¹, Maurício Reis Bogo², Tiana Tasca¹

¹UFRGS, Universidade Federal do Rio Grande do Sul, Faculdade de Farmácia, UFRGS, Av. Ipiranga 2752, Porto Alegre, RS, Brazil;²PUCRS, Pontifícia Universidade Católica do Rio Grande do Sul, Lab Biologia Genomica e Molecular, Fac Biociências, PUC, Porto Alegre, RS,Brazil;³PUCRS, Pontifícia Universidade Católica do Rio Grande do Sul, Instituto de Geriatria e Gerontologia, PUC, Porto Alegre, RS, Brazil.

The flagellated protozoan Trichomonas vaginalis causes trichomonosis, the most common non-viral STD in the world. The infection causes serious impact on public health; therefore, it is important to investigate the biochemical aspects of the parasite. Cells under stress, anoxia or injury release extracellular nucleotides and they can be inactivated by hydrolysis via ectonucleotidases. The members of E-NTPDase family hydrolyze nucleoside di- and tri-phosphates and the ecto-5′-nucleotidase is able to hydrolyze nucleoside monophosphate, producing adenosine. Important, T. vaginalis lacks the ability to synthesize purines and pyrimidines de novo, and the enzymes act on the salvage pathways generating the nucleosides. The aim of this study was to investigate the metabolism of ATP in trophozoites of T. vaginalis through of activities and gene expression analysis, under a condition of bovine serum limitation. For the assays, the trophozoites from ATCC 30236 and LACH1 isolates were grown in TYM medium supplemented with 1.0% inactivated bovine serum, while control cultures were maintained with 10% serum. The enzymatic tests were performed in the reaction mixture containing 50 mM Tris buffer, 5.0 mM CaCl2 or MgCl2, trophozoites and substrate (1.0 mM ATP, 1.0 mM ADP or 3.0 mM AMP), at 37°C. The specific activity was determined by colorimetric method and expressed as nmol Pi released/min/mg protein. The gene expression patterns were carried out by a RT-PCR assay using specific primers for NTPDase A and B. Genes responsible for AMP hydrolysis were searched by BLAST function in GenBank database and primers were designed for SurE (183), SurE(257), SurE(258) and 5′(3′) enzymes. Extracellular adenine nucleotide hydrolysis was estimated by HPLC analysis. The bovine serum limitation causes an increase on the hydrolysis of the nucleotides tested. In ATCC 30236, there was an increase of 154.4%, 176.1% and 302.5% on the hydrolysis of ATP, ADP and AMP, respectively, when compared with control cultures; in the clinical isolate, LACH1, the increase was to 14.4%, 22.5% and 162.5%. Transcript levels of NTPDase A were significantly increased in both isolates, while NTPDase B and SurE(183) mRNA transcript levels increased only on the LACH1 isolate. Extracellular ATP metabolism is increased in limiting serum, leading to higher ATP hydrolysis and formation of ADP and AMP. Our data suggest that in a situation of serum limitation these enzymes of T. vaginalis play an important role in the provision of adenosine for parasite growth and, moreover, the enzymes may contribute to escape mechanisms of the parasite by breaking down ATP.

Key words: Bovine serum, ectonucleotidases, nucleotide hydrolysis, Trichomonas vaginalis.

01-027

Hydrolysis of nucleotides di- and triphosphates in intact trophozoites ofGiardia lamblia

Muriel Primon de Barros¹, Patrícia de Brum Vieira¹, Camila Dorneles da Silva¹, Amanda Piccoli Frasson¹, Geraldo Attilio De Carli², Carla Denise Bonan², Tiana Tasca¹

¹UFRGS, Universidade Federal do Rio Grande do Sul, Avenida Ipiranga, 2752;²PUCRS, Pontifícia Universidade Católica do Rio Grande do Sul, Avenida Ipiranga, 6681, Porto Alegre, RS, Brazil.

Giardia lamblia, a parasitic flagellated protozoan, is the most common causative agent of diarrheal illness worldwide through waterborne outbreaks. The clinical manifestation of giardiosis is highly variable, since asymptomatic individuals to patients exhibiting severe symptoms. Considering the impact of giardiosis in public health, especially in children, it is very important to investigate the biochemical aspects of this parasite. Therefore, the aim of this study was to characterize the nucleotide hydrolysis in intact trophozoites of G. lamblia. The trophozoites of G. lamblia presented ATP and ADP hydrolysis. The parasites curve was linear between 1.0 and 5.0 × 106 trophozoites/mL. The time course of ATP and ADP hydrolysis was linear up to 60 min. The activity was influenced by divalent cations, Mg2+ and Ca2+, and the best activator was 5.0 mM Mg2+. We showed that the optimal pH for ATP and ADP hydrolysis was 7.5 and 7.0, respectively. The ability of G. lamblia trophozoites to hydrolyze other nucleotides was also evaluated. ATP and GTP were the preferred substrates; nonetheless, they also hydrolyzed ADP, GDP, and CDP. The parameters kinetics were determined for ATP and ADP, KM = 1.64 mM and Vmax = 0.49 nmol Pi/min/106 trophozoites and KM = 2,5 mM and Vmax = 0,14 nmol Pi/min/106 trophozoites, respectively. These results suggest a possible presence of a member of NTPDase family in trophozoites of G. lamblia. Further studies are necessary to confirm the characterization of this enzyme. The investigation on the hydrolysis activity of nucleotides in trophozoites of G. lamblia is important for modulation of the concentration of these compounds involved in cellular signalization.

Key words: cellular signalization, Giardia lamblia, nucleotide hydrolysis, NTPDase.

01-028

Morphine administration in early life alters e-NTPDase activity and gene expression pattern in spinal cord and cerebral cortex of rats

Joanna Ripoll Rozisky¹, Rosane Souza da Silva², Lauren Spezia Adachi¹, Katiúcia Marques Capiotti², Denise Barboza Ramos¹, Mauricio Reis Bogo², Carla Denise Bonan², Iraci Lucena da Silva Torres¹

¹UFRGS, Universidade Federal do Rio Grande do Sul, Sarmento Leite, 500;²PUCRS, Pontifícia Universidade Católica do Rio Grande do Sul, Ipiranga, 6681, Porto Alegre, RS, Brazil.

Introduction: Studies have shown that exposure to opioids in early life can have implications for nervous system development. It has been proposed that adenosine is involved in opioid antinociception, and ATP is involved in central and peripheral mechanisms of nociception. Extracellular nucleotides can be hydrolyzed by E-NTPDases and ecto-5′nucleotidase, which present the functions of removing ATP and generating adenosine. In this study, we evaluated ATP, ADP, and AMP hydrolysis in synaptosomes from spinal cord and cerebral cortex after repeated morphine exposure in early life. Additionally, we evaluated E-NTPDases and ecto-5′nucleotidase gene expressions. Methods: were utilized male Wistar rats, which were divided into two groups: control (C) and morphine (M), which received saline (0.9% NaCl) or morphine (5 μg s.c. in the mid-scapular area) at postnatal day 8 (P8), once a day for 7 days. At P16 the animals were killed and the chosen structures were removed for enzyme assays and analysis of gene expression. The synaptosomes were isolated as described by Nagy et al. (Nagy, J Neurochem. 43:1114, 1984). The reaction medium used to assay ATP, ADP and AMP hydrolysis was performed as described by Battastini et al. (Battastini, Neurochem Res 16:1303, 1991). The enzyme assays were performed on spinal cord and cerebral cortex (6 animals per group). Analysis of E-NTPDase expression (E-NTPDase 1, E-NTPDase 2 and E-NTPDase 3) and of ecto-5′nucleotidase was carried out with a semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) assay, and it was analyzed in spinal cord and cerebral cortex (3 animals per group). Data were expressed as mean ± SEM for the enzyme activities and percentage for E-NTPDases expression. Enzyme activities were expressed as nmolPi.min-1.mg-1 of protein. Statistical analysis were made by Student’s t test, considering P < 0.05 as significant. The Ethics Committee at the Institution approved this study where the work was conducted. Results: we observed a decrease in ADP hydrolysis in spinal cord (ADP: C = 82.55 + 1.6, M = 51.85 + 10.3, Student’s test, P < 0.05; ATP: C = 155.6 + 10, M = 142.3 + 14; AMP: C = 8.6 + 1.9, M = 6.5 + 1.1; Student’s test, P > 0.05) and an increase in ATP hydrolysis in the cerebral cortex (ATP: C = 161.6 + 27.5, M = 213 + 30.9, Student’s test, P < 0.05; ADP: C = 95.9 + 19.5, M = 104.3 + 17.6; AMP: C = 12.4 + 1.9, M = 14.5 + 2.8, Student’s test, P > 0.05). Expression levels of E-NTPDase 1 decreased in cerebral cortex (28%, optical density for C = 230.08, and for M = 165.3; Student’s t test, P < 0.05) in the M group when compared to C group, and increased in spinal cord (23%, optical density for C = 120.61, and for M = 148.26; Student’s t test, P < 0.05) in the M group when compared to C group. There were no differences in other E-NTPDases (2 and 3) and ecto-5′nucleotidase expression between the groups in both structures. Discussion: Our findings highlight the importance of the purinergic system in young rats submitted to repeated morphine exposure by showing that in the neonatal period such exposure is capable of affecting the control system for nucleotide levels, which can promote changes in modulation or transmission of painful stimuli. Financial Support: CNPq, FAPERGS, Propesq-UFRGS.

Key words: ENTPDase, ecto-5′ nucleotidase, morphine, nociception, neonate rats.

01-029

Change in the hydrolysis of nucleotides in spinal cord of rats submitted to pharmacological and surgical procedure

Medeiros, L.F.^1,2,4; Souza, A.^1,2; Rozisky, J.R.^1,2; Santos, V.S.^1,2; Caumo W^1,2., Netto, C.A.², Battastini, A.M.O.³; Torres, I.L.S.^1,2,4

¹Hospital de Clínicas de Porto Alegre,²Departamento de Farmacologia , Departamento de Bioquímica, ICBS, UFRGS,³Programa de Pós-Graduação em Fisiologia⁴, ICBS, UFRGS, Porto Alegre, RS, Brazil.

In the CNS immature is able to respond to the stress caused by painful procedures, and it is essential that neonates, infants and children receive adequate anesthesia to perform such procedures. Although millions of children use of general anesthetics and it is considered safe, you should consider possible changes in the body of patients in the short and medium term. The challenge of anesthesia is the immaturity of the organs and systems, and the risk of this intervention to promote changes in biochemical and behavioral responses that can be observed until adulthood. The objective was to evaluate the enzymatic activity of ectonucleotidases in spinal cord of rats submitted to isoflurane or fentanyl/S(+)ketamine, alone or combined with surgery at P14. Fourtteen years olda Wistar rats were divided into two experimental designs (ED): 1^st ED: a general inhalational anesthetic divided into three groups: control (Ci), isoflurane (ISO), isoflurane + surgery (ISO + SUR); 2^nd ED: a general injectable anesthetic associated with opioid divided into three groups: control (Ckf), fentanyl/S(+)ketamine (KF), fentanyl/S(+)ketamine + surgery (KF + CIR). 1^st ED -induction of anesthesia: isoflurane 5% and 3% for maintenance; 2^nd ED - 0.09 mg / kg fentanyl and 20 mg / kg S(+)ketamine. We used surgical model described by Levine, modified by Rice et al. (Ann Neurol 9:131, 1981), without production of hypoxia-ischemia. For evaluating of ectonucleotidases used the method Battastini et al. (Neurochem Res 16:1303, 1991). Data were analyzed by one-way ANOVA followed by post-hoc Student-Newman-Keuls, the results were expressed in nmolPi/min/mg protein and presented as mean ± SEM, and considered significantly different with P < 0.05. 1^st ED: at P14, ISO group showed a decrease in the hydrolysis of ATP, ADP and AMP and surgery group reversed this effect in the hydrolysis of ATP and ADP [ATP (Ci: 80.05 ± 15.62; ISO: 47 , 17 ± 4.73, ISO + SUR: 75.47 ± 6.45), ADP (Ci: 20.19 ± 1.76; ISO: 12.21 ± 1.85, ISO + SUR: 18.84 ± 1.50), AMP (Ci: 3.73 ± 0.85; ISO: 1.72 ±, 41; ISO + SUR: 1.63 ± 0.50), ANOVA P < 0.05, n = 9–12]. At P30, no significant alteration in nucleotide hydrolysis (ANOVA P > 0.05, n = 4–6). 2nd ED: at P14, no significant difference between groups (ANOVA P < 0.05, n = 2–3) and at P30, the KF group showed an increase in ATP hydrolysis in relation to other groups (Ckf: 100 , 87 ± 0.91, KF: 146.18 ± 8.92; KF-SUR: 104.95 ± 1.83, ANOVA P < 0.05), the KF and KF + SUR groups showed an increase in AMP hydrolysis in relation to Ckf (Ckf: 6.0 ± 0.09, KF: 14.97 ± 1.71; KF + SUR: 12.72 ± 0.79, ANOVA P < 0.05, n = 2–3) . At 1^st ED, we can suggest that the decrease of all nucleotides after isoflurane is related to alteration in the fluidity of cell membranes, the main mechanism of action attributed to isoflurane, whereas ectonucleotidases are anchored to the membranes. The NTPDase 1 (ecto-apyrase), also hydrolyzes nucleotide triphosphates and diphosphate, suggesting a role in this effect. The increased activity of 5′nucleotidase observed in the 2^nd ED influences the levels of nucleotide and nucleoside that participate in neurogenic processes present in the development process. The observed increase in ATP hydrolysis, and no change in ADP hydrolysis suggest that the enzyme involved is the NTPDase2. Financial Support: CAPES; GPPG / HCPA, Propesq / UFRGS; FAPERGS.

Key words: ectonucleotidases, isoflurane, ketamine, fentanyl, medulla.

01-030

Study of plasma membrane bound NTPDases and ecto-5´-nucleotidase/CD73 in rat and human brain tumors

Elizandra Braganhol¹, Leticia S Bergamin¹, Caroline B. de Farias², Ana L. Albujamra², André Cerutti Franciscato², Thiago Torres D’Avila², Michel Faustherd³, Jean Sévigny³, Rafael Roesler², Algemir L. Brunetto², Gilberto Schwartsmann², Marco Antonio Stefani², Ana Maria O. Battastini¹

¹Departamento de Bioquímica, ICBS, UFRGS,²HCPA, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil;³Université Laval, Quebec, Canadá.

Background: Malignant gliomas are the most common type of primary brain tumors in adults. This class of glial tumors is composed of anaplastic astrocytoma (AA), anaplastic oligodendroglioma, and glioblastoma multiforme (GBM). Extracellular nucleotides and nucleosides might influence aspects of cancer biology. In that, ATP and adenosine regulate events related to cell death and proliferation, participate in angiogenesis and regulate tumor immunesupression. Nucleotide receptor-mediated cell communication in CNS is controlled by ectonucleotidases, such as ectonucleoside triphosphodiphosphohydrolases (E-NTPDases) and ecto-5′-nucleotidase/CD73 (ecto-5´-NT/CD73) that can efficiently hydrolyze ATP to adenosine in the extracellular space. Methods: The purpose of this study was to characterize the expression profile of the membrane-bound NTPDases1, 2 and 3 and ecto-5´-NT/CD73 in rat and human brain tumor tissues by immunohistochemistry and RT-PCR techniques. Results: The expression of NTPDase 1 and 2 were not detected; the NTPDase3 was poorly expressed, while the ecto-5´-NT/CD73 was prominently expressed in all tumors analyzed. This study suggests that disruption in the ectonucleotidase expression seems to favor the ATP and the adenosine in the tumor periphery, which could mediate the tumor advance via purinergic receptor activation. Conclusion: In conclusion, the main characteristic exhibited by brain tumors is the absence of NTPDase1 and 2 expression and a prominent presence of ecto-5´-NT/CD73. Our results support the notion that purinergic signaling is involved in glioma progression. Financial Support: FIPE-HCPA, CNPq.

Key words: brain, ecto-5′-nucleotidase/CD73, NTPDases, tumors.

01-031

Endotoxin-induced effects on ectonucleotidase activities in mice kidney: influence of adenosine A2A receptor activation

Luiz Eduardo Baggio Savio¹, Fernanda Cenci Vuaden¹, Denise Barbosa Ramos¹, Maurício Reis Bogo², Carla Denise Bonan²

¹UFRGS, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil;²PUCRS, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, RS, Brazil.

Severe acute kidney injury (AKI) is a frequently condition in hospital patients and there is strong evidence that sepsis is an important cause of AKI. Extracellular ATP mainly functions as a proinflammatory mediator. Adenosine, the final product of ATP breakdown, is an anti-inflammatory compound, acting mainly on adenosine A2A receptors. Considering that kidney is an organ strongly affected by inflammation and ectonucleotidases are responsible for the control of extracellular nucleotides and nucleosides levels, we investigated the effect of a specific agonist of the adenosine A2A receptor (CGS-21680) on ectonucleotidase activities and mRNA expression in kidney membrane preparations from endotoxemic mice. Animals were injected intraperitoneally with 12 mg/kg LPS and/or 0.5 mg/kg CGS-21680 or saline(control). Animals were killed 24 or 48 h after the endotoxemia induction. Nucleotidase activities were determined in kidney membrane preparations using ATP, ADP, AMP, and p-Nph-5′-TMP as substrates. Analysis of ectonucleotidase expression was carried out by a semi-quantitative RT-PCR assay. Exposure to endotoxemia promoted an increase in ATP and 5′-TMP hydrolysis after 48 h (48% and 47%, respectively; n = 5), and a decrease on AMP hydrolysis after 24 h (40%; n = 5). CGS-21680 exposure was not able to reverse these alterations (n = 5). The expression pattern of ectonucleotidases demonstrated an increase on Entpd3 and Enpp3 mRNA levels after LPS injection, but not for other enzymes analyzed, indicating that the transcriptional control was not the only involved on the effect exerted by LPS. These findings indicate that there is an alteration on nucleotide and nucleoside viability in mice kidney in different periods of endotoxemia model, in order to protect the integrity of this organ exposed to inflammation.

Key words: kidney, lipopolysaccharide, adenosine, ATP, ecto-5´-nucleotidase.

01-032

Effects of antiepileptic drugs on ectonucleotidase activities in Zebrafish (Danio rerio) brain

Anna Maria Siebel¹, Eduardo Pacheco Rico², Katiucia Marques Capiotti¹, Angelo Luis Piato¹, Mauricio Reis Bogo¹, Carla Denise Bonan¹

¹PUCRS, Pontifícia Universidade Católica do Rio Grande Do Sul, Av Ipiranga, 6681 Porto Alegre;²UFRGS, Universidade Federal do Rio Grande do Sul, Ramiro Barcelos 2600-anexo, Porto Alegre, RS, Brazil.

Epilepsy, a neurological disorder characterized by the occurrence of spontaneous recurrent seizures, is one of the most common pathologies of the central nervous system. Several studies suggest that epilepsy and this treatment show significant influence on dynamic and functional properties, cognition, and behavior. Carbamazepine (CBZ) and phenytoin (PHT) are classical antiepileptic drugs (AEDs) that act through a variety of mechanisms. Considering that zebrafish may be a model organism to study human diseases and drug mechanisms, the aim of this study was to evaluate the in vitro effects of CBZ and PHT on purinergic system of zebrafish brain. CBZ significantly decreased ATP hydrolysis at 1000 uM (32%) whereas PHT significantly increased AMP hydrolysis both at 500 uM (65%) and 1000 uM (64.8%). These results have shown that CBZ can reduce NTPDase and PHT enhance ecto-5′-nucleotidase activities increasing ATP and adenosine levels, respectively. In purinergic system, ectonucleotidases modulate activation of P2 or P1 receptors by controlling extracellular concentrations of ATP and adenosine. It is possible to suggest that these effects are involved, at least partially, in the neuromodulatory mechanisms of CBZ and PHT. NTPDase family comprises cell-surface-enzymes anchored to the membrane via two transmembrane domains whereas ecto-5´-nucleotidase is attached to the extracellular membrane by a glycosyl phosphatidylinositol anchor. Therefore, it is possible to propose that different AEDs induced-effects on ectonucleotidases are related to enzyme anchorage form.

Key words: antiepileptic drugs, ectonucleotidase, zebrafish.

01-033

Investigation of the purinergic system in differentiated and undifferentiated mesenchymal stem cells

Isabele Cristiana Iser¹, Paula Andreghetto Bracco¹, Guido Lenz², Nance Bayer Nardi³, Martin Hernán Bonamino⁴, Ana Maria Oliveira Battastini ⁵, Márcia Rosângela Wink¹

¹Laboratório de Biologia Celular, UFCSPA,²Departamento de Biofísica, UFRGS,³Departamento de Genética, UFRGS,⁴Instituto Nacional de Câncer (INCA) e⁵Departamento de Bioquímica, ICBS, UFRGS, Brazil.

Introduction: The use of mesenchymal stem cells (MSCs) requires a specific characterization of their properties. Extracellular nucleotides are signaling molecules, involved in important processes in the body. The events induced by these molecules are controlled by the action of enzymes, ectonucelotidases. Recently, it has been suggested that the purinergic system might be involved in the biology of stem cells. A particular interest has been focused on the role of this system in the proliferation and differentiation of these cells. Objectives: To analyze the presence and activity of the enzymes NTPDases, capable to degrade nucleotides on cells surface in MSCs differentiated and non-differentiated. Methods: The cultures of MSCs of BALB/c, differentiated and non-differentiated, underwent determination of enzymatic activity for hydrolysis of ATP, ADP and AMP, by measuring the release of inorganic phosphate colorimetrically by the method of Chan. Protein content was performed by the method of Comassie blue. The technique of RT-PCR to verify the presence of NTPDase was held, so far, in undifferentiated MSCs, using primers for the six types of NTPDase and CD73. The gene silencing of CD73 in MSCs is being conducted by a lentiviral vector containing RNAi silencing of specific protein CD73. Results and conclusions: The undifferentiated MSCs express mRNA NTPDase 1, 2, 3, 5 and 6 and CD73. Moreover, the data suggest that there is a considerable increase in AMP hydrolysis in adipocytes, which can lead to increased production of adenosine. To analyze the significance of the increase in AMP hydrolysis in the cells differentiated into adipocytes, is being performed gene silencing of CD73. We believe that adenosine generated might be involved in the process of differentiation of MSCs. This study opens perspectives for a more detailed investigation of the involvement of purinergic signaling in the differentiation of MSCs, focusing on the role of adenosine and CD73 in this process. Financial Support: CNPq.

Key words: mesenchymal cells, differentiation, NTPDases, CD73, adenosine.

P1 RECEPTORS

02-001

Evaluation of the effect of ERK1/2 inhibitors on adenosine uptake in cultures of avian retinal cells

Alexandre dos Santos Rodrigues, Jainne Martins Ferreira, Roberto Paes de Carvalho

UFF, Universidade Federal Fluminense, Dept. of Neurobiology, Program of Neurosciences, Outeiro São João Batista S/N Centro Niterói, RJ, Brazil.

Introduction and Objectives: Adenosine (Ado) is an important modulator of neuronal survival and differentiation in the CNS. Our previous work showed that Ado transporters are present in cultures of chick retinal cells and that activation of Ado A1 receptors promotes an increase in ERK phosphorylation in glial cultures. Our aim in this work was to study the participation of the ERK pathway on Ado uptake and release, as well as on adenosine kinase activity in different types of retinal cultures. Methods: The uptake was measured after incubation with (3 H)-adenosine for 15 min and cell lysis to determine intracellular radioactivity. Basal and glutamate-stimulated radioactivity released from cells was determined by liquid scintillation spectroscopy. Results: Ado uptake involves 2 components, one saturable and one linear, in mixed or glial cultures, but the saturable component is much smaller in glial cultures. In glial cultures, ERK inhibition with PD98059 (25 μM) reduced the uptake in 37.1 ± 4.3% (n = 4). In mixed cultures, PD98059 (25 μM) or UO126 (10 μM), two MEK inhibitors with different chemical structures, reduced the uptake in 39.1 ± 4.5% (n = 5) and 48.6 ± 2.9% (n = 3), respectively. U0124 (10 μM), an innactive UO126 analog, was much less effective in inhibiting Ado uptake (17.9 ± 3.4% vs control, n = 3). The analysis of inhibition curves with MEK inhibitors showed that the blockade is observed mainly on the saturable component at both types of culture. Glutamate (Glu) induced the release of purines from mixed cultures (136.9 ± 31.0% increase vs control, n = 3) and this effect was not significantly inhibited by PD98059 (79.6 ± 18.8% increase vs Glu, n = 3, p <0.05). Conclusions: MEK inhibition dramatically reduces adenosine uptake in mixed neuronal/glial or purified cultures of retinal glial cells, apparently not affecting purine release induced by Glu. Since ERK activity is regulated by stimulation of A1 receptors in the retina, the results suggest that adenosine regulates its own transporter activity via activation of the ERK pathway. Financial Support: CAPES, CNPq, FAPERJ and PRONEX-MCT.

Key words: adenosine, avian retinal cells, ERKs

02-002

In vivomodulation of adenosine A1 receptor expression in the chick embryo retina

Rafael Brito da Silva, Mariana Rodrigues Pereira, Roberto Paes de Carvalho, Karin da Costa Calaza

Laboratory of Cellular Neurobiology, Department of Neurobiology and Program of Neurosciences, Fluminense Federal University, Niterói, RJ, Brazil.

Introduction: The retina, responsible for the phototransduction process, has the same embryological origin as the CNS and present the majority of chemical mediators found in the CNS. Adenosine plays several important roles in the CNS, including in the retina, by activating A1, A2a, A2b or A3 receptors. Several published data indicate that a chronic stimulation or inhibition of A1 adenosine receptor (A1R) induces a respective decrease or increase in receptor levels. Recently it was shown that a prolonged activation of A2a receptors (A2aR) induces an increase in the amount of A1R in retinal cell cultures (Pereira et al, 2010). Thus, the main goal of this study was to investigate the effect of chronic treatment with adenosine agonists and/or antagonists on the expression of A1R in the in vivo chick embryo retina. Methods and Results: Fourteen day-old chick embryo (E14) eggs were injected with adenosine agonists and/or antagonists (estimated final concentration of 100nM) or DMSO (control). Two days later, E16 embryos were sacrificed and the retinas processed for [³ H]-DPCPX (A1R antagonist) binding and western blot using an antibody against A1R. Exposure to CHA (A1R agonist) or DPCPX respectively decreased or increased [³ H]-DPCPX binding to retinal membranes whereas the simultaneous injection of CHA and DPCPX showed binding levels similar to control (CTR = 100.00 n = 9 CHA = 72.8 ± 3.67 p < 0.001 n = 5, DPCPX = 144.9 ± 4.04 p < 0.001 n = 3, CHA + DPCPX = 107.0 ± 13.69 p > 0.05 n = 3). The A2a agonist CGS-21680 increased, whereas treatment with SCH-58251 or ZM-241385 (both A2aR antagonists) caused a decrease in binding levels. Treatment with CGS 21680 together with SCH-58251 or ZM-241385 showed binding levels similar to control (CTR = 100.00 n = 14, CGS = 133.6 ± 11.46 p < 0.01 n = 7, SCH = 67.92 ± 6.99 p < 0.01 n = 6, CGS + SCH = 107.3 ± 8.53 p > 0.05 n = 4, ZM = 70.52 ± 6.67 p < 0.05 n = 4, CGS + ZM = 92.3 ± 19.67 p > 0.05 n = 4). Treatment with CHA plus SCH-58251 decreased the binding but this decrease was similar to that observed after CHA or SCH-58251 alone (CTR = 100.00 n = 5, n = CHA + SCH = 69.40 ± 6.99 p < 0.001 n = 5). Similar results were obtained when receptor levels were measured by western blot (CTR = 100.00 n = 7 CHA = 70.14 ± 4.97 p < 0.05 n = 5, DPCPX = 127.4 p < 0.05 n = 4, CHA + DPCPX = 88.33 ± 3.66 p > 0.05 n = 4); (CTR = 100.00 n = 10 CGS = 134.00 ± 11.06 p < 0.05 n = 5; ZM = 72.49 ± 8.62 p < 0.05 n = 7; CGS + ZM = 108.9 ± 10.72 p < 0.05 n = 6); (CTR = 100.00 n = 7; SCH = 52.55 ± 5.97 p < 0.01 n = 4; CHA + SCH = 48.93 ± 14.5 p < 0.001 n = 4). Conclusion: These data indicate a regulation of A1R expression by the activation of A2aR or A1R in an opposite way, the first increasing and the latter decreasing A1R expression in chick embryo retina. Furthermore, the modulatory action of antagonists suggests an effect of endogenous adenosine via A1R and A2aR on the expression of A1R in the in vivo chick retina. Supported by: CNPq, CAPES, FAPERJ, PRONEX-MCT.

Key words: adenosine, A1 receptor, A2a receptor, chick embryo retina, development.

02-003

Adenosine receptor activation stimulates vitamin C release in cultured retinal cells

Camila Cabral Portugal, Thaísa Godinho Encarnação, Roberto Paes de Carvalho

Laboratory of Cellular Neurobiology, Department of Neurobiology and Program of Neurosciences, Fluminense Federal University, Niterói, RJ, Brazil.

Objectives: In CNS, ascorbate (Asc) is taken up by SVCT-2, which transports L-Asc stereospecifically in a concentration-dependent manner, supported by a sodium-dependent mechanism. In the present work we have investigated the mechanism of Asc release modulated by adenosine in cultured retinal cells. Methods: Mixed cultured retinal cells were used for [¹⁴C]Asc release experiments. First, cultures were subjected to [¹⁴C]Asc uptake. Subsequently, cultures were incubated for successive periods of 10 min with different treatments. After that, released fractions were collected and the radioactivity was measured. The results were normalized to the control in each experiment. Results: The non-selective adenosine receptor agonist NECA (10 μM) increased vitamin C release (95.1% ± 3.5;n = 3). On the other hand, the selective A1 receptor agonist CHA (1 μM) did not stimulate vitamin C release (-15.9% ± 5.9;n = 3) while the selective A2a receptor agonist DPMA was capable of releasing Asc (117.3% ± 15.4;n = 17). The selective A2a receptor antagonists ZM241385 (1 μM) (-21.6% ± 4.12;n = 3) and SCH58261 (600 nM) (6.2% ± 12.8;n = 5) were both capable of inhibiting DPMA-induced Asc release. It is described that A2a receptor activation lead to cAMP accumulation and activation of PKA. Therefore, we used the PKA inhibitor H-89 (15 μM) and observed that DPMA-induced vitamin C release was partially inhibited (59.1% ± 5.4;n = 6; in relation to DPMA). To confirm the involvement of the cAMP signaling pathway we used forskolin (50 μM) and the cAMP permeable analogue 8Br-AMPc (1 mM), and observed that both treatments stimulate vitamin C release (73.1% ± 13.5;n = 7 and 58.6% ± 9.5;n = 4). As DPMA-stimulated Asc release was not totally abolished when PKA was inhibited by H-89, we decided to evaluate whether part of this effect was mediated by the PLC/PKC pathway. Cultured cells were treated with the phospholipase C inhibitor U73 (10 μM) and DPMA-induced Asc release was partially abrogated (65.4% ± 8.4;n = 7; in relation to DPMA), the same occuring when cultured retinal cells were treated with the PKC inhibitor cheleritrin chloride 10 μM (54.3 ± 9.5;n = 5; in relation DPMA). Even when both inhibitors were administered together the same partial effect remained (73.5 ± 5.2;n = 3; in relation to DPMA). We have also evaluated the involvement of other protein kinases in DPMA-stimulated vitamin C release. We observed that when inhibiting both the Src family protein tyrosine kinases with PP1 (10 μM) (62.5% ± 5.7;n = 4; in relation to DPMA) and the MEK kinase with U0126 (10 μM) (63.8% ± 17.5; n = 4) DPMA-induced vitamin C release was partially blocked. Conclusion: A2a receptor stimulation promotes Asc release in cultured retinal cells. This could be a mechanism by which A2a receptor antagonism display positive effects in ischemic paradigms ever since blocking these receptors promotes intracellular accumulation of Asc, which in turn should assist the cellular redox balance. Financial Support: CNPq, CAPES, FAPERJ, PRONEX/MC.

Key words: vitamin C, A2a receptor, PKC, PKA, retina

02-005

Modulation of A1 adenosine receptor expression by chronic activation of A2A receptors: Regulatory mechanisms

Mariana Rodrigues Pereira, Roberto Paes de Carvalho

Laboratory of Cellular Neurobiology, Department of Neurobiology and Program of Neurosciences, Fluminense Federal University, Niterói, RJ, Brazil.

Objective: Adenosine is a neuromodulator in the CNS and its actions are promoted by activation of 4 metabotropic receptors: A1, A2a, A2b and A3. Whereas A1 and A3 receptors inhibit adenylyl cyclase and decrease intracellular cAMP levels, A2 receptors stimulate the enzyme and increase cyclic nucleotide levels. Previous results demonstrated that chronic activation of A2a receptors increases A1 receptor expression by a cAMP and PKA-dependent mechanism in mixed cultures of avian retina cells. In these cultures, A1 receptors are present in neurons but not in glial cells. Moreover, A2a receptor activation increases A1 receptor labeling, but no alteration in cellular localization was observed. In the present work we have studied the signaling pathways involved in this regulation. Methods and results: NFkB is a transcription factor involved in regulation of A1 receptor expression. In order to test the participation of this factor, cultures were treated with gliotoxin or sulfasalazin (NFkB inhibitors) in presence or not of DPMA (A2a agonist) and A1 receptor expression was analyzed by western blot. Gliotoxin as well as sulfasalazin inhibit the DPMA effect (control: 100%, DPMA: 130.3 ± 4.5, gliotoxin: 109.1 ± 2.5, DPMA + gliotoxin: 101.4 ± 2.7, n = 3, p < 0.001) (control: 100%, DPMA: 137.3 ± 9.1, sulfasalazin: 103.7 ± 7.8, DPMA + sulfasalazin: 101.0 ± 5.8, n = 3, p < 0.01). Moreover, U0126, a MEK inhibitor, promotes increase in A1 receptor levels and this effect was not additive with that of DPMA (control: 100%, DPMA: 136.5 ± 11.0, U0126: 142.7 ± 12.1, DPMA + U0126: 135.0 ± 7.9, n = 3, p < 0.05). Similar results were observed with K252a, a tyrosine kinase receptor inhibitor (control: 100%, DPMA: 136.3 ± 4.4, K252a: 135.7 ± 7.1, DPMA + K252a: 141.0 ± 14.6, n = 3, p < 0.05). DPMA effect is also blocked by PP1, a Src inhibitor (control: 100%, DPMA: 129.8 ± 2.3, PP1: 97.3 ± 8.5, DPMA + PP1: 73.0 ± 14.6, n = 4, p < 0.05). Conclusion: Regulation of A1 receptor expression by chronic activation of A2a receptors involves NFkB, Src and ERK inhibiton. Support: CNPq, CAPES, FAPERJ, PRONEX-MCT.

Key words: A1 adenosine receptors, A2a adenosine receptors, expression, retina.

02-006

Control of ERK 1/2 phosphorylation by P1 receptors in avian retina cultures

S.A. Rodrigues, A. dos Santos-Rodrigues, R. Paes-de-Carvalho, Mariana Rodrigues Pereira, Elizabeth Giestal de Araujo, Roberto Paes de Carvalho

Laboratory of Cellular Neurobiology, Department of Neurobiology and Program of Neurosciences, Fluminense Federal University, Niterói, RJ, Brazil.

Objective: The chick retina, characterized by the presence of different neuronal cell types and one kind of glial cell, the Muller cell, has been used as a model for studying the functioning of central nervous system. Adenosine is a neuromodulator agent present in the retina and is important in the regulating of proliferation, differentiation and neuronal survival. There are four classes of adenosine receptors (A1, A2a, A2b and A3) coupled to adenylyl cyclase, but that can also modulate other signaling pathways. The ERK 1/2 protein kinases are involved in the regulation of cellular differentiation and proliferation processes. In this work, our objective was to characterize the effect of activation of adenosine receptors in phosphorylation of ERK 1/2 in cultures of chick retina. Methods and Results: Retinas of 8 days embryo chicks had their cells dissociated and maintained in culture. The medium was changed in the next day, and the experiments were performed 3–4 days after seeding. The phospho-ERK (pERK) levels were analyzed by Western blot. Treatment of cultures with CHA, A1 receptor selective agonist, led to a dose-dependent increase in pERK, with maximum stimulation being observed with 100 nM CHA (209.7 ± 39.3% stimulation) or 1 μM (202.4 ± 12.5% stimulation). Moreover, DPMA, A2a receptor selective agonist, caused a biphasic effect, with a decrease in pERK in low concentrations (maximum inhibitory effect with 10nM; 64.9 ± 4.6% inhibition, n = 3) and an inhibition decrease at higher concentrations (100nM-1 μM). Conclusion: In retinal cell cultures, adenosine regulates in different ways ERK phosphorylation, promoting stimulation through A1 receptors activation and inhibition by A2a receptors.

Key words: adenosine, ERK, A1 and A2a receptors

02-007

IL-6 increases A1 receptors expression in mixed cultures of avian retinas

M.R Pereira, R. Paes-de-Carvalho.

Laboratory of Cellular Neurobiology, Department of Neurobiology and Program of Neurosciences, Fluminense Federal University, Niterói, RJ, Brazil.

Objective: Adenosine is an important nucleoside presenting some functions in the CNS such as modulation of neurotransmitter release and neuronal survival. Adenosine activates 4 types of metabotropic receptors: A1, A2a, A2b e A3. A1 e A3 receptors inhibit adenylyl cyclase decreasing intracellular cAMP levels while A2a e A2b receptor stimulate adenylyl cyclase and increase the cyclic nucleotide levels. Interleukin-6 (IL-6) is a pro-inflammatory cytokine that acts in CNS and promotes survival and inhibition of glutamate release. Moreover, IL-6 increases A1 receptor expression in mouse hippocampus and cortex. In this work we have investigated whether A1 receptor expression can be regulated by IL-6 in mixed cultures of avian retina cells. Methods and results: Western Blot experiments showed that chronic treatment with IL-6 increases A1 receptor expression. This effect is inhibited by the NFkB inhibitors gliotoxin (control: 100%, IL-6: 148.0 ± 6.2%, gliotoxin: 100.7 ± 6.4%, IL-6 + gliotoxin: 92.7 ± 18.5%, n = 3, p < 0.05), sulfasalazin (control: 100%, IL-6: 159.3 ± 18.9%, sulfasalazin: 112.0 ± 8.2%, IL-6 + sulfasalazin: 102.0 ± 12.9%, n = 3, p < 0.05) and PDTC (control: 100%, IL-6: 142.7 ± 9.0%, PDTC: 116.0 ± 6.0%, IL-6 + PDTC: 96.7 ± 16.8%, n = 3, p < 0.05). The IL-6 effect is also inhibited by AG490, a JAK inhibitor (control: 100%, IL-6: 132.3 ± 7.8%, AG490: 82.3 ± 7.7%, IL-6 + AG490: 77.7 ± 3.3%, n = 3, p < 0.05). Conclusion: Chronic treatment with IL-6 promotes an increase in A1 receptor expression in a NFkB and JAK-dependent manner. Financial support: CNPq, CAPES, FAPERJ, PRONEX-MCT.

Key words: adenosine, retina, culture, interleukin-6, A1 receptors

03 – P2 RECEPTORS

03-002

Regulated ATP release from human erythrocytes

Maria Florencia Leal Denis, N Montalbetti, Pablo Schwarzbaum

Univ. Bs. As, Instituto de Química y Físicoquímica Biológica (IQUIFIB). Fa, Junin 956 (1113) Ciudad Autónoma de Buenos Aires. Argentina.

In almost all vertebrate cells challenged by hypotonicity, swelling is followed by a regulatory volume decrease (RVD) that tends to recover the original isotonic cell volume. There is abundant information regarding the intracellular mechanisms mediating volume regulation, but relatively little on extracellular signaling events. Moreover, although several reports show that specific P receptors and ectonucleotidases are important for RVD, the issue has not been studied systematically. To understand nucleotide dependent RVD it is essential to analyse to what extent nucleotides and adenosine accumulate in the extracellular space (nucleotide transmembrane transport), how they are interconverted by ectonucleotidases (nucleotide extracellular metabolism), and activate different purine and pyrimidine receptors (P receptor signaling). Using goldfish hepatocytes challenged by hipotonicity as a cell model to study volume regulation, results will be presented showing how the interaction between extracellular ATP (and other nucleotides), P receptors and ectonucleotidases can modulate RVD. Moreover, since in goldfish hepatocytes RVD is a function of extracellular [ATP], and on the other hand hypotonic exposure leads to a non linear extracellular accumulation of the nucleotide, studying the factors governing the kinetic of extracellular [ATP] is important to characterize the nucleotide-dependent RVD response.

Key words: ATP, erythrocytes, purinergic receptors.

03-005

Involvement of the PI3K/AKT pathway in ATP- induced proliferation of developing retinal cells in culture

Isis Moraes Ornelas, Ana Lúcia Marques Ventura

UFF, Universidade Federal Fluminense, Outeiro São João Batista S/N Centro Niterói, Brazil.

Introduction: ATP induces the proliferation of chick retinal cells in culture through the activation of P2Y1 receptors, PKC and MAP kinases. Together with MAP kinases, the PI3K/Akt pathway has also been implicated as an important mediator in proliferative events during development. Here we investigated the participation of the PI3K/AKT signal pathway on ATP-induced proliferation of chick embryo retinal cells in culture. When retinal cultures obtained from 7-day-old embryos and cultivated for 1 day were treated with ATP, a transient and dose-dependent phosphorylation of both ERKs and Akt was observed, an effect that could be mimicked by 500 μM ADP and blocked by 100 μM PPADS, a P2 receptor antagonist. Maximal stimulation of both enzymes was obtained with 100 μM ATP in 5 min, decreasing thereafter. The activation of these pathways by ATP seemed to be independent, since LY294002 and U0126, inhibitors of PI3K and MEK, did not block the activation of ERK and Akt, respectively, although each compound blocked its respective target. Moreover, when the cultures were incubated with ATP in the presence of LY294002, a decreased incorporation of [3 H]-thymidine was observed, as compared to cultures treated only with ATP, a decline that was also obtained by incubating the cells with ATP plus 0,5 μM API-59CJ-Ome, an inhibitor of AKT. No decrease in cell viability was observed with this concentration of API-59CJ-Ome. An increase in cyclin D1 expression, that could be inhibited by 10 μM LY 294002 or 20 μM U0126, was observed when cells were incubated with 500 μM ADP. No effect of PI3K and MEK inhibitors was observed in the expression of p27kip1 the cultures. These results suggest that, besides the involvement of the MAP kinases pathway, ATP-induced cell cycling of late developing retinal progenitors in culture also involves the activation of the PI3K/Akt pathway.

Key words: proliferation, PI3K, ATP, retina

03-006

Assessment of the effects of A438079, a selective P2X7 receptor antagonist, in the hemorrhagic cystitis induced by cyclophosphamide in mice

Jerônimo Pietrobon Martins¹, Rodrigo Bradiccini Madeira da Silva¹, André Avelino dos Santos Júnior1, Robson Coutinho Silva³, Ana Maria Oliveira Battastini², Maria Martha Campos¹, Fernanda Bueno Morrone¹

¹PUCRS, Pontifícia Universidade Católica do Rio Grande do Sul, Av. Ipiranga, 6681 - Partenon - Porto Alegre/RS ;²UFRGS, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos 2600 - anexo Bairro Santa Cecília Porto Alegre – RS ;³UFRJ, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373 - Ilha do Fundão - Rio de Janeiro – RJ, , Brazil.

Introduction: Extracellular nucleotides are important signaling molecules that mediate many biological effects, through the purinergic receptors activation (Ralevic et al., Pharmacol. Rev., 50, 413, 1998). ATP is generated in response to cellular damage, and the P2X7 receptors have an essential role in the onset and maintenance of pathological changes (Chesselli et al., Pain, 114, 386, 2005). The hemorrhagic cystitis (HC) is a well known adverse effect of therapy with cyclophosphamide (CYP) used in patients in the treatment of many solid tumors (Mosque et al. 2007). These urotoxic effects are attributed to the toxic metabolic of the CYP, named acrolein, which can be partially prevented by 2-mercaptoetanosulfonato of sodium (Mesna) (Katz et al., J Cancer Res. Clin. Oncol., 121, 128, 1995). The present study aimed to determine the role of P2X7 receptors in the model of hemorrhagic cystitis induced by CYP in mice. Methods: Male Swiss mice (n = 5; 25–30 g) were used. HC was induced by a single administration of CYP (300 mg/kg, i.p.). Immediately after the i.p. injection of CYP, mice were housed in individual plastic cages to observe the spontaneous behavior for 4 h, for 2 min every half-hour. Three behavioral parameters were considered: (i) general activity (walking, rearing, climbing, grooming etc.); (ii) immobility time; and (iii) indicatives of visceral pain behavior (‘crises’). In addition, the spontaneous behavior of mice was also scored according to the following scale: 0 = normal; 1 = piloerection; 2 = strong piloerection; 3 = labored breathing; 4 = abdomen licking; and 5 = abdomen stretching and contractions (Olivar et al., Eur. J. Pain., 3, 141, 1999). We have also performed the gross examination of bladders at 6 h, in order to determine the presence of edema and hemorrhage. The wet weight of bladders (g per 100 g of body weight) and the myeloperoxidase actitvity (MPO - an indicative of neutrophil migration) were also evaluated at this time-point (Gray et al., J. Urol., 136, 497, 1986). Mice were treated with the selective P2X7 receptor antagonist A438079 (100 umol/kg) (McGaraughty et al., Neurosci., 146, 1817, 2007), given 30 min before and 4 h after the CYP. Control animals received saline at the same intervals of time. All the experimental procedures were approved by the Local Ethics Committee (08/00074, CEUA, PUCRS). Results: The results of the present study show that pretreatment with the selective P2X7 receptor antagonist A438079 inhibited the nociceptive behavior score induced by CYP (37 + - 8%). In addition, the administration of A438079 produced a reduction of both edema and hemorrhage indexes (29 + - 8% and 36 + - 11%) in the gross evaluation. A438079 treatment also displayed a partial inhibition of wet weight of bladders (24 + - 11%). Of note, the administration of A438079 practically abolished the increase of urinary bladder MPO activity induced by CYP. Discussion: In the recent years, the interest in the therapeutical potential of purinergic receptors has dramatically increased (Burnstock et al., Pharmacol. Rev., 58, 58, 2006). Our study revealed the importance of P2X7 receptors in the HC induced by CYP. It is tempting to suggest that pharmacological inhibition of these receptors might represent a new therapeutic alternative for this pathological condition. Financial support: CNPq, PROBOLSAS-PUCRS.

Key words: Hemorrhagic cystitis, cyclophosphamide, P2X7 receptor, A438079

03-007

Study of polymorphisms in the P2X7 receptor in individuals with tuberculosis

Carla Santos de Oliveira, Thereza Christina Benévolo, Márcia M. Quinhones Pires Lopes, Harrison Magdinier Gomes, Adalberto Rezende Santos, Luiz Anastacio Alves

FIOCRUZ, Fundação Oswaldo Cruz, Av. Brazil, 4365 - Manguinhos - CEP: 25984242, Brazil.

Introduction: Tuberculosis is a chronic, infectious disease which is considered, to this day, a global health problem. It is estimated that one-third of world population is infected with Mycobacterium tuberculosis. Among those who are infected, only an approximated 5–10% will develop the clinical disease. Macrophages are the principal host cells for intracellular mycobacterium replication, and they are ultimately responsible for the regulation of the growth and viability of this pathogen. P2X₇ receptors (P2X₇R) are highly expressed in macrophages - a purinergic receptor, which under prolonged activation and/or in the presence of high concentrations of extracellular ATP induces, in the plasma membrane, the formation of a pore permeable to hydrophilic solutes which may lead to apoptosis. Several studies have associated P2X₇R, expressed in macrophages, to the death of M. tuberculosis by different mechanisms. Some studies have suggested that genetic factors may affect susceptibility or resistance to TB, but the specific genes involved have not yet been totally characterized. Human macrophages with the polymorphism 1513A → C in the P2X7R gene, infected with M. tuberculosis, fail to induce cell apoptosis and the death of the M. tuberculosis. A study of a Gambian population showed that a polymorphism at the position -762 of the P2X₇R gene was involved with the individual resistance to TB. Based on the data shown above and considering that there are no studies in Brazil involving the P2X₇R polymorphisms with susceptibility or resistance to TB, and taking in account that the TB is the second cause of death from infectious diseases, the study of interactions between the P2X₇R and this pathogen becomes a priority. Therefore, the aim of our study is to find polymorphisms in the P2X₇R of mononuclear cells in patients with TB, within a population from Rio de Janeiro (patients of the Hospital Evandro Chagas - Fiocruz) by using the polymerase chain reaction allele-specific (PCR-AS). Initially, four primers are added to generate three different PCR products in case of heterozygous, and from automated sequencing of these products to determine more polymorphism at the P2X₇ gene. Once the presence of polymorphisms has been identified, functional tests, in vitro, will be performed to verify the alterations in the receptor function that could be related to susceptibility or resistance to disease. Our preliminary results of PCR-AS differ from the results for same sequences primers available in the literature. The PCR products described were 285 pb, 184 pb, and 102 pb, while we obtained products with 859 pb and 205 pb, n = 6 independent experiments. Moreover, analysis by BLAST show that the two primers, specific to polymorphism, work as Foward, indicating that the sequence described is possibly wrong and could not be used for PCR-AS. Considering these data, we could not analyze cases with heterozygous in a same reaction. As mentioned above, the DNA of patients with tuberculosis are being analyzed to verify the presence or absence of the polymorphism -762. We have already analyzed 79 samples from an initial total of 90 volunteer patients. We are currently in the process of purifying the PCR products and then, we will be sequencing all these samples. Therefore, solving the difficulties regarded to the observation of cases with heterozygous and other possible polymorphisms near the target region. After the described results, tests will be conducted to verify if these polymorphisms can modify the main functions of the receptor.

03-008

Spectrophotometric method for P2X7 antagonist screening in vitro

Rômulo José Soares-Bezerra, Anael Viana Pinto Alberto, André Gustavo Calvano Bonavita, Valber da Silva Frutuoso, Luiz Anastacio Alves

FIOCRUZ, Fundação do Instituto Oswaldo Cruz, Av.Brazil,4365 - Pav.108 - Sl: 36 - Manguinhos - RJ – Brazil.

Introduction: The P2 receptors (P2R) are wide expressed in the organism such as endocrine, cardiovascullar, nervous, reproductive and immune systems. These receptors are activated mainly by nucleotides. P2R are divided in two families, P2X (ionotropic receptors) and P2Y (metabotropic receptors). In mammalian there are seven subtypes of P2X receptors (P2X1, P2X2, P2X3, P2X4, P2X5, P2X6 e P2X7) and eight subtypes of P2Y (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, P2Y14). The P2X7 is involved with physiological and metabolic functions and has been implicated with many diseases. In this work we established a methodology to screen P2X7 receptor antagonists candidates obtained from Brazilian flora. This method considers the ability of P2X7 to form a pore in the plasma mebrane when activated by high concentrations of ATP. The opened pore allows the passage of propidium iodide which fluorescence can be detected by plate reader spectrophotometry. In our experimental approach, we used J774 macrophage cell line in a concentration of 4 × 10&weierp6cells/well in a 96 wells plate. After that we used the following scheme of treatment: presence or not of ATP [5 mM], treatment or not with the plant extracts (in this case we used 320&mug/mL of the aqueous extract of Rheedia longifolia (Clusiacea). For the positive control cells were treated with 6 &mul/ml of ATPox [10&weierp-6 M] (commercial antagonist). After the treatments propidium iodide was added [50 nM] and the fluorescence analyses were made using a plate reader spectrophotometer, &lambdaexcitation = 488 nM and &lambdaread = 590 nM. This natural compound showed a significant antagonist activity on the ATP pore formation when compared with the positive control. Moreover the metodology proved to be reproducible. These results were confirmed by fluorescent microscopy. Taking together we suggested an improved method for detection of possible new P2X7 receptors antagonists compounds, which could be usefull for clinical treatment of diseases related with this receptor.

Key words: antagonist, natural products, P2X7, screening

03-009

Nucleotides promote PKB activation on epithelial cell line LLC-PK1: possible involvement of P2Y1, P2Y2, P2Y4 and P2X7 receptors

Dilza Balteiro Pereira de Campos¹, Ana Acacia de Sá Pinheiro^1,3, Robson Coutinho-Silva¹, Celso Caruso Neves^1,2

¹IBCCF/UFRJ, Instituto de Biofísica Carlos Chagas Filho, Rio de Janeiro, Brazil;²INBEB/INCT, Institutos Nacionais de Ciência e Tecnologia, Rio de Janeiro, Brazil;³INPTAm/INCT, Institutos Nacionais de Ciência e Tecnologia, Rio de Janeiro, Brazil

Introduction: Adenosine triphosphate (ATP) and other nucleotides are released to extracellular space and activate different P2 receptors, modulating several long term cellular mechanisms such as proliferation, differentiation and cell death. P2 receptors activation converge to increased intracellular calcium, that can activate phospholipase C and PKC, triggering signaling cascades, including PKB, a serine-treonine kinase whose activation is related with survival and proliferation. The aim of this study was to investigate if purinergic receptors modulate PKB activation on proximal tubule cells. METHODS AND RESULTS: LLC-PK1 cells, a well-characterized porcine proximal tubule cell model, were grown in DMEM supplemented with 10% FBS, 1% antibiotic/antimycotic at 5% CO₂ and 37 ⁰ C. Confluent monolayers of LLC-PK1 cells were kept overnight in serum-free DMEM. Next, cell cultures were treated with different nucleotides, scrapped and PKB activation was assayed on cytosolic extracts through immunodetection of serine 473 residue phosphorylation. In addition, western blot analysis was performed to identify P2 receptors on this cell culture. Both ATP and UTP promoted PKB phosphorylation in a dose and time-dependent manner. ADP was also able to activate PKB in LLC-PK1 although to a lesser extend than that of ATP and neither AMP nor adenosine were able to induce any effect on this activation. Four ATP concentrations were tested: 1, 10 and 100 μM and 3 mM. Maximal activation was found to be during 3 mM ATP treatment (1.16 + 0.04 arbitrary units versus 0.38 + 0.07 in control cells). Furthermore, we observed that 100 μM BzATP promoted Akt activation (1.2 + 0.1 arbitrary units in BzATP-treated cells versus 0.2 + 0.1 in control) and this effect was totally abolished by pre incubation with P2X₇ antagonist oxidized ATP. 100 μM ATP-induced PKB phosphorylation was not abolished when LLC-PK1 cells were incubated in PBS containing 10 mM EGTA instead of serum-free DMEM, suggesting that this effect does not require extracellular calcium influx. The pre treatment with a non selective P2 antagonist (suramin) partially prevented 100 μM ATP or UTP-induced PKB phosphorylation and abolished 100 μM ADP effects. Immunodetection by western blot analysis was performed to verify if LLC-PK1 cells expressed P2Y1, P2Y2, P2Y4 and P2X7 receptors. It was observed a ∼40 kDa, ∼47 kDa and ∼50 kDa band expected for the presence of P2Y₄, P2Y₂ and P2X₇ receptors respectively. It was detected two bands: a ∼47 kDa and a ∼100 kDa in extracts expected for the presence of P2Y₁ receptor. This data suggests that this receptor exists on both forms on LLC-PK1 cells: as a monomer and as a dimer. The treatment of LLC-PK1 cells with 3 mM ATP during 12 h induced apoptosis (14 ± 2% of hipidiploid nucleus versus 7.8 ± 0.7% in control p < 0.05). CONCLUSION: Nucleotides can activate different purinergic and pirimidinergic receptors on LLC-PK1 cells and their activation promote PKB phosphorylation. Suramin-sensitive ADP induced PKB phosphorylation may be due to P2Y₁ receptor activation while ATP and UTP effects can be related to P2Y₂ and P2Y₄ receptors activation. High ATP concentration induced PKB phosphorylation mainly through P2X₇ activation and is able to induce apoptosis on this cell model.

Key words: purinergic receptors, PKB, kidney

03-010

Effect of conditioned medium of mixed cultures on the death of neurons of retina of chicken embryo induced by activation of P2X7 receptors

Roxana Mamani Anccasi, Ana Lúcia Marques Ventura

UFF, UNIVERSIDADE FEDERAL FLUMINENSE, Rua Outeiro s/n Centro Niterói, Brazil.

Introduction: Previous data showed that ATP is capable to induce cellular death in mixed cultures of retina of chicken embryos, but not in purified cultures of neurons of 7 embryonic days and 2 days in cultivation (E7C2). However that the induced death would be through the activation of P2X7 receptors, in this work, we decided to study in the middle of cultivation of the mixed cultures accumulate him/it of some death agent, or also some mechanism in the death induction for P2X7. Materials and Methods:. Western blot was made of purified cultures of neurons and mixed cultures of retina of the chicken embryo. The determination of the presence of the P2X7 receptor in the glial cells or neurons, was certain through imunocitochemistry for couple demarcation for P2X7/2 M6 or P2X7/TUJ. TUNEL was made for the death determination. The effect of MK-801, antagonist of NMDA receptors, as well as DTT (1,4′ dithiolthreitol), inhibitor of reactive oxygen species (ROS) was evaluated on the cellular death induced by ATP through of MTT. Half conditioned of mixed (E7C2) cultures it was applied in cultures of neurons and soon treated with ATP 3 mM, and continuously being evaluated the cellular viability by MTT. Results. In mixed cultures, the P2X7 receptors would be located just in neurons cells, this seen through the imunocitochemistry. The western blot showed the presence of P2X7 so much in mixed cultures as in purified of neurons. Demarcation for TUNEL was seen alone in neurons and increased in mixed cultures treated with ATP 3 mM (12,72 ± 0,28 vs controls 9,46 ± 1,04 n = 2 p < 0.05). Pre-incubation of the cultures with MK-801 (50 μM) and treatment with ATP 3 mM, allowed an inhibition of 62% of death induced by ATP (p < 0.05, n = 3). DTT (500 μM) got to inhibit the death in the neurons in a 93% (n = 3, p < 0.05). p < 0.01). Treatment of ATP 3 mM on cultures of purified of neurons previously treated with half conditioned of cultures mixed E7C2 permitierom to induce a death of up to 60%. Conclusions: The results suggest that P2X7 receptors would be present only in neurons cells and that the death induced by ATP 3 mM would be in this cellular type. Also seemingly the death could be caused simultaneously by glutamate and/or production of ROS. Finally these agents could be being accumulated in the half conditioned of the mixed culture, which would have relevant importance in the death induction for ATP.

Key words: death of neurons, P2X7, conditioned medium, mixed cultures.

03-011

Involvement of P2X7 receptor in mycobacterium tuberculosis-mice infection

André Avelino dos Santos Junior¹, Valnês da Silva Rodrigues Junior¹, Robson Coutinho Silva², Diógenes Santiago Santos¹, Maria Martha Campos¹, Fernanda Bueno Morrone¹

¹PUCRS, Pontifícia Universidade Católica do Rio Grande do Sul, Av. Ipiranga, 6681 – Partenon-PortoAlegre/RS-CEP:90619-90;²UFRJ, Universidade Federal do Rio e Janeiro, Av. Pedro Calmon, nº 550 - Rio de Janeiro- RJ CEP 21941-90, Brazil.

Introduction: Tuberculosis (TB) continues to be one of the deadliest diseases in the world. The emergence of multi-drug-resistant strains of M. tuberculosis, the unbearable side effects of the available drugs and the frequent patient non-compliance in completing the therapy have increased the need for the identification of new molecular targets and other pharmacological strategies to treat this disease. It was previously demonstrated that treatment of Mycobacterium bovis (BCG)-infected human macrophages with ATP induced P2X7 receptor-dependent killing of intracellular Mycobacteria (Biswas et al., BMC Immunology, 9, 35, 2008). The aim of this study was to investigate the role of the purinergic P2X7 receptor during Mycobacterium tuberculosis-infection in mice. Methods: Firstly, we have evaluated the expression of P2X7 in the lungs of infected and non-infected animals. Male Swiss mice (6 per group, 25–30 g) were used for these experiments. All the experimental protocols were approved by the Local Animal Ethics Committee (CEUA 09/00094-PUCRS). The infection model was accomplished according to the methodology described by Chambers et al. (Antimicrobial Agents Chemotherapy, 49, 2816, 2005). The animals were anesthetized and received an intravenous injection of 200 ul of a M. tuberculosis suspension (H37Rv strain; 5e8 CFU/ml). Control mice received the same volume of saline. The procedures were carried out in a level III security cabinet. The infection was confirmed in a lung homogenate, by using the specific Ziehl-Neelsen staining. Parafin-embedded lung sections obtained after 28 days of infection were used for both histological and immunohistochemical analysis. As a second approach, we evaluated the response to infection in P2X7 knockout mice (C57BL/6 P2X7(-/-)) and C57BL/6 wild type (WT), and compared the profiles in infected and non-infected groups. After 28 days of infection, these animals were sacrificed and the spleens and lungs were weighed and submitted to clinical evaluation. Results: M. tuberculosis inoculation in Swiss mice resulted in a striking inflammatory response of both lungs, characterized by marked macrophage infiltration and typical TB-related granulomes, according to the Ziehl-Neelsen staining. Of high interest, immunohistochemical analysis revealed a significant increase of P2X7 receptor detection, especially around the granuloma region. This augmentation corresponded to 180 ± 3%, in comparison to control non-infected animals. We have also found that infected WT mice showed a marked increase in the spleen weight, in comparison to non-infected animals. Of high interest, M. tuberculosis-infected P2X7(-/-) mice showed an increase of 56 ± 3% in the spleen weight when compared to infected WT mice. Discussion: The evidence presented herein points out, for the first time, the P2X7 receptor as one target molecule for understanding the pathogenesis of TB. Whether selective agonists or antagonists of this receptor might be useful for improving TB complications remains a matter to be investigated. Financial support: CNPq-INCT-TB, CAPES, BNDES, PUCRS.

Key words: immunohistochemical, Mycobacterium tuberculosis, P2X7.

03-015

The P2 purinergic receptors are increased in the hippocampus of patients with temporal lobe epilepsy: What is the relevance to the epileptogenesis?

¹Rebeca Amorim Padrão; ²Carolina Batista Ariza; ³Mauro Canzian; ²Marimélia Porcionatto; ¹Michelle Gasparetti Leão Araújo; ¹Esper Abrão Cavalheiro; ⁴Henning Ulrich; ⁵Henrique Carrete Jr.; ⁵Ricardo Silva Centeno; ⁵Elza Márcia Yacubian; ¹Maria José da Silva Fernandes

¹Departamento de Neurologia e Neurocirurgia – Disciplina de Neurologia Experimental – UNIFESP – São Paulo, Brazil;²Departamento de Bioquímica – Disciplina de Biologia Molecular – UNIFESP – São Paulo, Brazil;³Departamento de Anatomia Patológica – Incor/USP – São Paulo, Brazil;⁴Departamento de Bioquímica – Instituto de Química, USP – São Paulo, Brazil;⁵Departamento de Neurologia e Neurocirurgia – Disciplina de Neurologia Clínica – UNIFESP – São Paulo, Brazil.

Introduction: Although the involvement of P2 receptors remains speculative in neurodegenerative diseases, our previous experimental data suggests participation of P2X receptors in the pathophysiology of temporal lobe epilepsy (Vianna et al, Epilepsia, 43, 227, 2002; Dona et al., Epilepsy Res.,83, 157–167, 2009, Fernandes et al., The Open Neurosci J., 4:35–43, 2010). In the present work, we have employed immunohistochemistry, Western blot and RT-PCR to study changes in P2X4, P2X7 and P2Y1 in the hippocampus of patients with temporal lobe epilepsy (TLE) associated with hippocampal sclerosis subjected to surgical treatment for seizures control. Methods: Selected patients with TLE (N = 5, age 42 ± 9) had detailed anamnesis, video EEG and MRI, all procedures approved by the Institutional Ethics Committee of the University (Unifesp). Non-epileptic autopsy tissue served as controls (N = 5). Results: Immunoblots and RT-PCR analysis showed up-regulation of all studied purinoceptors in the hippocampus of patients with TLE compared to control tissues. The amount of P2X4, P2X7 and P2Y1 receptor were respectively 1.5, 3 and 2-fold greater than in control hippocampus (p < 0,0001, Student “t” test). Increased number of P2X7, P2X4 and P2Y1 immunostained cell was observed in the hippocampal formation, located specifically in the dentate gyrus, CA1, and CA3. Strong punctate staining for P2X7 was also detected in the stratum lucidum of CA3. Conclusions: Our data indicate that P2X4, P2X7 and P2Y1 receptors are up-regulated in the hippocampus of patients with TLE and these receptors may play a significant role in neuronal firing as regulators of calcium influx, glutamate release and inflammatory processes. The role of these purinoceptors in hyperexcitability and neuronal death is being studied. Financial support: Fapesp-Cinapce, CNPq, CAPES, INNT/MCT, FAP-Unifesp.

Key words: P2 purinoceptors, temporal lobe epilepsy, hippocampus of patients, epileptic process, hyperexcitability, cell death.

03-021

Oxidized ATP alters theleishmania amazonensisinternalization in peritoneal macrophages of C57Bl/6 mice and the ecto-ATPase activity in membrane of this parasite

¹Vanessa Ribeiro Figliuolo, ²André Luis Fonseca de Souza, ¹ Suzana Passos Chaves, ²Roberto Meyer Fernandes, ¹ Robson Coutinho Silva

¹Laboratório de Imunofisiologia – IBCCF,²Laboratório de Bioquímica Celular - Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro/RJ, Brazil..

Introduction: P2X7 Receptor (P2X7R) belongs to purinergic receptors family activated by extracellular ATP. This receptor has an important role described in physiological events as apoptosis and IL-1β release. Recently some studies have been demonstrated its activity in development of diseases caused by intracellular microorganisms. Effects of P2X7R receptor can be inhibit by it antagonist, oxidized ATP (oATP), a Schiff base described by affects the cellular endocytic machinery. Results of our group have demonstrated that oATP has an antimastigote activity in vivo, after the intralesional treatment in BALB/c mice infected by L. amazonensis, what motivated us to study the mechanisms responsible for this oATP acitivity. Methods and Results: Experiments were carried out in vitro to verify whether the oATP alters the entrance of L. amazonensis promastigotes in host cell. For this, peritoneal macrophages of C57BL/6 mice were obtained by peritoneal wash and used in assay after at least 24 h of adhesion. They were plated in 24 wells plate containing sterile glass slide. The macrophages were infected by promastigotes of L. amazonensis (10: 1) for 4 h at 37°C, in presence or not of 0.5 mM oATP. After this time, the cells were washed, stained by Panótico Rápido kit and the internalizated parasites were counted by optic microscopy to calculate the Infection Index. The results demonstrated that oATP significantly decreases the entrance of parasite in host cells (p < 0.05), when it was present. Later, it was verified whether this reduction was a result of an action in ecto-ATPases present in parasite membrane. In these experiments were performed ecto-ATPases activity tests proceeded with [³²P]ATP, after incubation of the parasite with 0.5 mM oATP for different times. We observed that the nucleotide induces an increased activity in the ecto-ATPases in this parasite form, in at least 30 min of incubation with oATP. This was checked by reading in scintillator. Finally, we have done the infection with the same protocol above using L. amazonensis promastigotes previously incubated with 0.5 mM oATP for 1 h followed by washing. We observed that oATP pre-treatment of the parasite increases significantly its capacity of internalization. Conclusion: oATP acts in promastigotes form of L. amazonensis and increases its membrane ecto-ATPase activity, what results in an increase of the parasite internalization. These data corroborate with findings that have been demonstrated the ecto-ATPases as an important virulence factor in L. amazonensis infection (Acta tropica; 115, 2010). However, this nucleotide inhibits the internalization of promastigotes when it is present during the interaction between parasite and host cell, what suggest its action on host cell resistance to infection. Financial Support: FAPERJ e CNPq.

Key words: ecto-ATPase, infection, oxidized ATP (oATP).

03-022

Purinergic receptor expression and its interrelation with nitric oxide production during differentiation of neural stem cells

Claudiana Lameu^1,2, Cleber Trujillo¹, Telma Tiemi Schwindt¹, Luiz Eugênio Mello³, Henning Ulrich¹

¹IQ-USP, Dep.Bioquímica-Inst. de Química-Universidade de São Paulo, Av Lineu Prestes 748 Butantã, São Paulo; Brazil;²CAT-CEPID, Center for Applied Toxinology, Av Vital Brazil 1500,³UNIFESP, Depto de Fisiologia Universidade Federal de São Paulo, Rua Botucatu, 862, Brazil.

Introduction: The brain develops from a specific set of precursor cells which divide within the neural epithelium, migrate to appropriate locations and differentiate into either neurons or glia. These precursors proliferate in vitro as neurospheres and still retain the capacity of multipotent neural stem cells (NSC). In recent years much effort has been undertaken to understand the molecular mechanism of neuronal differentiation. A large number of transcription factors are sequentially expressed during neurogenesis, suggesting the existence of a complex array of transcriptional events that control the course of development and the emergence of specific cell phenotypes, resulting in differentiated cells. However, neuronal differentiation depends not only on intrinsic factors but also on external signals. Recently, the biological effects of extracellular purine nucleotides acting through ionotropic P2X and metabotropic P2Y receptors have been studied in cells and tissues; however the participation of ATP in neuronal differentiation needs yet to be elucidated. The aim of our work was to verify the role of the purinergic system in the differentiation process of NSC isolated from rat fetal telencephalon (E14). We have studied the pattern of purinergic receptors subunit expression along differentiation. Notably, there was a differential expression of P2X2 and P2X6 ionotropic and P2Y6 metabotropic subunit expression together with an increase of nitric oxide (NO) production throughout neural differentiation. The progress of neural differentiation was inhibited in the presence of the NO synthase (NOS) inhibitor L-NAME. Moreover, the addition of L-NAME during resulted in changes of purinergic receptor expression. There was a slight, but significant increase of P2X2 and P2X6 receptor subunit expression while P2X5 receptor expression, characteristic for NOS containing neurons, was 50-fold augmented. The expression of metabotropic P2Y6 receptors, known to be involved in induction of proliferation and differentiation of neural progenitor cells was increased by a factor of 25 when neurospheres had been differentiated in the presence of L-NAME. Further studies will reveal how interference with purinergic receptor activity affects the nitrergic system and neural differentiation.

Key words: neural differentiation, purinergic receptor, neurospheres, nitric oxide.

03-023

Reduction in ATP-induced endothelial nitric oxide synthesis in murine schistosomiasis

Suellen D’Arc dos Santos Oliveira, Luciana Silva do Amaral, Luis Eduardo Menezes Quintas, François Germain Noel, Cláudia Lúcia Martins da Silva¹.

UFRJ, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373, Bloco J, sala J-17, Cidade Universitária – RJ, Brazil.

Introduction: Schistosomiasis, an intravascular disease caused by Schistosoma mansoni, causes morphological and functional vascular changes that reduces endothelial-dependent vascular relaxation in mice (Silva et al. Comp. Biochem. Physiol. 120; 417, 1998; Silva et al. Vasc. Pharmacol. 46; 122, 2007). The objective of this study was to evaluate nitric oxide (NO) production by murine endothelial cells in response to ATP. Methods: Primary endothelial cells culture: Mesenteric vessels of control and S. mansoni-infected mice were used for culture as previously described (Silva et al. Br. J. Pharmacol. 151; 195, 2007). The production of NO was measured in confluent cells through fluorimetric assay using the probe DAF-FM (2.5 μM) in absence (basal) and presence of 100 μM ATP. Data were expressed as % over basal. Western blotting: Confluent endothelial cells were lysed, centrifuged and 20 μg of protein were loaded on SDS-PAGE gels (7.5% and 12%). After electrophoresis, proteins were transferred to nitrocellulose membrane, incubated for 1 h with non-fat milk (5%) followed by primary (polyclonal anti-eNOS, 1:300, or monoclonal anti-caveolin-1, 1:3,000, Santa Cruz Biotechnology) and peroxidase-conjugated secondary antibodies (anti-rabbit IgG, 1:500, Santa Cruz Biotechnology). The bands were revealed by ECL (Enhanced Chemiluminescence) and quantified by Image J software. Results and discussion: In infected animals the ATP-induced production of NO was smaller (-6.1 ± 5.83%; n = 15 replicates using 4 cultures obtained from different animals) than in the control group (27.15 ± 9.86%; n = 18 replicates using 4 cultures obtained from different animals), which corroborates previously functional data of a reduced endothelium-dependent vascular relaxation (Silva et al. Vasc. Pharmacol. 46; 122, 2007). Next we evaluated the expression of endothelial nitric oxide synthase (eNOS) and its inhibitory protein, caveolin-1 (CAV-1). The expression of eNOS was reduced in endothelial cells from infected animals, with no alteration of CAV-1 expression, suggesting that the reduced NO production may be related to the reduced eNOS expression. Furthermore an alteration in the purinergic signaling pathway cannot be discarded by now. CONCLUSION: In schistosomiasis there is a reduction of both NO production induced by ATP and of eNOS expression, suggesting that the infection probably leads to an endothelial dysfunction that compromises the physiological response to purinergic signaling. Financial Support: CAPES, CNPq.

Key words: ATP, endothelial dysfunction, nitric oxide, schistosomiasis.

03-024

P2 purinergic and kinin-B2 receptors modulate neurosphere differentiation

Cleber Augusto Trujillo¹, Telma Tiemi Schwindt¹, Claudiana Lameu², Henning Ulrich¹

¹IQ-USP, Instituto de Química da Universidade de São Paulo, Av. Prof. Lineur Prestes, 748 - São Paulo,Brazil;²CAT/CEPID, Center for Applied Toxinology – Inst. Butantan, Instituto Butanta, São Paulo, Brazil.

Introduction: Kinins generated upon proteolytic cleavage of low and high molecular weight kininogens by kallikreins were originally characterized as vasoactive oligopeptides participating in control of arterial blood pressure and inflammation. Nevertheless, emerging evidence suggests that bradykinin (Bk) and other kinins are stored in the central nervous system and act as neuromediators in the control of nociceptive response. Functions of Bk and its receptor were further characterized during in vitro differentiation of neural stem cells isolated from fetal rat telencephalon (E 14) by calcium imaging and flow cytometry. These cells proliferated as neurospheres, and their differentiation into neuronal and glial phenotypes closely resembled events occurring during in vivo development. Kinin-B2 receptor activity, determined by single-cell calcium imaging, and liberation of BK into the culture medium suggested the existence of an autocrine loop during neurosphere differentiation into neurons, astrocytes and oligodendrocytes. The presence of the kinin-B2 receptor antagonist HOE-140 during neurosphere differentiation resulted in inhibition of neuron-specific b-3 tubulin and enolase expression and at the same time an increase in expression of glial-specific GFAP, indicating that neurogenesis depended on Bk-induced receptor activity. In agreement with these data, neurogenesis was augmented in the presence of Bk or captopril preventing Bk degradation. Chronic HOE-140 treatment also diminished M1–M4 muscarinic, P2X5, P2X6, P2Y1, P2Y2, P2Y4 and P2Y6 purinergic receptor as well as eNOS and nNOS expression in differentiating neurospheres. Besides, alterations in the expression levels of purinergic receptors have also been found in whole brain extracts of kinin-B2 receptor knock-out mice and in differentiated P19 embryonal carcinoma cells. We conclude that Bk-induced signaling is essential for early fate determination whether a neural stem cell will undergo neuronal or glial differentiation as well for the specification of neurotransmitter receptor expression in differentiated cells.

Key words: Bradykinin, Neurogenesis, Purinergic Receptors.

03-025

Adenosine triphosphate at the PVN level increases sympathetic nerve activity in a dose-dependent manner

Hildebrando Candido Ferreira Neto¹, Song Tieng Yao², Vagner Roberto Antunes¹

¹ICB - USP, Instituto de Ciências Biomédicas - Universidade de São Paulo, Av. Prof. Lineu Prestes, 1524 - Cidade Universitária - Butantã - São Paulo – SP, Brazil;²BHI - UB, Bristol Heart Institute - University of Bristol, Whitson Street, Bristol BS1 3NY, U.S.A.

Introduction: The paraventricular nucleus of hypothalamus (PVN) plays a pivotal role in the control of autonomic function that involves a complex neuronal connection with both the rostroventrolateral medulla (RVLM) and the sympathetic preganglionic neurones of the intermediolateral cell column (IML) in the spinal cord. Studies have demonstrated that PVN neurons projecting to RVLM express P2X purinoreceptors and, via these connections, ATP could be released by neurons or glia to control sympathetic outflow. The aim of the current study is to investigate the effect of ATP injected into PVN on lumbar sympathetic nerve activity (LSNA) in an in situ rat preparation. METHODS AND RESULTS: Male Wistar rats (50–70 g) were deeply anesthetized with halothane, decorticated and arterially perfused with a Ringer’s solution containing Ficoll (1.25%) or Polyethylene glycol (PEG – 1.5%) at 31oC. Recordings of phrenic (PNA) and lumbar sympathetic nerve activities (LSNA) as well as heart rate (HR) and perfusion pressure (PP) were monitored (for details see J Physiol 576, pp 569, 2006). Three-barrelled glass pipettes was filled with L-glutamate (10 mM), ATP (10, 25 and 50 mM) and Evans’ blue (2%) and the volume injected was 100 nL determined by viewing the movement of the meniscus through a binocular microscope fitted with a precalibrated eyepiece reticule. The pipettes were placed in the PVN according to the stereotaxic coordinates from superior colliculus previously established (2.7 mm rostral, 0.3 mm lateral to midline and 3.4 mm below the brain surface). PVN injection sites were marked with Evans’ blue and only data in which the injections were confirmed to be within the PVN were analyzed in this study. A functional test was made by injection of L-glutamate that increases LSNA. Unilateral injection of ATP (10 mM) into the PVN (n = 6) elicited a peak of increasing in basal LSNA (40%) in the first second and 20% five seconds later. LSNA returned to basal values 20 s after ATP injection. Furthermore, injection of ATP at 25 mM induced an augmentation in baseline values of LSNA (68%) in the first second; five seconds later LSNA was still elevated (53%). This increase was maintained for 90 s after the injection, when LSNA returned to basal values. ATP (50 mM) injected into the PVN increases 100% LSNA above basal values in the first second following the injection (n = 3) and returned to the baseline 2 min later. Any significant change was observed in the PNA and HR after ATP injected into the PVN. CONCLUSION: Exogenous ATP applied to the PVN elicits an increase in sympathetic nerve activity in a dose-dependent manner, which suggests that this purine plays a functional role in the control of autonomic function; however elucidation of its precise role and the purine receptor subtypes involved await further investigation. FINANCIAL SUPPORT: CAPES, CNPq and FAPESP.

Key words: adenosine triphosphate, paraventricular nucleus, purinergic receptors, sympathetic activity.

03-026

The absence of P2X7 receptor changes the lesional profile ofLeishmania amazonensisinfection, but does not alter the parasite burden

Suzana Passos Chaves^1,2, Vanessa Ribeiro Figliuolo¹, Herbert Leonel de Matos Guedes², Bartira Rossi Bergmann², Robson Coutinho Silva¹

¹UFRJ, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho 373 Bloco G sala17,CCS, Ilha do Fundão²UFRJ, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho 373 Bloco I sala 38/2 CCS, Ilha do Funda, Brazil.

Introduction: Leishmania parasites have evolved different mechanisms to penetrate and survive inside the macrophages. Recently, we demonstrated the importance of macrophage P2X7 receptor (P2X7R) on the intracellular elimination of Leishmania amazonensis (Microbes and Infection, 11,10–11; 842–849, 2009), In this work, we evaluated the role of P2X7R in cutaneous leishmaniasis using P2X7R wild type (WT) and knockout (KO) C57Bl/6 mice. Methods and Results: P2X7R-WT and KO were infected with 2 × 106 promastigotes of GFP-transfected L. amazonensis in the footpad. The lesion size was measured every 3–4 days. After 78 days, the animals were euthanized and the infected paws and draining popliteal lymph nodes (LNs) were removed for fluorimetric analysis of parasite loads and lymphocyte activation. We found that the lesion onset was delayed in animals lacking P2X7R, but on day 78 of infection these animals had bigger lesions than control mice, although their parasite load in the footpads was similar to WT controls (4680 ± 743 and 5921 ± 695 UF, respectively). Also, the production of IFNγ was significantly higher in KO than in WT mice (4,3 ± 0,6 and 1,2 ± 0,6 ng/ml), whereas the production of IL-10 was similar (0,7 ± 0,5 and 0,7 ± 0,4 ng/ml). Last, we analyzed the proliferation of lymphocytes from infected and uninfected mice in response to Con A using MTT assay. The absence of P2X7R diminished the capacity of lymphocytes to respond to the mitogen in both infected and uninfected mice (1805 ± 621 vs 3766 ± 45 and 1300 ± 579 vs 2248 ± 181 OD, respectively). Conclusion: These results indicate that P2X7R are required for the optimal proliferative response of lymphocytes, which can account for the slower lesion development in KO mice. At the end of the experiment, the parasite burdens between mice P2X7R-WT and KO were similar, even with the bigger lesion in P2X7R-KO animals that may be due to IFNγ-associated inflammatory reaction. Financial Support: CNPq, CAPES and FAPERJ.

Key words:Leishmania amazonensis, Leishmaniasis, P2X7 receptor

03-027

Mechanical lesion of monolayer cultures of chick embryo retinal cells: role of P2 receptors in the growth of glial cells.

Mariana Siqueira de Oliveira, Karina Lima Tosto, Guilherme Rapozeiro França, Ana Lúcia Marques Ventura

Neurobiology Department, Universidade Federal Fluminense (UFF) – Niterói, RJ – Brazil

Introduction: After injury, the Müller glia can proliferate and undergo morphological and gene expression changes through a process known as reactive gliosis (Nature Neurosci. 3(9), 2000). In this work, we investigated nucleotide-dependent cell proliferation and morphological changes after mechanical injury of retinal cell monolayer cultures. Methods and Results: Retinal cell cultures from 8-day-old embryos (E8) cultivated for 7 days (E8C7) were injured with a micropipette tip, generating a region devoid of cells. Significant cell overlaying of the injured area was detected between the 1st and 4th day after injury. In days 1, 2, 3 and 4 after injury, the areas devoid of cells in mm² × 10² ± EPM were 22.38 ± 0.99, 18.11 ± 0.87, 13.56 ± 0.69, 7.57 ± 0.89 e 5.35 ± 1.24, respectively. Three days after injury in E8C7 cultures, only cells with glial morphology were detected in the area of lesion. Treatment of injured cultures with apyrase (5 U/mL), an enzyme that promotes hydrolysis of ATP to AMP, significantly inhibited cell growth over the cell-free area by 60, 57 and 73%, after 1, 2 and 3 days of treatment, respectively. Treatment of injured cultures at E8C7 with P2 receptor antagonists also resulted in the inhibition of cell growth over the lesion after 3 days. The areas devoid of cells, in mm² × 10² ± EPM were: Control = 3,37 ± 0,29; suramin = 13,4 ± 0,61; PPADS = 7,95 ± 0,90; RB-2 = 15,9 ± 0,37. An increase in the incorporation of BrdU was observed in cultures subjected to injury. In % nuclei BrdU⁺ / DAPI⁺ ± EPM: Uninjured cultures = 100 ± 9.12; Injured area = 310 ± 23.3; 5 mm distant from injured area = 156 ± 13.44. This increase was inhibited by the antagonist RB-2, both at the edge of area free of cells, as in 5 mm distant areas (number of cells BRDU⁺ / DAPI⁺ ± EPM: Control = 0.03 ± 0.0027; 5 mm = 0.045 ± 0.005; Injured area 0.09 ± 0.007; 5 mm + RB-2 = 0.027 ± 0.002; Injured area + RB-2 = 0.04 ± 0.006; n = 2). In addition to P2 antagonists, dantrolene (50 μM), an inhibitor of intracellular calcium mobilization, was also able to inhibit cell growth over the area of lesion. The areas devoid of cells in mm² × 10² ± EPM were: Control, Day 0 = 21,60 ± 0,3883; day 1 = 16,43 ± 0,2779; day 2 = 7,365 ± 0,3012; day 3 = 1,646 ± 0,2361; Dantrolene, Day 0 = 22,73 ± 0,2503; day 1 = 19,44 ± 0,4121; day 2 = 15,30 ± 0,4767; day 3 = 7,577 ± 0,6322; n = 4). Experiments of immunocytochemistry revealed a consistent labeling of P2Y1 receptors in glial cells located in the area of lesion in cultures subjected to injury at E8C7 and cultured for 2 days. Conclusions: These results suggest that injury of retinal cell cultures in the form of monolayer is able to induce the growth and proliferation of glial cells over the injured area through a mechanism dependent on activation of nucleotide receptors and intracellular calcium mobilization. The participation of P2Y1 receptors in the growth of glial cells over the injured area will be further investigated.Financial support: CNPq, FAPERJ, PROPPi-UFF, Capes.

Key words: Chicken retina, cellular lesion, P2 receptor, proliferation.

03-028

ATP and oxATP inhibit HIV-1 replication in human primary macrophages

Julieta Schachter¹, Victor Barreto-de-Souza², Renato Santana de Aguiar³, Dumith Chequer Bou-Habib², Pedro Muanis Persechini¹

¹IBCCF, Instituto de Biofisica Carlos Chagas Filho, Av Carlos Chagas s/n, Cidade Universitaria, Fundão. Rio de Janeiro, Brazil²Fiocruz, Fundação Oswaldo Cruz/Fiocruz, Av. Brazil, 4365 - Manguinhos, Rio de Janeiro³IB, UFRJ, Instituto de Biologia, UFRJ, Av. Carlos Chagas Filho s/n, Cidade Universitaria, Fundão, Brazil.

Introduction: Macrophages play a critical role in HIV-1 infection because they produce virus continuously, functioning as an HIV-1 reservoir. Extracellular nucleotides are important regulators of the immune response. An increase in extracellular ATP (eATP) is seen in situations of tissue injury or microbial invasion. In addition, eATP can induce the death of intracellular pathogens, such as Mycobacterium, Chlamydia and Leishmania, via activation of P2 receptors. Objectives: In this work we investigated the effect of eATP and oxidized ATP (oxATP) on HIV-1infection in macrophages. Material and Methods: Human monocyte-derived macrophages (MDM) were obtained from peripheral blood mononuclear cells (PBMC) obtained by density gradient centrifugation from buffy coat preparations of healthy blood donors. PBMCs were plated in 48-well culture plates and maintained at 37oC in 5% CO2 during 7 days for monocyte differentiation to macrophages. MDM were then infected with HIV-1 using 10 ng/mL HIV-1 p24 antigen and, after 3 h excess virus was washed out, fresh medium was added and infected macrophages were maintained under standard culture conditions. After or during infection, MDM were treated or not with eATP or oxATP. In some experiments, HIV-1 virions were pre-treated with eATP or ATPox prior to cellular infection. To measure HIV-1 production, supernatants and cells were collected, and HIV-1 p24 antigen was assessed using a commercial ELISA kit (ZeptoMetrix Co., Buffalo, NY). To evaluate the ATP and oxATP effects in the early steps of HIV infection (until HIV genome integration), human osteosarcoma (HOS) cells expressing hCCR5, hCXCR4 and hCD4 were infected with NL43-Luc viruses. Cells were seeded in 6-well dishes in culture medium and infected with luciferase reporter virus (50 ng of p24) in a total volume of 0,5 ml, treated or not with eATP or oxATP by 5 h. After 48 h of culture, 100 μl lysates were prepared and luciferase activity in 20 μl was measured by using commercially available reagents (Promega). Results and Discussion: We analyzed HIV-1 replication at 10 days after infection in cells treated with eATP or oxATP (20, 100 e 500 μM). At 100 μM, eATP and oxATP exerted a pronounced inhibition of HIV-1 replication. Viral replication at 10 days was also inhibited when either macrophages or virus were pre-treated with 500 μM of oxATP or eATP for 3 h prior to the infection. These results suggest that other non-P2 associated phenomena are involved in the inhibition of HIV-1 replication by eATP and oxATP. We also investigated whether nucleotide treatment affects early steps of HIV infection using a luciferase infectious clone. We observed that oxATP but not eATP inhibited HIV-1 cDNA integration into the host cell genome. We conclude that the effects of these nucleotides on virus replication are mediated by distinct mechanisms, which are currently under investigation.

Key words: ATP, oxATP, HIV, macrophage.

03-029

Estimation of the channel diameter formed by the consensus segment – part of the TM2 domain - of the P2X7 receptor in planar lipid bilayer

Cristina Alves Magalhães de Souza, Pedro Celso Nogueira Teixeira, Dinarte Neto Morreira Ferreira, Luiz Anastacio Alves

FIOCRUZ, Fundação Oswaldo Cruz, Rua Leopoldo Bulhões 1480-Manguinhos CEP: 21041-21, Brazil.

Introduction: The P2X₇ receptors (P2XRs) are ATP activated cationic channels, which have a unique structure among the P2X family due to their longer carboxyl terminal in the intracellular milieu. The P2X₇Rs show uncommon biophysical proprieties, such as their capacity to change from a low conductance pore (∼ 10 pS) to a non-selective large conductance pore (∼ 400 pS), which allows for the passage of 900 Da molecules, in response to repeated or prolonged agonist applications. The P2X₇Rs are mainly expressed in immune and epithelial cells and have been associated to some pathologies, such as cystic fibrosis and tuberculosis. Furthermore, the P2X₇ receptors take part in physiological events, with cytokine secretion and apoptosis being two examples. The P2X₇Rs show a trimeric arrangement, with two transmembrane domains, an intracellular N and C-terminals space facing the cytoplasm, and a large extracellular loop. However, at this point in time, there has been no way to obtain their structural resolution, neither with RMN nor X-ray crystallography. The P2X₇ structural resolution would facilitate the creation of new agonists and antagonists, which would be useful in basic and clinical research. In clinical research, the P2X7Rs have been the target of studies on the treatment of cystic fibrosis, tuberculosis and myeloid leukemia. As such, UTP analogs are already used in cystic fibrosis treatment, in phase 3 trials. Moreover, other P2Xs antagonists analogs that have already been developed, such as Prasugrel^®, Ticlopidin^® and Clopidogrel^®, which interfere in platelet aggregation, have been used as anti-thrombotic drugs. In vitro assays have shown that different ATP concentrations were able to induce apoptosis and allowed for the control of the cellular cycle of human cells in myeloid leukemia. Our group has been working with a synthetic peptide consisting of a part of the TM2 dominion of P2X₇R (peptide ADSEG), which showed similar P2X₇ channel activity both in artificial lipid bilayers and patch-clamp. The ADSEG ion channels obtained were cation-selective, voltage-independent and with a probability of opening similar to that of the P2X₇R. The aim of this work is to estimate the pore diameter of ADSEG peptide, in artificial bilayer systems, and to compare the data to the P2X₇ in different approaches: 1) Measuring the channel conductance in the presence of different ions, which have different hydration radiuses, 2) Through the equation G = s p r2 /l for an ohmic conductor (where G = channel conductance; s = KCl buffer conductance ; l = membrane thickness), 3) Through the channel conductance cut off measured when adding PEGs of different molecular weights. After the addition of different ions with different hydration radiuses we observed that when adding ions with radiuses above 4 Å the conductance was approximately zero, with this result suggesting use of a pore diameter <4 Å. The “r” estimative used throughout the above equation shows a 3.5 Å pore diameter for the channels. However, PEG additions showed conflicting values when compared to previous approaches. The estimated value for the pore diameter obtained with a peptide ADSEG is similar to the P2X₇ pore diameter described in the literature.

03-030

Vesicular release of ATP from müller cells of the embryonic chick retina in culture

Erick Correia Loiola, Ana Lúcia Marques Ventura

Departamento de Neurobiologia, Univ. Fed. Fluminense, Niterói/R, Brazil.

Introduction: Signaling between glial cells and neurons has been demonstrated in cerebral slices and gliotransmitters released such as ATP can modulate synaptic transmission in CNS, including the retina. Cortical astrocytes are capable of releasing ATP from acidic vesicles by a calcium-dependent process. In this study, we investigated the effect of glutamate receptor activation, KCl-induced depolarization and intracellular calcium rise on vesicular release of ATP in cultured Müller cells of the embryonic chick retina. Methods: Exocytosis of ATP-filled vesicles was estimated using a modification of the method of Bodin and Burnstock (J. Cardiov. Pharmacol. 38 (6): 900, 2001). Glia enriched cultures, obtained from 8-day-old chick embryo retinas, were incubated, for 5 min, at 37°C, with quinacrine (5 μM), a fluorescent dye known to selectively label vesicles with high levels of ATP. Cultures were washed with saline, stimulated and the fluorescence analyzed by microscopy. The release of ATP by the cells was estimated by luminescence, using the ATP determination kit of Invitrogen. Results: Quinacrine staining revealed fluorescent vesicles in the soma of Müller cells that were not observed when cultures were pre-treated with bafilomycin A1 (1 μM). Whilst there was no modification in quinacrine staining for at least 10 min of incubation of the cells in Hanks’ solution, their treatment with KCl (50 mM), glutamate (1 mM) or kainate (0.1 mM) resulted in reduced intracellular levels of quinacrine fluorescence at the same period of time. Treatment with NMDA had a very discrete effect but glutamate-induced decrease in quinacrine staining was inhibited by both DNQX (50 μM) and MK-801 (50 μM). Treatment with ionomycin (5 μM) induced a reduction in intracellular quinacrine levels after 10 min, an effect that was blocked by co-incubation with EGTA (1 mM). The KCl-induced quinacrine staining reduction was blocked by the calcium chelator BAPTA-AM (30 μM). Luminescence assays for ATP detection showed that KCl (50 mM), glutamate (1 mM) or kainate (0.1 mM), but not NMDA (0.1 mM) induced a significant release of this nucleotide in enriched glial cell cultures after 5 min of incubation (% of control: KCl = 246 ± 51%; glutamate = 197 ± 19%; kainate = 204 ± 29 NMDA = 116 ± 22%; p < 0.001, n > 3). Incubation with bafilomycin A1 (1 μM) and BAPTA-AM (30 μM) blocked the release of ATP in response to glutamate (% of control: glutamate + bafilomycin A1 = 92 ± 22%; glutamate + BAPTA-AM = 86 ± 17%). A similar effect was achieved by incubation with DNQX (50 μM) and MK-801 (50 μM) (% of control: glutamate + DNQX = 94 ± 25%; glutamate + MK-801 = 57 ± 15%; p < 0.01; n > 5). Conclusions: These results suggest that cultured glial cells from embryonic chick retina present ATP-containing vesicles that can be released in a calcium-dependent way by activation of glutamate receptors and KCl depolarization. Both NMDA and non-NMDA glutamate receptors seem to be involved in the release of ATP-containing vesicles, although NMDA alone could not induce ATP release. Financial Support: CNPq, Faperj, Proppi- UFF, Pronex-MCT.

03-031

Role of P2X receptors and glia in the synaptic transmission of NTS neurons projecting to RVLM

Daniela Accorsi-Mendonça, Leni Gomes Heck Bonagamba, Ricardo Mauricio Xavier Leao, Benedito Honorio Machado

USP, Universidade de São Paulo, Av. Bandeirantes, 390, Ribeirão Preto, Brazil.

Introduction: The peripheral chemoreflex activation produces autonomic and respiratory adjustments. The NTS is the primary site of peripheral chemoreceptor afferents and NTS neurons projecting to RVLM are related to the sympathoexcitatory component of this reflex. In a previous study we demonstrated that ATP modulates the spontaneous and evoked glutamate release on these NTS neurons. Herein, using a pharmacological approach, we evaluated the subtypes of purinergic receptors involved with this modulation. Adults Wistar rats received microinjection of DiI tracer into RVLM and 24 h later the labeled NTS neurons projecting to RVLM were identified in transversal brainstem slices. Spontaneous and TS evoked excitatory pos-synaptic currents (sEPSCs and TS-eEPSCs) were recorded using whole cell patch clamp. PPADS (100 μM, P2X1/2/3/5/7 receptor antagonist) decreased the frequency of sEPSCs (control: 3.39 ± 0.1 vs PPADS: 1.21 ± 0.1 Hz, n = 7), but did not change the amplitude or half-width of events. TNP-ATP (1 μM, P2X1/2/3/4/7 receptor antagonist) also decreased the frequency of currents (control: 4.3 ± 1.1 Hz vs TNP-ATP: 1.6 ± 0.5 Hz, n = 8) and did not alter the amplitude or half-width of events. FAC (1 mM), an inhibitor of glia, reduced the sEPSCs (control: 2.1 ± 0.33 Hz vs FAC: 1.2 ± 0.43 Hz, n = 7). PPADS, TNP-ATP and FAC also decreased the amplitude of TS-eEPSC (23%, 32% and 42% respectively). These data suggest that glia is involved in the endogenous ATP release and also that modulation of glutamate release onto NTS neurons projecting to RVLM is due to the activation of P2X1, P2X2, P2X3 or P2X7 subtypes receptor. Supported by FAPESP and CNPQ.

Key words: chemoreflex, NTS, glia, P2 receptor, patch clamp.

03-033

Purinergic signaling is altered in rostral ventrolateral medulla after chronic intermittent hypoxia in rats

Daniel Zoccal¹, Juan Pablo Huidobro-Toro², Benedito Machado¹

¹USP, School of Medicine of Ribeirão Preto,University of São Paulo, Ribeirão Preto, Brazil;²PUC, Faculty of Biological Sciences, P. Catholic University, Santiago, Chile

Introduction: Hypertension and sympathetic overactivity observed after chronic exposure to intermittent hypoxia (CIH) in rats involve alterations in neurochemical mechanisms underlying central coupling of respiratory and sympathetic activities. In the present study, we evaluated possible alterations in purinergic signaling in the rostral ventrolateral medulla (RVL), a critical region controlling respiratory and sympathetic activities, of juvenile rats submitted to CIH. Methods and Results: Juvenile rats (P19-21) were submitted to chronic intermittent hypoxia (CIH, 6% O2 for 40 s, every 9 min, 8 h/day) for 10 days while control rats were maintained in normoxia (control). On the 11th day using the in situ working heart-brainstem preparation, changes in thoracic sympathetic (tSN), abdominal (Abd) and phrenic nerve (PN) activities were recorded in response to ATP microinjections (1, 10 and 50 mM) into the RVL of CIH (n = 8) and control rats (n = 8). In separate groups of CIH (n = 8) and control rats (n = 8), the density of P2X1, P2X3, P2X4 and P2Y2 receptors were examined in RVL punches by using the western blot technique. Microinjections of ATP into the RVL evoked similar responses of excitation of Abd and reduction of PN frequency in both groups. On the other hand, microinjections of ATP into RVL caused larger responses of tSN excitation in CIH rats than in controls (P < 0.01). Consonant to the functional changes, the density of P2X3 and P2X4, but not P2X1 or P2Y2, increased 20% (P < 0.05) in CIH rats in comparison to control rats. Conclusion: Altogether, these findings indicate that purinergic mechanisms are up-regulated in the RVL of rats submitted to CIH, which may contribute to the changes in respiratory and sympathetic activities observed in this experimental model. Financial support: FAPESP and CNPq (Brazil); FONDAP 13980001, MIFAB and PFB-12 (Chile).

Key words: Intermittent hypoxia, Sympathetic activity, Respiration, ATP

03-035

The role of P2X7 receptor in streptozotocin inducing diabetes

Flávia Sarmento Vieira, Hayandra Ferreira Nanini, Robson Coutinho-Silva

¹UFRJ, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373 - CCS bl. C sl. C-17, Brazil.

Introduction: Type 1 diabetes (T1D) is a debilitating autoimmune disease that results from T-cell-mediated destruction of insulin-producing beta-cells in the pancreatic islets. The process of immune cells infiltration begins early before the clinical manifestation of T1D. Macrophages are potent immune regulators and are critical in the development and pathogenesis of autoimmune diabetes. They are said to be the first cell type to infiltrate the pancreatic islet, serve as antigen-presenting cells, and are important as effector cells during diabetogenesis. After infiltration, lymphocytes begin to produce proinflammatory cytokines that destruct mainly beta-cells within the islets of Langerhans. Extracellular nucleotides such as ATP may function as pro-inflammatory and danger signals modulating immune responses. Material and Methods. To study the purinergic signaling in an autoimmune diabetes model we induced autoimmune diabetes in 8 weeks old C57Bl/6 wild type or P2X7R-/- mice by daily injection of 60 mg/Kg streptozotocin (STZ) for 5 consecutive days. After that the blood glucose and body weight were measured twice a week during 5 weeks and animals were killed to perform the histological analysis of the pancreas. In another approach, we treated C57Bl/6 wild type mice with 40 mg/Kg streptozotocin which were treated or not with P2X7-antagonist Brilliant Blue-G (BBG) 45.5 mg/Kg for 5 days and the diabetic status (glycemia and body weight) was followed over time. Results. With respect to STZ-treated animals, We observed that P2X7R-/- mice were resistant to diabetes inducing (P2X7-/- had glucose 286Â ± 71 mg/dL and WT 580Â ± 12 mg/dL on week 4.5, n = 4) and to the body weight loss (P2X7-/- had gain 5Â ± 2% of body weight n = 4 and WT had lost 18Â ± 5% of body weight n = 5 on week 5). We also observed that BBG administration prevented diabetes induction with respect to STZ-treated animals, mice previously injected with BBG + STZ were characterized by 1) lower levels of glycemia, 2) higher levels of body weight, 3) a partial preservation of pancreatic islets with normal morphology, 4) a lower level of pancreatic nitric oxide secretion, and 5) normal production of pancreatic insulin. Overall, these data demonstrate that the absence of P2X7 receptor or the administration of BBG ameliorates the severity of STZ-induced type 1 diabetes. Thus altered P2X7-receptor level and function contribute to T1D pathogenesis and highlight the therapeutic potential of P2X7 receptor antagonists. Grants: CNPq, FAPERJ, PRONEX.

Key words: Diabetes, P2X7 receptor, streptozotocin

03-036

Directed differentiation of neural progenitors into neurons is accompanied by altered expression of P2X purinergic receptors

Telma Tiemi Schwindt¹, Cleber Augusto Trujillo¹, Priscilla Davidson Negraes², Claudiana Lameu², Henning Ulrich¹

¹IQ-USP, Instituto de Química - Universidade de São Paulo, Av. Prof. Lineu Prestes, 748;²CAT/CEPID, CAT/CEPID, Instituto Butantan, Av. Vital Brazil 1500, São Paulo, Brazil.

Introduction: Neural differentiation has been extensively studied in vitro in a model termed neurospheres, which consists of aggregates of neural progenitor cells. Previous studies suggest that they have a great potential for the treatment of neurological disorders. One of the major challenges for scientists is to control cell fate and develop ideal culture conditions for neurospheres expansion in vitro, without altering their features. Similar to human neural progenitors, rat neurospheres cultured in the absence of EGF and FGF-2 for a short period, increase the levels of beta-3 tubulin and decrease the expression of GFAP and Nestin compared to neurospheres cultured in the presence of these factors. In this manuscript we demonstrate significant differences in cell migration after plating and in the expression of neural proteins in rNPC subjected to deprivation of EGF and FGF2, when compared with those cultured in standard conditions. Cell migration after plating is clearly favored in the EFless group. Moreover, immunofluorescence studies performed following 7 days of plating and differentiation revealed increased NPC differentiation into neurons, when neurospheres had been cultured in the absence of growth factors (EFless group), compared to neurospheres from the Ctr group. Flow cytometry analysis showed that, when rNPC are cultured in the absence of FGF2 and EGF for a short period, they express significant higher levels of the neuronal marker beta-3 tubulin. Cultures contained 60.5% of beta-3 tubulin positive cells following short-term mitogen deprivation of rNPC cultures, while cells cultured in standard condition displayed 26.0% of neurons. The percentage of GFAP positive cells decreased from 21.4% in the Ctr group to 12.5% in the EFless group, and the percentage of Nestin positive cells did not significantly change between Ctr and EFless groups, from 11.3 to 14.2%. Western blot experiments confirmed increased beta-3 tubulin and reduced GFAP protein expression in the EFless group. Real-time PCR analysis revealed that beta-3 tubulin gene expression was seven times higher in its expression when compared to the control group. Different from protein expression levels, gene expression of GFAP was four-times enhanced, while mRNA transcription of the nestin gene was reduced by a factor of 1.5. Further studies may now concentrate on the participation of P2X2 and P2X2/6 receptors in the progress of neurogenesis using pharmacological approaches and/or specific suppression of purinergic receptor subunit expression.

Key words: neurogenesis, growth factors, neurospheres, P2X receptors

03-037

Comparative action of P2X7 receptor agonists on human glioma cell lines proliferation

Marina Petersen Gehring¹, Maria Martha Campos¹, Ana Maria Oliveira Battastini², Fernanda Bueno Morrone¹

¹PUCRS, Pontifícia Universidade Católica do Rio Grande do Sul, Av. Ipiranga, 6681 P. 12Csala148;²UFRGS, Universidade Federal do Rio Grande do Sul, Ramiro Barcelos, 2600 – Anex, Brazil.

Introduction: ATP is an important signaling molecule in the peripheral and central nervous system (CNS). Studies have shown that extracellular nucleotides/nucleosides induce proliferation of different glioma cell lines through mechanisms involving P1 and P2 receptors, and treatment with ATP promotes cell cycle progression in human glioma cells. Furthermore, P2X7 expression was found up-regulated under several pathological conditions, including cancer. Recent studies have shown that C6 and U138-MG glioma cells resist to death induced by high concentrations of ATP (5 mM) and BzATP (P2X7 receptor agonist). The present study aimed to compare the effects of P2X7 agonists in the proliferation of human glioma radiosensitive M059J and radioresistant U138-MG cell lines. Methods: Glioma cells were obtained from the American Type Culture Collection (ATCC, Rockville, Maryland, USA). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 15% (U138-MG) and 10% (M059J) fetal bovine serum (FBS), under ideal conditions of cultivation. Cells were plated in 24 and 96 wells in densities of 20 × 103 and 5 × 103 cells/well, respectively. After reaching semi-confluence, cells were treated with ATP (0.5, 3 and 5 mM) and BzATP (5 to 100 μM). To assess cell proliferation, cells were counted in hemocytometer, and cell viability was assessed by the MTT assay, at 24, 48 and 72 h. All experiments were carried out at least three times in triplicate. Data were analyzed by one-way analysis of variance (ANOVA), followed by Tukey–Kramer test. Results: The human glioma line U138-MG presented resistance to death when treated with either ATP or BzATP, whereas the high ATP concentration (5 mM) and the selective P2X7 agonist BzATP, in a concentration as low as 10 μM, significantly diminished the cell viability (61.7% and 17.8%, respectively) of the glioma line M059J. Discussion: Our results showed, for the first time, that treatment with high ATP concentration and BzATP induced cell death of the human glioma cell line M059J. In agreement to previous studies, the human cell line U138-MG showed resistance to death when treated with the same agonists. Our data indicate the possible participation of the receptor P2X7 on the cell death of radiosensitive glioma cells.

Key words: cell proliferation, glioma, M059J cell line, P2X7 receptor , U138-MG cell line

03-038

Inhibition by P2X7 receptors of nucleotide-dependent proliferation of chick embryo retinal cells in culture

Thayane Martins Silva, Ana Lúcia Marques Ventura.

¹Departamento de Neurobiologia, Univ. Fed. Fluminense, Niterói/RJ, Brazil.

Introduction: Activation of P2Y1 receptors by ATP is capable of inducing proliferation of glial cell progenitors in the embryonic chick retina, a response that occurs only during early periods of development of this tissue (Intl. J. Devl. Neurosci., 20:21–27, 2002). Since activation of P2X7 receptors by ATP inhibits the proliferation of astrocytes (J. Neuroci. Res. 86:3096, 2008), we decided to evaluate whether activation of these receptors was also able to modulate ATP-induced proliferation of retinal cells in culture. Methods: Cell proliferation was evaluated by [³ H]-thymidine incorporation assays in retinal cell cultures obtained from 7- or 8-day-old chick embryos and maintained for various periods of time. Results: When retina cell cultures at E8C8 were treated for 48 h with increasing concentrations of BBG, a P2X7 receptor antagonist, a dose-dependent increase in the incorporation of [³ H]-thymidine in cells was observed. Maximal stimulation of ∼ 54% was observed at concentrations equal or greater than 25 μM (control = 100 ± 7%; BBG 25 μM = 154.7 ± 3%, n = 2), and the EC₅₀ was estimated to be 5.3 μM. This increase in [³ H]-thymidine incorporation was also dependent on the duration of BBG treatment. With 10 μM BBG, maximal stimulation was observed after 8 h of treatment (basal = 646.4 ± 124.3 cpm/culture; BBG = 1426 ± 216.5 cpm/culture, p < 0.05, n = 3). Treatment for 48 h with 200 μM oxidized ATP, another P2X7 receptor antagonist, also increased by 72.5% above basal levels the incorporation of [³ H]-thymidine in the cultures. Incubation of the cultures at E8C8 for 24 h with 500 μM ADP + 10 μM BBG promoted an increase of 59% above the basal incorporation of [³ H]-thymidine (basal = 252.2 ± 28.7; ADP + BBG = 402.2 ± 50.8 cpm/culture, n = 4). No effect of UTP was observed. As expected, in cultures of early stages of development (E7C2), the addition of 500 μM ADP induced an increase of 162% in [³ H]-thymidine incorporation. Treatment with increasing concentrations of Bz-ATP, a selective agonist for the P2X7 receptor, completely inhibited the proliferative effect of ADP at this developmental stage of the cells (cpm/culture: basal = 905.1 ± 124.8; ADP = 2373 ± 159; ADP + Bz-ATP 10 μM = 1456 ± 154.7; ADP + Bz-ATP 30 μM = 1118 ± 190.9; ADP + Bz-ATP 100 μM = 692 ± 89.6, p < 0.001, N = 3). No effect of Bz-ATP on the viability of retinal progenitors was detected as no decrease in the radioactivity of cultures that incorporated [³ H]-thymidine before Bz-ATP treatment was observed. Finally, although Bz-ATP reduced the incorporation of [³H]-thymidine induced by ADP in the cultures, no blockade of Bz-ATP inhibition was observed with either BBG or oxidized ATP. Conclusions: These results suggest that activation of P2 receptors by Bz-ATP has an inhibitory effect on the proliferation of late developing glial progenitors induced by activation of P2Y1 receptors. Considering that P2X7 receptor blockade was able to induce proliferation of retinal cells in cultures at more differentiated stages (E8C8), our data suggest that P2X7 are the receptors involved in the decrease of glial proliferation that occurs during development of the embryonic chick retina. However, involvement of other receptor subtypes such as the P2Y11 receptor cannot be discarded, since P2X7 receptor antagonists did not prevent the inhibitory effect of Bz-ATP in early developing cultures. Financial support: Capes, CNPq, PROPPi-UFF, Faperj.

Key words: ATP, development, proliferation, P2X7 receptor, retina.

03-039

Involvement of purinergic mechanisms on respiratory and cardiovascular modulation in different subregions of rostral ventrolateral medulla of awake rats

Davi J. A. Moraes, Bendito H. Machado

FMRP, School of Medicine of Ribeirão Preto/University of São Paulo, Av. Bandeirantes, 3900 - 14049-900 Ribeirão Preto/SP, Brazil.

Introduction: To evaluate the effects of the P2X receptors antagonism in different subregions of RVLM on baseline cardiovascular and respiratory parameters as well as on chemoreflex responses in awake rats. Methods and Results: Under anesthesia rats received bilateral guide-cannulas in direction of RVLM subregions and the femoral artery and vein were cannulated for cardiovascular recordings and chemoreflex activation by KCN. Changes in respiratory parameters were evaluated by the plethysmographic method. Unilateral microinjections of DE50 (0,25 nmol/50 nl) of ATP into the rostral aspect of RVLM (rRVLM; n = 7) caused a reduction of ventilation (VE)(- 780 ± 79 ml/kg/min) and heart rate (HR) (-111 ± 6 bpm) and an increase in mean arterial pressure (MAP)(47 ± 1). Unilateral microinjections of DE50 of ATP into the caudal aspect of RVLM (cRVLM; n = 6) caused a reduction of VE (-790 ± 98 ml/kg/min) and HR (-114 ± 10 bpm) and an increase in MAP (50 ± 9 mmHg). Selective antagonism of purinergic P2X receptors by PPADS (0.25 nmol/50 nl) in the rRVLM or cRVLM produced no changes in the baseline VE, HR and MAP (n = 12). Bilateral microinjections of PPADS into cRVLM produced no significant changes in tachypneic and pressor responses to chemoreflex activation (n = 6). On the other hand, bilateral microinjections of PPADS into rRVLM significantly reduced the tachypneic response (56 ± 4 vs 192 ± 13 cpm; p < 0.001; n = 6) but did not change the pressor response to chemoreflex activation. Conclusions: Activation of purinergic receptors in different VLM subregions elicited the same pattern of the cardiovascular and respiratory responses. In addition, purinergic P2X receptors at the rRVLM level are very important for the processing of the tachypneic component of chemoreflex in awake rats.

Key words: ATP, chemoreflex, ventrolateral medulla, awake rats.

03-041

The Involvement of P2Y Receptors in the control ofT.gondiiInfection in Murine Macrophages

Moreira-Souza, A.C.A., Vommaro, R.C., Coutinho-Silva, R.,

Programa de Imunobiologia, Instituto de Biofísica Carlos Chagas Filho, UFRJ, Rio de Janeiro; RJ Instituto Nacional de Ciência e Tecnologia para Pesquisa Translacional em Saúde e Ambiente na Região Amazônica (INPeTAm/UFRJ, Brazil.

Introduction: The nucleotide G-protein coupled P2Y receptors are involved in several cellular events such as calcium release from intracellular stores and PLC activation. Recently our group has been studying the implication of the P2Y receptors in the context of the pathogens which subvert the host response, such a Leishmania amazonensis. T. gondii is a protozoan parasite known to escape from lisossomal fusion and survive and replicate in the parasitophorous vacuole. Therefore,the study of the P2Y receptors activation in T. gondii interaction with macrophages can be crucial to understand their role in the infection. In vitro experiments were carried out using peritoneal macrophages (MΦ) from Balb /c or Swiss Webster or C57BL / 6 mice plated for 48 h, infected with T. gondii (RH strain) in the ratio 5:1 for 2 h and incubated with 100 μM UTP for 30 min. Treatment with UTP reduced the parasite burden compared with controls (Balbc, Swiss and C57BL / 6, 36 ± 8%, 70 ± 4%, 28 ± 5%, n = 4, respectively). Then, peritoneal MΦ from BALB / c were plated for 48 h, infected with T. gondii for 2 h and incubated with other nucleotides that activate P2Y receptors, namely: ATP, ADP, UDP, all at 100 μM for 30 min. All nucleotides studied reduced the parasitic load significantly (ATP, ADP, UTP and UDP: 32 ± 7%, 24 ± 7%, 26 ± 8% and ± 32%, respectively). Our data suggest the involvement of different P2Y receptors in the control of T. gondii infection.

Key words: P2Y Receptors, T. gondii , UTP.

03-042

Lysosomes and reactive oxygen species pathways in macrophages: the possible routes forToxoplasma gondiielimination after ATP incubation

Gladys Corrêa, Camila Marques-da-Silva, Aline Cristina de Abreu Moreira-Souza, Rossiane Claudia Vommaro, Robson Coutinho-Silva

UFRJ, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373.Bl C. Sl C17, Brazil.

Introduction: The P2X7 receptor, a purinergic receptor activated by ATP, is widely involved in immune response and inflammatory processes (J. Inflamm. 4–5, 2007). A wide range of works has demonstrated the involvement of the P2X7 receptor with pathogen clearance (Purinergic. Signal. 3; 83, 2007; Purinergic. Signal. 5; 197, 2009). The parasite survival and replication within host cell can occur by evolutionary strategies developed to permit the perpetuation, which include the avoidance of reactive oxygen species and the well known inhibition of lysosomal pathway (Purinergic. Signal. 5; 197, 2009). T. gondii tachyzoites successfully invade target cells and develop a hospitable environment, where they acquire nutrients and avoid the host immune response (Kim and Weiss, 2008). The active entrance of this parasite leads to the formation of a membrane-bound parasitophorous vacuole that avoids fusion with the endolysosomal system and maintains a neutral pH (Traffic 4; 581, 2003). Objectives: To identify the possible mechanisms of elimination of Toxoplasma gondii tachyzoites by macrophages after ATP incubation. Methods and Results: Acridine orange stain: infected J774 macrophages stained with 10ug/ml Acridine Orange for 10 min at room temperature (RT), after 22 h of infection, showed no fusion of acidic vesicles with tachyzoites in untreated cells, however after the incubation with 3 mM ATP for 30 min, treated macrophages presented lysosomal fusion with the parasitophorous vacuoles. Anti-LAMP1 and anti-SAG1 co-immunofluorescence: After treatment with ATP or BzATP for 30 min, Balb/c infected macrophages showed a co-localization between LAMP-1 (lysosome associated membrane protein-1) and SAG-1 (surface antigen of T. gondii-1) 6 h post incubation with the nucleotides mentioned above in comparison with the cells untreated. 16 h post ATP or BzATP treatment an increase of positive tachyzoites SAG-1 and LAMP-1 labeling was observed. Reactive oxygen species (ROS) detection: ROS assays evaluated by flow cytometry was performed using 20uM DCFDA (dichlorofluorescein diacetate) solution at 37°C for 30 min. The infection did not reduce ROS production induced by ATP, once that infected macrophages after a pulse of 5 mM ATP produced values of ROS similar to the uninfected cells. Oxide nitric (NO) assay: The supernatant of infected macrophages 16 h post ATP incubation (30 min) was collected and the samples were subjected to the Griess method by spectrophotometry for indirect NO measurement. We observed in our experiments that infected macrophages even after incubation with ATP are not able to release more NO than untreated cells. These results suggest that NO participation it is not essential for T. gondii elimination after ATP incubation. Conclusion: Our results indicate that after ATP treatment, infected macrophages could eliminate the tachyzoites of T. gondii via lysosomal and ROS pathways, but not through NO action. Financial support: FAPERJ, PRONEX, CAPES, CNPq.

Key words: ATP, P2X7 receptors, purinergic signalling, Toxoplasma gondii.

03-044

Leishmania amazonensisinfection induces differential modulation of P2X7-associated pores in macrophages

Mariana Chaves, Camila Marques da Silva, Bartira Rossi Bergman, Pedro Persechini, Robson Coutinho-Silva

UFRJ, Universidade Federal do Rio de Janeiro, Avenida Carlos Chagas Filho, 373 - Bloco C - sala 17 - Ilha do Fundão – RJ, Brazil.

Introduction: Extracellular ATP has been reported as an important signaling molecule in the immune system through its coupling to P2X7 receptor. Once these receptors are activated, they promote several intracellular mechanisms such as apoptosis, release of cytokines like IL-1beta, cell permeabilization, among others. Additionally, the P2X7 receptor is a nonselective ion channel that, when exposed to its agonists for prolonged minutes, induces a pores opening in the plasma membrane. Recently, Schachter et al (J Cell Sci. 121, 3261, 2008) showed that the P2X7 receptor activation induces anions and cations uptake by different mechanisms. Our group has demonstrated the influence of L.amazonensis infection on the P2X7 receptor expression and functionality. So we investigated the effect of L.amazonensis infection in the capture of fluorescence dyes induced by P2X7 receptors activation in murine macrophages. Methods. We used intraperitoneal macrophages of Balb/c or RAW 267.4 macrophage lineage infected or not with L.amazonensis at a ratio of 10:1. These were tested for uptake of fluorescent dyes (assay of permeabilization) in the presence and absence of 5 mM ATP for 15 min at 37°C. We used the dyes 2.5 μM ethidium bromide (EB) and 300 μM sulforhodamine (cationic), 5 mM Lucifer Yellow (LY) and 5 mM carboxiflurescein (anionic). Then, labeled cells were direct counted in a fluorescence microscope (counting was done in five random fields) and also the permeabilization in fluorimeter plate. The graphs were generated and analyzed using the GraphPad Prism 4.0. Results: We observed that anionic and cationic dyes have different patterns of permeabilization when macrophages were infected with L.amazonensis. That is, infection with L.amazonensis positively modulate the uptake of LY (anionic dye) in cells treated with ATP (52% of increase in infected, n = 3). The analysis of carboxyfluorescein uptake in infected cells was also higher (difference between means 46% ± 17, n = 3). However, permeabilization using EB (cationic dye) showed that the infection induced negative modulation of dye uptake (difference between means 62% ± 4, n = 3). In RAW infected cells the sulforhodamine displayed a decreased of dye uptake (difference between means 66% ± 14, n = 3). When we analyzed the permeabilization in macrophages subjected to contact with dead L.amazonensis we did not observe inhibition of the cationic dyes uptake showing the need of active parasite. Conclusion: Our results suggest that activation of P2X7 receptor is indeed associated with more than one type of dye uptake mechanism (at least one for anion and other for cation). Additionally, during infection, concomitantly with a positive modulation of P2X7 receptor, there is a raise of anions uptake in macrophages, while the infection reduce the cationic cell uptake. This study allowed us to better understand the mechanism of drugs entry/extrusion induced by the parasites. Support: CNPq, CAPES, FAPERJ and PRONEX

Key words: P2X7 receptor, Leishmania, pore

03-045

Role of ATP and P2X7 receptor in the CD4+ T CELL response to blood-stageplamodium chabaudimalaria.

Érika Machado de Salles¹, Claudia Augusta Zago¹, Henrique Borges da Silva¹, Jose Maria Alvarez Mosig¹, Robson Coutinho Silva^2,3, Maria Regina D’Império Lima¹

¹USP, Universidade de São Paulo, AV. Profº Lineu Prestes, 1730, São Paulo/SP;²UFRJ, Instituto de Biofísica Carlos Chagas Filho, Av. Carlos Chagas Filho, 373, Ilha do Fundão, Rio de Janeiro/RJ;³INPeTAm/UFRJ, Instituto Nacional de Ciência e Tecnologia , Avenida Carlos Chagas Filho, 373,Ilha do Fundão, Rio de Janeiro/RJ, Brazil.

Introduction: Malaria is responsible for one million deaths yearly, mostly affecting children. The immune system participates in the protection against Plasmodium infection, such as malaria syndromes like anemia, cerebral malaria, metabolic acidosis and systemic shock. Recently, it has been shown that innate immunity is able to detect signals such as ATP and uric acid released by damaged cells or tissues. These danger signs appear to be important to promote the regulation of inflammation after trauma or injury caused by pathogens. However, the physiological relevance of these signals in the immune response and its mechanism are not yet clear. Extracellular ATP has been suggested to act as a costimulatory molecule in T cell response although in high concentrations can lead to cell death. In malaria, it is likely that ATP could be released upon rupture of infected erythrocytes or cells of the vascular endothelium damaged by the action of effector molecules of the immune system. Thus, this study aims to assess the role of ATP and P2X7 receptor in the early CD4+ T cell response to blood-stage P. chabaudi malaria. Methods: C57BL/6 mice (6–8-week old) were infected by intraperitoneal injection with parasitized erythrocytes. Four and 7 days later, spleen cells from infected mice and controls were stained with FITC-conjugated anti-mouse CD4 monoclonal antibodies, subjected to different concentrations of ATP and then evaluated according to permeability using ethidium bromide. Erythrocytes were removed from spleen cell preparations either by lysis buffer (40 m NH4Cl, 4,2 mM Tris base, pH 7.4) or by Percoll gradient separation (70%), to assess whether the ATP released from erythrocytes is able to permeabilize CD4+ T cells. Results: By analyzing ATP-induced cell permeability, we observed that infection by P. chabaudi is capable of up-modulating the P2X7 receptor expression on CD4+ T cells (p < 0.001) and that this modulation is greater during the afternoon, where there is the rupture of erythrocytes. The presence of the P2X7 receptor antagonist Brilliant Blue G (BBG) decreases the cell permeability induced by infection (p < 0.01). In addition, the ATP released from erythrocytes is able to induce permeabilization in CD4+ T cells (p < 0.001). The results are representative of two experiments with three mice. Conclusion: Our results suggest that ATP released by the rupture of erythrocytes during the blood-stage of P. chabaudi induces increase in the expression of P2X7 receptor in CD4+ T cells. The outcomes in this study should contribute to the understanding of the mechanisms involved in the immune response to malaria. Financial support: FAPESP, CNPq and FAPERJ

Key words: ATP, CD4+ T cell, P2X7, Plasmodium chabaudi.